TMT-based quantitative proteomics analyses reveal novel defense mechanisms of Brassica napus against the devastating necrotrophic pathogen Sclerotinia sclerotiorum

Abstract

The white mould disease, caused by Sclerotinia sclerotiorum, is one of the most important diseases in the vital oil crop Brassica napus. Nevertheless, the defense mechanisms of B. napus against S. sclerotiorum are poorly understood. In this study, we performed comparative quantitative proteomics analyses to reveal B. napus defense mechanisms against S. sclerotiorum. The proteomes of B. napus leaves inoculated with S. sclerotiorum wild-type strain 1980 and nonpathogenic mutant strain Ep-1PB as well as empty agar plug as the control were analyzed using TMT label-based quantitative analysis technique. A total of 79, 299 and 173 proteins consistently differentially expressed between Ep-1PB- and mock-inoculated leaves, 1980- and mock-inoculated leaves, as well as 1980- and Ep-1PB-inoculated leaves, respectively, were identified. The differential expression of 12 selected proteins was confirmed by qRT-PCR analyses. The Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction prediction analyses revealed that redox homeostasis, lipid signaling, calcium signaling, histone and DNA methylation-mediated transcription regulation and defense-related proteins such as defensin and defensin-like proteins and cyanate lyase, contribute to defense against S. sclerotiorum. Our results provide new insights into molecular mechanisms that may be involved in defense responses of B. napus to S. sclerotiorum. SignificanceThe Sclerotinia white mould disease is one of the most important diseases in the significant oil crop Brassica napus. Nevertheless, the defense mechanisms of B. napus against S. sclerotiorum are still largely unknown to date. In this study, we addressed this issue by performing TMT label-based comparative quantitative analyses of the proteomes of B. napus leaves inoculated with S. sclerotiorum wild-type strain 1980 and nonpathogenic mutant strain Ep-1PB as well as empty agar plug as the control. Through comparative analyses on 79, 299, and 173 proteins that are consistently differentially expressed in between Ep-1PB-inoculated and the control leaves, 1980-inoculated and the control leaves, as well as 1980-inoculated and Ep-1PB-inoculated leaves, respectively, we revealed that redox homeostasis, lipid signaling, calcium signaling, histone and DNA methylation-mediated transcription regulation and defense-related proteins such as defensin and defensin-like proteins as well as cyanate lyase, contribute to B. napus defenses against S. sclerotiorum. Notably, the potential role of lipid signaling, calcium signaling, histone and DNA methylation-mediated transcription regulation and cyanate lyase in B. napus defense against S. sclerotiorum are not reported previously but rather unveiled for the first time in this study. The current study represents the most extensive analysis of the protein profile of B. napus in response to S. sclerotiorum inoculation and includes for the first time the results from comparison between plants inoculated with the wild-type strain and a nonpathogenic mutant strain of S. sclerotiorum. Collectively, our results provide new insights into the molecular mechanisms of interactions between B. napus and S. sclerotiorum.