Molecular Responses to Small Regulating Molecules against Huanglongbing Disease
Identifieur interne : 000398 ( Pmc/Corpus ); précédent : 000397; suivant : 000399Molecular Responses to Small Regulating Molecules against Huanglongbing Disease
Auteurs : Federico Martinelli ; David Dolan ; Veronica Fileccia ; Russell L. Reagan ; My Phu ; Timothy M. Spann ; Thomas G. Mccollum ; Abhaya M. DandekarSource :
- PLoS ONE [ 1932-6203 ] ; 2016.
Abstract
Huanglongbing (HLB; citrus greening) is the most devastating disease of citrus worldwide. No cure is yet available for this disease and infected trees generally decline after several months. Disease management depends on early detection of symptoms and chemical control of insect vectors. In this work, different combinations of organic compounds were tested for the ability to modulate citrus molecular responses to HLB disease beneficially. Three small-molecule regulating compounds were tested: 1) L-arginine, 2) 6-benzyl-adenine combined with gibberellins, and 3) sucrose combined with atrazine. Each treatment contained K-phite mineral solution and was tested at two different concentrations. Two trials were conducted: one in the greenhouse and the other in the orchard. In the greenhouse study, responses of 42 key genes involved in sugar and starch metabolism, hormone-related pathways, biotic stress responses, and secondary metabolism in treated and untreated mature leaves were analyzed.
Url:
DOI: 10.1371/journal.pone.0159610
PubMed: 27459099
PubMed Central: 4961454
Links to Exploration step
PMC:4961454Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Molecular Responses to Small Regulating Molecules against Huanglongbing Disease</title><author><name sortKey="Martinelli, Federico" sort="Martinelli, Federico" uniqKey="Martinelli F" first="Federico" last="Martinelli">Federico Martinelli</name><affiliation><nlm:aff id="aff001"><addr-line>Dipartimento di Scienze Agrarie e Forestali, Palermo, Italy</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff002"><addr-line>Euro-Mediterranean Institute of Science and Technology (IEMEST), Palermo, Italy</addr-line></nlm:aff></affiliation></author><author><name sortKey="Dolan, David" sort="Dolan, David" uniqKey="Dolan D" first="David" last="Dolan">David Dolan</name><affiliation><nlm:aff id="aff003"><addr-line>Department of Plant Sciences, University of California Davis, Davis, California, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Fileccia, Veronica" sort="Fileccia, Veronica" uniqKey="Fileccia V" first="Veronica" last="Fileccia">Veronica Fileccia</name><affiliation><nlm:aff id="aff001"><addr-line>Dipartimento di Scienze Agrarie e Forestali, Palermo, Italy</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff002"><addr-line>Euro-Mediterranean Institute of Science and Technology (IEMEST), Palermo, Italy</addr-line></nlm:aff></affiliation></author><author><name sortKey="Reagan, Russell L" sort="Reagan, Russell L" uniqKey="Reagan R" first="Russell L." last="Reagan">Russell L. Reagan</name><affiliation><nlm:aff id="aff003"><addr-line>Department of Plant Sciences, University of California Davis, Davis, California, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Phu, My" sort="Phu, My" uniqKey="Phu M" first="My" last="Phu">My Phu</name><affiliation><nlm:aff id="aff003"><addr-line>Department of Plant Sciences, University of California Davis, Davis, California, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Spann, Timothy M" sort="Spann, Timothy M" uniqKey="Spann T" first="Timothy M." last="Spann">Timothy M. Spann</name><affiliation><nlm:aff id="aff004"><addr-line>California Avocado Commission, Irvine, California, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Mccollum, Thomas G" sort="Mccollum, Thomas G" uniqKey="Mccollum T" first="Thomas G." last="Mccollum">Thomas G. Mccollum</name><affiliation><nlm:aff id="aff005"><addr-line>United States Department of Agriculture-The Agricultural Research Service, U.S. Horticultural Research Laboratory, Fort Pierce, Florida, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Dandekar, Abhaya M" sort="Dandekar, Abhaya M" uniqKey="Dandekar A" first="Abhaya M." last="Dandekar">Abhaya M. Dandekar</name><affiliation><nlm:aff id="aff003"><addr-line>Department of Plant Sciences, University of California Davis, Davis, California, United States of America</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">27459099</idno><idno type="pmc">4961454</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961454</idno><idno type="RBID">PMC:4961454</idno><idno type="doi">10.1371/journal.pone.0159610</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000398</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Molecular Responses to Small Regulating Molecules against Huanglongbing Disease</title><author><name sortKey="Martinelli, Federico" sort="Martinelli, Federico" uniqKey="Martinelli F" first="Federico" last="Martinelli">Federico Martinelli</name><affiliation><nlm:aff id="aff001"><addr-line>Dipartimento di Scienze Agrarie e Forestali, Palermo, Italy</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff002"><addr-line>Euro-Mediterranean Institute of Science and Technology (IEMEST), Palermo, Italy</addr-line></nlm:aff></affiliation></author><author><name sortKey="Dolan, David" sort="Dolan, David" uniqKey="Dolan D" first="David" last="Dolan">David Dolan</name><affiliation><nlm:aff id="aff003"><addr-line>Department of Plant Sciences, University of California Davis, Davis, California, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Fileccia, Veronica" sort="Fileccia, Veronica" uniqKey="Fileccia V" first="Veronica" last="Fileccia">Veronica Fileccia</name><affiliation><nlm:aff id="aff001"><addr-line>Dipartimento di Scienze Agrarie e Forestali, Palermo, Italy</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff002"><addr-line>Euro-Mediterranean Institute of Science and Technology (IEMEST), Palermo, Italy</addr-line></nlm:aff></affiliation></author><author><name sortKey="Reagan, Russell L" sort="Reagan, Russell L" uniqKey="Reagan R" first="Russell L." last="Reagan">Russell L. Reagan</name><affiliation><nlm:aff id="aff003"><addr-line>Department of Plant Sciences, University of California Davis, Davis, California, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Phu, My" sort="Phu, My" uniqKey="Phu M" first="My" last="Phu">My Phu</name><affiliation><nlm:aff id="aff003"><addr-line>Department of Plant Sciences, University of California Davis, Davis, California, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Spann, Timothy M" sort="Spann, Timothy M" uniqKey="Spann T" first="Timothy M." last="Spann">Timothy M. Spann</name><affiliation><nlm:aff id="aff004"><addr-line>California Avocado Commission, Irvine, California, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Mccollum, Thomas G" sort="Mccollum, Thomas G" uniqKey="Mccollum T" first="Thomas G." last="Mccollum">Thomas G. Mccollum</name><affiliation><nlm:aff id="aff005"><addr-line>United States Department of Agriculture-The Agricultural Research Service, U.S. Horticultural Research Laboratory, Fort Pierce, Florida, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Dandekar, Abhaya M" sort="Dandekar, Abhaya M" uniqKey="Dandekar A" first="Abhaya M." last="Dandekar">Abhaya M. Dandekar</name><affiliation><nlm:aff id="aff003"><addr-line>Department of Plant Sciences, University of California Davis, Davis, California, United States of America</addr-line></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Huanglongbing (HLB; citrus greening) is the most devastating disease of citrus worldwide. No cure is yet available for this disease and infected trees generally decline after several months. Disease management depends on early detection of symptoms and chemical control of insect vectors. In this work, different combinations of organic compounds were tested for the ability to modulate citrus molecular responses to HLB disease beneficially. Three small-molecule regulating compounds were tested: 1) L-arginine, 2) 6-benzyl-adenine combined with gibberellins, and 3) sucrose combined with atrazine. Each treatment contained K-phite mineral solution and was tested at two different concentrations. Two trials were conducted: one in the greenhouse and the other in the orchard. In the greenhouse study, responses of 42 key genes involved in sugar and starch metabolism, hormone-related pathways, biotic stress responses, and secondary metabolism in treated and untreated mature leaves were analyzed. <italic>TGA5</italic> was significantly induced by arginine. Benzyladenine and gibberellins enhanced two important genes involved in biotic stress responses: <italic>WRKY54</italic> and <italic>WRKY59</italic>. Sucrose combined with atrazine mainly upregulated key genes involved in carbohydrate metabolism such as sucrose-phosphate synthase, sucrose synthase, starch synthase, and α-amylase. Atrazine also affected expression of some key genes involved in systemic acquired resistance such as <italic>EDS1</italic>, <italic>TGA6</italic>, <italic>WRKY33</italic>, and <italic>MYC2</italic>. Several treatments upregulated <italic>HSP82</italic>, which might help protect protein folding and integrity. A subset of key genes was chosen as biomarkers for molecular responses to treatments under field conditions. <italic>GPT2</italic> was downregulated by all small-molecule treatments. Arginine-induced genes involved in systemic acquired resistance included PR1, <italic>WRKY70</italic>, and <italic>EDS1</italic>. These molecular data encourage long-term application of treatments that combine these regulating molecules in field trials.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Bove, Jm" uniqKey="Bove J">JM Bove’</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jagoueix, S" uniqKey="Jagoueix S">S Jagoueix</name></author><author><name sortKey="Bove, Jm" uniqKey="Bove J">JM Bové</name></author><author><name sortKey="Garnier, M" uniqKey="Garnier M">M Garnier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Folimonova, Sy" uniqKey="Folimonova S">SY Folimonova</name></author><author><name sortKey="Robertson, Cj" uniqKey="Robertson C">CJ Robertson</name></author><author><name sortKey="Garnsey, Sm" uniqKey="Garnsey S">SM Garnsey</name></author><author><name sortKey="Gowda, S" uniqKey="Gowda S">S Gowda</name></author><author><name sortKey="Dawson, Wo" uniqKey="Dawson W">WO Dawson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Folimonova, Sy" uniqKey="Folimonova S">SY Folimonova</name></author><author><name sortKey="Achor, D" uniqKey="Achor D">D Achor</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Albrecht, U" uniqKey="Albrecht U">U Albrecht</name></author><author><name sortKey="Bowman, Kd" uniqKey="Bowman K">KD Bowman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kim, J S" uniqKey="Kim J">J-S Kim</name></author><author><name sortKey="Sagaram, Us" uniqKey="Sagaram U">US Sagaram</name></author><author><name sortKey="Burns, Jk" uniqKey="Burns J">JK Burns</name></author><author><name sortKey="Li, J L" uniqKey="Li J">J-L Li</name></author><author><name sortKey="Wang, N" uniqKey="Wang N">N Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Martinelli, F" uniqKey="Martinelli F">F Martinelli</name></author><author><name sortKey="Uratsu, Sl" uniqKey="Uratsu S">SL Uratsu</name></author><author><name sortKey="Albrecht, U" uniqKey="Albrecht U">U Albrecht</name></author><author><name sortKey="Reagan, Rl" uniqKey="Reagan R">RL Reagan</name></author><author><name sortKey="Phu, Ml" uniqKey="Phu M">ML Phu</name></author><author><name sortKey="Britton, M" uniqKey="Britton M">M Britton</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dandekar, Am" uniqKey="Dandekar A">AM Dandekar</name></author><author><name sortKey="Martinelli, F" uniqKey="Martinelli F">F Martinelli</name></author><author><name sortKey="Davis, Ce" uniqKey="Davis C">CE Davis</name></author><author><name sortKey="Bhushan, A" uniqKey="Bhushan A">A Bhushan</name></author><author><name sortKey="Zhao, W" uniqKey="Zhao W">W Zhao</name></author><author><name sortKey="Fiehn, O" uniqKey="Fiehn O">O Fiehn</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Martinelli, F" uniqKey="Martinelli F">F Martinelli</name></author><author><name sortKey="Reagan, Rl" uniqKey="Reagan R">RL Reagan</name></author><author><name sortKey="Uratsu, Sl" uniqKey="Uratsu S">SL Uratsu</name></author><author><name sortKey="Phu, Ml" uniqKey="Phu M">ML Phu</name></author><author><name sortKey="Albrecht, U" uniqKey="Albrecht U">U Albrecht</name></author><author><name sortKey="Zhao, W" uniqKey="Zhao W">W Zhao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nwugo, Cc" uniqKey="Nwugo C">CC Nwugo</name></author><author><name sortKey="Lin, H" uniqKey="Lin H">H Lin</name></author><author><name sortKey="Duan, Y" uniqKey="Duan Y">Y Duan</name></author><author><name sortKey="Civerolo, El" uniqKey="Civerolo E">EL Civerolo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fan, J" uniqKey="Fan J">J Fan</name></author><author><name sortKey="Chen, C" uniqKey="Chen C">C Chen</name></author><author><name sortKey="Yu, Q" uniqKey="Yu Q">Q Yu</name></author><author><name sortKey="Brlansky, Rh" uniqKey="Brlansky R">RH Brlansky</name></author><author><name sortKey="Li, Z G" uniqKey="Li Z">Z-G Li</name></author><author><name sortKey="Gmitter, Fg" uniqKey="Gmitter F">FG Gmitter</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cong, Q" uniqKey="Cong Q">Q Cong</name></author><author><name sortKey="Kinch, Ln" uniqKey="Kinch L">LN Kinch</name></author><author><name sortKey="Kim, B H" uniqKey="Kim B">B-H Kim</name></author><author><name sortKey="Grishin, Nv" uniqKey="Grishin N">NV Grishin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tyler, Hl" uniqKey="Tyler H">HL Tyler</name></author><author><name sortKey="Roesch, Lfw" uniqKey="Roesch L">LFW Roesch</name></author><author><name sortKey="Gowda, S" uniqKey="Gowda S">S Gowda</name></author><author><name sortKey="Dawson, Wo" uniqKey="Dawson W">WO Dawson</name></author><author><name sortKey="Triplett, Ew" uniqKey="Triplett E">EW Triplett</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Khan, Aa" uniqKey="Khan A">AA Khan</name></author><author><name sortKey="Afzal, M" uniqKey="Afzal M">M Afzal</name></author><author><name sortKey="Qureshi, Ja" uniqKey="Qureshi J">JA Qureshi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhang, M" uniqKey="Zhang M">M Zhang</name></author><author><name sortKey="Powell, Ca" uniqKey="Powell C">CA Powell</name></author><author><name sortKey="Zhou, L" uniqKey="Zhou L">L Zhou</name></author><author><name sortKey="He, Z" uniqKey="He Z">Z He</name></author><author><name sortKey="Stover, E" uniqKey="Stover E">E Stover</name></author><author><name sortKey="Duan, Y" uniqKey="Duan Y">Y Duan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhang, M" uniqKey="Zhang M">M Zhang</name></author><author><name sortKey="Powell, Ca" uniqKey="Powell C">CA Powell</name></author><author><name sortKey="Guo, Y" uniqKey="Guo Y">Y Guo</name></author><author><name sortKey="Doud, Ms" uniqKey="Doud M">MS Doud</name></author><author><name sortKey="Duan, Y" uniqKey="Duan Y">Y Duan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yang, C" uniqKey="Yang C">C Yang</name></author><author><name sortKey="Powell, Ca" uniqKey="Powell C">CA Powell</name></author><author><name sortKey="Duan, Y" uniqKey="Duan Y">Y Duan</name></author><author><name sortKey="Shatters, R" uniqKey="Shatters R">R Shatters</name></author><author><name sortKey="Zhang, M" uniqKey="Zhang M">M Zhang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Akula, N" uniqKey="Akula N">N Akula</name></author><author><name sortKey="Trivedi, P" uniqKey="Trivedi P">P Trivedi</name></author><author><name sortKey="Han, Fq" uniqKey="Han F">FQ Han</name></author><author><name sortKey="Wang, N" uniqKey="Wang N">N Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, W" uniqKey="Li W">W Li</name></author><author><name sortKey="Hartung, Js" uniqKey="Hartung J">JS Hartung</name></author><author><name sortKey="Levy, L" uniqKey="Levy L">L Levy</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pieterse, Cmj" uniqKey="Pieterse C">CMJ Pieterse</name></author><author><name sortKey="Leon Reyes, A" uniqKey="Leon Reyes A">A Leon-Reyes</name></author><author><name sortKey="Van Der Ent, S" uniqKey="Van Der Ent S">S Van der Ent</name></author><author><name sortKey="Van Wees, Scm" uniqKey="Van Wees S">SCM Van Wees</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ramel, F" uniqKey="Ramel F">F Ramel</name></author><author><name sortKey="Sulmon, C" uniqKey="Sulmon C">C Sulmon</name></author><author><name sortKey="Cabello Hurtado, F" uniqKey="Cabello Hurtado F">F Cabello-Hurtado</name></author><author><name sortKey="Tacconat, L" uniqKey="Tacconat L">L Tacconat</name></author><author><name sortKey="Martin Magniette, M L" uniqKey="Martin Magniette M">M-L Martin-Magniette</name></author><author><name sortKey="Renou, J P" uniqKey="Renou J">J-P Renou</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ramel, F" uniqKey="Ramel F">F Ramel</name></author><author><name sortKey="Sulmon, C" uniqKey="Sulmon C">C Sulmon</name></author><author><name sortKey="Gouesbet, G" uniqKey="Gouesbet G">G Gouesbet</name></author><author><name sortKey="Couee, I" uniqKey="Couee I">I Couee</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zheng, Y" uniqKey="Zheng Y">Y Zheng</name></author><author><name sortKey="Sheng, J" uniqKey="Sheng J">J Sheng</name></author><author><name sortKey="Zhao, R" uniqKey="Zhao R">R Zhao</name></author><author><name sortKey="Zhang, J" uniqKey="Zhang J">J Zhang</name></author><author><name sortKey="Lv, S" uniqKey="Lv S">S Lv</name></author><author><name sortKey="Liu, L" uniqKey="Liu L">L Liu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Navarro, L" uniqKey="Navarro L">L Navarro</name></author><author><name sortKey="Bari, R" uniqKey="Bari R">R Bari</name></author><author><name sortKey="Achard, P" uniqKey="Achard P">P Achard</name></author><author><name sortKey="Lison, P" uniqKey="Lison P">P Lison</name></author><author><name sortKey="Nemri, A" uniqKey="Nemri A">A Nemri</name></author><author><name sortKey="Harberd, N P" uniqKey="Harberd N">N.P Harberd</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Shen, W" uniqKey="Shen W">W Shen</name></author><author><name sortKey="Cevallos Cevallos, Jm" uniqKey="Cevallos Cevallos J">JM Cevallos-Cevallos</name></author><author><name sortKey="Nunes Da Rocha, U" uniqKey="Nunes Da Rocha U">U Nunes da Rocha</name></author><author><name sortKey="Arevalo, Ha" uniqKey="Arevalo H">HA Arevalo</name></author><author><name sortKey="Stansly, Pa" uniqKey="Stansly P">PA Stansly</name></author><author><name sortKey="Roberts, Pd" uniqKey="Roberts P">PD Roberts</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gottwald, Tr" uniqKey="Gottwald T">TR Gottwald</name></author><author><name sortKey="Graham, Jh" uniqKey="Graham J">JH Graham</name></author><author><name sortKey="Irey, M" uniqKey="Irey M">M Irey</name></author><author><name sortKey="Mccollum, G" uniqKey="Mccollum