Dussart, F

Hoebe, PN

McGrann, GRD

Spoel, Steven H

English

SRUC - Scotland's Rural College

Scotland's Rural CollegeDetermining the role of secondary metabolism in the pathology of Ramularia collo-cygni, the fungus responsible for Ramularia leaf spot disease of barley.Dussart, F; Spoel, Steven H; Hoebe, PN; McGrann, GRDPrint publication: 01/01/2017Link to publicationCitation for pulished version (APA):Dussart, F., Spoel, S. H., Hoebe, PN., & McGrann, GRD. (2017). Determining the role of secondary metabolismin the pathology of Ramularia collo-cygni, the fungus responsible for Ramularia leaf spot disease of barley..Poster session presented at International Workshop on Barley Leaf Diseases, Rabbat, Morocco.General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            • Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            • You may not further distribute the material or use it for any profit-making activity or commercial gain            • You may freely distribute the URL identifying the publication in the public portal ?Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Download date: 27. Nov. 2022Leading the way in Agriculture and Rural Research, Education and Consulting SRUC is a charity registered in Scotland, No. SC003712www.sruc.ac.ukFrancois Dussart1,2, Steven H. Spoel2, Peter N. Hoebe1, Graham R.D. McGrann11Scotland’s Rural College, West Mains Road, Edinburgh, EH9 3JG, UK2 School of Biological Sciences, University of Edinburgh,  Edinburgh EH9 3JR, UKE-mail: Francois.Dussart@sruc.ac.ukRamularia leaf spotConclusionsAcknowledgementsReferencesIn silico identification of PKSs in Ramularia collo-cygniIn planta expression of PKSs in Ramularia collo-cygniIdentification and characterisation of polyketide synthases in the barley pathogen Ramularia collo-cygni.Ramularia collo-cygni (Rcc) is the causative agent of Ramularia leaf spot (RLS), a late season disease of barley occurringin temperate regions worldwide (Figure 1; Havis et al., 2015). RLS can cause substantial yield losses estimated to be ashigh as 70% but typically around 5-20%. Development of RLS is thought to be linked with the production and release inplanta of a set of anthraquinone-derived phytotoxic fungal secondary metabolites (SMs) called rubellins (Miethbauer et al.,2003; Walters et al., 2008). Rubellins are predicted to be synthesised through the polyketide synthase (PKS) pathway(Miethbauer et al., 2006). This study aimed at identifying putative SM gene clusters located near Rcc PKS core genes andto assess the expression of PKSs and SM-related genes during RLS development.This project is funded by SRUCHavis, N.D., et al., 2015. Phytopathol. 105(7), 895-904.Miethbauer, S. et al., 2003. J. Phytopathol. 151, 665-668. Walter, D.R., et al., 2008. FEMS Microbiol. Lett. 279(1), 1-7.Miethbauer, S. et al., 2006. Phytochem. 67(12), 1206-1213.McGrann, G.R.D., et al., 2016. BMC Genomics, 17(1), 584.Figure 1: Ramularia leaf spotsymptoms on barley leaf andstemUsing in silico genome walking, Rcc genes with a predicted functionassociated with secondary metabolism were identified near PKS coregenes. Eight putative SM gene clusters containing at least one PKSwere discovered in the genome of Rcc (Figure 3). Several clustersexhibited similarity to known SM biosynthetic clusters in other fungi.I) Identification of putatively functional PKSs in Rcc II) Identification of putative PKS-based SM gene clusters in RccProtein BLAST (pBLAST) analysis using known fungal PKSs sequencesas queries identified 30 gene models in the Rcc genome (McGrann etal., 2016) as putative PKS candidates. Only 10 of these Rcc genemodels encoded proteins containing the three domains KS-AT-ACPrequired for PKS function (Figure 2).Figure 2: Domain organisation of R. collo-cygni putatively functional PKSsSAT: starter unit: acyl carrier protein transacylase, KS: keto-synthase, AT: acyl-transferase, DH:dehydrogenase, PT: product template, MT: methyl-transferase, ER: enoyl-reductase, KR: keto-reductase, ACP: acyl carrier protein, TE: thiol-esterase, R: reductaseI) Expression of Rcc PKSs during RLS development II) Rcc PKSs and SM-related genes appear to be co-regulated051015202530354001020304050607080901005 7 10 12 15 18 21Area Under Disease Progression Curve (%max)Green Leaf Area (%)Days post inoculation00.511.522.533.55 7 10 12 15 18 21Mean normalised expressionDays post inoculationPks30-1.114Pks17-1.203Pks282-0.161Pks118-3.254Pksm46-0.119Pksm24-1.219A) B)Figure 5: R. collo-cygni PKSs are preferentially expressed during the asymptomatic andearly lesion development stages of RLS development.A) RLS disease development over 21 day post inoculation time course. B) Transcript levels ofRcc PKSs during disease development.00.511.522.535d 7d 10d 12d 15d 18d 21dMean normalised expressionDays post inoculationPksm46Pksm24AflRAflJStcQFigure 6: Co-regulation of R. collo-cygni PKSs with SM-related genesThe putative homologs of A. flavus AflR, AflJ and StcQ identified in aputative SM gene cluster containing no core gene in Rcc appear to beco-regulated with several Rcc PKSs, including the AfPksA putativehomologs Pksm24 and Pksm46. Transcript levels were highest duringthe asymptomatic and early symptoms formation stages of RLSdevelopment (Figure 6).Five of the putative PKSs identified in the Rcc genome were mosthighly expressed during the early stages of disease development(Figure 5).• Rcc appears to have the genetic equipment to produce severalpolyketide-derived SMs.• Most of Rcc PKSs are expressed early during RLS developmentquestioning the role of these genes in disease development butsuggesting a putative role in host manipulation.Future work• Identifying and disrupting candidate Rcc PKS genes to determinetheir role in rubellin biosynthesis to characterise the function of RccPKSs in RLS development.• Characterisation of new SMs produced by Rcc may help relate theircore gene expression to the biology of RLS.Figure 3: Putative SM gene clusters located near PKS genes identified in R. collo-cygni

