Functional Analysis Of Two Putative Secreted Proteins In Botrytis Cinerea

Botrytis cinerea is a necrotrophic plant pathogenic fungus with a wide range of non-specific hosts.Indeed,this pathogen has been considered as one of the top ten fungal pathogens since it can infect more than 1,000 plant species and cause significant losses in crop production and post-harvest storage.Botrytis cinerea and Sclerotinia sclerotiorum are closely related plant pathogens,and they are the best-studied necrotrophic fungal plant pathogens due to their phylogenetic proximity and similarities in lifestyle.B.cinerea can secretes a great number of extracellular proteins,which play an important role in the process of invading plants,while the functions and mechanisms of many secreted proteins remain unknown to date.Therefore,the related research on the secreted proteins of B.cinerea is important for further revealing the pathogenic mechanisms of B.cinerea and the interaction with host plants.In the previous study,our group found that SS1G₀8163 in S.sclerotiorum encoding a putative secreted protein had a certain role in inhibiting plant defense responses and promoting S.sclerotiorum infection towards plants.Then we suspect that the SS1G₀8163 homologous genes in B.cinerea could also involve in the host-pathogen interactions.Through amino acid sequence alignment,we identified two homologous genes: BCIN₀4g00360 and BCIN₀9g07100,which have 50.00% and 60.87% identity with SS1G₀8163,respectively.Using homologous recombination strategy,we knocked out BCIN₀4g00360 and BCIN₀9g07100,and generated 5 BCIN₀4g00360 deletion mutants and 4 BCIN₀9g07100 deletion mutants.Compared with wild type B05.10,?BCIN₀4g00360 and ?BCIN₀9g07100 showed no significant difference in the vegetative growth and the virulence on tomato leaves.We constructed BCIN₀4g00360 and BCIN₀9g07100 into plant virus expression vectors containing GFP tag,and found that they located on the plant cell membranes.In addition,Agrobacterium-mediated transient expression assays in N.benthamiana exhibited that neither BCIN₀4g00360 or BCIN₀9g071002 could induce N.benthamiana hypersensitive response,and none of them could inhibit the plant defense response triggered by elicitor INF1.The results of this study indicate that BCIN₀4g00360 and BCIN₀9g07100 are unnecessary for the vegetative development and pathogenicity in B.cinerea,which may be related to the redundance of the secreted proteins of B.cinerea.Based on the results of subcellular localization,we speculate that these two putative secreted proteins might play a certain role in plant cell membranes.BCIN₀4g00360 and BCIN₀9g07100 were unable to induce plant cell death and inhibit plant defense responses,showing that they might not have specific functions in host-pathogen interaction.However,this might be related to the fact that the transient expression levels of these two putative secreted proteins in N.benthamiana are not high enough,or they do not participate in the immune response pathway initiated by INF1.The relevant experiments need to be further carried out.