Comparative genomic and transcriptional analyses of the carbohydrate-active enzymes and secretomes of phytopathogenic fungi reveal their significant roles during infection and development
Identifieur interne : 000724 ( Pmc/Checkpoint ); précédent : 000723; suivant : 000725Comparative genomic and transcriptional analyses of the carbohydrate-active enzymes and secretomes of phytopathogenic fungi reveal their significant roles during infection and development
Auteurs : Xueliang Lyu [République populaire de Chine] ; Cuicui Shen [République populaire de Chine] ; Yanping Fu [République populaire de Chine] ; Jiatao Xie [République populaire de Chine] ; Daohong Jiang [République populaire de Chine] ; Guoqing Li [République populaire de Chine] ; Jiasen Cheng [République populaire de Chine]Source :
- Scientific Reports [ 2045-2322 ] ; 2015.
Abstract
Our comparative genomic analysis showed that the numbers of plant cell wall (PCW)- and fungal cell wall (FCW)-degradation-associated carbohydrate-active enzymes (CAZymes) in necrotrophic and hemibiotrophic fungi are significantly larger than that in most biotrophic fungi. However, our transcriptional analyses of CAZyme-encoding genes in
Url:
DOI: 10.1038/srep15565
PubMed: 26531059
PubMed Central: 4632110
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Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Shen, Cuicui" sort="Shen, Cuicui" uniqKey="Shen C" first="Cuicui" last="Shen">Cuicui Shen</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a2"><institution>The Provincial Key Lab of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Fu, Yanping" sort="Fu, Yanping" uniqKey="Fu Y" first="Yanping" last="Fu">Yanping Fu</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>The Provincial Key Lab of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Xie, Jiatao" sort="Xie, Jiatao" uniqKey="Xie J" first="Jiatao" last="Xie">Jiatao Xie</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>The Provincial Key Lab of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei 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de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Li, Guoqing" sort="Li, Guoqing" uniqKey="Li G" first="Guoqing" last="Li">Guoqing Li</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a2"><institution>The Provincial Key Lab of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Cheng, Jiasen" sort="Cheng, Jiasen" uniqKey="Cheng J" first="Jiasen" last="Cheng">Jiasen Cheng</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a2"><institution>The Provincial Key Lab of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></titleStmt><publicationStmt><idno 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sortKey="Lyu, Xueliang" sort="Lyu, Xueliang" uniqKey="Lyu X" first="Xueliang" last="Lyu">Xueliang Lyu</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a2"><institution>The Provincial Key Lab of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Shen, Cuicui" sort="Shen, Cuicui" uniqKey="Shen C" first="Cuicui" last="Shen">Cuicui Shen</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a2"><institution>The Provincial Key Lab of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Fu, Yanping" sort="Fu, Yanping" uniqKey="Fu Y" first="Yanping" last="Fu">Yanping Fu</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>The Provincial Key Lab of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Xie, Jiatao" sort="Xie, Jiatao" uniqKey="Xie J" first="Jiatao" last="Xie">Jiatao Xie</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>The Provincial Key Lab of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Jiang, Daohong" sort="Jiang, Daohong" uniqKey="Jiang D" first="Daohong" last="Jiang">Daohong Jiang</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a2"><institution>The Provincial Key Lab of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Li, Guoqing" sort="Li, Guoqing" uniqKey="Li G" first="Guoqing" last="Li">Guoqing Li</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a2"><institution>The Provincial Key Lab of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Cheng, Jiasen" sort="Cheng, Jiasen" uniqKey="Cheng J" first="Jiasen" last="Cheng">Jiasen Cheng</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a2"><institution>The Provincial Key Lab