A Forward Genetic Screen in Sclerotinia sclerotiorum Revealed the Transcriptional Regulation of Its Sclerotial Melanization Pathway

Abstract

Most plant fungal pathogens that cause worldwide crop losses are understudied due to various technical challenges. With the increasing availability of sequenced whole genomes of these non-model fungi, effective genetic analysis methods are highly desirable. Here we describe a newly developed pipeline, which combines forward genetic screening with high-throughput next-generation sequencing to enable quick gene discovery. We applied this pipeline in the notorious soilborne phytopathogen, Sclerotinia sclerotiorum, and identified 32 mutants with various developmental and growth deficiencies. Detailed molecular studies of three melanisation-deficient mutants provide a proof of concept for the effectiveness of our method. A master transcription factor was found to regulate melanisation of sclerotia through the DHN (1,8-dihydroxynaphthalene) melanin biosynthesis pathway. In addition, these mutants revealed that sclerotial melanisation is important for sclerotia survival under abiotic stresses, sclerotial surface structure, and sexual reproduction. Foreseeably, this pipeline can be applied to facilitate efficient in-depth studies of other non-model fungal species in the future.

An Amidase Contributes to Full Virulence of Sclerotinia sclerotiorum

“…Using a UV-mutagenesis based forward genetics pipeline [ 26 ], we generated a mutagenized population originated from single haploid ascospores. Our initial screen aimed for mutants deficient in sclerotial formation and melanization.…”

“…To identify the mutation responsible for the observed phenotypes of 5–10, the full genome of 5–10 was subjected to whole genome next-generation sequencing (NGS). The genomic sequence of an unrelated mutant pnk1 was used as negative control to exclude background mutations that occured in both 5–10 and pnk1 [ 26 ]. From whole genome sequence comparisons, a total of 77 SNPs (single nucleotide polymorphisms) and 689 INDELs (insertions and deletions) were captured in 5–10, including mutations in exonic, intronic, upstream, downstream, and intergenic regions.…”

“…al. [ 26 ] provided an effective pipeline, which combines forward genetic screening with high-throughput next-generation sequencing (NGS) to enable quick gene discovery and confirmed the reliability of this method in S. sclerotiorum .…”

“…al. [ 26 ]. Mutants defective in virulence were isolated and NGS was used to identify the candidate genes.…”

SsNEP2 Contributes to the Virulence of Sclerotinia sclerotiorum

“…Full-length SsNEP2 gene fragment and pCH-EF-1 vector were digested by using restriction enzyme XhoI and SacI, then linked by homologous recombinase (ClonExpress ® II One Step Cloning Kit, Vazyme, Nanjing, China) to generate the pCH-EF-1-NEP2 construct. Then the plasmid was used for SsNEP2 transformation using the polyethylene glycol (PEG)-mediated transformation method [47,48].…”