Mining And Functional Research Of Sclerotial Development Associated Genes In Botrytis Cinerea

Botrytis cinerea,the causal agent of grey mould disease in more than 1000 plant species worldwide,is considered a typical necrotrophic plant-pathogenic fungus and causes significant losses to agricultural production every year.Due to its great economic impact and scientific significance,B.cinerea has become an improtant model fungus for the studing the necrotrophic pathogens.Sclerotia play crucial roles in the lifecycle of B.cinerea and may either serve as asexual survival structure for local colonization or as the maternal parent in sexual reproduction to initiate the sexual cycle.It is also an important source of initial infection of B.cinerea.Inhibiting the formation of sclerotia will cut off the disease cycle of B.cinerea.Therefore,to study the function of sclerotial formation associated genes and to reveal the molecular mechanism of sclerotial development will provide a new theoretical basis for the effective control of grey mould disease.In order to explore the sclerotial formation associated genes of B.cinerea,the differentially expressed genes in the growth stage and the sclerotial development stage were analyzed by transcriptome sequencing technology.Two biological repeats showed that 1,318 and 1,249 genes were significantly up-regulated during sclerotial formation,and 853 and 943 genes were significantly down-regulated.Therefore,we speculated that some of these genes might play important roles in sclerotial development of B.cinerea.Based on the RNA-seq sequencing data of B.cinerea,10 genes were significantly up-regulated in sclerotial formation stage and one gene was significantly down-regulated in sclerotial formation were screened for further study.Firstly,the expression level of these genes was detected by RT-PCR,and the results were consistent with the sequencing results.Meanwhile,genes such as BC1G?04708,BC1G?00896,BC1G?08642 and BC1G?04810 were found to be up-regulated in the late stages of infection,while BC1G?14271,BC1G?08490 and BC1G?10623 were not detected in the late stages of infection.To further study the function of sclerotial formation associated genes in B.cinerea,knockout transformants of these candidate genes were obtained,and the biological characteristics of each knockout transformant were determined.The results indicated that only the phenotype of BC1G?08490 knockout transformant changes significantly,and the gene encodes a putative protein.The colony of the knockout transformant ?BC1G?08490-2 was smooth on the PDA plate,and lost the ability to form conidia and sclerotia.Although BC1G?08490 was not expressed during infection,?BC1G?08490-2 was lost virulence on Arabidopsis leaves,and this may be due to the significant influence of acid production ability.The results indicate that the BC1G?08490 gene plays an important role in the sclerotia development and pathogenesis of Botrytis cinerea.Howere,the results also showed that the knockout of BC1G?14271,BC1G?07408,BC1G?08642,BC1G?00896,BC1G?12892,BC1G?13021,BC1G?00448,BC1G?08931 and BC1G?10623 had no significant effects on the colony morphology,growth rate,sclerotial formation,virulence and acid production ability of B.cinerea.We speculated that although these genes were significantly up-regulated during sclerotial formation and late stages of infection,they do not play a decisive role in this process,or there are functionally redundant genes.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 upregulation 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 sporulation of B.cinerea.We also found that ?Bcser2 and ?Bcser1/2 could not form complete infection cushions on glass slides and onion epidermal cell layers,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 and virulence of B.cinerea.In summary,this study used transcriptome sequencing technology to analyze and screen out a number of candidate genes related to sclerotial formation in B.cinerea.Some genes were knocked out,and the biological phenotype of each gene knockout transformant was preliminarily studied.The functions of subtilisin-like serine protease encoded genes Bcser1 and Bcser2 in B.cinerea were studied in detail.We found that Bcser2 is essential for the sclerotial development and infection of B.cinerea.This study not only provides a new theoretical basis for further analysis of the molecular mechanism of sclerotial development of B.cinerea,but also offers new clues for the control of grey mould disease.