Determining the role of secondary metabolism in the pathology of Ramularia collo-cygni, the fungus responsible for Ramularia leaf spot disease of barley.

Scotland's Rural CollegeDetermining the role of secondary metabolism in the pathology of Ramularia collo-cygni, the fungus responsible for Ramularia leaf spot disease of barley.Dussart, F; Spoel, Steven H; Hoebe, PN; McGrann, GRDPrint publication: 01/01/2017Link to publicationCitation for pulished version (APA):Dussart, F., Spoel, S. H., Hoebe, PN., & McGrann, GRD. (2017). Determining the role of secondary metabolismin the pathology of Ramularia collo-cygni, the fungus responsible for Ramularia leaf spot disease of barley..Poster session presented at International Workshop on Barley Leaf Diseases, Rabbat, Morocco.General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            • Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            • You may not further distribute the material or use it for any profit-making activity or commercial gain            • You may freely distribute the URL identifying the publication in the public portal ?Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Download date: 05. Nov. 2022Leading the way in Agriculture and Rural Research, Education and Consulting SRUC is a charity registered in Scotland, No. SC003712www.sruc.ac.ukFrancois Dussart1,2, Steven H. Spoel2, Peter N. Hoebe1, Graham R.D. McGrann11Scotland’s Rural College, West Mains Road, Edinburgh, EH9 3JG, UK2 School of Biological Sciences, University of Edinburgh,  Edinburgh EH9 3JR, UKE-mail: Francois.Dussart@sruc.ac.ukRamularia leaf spotConclusionsAcknowledgementsReferencesIn silico identification of PKSs in Ramularia collo-cygniIn planta expression of PKSs in Ramularia collo-cygniIdentification and characterisation of polyketide synthases in the barley pathogen Ramularia collo-cygni.Ramularia collo-cygni (Rcc) is the causative agent of Ramularia leaf spot (RLS), a late season disease of barley occurringin temperate regions worldwide (Figure 1; Havis et al., 2015). RLS can cause substantial yield losses estimated to be ashigh as 70% but typically around 5-20%. Development of RLS is thought to be linked with the production and release inplanta of a set of anthraquinone-derived phytotoxic fungal secondary metabolites (SMs) called rubellins (Miethbauer et al.,2003; Walters et al., 2008). Rubellins are predicted to be synthesised through the polyketide synthase (PKS) pathway(Miethbauer et al., 2006). This study aimed at identifying putative SM gene clusters located near Rcc PKS core genes andto assess the expression of PKSs and SM-related genes during RLS development.This project is funded by SRUCHavis, N.D., et al., 2015. Phytopathol. 105(7), 895-904.Miethbauer, S. et al., 2003. J. Phytopathol. 151, 665-668. Walter, D.R., et al., 2008. FEMS Microbiol. Lett. 279(1), 1-7.Miethbauer, S. et al., 2006. Phytochem. 