Abstract
Small, secreted proteins have been found to play crucial roles in interactions between biotrophic/hemi-biotrophic pathogens and plants. However, little is known about the roles of these proteins produced by broad host-range necrotrophic phytopathogens during infection. Here, we report that a cysteine-rich, small protein SsSSVP1 in the necrotrophic phytopathogen Sclerotinia sclerotiorum was experimentally confirmed to be a secreted protein, and the secretion of SsSSVP1 from hyphae was followed by internalization and cell-to-cell movement independent of a pathogen in host cells. SsSSVP1∆SP could induce significant plant cell death and targeted silencing of SsSSVP1 resulted in a significant reduction in virulence. Through yeast two-hybrid (Y2H), coimmunoprecipitation (co-IP) and bimolecular fluorescence complementation (BiFC) assays, we demonstrated that SsSSVP1∆SP interacted with QCR8, a subunit of the cytochrome b-c1 complex of mitochondrial respiratory chain in plants. Double site-directed mutagenesis of two cysteine residues (C38 and C44) in SsSSVP1∆SP had significant effects on its homo-dimer formation, SsSSVP1∆SP-QCR8 interaction and plant cell death induction, indicating that partial cysteine residues surely play crucial roles in maintaining the structure and function of SsSSVP1. Co-localization and BiFC assays showed that SsSSVP1∆SP might hijack QCR8 to cytoplasm before QCR8 targeting into mitochondria, thereby disturbing its subcellular localization in plant cells. Furthermore, virus induced gene silencing (VIGS) of QCR8 in tobacco caused plant abnormal development and cell death, indicating the cell death induced by SsSSVP1∆SP might be caused by the SsSSVP1∆SP-QCR8 interaction, which had disturbed the QCR8 subcellular localization and hence disabled its biological functions. These results suggest that SsSSVP1 is a potential effector which may manipulate plant energy metabolism to facilitate the infection of S. sclerotiorum. Our findings indicate novel roles of small secreted proteins in the interactions between host-non-specific necrotrophic fungi and plants, and highlight the significance to illuminate the pathogenic mechanisms of this type of interaction.
Highlights
Sclerotinia sclerotiorum (Lib.) de Bary is an exemplary necrotrophic phytopathogenic fungus with a broad host range
SsSSVP1ΔSP interacts with QCR8, a subunit of cytochrome b-c1 complex, and disturbs the localization of QCR8 in mitochondria, which may disable its biological function
This is a completely different interaction model from the gene-for-gene or inversed gene-for-gene paradigm. These findings suggest that the pathogenesis of S. sclerotiorum is more subtle and complex than previously appreciated and highlight the significance to investigate the interaction models between the host-non-specific necrotrophs and their hosts
Summary
Sclerotinia sclerotiorum (Lib.) de Bary is an exemplary necrotrophic phytopathogenic fungus with a broad host range. The nutrient acquisition of necrotrophic pathogens is based on host cell killing [2]. Biotrophic and hemibiotrophic fungi secrete effectors that manipulate host cell structure and function to obtain nutrients and suppress plant defenses, thereby facilitating infection [3]. The secretion and transfer of effectors into plant host cells are essential for the pathogenesis of many biotrophic and hemibiotrophic fungi [4,5,6,7]. Plant cell death triggered through hypersensitive responses (HRs) is a major obstacle for the further expansion of biotrophic and hemibiotrophic fungi during the initial stage of infection. For necrotrophic fungi, host cell death might be beneficial rather than detrimental for pathogenesis; the canonical necrotrophic fungus S. sclerotiorum secretes a wide array of cell-wall-degrading enzymes (CWDEs) to
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