Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes.
Identifieur interne : 000064 ( Main/Exploration ); précédent : 000063; suivant : 000065Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes.
Auteurs : Liz M. Florez [Nouvelle-Zélande] ; Reiny W A. Scheper [Nouvelle-Zélande] ; Brent M. Fisher [Nouvelle-Zélande] ; Paul W. Sutherland [Nouvelle-Zélande] ; Matthew D. Templeton [Nouvelle-Zélande] ; Joanna K. Bowen [Nouvelle-Zélande]Source :
- PloS one [ 1932-6203 ] ; 2020.
Abstract
European canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Reverse transcription quantitative real-time PCR (RT-qPCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate RT-qPCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.
DOI: 10.1371/journal.pone.0238157
PubMed: 33186359
PubMed Central: PMC7665675
Affiliations:
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Florez</name><affiliation wicri:level="1"><nlm:affiliation>Bioprotection, The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.</nlm:affiliation><country xml:lang="fr">Nouvelle-Zélande</country><wicri:regionArea>Bioprotection, The New Zealand Institute for Plant & Food Research Limited, Auckland</wicri:regionArea><wicri:noRegion>Auckland</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:affiliation>School of Biological Sciences, University of Auckland, Auckland, New Zealand.</nlm:affiliation><country xml:lang="fr">Nouvelle-Zélande</country><wicri:regionArea>School of Biological Sciences, University of Auckland, Auckland</wicri:regionArea><wicri:noRegion>Auckland</wicri:noRegion></affiliation></author><author><name sortKey="Scheper, Reiny W A" sort="Scheper, Reiny W A" uniqKey="Scheper R" first="Reiny W A" last="Scheper">Reiny W A. 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Fisher</name><affiliation wicri:level="1"><nlm:affiliation>Bioprotection, The New Zealand Institute for Plant & Food Research Limited, Havelock North, New Zealand.</nlm:affiliation><country xml:lang="fr">Nouvelle-Zélande</country><wicri:regionArea>Bioprotection, The New Zealand Institute for Plant & Food Research Limited, Havelock North</wicri:regionArea><wicri:noRegion>Havelock North</wicri:noRegion></affiliation></author><author><name sortKey="Sutherland, Paul W" sort="Sutherland, Paul W" uniqKey="Sutherland P" first="Paul W" last="Sutherland">Paul W. Sutherland</name><affiliation wicri:level="1"><nlm:affiliation>Food Innovation, The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.</nlm:affiliation><country xml:lang="fr">Nouvelle-Zélande</country><wicri:regionArea>Food Innovation, The New Zealand Institute for Plant & Food Research Limited, Auckland</wicri:regionArea><wicri:noRegion>Auckland</wicri:noRegion></affiliation></author><author><name sortKey="Templeton, Matthew D" sort="Templeton, Matthew D" uniqKey="Templeton M" first="Matthew D" last="Templeton">Matthew D. Templeton</name><affiliation wicri:level="1"><nlm:affiliation>Bioprotection, The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.</nlm:affiliation><country xml:lang="fr">Nouvelle-Zélande</country><wicri:regionArea>Bioprotection, The New Zealand Institute for Plant & Food Research Limited, Auckland</wicri:regionArea><wicri:noRegion>Auckland</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:affiliation>School of Biological Sciences, University of Auckland, Auckland, New Zealand.</nlm:affiliation><country xml:lang="fr">Nouvelle-Zélande</country><wicri:regionArea>School of Biological Sciences, University of Auckland, Auckland</wicri:regionArea><wicri:noRegion>Auckland</wicri:noRegion></affiliation></author><author><name sortKey="Bowen, Joanna K" sort="Bowen, Joanna K" uniqKey="Bowen J" first="Joanna K" last="Bowen">Joanna K. Bowen</name><affiliation wicri:level="1"><nlm:affiliation>Bioprotection, The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.</nlm:affiliation><country xml:lang="fr">Nouvelle-Zélande</country><wicri:regionArea>Bioprotection, The New Zealand Institute for Plant & Food Research Limited, Auckland</wicri:regionArea><wicri:noRegion>Auckland</wicri:noRegion></affiliation></author></analytic><series><title level="j">PloS one</title><idno type="eISSN">1932-6203</idno><imprint><date when="2020" type="published">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">European canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Reverse transcription quantitative real-time PCR (RT-qPCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate RT-qPCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.