G">G McCollum</name></author><author><name sortKey="Wood, B" uniqKey="Wood B">B Wood</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cao, J" uniqKey="Cao J">J Cao</name></author><author><name sortKey="Cheng, C" uniqKey="Cheng C">C Cheng</name></author><author><name sortKey="Yang, J" uniqKey="Yang J">J Yang</name></author><author><name sortKey="Wang, Q" uniqKey="Wang Q">Q Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Stansly, Pa" uniqKey="Stansly P">PA Stansly</name></author><author><name sortKey="Arevalo, Ha" uniqKey="Arevalo H">HA Arevalo</name></author><author><name sortKey="Qureshi, Ja" uniqKey="Qureshi J">JA Qureshi</name></author><author><name sortKey="Jones, Mm" uniqKey="Jones M">MM Jones</name></author><author><name sortKey="Hendricks, K" uniqKey="Hendricks K">K Hendricks</name></author><author><name sortKey="Roberts, Pd" uniqKey="Roberts P">PD Roberts</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Xu, M" uniqKey="Xu M">M Xu</name></author><author><name sortKey="Liang, M" uniqKey="Liang M">M Liang</name></author><author><name sortKey="Chen, J" uniqKey="Chen J">J Chen</name></author><author><name sortKey="Xia, Y" uniqKey="Xia Y">Y Xia</name></author><author><name sortKey="Zheng, Z" uniqKey="Zheng Z">Z Zheng</name></author><author><name sortKey="Zhu, Q" uniqKey="Zhu Q">Q Zhu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Flores, F" uniqKey="Flores F">F Flores</name></author><author><name sortKey="Collier, Cj" uniqKey="Collier C">CJ Collier</name></author><author><name sortKey="Mercurio, P" uniqKey="Mercurio P">P Mercurio</name></author><author><name sortKey="Negri, Ap" uniqKey="Negri A">AP Negri</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Aritua, V" uniqKey="Aritua V">V Aritua</name></author><author><name sortKey="Achor, D" uniqKey="Achor D">D Achor</name></author><author><name sortKey="Gmitter, Fg" uniqKey="Gmitter F">FG Gmitter</name></author><author><name sortKey="Albrigo, G" uniqKey="Albrigo G">G Albrigo</name></author><author><name sortKey="Wang, N" uniqKey="Wang N">N Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wang, Z" uniqKey="Wang Z">Z Wang</name></author><author><name sortKey="Fang, H" uniqKey="Fang H">H Fang</name></author><author><name sortKey="Chen, Y" uniqKey="Chen Y">Y Chen</name></author><author><name sortKey="Chen, K" uniqKey="Chen K">K Chen</name></author><author><name sortKey="Li, G" uniqKey="Li G">G Li</name></author><author><name sortKey="Gu, S" uniqKey="Gu S">S Gu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wang, X M" uniqKey="Wang X">X-M Wang</name></author><author><name sortKey="Gaudet, Da" uniqKey="Gaudet D">DA Gaudet</name></author><author><name sortKey="Liu, W" uniqKey="Liu W">W Liu</name></author><author><name sortKey="Frick, M" uniqKey="Frick M">M Frick</name></author><author><name sortKey="Puchalski, B" uniqKey="Puchalski B">B Puchalski</name></author><author><name sortKey="Lu, Z X" uniqKey="Lu Z">Z-X Lu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Birkenbihl, Rp" uniqKey="Birkenbihl R">RP Birkenbihl</name></author><author><name sortKey="Diezel, C" uniqKey="Diezel C">C Diezel</name></author><author><name sortKey="Somssich, Ie" uniqKey="Somssich I">IE Somssich</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lorenzo, O" uniqKey="Lorenzo O">O Lorenzo</name></author><author><name sortKey="Chico, Jm" uniqKey="Chico J">JM Chico</name></author><author><name sortKey="Sanchez Serrano, Jj" uniqKey="Sanchez Serrano J">JJ Sánchez-Serrano</name></author><author><name sortKey="Solano, R" uniqKey="Solano R">R Solano</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Abe, H" uniqKey="Abe H">H Abe</name></author><author><name sortKey="Urao, T" uniqKey="Urao T">T Urao</name></author><author><name sortKey="Ito, T" uniqKey="Ito T">T Ito</name></author><author><name sortKey="Seki, M" uniqKey="Seki M">M Seki</name></author><author><name sortKey="Shinozaki, K" uniqKey="Shinozaki K">K Shinozaki</name></author><author><name sortKey="Yamaguchi Shinozaki, K" uniqKey="Yamaguchi Shinozaki K">K Yamaguchi-Shinozaki</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Laurie Berry, N" uniqKey="Laurie Berry N">N Laurie-Berry</name></author><author><name sortKey="Joardar, V" uniqKey="Joardar V">V Joardar</name></author><author><name sortKey="Street, Ih" uniqKey="Street I">IH Street</name></author><author><name sortKey="Kunkel, Bn" uniqKey="Kunkel B">BN Kunkel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Falk, A" uniqKey="Falk A">A Falk</name></author><author><name sortKey="Feys, Bj" uniqKey="Feys B">BJ Feys</name></author><author><name sortKey="Frost, Ln" uniqKey="Frost L">LN Frost</name></author><author><name sortKey="Jones, Jdg" uniqKey="Jones J">JDG Jones</name></author><author><name sortKey="Daniels, Mj" uniqKey="Daniels M">MJ Daniels</name></author><author><name sortKey="Parker, Je" uniqKey="Parker J">JE Parker</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lipka, V" uniqKey="Lipka V">V Lipka</name></author><author><name sortKey="Dittgen, J" uniqKey="Dittgen J">J Dittgen</name></author><author><name sortKey="Bednarek, P" uniqKey="Bednarek P">P Bednarek</name></author><author><name sortKey="Bhat, R" uniqKey="Bhat R">R Bhat</name></author><author><name sortKey="Wiermer, M" uniqKey="Wiermer M">M Wiermer</name></author><author><name sortKey="Stein, M" uniqKey="Stein M">M Stein</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Feys, Bj" uniqKey="Feys B">BJ Feys</name></author><author><name sortKey="Wiermer, M" uniqKey="Wiermer M">M Wiermer</name></author><author><name sortKey="Bhat, Ra" uniqKey="Bhat R">RA Bhat</name></author><author><name sortKey="Moisan, Lj" uniqKey="Moisan L">LJ Moisan</name></author><author><name sortKey="Medina Escobar, N" uniqKey="Medina Escobar N">N Medina-Escobar</name></author><author><name sortKey="Neu, C" uniqKey="Neu C">C Neu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Moreau, M" uniqKey="Moreau M">M Moreau</name></author><author><name sortKey="Degrave, A" uniqKey="Degrave A">A Degrave</name></author><author><name sortKey="Vedel, R" uniqKey="Vedel R">R Vedel</name></author><author><name sortKey="Bitton, F" uniqKey="Bitton F">F Bitton</name></author><author><name sortKey="Patrit, O" uniqKey="Patrit O">O Patrit</name></author><author><name sortKey="Renou, J P" uniqKey="Renou J">J-P Renou</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Besseau, S" uniqKey="Besseau S">S Besseau</name></author><author><name sortKey="Li, J" uniqKey="Li J">J Li</name></author><author><name sortKey="Palva, Et" uniqKey="Palva E">ET Palva</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ibanez, Am" uniqKey="Ibanez A">AM Ibanez</name></author><author><name sortKey="Martinelli, F" uniqKey="Martinelli F">F Martinelli</name></author><author><name sortKey="Reagan, Rl" uniqKey="Reagan R">RL Reagan</name></author><author><name sortKey="Uratsu, Sl" uniqKey="Uratsu S">SL Uratsu</name></author><author><name sortKey="Vo, A" uniqKey="Vo A">A Vo</name></author><author><name sortKey="Tinoco, Ma" uniqKey="Tinoco M">MA Tinoco</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Van Loon, Lc" uniqKey="Van Loon L">LC van Loon</name></author><author><name sortKey="Rep, M" uniqKey="Rep M">M Rep</name></author><author><name sortKey="Pieterse, Cm" uniqKey="Pieterse C">CM Pieterse</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kiba, A" uniqKey="Kiba A">A Kiba</name></author><author><name sortKey="Maimbo, M" uniqKey="Maimbo M">M Maimbo</name></author><author><name sortKey="Kanda, A" uniqKey="Kanda A">A Kanda</name></author><author><name sortKey="Tomiyama, H" uniqKey="Tomiyama H">H Tomiyama</name></author><author><name sortKey="Ohnishi, K" uniqKey="Ohnishi K">K Ohnishi</name></author><author><name sortKey="Hikichi, Y" uniqKey="Hikichi Y">Y Hikichi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Martinelli, F" uniqKey="Martinelli F">F Martinelli</name></author><author><name sortKey="Scalenghe, R" uniqKey="Scalenghe R">R Scalenghe</name></author><author><name sortKey="Giovino, A" uniqKey="Giovino A">A Giovino</name></author><author><name sortKey="Pasquale, M" uniqKey="Pasquale M">M Pasquale</name></author><author><name sortKey="Aksenov, Aa" uniqKey="Aksenov A">AA Aksenov</name></author><author><name sortKey="Pasamontes, A" uniqKey="Pasamontes A">A Pasamontes</name></author><author><name sortKey="Peirano, Dj" uniqKey="Peirano D">DJ Peirano</name></author><author><name sortKey="Davis, Ce" uniqKey="Davis C">CE Davis</name></author><author><name sortKey="Dandekar, Am" uniqKey="Dandekar A">AM Dandekar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Martinelli, F" uniqKey="Martinelli F">F Martinelli</name></author><author><name sortKey="Scalenghe, R" uniqKey="Scalenghe R">R Scalenghe</name></author><author><name sortKey="Davino, Sw" uniqKey="Davino S">SW Davino</name></author><author><name sortKey="Panno, S" uniqKey="Panno S">S Panno</name></author><author><name sortKey="Suderi, G" uniqKey="Suderi G">G Suderi</name></author><author><name sortKey="Ruisi, P" uniqKey="Ruisi P">P Ruisi</name></author><author><name sortKey="Villa, P" uniqKey="Villa P">P Villa</name></author><author><name sortKey="Stroppiana, D" uniqKey="Stroppiana D">D Stroppiana</name></author><author><name sortKey="Boschetti, M" uniqKey="Boschetti M">M Boschetti</name></author><author><name sortKey="Goulart, Lr" uniqKey="Goulart L">LR Goulart</name></author><author><name sortKey="Davis, Ce" uniqKey="Davis C">CE Davis</name></author><author><name sortKey="Dandekar, Am" uniqKey="Dandekar A">AM Dandekar</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, CA USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">27459099</article-id><article-id pub-id-type="pmc">4961454</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0159610</article-id><article-id pub-id-type="publisher-id">PONE-D-16-12269</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Physical Sciences</subject><subj-group><subject>Chemistry</subject><subj-group><subject>Chemical Compounds</subject><subj-group><subject>Organic Compounds</subject><subj-group><subject>Carbohydrates</subject><subj-group><subject>Disaccharides</subject><subj-group><subject>Sucrose</subject></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Physical Sciences</subject><subj-group><subject>Chemistry</subject><subj-group><subject>Organic Chemistry</subject><subj-group><subject>Organic Compounds</subject><subj-group><subject>Carbohydrates</subject><subj-group><subject>Disaccharides</subject><subj-group><subject>Sucrose</subject></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Plant Science</subject><subj-group><subject>Plant Anatomy</subject><subj-group><subject>Leaves</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Biochemistry</subject><subj-group><subject>Hormones</subject><subj-group><subject>Plant Hormones</subject><subj-group><subject>Gibberellins</subject></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Biochemistry</subject><subj-group><subject>Plant Biochemistry</subject><subj-group><subject>Plant Hormones</subject><subj-group><subject>Gibberellins</subject></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Plant Science</subject><subj-group><subject>Plant Biochemistry</subject><subj-group><subject>Plant Hormones</subject><subj-group><subject>Gibberellins</subject></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Physical Sciences</subject><subj-group><subject>Chemistry</subject><subj-group><subject>Chemical Compounds</subject><subj-group><subject>Organic Compounds</subject><subj-group><subject>Carbohydrates</subject><subj-group><subject>Starches</subject></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Physical Sciences</subject><subj-group><subject>Chemistry</subject><subj-group><subject>Organic Chemistry</subject><subj-group><subject>Organic Compounds</subject><subj-group><subject>Carbohydrates</subject><subj-group><subject>Starches</subject></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Biochemistry</subject><subj-group><subject>Metabolism</subject><subj-group><subject>Carbohydrate Metabolism</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Physical Sciences</subject><subj-group><subject>Chemistry</subject><subj-group><subject>Chemical Compounds</subject><subj-group><subject>Organic Compounds</subject><subj-group><subject>Amino Acids</subject><subj-group><subject>Basic Amino Acids</subject><subj-group><subject>Arginine</subject></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Physical Sciences</subject><subj-group><subject>Chemistry</subject><subj-group><subject>Organic Chemistry</subject><subj-group><subject>Organic Compounds</subject><subj-group><subject>Amino Acids</subject><subj-group><subject>Basic Amino Acids</subject><subj-group><subject>Arginine</subject></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Biochemistry</subject><subj-group><subject>Proteins</subject><subj-group><subject>Amino Acids</subject><subj-group><subject>Basic Amino Acids</subject><subj-group><subject>Arginine</subject></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Genetics</subject><subj-group><subject>Gene Expression</subject></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Immunology</subject><subj-group><subject>Immune Response</subject></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Medicine and Health Sciences</subject><subj-group><subject>Immunology</subject><subj-group><subject>Immune Response</subject></subj-group></subj-group></subj-group></article-categories><title-group><article-title>Molecular Responses to Small Regulating Molecules against Huanglongbing Disease</article-title><alt-title alt-title-type="running-head">Therapy against HLB Disease</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Martinelli</surname><given-names>Federico</given-names></name><xref ref-type="aff" rid="aff001"><sup>1</sup></xref><xref ref-type="aff" rid="aff002"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Dolan</surname><given-names>David</given-names></name><xref ref-type="aff" rid="aff003"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Fileccia</surname><given-names>Veronica</given-names></name><xref ref-type="aff" rid="aff001"><sup>1</sup></xref><xref ref-type="aff" rid="aff002"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Reagan</surname><given-names>Russell L.</given-names></name><xref ref-type="aff" rid="aff003"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Phu</surname><given-names>My</given-names></name><xref ref-type="aff" rid="aff003"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Spann</surname><given-names>Timothy M.</given-names></name><xref ref-type="aff" rid="aff004"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>McCollum</surname><given-names>Thomas G.</given-names></name><xref ref-type="aff" rid="aff005"><sup>5</sup></xref></contrib><contrib contrib-type="author"><name><surname>Dandekar</surname><given-names>Abhaya M.</given-names></name><xref ref-type="aff" rid="aff003"><sup>3</sup></xref><xref ref-type="corresp" rid="cor001">*</xref></contrib></contrib-group><aff id="aff001"><label>1</label><addr-line>Dipartimento di Scienze Agrarie e Forestali, Palermo, Italy</addr-line></aff><aff id="aff002"><label>2</label><addr-line>Euro-Mediterranean Institute of Science and Technology (IEMEST), Palermo, Italy</addr-line></aff><aff id="aff003"><label>3</label><addr-line>Department of Plant Sciences, University of California Davis, Davis, California, United States of America</addr-line></aff><aff id="aff004"><label>4</label><addr-line>California Avocado Commission, Irvine, California, United States of America</addr-line></aff><aff id="aff005"><label>5</label><addr-line>United States Department of Agriculture-The Agricultural Research Service, U.S. Horticultural Research Laboratory, Fort Pierce, Florida, United States of America</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Wang</surname><given-names>Zonghua</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>Fujian Agriculture and Forestry University, CHINA</addr-line></aff><author-notes><fn fn-type="conflict" id="coi001"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type="con" id="contrib001"><p>Conceived and designed the experiments: AMD FM DD. Performed the experiments: FM DD MP TMS TGM. Analyzed the data: FM VF RLR TGM AMD. Contributed reagents/materials/analysis tools: VF RLR TMS TGM. Wrote the paper: FM AMD.</p></fn><corresp id="cor001">* E-mail: <email>amdandekar@ucdavis.edu</email></corresp></author-notes><pub-date pub-type="epub"><day>26</day><month>7</month><year>2016</year></pub-date><pub-date pub-type="collection"><year>2016</year></pub-date><volume>11</volume><issue>7</issue><elocation-id>e0159610</elocation-id><history><date date-type="received"><day>24</day><month>3</month><year>2016</year></date><date date-type="accepted"><day>6</day><month>7</month><year>2016</year></date></history><permissions><copyright-statement>© 2016 Martinelli et al</copyright-statement><copyright-year>2016</copyright-year><copyright-holder>Martinelli et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="pone.0159610.pdf"></self-uri><abstract><p>Huanglongbing (HLB; citrus greening) is the most devastating disease of citrus worldwide. No cure is yet available for this disease and infected trees generally decline after several months. Disease management depends on early detection of symptoms and chemical control of insect vectors. In this work, different combinations of organic compounds were tested for the ability to modulate citrus molecular responses to HLB disease beneficially. Three small-molecule regulating compounds were tested: 1) L-arginine, 2) 6-benzyl-adenine combined with gibberellins, and 3) sucrose combined with atrazine. Each treatment contained K-phite mineral solution and was tested at two different concentrations. Two trials were conducted: one in the greenhouse and the other in the orchard. In the greenhouse study, responses of 42 key genes involved in sugar and starch metabolism, hormone-related pathways, biotic stress responses, and secondary metabolism in treated and untreated mature leaves were analyzed. <italic>TGA5</italic> was significantly induced by arginine. Benzyladenine and gibberellins enhanced two important genes involved in biotic stress responses: <italic>WRKY54</italic> and <italic>WRKY59</italic>. Sucrose combined with atrazine mainly upregulated key genes involved in carbohydrate metabolism such as sucrose-phosphate synthase, sucrose synthase, starch synthase, and α-amylase. Atrazine also affected expression of some key genes involved in systemic acquired resistance such as <italic>EDS1</italic>, <italic>TGA6</italic>, <italic>WRKY33</italic>, and <italic>MYC2</italic>. Several treatments upregulated <italic>HSP82</italic>, which might help protect protein folding and integrity. A subset of key genes was chosen as biomarkers for molecular responses to treatments under field conditions. <italic>GPT2</italic> was downregulated by all small-molecule treatments. Arginine-induced genes involved in systemic acquired resistance included PR1, <italic>WRKY70</italic>, and <italic>EDS1</italic>. These molecular data encourage long-term application of treatments that combine these regulating molecules in field trials.