Secretome analysis reveals effector candidates associated with broad host range necrotrophy in the fungal plant pathogen Sclerotinia sclerotiorum

Abstract Background The broad host range pathogen Sclerotinia sclerotiorum infects over 400 plant species and causes substantial yield losses in crops worldwide. Secondary metabolites are known to play important roles in the virulence of plant pathogens, but little is known about the secondary metabolite repertoire of S. sclerotiorum. In this study, we predicted secondary metabolite biosynthetic gene clusters in the genome of S. sclerotiorum and analysed their expression during infection of Brassica napus using an existing transcriptome data set. We also investigated their sequence diversity among a panel of 25 previously published S. sclerotiorum isolate genomes.Results We identified 80 putative secondary metabolite clusters. Over half of the clusters contained at least three transcriptionally coregulated genes. Comparative genomics revealed clusters homologous to clusters in the closely related plant pathogen Botrytis cinerea for production of carotenoids, hydroxamate siderophores, DHN melanin and botcinic acid. We also identified putative phytotoxin clusters that can potentially produce the polyketide sclerin and an epipolythiodioxopiperazine. Secondary metabolite clusters were enriched in subtelomeric genomic regions, and those containing paralogues showed a particularly strong association with repeats. The positional bias we identified was borne out by intraspecific comparisons that revealed putative secondary metabolite genes suffered more presence / absence polymorphisms and exhibited a significantly higher sequence diversity than other genes.Conclusions These data suggest that S. sclerotiorum produces numerous secondary metabolites during plant infection and that their gene clusters undergo enhanced rates of mutation, duplication and recombination in subtelomeric regions. The microevolutionary regimes leading to S. sclerotiorum secondary metabolite diversity have yet to be elucidated. Several potential phytotoxins documented in this study provide the basis for future functional analyses.

Download Full-text

A novel alphahypovirus that infects the fungal plant pathogen Sclerotinia sclerotiorum

Archives of Virology ◽

10.1007/s00705-021-05315-4 ◽

2021 ◽

Author(s):

Weibo Liang ◽

Zhongbo Lu ◽

Jie Duan ◽

Daohong Jiang ◽

Jiatao Xie ◽

...

Keyword(s):

Sclerotinia Sclerotiorum ◽

Plant Pathogen ◽

Fungal Plant Pathogen

Download Full-text

Effects of Sublethal Fungicides on Mutation Rates and Genomic Variation in Fungal Plant Pathogen, Sclerotinia sclerotiorum

PLoS ONE ◽

10.1371/journal.pone.0168079 ◽

2016 ◽

Vol 11 (12) ◽

pp. e0168079 ◽

Cited By ~ 12

Author(s):

B. Sajeewa Amaradasa ◽

Sydney E. Everhart

Keyword(s):

Sclerotinia Sclerotiorum ◽

Plant Pathogen ◽

Genomic Variation ◽

Mutation Rates ◽

Fungal Plant Pathogen

Download Full-text

Introduction of Large Sequence Inserts by CRISPR-Cas9 To Create Pathogenicity Mutants in the Multinucleate Filamentous PathogenSclerotinia sclerotiorum

mBio ◽

10.1128/mbio.00567-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 21

Author(s):

Jingtao Li ◽

Yanhua Zhang ◽

Yucheng Zhang ◽

Pei-Ling Yu ◽

Hongyu Pan ◽

...

Keyword(s):

