Abstract
Botrytis cinerea, a ubiquitous necrotrophic plant-pathogenic fungus, is responsible for grey mold and rot disease in a very wide range of plant species. Subtilisin-like proteases (or subtilases) are a very diverse family of serine proteases present in many organisms and are reported to have a broad spectrum of biological functions. Here, we identified two genes encoding subtilisin-like proteases (Bcser1 and Bcser2) in the genome of B. cinerea, both of which contain an inhibitor I9 domain and a peptidase S8 domain. The expression levels of Bcser1 and Bcser2 increased during the sclerotial forming stage, as well as during a later stage of hyphal infection on Arabidopsis thaliana leaves, but the up-regulation of Bcser1 was significantly higher than that of Bcser2. Interestingly, deletion of Bcser1 had no effect on the fungal development or virulence of B. cinerea. However, deletion of Bcser2 or double deletion of Bcser1 and Bcser2 severely impaired the hyphal growth, sclerotial formation and conidiation of B. cinerea. We also found that ∆Bcser2 and ∆Bcser1/2 could not form complete infection cushions and then lost the ability to infect intact plant leaves of Arabidopsis and tomato but could infect wounded plant tissues. Taken together, our results indicate that the subtilisin-like protease Bcser2 is crucial for the sclerotial formation, conidiation, and virulence of B. cinerea.
Highlights
Botrytis cinerea, the causal agent of grey mold, is considered a typical necrotrophic plant-pathogenic fungus and causes significant losses in more than 1000 plant species worldwide [1,2,3]
Our results indicate that the subtilisin-like protease Bcser2 is crucial for the sclerotial formation, conidiation, and virulence of B. cinerea
Two genes that encoded subtilisin-like serine proteases, Bcser1 (Gene ID: Bcin10g02530) and Bcser2 (Gene ID: Bcin08g02990), were found in B. cinerea by using the program BlastP [27]
Summary
The causal agent of grey mold, is considered a typical necrotrophic plant-pathogenic fungus and causes significant losses in more than 1000 plant species worldwide [1,2,3]. Many recent studies have shown that subtilases play important roles in development and virulence of pathogenic fungi [20]. Deletion of a subtilisin-like protease encoded gene prb results in lower level of sporulation and aerial hyphae, and a remarkable reduction in virulence in the chestnut blight fungus Cryphonectria parasitica. In Magnaporthe grisea, a subtilase encoding gene spm has been shown to be involved in sporulation, appressorium formation and pathogenicity and its function is related to autophagy [25]. In B. cinerea, two genes (Bcser and Bcser2) encoding subtilisin-like serine proteases showed a significant level of expression during the colonization of sunflower cotyledons [26]. To better understand the biological function of subtilisin-like proteases in B. cinerea, we constructed and characterized mutants of Bcser and Bcser. Our results suggest that Bcser is essential for the development and pathogenesis of B. cinerea
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