The Roles Of Two Target Genes(Bas2a/b) Of Bbste12 In The Fungal Pathogen Beauveria Bassiana-host Insect Interaction

Fungal pathogens are sensed by host insects during early stage of infection and initiate immune defense response.However,little is known about the mechanism that fungal pathogens evade the early immune defense response.Our previous study demonstrated that Fus3/Kss1 MAPK（Bbmpk1）and Ste12-like（Bbste12）are crucial factors that regulate the penetration of insect cuticle in the insect fungal pathogen Beauveria bassiana,suggesting an important clue to reveal the regulatory mechanism of penetration of pathogenic fungus into insect cuticle.The target genes mediated by Bbmpk1-Bbste12 during penetration of cuticle were identified using comparative transcriptomics,in order to understant the molecular basis of Bbmpk1-Bbste12regulating penetration of cuticle.The proteins which encoded by the two target genes BBA＿02608（BbBas2a）and BBA＿08405（BbBas2b）were homologous to Bas2（Biotrophy-associated secreted proteins 2）of plant pathogenic fungi.Bas-like proteins are highly expressed in the early stage of Magnaporthe oryzae and Colletorichum gloeosporioides infection by report,which is involved in growth of infected hypha and pathogenicity,but the underlying mechanism is not clear.In this study,the two BAS2 homologous proteins,named BbBas2a and BbBas2b,were characterized in B.bassiana during infection process by adoption of a series of approaches,including gene expression patterns during fungal infection,gene knockout and overexpression,as well as in vitro expression technology.The main results are as follows:1.BbBas2a and BbBas2b are the target genes of Bbste12Bioinformatics analysis showed that the promoter sequences of BbBas2a and BbBas2b contained typical Ste12 binding sites.Yeast one-hybrid assays confirmed that BbBas2a and BbBas2b were target genes of Bbste12.The amino acid sequence similarity between BbBas2a and BbBas2b was 80.82%.Searching the fungal genome revealed BAS2 homologous protein mainly exists in filamentous ascomycetes.Phylogenetic tree analysis showed that both BbBas2a and BbBas2b were clustered into a clade with the homologous proteins of Cordyceps,Nematodes and Torrubiella.2.Expression of BbBas2a and BbBas2b in the process of penetrating the insect cuticleTo investigate expression patterns,GFP gene was fused to 3’-terminus or controlled by the gene promoters.Insects were sectioned during fungal infection to examine GFP signals.The results indicated that the BbBas2a/b were highly expressed in fungal cells during penetration of the insect cuticle,while no obvious GFP fluorescence was detected in fungal cells,hyphal bodies,in insect haemodocoele.These results suggested that the two Bas2 secreted proteins mightly participate in penetration of insect cuticle.3.BbBas2a and BbBas2b are involved in the process of pathogens penetrating the insect cuticleIn order to reveal roles of BbBas2a/b in penetration of the insect cuticle,gene disruption and overespression strains,as well as strain overespressing two genes,were constructed.The results showed that fungal growth and development were not affected by disruption and overexpression of single and double genes.The typical cuticle inoculation bioassay indicated that the either disruption BbBas2a or BbBas2b resulted in a significantly decrease in virulence reduced,while overexpression of single gene or two gene strain significantly increased fungal virulence.The virulence of the strain overexpressing two genes(BbBas2a/b ^OE)was significantly higher than thse strains overexpressing single gene,suggesting synergistic effects of BbBas2a and BbBas2b in fungal pathogenicity.It was noticed that lethal insect caused by BbBas2b ^OEwas faster that those by BbBas2a ^OE,suggesting that contribution of BbBas2b to fungal virulence was more than BbBas2a.Intrahemocoel injection（avoiding the penetration of the insect cuticle）bioassay showed that no obvious difference in lethal of of the insect in the two gene knockout mutants,BbBas2a overexpression strain and the parental strain.However,the lethal of the insect caused by BbBas2b ^OEor BbBas2a/b ^OEwas significantly higher than the wild type strain-innoculated insect,in which obviously melanization reaction was seen,accompaning with inincreased phenol oxidase activities.Thus,we speculate that the lethal effect of BbBas2a/b ^OEstrain might be due to overexpression of BbBas2b.These data suggested that BbBas2a was only involved in penetrating insect cuticle,while BbBas2b either participated in penetration of insect cuticle or acted in the host intrahemocoel.4.BbBas2a and BbBas2b interfere with host insect immune responseTo reveal roles of the two proteins during fungal infection,the phenoloxidase（PO）enzyme activities were assayed after inoculation by spraying conidia.The results showed that inoculation of BbBas2a ^OE,BbBas2b ^OEor BbBas2a/b ^OEsignificantly activated the phenoloxidase activity,while no obvious difference in PO activities betweenΔBbBas2a orΔBbBas2b-inoculated insects and wild-type treatment.These data suggested that the overexpression strains interfere with insect immune response.After hyphae penetrated the insect cuticle into the blood cavity,the emergence and proliferation rate of single gene and two gene overexpression strains were significantly faster than wild strains,while the proliferation rate of gene knockout strains was significantly lower than wild strains.The changes in the number of insects hemocytes showed the opposite trend with the changes in insect cells.This indicated that overexpression of BbBas2a,BbBas2a and BbBas2a/b ^OEpromoted penetration of insect cuticle,and interfered with the immune response in insects.Overall,our results demonstrated that two biotrophic-associated secreted protein2（BbBas2a and BbBas2b）were involved in cuticle penetration via interfering with host immune response.