of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Scientific Reports</title><idno type="eISSN">2045-2322</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Our comparative genomic analysis showed that the numbers of plant cell wall (PCW)- and fungal cell wall (FCW)-degradation-associated carbohydrate-active enzymes (CAZymes) in necrotrophic and hemibiotrophic fungi are significantly larger than that in most biotrophic fungi. However, our transcriptional analyses of CAZyme-encoding genes in <italic>Melampsora larici-populina</italic>, <italic>Puccinia graminis</italic> and <italic>Sclerotinia sclerotiorum</italic> showed that many genes encoding PCW- and FCW-degradation-associated CAZymes were significantly up-regulated during the infection of both necrotrophic fungi and biotrophic fungi, indicating an existence of a universal mechanism underlying PCW degradation and FCW reorganization or modification, which are both intimately involved in necrotrophic and biotrophic fungal infection. Furthermore, our results showed that the FCW reorganization or modification was also related to the fungal development. Additionally, our transcriptional analysis of the secretome of <italic>S. sclerotiorum</italic> showed that many secreted protein-encoding genes were dramatically induced during infection. Among them, a small, cysteine-rich protein SsCVNH was experimentally confirmed to be essential for the virulence and sclerotial development, indicating that the small secreted proteins might also play crucial roles as potential effectors in host-non-specific necrotrophic fungi.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Park, B H" uniqKey="Park B">B. H. Park</name></author><author><name sortKey="Karpinets, T V" uniqKey="Karpinets T">T. V. Karpinets</name></author><author><name sortKey="Syed, M H" uniqKey="Syed M">M. H. Syed</name></author><author><name sortKey="Leuze, M R" uniqKey="Leuze M">M. R. Leuze</name></author><author><name sortKey="Uberbacher, E C" uniqKey="Uberbacher E">E. C. Uberbacher</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cantarel, B L" uniqKey="Cantarel B">B. L. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Sci Rep</journal-id><journal-id journal-id-type="iso-abbrev">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type="epub">2045-2322</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26531059</article-id><article-id pub-id-type="pmc">4632110</article-id><article-id pub-id-type="pii">srep15565</article-id><article-id pub-id-type="doi">10.1038/srep15565</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Comparative genomic and transcriptional analyses of the carbohydrate-active enzymes and secretomes of phytopathogenic fungi reveal their significant roles during infection and development</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Lyu</surname><given-names>Xueliang</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Shen</surname><given-names>Cuicui</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Fu</surname><given-names>Yanping</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Xie</surname><given-names>Jiatao</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Jiang</surname><given-names>Daohong</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Guoqing</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Cheng</surname><given-names>Jiasen</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><aff id="a1"><label>1</label><institution>State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></aff><aff id="a2"><label>2</label><institution>The Provincial Key Lab of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University</institution>, Wuhan 430070, Hubei Province,<country>China</country></aff></contrib-group><author-notes><corresp id="c1"><label>a</label><email>jiasencheng@mail.hzau.edu.cn</email></corresp></author-notes><pub-date pub-type="epub"><day>04</day><month>11</month><year>2015</year></pub-date><pub-date pub-type="collection"><year>2015</year></pub-date><volume>5</volume><elocation-id>15565</elocation-id><history><date date-type="received"><day>26</day><month>06</month><year>2015</year></date><date date-type="accepted"><day>29</day><month>09</month><year>2015</year></date></history><permissions><copyright-statement>Copyright © 2015, Macmillan Publishers