67(12), 1206-1213.McGrann, G.R.D., et al., 2016. BMC Genomics, 17(1), 584.Figure 1: Ramularia leaf spotsymptoms on barley leaf andstemUsing in silico genome walking, Rcc genes with a predicted functionassociated with secondary metabolism were identified near PKS coregenes. Eight putative SM gene clusters containing at least one PKSwere discovered in the genome of Rcc (Figure 3). Several clustersexhibited similarity to known SM biosynthetic clusters in other fungi.I) Identification of putatively functional PKSs in Rcc II) Identification of putative PKS-based SM gene clusters in RccProtein BLAST (pBLAST) analysis using known fungal PKSs sequencesas queries identified 30 gene models in the Rcc genome (McGrann etal., 2016) as putative PKS candidates. Only 10 of these Rcc genemodels encoded proteins containing the three domains KS-AT-ACPrequired for PKS function (Figure 2).Figure 2: Domain organisation of R. collo-cygni putatively functional PKSsSAT: starter unit: acyl carrier protein transacylase, KS: keto-synthase, AT: acyl-transferase, DH:dehydrogenase, PT: product template, MT: methyl-transferase, ER: enoyl-reductase, KR: keto-reductase, ACP: acyl carrier protein, TE: thiol-esterase, R: reductaseI) Expression of Rcc PKSs during RLS development II) Rcc PKSs and SM-related genes appear to be co-regulated051015202530354001020304050607080901005 7 10 12 15 18 21Area Under Disease Progression Curve (%max)Green Leaf Area (%)Days post inoculation00.511.522.533.55 7 10 12 15 18 21Mean normalised expressionDays post inoculationPks30-1.114Pks17-1.203Pks282-0.161Pks118-3.254Pksm46-0.119Pksm24-1.219A) B)Figure 5: R. collo-cygni PKSs are preferentially expressed during the asymptomatic andearly lesion development stages of RLS development.A) RLS disease development over 21 day post inoculation time course. B) Transcript levels ofRcc PKSs during disease development.00.511.522.535d 7d 10d 12d 15d 18d 21dMean normalised expressionDays post inoculationPksm46Pksm24AflRAflJStcQFigure 6: Co-regulation of R. collo-cygni PKSs with SM-related genesThe putative homologs of A. flavus AflR, AflJ and StcQ identified in aputative SM gene cluster containing no core gene in Rcc appear to beco-regulated with several Rcc PKSs, including the AfPksA putativehomologs Pksm24 and Pksm46. Transcript levels were highest duringthe asymptomatic and early symptoms formation stages of RLSdevelopment (Figure 6).Five of the putative PKSs identified in the Rcc genome were mosthighly expressed during the early stages of disease development(Figure 5).• Rcc appears to have the genetic equipment to produce severalpolyketide-derived SMs.• Most of Rcc PKSs are expressed early during RLS developmentquestioning the role of these genes in disease development butsuggesting a putative role in host manipulation.Future work• Identifying and disrupting candidate Rcc PKS genes to determinetheir role in rubellin biosynthesis to characterise the function of RccPKSs in RLS development.• Characterisation of new SMs produced by Rcc may help relate theircore gene expression to the biology of RLS.Figure 3: Putative SM gene clusters located near PKS genes identified in R. collo-cygni

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Determining the role of secondary metabolism in the pathology of Ramularia collo-cygni, the fungus responsible for Ramularia leaf spot disease of barley.

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