</div></front></TEI><pubmed><MedlineCitation Status="In-Data-Review" Owner="NLM"><PMID Version="1">33186359</PMID><DateRevised><Year>2020</Year><Month>11</Month><Day>19</Day></DateRevised><Article PubModel="Electronic-eCollection"><Journal><ISSN IssnType="Electronic">1932-6203</ISSN><JournalIssue CitedMedium="Internet"><Volume>15</Volume><Issue>11</Issue><PubDate><Year>2020</Year></PubDate></JournalIssue><Title>PloS one</Title><ISOAbbreviation>PLoS One</ISOAbbreviation></Journal><ArticleTitle>Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes.</ArticleTitle><Pagination><MedlinePgn>e0238157</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1371/journal.pone.0238157</ELocationID><Abstract><AbstractText>European canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Reverse transcription quantitative real-time PCR (RT-qPCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate RT-qPCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Florez</LastName><ForeName>Liz M</ForeName><Initials>LM</Initials><AffiliationInfo><Affiliation>Bioprotection, The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>School of Biological Sciences, University of Auckland, Auckland, New Zealand.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Scheper</LastName><ForeName>Reiny W A</ForeName><Initials>RWA</Initials><AffiliationInfo><Affiliation>Bioprotection, The New Zealand Institute for Plant & Food Research Limited, Havelock North, New Zealand.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Fisher</LastName><ForeName>Brent M</ForeName><Initials>BM</Initials><AffiliationInfo><Affiliation>Bioprotection, The New Zealand Institute for Plant & Food Research Limited, Havelock North, New Zealand.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Sutherland</LastName><ForeName>Paul W</ForeName><Initials>PW</Initials><AffiliationInfo><Affiliation>Food Innovation, The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Templeton</LastName><ForeName>Matthew D</ForeName><Initials>MD</Initials><Identifier Source="ORCID">https://orcid.org/0000-0003-0192-9041</Identifier><AffiliationInfo><Affiliation>Bioprotection, The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>School of Biological Sciences, University of Auckland, Auckland, New Zealand.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Bowen</LastName><ForeName>Joanna K</ForeName><Initials>JK</Initials><Identifier Source="ORCID">https://orcid.org/0000-0001-9863-1078</Identifier><AffiliationInfo><Affiliation>Bioprotection, The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2020</Year><Month>11</Month><Day>13</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>PLoS One</MedlineTA><NlmUniqueID>101285081</NlmUniqueID><ISSNLinking>1932-6203</ISSNLinking></MedlineJournalInfo><CitationSubset>IM</CitationSubset><CoiStatement>The authors have declared that no competing interests exist.</CoiStatement></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2020</Year><Month>08</Month><Day>09</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2020</Year><Month>11</Month><Day>01</Day></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2020</Year><Month>11</Month><Day>13</Day><Hour>17</Hour><Minute>8</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2020</Year><Month>11</Month><Day>14</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2020</Year><Month>11</Month><Day>14</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>epublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">33186359</ArticleId><ArticleId IdType="doi">10.1371/journal.pone.0238157</ArticleId><ArticleId IdType="pii">PONE-D-20-24931</ArticleId><ArticleId IdType="pmc">PMC7665675</ArticleId></ArticleIdList><ReferenceList><Reference><Citation>Curr Opin Plant Biol. 2010 Aug;13(4):415-9</Citation><ArticleIdList><ArticleId 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Florez</name></noRegion><name sortKey="Bowen, Joanna K" sort="Bowen, Joanna K" uniqKey="Bowen J" first="Joanna K" last="Bowen">Joanna K. Bowen</name><name sortKey="Fisher, Brent M" sort="Fisher, Brent M" uniqKey="Fisher B" first="Brent M" last="Fisher">Brent M. Fisher</name><name sortKey="Florez, Liz M" sort="Florez, Liz M" uniqKey="Florez L" first="Liz M" last="Florez">Liz M. Florez</name><name sortKey="Scheper, Reiny W A" sort="Scheper, Reiny W A" uniqKey="Scheper R" first="Reiny W A" last="Scheper">Reiny W A. Scheper</name><name sortKey="Sutherland, Paul W" sort="Sutherland, Paul W" uniqKey="Sutherland P" first="Paul W" last="Sutherland">Paul W. Sutherland</name><name sortKey="Templeton, Matthew D" sort="Templeton, Matthew D" uniqKey="Templeton M" first="Matthew D" last="Templeton">Matthew D. Templeton</name><name sortKey="Templeton, Matthew D" sort="Templeton, Matthew D" uniqKey="Templeton M" first="Matthew D" last="Templeton">Matthew D. 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