Limited</copyright-statement><copyright-year>2015</copyright-year><copyright-holder>Macmillan Publishers Limited</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/4.0/"><pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link></license-p></license></permissions><abstract><p>Our comparative genomic analysis showed that the numbers of plant cell wall (PCW)- and fungal cell wall (FCW)-degradation-associated carbohydrate-active enzymes (CAZymes) in necrotrophic and hemibiotrophic fungi are significantly larger than that in most biotrophic fungi. However, our transcriptional analyses of CAZyme-encoding genes in <italic>Melampsora larici-populina</italic>, <italic>Puccinia graminis</italic> and <italic>Sclerotinia sclerotiorum</italic> showed that many genes encoding PCW- and FCW-degradation-associated CAZymes were significantly up-regulated during the infection of both necrotrophic fungi and biotrophic fungi, indicating an existence of a universal mechanism underlying PCW degradation and FCW reorganization or modification, which are both intimately involved in necrotrophic and biotrophic fungal infection. Furthermore, our results showed that the FCW reorganization or modification was also related to the fungal development. Additionally, our transcriptional analysis of the secretome of <italic>S. sclerotiorum</italic> showed that many secreted protein-encoding genes were dramatically induced during infection. Among them, a small, cysteine-rich protein SsCVNH was experimentally confirmed to be essential for the virulence and sclerotial development, indicating that the small secreted proteins might also play crucial roles as potential effectors in host-non-specific necrotrophic fungi.</p></abstract></article-meta></front><floats-group><fig id="f1"><label>Figure 1</label><caption><title>Comparative analysis of CAZymes and related CBMs in necrotrophic and hemibiotrophic fungi, biotrophic fungi and two yeasts.</title><p>The numbers of PCW- (<bold>a</bold>) and FCW- (<bold>c</bold>) degradation-associated CAZymes and respective related CBMs ((<bold>b</bold>,<bold>d</bold>) respectively) were plotted for necrotrophic and hemibiotrophic fungi, biotrophic fungi and two yeasts. The numbers of PCW- and FCW-degradation-associated CAZymes and related CBMs showed significant differences among necrotrophic and hemibiotrophic fungi, biotrophic fungi and two yeasts. Differentiation was supported by the t-test at a significance level of P < 0.05.</p></caption><graphic xlink:href="srep15565-f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><title>Transcriptional analysis of genes encoding CAZymes and related CBMs during different developmental stages of <italic>S. sclerotiorum</italic>.</title><p>(<bold>a</bold>) The expression levels of genes encoding PCW- and FCW-degradation-associated CAZymes and respective related CBMs during different developmental stages. The expression levels were measured using the geometric mean of the TPM values of all genes in corresponding groups. To calculate the geometric mean of TPM values, all the TPM values of 0 were replaced by 0.001. (<bold>b</bold>) Gene expression cluster analysis of genes encoding PCW-degradation-associated CAZymes and related CBMs. (<bold>c</bold>) Gene expression cluster analysis of genes encoding FCW-degradation-associated CAZymes and related CBMs. The TPM values were used for the gene expression cluster analysis. Red, green and grey indicate high expression, low expression and no expression, respectively; For each heat map: top, stage tree; left, gene tree. Expression values are indicated in log2 scale. Column diagrams show the proportions of differentially expressed genes and unchanged expressed genes encoding PCW- (<bold>d</bold>) and FCW- (<bold>e</bold>) degradation-associated CAZymes and respective related CBMs, during different developmental stages, compared with the vegetative growth stage of <italic>S. sclerotiorum</italic>. The differentially expressed genes in the DGE data were identified with the threshold of |log<sub>2</sub>Ratio| ≥ 1 and FDR ≤ 0.001. Up, Down and Un indicate the up-regulated, down-regulated and unchanged expressed genes, respectively.</p></caption><graphic xlink:href="srep15565-f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><title>Morphology of the vegetative and invasive hyphal tips of <italic>S. sclerotiorum</italic>.