</p></abstract><funding-group><award-group id="award001"><funding-source><institution-wrap><institution-id institution-id-type="funder-id">http://dx.doi.org/10.13039/100007565</institution-id><institution>Citrus Research and Development Foundation</institution></institution-wrap></funding-source><award-id>CATP09-305</award-id><principal-award-recipient><name><surname>Dandekar</surname><given-names>Abhaya M</given-names></name></principal-award-recipient></award-group><funding-statement>The research reported in this publication was supported by grant# CATAP09-305 received from the Citrus Research and Development Foundation and supported the work conducted by all of the authors except Veronica Fileccia who was supported by the University of Palermo. It should be noted that in either case the funders had no role in study design, data collection and analysis, decision to publish, or preparation of this manuscript.</funding-statement></funding-group><counts><fig-count count="4"></fig-count><table-count count="5"></table-count><page-count count="17"></page-count></counts><custom-meta-group><custom-meta id="data-availability"><meta-name>Data Availability</meta-name><meta-value>All relevant data are within the paper and its Supporting Information files.</meta-value></custom-meta></custom-meta-group></article-meta><notes><title>Data Availability</title><p>All relevant data are within the paper and its Supporting Information files.</p></notes></front><body><sec sec-type="intro" id="sec001"><title>Introduction</title><p>Plant pests and diseases threaten agricultural systems. Huanglongbing (HLB) disease endangers the citrus industry and citrus cultivation worldwide [<xref rid="pone.0159610.ref001" ref-type="bibr">1</xref>]. Neither genetic resistance nor short- or long-term therapeutic strategies to mitigate HLB has been found. Huanglongbing disease is associated with three <italic>Candidatus liberibacter</italic> (C. Las) species: <italic>asiaticus</italic>, <italic>americanus</italic>, and <italic>africanus</italic>. C. Las is a member of the alpha subdivision of the proteobacteria based on ribosomal region sequence data [<xref rid="pone.0159610.ref002" ref-type="bibr">2</xref>]. Symptoms have been extensively described and all citrus species are susceptible to HLB to varying degrees [<xref rid="pone.0159610.ref003" ref-type="bibr">3</xref>,<xref rid="pone.0159610.ref004" ref-type="bibr">4</xref>].</p><p>Microarray analysis identified key genes and pathways affected by HLB at the transcriptomic level in mature, symptomatic leaves [<xref rid="pone.0159610.ref005" ref-type="bibr">5</xref>,<xref rid="pone.0159610.ref006" ref-type="bibr">6</xref>]. RNA-seq was applied to describe molecular responses in fruit with different degrees of HLB symptoms [<xref rid="pone.0159610.ref007" ref-type="bibr">7</xref>]. This allowed a comprehensive analysis of gene regulatory networks on source and sink tissues at different developmental stages [<xref rid="pone.0159610.ref008" ref-type="bibr">8</xref>,<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>]. Effects of C. Las infection on key genes involved in sugar and starch metabolism, disrupting source-sink relationships, was a key cause of the metabolic dysfunction. Fruits of infected plants remained immature and photosynthesizing while mature leaves became yellow and accumulated starch [<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>]. Protein expression has been linked to the nutritional condition of grapefruit plants before and after symptom appearance. C. Las-upregulated proteins were involved in redox reactions, cell wall modification, and biotic stress responses [<xref rid="pone.0159610.ref010" ref-type="bibr">10</xref>]. Isobaric tags for relative and absolute quantitation (iTRAQ) identified which pathways are affected post-transcriptionally by pathogen infections [<xref rid="pone.0159610.ref011" ref-type="bibr">11</xref>]. A predictive proteome analysis of C. Las has been conducted [<xref rid="pone.0159610.ref012" ref-type="bibr">12</xref>]. Because no toxins or other pathogenic substances were clearly identified in the genome [<xref rid="pone.0159610.ref013" ref-type="bibr">13</xref>], the pathogenetic mechanisms of HLB disease are still unclear, nor is it clear whether the HLB-associated changes in sugar and starch metabolism are a cause or an effect of the disease [<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>].</p><p>Current management procedures consist mainly of visual scouting for symptoms, PCR-based detection of the pathogen, and insecticides for vector control [<xref rid="pone.0159610.ref014" ref-type="bibr">14</xref>]. Although application of insecticides can reduce disease spread, the disease can spread with only a few infected psyllids in the orchard. Early disease detection and psyllid control are critical practices in areas where neither disease nor vector has yet been discovered [<xref rid="pone.0159610.ref001" ref-type="bibr">1</xref>]. At present, no chemical compounds have been tested to beneficially modulate Citrus host responses and eventually extend the life and production of HLB-infected trees, reducing high economic costs due to lost production. Research has focused only on testing compounds targeting the pathogen. A combination of two antibiotics (penicillin and streptomycin) applied by either root soaking or foliar spray decreased C. Las titer in infected plants [<xref rid="pone.0159610.ref015" ref-type="bibr">15</xref>]. Antimicrobial compounds have been delivered through graft-based chemotherapy [<xref rid="pone.0159610.ref016" ref-type="bibr">16</xref>]. Nanoemulsion formulations were evaluated for their ability to increase permeability of antimicrobial molecules with success dependent on the citrus cultivar and degree of HLB symptoms [<xref rid="pone.0159610.ref017" ref-type="bibr">17</xref>]. Small-molecule inhibitors designed by molecular docking were significantly effective in inhibiting SecA ATPase in vitro [<xref rid="pone.0159610.ref018" ref-type="bibr">18</xref>]. Extensive use of antibiotics in open fields is not desirable due to environmental and human health concerns. Host-based treatments that modulate key genes involved in metabolic HLB syndrome are highly desirable. Data on citrus molecular responses to HLB can now be exploited to design small-molecule combinations to ameliorate the devastating symptoms. The aim of this study was to determine if small molecules are effective in modulating expression of key HLB-regulated or innate response genes after three to six days of treatment.</p></sec><sec sec-type="materials|methods" id="sec002"><title>Materials and Methods</title><sec id="sec003"><title>Plant material</title><sec id="sec004"><title>Greenhouse trial</title><p>In 2011, Valencia orange scions on Kuharske Carrizo rootstocks were grown in one-gallon plastic nursery containers and kept in the greenhouse under natural light at 17 to 25°C. Graft inoculations were performed using a standard inverted “T” budding technique with C. Las-infected budwood tested as described [<xref rid="pone.0159610.ref019" ref-type="bibr">19</xref>]. Starting three months after budding, each plant was tested monthly using quantitative RT-PCR for C. Las species as described [<xref rid="pone.0159610.ref019" ref-type="bibr">19</xref>]. Each control or treatment was represented by nine to 10 trees. The control consisted of trees sprayed with distilled water. The first treatment consisted of a Silwet (0.12%), DKP3XTRA (32.5 mL/20 L) and LK-phite spray (2 mL/20 L). The other six spray treatments were composed of three different small-molecule combinations at two different concentrations each: 1) L-arginine at 1 mM or 0.5 mM, 2) 120 μM 6-benzyl adenine in combination with 15 or 30 μM gibberellin, and 3) 80 mM sucrose combined with the herbicide atrazine (2 μM or 1 μM). All seven treatments contained the surfactant Silwet and LK-phite at the same concentration used for the first treatment. Phenotypes were evaluated to determine any phytotoxic effects of these sprays. All treatments were sprayed on the citrus foliage; the volume sprayed per tree was enough to wet both upper and lower leaf surfaces just to the point of runoff. Gene expression analyses were conducted on RNA extracted three days following treatment. Three biological replicates of nine mature leaves harvested from three trees (three leaves per tree) were analyzed for each treatment. Collected leaves were immediately frozen in liquid nitrogen and kept at -80°C until RNA was extracted. Forty-two host genes were analyzed in mature symptomatic leaves of treated and untreated trees.</p></sec><sec id="sec005"><title>Field trial</title><p>The same treatments were applied during the field experiments, which were conducted in a commercial citrus orchard in Indian Ricer County, FL, composed of Valencia orange scions on Swingle rootstocks. The study was conducted on private land and the owner of the land gave permission to conduct the study on the site of our experiments. We also confirm that the field studies did not involve endangered or protected species. Twenty-four trees of medium height, 12 trees per row, were selected. These trees were mildly HLB-symptomatic and confirmed infected by C. Las through the same qPCR assay used for the greenhouse trial. The experimental design was a completely randomized blocks. There were eight treatments with three single-tree replicates. Samples were collected at three and six days following treatment. Each replicate was a pool of 10 mature symptomatic leaves per tree.</p></sec><sec id="sec006"><title>RNA extraction and qRT-PCR analysis</title><p>Total RNA was extracted from mature, fully-expanded leaves of plants grown in the greenhouse or orchard using the Rneasy Plant RNA Isolation kit (Qiagen Inc., Germany). The RNA concentration and purity were assessed by Nanodrop (Thermo Fischer Scientific Inc., MA, USA). RNA was stored at −80°C until analyzed. For each target gene, PCR primers were designed using Primer Express software (Applied Biosystems, Foster City, CA; <xref ref-type="supplementary-material" rid="pone.0159610.s001">S1 Table</xref>). DNase treatment and cDNA synthesis were completed following a combined protocol based on the Quantitect Reverse Transcription Kit (Qiagen Inc., Germany). A standard curve determined the linearity of amplicon quantity vs. initial cDNA quantity for each gene. Five μL cDNA at five ng/μL was diluted to a 12-μL final volume using Sybr Green Master Mix (Bio Rad Laboratories, Hercules, CA, USA). Amplifications were performed using standard conditions: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C, and 60 s at 60°C. Fluorescent signals were collected during the annealing temperature and ΔCT was calculated. Elongation factor 1 alpha (EF-1a, accession AY498567) was used as reference gene. ΔΔCT was determined by subtracting the average EF-1a CT from the average CT of the studied gene [<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>].</p></sec></sec><sec id="sec007"><title>Phenotypic measurements</title><p>Four phenotypic parameters for all treated and untreated trees were measured in the field: trunk diameter (mm), trunk height (m), width drill (m), and width row (m). These measurements were taken on 5/21/2014 and 6/28/2014 during the vegetative season.</p></sec><sec id="sec008"><title>Statistical analysis</title><p>All statistical analyses of gene expression and phenotypic data were performed using SAS II (2008) SAS/STAT software (SAS Institute). Gene expression and phenotypic data were analyzed using ANOVA and a post-hoc test to identify significant differences among treatments. Principal component analysis (PCA) was used to reduce the dimensionality of the gene expression data. Data analysis was performed to alleviate possible bias caused by the collected material for each class or by other confounding factors. Principal component analysis was applied to the ratio matrix of gene expression data to examine the contribution of each target parameter to the separation of the sample classes. A biplot was constructed based on the first two principal components.</p></sec></sec><sec sec-type="results" id="sec009"><title>Results</title><p>Gene expression analyses were conducted in the greenhouse and orchard. First, in greenhouse-grown plants, we quantified transcript abundance of 42 genes selected for their link with for being strongly up- or down-regulated by HLB syndrome [<xref rid="pone.0159610.ref007" ref-type="bibr">7</xref>,<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>] or for having a well-known role in plant responses to pathogen attacks [<xref rid="pone.0159610.ref020" ref-type="bibr">20</xref>]. Second, we selected a subset of five particularly representative genes to be analyzed in field-grown trees, to which we added an additional two genes previously linked with HLB syndrome in published data [<xref rid="pone.0159610.ref005" ref-type="bibr">5</xref>,<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>]. The presence of leaf drop or discoloration and other morphological features of trees were measured to check whether these treatments had deleterious effects on important vegetative parameters.</p><p>At advanced stages, HLB blocks sugar transport out of leaves, leading to starch accumulation in leaves, reduced photosynthesis and disrupted source-sink relationships [<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>]. Anatomical analysis showed that HLB caused phloem disruption, increased sucrose, and plugged sieve pores [<xref rid="pone.0159610.ref006" ref-type="bibr">6</xref>]. The disease also negatively modifies JA-SA crosstalk, leading to an ineffective innate immune response [<xref rid="pone.0159610.ref007" ref-type="bibr">7</xref>,<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>]. The three small-molecule treatments were selected for the potential to beneficially modulate these negative HLB-regulated responses. The combination of atrazine and sucrose upregulates genes associated with reactive-oxygen-species (ROS) defense mechanisms and sucrose metabolism [<xref rid="pone.0159610.ref021" ref-type="bibr">21</xref>,<xref rid="pone.0159610.ref022" ref-type="bibr">22</xref>]. We postulated that this treatment might upregulate genes that reduce sucrose and starch accumulation. Because L-arginine is the precursor of nitric oxide, which is involved in the SAR response and upregulates genes involved in secondary metabolism [<xref rid="pone.0159610.ref023" ref-type="bibr">23</xref>], we designed a second treatment consisting of two concentrations of L-arginine to induce upregulation of genes for secondary metabolic pathways such as phenols and terpenoids. Gibberellins boost systemic acquired resistance [<xref rid="pone.0159610.ref020" ref-type="bibr">20</xref>], favoring the resistance response against biotrophs such as C. Las [<xref rid="pone.0159610.ref020" ref-type="bibr">20</xref>]. Benzyladenine downregulates hexose transport in leaves, based on data deposited in the Genevestigator database. Indeed, we postulated that the combination of gybberellins and L-benzyladenine should have two synergistic effecs: 1) it should beneficially induce genes involved in the innate response against C. Las such as <italic>WRKYs</italic>, <italic>MYC2</italic> and salycilic acid methyl transferase, and 2) it should repress the expression of <italic>GPT2</italic> in symptomatic leaves, mitigating the deleterious HLB-driven upregulation [<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>].</p><sec id="sec010"><title>Greenhouse trial</title><sec id="sec011"><title>Atrazine combined with sucrose treatment</title><p>The transcript abundance of genes related to carbohydrate metabolism varied significantly in response to atrazine combined with sucrose (<xref ref-type="table" rid="pone.0159610.t001">Table 1</xref>).</p><table-wrap id="pone.0159610.t001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0159610.t001</object-id><label>Table 1</label><caption><title>Relative transcript abundance of genes involved in carbohydrate metabolism.</title></caption><alternatives><graphic id="pone.0159610.t001g" xlink:href="pone.0159610.t001"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col></colgroup><thead><tr><th align="left" rowspan="1" colspan="1">Genes</th><th align="left" rowspan="1" colspan="1">Untreated</th><th align="left" rowspan="1" colspan="1">Control K-phite</th><th align="left" rowspan="1" colspan="1">1 μM Atrazine + sucrose</th><th align="left" rowspan="1" colspan="1">2 μM Atrazine + sucrose</th><th align="left" rowspan="1" colspan="1">120 μM BA + 30 μM GA</th><th align="left" rowspan="1" colspan="1">120 μM BA + 15 μM GA</th><th align="left" rowspan="1" colspan="1">1 mM Arginine</th><th align="left" rowspan="1" colspan="1">0.5 mM Arginine</th></tr></thead><tbody><tr><td align="center" colspan="9" rowspan="1">Starch metabolism</td></tr><tr><td align="justify" rowspan="1" colspan="1">Alpha- amylase</td><td align="justify" rowspan="1" colspan="1">0.470 b</td><td align="justify" rowspan="1" colspan="1">4.950 ab</td><td align="justify" rowspan="1" colspan="1">3.320 ab</td><td align="justify" rowspan="1" colspan="1">4.860 ab</td><td align="justify" rowspan="1" colspan="1">87.140 a</td><td align="justify" rowspan="1" colspan="1">5.210 ab</td><td align="justify" rowspan="1" colspan="1">8.520 ab</td><td align="justify" rowspan="1" colspan="1">3.880 ab</td></tr><tr><td align="justify" rowspan="1" colspan="1">Water dikinase starch degrad.