In order to gain a more thorough understanding of the phytopathogenic fungus, Sclerotinia sclerotiorum, we initiated a proteome-level study of the fungal mycelia and secretome. To our knowledge, this is the first comprehensive proteome-level study of this fungus. Extracted mycelial proteins and secreted proteins collected from liquid culture were separated using 2-DE and annotated following ESI-q-TOF MS/MS. Fifty-two secreted proteins were reproducibly present in three biological replicates and 18 of them were identified by MS/MS while over 200 mycelial proteins were reproducibly present in three independent extractions and approximately half of them were identified. Many of the annotated secreted proteins were cell wall degrading enzymes that had been previously identified as pathogenicity or virulence factors of S. sclerotiorum; however, the contribution to the virulence of S. sclerotiorum of one of the identified proteins, alpha-L-arabinofuranosidase, is yet to be analyzed. Furthermore, previous comprehensive EST studies did not detect the presence of the alpha-L-arabinofuranosidase transcript, which demonstrates the merit of performing proteome-level research. All of the secreted and mycelial proteins identified were functionally classified, and the known and proposed roles in disease initiation or progression for many of them are discussed.

Sclerotinia stem rot is an economically important disease of canola (Brassica napus) and is caused by the fungal pathogen Sclerotinia sclerotiorum. This study evaluated the differential gene expression patterns of S. sclerotiorum during disease development on two canola lines differing in susceptibility to this pathogen. Sequencing of the mRNA libraries derived from inoculated petioles and mycelium grown on liquid medium generated approximately 164 million Illumina reads, including 95 million 75-bp-single reads, and 69 million 50-bp-paired end reads. Overall, 36% of the quality filter-passed reads were mapped to the S. sclerotiorum reference genome. On the susceptible line, 1301 and 1214 S. sclerotiorum genes were differentially expressed at early (8-16 hours post inoculation (hpi)) and late (24-48 hpi) infection stages, respectively, while on the resistant line, 1311 and 1335 genes were differentially expressed at these stages, respectively. Gene ontology (GO) categories associated with cell wall degradation, detoxification of host metabolites, peroxisome related activities like fatty acid ß-oxidation, glyoxylate cycle, oxidoreductase activity were significantly enriched in the up-regulated gene sets on both susceptible and resistant lines. Quantitative RT-PCR of six selected DEGs further validated the RNA-seq differential gene expression analysis. The regulation of effector genes involved in host defense suppression or evasion during the early infection stage, and the expression of effectors involved in host cell death in the late stage of infection provide supporting evidence for a two-phase infection model involving a brief biotrophic phase during early stages of infection. The findings from this study emphasize the role of peroxisome related pathways along with cell wall degradation and detoxification of host metabolites as the key mechanisms underlying pathogenesis of S. sclerotiorum on B. napus.

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Canola (Brassica napus), an agriculturally important oilseed crop, can be significantly affected by diseases such as sclerotinia stem rot, blackleg, and alternaria black spot resulting in significant loss of crop productivity and quality. Cysteine-rich antimicrobial peptides isolated from plants have emerged as a potential resource for protection of plants against phytopathogens. Here we report the significance of an antimicrobial peptide, PmAMP1, isolated from western white pine (Pinus monticola), in providing canola with resistance against multiple phytopathogenic fungi. The cDNA encoding PmAMP1 was successfully incorporated into the genome of B. napus, and it's in planta expression conferred greater protection against Alternaria brassicae, Leptosphaeria maculans and Sclerotinia sclerotiorum. In vitro experiments with proteins extracted from transgenic canola expressing Pm-AMP1 demonstrated its inhibitory activity by reducing growth of fungal hyphae. In addition, the in vitro synthesized peptide also inhibited the growth of the fungi. These results demonstrate that generating transgenic crops expressing PmAMP1 may be an effective and versatile method to protect susceptible crops against multiple phytopathogens.

Although Sclerotinia sclerotiorum (Lib.) de Bary has been studied extensively, there are still aspects of this important phytopathogen's ability to cause disease in susceptible plants that remain unclear. A recent comprehensive proteome-level investigation of this fungus identified a number of proteins whose functions in disease initiation and progression have not been clearly established. Included among these proteins was an arabinofuranosidase/beta-xylosidase precursor whose role as a potential virulence factor had not been investigated previously. This article describes the generation of gene-disrupted mutant S. sclerotiorum unable to produce this arabinofuranosidase/beta-xylosidase precursor as well as the comparison of the virulence of this mutant with that of wild-type mycelia on susceptible canola leaves and stems. At all time points tested, the degree of necrosis was observed to be significantly greater on the plant tissue inoculated with wild-type mycelia. To our knowledge, this is the first report that clearly demonstrates that this arabinofuranosidase/beta-xylosidase precursor is a virulence factor for S. sclerotiorum.