</title><p>Cellophane on PDA plates and onion bulb epidermis were inoculated with the <italic>S. sclerotiorum</italic> wild-type strain for 36 h at 20 °C prior to microscopic examination. Microscopic observation showed that the invasive hyphal tips were thicker than the vegetative hyphal tips and that their morphology was blistered, swollen and malformed. Scale bar = 100 μm.</p></caption><graphic xlink:href="srep15565-f3"></graphic></fig><fig id="f4"><label>Figure 4</label><caption><title>Transcriptional analysis of genes encoding CAZymes and related CBMs in <italic>M. larici-populina</italic> and <italic>P. graminis</italic> during urediniospore germination and infection.</title><p>Expression cluster analysis of genes encoding PCW- (<bold>a</bold>) and FCW- (<bold>b</bold>) degradation-associated CAZymes and respective related CBMs in <italic>M. larici-populina</italic> and expression cluster analysis of genes encoding PCW- (<bold>c</bold>) and FCW- (<bold>d</bold>) degradation-associated CAZymes and respective related CBMs in <italic>P. graminis</italic> were performed based on the expression average values of three independently repeated tests<xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref>. Red and green indicate high expression and low expression respectively. For each heat map: top, stage tree; left, gene tree. Expression values are indicated in log2 scale. Column diagrams show the proportions of differentially expressed genes encoding PCW- (<bold>e</bold>) and FCW- (<bold>f</bold>) degradation-associated CAZymes and respective related CBMs, during <italic>M. larici-populina</italic> urediniospore germination and different infection stages on poplar leaves, compared with dried-urediniospores <italic>in vitro</italic>. Column diagrams show the proportions of differentially expressed genes encoding PCW- (<bold>g</bold>) and FCW- (<bold>h</bold>) degradation-associated CAZymes and respective related CBMs during <italic>P. graminis</italic> urediniospore germination and infection on wheat and barley, respectively, compared with urediniospores <italic>in vitro</italic>. The differentially expressed genes in the microarray data were identified with the threshold of |Fold change | 2 and an adjusted p-value < 0.05. Up, Down and Un indicate the up-regulated, down-regulated and unchanged expressed genes, respectively.</p></caption><graphic xlink:href="srep15565-f4"></graphic></fig><fig id="f5"><label>Figure 5</label><caption><title>SsCVNH has a conserved CVNH domain.</title><p>(<bold>a</bold>) Domain architecture of SsCVNH. Protein sequences are indicated by thick lines. Signal peptide (sp) and CVNH domain are indicated by boxes. (<bold>b</bold>) Representation of the three-dimensional structure of cyanovirin-N<xref ref-type="bibr" rid="b33">33</xref> and predicted SsCVNH. A total of 85 residues (56% of the SsCVNH) were modelled with 98.8% confidence by the single highest scoring template cyanovirin-N (d1n02a) in the Phyre database. (<bold>c</bold>) Multiple sequence alignment of amino acid sequences of CVNHs from <italic>Sclerotinia</italic> and <italic>Botryotinia</italic>. Protein sequences from top to bottom are derived from <italic>B. cinerea</italic> T4, <italic>B. cinerea</italic> B05.10, <italic>B. cinerea</italic> BcDW1, <italic>S. sclerotiorum</italic> Ep-1PNA367 and <italic>Sclerotinia borealis</italic> F-4157, respectively.</p></caption><graphic xlink:href="srep15565-f5"></graphic></fig><fig id="f6"><label>Figure 6</label><caption><title>The expression patterns of <italic>SsCVNH</italic> in the wild-type strain.</title><p>(<bold>a</bold>) The relative expression of <italic>SsCVNH</italic> during different developmental stages in the DGE data. (<bold>b</bold>) The relative expression of <italic>SsCVNH</italic> on PDA at 20 °C for 1–9 days. The expression level on the first day was set at 1.0. (<bold>c</bold>) The relative expression of <italic>SsCVNH</italic> post inoculation of the wild-type strain on <italic>A. thaliana</italic> leaves (red columns) and on PDA (dark columns) for 0–12 h. The expression level at 0 hpi on PDA was set at 1.0. The expression level of β-tubulin was used to normalize <italic>SsCVNH</italic> expression. The values are presented as the means ± s.d.