</td><td align="justify" rowspan="1" colspan="1">0.317 d</td><td align="justify" rowspan="1" colspan="1">0.087 d</td><td align="justify" rowspan="1" colspan="1">0.063 d</td><td align="justify" rowspan="1" colspan="1">1.096 a</td><td align="justify" rowspan="1" colspan="1">0.185 d</td><td align="justify" rowspan="1" colspan="1">0.732 b</td><td align="justify" rowspan="1" colspan="1">0.635 bc</td><td align="justify" rowspan="1" colspan="1">0.364 cd</td></tr><tr><td align="justify" rowspan="1" colspan="1">GPT2</td><td align="justify" rowspan="1" colspan="1">0.569 a</td><td align="justify" rowspan="1" colspan="1">0.012 b</td><td align="justify" rowspan="1" colspan="1">0.219 b</td><td align="justify" rowspan="1" colspan="1">0.171 b</td><td align="justify" rowspan="1" colspan="1">0.025 b</td><td align="justify" rowspan="1" colspan="1">0.107 b</td><td align="justify" rowspan="1" colspan="1">0.243 b</td><td align="justify" rowspan="1" colspan="1">0.119 b</td></tr><tr><td align="justify" rowspan="1" colspan="1">Starch synthase</td><td align="justify" rowspan="1" colspan="1">0.672 b</td><td align="justify" rowspan="1" colspan="1">2.429 ab</td><td align="justify" rowspan="1" colspan="1">4.256 a</td><td align="justify" rowspan="1" colspan="1">1.886 ab</td><td align="justify" rowspan="1" colspan="1">0.792 b</td><td align="justify" rowspan="1" colspan="1">2.870 ab</td><td align="justify" rowspan="1" colspan="1">2.016 ab</td><td align="justify" rowspan="1" colspan="1">3.029 ab</td></tr><tr><td align="center" colspan="9" rowspan="1">Sucrose metabolism</td></tr><tr><td align="justify" rowspan="1" colspan="1">Invertase</td><td align="justify" rowspan="1" colspan="1">0.839 a</td><td align="justify" rowspan="1" colspan="1">0.766 a</td><td align="justify" rowspan="1" colspan="1">0.351 a</td><td align="justify" rowspan="1" colspan="1">0.274 a</td><td align="justify" rowspan="1" colspan="1">0.296 a</td><td align="justify" rowspan="1" colspan="1">0.851 a</td><td align="justify" rowspan="1" colspan="1">0.758 a</td><td align="justify" rowspan="1" colspan="1">0.611 a</td></tr><tr><td align="justify" rowspan="1" colspan="1">Sugar signaling</td><td align="justify" rowspan="1" colspan="1">0.546 a</td><td align="justify" rowspan="1" colspan="1">2.116 a</td><td align="justify" rowspan="1" colspan="1">0.937 a</td><td align="justify" rowspan="1" colspan="1">2.769 a</td><td align="justify" rowspan="1" colspan="1">7.359 a</td><td align="justify" rowspan="1" colspan="1">1.763 a</td><td align="justify" rowspan="1" colspan="1">1.750 a</td><td align="justify" rowspan="1" colspan="1">3.209 a</td></tr><tr><td align="justify" rowspan="1" colspan="1">Susy</td><td align="justify" rowspan="1" colspan="1">2.310 b</td><td align="justify" rowspan="1" colspan="1">4.218 ab</td><td align="justify" rowspan="1" colspan="1">5.573 a</td><td align="justify" rowspan="1" colspan="1">2.131 b</td><td align="justify" rowspan="1" colspan="1">2.795 b</td><td align="justify" rowspan="1" colspan="1">3.412 ab</td><td align="justify" rowspan="1" colspan="1">4.296 ab</td><td align="justify" rowspan="1" colspan="1">2.022 b</td></tr><tr><td align="justify" rowspan="1" colspan="1">Sps</td><td align="justify" rowspan="1" colspan="1">0.729 b</td><td align="justify" rowspan="1" colspan="1">2.023 b</td><td align="justify" rowspan="1" colspan="1">5.700 b</td><td align="justify" rowspan="1" colspan="1">13.486 a</td><td align="justify" rowspan="1" colspan="1">1.031 b</td><td align="justify" rowspan="1" colspan="1">3.393 b</td><td align="justify" rowspan="1" colspan="1">2.637 b</td><td align="justify" rowspan="1" colspan="1">5.966 b</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="t001fn001"><p>Means of three replicates were indicated. Letters means significant differences using ANOVA (P < = 0.05) and post-hoc test.</p></fn></table-wrap-foot></table-wrap><p>The <italic>sucrose synthase</italic> (Susy) and starch synthase transcripts were more abundant in 1 μM atrazine-treated plants. <italic>Sucrose-phosphate-synthase</italic> and <italic>water dikinase starch degradation</italic> (<italic>GW</italic>D) gene were upregulated by 2 μM atrazine + sucrose. Taken together, these findings highlight that activation of <italic>sucrose synthase</italic> should counter the accumulation of sucrose in symptomatic leaves, while the upregulation of <italic>GWD</italic> should promote degradation of accumulated starch.</p><p>Defense responses and hormone-related genes were affected by atrazine + sucrose treatments. <italic>WRKY33</italic> was upregulated by 1 μM atrazine + sucrose (<xref ref-type="table" rid="pone.0159610.t002">Table 2</xref>).</p><table-wrap id="pone.0159610.t002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0159610.t002</object-id><label>Table 2</label><caption><title>Relative transcript abundance of genes involved in plant innate immune responses.</title></caption><alternatives><graphic id="pone.0159610.t002g" xlink:href="pone.0159610.t002"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col></colgroup><thead><tr><th align="left" rowspan="1" colspan="1">Genes</th><th align="left" rowspan="1" colspan="1">Untreated</th><th align="left" rowspan="1" colspan="1">Control K-phite</th><th align="left" rowspan="1" colspan="1">1 μM Atrazine + sucrose</th><th align="left" rowspan="1" colspan="1">2 μM Atrazine + sucrose</th><th align="left" rowspan="1" colspan="1">120 μM BA + 30 μM GA</th><th align="left" rowspan="1" colspan="1">120 μM BA + 15 μM GA</th><th align="left" rowspan="1" colspan="1">1 mM Arginine</th><th align="left" rowspan="1" colspan="1">0.5 mM Arginine</th></tr></thead><tbody><tr><td align="justify" rowspan="1" colspan="1">RAD51 D</td><td align="justify" rowspan="1" colspan="1">0.197 b</td><td align="justify" rowspan="1" colspan="1">0.175 b</td><td align="justify" rowspan="1" colspan="1">0.690 ab</td><td align="justify" rowspan="1" colspan="1">1.878 a</td><td align="justify" rowspan="1" colspan="1">0.335 b</td><td align="justify" rowspan="1" colspan="1">0.350 b</td><td align="justify" rowspan="1" colspan="1">0.272 b</td><td align="justify" rowspan="1" colspan="1">1.362 ab</td></tr><tr><td align="justify" rowspan="1" colspan="1">BZIP45</td><td align="justify" rowspan="1" colspan="1">5.781 ab</td><td align="justify" rowspan="1" colspan="1">7.650 ab</td><td align="justify" rowspan="1" colspan="1">10.216 ab</td><td align="justify" rowspan="1" colspan="1">11.644 ab</td><td align="justify" rowspan="1" colspan="1">13.370 a</td><td align="justify" rowspan="1" colspan="1">5.197 a</td><td align="justify" rowspan="1" colspan="1">8.997 ab</td><td align="justify" rowspan="1" colspan="1">4.983 ab</td></tr><tr><td align="justify" rowspan="1" colspan="1">TGA5</td><td align="justify" rowspan="1" colspan="1">2.391 b</td><td align="justify" rowspan="1" colspan="1">3.107 ab</td><td align="justify" rowspan="1" colspan="1">4.370 ab</td><td align="justify" rowspan="1" colspan="1">3.366 ab</td><td align="justify" rowspan="1" colspan="1">2.203 b</td><td align="justify" rowspan="1" colspan="1">3.026 ab</td><td align="justify" rowspan="1" colspan="1">6.010 a</td><td align="justify" rowspan="1" colspan="1">2.201 b</td></tr><tr><td align="justify" rowspan="1" colspan="1">RGA1</td><td align="justify" rowspan="1" colspan="1">3.537 a</td><td align="justify" rowspan="1" colspan="1">4.092 a</td><td align="justify" rowspan="1" colspan="1">5.953 a</td><td align="justify" rowspan="1" colspan="1">5.301 a</td><td align="justify" rowspan="1" colspan="1">5.540 a</td><td align="justify" rowspan="1" colspan="1">4.968 a</td><td align="justify" rowspan="1" colspan="1">3.562 a</td><td align="justify" rowspan="1" colspan="1">13.320 a</td></tr><tr><td align="justify" rowspan="1" colspan="1">WRKY33</td><td align="justify" rowspan="1" colspan="1">1.752 b</td><td align="justify" rowspan="1" colspan="1">1.099 b</td><td align="justify" rowspan="1" colspan="1">6.372 a</td><td align="justify" rowspan="1" colspan="1">2.891 b</td><td align="justify" rowspan="1" colspan="1">2.425 b</td><td align="justify" rowspan="1" colspan="1">1.128 b</td><td align="justify" rowspan="1" colspan="1">1.510 b</td><td align="justify" rowspan="1" colspan="1">1.074 b</td></tr><tr><td align="justify" rowspan="1" colspan="1">WRKY48</td><td align="justify" rowspan="1" colspan="1">8.133 ab</td><td align="justify" rowspan="1" colspan="1">4.783 b</td><td align="justify" rowspan="1" colspan="1">6.387 ab</td><td align="justify" rowspan="1" colspan="1">13.873 a</td><td align="justify" rowspan="1" colspan="1">10.300 ab</td><td align="justify" rowspan="1" colspan="1">3.918 b</td><td align="justify" rowspan="1" colspan="1">3.405 b</td><td align="justify" rowspan="1" colspan="1">1.928 b</td></tr><tr><td align="justify" rowspan="1" colspan="1">WRKY54</td><td align="justify" rowspan="1" colspan="1">1.701 a</td><td align="justify" rowspan="1" colspan="1">4.708 a</td><td align="justify" rowspan="1" colspan="1">4.753 a</td><td align="justify" rowspan="1" colspan="1">6.452 a</td><td align="justify" rowspan="1" colspan="1">3.576 a</td><td align="justify" rowspan="1" colspan="1">6.850 a</td><td align="justify" rowspan="1" colspan="1">2.723 a</td><td align="justify" rowspan="1" colspan="1">1.808 a</td></tr><tr><td align="justify" rowspan="1" colspan="1">EDS1</td><td align="justify" rowspan="1" colspan="1">1.491 a</td><td align="justify" rowspan="1" colspan="1">1.905 a</td><td align="justify" rowspan="1" colspan="1">11.022 a</td><td align="justify" rowspan="1" colspan="1">9.360 a</td><td align="justify" rowspan="1" colspan="1">6.914 a</td><td align="justify" rowspan="1" colspan="1">8.289 a</td><td align="justify" rowspan="1" colspan="1">6.561 a</td><td align="justify" rowspan="1" colspan="1">4.319 a</td></tr><tr><td align="justify" rowspan="1" colspan="1">ERF1</td><td align="justify" rowspan="1" colspan="1">7.064 b</td><td align="justify" rowspan="1" colspan="1">6.616 b</td><td align="justify" rowspan="1" colspan="1">17.691 ab</td><td align="justify" rowspan="1" colspan="1">29.435 a</td><td align="justify" rowspan="1" colspan="1">18.566 ab</td><td align="justify" rowspan="1" colspan="1">9.641 b</td><td align="justify" rowspan="1" colspan="1">5.636 b</td><td align="justify" rowspan="1" colspan="1">11.654 b</td></tr><tr><td align="justify" rowspan="1" colspan="1">MYC2</td><td align="justify" rowspan="1" colspan="1">5.369 cd</td><td align="justify" rowspan="1" colspan="1">3.872 d</td><td align="justify" rowspan="1" colspan="1">10.282 abcd</td><td align="justify" rowspan="1" colspan="1">6.505 bcd</td><td align="justify" rowspan="1" colspan="1">17.029 ab</td><td align="justify" rowspan="1" colspan="1">18.585 a</td><td align="justify" rowspan="1" colspan="1">4.134 d</td><td align="justify" rowspan="1" colspan="1">6.320 bcd</td></tr><tr><td align="justify" rowspan="1" colspan="1">PR1</td><td align="justify" rowspan="1" colspan="1">0.053 b</td><td align="justify" rowspan="1" colspan="1">0.229 ab</td><td align="justify" rowspan="1" colspan="1">0.078 b</td><td align="justify" rowspan="1" colspan="1">0.970 ab</td><td align="justify" rowspan="1" colspan="1">0.454 ab</td><td align="justify" rowspan="1" colspan="1">0.119 ab</td><td align="justify" rowspan="1" colspan="1">0.832 ab</td><td align="justify" rowspan="1" colspan="1">2.27 ab</td></tr><tr><td align="justify" rowspan="1" colspan="1">SR1</td><td align="justify" rowspan="1" colspan="1">1.670 a</td><td align="justify" rowspan="1" colspan="1">2.690 a</td><td align="justify" rowspan="1" colspan="1">6.680 a</td><td align="justify" rowspan="1" colspan="1">4.720 a</td><td align="justify" rowspan="1" colspan="1">46.630 a</td><td align="justify" rowspan="1" colspan="1">5.590 a</td><td align="justify" rowspan="1" colspan="1">6.950 a</td><td align="justify" rowspan="1" colspan="1">5.210 a</td></tr><tr><td align="justify" rowspan="1" colspan="1">WRKY59</td><td align="justify" rowspan="1" colspan="1">1.376 b</td><td align="justify" rowspan="1" colspan="1">1.653 b</td><td align="justify" rowspan="1" colspan="1">1.709 b</td><td align="justify" rowspan="1" colspan="1">2.632 b</td><td align="justify" rowspan="1" colspan="1">8.332 a</td><td align="justify" rowspan="1" colspan="1">1.366b</td><td align="justify" rowspan="1" colspan="1">2.996 ab</td><td align="justify" rowspan="1" colspan="1">1.035 b</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="t002fn001"><p>Means of three replicates were indicated. Letters means significant differences using ANOVA (P < = 0.05) and post-hoc test.</p></fn></table-wrap-foot></table-wrap><p>A <italic>zinc ion binding transcription factor</italic>, <italic>heat shock protein 82</italic> (<italic>HSP82</italic>) and <italic>ERF1</italic> were strongly induced by 2 μM atrazine (Tables <xref ref-type="table" rid="pone.0159610.t002">2</xref> and <xref ref-type="table" rid="pone.0159610.t003">3</xref>).</p><table-wrap id="pone.0159610.t003" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0159610.t003</object-id><label>Table 3</label><caption><title>Relative transcript abundance of genes involved in stress responses and secondary metabolism.</title></caption><alternatives><graphic id="pone.0159610.t003g" xlink:href="pone.0159610.t003"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col></colgroup><thead><tr><th align="justify" rowspan="1" colspan="1">Genes</th><th align="justify" rowspan="1" colspan="1">Untreated</th><th align="justify" rowspan="1" colspan="1">Control K-phite</th><th align="justify" rowspan="1" colspan="1">1 μM Atrazine + sucrose</th><th align="justify" rowspan="1" colspan="1">2 μM Atrazine + sucrose</th><th align="justify" rowspan="1" colspan="1">120 μM BA + 30 μM GA</th><th align="justify" rowspan="1" colspan="1">120 μM BA + 15 μM GA</th><th align="justify" rowspan="1" colspan="1">1 mM Arginine</th><th align="left" rowspan="1" colspan="1">0.5 mM Arginine</th></tr></thead><tbody><tr><td align="center" colspan="9" rowspan="1">Stress-related and cell wall</td></tr><tr><td align="justify" rowspan="1" colspan="1">HSP21</td><td align="justify" rowspan="1" colspan="1">23.350 c</td><td align="justify" rowspan="1" colspan="1">308.390 a</td><td align="justify" rowspan="1" colspan="1">24.110 c</td><td align="justify" rowspan="1" colspan="1">106.950 bc</td><td align="justify" rowspan="1" colspan="1">27.670 c</td><td align="justify" rowspan="1" colspan="1">83.290 bc</td><td align="justify" rowspan="1" colspan="1">97.860 bc</td><td align="justify" rowspan="1" colspan="1">288.346 a</td></tr><tr><td align="justify" rowspan="1" colspan="1">HSP82</td><td align="justify" rowspan="1" colspan="1">1.688 c</td><td align="justify" rowspan="1" colspan="1">5.208 ab</td><td align="justify" rowspan="1" colspan="1">0.729 c</td><td align="justify" rowspan="1" colspan="1">5.585 a</td><td align="justify" rowspan="1" colspan="1">3.364 abc</td><td align="justify" rowspan="1" colspan="1">5.448 ab</td><td align="justify" rowspan="1" colspan="1">4.332 ab</td><td align="justify" rowspan="1" colspan="1">0.920 c</td></tr><tr><td align="justify" rowspan="1" colspan="1">ABC Transporter</td><td align="justify" rowspan="1" colspan="1">2.189 de</td><td align="justify" rowspan="1" colspan="1">1.110 de</td><td align="justify" rowspan="1" colspan="1">0.537 e</td><td align="justify" rowspan="1" colspan="1">15.798 b</td><td align="justify" rowspan="1" colspan="1">6.580 cd</td><td align="justify" rowspan="1" colspan="1">24.822 a</td><td align="justify" rowspan="1" colspan="1">9.245 c</td><td align="justify" rowspan="1" colspan="1">1.229 de</td></tr><tr><td align="justify" rowspan="1" colspan="1">ATPtranslocase2</td><td align="justify" rowspan="1" colspan="1">0.022 a</td><td align="justify" rowspan="1" colspan="1">0.052 a</td><td align="justify" rowspan="1" colspan="1">0.032 a</td><td align="justify" rowspan="1" colspan="1">11.499 a</td><td align="justify" rowspan="1" colspan="1">0.012 a</td><td align="justify" rowspan="1" colspan="1">0.148 a</td><td align="justify" rowspan="1" colspan="1">0.125 a</td><td align="justify" rowspan="1" colspan="1">0.029 a</td></tr><tr><td align="justify" rowspan="1" colspan="1">Sulfotransfer. 1</td><td align="justify" rowspan="1" colspan="1">0.298 c</td><td align="justify" rowspan="1" colspan="1">0.008 c</td><td align="justify" rowspan="1" colspan="1">0.6987 c</td><td align="justify" rowspan="1" colspan="1">0.6015 c</td><td align="justify" rowspan="1" colspan="1">2.678 b</td><td align="justify" rowspan="1" colspan="1">6.932 a</td><td align="justify" rowspan="1" colspan="1">0.483 c</td><td align="justify" rowspan="1" colspan="1">0.136 c</td></tr><tr><td align="justify" rowspan="1" colspan="1">NNLTP</td><td align="justify" rowspan="1" colspan="1">0.022 a</td><td align="justify" rowspan="1" colspan="1">0.052 a</td><td align="justify" rowspan="1" colspan="1">0.032 a</td><td align="justify" rowspan="1" colspan="1">11.499 a</td><td align="justify" rowspan="1" colspan="1">0.012 a</td><td align="justify" rowspan="1" colspan="1">0.148 a</td><td align="justify" rowspan="1" colspan="1">0.125 a</td><td align="justify" rowspan="1" colspan="1">0.029 a</td></tr><tr><td align="justify" rowspan="1" colspan="1">PSBW</td><td align="justify" rowspan="1" colspan="1">2.394 c</td><td align="justify" rowspan="1" colspan="1">3.178 c</td><td align="justify" rowspan="1" colspan="1">8.020 b</td><td align="justify" rowspan="1" colspan="1">6.470 bc</td><td align="justify" rowspan="1" colspan="1">2.287 c</td><td align="justify" rowspan="1" colspan="1">6.226 bc</td><td align="justify" rowspan="1" colspan="1">2.539 c</td><td align="justify" rowspan="1" colspan="1">12.839 a</td></tr><tr><td align="justify" rowspan="1" colspan="1">Pectate lyase 5</td><td align="justify" rowspan="1" colspan="1">0.427 b</td><td align="justify" rowspan="1" colspan="1">0.242 b</td><td align="justify" rowspan="1" colspan="1">0.169 b</td><td align="justify" rowspan="1" colspan="1">0.919 ab</td><td align="justify" rowspan="1" colspan="1">0.231 b</td><td align="justify" rowspan="1" colspan="1">4.233 a</td><td align="justify" rowspan="1" colspan="1">0.817 ab</td><td align="justify" rowspan="1" colspan="1">0.589 ab</td></tr><tr><td align="center" colspan="9" rowspan="1">Secondary metabolism</td></tr><tr><td align="justify" rowspan="1" colspan="1">Terpene synthase 14</td><td align="justify" rowspan="1" colspan="1">0.198 b</td><td align="justify" rowspan="1" colspan="1">0.184 b</td><td align="justify" rowspan="1" colspan="1">0.402 b</td><td align="justify" rowspan="1" colspan="1">0.112 b</td><td align="justify" rowspan="1" colspan="1">0.101 b</td><td align="justify" rowspan="1" colspan="1">0.345 b</td><td align="justify" rowspan="1" colspan="1">0.051 b</td><td align="justify" rowspan="1" colspan="1">1.944 a</td></tr><tr><td align="justify" rowspan="1" colspan="1">Terpene synthase 21</td><td align="justify" rowspan="1" colspan="1">3.472 a</td><td align="justify" rowspan="1" colspan="1">1.309 a</td><td align="justify" rowspan="1" colspan="1">1.026 a</td><td align="justify" rowspan="1" colspan="1">5.870 a</td><td align="justify" rowspan="1" colspan="1">4.891 a</td><td align="justify" rowspan="1" colspan="1">1.849 a</td><td align="justify" rowspan="1" colspan="1">1.543 a</td><td align="justify" rowspan="1" colspan="1">6.564 a</td></tr><tr><td align="justify" rowspan="1" colspan="1">Terpene synthase 3</td><td align="justify" rowspan="1" colspan="1">0.76 a</td><td align="justify" rowspan="1" colspan="1">0.53 a</td><td align="justify" rowspan="1" colspan="1">4.92 a</td><td align="justify" rowspan="1" colspan="1">38.40 a</td><td align="justify" rowspan="1" colspan="1">0.64 a</td><td align="justify" rowspan="1" colspan="1">1.37 a</td><td align="justify" rowspan="1" colspan="1">0.32 a</td><td align="justify" rowspan="1" colspan="1">0.19 a</td></tr><tr><td align="justify" rowspan="1" colspan="1">B-Amyrin</td><td align="justify" rowspan="1" colspan="1">1.457 b</td><td align="justify" rowspan="1" colspan="1">2.300 ab</td><td align="justify" rowspan="1" colspan="1">2.178 b</td><td align="justify" rowspan="1" colspan="1">3.504 ab</td><td align="justify" rowspan="1" colspan="1">22.614 a</td><td align="justify" rowspan="1" colspan="1">9.651 ab</td><td align="justify" rowspan="1" colspan="1">3.253 ab</td><td align="justify" rowspan="1" colspan="1">6.649 ab</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="t003fn001"><p>Means of three replicates were indicated. Letters means significant differences using ANOVA (P < = 0.05) and post-hoc test.</p></fn></table-wrap-foot></table-wrap><p><italic>JIN1</italic> was induced by 1 μM atrazine + sucrose (<xref ref-type="table" rid="pone.0159610.t004">Table 4</xref>).