Functional Analysis Of The Gβ-like Protein Bcgbl1 In Botrytis Cinerea

Botrytis cinerea is considered as an efficient and main pathogen which causing plant grey mould in a wide variety of plant species,leading great economic loss.This pathogen infects plants by producing infection structures such as infection cushions and appressorium-like structures.However,the molecular mechanism of infection structures formation and development is still not clear.Here,we screened the T-DNA insertion mutant library of B.cinerea strain RoseBC-3,achieving an infection cushions formation deficient mutant AT19,which served as a clue to explore the mechanism of infection cushions formation and pathogenicity of B.cinerea.The main results are as follows:1.Achieved the Gβ-like gene Bcgbl1 deficient mutant AT19 from T-DNA muant library.Through the virulence test on oilseed rape leaves,we obtained seven virulence deficient T-DNA mutants.Mutant AT19 was defect in infection cushions formation.In comparison with the parent strain RoseBC-3,AT19 showed the reduced mycelium growth rate and conidiation,more sensitive to hypersalt and hyperosmotic and oxidative stress.Moreover,AT19 was nonpathogenic on tobacco leaves.Southern blot and hiTAIL-PCR showed that the single-copy T-DNA insertion in the promoter region suppressed the expression of Gβ-like protein encoding gene Bcgbl1 in AT19.2.Deletion of Bcgbl1 caused mycelium growth,sclerotia and conidia development,virulence defects of B.cinerea.We knocked out Bcgbl1 gene in B.cinerea strain B05.10.The phenotype of theΔBcgbl1 was similar to that of AT19.Deletion of Bcgbl1 severely impaired mycelium radial growth,sclerotia formation,conidiation but not conidial germination.Meanwhile,conidia size changed from circular or oblonged to long-oblonged.In addition,ΔBcgbl1 displayed more sensitive to hypersalt,cell wall stress and oxidative stresss.We found thatΔBcgbl1could not form infection structures on onion epidermis,failed to penetrate the unwounded tobacco leaves,but could cause small lesions on wounded tobacco leaves and the lesion diameter ofΔBcgbl1 was just three tenths of that of the WT.3.Deletion of Bcgbl1 reduced phosphorylation level of MAPK（Bmp1 and Bmp3）thus blocked infection structures formation inΔBcgbl1.Exogenous cAMP could not rescue the deficient in colony morphology and infection cushions formation ofΔBcgbl1.There was no significant difference between the WT andΔBcgbl1 in intracellular cAMP levels.Yeast two-hybrid assay showed that Bcgbl1 did not directly interact with cAMP-dependent signaling components.However,Western bolt analysis showed the phosphorylation level of Bmp1 and Bmp3 were both decreased inΔBcgbl1.And Bcgbl1 could directly interact with the Bmp3 upstream MAPKKK BcBck1and the scaffold protein BcSte50 of BcSte11-BcSte7-Bmp1.Moreover,BcSte50 could directly interact with BcSte11 and BcBck1.These results indicated that Bcgbl1 regulated infection structures formation and colonization via regulating the phosphorylation level of Bmp1 and Bmp3 in B.cinerea.4.Tanscriptomic profiling revealed the regulatory network of infection cushions formation mediated by Bcgbl1 in B.cinerea.Under hydrophobic surface induction,3001 genes were up-regulated and 1695 genes were down-regulated in the WT compared with expression in the WT grown on PDA.There are 3668 candidate differential expression genes（DEGs）involved in infection cushions formation dependent on Bcgbl1,including 2499 genes up-regulated and 1169 genes down-regulated.GO enrich and KEGG pathway analysis indicated that these DEGs were mainly enriched at stimulus response,substance synthesis and metabolism,energy supply and transporter activity.These results indicated that infection cushions formation and development need lots of substances and energy,which may be seriously prevented inΔBcgbl1 and then led to infection structures formation defects.We screened the putative Bcgbl1-regulated genes based on their functions previously reported and identified eleven genes that were previousely proved to be involed in virulence.Consistent with RNA-seq analysis,qRT-PCR showed that these genes were significantly down-regulated in theΔBcgbl1 mutant compared with that in the WT.5.Function of three hydrophobic surface binding proteins encoding genes（BcHsbA1,BcHsbA2 and BcHsbA3）was determined.Due to the decreased adhesion on plant surface ofΔBcgbl1 knockout mutants,three hydrophobic surface binding protein genes（BcHsbA1,BcHsbA2 and BcHsbA3）were screened from the transcriptome data.The expression of these three genes inΔBcgbl1 was significantly lower than that in WT,and the promoter regions contained the binding site of the transcription factor Ste12,which is the downstream target of Bmp1.Amino acid sequence analysis showed that all the three proteins contained the singal peptide and hydrophobic surface binding protein A motif.Deletion of these three genes had no significant effects on mycelium growth,sclerotia formation,conidia production,colony morphology and infection cushions formation.However,the virulence ofΔBcHsbA2 andΔBcHsbA3 were significantly reduced.Additionally,in order to clarify the difference between Gβ-like protein Bcgbl1 and Gβprotein Bcgb1 in development and virulence of B.cinerea,we also knocked out Bcgb1 gene.TheΔBcgb1 mutants showed defects in mycelial growth,more aerial hyphae,lack of conidia and sclerotia formation,increased sensitivity to H₂O₂ and significantly increased intracellular cAMP levels.ΔBcgb1 performed significantly reduced virulence on tobacco leaves.Meanwhile,ΔBcgb1 delayed in infection cushions formation,resulted in the decrease of penetration efficiency and virulence.Yeast two-hybrid assay showed that Bcgb1 directly interacts with BcSte11,BcBck1,BcMkk1 and BcSte50.Phosphorylation level of Bmp1 and Bmp3 were both markedly increased inΔBcgb1.Moreover,qRT-PCR result showed that Bcgas2,the downstream target gene of Bmp1 and BcSte12,was remarkably up-regulated inΔBcgb1.These results suggested that Gβprotein Bcgb1 was involved in regulation of the development and virulence via both cAMP and MAPK（Bmp1and Bmp3）signaling in B.cinerea.In conclusion,this study demonstrated that the Gβ-like gene Bcgbl1 plays important roles in mycelium growth,sclerotia and condia development,and it regulates infection structures formation and colonization of B.cinerea via regulating the phosphorylation level of MAPK（Bmp1 and Bmp3）.The relationship between Bcgbl1 and Gβsubunit Bcgb1 was revealed.This study advanced our understanding about the mechanism of development,infection structures formation and virulence in B.cinerea,provided potential strategy for control of grey mould.