Host Range ◽

Sclerotinia Sclerotiorum ◽

Host Resistance ◽

Gene Disruption ◽

Plant Diseases ◽

Functional Gene ◽

Broad Host Range ◽

Content Type ◽

Gene Characterization ◽

Quantitative Nature

ABSTRACTThe necrotrophic fungal plant pathogenSclerotinia sclerotiorumis responsible for substantial global crop losses annually resulting in localized food insecurity and loss of livelihood. Understanding the basis of this broad-host-range and aggressive pathogenicity is hampered by the quantitative nature of both host resistance and pathogen virulence. To improve this understanding, methods for efficient functional gene characterization that build upon the existing completeS. sclerotiorumgenome sequence are needed. Here, we report on the development of a clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (CRISPR-Cas9)-mediated strategy for creating gene disruption mutants and the application of this technique for exploring roles of known and hypothesized virulence factors. A key finding of this research is that transformation with a circular plasmid encoding Cas9, target single guide RNA (sgRNA), and a selectable marker resulted in a high frequency of targeted, insertional gene mutation. We observed that 100% of the mutants integrated large rearranged segments of the transforming plasmid at the target site facilitated by the nonhomologous end joining (NHEJ) repair pathway. This result was confirmed in multiple target sites within the same gene in three independent wild-type isolates ofS. sclerotiorumand in a second independent gene. Targeting the previously characterizedSsoah1gene allowed us to confirm the loss-of-function nature of the CRISPR-Cas9-mediated mutants and explore new aspects of the mutant phenotype. Applying this technology to create mutations in a second previously uncharacterized gene allowed us to determine the requirement for melanin accumulation in infection structure development and function.IMPORTANCEFungi that cause plant diseases by rotting or blighting host tissue with limited specificity remain among the most difficult to control. This is largely due to the quantitative nature of host resistance and a limited understanding of fungal pathogenicity. A mechanistic understanding of pathogenicity requires the ability to manipulate candidate virulence genes to test hypotheses regarding their roles in disease development.Sclerotinia sclerotiorumis among the most notorious of these so-called broad-host-range necrotrophic plant pathogens. The work described here provides a new method for rapidly constructing gene disruption vectors to create gene mutations with high efficiency compared with existing methods. Applying this method to characterize gene functions inS. sclerotiorum, we confirm the requirement for oxalic acid production as a virulence factor in multiple isolates of the fungus and demonstrate that melanin accumulation is not required for infection. Using this approach, the pace of functional gene characterization and the understanding of pathogenicity and related disease resistance will increase.

Download Full-text

Characterization of microsatellites in the fungal plant pathogen, Sclerotinia sclerotiorum

Molecular Ecology Notes ◽

10.1046/j.1471-8278.2001.00102.x ◽

2001 ◽

Vol 1 (4) ◽

pp. 267-269 ◽

Cited By ~ 60

Author(s):

Caroline Sirjusingh ◽

Linda M. Kohn

Keyword(s):

Sclerotinia Sclerotiorum ◽

Plant Pathogen ◽

Fungal Plant Pathogen

Download Full-text

Identification of virulence associated loci in the emerging broad host range plant pathogen Pseudomonas fuscovaginae

BMC Microbiology ◽

10.1186/s12866-014-0274-7 ◽

2014 ◽

Vol 14 (1) ◽

Cited By ~ 9

Author(s):

Hitendra Kumar Patel ◽

Maura Matiuzzo ◽

Iris Bertani ◽

Vincent de Paul Bigirimana ◽

Gavin J Ash ◽

...

Keyword(s):

Host Range ◽

Plant Pathogen ◽

Broad Host Range

Download Full-text

Rapid identification of an Arabidopsis NLR gene conferring susceptibility to Sclerotinia sclerotiorum using time-resolved automated phenotyping

10.1101/488171 ◽

2018 ◽

Cited By ~ 1

Author(s):

Adelin Barbacci ◽

Olivier Navaud ◽

Malick Mbengue ◽

Rémy Vincent ◽

Marielle Barascud ◽

...