</p><table-wrap id="pone.0159610.t004" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0159610.t004</object-id><label>Table 4</label><caption><title>Relative transcript abundance of genes involved in hormone-related pathways.</title></caption><alternatives><graphic id="pone.0159610.t004g" xlink:href="pone.0159610.t004"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col></colgroup><thead><tr><th align="left" rowspan="1" colspan="1">Genes</th><th align="left" rowspan="1" colspan="1">Untreated</th><th align="left" rowspan="1" colspan="1">Control K-phite</th><th align="left" rowspan="1" colspan="1">1 μM Atrazine + sucrose</th><th align="left" rowspan="1" colspan="1">2 μM Atrazine + sucrose</th><th align="left" rowspan="1" colspan="1">120 μM BA + 30 μM GA</th><th align="left" rowspan="1" colspan="1">120 μM BA + 15 μM GA</th><th align="left" rowspan="1" colspan="1">1 mM Arginine</th><th align="left" rowspan="1" colspan="1">0.5 mM Arginine</th></tr></thead><tbody><tr><td align="center" colspan="9" rowspan="1">Gibberellins</td></tr><tr><td align="left" rowspan="1" colspan="1">GA2-oxidase</td><td align="left" rowspan="1" colspan="1">0.395 a</td><td align="left" rowspan="1" colspan="1">0.676 a</td><td align="left" rowspan="1" colspan="1">0.556 a</td><td align="left" rowspan="1" colspan="1">5.276 a</td><td align="left" rowspan="1" colspan="1">2.718 a</td><td align="left" rowspan="1" colspan="1">1.752 a</td><td align="left" rowspan="1" colspan="1">0.199 a</td><td align="left" rowspan="1" colspan="1">1.379 a</td></tr><tr><td align="left" rowspan="1" colspan="1">Gibberelin-2-oxygenase</td><td align="left" rowspan="1" colspan="1">0.765 a</td><td align="left" rowspan="1" colspan="1">0.357 a</td><td align="left" rowspan="1" colspan="1">0.793 a</td><td align="left" rowspan="1" colspan="1">1.367 a</td><td align="left" rowspan="1" colspan="1">2.629 a</td><td align="left" rowspan="1" colspan="1">3.965 a</td><td align="left" rowspan="1" colspan="1">1.158 a</td><td align="left" rowspan="1" colspan="1">0.487 a</td></tr><tr><td align="center" colspan="9" rowspan="1">Auxins and Benzyl-adenine</td></tr><tr><td align="left" rowspan="1" colspan="1">GH3.1</td><td align="left" rowspan="1" colspan="1">0.077 b</td><td align="left" rowspan="1" colspan="1">0.021 b</td><td align="left" rowspan="1" colspan="1">0.135 b</td><td align="left" rowspan="1" colspan="1">0.511 a</td><td align="left" rowspan="1" colspan="1">0.048 b</td><td align="left" rowspan="1" colspan="1">0.070 b</td><td align="left" rowspan="1" colspan="1">0.118 b</td><td align="left" rowspan="1" colspan="1">0.255 ab</td></tr><tr><td align="left" rowspan="1" colspan="1">Ka02</td><td align="left" rowspan="1" colspan="1">2.837 ab</td><td align="left" rowspan="1" colspan="1">3.661 a</td><td align="left" rowspan="1" colspan="1">2.532 ab</td><td align="left" rowspan="1" colspan="1">3.608 a</td><td align="left" rowspan="1" colspan="1">0.139 b</td><td align="left" rowspan="1" colspan="1">4.164 a</td><td align="left" rowspan="1" colspan="1">0.929 ab</td><td align="left" rowspan="1" colspan="1">2.794 ab</td></tr><tr><td align="center" colspan="9" rowspan="1">Jasmonic acid</td></tr><tr><td align="left" rowspan="1" colspan="1">12-oxophytodi. reductase 1-like</td><td align="left" rowspan="1" colspan="1">0.444 b</td><td align="left" rowspan="1" colspan="1">0.290 b</td><td align="left" rowspan="1" colspan="1">0.322 b</td><td align="left" rowspan="1" colspan="1">1.986 a</td><td align="left" rowspan="1" colspan="1">0.174 b</td><td align="left" rowspan="1" colspan="1">1.063 ab</td><td align="left" rowspan="1" colspan="1">1.964 a</td><td align="left" rowspan="1" colspan="1">0.732 ab</td></tr><tr><td align="center" colspan="9" rowspan="1">Salicylic acid methyl transferase</td></tr><tr><td align="left" rowspan="1" colspan="1">SA-methyl transferase</td><td align="left" rowspan="1" colspan="1">0.033 b</td><td align="left" rowspan="1" colspan="1">0.015 b</td><td align="left" rowspan="1" colspan="1">0.052 b</td><td align="left" rowspan="1" colspan="1">0.277 a</td><td align="left" rowspan="1" colspan="1">0.314 a</td><td align="left" rowspan="1" colspan="1">0.091 a</td><td align="left" rowspan="1" colspan="1">0.360 a</td><td align="left" rowspan="1" colspan="1">0.434 b</td></tr><tr><td align="center" colspan="9" rowspan="1">Ethylene</td></tr><tr><td align="left" rowspan="1" colspan="1">ACS-1</td><td align="left" rowspan="1" colspan="1">3.960 a</td><td align="left" rowspan="1" colspan="1">3.580 a</td><td align="left" rowspan="1" colspan="1">5.039 a</td><td align="left" rowspan="1" colspan="1">6.202 a</td><td align="left" rowspan="1" colspan="1">2.025 a</td><td align="left" rowspan="1" colspan="1">1.723 a</td><td align="left" rowspan="1" colspan="1">4.595 a</td><td align="left" rowspan="1" colspan="1">4.247 a</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="t004fn001"><p>Means of three replicates were indicated. Letters means significant differences using ANOVA (P < = 0.05) and post-hoc test.</p></fn></table-wrap-foot></table-wrap><p><italic>Salicylic acid methyl transferase</italic> and <italic>12-oxophytodienoate reductase 1-like</italic> were upregulated by 2 μM atrazine + sucrose.</p></sec><sec id="sec012"><title>Gibberellin and benzyl-adenine treatment</title><p>GA + BA treatments were tested to determine their effects on key genes involved in plant innate immune responses and carbohydrate metabolism. Among carbohydrate metabolism genes, alpha-amylase was significantly induced by 120 μM gibberellins combined with 30 μM benzyl-adenine (<xref ref-type="table" rid="pone.0159610.t001">Table 1</xref>). Among innate immune response genes, <italic>WRKY54</italic> was upregulated in response to 120 μM gibberellins + 15 μM benzyl-adenine while <italic>WRKY59</italic> was enhanced by 120 μM gibberellins + 30 μM benzyl-adenine (<xref ref-type="table" rid="pone.0159610.t002">Table 2</xref>). <italic>Sulfotransferase1</italic> was enhanced by both gibberellin and benzyl-adenine treatments. <italic>MYC2</italic> was upregulated by benzyladenine + gibberellin.</p><p>Among the secondary metabolism and stress response genes, 120 μM gibberellins + 15 μM benzyl-adenine enhanced expression of <italic>HSP82</italic> and two genes encoding <italic>pectate lyases</italic> involved in cell wall metabolism (<xref ref-type="table" rid="pone.0159610.t003">Table 3</xref>). <italic>β-amyrin</italic> was enhanced by 30 μM gibberellins combined with benzyl-adenine.</p><p>BA + GA treatments induced <italic>SA methyl transferase</italic> (<xref ref-type="table" rid="pone.0159610.t004">Table 4</xref>). BA + GA partially induced HLB-related changes to SA-mediated defense response, but <italic>EDS1</italic> was not altered by the treatment, so the plant probably still can’t downregulate jasmonic antagonistic signaling. <italic>Ka02</italic> involved in cytokinin metabolism was higher in response to 15 μM than to 30 μM benzyl-adenine.</p></sec><sec id="sec013"><title>Arginine treatment and K-phite treatments</title><p>0.5 mM L-arginine upregulated <italic>HSP21</italic> and <italic>terpene synthase3</italic> (<xref ref-type="table" rid="pone.0159610.t003">Table 3</xref>). Arginine treatments did not alter the expression of <italic>terpene synthase14</italic> and <italic>terpene synthase21</italic>, so the treated plant is still unable produce important terpene compounds. <italic>TGA5</italic> and <italic>HSP82</italic> were enhanced by 1 mM L-arginine. K-phite treatment repressed <italic>glucose-phosphate-transporter2</italic> (<italic>GPT2</italic>). This downregulation may have a beneficial effect since this gene allows glucose import into the chloroplast and starch accumulation. K-phite significantly induced <italic>HSP21</italic>, a chaperone involved in functional protein stability.</p></sec><sec id="sec014"><title>Principal component analysis</title><p>Two principal component analyses were conducted to independently assess two gene subsets: 1) genes involved in sucrose and starch metabolism (PCA-1; Figs <xref ref-type="fig" rid="pone.0159610.g001">1</xref> and <xref ref-type="fig" rid="pone.0159610.g002">2</xref>) genes involved in hormone-related proteins and biotic stress responses (PCA-2; <xref ref-type="fig" rid="pone.0159610.g002">Fig 2</xref>).</p><fig id="pone.0159610.g001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0159610.g001</object-id><label>Fig 1</label><caption><title>Overall analysis of HLB-regulated changes in carbohydrate metabolism.</title><p>Principal component analysis of treated and untreated Citrus categories in relation to genes involved in sucrose and starch pathways. UNT = Untreated with hormones (Control), CK = treated with K-phite, 30G = 30 μM gibberellin, 15G = 15 μM gibberellin, 1R = 1 mM L-arginine, 0.5R = 0.5 mM L-arginine, 2AS = sucrose combined with 2 μM atrazine, 1AS = sucrose combined with 2 μM atrazine. SPS = sucrose-phosphate-synthase.</p></caption><graphic xlink:href="pone.0159610.g001"></graphic></fig><fig id="pone.0159610.g002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0159610.g002</object-id><label>Fig 2</label><caption><title>Overall analysis of HLB-regulated changes in biotic stress response.</title><p>Principal component analysis of treated and untreated Citrus categories in relation to genes involved in biotic stress responses. UNT = Untreated with hormones (Control), CK = treated with K-phite, 30G = 30 μM gibberellin, 15G = 15 μM gibberellin, 1R = 1 mM L-arginine, 0.5R = 0.5 mM L-arginine, 2AS = sucrose combined with 2 μM atrazine, 1AS = sucrose combined with 2 μM atrazine. GA20 = Ga2-oxidase, HSP21 = Heat shock protein 21.</p></caption><graphic xlink:href="pone.0159610.g002"></graphic></fig><p>In PCA-1, the first two principle components explained 47 and 28% of data variability, respectively. The 2 μM atrazine + sucrose treatment separated from the rest of the treatments. <italic>SPS</italic> greatly contributed to this separation. 120 μM BA + 30 μM GA was also highly discriminated from the rest of the treatments. The other treatments were not distinct from the untreated controls.</p><p>PC 1 and PC 2 of PCA-2 (<xref ref-type="fig" rid="pone.0159610.g002">Fig 2</xref>) explained 36 and 17% of data variability, respectively. The 1 mM L-arginine treatment was not distinct from untreated conditions, but the 2 μM atrazine + sucrose treatment was highly distant. <italic>GA2-oxidase</italic>, <italic>zinc ion binding</italic>, and <italic>cysteine-histine rich domain C1</italic> gene contributed significantly to the separation of 2 μM atrazine + sucrose treatment. <italic>WRKY33</italic> highly contributed to the separation of the 1 μM atrazine + sucrose treatment.</p></sec></sec><sec id="sec015"><title>Field trial</title><p>Small-molecule regulating treatments investigated in the greenhouse were also applied to HLB-symptomatic trees in a young orchard where disease symptoms were frequently present. The aim of this trial was to determine whether the same treatments used in the greenhouse could modulate the expression of key host genes at three and six days after their application. Seven key genes were selected to monitor the transcriptomic regulation of the treatments for two reasons: 1) previous data found them highly characteristic of an HLB-induced response [<xref rid="pone.0159610.ref007" ref-type="bibr">7</xref>,<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>] and 2) they play a key role in innate immune responses. A gibberellin responsive gene was used as a marker to determine the efficacy of GA + BA treatments to modulate gene expression under field conditions. <italic>GPT2</italic> is a key HLB-regulated gene involved in glucose import into the chloroplast and is linked to the increased accumulation of starch in symptomatic leaves. The other genes were involved in plant defense and hormonal-mediated innate responses. <italic>WRKY70</italic> and <italic>EDS1</italic> are key points of regulation of JA-SA crosstalk. <italic>PR1</italic> upregulation is a beneficial against C. Las since this gene is involved in the systemic acquired resistance response. <italic>WRKY48</italic> and <italic>WRKY54</italic> were induced by HLB in previous studies [<xref rid="pone.0159610.ref007" ref-type="bibr">7</xref>,<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>].</p><p>At three days after treatment, 1 μM atrazine + sucrose induced the gibberellin-responsive protein and <italic>PR1</italic> and repressed <italic>WRKY48</italic> (<xref ref-type="fig" rid="pone.0159610.g003">Fig 3</xref>).</p><fig id="pone.0159610.g003" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0159610.g003</object-id><label>Fig 3</label><caption><title>Expression of seven host genes in response to spray treatments in field conditions.</title><p>Relative expression of each gene and treatment was shown as average of three biological replicates. Standard deviations were indicated.</p></caption><graphic xlink:href="pone.0159610.g003"></graphic></fig><p>In addition, 30 μM GA + 120 μM BA induced <italic>WRKY48</italic> and <italic>WRKY54</italic>. This finding may have positive effects on infected Citrus since the two genes are involved in salicylic acid-mediated responses against biotrophs. 0.5 mM L-Arginine upregulated <italic>WRKY48</italic> and <italic>EDS1</italic>. <italic>WRKY70</italic> was enhanced by 1 mM arginine and 15 μM GA combined with 120 μM BA.</p><p>At six days following treatment, some important changes in expression of the seven host biomarkers were observed. 1 μM atrazine upregulated <italic>PR1</italic>. 2 μM atrazine and 0.5 mM L-arginine repressed <italic>WRKY48</italic>. A general inhibition of <italic>GPT2</italic> was observed in all treated trees at both three and six days after treatment. This repression was particularly evident in leaves sprayed with gibberellins + benzyladenine.</p><sec id="sec016"><title>Phenotypic measurements and pathogen qPCR detection</title><p>No obvious symptoms of a harmful spray as leaf drop or discoloration was observed in treated trees. The tree trunk diameter, height, width drill, and width row of the treated trees grown under field conditions were measured in May and June 2014 (<xref ref-type="table" rid="pone.0159610.t005">Table 5</xref>).</p><table-wrap id="pone.0159610.t005" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0159610.t005</object-id><label>Table 5</label><caption><title>Phenotypic measurements in response to the seven treatments and control (untreated).</title></caption><alternatives><graphic id="pone.0159610.t005g" xlink:href="pone.0159610.t005"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col><col align="left" valign="middle" span="1"></col></colgroup><thead><tr><th align="left" rowspan="1" colspan="1">1st Measurement (5.21.14)</th><th align="left" rowspan="1" colspan="1">Untreated</th><th align="left" rowspan="1" colspan="1">120 μM BA + 30 μM GA</th><th align="left" rowspan="1" colspan="1">120 GA + 15 μM BA</th><th align="left" rowspan="1" colspan="1">1 μM Atrazine</th><th align="left" rowspan="1" colspan="1">2 μM Atrazine</th><th align="left" rowspan="1" colspan="1">.12% Siluet K-Phite +</th><th align="left" rowspan="1" colspan="1">0.5 mM L-Arginine</th><th align="left" rowspan="1" colspan="1">1.0 mM L-Arginine</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Trunk Diameter (mm)</td><td align="left" rowspan="1" colspan="1">90.770 a</td><td align="left" rowspan="1" colspan="1">91.537 a</td><td align="left" rowspan="1" colspan="1">82.280 a</td><td align="left" rowspan="1" colspan="1">96.097 a</td><td align="left" rowspan="1" colspan="1">88.197 a</td><td align="left" rowspan="1" colspan="1">83.827 a</td><td align="left" rowspan="1" colspan="1">96.427 a</td><td align="left" rowspan="1" colspan="1">98.747 a</td></tr><tr><td align="left" rowspan="1" colspan="1">Tree Height (m)</td><td align="left" rowspan="1" colspan="1">2.9533 ab</td><td align="left" rowspan="1" colspan="1">3.0000 ab</td><td align="left" rowspan="1" colspan="1">2.5533 b</td><td align="left" rowspan="1" colspan="1">3.1667 ab</td><td align="left" rowspan="1" colspan="1">3.1900 ab</td><td align="left" rowspan="1" colspan="1">2.8133 ab</td><td align="left" rowspan="1" colspan="1">3.2567 ab</td><td align="left" rowspan="1" colspan="1">3.3200 a</td></tr><tr><td align="left" rowspan="1" colspan="1">Width Drill (m)</td><td align="left" rowspan="1" colspan="1">2.4167 a</td><td align="left" rowspan="1" colspan="1">2.7400 a</td><td align="left" rowspan="1" colspan="1">2.4733 a</td><td align="left" rowspan="1" colspan="1">2.7000 a</td><td align="left" rowspan="1" colspan="1">2.6767 a</td><td align="left" rowspan="1" colspan="1">2.4433 a</td><td align="left" rowspan="1" colspan="1">2.7467 a</td><td align="left" rowspan="1" colspan="1">2.6233 a</td></tr><tr><td align="left" rowspan="1" colspan="1">Width Row (m)</td><td align="left" rowspan="1" colspan="1">2.4767 a</td><td align="left" rowspan="1" colspan="1">2.7667 a</td><td align="left" rowspan="1" colspan="1">2.4400 a</td><td align="left" rowspan="1" colspan="1">2.7400 a</td><td align="left" rowspan="1" colspan="1">2.5267 a</td><td align="left" rowspan="1" colspan="1">2.5533 a</td><td align="left" rowspan="1" colspan="1">2.7400 a</td><td align="left" rowspan="1" colspan="1">2.6800 a</td></tr><tr><td align="left" rowspan="1" colspan="1">2nd Measurement (6.28.13)</td><td align="left" rowspan="1" colspan="1">Untreated</td><td align="left" rowspan="1" colspan="1">120 μM BA + 30 μM GA</td><td align="left" rowspan="1" colspan="1">120 GA + 15 μM BA</td><td align="left" rowspan="1" colspan="1">1 μM Atrazine</td><td align="left" rowspan="1" colspan="1">2 μM Atrazine</td><td align="left" rowspan="1" colspan="1">.12% Siluet K-Phite +</td><td align="left" rowspan="1" colspan="1">0.5 mM L-Arginine</td><td align="left" rowspan="1" colspan="1">1.0 mM L-Arginine</td></tr><tr><td align="left" rowspan="1" colspan="1">Trunk Diameter (mm)</td><td align="left" rowspan="1" colspan="1">70.940 a</td><td align="left" rowspan="1" colspan="1">77.427 a</td><td align="left" rowspan="1" colspan="1">70.503 a</td><td align="left" rowspan="1" colspan="1">80.813 a</td><td align="left" rowspan="1" colspan="1">75.207 a</td><td align="left" rowspan="1" colspan="1">72.513 a</td><td align="left" rowspan="1" colspan="1">82.293 a</td><td align="left" rowspan="1" colspan="1">83.617 a</td></tr><tr><td align="left" rowspan="1" colspan="1">Tree Height (m)</td><td align="left" rowspan="1" colspan="1">2.5500 abc</td><td align="left" rowspan="1" colspan="1">2.6833 ab</td><td align="left" rowspan="1" colspan="1">2.3533 bc</td><td align="left" rowspan="1" colspan="1">2.7067 ab</td><td align="left" rowspan="1" colspan="1">2.6733 abc</td><td align="left" rowspan="1" colspan="1">2.3100 c</td><td align="left" rowspan="1" colspan="1">2.7267 a</td><td align="left" rowspan="1" colspan="1">2.7700 a</td></tr><tr><td align="left" rowspan="1" colspan="1">Width Drill (m)</td><td align="left" rowspan="1" colspan="1">2.1767 a</td><td align="left" rowspan="1" colspan="1">2.5400 a</td><td align="left" rowspan="1" colspan="1">2.3133 a</td><td align="left" rowspan="1" colspan="1">2.4233 a</td><td align="left" rowspan="1" colspan="1">2.4000 a</td><td align="left" rowspan="1" colspan="1">2.4200 a</td><td align="left" rowspan="1" colspan="1">2.6533 a</td><td align="left" rowspan="1" colspan="1">2.4400 a</td></tr><tr><td align="left" rowspan="1" colspan="1">Width Row (m)</td><td align="left" rowspan="1" colspan="1">2.0700 c</td><td align="left" rowspan="1" colspan="1">2.6167 ab</td><td align="left" rowspan="1" colspan="1">2.2733 bc</td><td align="left" rowspan="1" colspan="1">2.4900 abc</td><td align="left" rowspan="1" colspan="1">2.3033 abc</td><td align="left" rowspan="1" colspan="1">2.2667 bc</td><td align="left" rowspan="1" colspan="1">2.7267 a</td><td align="left" rowspan="1" colspan="1">2.4267 abc</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="t005fn001"><p>Means of three replicates were indicated. Letters means significant differences using ANOVA (P < = 0.05) and post-hoc test.