Dataset Information

Impacts of fludioxonil resistance on global gene expression in the necrotrophic fungal plant pathogen Sclerotinia sclerotiorum.

ABSTRACT:

Background

The fungicide fludioxonil over-stimulates the fungal response to osmotic stress, leading to over-accumulation of glycerol and hyphal swelling and bursting. Fludioxonil-resistant fungal strains that are null-mutants for osmotic stress response genes are easily generated through continual sub-culturing on sub-lethal fungicide doses. Using this approach combined with RNA sequencing, we aimed to characterise the effects of mutations in osmotic stress response genes on the transcriptional profile of the important agricultural pathogen Sclerotinia sclerotiorum under standard laboratory conditions. Our objective was to understand the impact of disruption of the osmotic stress response on the global transcriptional regulatory network in an important agricultural pathogen.

Results

We generated two fludioxonil-resistant S. sclerotiorum strains, which exhibited growth defects and hypersensitivity to osmotic stressors. Both had missense mutations in the homologue of the Neurospora crassa osmosensing two component histidine kinase gene OS1, and one had a disruptive in-frame deletion in a non-associated gene. RNA sequencing showed that both strains together differentially expressed 269 genes relative to the parent during growth in liquid broth. Of these, 185 (69%) were differentially expressed in both strains in the same direction, indicating similar effects of the different point mutations in OS1 on the transcriptome. Among these genes were numerous transmembrane transporters and secondary metabolite biosynthetic genes.

Conclusions

Our study is an initial investigation into the kinds of processes regulated through the osmotic stress pathway in S. sclerotiorum. It highlights a possible link between secondary metabolism and osmotic stress signalling, which could be followed up in future studies.

SUBMITTER: Taiwo AO

PROVIDER: S-EPMC7847169 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Publications

Impacts of fludioxonil resistance on global gene expression in the necrotrophic fungal plant pathogen Sclerotinia sclerotiorum.

Taiwo Akeem O AO Harper Lincoln A LA Derbyshire Mark C MC

BMC genomics 20210130 1

<h4>Background</h4>The fungicide fludioxonil over-stimulates the fungal response to osmotic stress, leading to over-accumulation of glycerol and hyphal swelling and bursting. Fludioxonil-resistant fungal strains that are null-mutants for osmotic stress response genes are easily generated through continual sub-culturing on sub-lethal fungicide doses. Using this approach combined with RNA sequencing, we aimed to characterise the effects of mutations in osmotic stress response genes on the transcri ...[more]

PMID: 33516198

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Project description:Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.

Project description:Pathogen exposure to sublethal doses of fungicides may result in mutations that may represent an important and largely overlooked mechanism of introducing new genetic variation into strictly clonal populations, including acquisition of fungicide resistance. We tested this hypothesis using the clonal plant pathogen, Sclerotinia sclerotiorum. Nine susceptible isolates were exposed independently to five commercial fungicides with different modes of action: boscalid (respiration inhibitor), iprodione (unclear mode of action), thiophanate methyl (inhibition of microtubulin synthesis) and azoxystrobin and pyraclostrobin (quinone outside inhibitors). Mycelium of each isolate was inoculated onto a fungicide gradient and sub-cultured from the 50-100% inhibition zone for 12 generations and experiment repeated. Mutational changes were assessed for all isolates at six neutral microsatellite (SSR) loci and for a subset of isolates using amplified fragment length polymorphisms (AFLPs). SSR analysis showed 12 of 85 fungicide-exposed isolates had a total of 127 stepwise mutations with 42 insertions and 85 deletions. Most stepwise deletions were in iprodione- and azoxystrobin-exposed isolates (n = 40/85 each). Estimated mutation rates were 1.7 to 60-fold higher for mutated loci compared to that expected under neutral conditions. AFLP genotyping of 33 isolates (16 non-exposed control and 17 fungicide exposed) generated 602 polymorphic alleles. Cluster analysis with principal coordinate analysis (PCoA) and discriminant analysis of principal components (DAPC) identified fungicide-exposed isolates as a distinct group from non-exposed control isolates (PhiPT = 0.15, P = 0.001). Dendrograms based on neighbor-joining also supported allelic variation associated with fungicide-exposure. Fungicide sensitivity of isolates measured throughout both experiments did not show consistent trends. For example, eight isolates exposed to boscalid had higher EC50 values at the end of the experiment, and when repeated, only one isolate had higher EC50 while most isolates showed no difference. Results of this support the hypothesis that sublethal fungicide stress increases mutation rates in a largely clonal plant pathogen under in vitro conditions. Collectively, this work will aid our understanding how non-lethal fungicide exposure may affect genomic variation, which may be an important mechanism of novel trait emergence, adaptation, and evolution for clonal organisms.

Dataset Information

Impacts of fludioxonil resistance on global gene expression in the necrotrophic fungal plant pathogen Sclerotinia sclerotiorum.

Background

Results

Conclusions

Publications

Impacts of fludioxonil resistance on global gene expression in the necrotrophic fungal plant pathogen Sclerotinia sclerotiorum.

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