Sclerotinia sclerotiorum necrosis-inducing effectors
Stem rot disease in canola caused by Sclerotinia sclerotiorum leads to lodging and severe yield losses in Canada. Sclerotinia sclerotiorum is equipped with small, secreted proteins (effectors) to induce plant cell death to facilitate nutrient uptake. Characterizing cell death/necrosis-inducing effectors might enable devising strategies to identify disease tolerant germplasm that is impervious to select necrosis-inducing effectors. In this study, RNA-Seq analysis was performed with a focus on the events occurring through the early (1 hour) to the middle (48 hours) stages of infection to reveal the gene expression patterns during the course of S. sclerotiorum infection on B. napus. The differentially expressed genes including those encoding hydrolytic enzymes, secreted effectors, enzymes involved in the synthesis of secondary metabolites or their detoxification, signaling, development, as well as oxalic acid and reactive oxygen species production. This investigation provides a broad overview of the sequential expression of virulence/pathogenicity-associated genes during infection of B. napus. To identify candidate necrosis-inducing effectors, the genome of S. sclerotiorum was searched for genes encoding small, secreted, cysteine-rich proteins. These effectors were tested for their ability to induce necrosis in Nicotiana benthamiana via Agrobacterium tumefaciens-mediated transient expression and for their host cellular localization. Six novel necrosis-inducing effectors were discovered, of which all but one required a signal peptide and secretion to the extracellular space for necrotic activity. These five effectors were localized to the endoplasmic reticulum and nucleus, while one that did not require signal peptide for necrotizing activity was localized in cytoplasm and nucleus. Virus-induced gene silencing (VIGS) experiments were conducted to reveal the participation of plant receptor-like kinases (RLK) in the induction of cell death. VIGS revealed that these five effectors required the RLKs, BAK1 and SOBIR1, for the induction of necrosis. These results illustrated the importance of necrosis-inducing effectors for S. sclerotiorum virulence and the potential role of host extra-cellular receptor(s) in the perception of S. sclerotiorum effectors. Substitution of cysteine residues with alanine and examination of truncated peptides for one of these effectors suggested that the native protein is necessary for necrotizing activity. These effectors could be applied for effector-assisted breeding of resistance to stem rot disease in canola.
Sclerotinia sclerotiorum, necrosis, cell death, receptors, virus-induced gene silencing, RNA-Seq, effector
Doctor of Philosophy (Ph.D.)