</p></fn></table-wrap-foot></table-wrap><p>The aim of this analysis was to determine whether treatments were detrimental to tree growth or had undesirable phenotypic effects. No significant phenotypic differences were observed among untreated and treated trees except width row, which was significantly lower in untreated trees than in trees treated with 30 μM GA + 120 μM BA. There were no visible discolorations or other signs of plant distress from the spraying. No set of trees had visibly different vegetative vigor.</p><p>The quantification of pathogen titer was performed after three months from treatments as previously indicated to check if it was not changed in response to treatments.</p></sec></sec></sec><sec sec-type="conclusions" id="sec017"><title>Discussion</title><p>Our objective was to test the ability of six combinations of small-molecule compounds to modulate expression of key genes involved in HLB syndrome and innate immune responses shortly after treatment. We did not pretend to reduce pathogen titers or cure the plants with only one treatment. Before performing a long-term study, we wanted to evaluate the ability of the treatments to modulate expression of a subset of key genes that are altered during HLB syndrome. As we expected pathogen titers did not significantly differ among treated and control trees. A long-term study will reveal if repetitive and continuous applications will reduce pathogen concentrations and symptom severity.</p><p>Treatments were designed based on previously proposed hypotheses. Sucrose-induced protection against atrazine effects was linked to upregulation of reactive oxygen species (ROS) defence and repair mechanisms [<xref rid="pone.0159610.ref021" ref-type="bibr">21</xref>].</p><p>Nitric oxide (NO) is produced by l-arginine. Treatment with arginine provoked resistance against <italic>Botrytis cinerea</italic> in tomato at three to six days after treatment. Endogenous NO concentrations correlated positively with induction of key enzymes involved in biotic stress responses such as phenylalanine ammonia-lyase, chitinase, β-1,3-glucanase and polyphenoloxidase [<xref rid="pone.0159610.ref023" ref-type="bibr">23</xref>].</p><p>Gibberellins regulate plant growth by modulating degradation of growth-repressing DELLA proteins that promote susceptibility to biotrophic pathogens and resistance to necrotrophic pathogens [<xref rid="pone.0159610.ref024" ref-type="bibr">24</xref>]. This is accomplished by modulating the relative strength of the SA and JA signaling pathways [<xref rid="pone.0159610.ref024" ref-type="bibr">24</xref>]. Through regulation of DELLA stability, gibberellins affect the SA-JA-ET network and plant immune response. Genevestigator showed that benzyl-adenine downregulated the glucose-phosphate transporter in <italic>Arabidopsis</italic>. Since this gene is induced by HLB metabolic syndrome [<xref rid="pone.0159610.ref005" ref-type="bibr">5</xref>,<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>], benzyl-adenine treatments might help mitigate the negative effects of HLB on leaf metabolism. In combination, the two hormones may beneficially modulate key HLB-regulated genes involved in carbohydrate metabolism [<xref rid="pone.0159610.ref007" ref-type="bibr">7</xref>,<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>].</p><p>K-phite mineral solution was also tested, alone or in combination with the three small molecule compounds. This treatment was considered because of contrasting published reports on the effects of nutrient solutions such as K-phite [<xref rid="pone.0159610.ref025" ref-type="bibr">25</xref>]. Mineral solutions increased the concentrations of important N, Mn, Zn and B ions in leaves and long-term application reduced pathogen titer, leaf size, and leaf weight [<xref rid="pone.0159610.ref025" ref-type="bibr">25</xref>]. Although enhanced nutritional solutions composed of essential micronutrients did not improve fruit production and quality of C. Las-infected trees [<xref rid="pone.0159610.ref026" ref-type="bibr">26</xref>], others results support the hypothesis that the pathogen severely affects nutrient patterns [<xref rid="pone.0159610.ref027" ref-type="bibr">27</xref>]. In addition, foliar nutrition and soil conditioners helped reduce economic and production losses due to HLB [<xref rid="pone.0159610.ref028" ref-type="bibr">28</xref>,<xref rid="pone.0159610.ref029" ref-type="bibr">29</xref>].</p><p>To determine how the treatments affected the metabolism of infected trees, 42 genes were selected from previously published Citrus transcriptome data [<xref rid="pone.0159610.ref007" ref-type="bibr">7</xref>,<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>]. These genes fell into three subsets involved in: 1) carbohydrate metabolism and signaling, 2) innate immune responses, including key players in JA-SA signaling, crosstalk and induced responses, or 3) other genes involved in biotic stress responses such as those involved in hormone-related pathways, secondary metabolism and stress-preventing factors. From these biomarkers, we chose seven representative biomarkers to be followed under field conditions in response to the same treatments. Treated plants were tested for the presence of C. Las using qPCR and showed clear HLB symptoms.</p><p>QRT-PCR analyses were conducted at three to six days after treatment. As expected, we observed no significant changes in pathogen titer. Repeated applications of treatments (at least weekly) should eventually affect the titer. Since we only treated infected trees once, an analysis of pathogen titer after treaments was outside the scope of this study. Long-term studies on the effects of repeated applications of these treatments should test pathogen titer using qPCR.</p><sec id="sec018"><title>Atrazine combined with sucrose</title><p>The first small-molecule treatment tested was the combination of sucrose and the herbicide atrazine. Atrazine is a well-known photosystem II inhibitor that affects plant gene expression, seedling physiology, and potentiality impairs protein translation and the ROS defense mechanism [<xref rid="pone.0159610.ref030" ref-type="bibr">30</xref>]. However, in combination with sucrose, atrazine induces xenobiotic and ROS signaling. In addition, this treatment upregulated important classes of antioxidant enzymes [<xref rid="pone.0159610.ref021" ref-type="bibr">21</xref>,<xref rid="pone.0159610.ref022" ref-type="bibr">22</xref>]. These findings were consistent with our unpublished findings that <italic>glutathione-S-transferases</italic> are upregulated in more tolerant Citrus genotypes.</p><p>Here we observed that atrazine combined with sucrose drastically affected some key genes responsible for HLB-induced carbohydrate changes. Increased sucrose concentrations have been found in in C. Las-infected leaves [<xref rid="pone.0159610.ref006" ref-type="bibr">6</xref>,<xref rid="pone.0159610.ref011" ref-type="bibr">11</xref>]. 1 μM atrazine + sucrose enhanced sucrose synthase while 2 μM atrazine + sucrose upregulated the water dikinase starch degradation gene and sucrose-phosphate-synthase. Atrazine upregulated <italic>alpha-amylase</italic>, which was repressed in mature HLB-infected Citrus leaves [<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>] but upregulated in infected Citrus stems [<xref rid="pone.0159610.ref031" ref-type="bibr">31</xref>]. Taken together, these findings lead us to speculate that atrazine + sucrose might help sucrose degradation by activating <italic>sucrose synthase</italic>. In addition, upregulation of <italic>alpha-amylase</italic> may increase starch degradation in HLB-infected plants where its accumulation is advanced.</p><p>Atrazine (1 μM) combined with sucrose upregulated <italic>WRKY33</italic>. <italic>Brassica napus</italic> plants overexpressing <italic>BnWRKY33</italic> had increased resistance to <italic>Sclerotinia sclerotiorum</italic> infection [<xref rid="pone.0159610.ref032" ref-type="bibr">32</xref>]. This effect was mediated by SA [<xref rid="pone.0159610.ref032" ref-type="bibr">32</xref>]. <italic>WRKY33</italic> upregulation allowed resistance to the necrotroph <italic>Botrytis cinerea</italic> in <italic>Arabidopsis</italic> [<xref rid="pone.0159610.ref033" ref-type="bibr">33</xref>]. Loss of <italic>WRKY33</italic> function induces salicylic acid (SA)-mediated responses, increases salicylic acid and represses jasmonic acid (JA)-mediated responses [<xref rid="pone.0159610.ref034" ref-type="bibr">34</xref>].</p><p>Overall, our results support the hypothesis that this treatment could beneficially modulate key HLB-regulated genes associated with the well-known HLB carbohydrate metabolic syndrome [<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>]. The changes to expression of some key genes involved in sugar and starch metabolism could beneficially modulate the metabolic responses of HLB disease in photosynthesizing Citrus leaves, restoring a more normal source-sink relationship and potentially inhibiting the characteristic and deleterious syndrome in the fruit.</p></sec><sec id="sec019"><title>Gibberelllins combined with benzyl adenine treatments</title><p>A mixture of gibberellin (GA3) and 6-benzyladenine (BA) was tested to modulate jasmonic acid-salicylic acid (JA-SA) crosstalk in favor of responses to biotrophs such as C. Las. Our hypothesis was that gibberellin treatments may activate SAR responses through positive regulation of hormone-mediated crosstalk regulating biotic stress responses [<xref rid="pone.0159610.ref019" ref-type="bibr">19</xref>]. Some key genes in hormone-related pathways and JA-SA crosstalk were chosen as indicators of treatment effects.</p><p><italic>MYC2</italic> was significantly induced by both 15 and 30 μM gibberellin treatments. <italic>MYC2</italic> is a transcription factor composed of a basic helix-loop-helix (bHLH) domain that activates and represses specific JA-responsive gene expression in <italic>Arabidopsis</italic> [<xref rid="pone.0159610.ref035" ref-type="bibr">35</xref>]. <italic>MYC2</italic> also induced responses to abiotic stress mediated by abscissic acid in Arabidopsis [<xref rid="pone.0159610.ref036" ref-type="bibr">36</xref>] and suppressed salicylic acid-mediated responses in Arabidopsis [<xref rid="pone.0159610.ref037" ref-type="bibr">37</xref>]. Upregulation of <italic>WRKY54</italic> in gibberellin-treated field trees is also interesting because this gene is a positive regulator of resistance against <italic>Erwinia amylovora</italic>, the agent of fire blight in the Rosaceae family.</p><p>The plant immune regulator <italic>EDS1</italic> (Enhanced Disease Susceptibility1) plays a fundamental role in resistance mechanisms to biotrophs and hemi-biotrophs [<xref rid="pone.0159610.ref038" ref-type="bibr">38</xref>,<xref rid="pone.0159610.ref039" ref-type="bibr">39</xref>]. This role is due to the formation of complexes with <italic>PAD4</italic> and <italic>SAD101</italic> in both cytoplasm and nucleus [<xref rid="pone.0159610.ref040" ref-type="bibr">40</xref>]. The 15 μM GA + BA treatment enhanced EDS1 at six days after treatment in the field. The increase in <italic>EDS1</italic> transcripts after application of these small molecules could help activate SAR response against pathogen infections. Mutant screening showed that upregulation of <italic>EDS1</italic> induces non-host resistance against <italic>E</italic>. <italic>amylovora</italic> in <italic>Arabidopsis</italic> by activating <italic>WRKY46</italic> and <italic>WRKY54</italic> genes [<xref rid="pone.0159610.ref041" ref-type="bibr">41</xref>].</p><p>Our hypothesis was partially confirmed. GA + BA may beneficially increase innate responses by inducing <italic>EDS1</italic> and <italic>MYC2</italic>. Although long-term field trials are required, we speculate that continuous application of this small molecule mixture could stimulate improved SA-JA crosstalk</p></sec><sec id="sec020"><title>L-arginine treatments</title><p>The third small-molecule treatment was composed of L-arginine, used in two concentrations. L-arginine positively regulates key genes involved in innate immune responses [<xref rid="pone.0159610.ref023" ref-type="bibr">23</xref>]. L-arginine may act on nitric oxide and directly upregulate key genes in salicylic acid signaling. Increased endogenous NO concentrations after L-arginine treatment in pre-harvest tomatoes correlated positively with increased defensive enzyme activity and postharvest disease resistance [<xref rid="pone.0159610.ref015" ref-type="bibr">15</xref>]. <italic>PR1</italic>, <italic>WRKY70</italic> and <italic>WRKY54</italic> were upregulated by 1 mM L-arginine under field conditions. <italic>WRKY</italic> transcription factors are important regulators of responses to abiotic and biotic stresses. <italic>WRKY54</italic> and <italic>WRKY70</italic> play a key role in a regulatory network that affects leaf senescence by interacting with another WRKY factor [<xref rid="pone.0159610.ref042" ref-type="bibr">42</xref>].</p><p>In our field trial, arginine induced some key important gene regulation that should benefit SAR responses.</p></sec><sec id="sec021"><title>Common effects among treatments</title><p>Glucose accumulation induced by C. Las infection is transported to the plastid by hexose transporters [<xref rid="pone.0159610.ref005" ref-type="bibr">5</xref>,<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>,<xref rid="pone.0159610.ref043" ref-type="bibr">43</xref>]. GPT2 is a key player in HLB-mediated starch accumulation in leaves because this gene mediates glucose import into the chloroplast in infected leaves [<xref rid="pone.0159610.ref005" ref-type="bibr">5</xref>,<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>]. <italic>GPT2</italic> was significantly repressed by all spray applications at both three and six days after treatment under field conditions. This inhibition may reduce the amount of glucose carried into the plastid and thus starch accumulation, with consequent improvement of disrupted source-sink relationships.</p><p>Gene expression changes observed in this work corroborated the hypothesis that these spray treatments may help stimulate systemic acquired resistance responses by activating key genes involved in innate responses (<xref ref-type="fig" rid="pone.0159610.g004">Fig 4</xref>).</p><fig id="pone.0159610.g004" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0159610.g004</object-id><label>Fig 4</label><caption><title>Key differentially regulated genes in response to treatments involved in biotic stress responses.</title></caption><graphic xlink:href="pone.0159610.g004"></graphic></fig><p>PR gene induction is mediated through interaction with TGA transcription factors [<xref rid="pone.0159610.ref015" ref-type="bibr">15</xref>]. The observed upregulation of <italic>TGA5</italic> and <italic>TGA6</italic> by L-arginine and 2 μM atrazine + sucrose, respectively, might help stimulate defense responses against C. Las infection.</p><p>Arginine and atrazine sprays upregulated the <italic>PR1</italic> gene under field conditions. PR1 protein is the hallmark of the defense response induction mediated by salicylic acid through systemic acquired resistance [<xref rid="pone.0159610.ref044" ref-type="bibr">44</xref>]. Molecular action of this protein against pathogens is still unclear, although antifungal properties have been attributed to it [<xref rid="pone.0159610.ref045" ref-type="bibr">45</xref>]. <italic>PR1</italic> also interacted with fungal toxin activities, mediating necrosis in sensitive wheat [<xref rid="pone.0159610.ref031" ref-type="bibr">31</xref>]. This gene was not activated in response to C. Las infections in orchard trees [<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>].</p><p>Interestingly, all three treatments upregulated <italic>HSP82</italic>. Previous data on C. Las-infected citrus leaves and fruits showed that C. Las caused a significant repression of genes encoding chaperones [<xref rid="pone.0159610.ref007" ref-type="bibr">7</xref>–<xref rid="pone.0159610.ref009" ref-type="bibr">9</xref>]. Modified expression of these genes plays a key role in general stress conditions [<xref rid="pone.0159610.ref046" ref-type="bibr">46</xref>, <xref rid="pone.0159610.ref047" ref-type="bibr">47</xref>]. A link between reduced HSP protein amount and HLB symptoms was also confirmed by analysis [<xref rid="pone.0159610.ref010" ref-type="bibr">10</xref>].</p></sec></sec><sec sec-type="conclusions" id="sec022"><title>Conclusions</title><p>Present data confirmed our hypothesis that these small-molecule sprays may affect transcript abundance of key genes involved in HLB carbohydrate metabolic syndrome and innate responses. As expected, there were no phenotypic changes in response to treatments at one to two months after treatment. Treatment sprays did not cause negative effects such as leaf drop or discoloration. As expected, tree measurements showed almost no differences between treated and untreated trees in field conditions. We believe that beneficial effects are likely to be seen only if treatments are applied frequently before or at the onset of visible HLB symptoms. Here, our aim was to analyze the molecular effects of these treatments on gene expression several days after treatment. Future studies should examine long-term molecular and phenotypic improvements associated with ongoing applications to young trees infected with C. Las.</p></sec><sec sec-type="supplementary-material" id="sec023"><title>Supporting Information</title><supplementary-material content-type="local-data" id="pone.0159610.s001"><label>S1 Table</label><caption><title>List of primers used for each gene analyzed using qRT-PCR.</title><p>(DOCX)</p></caption><media xlink:href="pone.0159610.s001.docx"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack><p>The authors wish to thank Mary Lou Mendum for her editorial review of the manuscript.