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Host Range ◽

Sclerotinia Sclerotiorum ◽

Complex Traits ◽

Functional Characterization ◽

Broad Host Range ◽

Vegetable Crops ◽

Phenotypic Effect ◽

Time Resolved

ABSTRACTThe broad host range necrotrophic fungus Sclerotinia sclerotiorum is a devastating pathogen of many oil and vegetable crops. Plant genes conferring complete resistance against S. sclerotiorum have not been reported. Instead, plant populations challenged by S. sclerotiorum exhibit a continuum of partial resistance designated as quantitative disease resistance (QDR). Because of their complex interplay and their small phenotypic effect, the functional characterization of QDR genes remains limited. How broad host range necrotrophic fungi manipulate plant programmed cell death is for instance largely unknown. Here, we designed a time-resolved automated disease phenotyping pipeline and assessed the kinetics of disease symptoms caused by seven S. sclerotiorum isolates on six A. thaliana natural accessions with unprecedented resolution. We hypothesized that large effect polymorphisms common to the most resistant A. thaliana accessions, but absent from the most susceptible ones, would point towards disease susceptibility genes. This identified highly divergent alleles of the nucleotide-binding site leucine-rich repeat gene LAZ5 in the resistant accessions Rubenzhnoe and Lip-0. Two LAZ5-deficient mutant lines in the Col-0 genetic background showed enhanced QDR to S. sclerotiorum, whereas plants mutated in the closely related CSA1 gene responded like the wild type. These findings illustrate the value of time-resolved image-based phenotyping for unravelling the genetic bases of complex traits such as QDR. Our results suggest that S. sclerotiorum manipulates plant sphingolipid pathways guarded by LAZ5 to trigger programmed cell death and cause disease.

Download Full-text

Integrated soybean transcriptomics, metabolomics, and chemical genomics reveal the importance of the phenylpropanoid pathway and antifungal activity in resistance to the broad host range pathogenSclerotinia sclerotiorum

10.1101/363895 ◽

2018 ◽

Cited By ~ 2

Author(s):

Ashish Ranjan ◽

Nathaniel M. Westrick ◽

Sachin Jain ◽

Jeff S. Piotrowski ◽

Manish Ranjan ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Antifungal Activity ◽

Host Range ◽

Sclerotinia Sclerotiorum ◽

Phenylpropanoid Pathway ◽

Broad Host Range ◽

Oxygen Species ◽

Resistant Line ◽

Chemical Genomics ◽

Antifungal Activities

AbstractSclerotinia sclerotiorum, a predominately necrotrophic fungal pathogen with a broad host range, causes a significant yield limiting disease of soybean called Sclerotinia stem rot (SSR). Resistance mechanisms against SSR are poorly understood, thus hindering the commercial deployment of SSR resistant varieties. We used a multiomic approach utilizing RNA-sequencing, Gas chromatography-mass spectrometry-based metabolomics and chemical genomics in yeast to decipher the molecular mechanisms governing resistance toS.sclerotiorumin soybean. Transcripts and metabolites of two soybean recombinant inbred lines, one resistant, and one susceptible toS.sclerotiorumwere analyzed in a time course experiment. The combined results show that resistance toS.sclerotiorumin soybean is associated in part with an early accumulation of JA-Ile ((+)-7-iso-Jasmonoyl-L-isoleucine), a bioactive jasmonate, increased ability to scavenge reactive oxygen species (ROS), and importantly, a reprogramming of the phenylpropanoid pathway leading to increased antifungal activities. Indeed, we noted that phenylpropanoid pathway intermediates such as, 4-hydroxybenzoate, ferulic acid and caffeic acid were highly accumulated in the resistant line.In vitroassays show that these metabolites and total stem extracts from the resistant line clearly affectS.sclerotiorumgrowth and development. Using chemical genomics in yeast, we further show that this antifungal activity targets ergosterol biosynthesis in the fungus, by disrupting enzymes involved in lipid and sterol biosynthesis. Overall, our results are consistent with a model where resistance toS.sclerotiorumin soybean coincides with an early recognition of the pathogen, leading to the modulation of the redox capacity of the host and the production of antifungal metabolites.Author SummaryResistance to plant fungal pathogens with predominately necrotrophic lifestyles is poorly understood. In this study, we useSclerotinia sclerotiorumand soybean as a model system to identify key resistance components in this crop plant. We employed a variety of omics approaches in combination with functional studies to identify plant processes associated with resistance toS.sclerotiorum. Our results suggest that resistance to this pathogen is associated in part with an earlier induction of jasmonate signaling, increased ability to scavenge reactive oxygen species, and importantly, a reprogramming of the phenylpropanoid pathway resulting in increased antifungal activities. These findings provide specific plant targets that can exploited to confer resistance toS.sclerotiorumand potentially other pathogens with similar lifestyle.

Download Full-text