</p></caption><graphic xlink:href="srep15565-f6"></graphic></fig><fig id="f7"><label>Figure 7</label><caption><title>SsCVNH was secreted from the hyphal tips during infection.</title><p>(<bold>a</bold>) Western blot analysis showed that SsCVNH-FLAG could be detected in the engineered strains. SDS-polyacrylamide gel electrophoresis showed the equal loading amounts of total proteins used for the western blot analysis. Alkaline phosphatase-conjugated secondary antibody detected an approximately 17 kDa band in the SsCVNH-FLAG-engineered strains but not in the wild-type strain. (<bold>b</bold>) Immunofluorescence detection of SsCVNH-FLAG during infection. Onion bulb epidermis was inoculated with SsCVNH-FLAG-engineered strains for 36 h at 20 °C. Two different secondary antibodies conjugated with RRX or FITC respectively, were used independently to exhibit the specificity of fluorescence signal. The fluorescence of both RRX (red) and FITC (green) was detected in the SsCVNH-FLAG-engineered strains, but not in the wild-type strain. Arrows show the concentrated distribution of SsCVNH in the hyphal tips during infection. Scale bar = 50 μm.</p></caption><graphic xlink:href="srep15565-f7"></graphic></fig><fig id="f8"><label>Figure 8</label><caption><title>The phenotypes of <italic>SsCVNH</italic>-silenced transformants.</title><p>(<bold>a</bold>) Colony morphology in <italic>SsCVNH</italic>-silenced transformants. Colonies were grown on PDA for 10 days at 20 °C. (<bold>b</bold>) <italic>SsCVNH</italic>-silenced transformants showing significantly reduced virulence on detached rapeseed (<italic>Brassica napus</italic>) leaves. Virulence was evaluated based on the lesion diameter at 20 °C for 48 h. (<bold>c</bold>) Comparison of the hyphal tips of <italic>SsCVNH</italic>-silenced transformants and the wild-type strain. Scale bar = 100 μm. (<bold>d</bold>) Expression of <italic>SsCVNH</italic> in silenced transformants was determined by qRT-PCR. The expression levels of β-tubulin were used to normalize <italic>SsCVNH</italic> expression. The expression level of <italic>SsCVNH</italic> in the wild-type strain was set at 1.0. (<bold>e</bold>) Comparison of the lesion diameters of the silenced transformants and the wild-type strain. (<bold>f</bold>) Comparison of the growth rates of the silenced transformants and the wild-type strain. In all experiments, three independent replications were performed. The values are presented as the means ± s.d. Different letters in the graph indicate significant differences while same letters indicate no significant differences, P = 0.05.</p></caption><graphic xlink:href="srep15565-f8"></graphic></fig></floats-group></pmc><affiliations><list><country><li>République populaire de Chine</li></country></list><tree><country name="République populaire de Chine"><noRegion><name sortKey="Lyu, Xueliang" sort="Lyu, Xueliang" uniqKey="Lyu X" first="Xueliang" last="Lyu">Xueliang Lyu</name></noRegion><name sortKey="Cheng, Jiasen" sort="Cheng, Jiasen" uniqKey="Cheng J" first="Jiasen" last="Cheng">Jiasen Cheng</name><name sortKey="Cheng, Jiasen" sort="Cheng, Jiasen" uniqKey="Cheng J" first="Jiasen" last="Cheng">Jiasen Cheng</name><name sortKey="Fu, Yanping" sort="Fu, Yanping" uniqKey="Fu Y" first="Yanping" last="Fu">Yanping Fu</name><name sortKey="Jiang, Daohong" sort="Jiang, Daohong" uniqKey="Jiang D" first="Daohong" last="Jiang">Daohong Jiang</name><name sortKey="Jiang, Daohong" sort="Jiang, Daohong" uniqKey="Jiang D" first="Daohong" last="Jiang">Daohong Jiang</name><name sortKey="Li, Guoqing" sort="Li, Guoqing" uniqKey="Li G" first="Guoqing" last="Li">Guoqing Li</name><name sortKey="Li, Guoqing" sort="Li, Guoqing" uniqKey="Li G" first="Guoqing" last="Li">Guoqing Li</name><name sortKey="Lyu, Xueliang" sort="Lyu, Xueliang" uniqKey="Lyu X" first="Xueliang" last="Lyu">Xueliang Lyu</name><name sortKey="Shen, Cuicui" sort="Shen, Cuicui" uniqKey="Shen C" first="Cuicui" last="Shen">Cuicui Shen</name><name sortKey="Shen, Cuicui" sort="Shen, Cuicui" uniqKey="Shen C" first="Cuicui" last="Shen">Cuicui Shen</name><name sortKey="Xie, Jiatao" sort="Xie, Jiatao" uniqKey="Xie J" first="Jiatao" last="Xie">Jiatao Xie</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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