</p></ack><ref-list><title>References</title><ref id="pone.0159610.ref001"><label>1</label><mixed-citation publication-type="journal"><name><surname>Bove’</surname><given-names>JM</given-names></name>. <article-title>Huanglongbing: A destructive, newly-emerging, century-old disease of citrus</article-title>. <source>J Plant Pathol</source>. <year>2006</year>;<volume>88</volume>:<fpage>7</fpage>–<lpage>37</lpage>.</mixed-citation></ref><ref id="pone.0159610.ref002"><label>2</label><mixed-citation publication-type="journal"><name><surname>Jagoueix</surname><given-names>S</given-names></name>, <name><surname>Bové</surname><given-names>JM</given-names></name>, <name><surname>Garnier</surname><given-names>M</given-names></name>. <article-title>The phloem-limited bacterium of greening disease of citrus is a member of the alpha subdivision of the Proteobacteria</article-title>. <source>Int J of Syst Bacteriol</source>. <year>1994</year>;<volume>44</volume>:<fpage>397</fpage>–<lpage>86</lpage>.</mixed-citation></ref><ref id="pone.0159610.ref003"><label>3</label><mixed-citation publication-type="journal"><name><surname>Folimonova</surname><given-names>SY</given-names></name>, <name><surname>Robertson</surname><given-names>CJ</given-names></name>, <name><surname>Garnsey</surname><given-names>SM</given-names></name>, <name><surname>Gowda</surname><given-names>S</given-names></name>, <name><surname>Dawson</surname><given-names>WO</given-names></name>. <article-title>Examination of the responses of different genotypes of Citrus to Huanglongbing (Citrus greening) under different conditions</article-title>. <source>Phytopathology</source>. <year>2009</year>;<volume>99</volume>:<fpage>1346</fpage>–<lpage>54</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1094/PHYTO-99-12-1346">10.1094/PHYTO-99-12-1346</ext-link></comment><pub-id pub-id-type="pmid">19900000</pub-id></mixed-citation></ref><ref id="pone.0159610.ref004"><label>4</label><mixed-citation publication-type="journal"><name><surname>Folimonova</surname><given-names>SY</given-names></name>, <name><surname>Achor</surname><given-names>D</given-names></name>. <article-title>Early events of citrus greening (Huanglongbing) disease development at the ultrastructural level</article-title>. <source>Bacteriology</source>. <year>2010</year>;<volume>100</volume>:<fpage>949</fpage>–<lpage>58</lpage>.</mixed-citation></ref><ref id="pone.0159610.ref005"><label>5</label><mixed-citation publication-type="journal"><name><surname>Albrecht</surname><given-names>U</given-names></name>, <name><surname>Bowman</surname><given-names>KD</given-names></name>. <article-title>Gene expression in <italic>Citrus sinensis</italic> (L.) Osbeck following infection with the bacterial pathogen <italic>Candidatus</italic> Liberibacter asiaticus causing Huanglongbing in Florida</article-title>. <source>Plant Sci</source>. <year>2008</year>;<volume>175</volume>:<fpage>291</fpage>–<lpage>306</lpage>.</mixed-citation></ref><ref id="pone.0159610.ref006"><label>6</label><mixed-citation publication-type="journal"><name><surname>Kim</surname><given-names>J-S</given-names></name>, <name><surname>Sagaram</surname><given-names>US</given-names></name>, <name><surname>Burns</surname><given-names>JK</given-names></name>, <name><surname>Li</surname><given-names>J-L</given-names></name>, <name><surname>Wang</surname><given-names>N</given-names></name>. <article-title>Response of sweet orange (<italic>Citrus sinensis</italic>) to ‘<italic>Candidatus</italic> Liberibacter asiaticus’ infection: microscopy and microarray analyses</article-title>. <source>Phytopathology</source>. <year>2009</year>;<volume>99</volume>:<fpage>50</fpage>–<lpage>57</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1094/PHYTO-99-1-0050">10.1094/PHYTO-99-1-0050</ext-link></comment><pub-id pub-id-type="pmid">19055434</pub-id></mixed-citation></ref><ref id="pone.0159610.ref007"><label>7</label><mixed-citation publication-type="journal"><name><surname>Martinelli</surname><given-names>F</given-names></name>, <name><surname>Uratsu</surname><given-names>SL</given-names></name>, <name><surname>Albrecht</surname><given-names>U</given-names></name>, <name><surname>Reagan</surname><given-names>RL</given-names></name>, <name><surname>Phu</surname><given-names>ML</given-names></name>, <name><surname>Britton</surname><given-names>M</given-names></name>, <etal>et al</etal><article-title>Transcriptome profiling of Citrus fruit response to Huanglongbing disease</article-title>. <source>PLoS One</source>. <year>2012</year>;<volume>7</volume>:<fpage>e38039</fpage><comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1371/journal.pone.0038039">10.1371/journal.pone.0038039</ext-link></comment><pub-id pub-id-type="pmid">22675433</pub-id></mixed-citation></ref><ref id="pone.0159610.ref008"><label>8</label><mixed-citation publication-type="journal"><name><surname>Dandekar</surname><given-names>AM</given-names></name>, <name><surname>Martinelli</surname><given-names>F</given-names></name>, <name><surname>Davis</surname><given-names>CE</given-names></name>, <name><surname>Bhushan</surname><given-names>A</given-names></name>, <name><surname>Zhao</surname><given-names>W</given-names></name>, <name><surname>Fiehn</surname><given-names>O</given-names></name>, <etal>et al</etal><article-title>Analysis of Early Host Responses for Asymptomatic Disease Detection and Management of Specialty Crops</article-title>. <source>Crit Rev Immunol</source>. <year>2010</year>;<volume>30</volume>:<fpage>277</fpage>–<lpage>89</lpage>. <pub-id pub-id-type="pmid">20370635</pub-id></mixed-citation></ref><ref id="pone.0159610.ref009"><label>9</label><mixed-citation publication-type="journal"><name><surname>Martinelli</surname><given-names>F</given-names></name>, <name><surname>Reagan</surname><given-names>RL</given-names></name>, <name><surname>Uratsu</surname><given-names>SL</given-names></name>, <name><surname>Phu</surname><given-names>ML</given-names></name>, <name><surname>Albrecht</surname><given-names>U</given-names></name>, <name><surname>Zhao</surname><given-names>W</given-names></name>, <etal>et al</etal><article-title>Gene Regulatory Networks Elucidating Huanglongbing Disease Mechanisms</article-title>. <source>Plos One</source>. <year>2013</year>;<volume>8</volume>:<fpage>e74256</fpage><comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1371/journal.pone.0074256">10.1371/journal.pone.0074256</ext-link></comment><pub-id pub-id-type="pmid">24086326</pub-id></mixed-citation></ref><ref id="pone.0159610.ref010"><label>10</label><mixed-citation publication-type="journal"><name><surname>Nwugo</surname><given-names>CC</given-names></name>, <name><surname>Lin</surname><given-names>H</given-names></name>, <name><surname>Duan</surname><given-names>Y</given-names></name>, <name><surname>Civerolo</surname><given-names>EL</given-names></name>. <article-title>The effect of ‘<italic>Candidatus</italic> Liberibacter asiaticus’ infection on the proteomic profiles and nutritional status of pre-symptomatic and symptomatic grapefruit (<italic>Citrus paradisi</italic>) plants</article-title>. <source>BMC Plant Biology</source>. <year>2013</year>;<volume>13</volume>:<fpage>59</fpage><comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1186/1471-2229-13-59">10.1186/1471-2229-13-59</ext-link></comment><pub-id pub-id-type="pmid">23578104</pub-id></mixed-citation></ref><ref id="pone.0159610.ref011"><label>11</label><mixed-citation publication-type="journal"><name><surname>Fan</surname><given-names>J</given-names></name>, <name><surname>Chen</surname><given-names>C</given-names></name>, <name><surname>Yu</surname><given-names>Q</given-names></name>, <name><surname>Brlansky</surname><given-names>RH</given-names></name>, <name><surname>Li</surname><given-names>Z-G</given-names></name>, <name><surname>Gmitter</surname><given-names>FG</given-names></name>. <article-title>Comparative iTRAQ proteome and transcriptome analyses of sweet orange infected by “Candidatus Liberibacter asiaticus”</article-title>. <source>Physiol Plant</source>. <year>2011</year>;<volume>143</volume>:<fpage>235</fpage>–<lpage>45</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1111/j.1399-3054.2011.01502.x">10.1111/j.1399-3054.2011.01502.x</ext-link></comment><pub-id pub-id-type="pmid">21838733</pub-id></mixed-citation></ref><ref id="pone.0159610.ref012"><label>12</label><mixed-citation publication-type="journal"><name><surname>Cong</surname><given-names>Q</given-names></name>, <name><surname>Kinch</surname><given-names>LN</given-names></name>, <name><surname>Kim</surname><given-names>B-H</given-names></name>, <name><surname>Grishin</surname><given-names>NV</given-names></name>. <article-title>Predictive sequence analysis of the Candidatus liberibacter asiaticu proteome</article-title>. <source>Plos One</source>. <year>2012</year>;<volume>7</volume>(<issue>7</issue>):<fpage>e41071</fpage><comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1371/journal.pone.0041071">10.1371/journal.pone.0041071</ext-link></comment><pub-id pub-id-type="pmid">22815919</pub-id></mixed-citation></ref><ref id="pone.0159610.ref013"><label>13</label><mixed-citation publication-type="journal"><name><surname>Tyler</surname><given-names>HL</given-names></name>, <name><surname>Roesch</surname><given-names>LFW</given-names></name>, <name><surname>Gowda</surname><given-names>S</given-names></name>, <name><surname>Dawson</surname><given-names>WO</given-names></name>, <name><surname>Triplett</surname><given-names>EW</given-names></name>. <article-title>Confirmation of the sequence of ‘Candidatus Liberibacter asiaticus’ and assessment of microbial diversity in Huanglongbing-infected Citrus phloem using a metagenomic approach</article-title>. <source>Mol Plant Microbe Interact</source>. <year>2009</year>;<volume>22</volume>:<fpage>1624</fpage>–<lpage>34</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1094/MPMI-22-12-1624">10.1094/MPMI-22-12-1624</ext-link></comment><pub-id pub-id-type="pmid">19888827</pub-id></mixed-citation></ref><ref id="pone.0159610.ref014"><label>14</label><mixed-citation publication-type="journal"><name><surname>Khan</surname><given-names>AA</given-names></name>, <name><surname>Afzal</surname><given-names>M</given-names></name>, <name><surname>Qureshi</surname><given-names>JA</given-names></name>. <article-title>Botanicals, selective insecticides, and predators to control Diaphorina citri (Hemiptera: Liviidae) in citrus orchards</article-title>. <source>Insect Sci</source>. <year>2014</year>;<volume>21</volume>:<fpage>717</fpage>–<lpage>26</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1111/1744-7917.12173">10.1111/1744-7917.12173</ext-link></comment><pub-id pub-id-type="pmid">25205398</pub-id></mixed-citation></ref><ref id="pone.0159610.ref015"><label>15</label><mixed-citation publication-type="journal"><name><surname>Zhang</surname><given-names>M</given-names></name>, <name><surname>Powell</surname><given-names>CA</given-names></name>, <name><surname>Zhou</surname><given-names>L</given-names></name>, <name><surname>He</surname><given-names>Z</given-names></name>, <name><surname>Stover</surname><given-names>E</given-names></name>, <name><surname>Duan</surname><given-names>Y</given-names></name>. <article-title>Chemical compounds effective against the citrus Huanglongbing bacterium ‘Candidatus Liberibacter asiaticus’ in planta</article-title>. <source>Phytopathology</source>. <year>2011</year>;<volume>101</volume>:<fpage>1097</fpage>–<lpage>1103</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1094/PHYTO-09-10-0262">10.1094/PHYTO-09-10-0262</ext-link></comment><pub-id pub-id-type="pmid">21834727</pub-id></mixed-citation></ref><ref id="pone.0159610.ref016"><label>16</label><mixed-citation publication-type="journal"><name><surname>Zhang</surname><given-names>M</given-names></name>, <name><surname>Powell</surname><given-names>CA</given-names></name>, <name><surname>Guo</surname><given-names>Y</given-names></name>, <name><surname>Doud</surname><given-names>MS</given-names></name>, <name><surname>Duan</surname><given-names>Y</given-names></name>. <article-title>A graft-based chemotherapy method for screening effective molecules and rescuing huanglongbing-affected citrus plants</article-title>. <source>Phytopathology</source>. <year>2012</year>;<volume>102</volume>:<fpage>567</fpage>–<lpage>74</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1094/PHYTO-09-11-0265">10.1094/PHYTO-09-11-0265</ext-link></comment><pub-id pub-id-type="pmid">22568814</pub-id></mixed-citation></ref><ref id="pone.0159610.ref017"><label>17</label><mixed-citation publication-type="journal"><name><surname>Yang</surname><given-names>C</given-names></name>, <name><surname>Powell</surname><given-names>CA</given-names></name>, <name><surname>Duan</surname><given-names>Y</given-names></name>, <name><surname>Shatters</surname><given-names>R</given-names></name>, <name><surname>Zhang</surname><given-names>M</given-names></name>. <article-title>Antimicrobial nanoemulsion formulation with improved penetration of foliar spray through citrus leaf cuticles to control citrus huanglongbing</article-title>. <source>Plos One</source>. <year>2015</year>;<volume>10</volume>(<issue>7</issue>):<fpage>e0133826</fpage><comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1371/journal.pone.0133826">10.1371/journal.pone.0133826</ext-link></comment><pub-id pub-id-type="pmid">26207823</pub-id></mixed-citation></ref><ref id="pone.0159610.ref018"><label>18</label><mixed-citation publication-type="journal"><name><surname>Akula</surname><given-names>N</given-names></name>, <name><surname>Trivedi</surname><given-names>P</given-names></name>, <name><surname>Han</surname><given-names>FQ</given-names></name>, <name><surname>Wang</surname><given-names>N</given-names></name>. <article-title>Identification of small molecule inhibitors against SecA of Candidatus Liberibacter asiaticus by structure based design</article-title>. <source>Eur J Med Chem</source>. <year>2012</year>;<volume>54</volume>:<fpage>919</fpage>–<lpage>24</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1016/j.ejmech.2012.05.035">10.1016/j.ejmech.2012.05.035</ext-link></comment><pub-id pub-id-type="pmid">22703844</pub-id></mixed-citation></ref><ref id="pone.0159610.ref019"><label>19</label><mixed-citation publication-type="journal"><name><surname>Li</surname><given-names>W</given-names></name>, <name><surname>Hartung</surname><given-names>JS</given-names></name>, <name><surname>Levy</surname><given-names>L</given-names></name>. <article-title>Quantitative real-time PCR for detection and identification of <italic>Candidatus Liberibacter</italic> species associated with citrus huanglongbing</article-title>. <source>J Microbiol Methods</source>. <year>2006</year>;<volume>66</volume>:<fpage>104</fpage>–<lpage>15</lpage>. <pub-id pub-id-type="pmid">16414133</pub-id></mixed-citation></ref><ref id="pone.0159610.ref020"><label>20</label><mixed-citation publication-type="journal"><name><surname>Pieterse</surname><given-names>CMJ</given-names></name>, <name><surname>Leon-Reyes</surname><given-names>A</given-names></name>, <name><surname>Van der Ent</surname><given-names>S</given-names></name>, <name><surname>Van Wees</surname><given-names>SCM</given-names></name>. <article-title>Networking by small-molecule hormones in plant immunity</article-title>. <source>Nat Chem Biol</source>. <year>2009</year>;<volume>5</volume>:<fpage>308</fpage>–<lpage>16</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1038/nchembio.164">10.1038/nchembio.164</ext-link></comment><pub-id pub-id-type="pmid">19377457</pub-id></mixed-citation></ref><ref id="pone.0159610.ref021"><label>21</label><mixed-citation publication-type="journal"><name><surname>Ramel</surname><given-names>F</given-names></name>, <name><surname>Sulmon</surname><given-names>C</given-names></name>, <name><surname>Cabello-Hurtado</surname><given-names>F</given-names></name>, <name><surname>Tacconat</surname><given-names>L</given-names></name>, <name><surname>Martin-Magniette</surname><given-names>M-L</given-names></name>, <name><surname>Renou</surname><given-names>J-P</given-names></name>, <etal>et al</etal><article-title>Genome-wide interacting effects of sucrose and herbicide-mediated stress in <italic>Arabidopsis thaliana</italic>: Novel insights into atrazine toxicity and sucrose-induced tolerance</article-title>. <source>BMC Genomics</source>. <year>2007</year>;<volume>8</volume>:<fpage>450</fpage><pub-id pub-id-type="pmid">18053238</pub-id></mixed-citation></ref><ref id="pone.0159610.ref022"><label>22</label><mixed-citation publication-type="journal"><name><surname>Ramel</surname><given-names>F</given-names></name>, <name><surname>Sulmon</surname><given-names>C</given-names></name>, <name><surname>Gouesbet</surname><given-names>G</given-names></name>, <name><surname>Couee</surname><given-names>I</given-names></name>. <article-title>Regulatory effects of atrazine differentially override sucrose repression of amino acid catabolism</article-title>. <source>Acta Physiol Plant</source>. <year>2013</year>;<volume>35</volume>:<fpage>2329</fpage>–<lpage>37</lpage>.</mixed-citation></ref><ref id="pone.0159610.ref023"><label>23</label><mixed-citation publication-type="journal"><name><surname>Zheng</surname><given-names>Y</given-names></name>, <name><surname>Sheng</surname><given-names>J</given-names></name>, <name><surname>Zhao</surname><given-names>R</given-names></name>, <name><surname>Zhang</surname><given-names>J</given-names></name>, <name><surname>Lv</surname><given-names>S</given-names></name>, <name><surname>Liu</surname><given-names>L</given-names></name>, <etal>et al</etal><article-title>Preharvest l -arginine treatment induced postharvest disease resistance to botrysis cinerea in tomato fruits</article-title>. <source>J Agric Food Chem</source>. <year>2011</year>;<volume>59</volume>:<fpage>6543</fpage>–<lpage>49</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1021/jf2000053">10.1021/jf2000053</ext-link></comment><pub-id pub-id-type="pmid">21574662</pub-id></mixed-citation></ref><ref id="pone.0159610.ref024"><label>24</label><mixed-citation publication-type="journal"><name><surname>Navarro</surname><given-names>L</given-names></name>, <name><surname>Bari</surname><given-names>R</given-names></name>, <name><surname>Achard</surname><given-names>P</given-names></name>, <name><surname>Lison</surname><given-names>P</given-names></name>, <name><surname>Nemri</surname><given-names>A</given-names></name>, <name><surname>Harberd</surname><given-names>N.P</given-names></name>, <etal>et al</etal><article-title>DELLAs control plant immune responses by modulating the balance of jasmonic acid and salicylic acid signaling</article-title>. <source>Curr Biol</source>. <year>2008</year>;<volume>18</volume>:<fpage>650</fpage>–<lpage>55</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1016/j.cub.2008.03.060">10.1016/j.cub.2008.03.060</ext-link></comment><pub-id pub-id-type="pmid">18450451</pub-id></mixed-citation></ref><ref id="pone.0159610.ref025"><label>25</label><mixed-citation publication-type="journal"><name><surname>Shen</surname><given-names>W</given-names></name>, <name><surname>Cevallos-Cevallos</surname><given-names>JM</given-names></name>, <name><surname>Nunes da Rocha</surname><given-names>U</given-names></name>, <name><surname>Arevalo</surname><given-names>HA</given-names></name>, <name><surname>Stansly</surname><given-names>PA</given-names></name>, <name><surname>Roberts</surname><given-names>PD</given-names></name>, <etal>et al</etal><article-title>Relation between plant nutrition, hormones, insecticide applications, bacterial endophytes, and Candidatus Liberibacter Ct values in citrus trees infected with Huanglongbing</article-title>. <source>Eur J Plant Pathol</source>. <year>2013</year>;<volume>137</volume>:<fpage>727</fpage>–<lpage>42</lpage>.</mixed-citation></ref><ref id="pone.0159610.ref026"><label>26</label><mixed-citation publication-type="journal"><name><surname>Gottwald</surname><given-names>TR</given-names></name>, <name><surname>Graham</surname><given-names>JH</given-names></name>, <name><surname>Irey</surname><given-names>M</given-names></name>, <name><surname>McCollum</surname><given-names>G</given-names></name>, <name><surname>Wood</surname><given-names>B</given-names></name>. <article-title>Inconsequential effect of nutritional treatments on huanglongbing control, fruit quality, bacterial titer and disease progress</article-title>. <source>Crop Protection</source>. <year>2012</year>;<volume>36</volume>: <fpage>72</fpage>–<lpage>82</lpage>.</mixed-citation></ref><ref id="pone.0159610.ref027"><label>27</label><mixed-citation publication-type="journal"><name><surname>Cao</surname><given-names>J</given-names></name>, <name><surname>Cheng</surname><given-names>C</given-names></name>, <name><surname>Yang</surname><given-names>J</given-names></name>, <name><surname>Wang</surname><given-names>Q</given-names></name>. <article-title>Pathogen infection drives patterns of nutrient resorption in citrus plants</article-title>. <source>Scientific Reports</source>. <year>2015</year>;<volume>5</volume>:<fpage>e14675</fpage>.</mixed-citation></ref><ref id="pone.0159610.ref028"><label>28</label><mixed-citation publication-type="journal"><name><surname>Stansly</surname><given-names>PA</given-names></name>, <name><surname>Arevalo</surname><given-names>HA</given-names></name>, <name><surname>Qureshi</surname><given-names>JA</given-names></name>, <name><surname>Jones</surname><given-names>MM</given-names></name>, <name><surname>Hendricks</surname><given-names>K</given-names></name>, <name><surname>Roberts</surname><given-names>PD</given-names></name>, <etal>et al</etal><article-title>Vector control and foliar nutrition to maintain economic sustainability of bearing citrus in Florida groves affected by huanglongbing</article-title>. <source>Pest Man Sci</source>. <year>2014</year>;<volume>70</volume>:<fpage>415</fpage>–<lpage>26</lpage>.</mixed-citation></ref><ref id="pone.0159610.ref029"><label>29</label><mixed-citation publication-type="journal"><name><surname>Xu</surname><given-names>M</given-names></name>, <name><surname>Liang</surname><given-names>M</given-names></name>, <name><surname>Chen</surname><given-names>J</given-names></name>, <name><surname>Xia</surname><given-names>Y</given-names></name>, <name><surname>Zheng</surname><given-names>Z</given-names></name>, <name><surname>Zhu</surname><given-names>Q</given-names></name>, <etal>et al</etal><article-title>Preliminary research on soil conditioner mediated citrus Huanglongbing mitigation in the field in Guangdong, China</article-title>. <source>Eur J of Plant Pathol</source>. <year>2013</year>;<volume>137</volume>:<fpage>283</fpage>–<lpage>93</lpage>.</mixed-citation></ref><ref id="pone.0159610.ref030"><label>30</label><mixed-citation publication-type="journal"><name><surname>Flores</surname><given-names>F</given-names></name>, <name><surname>Collier</surname><given-names>CJ</given-names></name>, <name><surname>Mercurio</surname><given-names>P</given-names></name>, <name><surname>Negri</surname><given-names>AP</given-names></name>. <article-title>Phytotoxicity of Four Photosystem II Herbicides to Tropical Seagrasses</article-title>. <source>PLoS ONE</source>. <year>2013</year>;<volume>8</volume>:<fpage>e75798</fpage><comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1371/journal.pone.0075798">10.1371/journal.pone.0075798</ext-link></comment><pub-id pub-id-type="pmid">24098726</pub-id></mixed-citation></ref><ref id="pone.0159610.ref031"><label>31</label><mixed-citation publication-type="journal"><name><surname>Aritua</surname><given-names>V</given-names></name>, <name><surname>Achor</surname><given-names>D</given-names></name>, <name><surname>Gmitter</surname><given-names>FG</given-names></name>, <name><surname>Albrigo</surname><given-names>G</given-names></name>, <name><surname>Wang</surname><given-names>N</given-names></name>. <article-title>Transcriptional and Microscopic Analyses of Citrus Stem and Root Responses to <italic>Candidatus</italic> Liberibacter asiaticus Infection</article-title>. <source>PLoS ONE</source>. <year>2013</year>;<volume>8</volume>, <fpage>e73742</fpage><comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1371/journal.pone.0073742">10.1371/journal.pone.0073742</ext-link></comment><pub-id pub-id-type="pmid">24058486</pub-id></mixed-citation></ref><ref id="pone.0159610.ref032"><label>32</label><mixed-citation publication-type="journal"><name><surname>Wang</surname><given-names>Z</given-names></name>, <name><surname>Fang</surname><given-names>H</given-names></name>, <name><surname>Chen</surname><given-names>Y</given-names></name>, <name><surname>Chen</surname><given-names>K</given-names></name>, <name><surname>Li</surname><given-names>G</given-names></name>, <name><surname>Gu</surname><given-names>S</given-names></name>, <etal>et al</etal><article-title>Overexpression of BnWRKY33 in oilseed rape enhances resistance to <italic>Sclerotinia sclerotiorum</italic></article-title>. <source>Mol Plant Pathol</source>. <year>2014</year>;<volume>15</volume>:<fpage>677</fpage>–<lpage>89</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1111/mpp.12123">10.1111/mpp.12123</ext-link></comment><pub-id pub-id-type="pmid">24521393</pub-id></mixed-citation></ref><ref id="pone.0159610.ref033"><label>33</label><mixed-citation publication-type="journal"><name><surname>Wang</surname><given-names>X-M</given-names></name>, <name><surname>Gaudet</surname><given-names>DA</given-names></name>, <name><surname>Liu</surname><given-names>W</given-names></name>, <name><surname>Frick</surname><given-names>M</given-names></name>, <name><surname>Puchalski</surname><given-names>B</given-names></name>, <name><surname>Lu</surname><given-names>Z-X</given-names></name>, <etal>et al</etal><article-title>Defence responses including hypersensitive cell death, oxidative burst and defence gene expression in Moro wheat inoculated with Puccinia striiformis</article-title>. <source>Can J Plant Pathol</source>. <year>2014</year>;<volume>36</volume>:<fpage>202</fpage>–<lpage>15</lpage>.</mixed-citation></ref><ref id="pone.0159610.ref034"><label>34</label><mixed-citation publication-type="journal"><name><surname>Birkenbihl</surname><given-names>RP</given-names></name>, <name><surname>Diezel</surname><given-names>C</given-names></name>, <name><surname>Somssich</surname><given-names>IE</given-names></name>. <article-title>Arabidopsis WRKY33 is a key transcriptional regulator of hormonal and metabolic responses toward Botrytis cinerea infection</article-title>. <source>Plant Physiol</source>. <year>2012</year>; <volume>159</volume>:<fpage>266</fpage>–<lpage>85</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1104/pp.111.192641">10.1104/pp.111.192641</ext-link></comment><pub-id pub-id-type="pmid">22392279</pub-id></mixed-citation></ref><ref id="pone.0159610.ref035"><label>35</label><mixed-citation publication-type="journal"><name><surname>Lorenzo</surname><given-names>O</given-names></name>, <name><surname>Chico</surname><given-names>JM</given-names></name>, <name><surname>Sánchez-Serrano</surname><given-names>JJ</given-names></name>, <name><surname>Solano</surname><given-names>R</given-names></name>. <article-title><italic>JASMONATE- INSENSITIVE1</italic> encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis</article-title>. <source>Plant Cell</source>. <year>2004</year>;<volume>16</volume>:<fpage>1938</fpage>–<lpage>50</lpage>. <pub-id pub-id-type="pmid">15208388</pub-id></mixed-citation></ref><ref id="pone.0159610.ref036"><label>36</label><mixed-citation publication-type="journal"><name><surname>Abe</surname><given-names>H</given-names></name>, <name><surname>Urao</surname><given-names>T</given-names></name>, <name><surname>Ito</surname><given-names>T</given-names></name>, <name><surname>Seki</surname><given-names>M</given-names></name>, <name><surname>Shinozaki</surname><given-names>K</given-names></name>, <name><surname>Yamaguchi-Shinozaki</surname><given-names>K</given-names></name>. <article-title>Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling</article-title>. <source>Plant Cell</source>. <year>2003</year>;<volume>15</volume>:<fpage>63</fpage>–<lpage>78</lpage>. <pub-id pub-id-type="pmid">12509522</pub-id></mixed-citation></ref><ref id="pone.0159610.ref037"><label>37</label><mixed-citation publication-type="journal"><name><surname>Laurie-Berry</surname><given-names>N</given-names></name>, <name><surname>Joardar</surname><given-names>V</given-names></name>, <name><surname>Street</surname><given-names>IH</given-names></name>, <name><surname>Kunkel</surname><given-names>BN</given-names></name>. <article-title>The <italic>Arabidopsis thaliana JASMONATE INSENSITIVE 1</italic> gene is required for suppression of salicylic acid–dependent defenses during infection by <italic>Pseudomonas syringae</italic></article-title>. <source>Mol Plant Microbe Interact</source>. <year>2006</year>;<volume>19</volume>:<fpage>789</fpage>–<lpage>800</lpage>. <pub-id pub-id-type="pmid">16838791</pub-id></mixed-citation></ref><ref id="pone.0159610.ref038"><label>38</label><mixed-citation publication-type="journal"><name><surname>Falk</surname><given-names>A</given-names></name>, <name><surname>Feys</surname><given-names>BJ</given-names></name>, <name><surname>Frost</surname><given-names>LN</given-names></name>, <name><surname>Jones</surname><given-names>JDG</given-names></name>, <name><surname>Daniels</surname><given-names>MJ</given-names></name>, <name><surname>Parker</surname><given-names>JE</given-names></name>. <article-title>EDS1, an essential component of R gene-mediated disease resistance in Arabidopsis has homology to eukaryotic lipases</article-title>. <source>Proc Natl Acad Sci USA</source>. <year>1999</year>;<volume>96</volume>:<fpage>3292</fpage>–<lpage>97</lpage>. <pub-id pub-id-type="pmid">10077677</pub-id></mixed-citation></ref><ref id="pone.0159610.ref039"><label>39</label><mixed-citation publication-type="journal"><name><surname>Lipka</surname><given-names>V</given-names></name>, <name><surname>Dittgen</surname><given-names>J</given-names></name>, <name><surname>Bednarek</surname><given-names>P</given-names></name>, <name><surname>Bhat</surname><given-names>R</given-names></name>, <name><surname>Wiermer</surname><given-names>M</given-names></name>, <name><surname>Stein</surname><given-names>M</given-names></name>, <etal>et al</etal><article-title>Pre- and Postinvasion Defenses Both Contribute to Nonhost Resistance in Arabidopsis</article-title>. <source>Science</source>. <year>2005</year>;<volume>310</volume>:<fpage>1180</fpage>–<lpage>83</lpage>. <pub-id pub-id-type="pmid">16293760</pub-id></mixed-citation></ref><ref id="pone.0159610.ref040"><label>40</label><mixed-citation publication-type="journal"><name><surname>Feys</surname><given-names>BJ</given-names></name>, <name><surname>Wiermer</surname><given-names>M</given-names></name>, <name><surname>Bhat</surname><given-names>RA</given-names></name>, <name><surname>Moisan</surname><given-names>LJ</given-names></name>, <name><surname>Medina-Escobar</surname><given-names>N</given-names></name>, <name><surname>Neu</surname><given-names>C</given-names></name>, <etal>et al</etal><article-title>Arabidopsis Senescence-associated gene101 stabilizes and signals within an enhanced disease susceptibility1 complex in plant innate immunity</article-title>. <source>Plant Cell</source>. <year>2005</year>;<volume>17</volume>:<fpage>2601</fpage>–<lpage>13</lpage>. <pub-id pub-id-type="pmid">16040633</pub-id></mixed-citation></ref><ref id="pone.0159610.ref041"><label>41</label><mixed-citation publication-type="journal"><name><surname>Moreau</surname><given-names>M</given-names></name>, <name><surname>Degrave</surname><given-names>A</given-names></name>, <name><surname>Vedel</surname><given-names>R</given-names></name>, <name><surname>Bitton</surname><given-names>F</given-names></name>, <name><surname>Patrit</surname><given-names>O</given-names></name>, <name><surname>Renou</surname><given-names>J-P</given-names></name>, <etal>et al</etal><article-title>EDS1 contributes to nonhost resistance of Arabidopsis thaliana against Erwinia amylovora</article-title>. <source>Mol Plant Microbe In</source>. <year>2012</year>;<volume>25</volume>:<fpage>421</fpage>–<lpage>30</lpage>.</mixed-citation></ref><ref id="pone.0159610.ref042"><label>42</label><mixed-citation publication-type="journal"><name><surname>Besseau</surname><given-names>S</given-names></name>, <name><surname>Li</surname><given-names>J</given-names></name>, <name><surname>Palva</surname><given-names>ET</given-names></name>. <article-title>WRKY54 and WRKY70 co-operate as negative regulators of leaf senescence in <italic>Arabidopsis thaliana</italic></article-title>. <source>J Exp Bot</source>. <year>2012</year>; <volume>63</volume>:<fpage>2667</fpage>–<lpage>679</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1093/jxb/err450">10.1093/jxb/err450</ext-link></comment><pub-id pub-id-type="pmid">22268143</pub-id></mixed-citation></ref><ref id="pone.0159610.ref043"><label>43</label><mixed-citation publication-type="journal"><name><surname>Ibanez</surname><given-names>AM</given-names></name>, <name><surname>Martinelli</surname><given-names>F</given-names></name>, <name><surname>Reagan</surname><given-names>RL</given-names></name>, <name><surname>Uratsu</surname><given-names>SL</given-names></name>, <name><surname>Vo</surname><given-names>A</given-names></name>, <name><surname>Tinoco</surname><given-names>MA</given-names></name>, <etal>et al</etal><article-title>Transcriptome and metabolome analysis of Citrus fruit to elucidate puffing disorder</article-title>. <source>Plant Science</source>. <year>2014</year>;<volume>217</volume>:<fpage>87</fpage>–<lpage>98</lpage>. <comment>doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1016/j.plantsci.2013.12.003">10.1016/j.plantsci.2013.12.003</ext-link></comment><pub-id pub-id-type="pmid">24467900</pub-id></mixed-citation></ref><ref id="pone.0159610.ref044"><label>44</label><mixed-citation publication-type="journal"><name><surname>van Loon</surname><given-names>LC</given-names></name>, <name><surname>Rep</surname><given-names>M</given-names></name>, <name><surname>Pieterse</surname><given-names>CM</given-names></name>. <article-title>Significance of inducible defense-related proteins in infected plants</article-title>. <source>Annu Rev Phytopathol</source>. <year>2006</year>;<volume>44</volume>:<fpage>135</fpage>–<lpage>62</lpage>. <pub-id pub-id-type="pmid">16602946</pub-id></mixed-citation></ref><ref id="pone.0159610.ref045"><label>45</label><mixed-citation publication-type="journal"><name><surname>Kiba</surname><given-names>A</given-names></name>, <name><surname>Maimbo</surname><given-names>M</given-names></name>, <name><surname>Kanda</surname><given-names>A</given-names></name>, <name><surname>Tomiyama</surname><given-names>H</given-names></name>, <name><surname>Ohnishi</surname><given-names>K</given-names></name>, <name><surname>Hikichi</surname><given-names>Y</given-names></name>. <article-title>Isolation and expression analysis of candidate genes related to Ralstonia solanacearum-tobacco interaction</article-title>. <source>Plant Biotechnol</source>. <year>2007</year>;<volume>24</volume>:<fpage>409</fpage>–<lpage>16</lpage>.</mixed-citation></ref><ref id="pone.0159610.ref046"><label>46</label><mixed-citation publication-type="journal"><name><surname>Martinelli</surname><given-names>F</given-names></name>, <name><surname>Scalenghe</surname><given-names>R</given-names></name>, <name><surname>Giovino</surname><given-names>A</given-names></name>, <name><surname>Pasquale</surname><given-names>M</given-names></name>, <name><surname>Aksenov</surname><given-names>AA</given-names></name>, <name><surname>Pasamontes</surname><given-names>A</given-names></name>, <name><surname>Peirano</surname><given-names>DJ</given-names></name>, <name><surname>Davis</surname><given-names>CE</given-names></name>, <name><surname>Dandekar</surname><given-names>AM</given-names></name>. <article-title>Proposal of a Citrus translational genomic approach for early and infield detection of Flavescence dorée in Vitis</article-title>. <source>Plant Biosystems</source><year>2016</year>;<volume>150</volume>:<fpage>43</fpage>–<lpage>53</lpage>.</mixed-citation></ref><ref id="pone.0159610.ref047"><label>47</label><mixed-citation publication-type="journal"><name><surname>Martinelli</surname><given-names>F</given-names></name>, <name><surname>Scalenghe</surname><given-names>R</given-names></name>, <name><surname>Davino</surname><given-names>SW</given-names></name>, <name><surname>Panno</surname><given-names>S</given-names></name>, <name><surname>Suderi</surname><given-names>G</given-names></name>, <name><surname>Ruisi</surname><given-names>P</given-names></name>, <name><surname>Villa</surname><given-names>P</given-names></name>, <name><surname>Stroppiana</surname><given-names>D</given-names></name>, <name><surname>Boschetti</surname><given-names>M</given-names></name>, <name><surname>Goulart</surname><given-names>LR</given-names></name>, <name><surname>Davis</surname><given-names>CE</given-names></name>, <name><surname>Dandekar</surname><given-names>AM</given-names></name>. <article-title>Advanced methods of plant disease detection</article-title>. <source>A Review. Agronomy for Sustainable Development</source><year>2015</year>;<volume>35</volume>:<fpage>1</fpage>–<lpage>25</lpage>.</mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Bois/explor/OrangerV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000398 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000398 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Bois
|area= OrangerV1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé= PMC:4961454
|texte= Molecular Responses to Small Regulating Molecules against Huanglongbing Disease
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:27459099" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \
| NlmPubMed2Wicri -a OrangerV1
| This area was generated with Dilib version V0.6.25. |  |