Role Of Two Zn2Cys6 Transcription Factors In Colony Growth And Stress Tolerance Of Beauveria Bassiana

Entomopathogenic fungi are one of the important factors in regulating insect population in nature and have been developed as biocontrol agents.These fungi possess several advantages as biopesticides,such as broad host range,easy cultivation and low risk of resistance development.However,slow death rate of pests and unstable control effect arose when fungal pesticides are used in field,which may be resulted from the effects of environmental factors and various host immune reactions.In the infection process of entomopathogenic fungi,environmental factors,e.g.,temperature,humidity and ultraviolet radiation,greatly affected fungal survival and germination on insect cuticle.Therefore,the tolerance of insect pathogenic fungi to these environmental factors determines their biocontrol efficiency.Studies have found that zinc finger proteins play a vital role in various biological processes such as fungal growth and stress tolerance.It has been shown that the global regulator LaeA regulates mycelial growth and conidial development of Beauveria bassiana in insects.In the LaeA deletion mutant,two Zn2Cys6transcription factors?designated Bbstr1 and Bbstr2?were expressed at a high level compared to wild type strain by transcriptome analysis.To analyze the functions of the two genes in colony growth and stress-resistance of B.bassiana,we constructed their deletion mutants,and analyzed their phenotypic differences in growth,development,and stress-resistance.The main results are as follows:1.Sequence analysis of Bbstr1 and Bbstr2The open reading frame of Bbstr1 is 2250 bp,encoding 749 amino acids.The open reading frame of Bbstr2 is 2013 bp,encoding 670 amino acids.Bbstr1 and Bbstr2 contain a conserved Zn?2?-Cys?6?domain at the C-terminus,which are conserved in other filamentous fungi.To analyze the expression pattern of Bbstr1 and Bbstr2,their promoters are fused with the encoding sequence of green fluorescence protein eGFP and the constructions were transformed into B.bassiana?designated PBbstr1::eGFP and PBbstr2::eGFP?.The green fluorescent signals were observed in both PBbstr1::eGFP and PBbstr2::eGFP cultured in PDB,SDB or CZM media.The PBbstr1::eGFP fluorescence signal was also observed in the hyphal body.2.Functional analysis of Bbstr1 and Bbstr2 in B.bassianaTo study the functions of Bbstr1 and Bbstr2,we constructed knockout strains,overexpression strains and complementation mutant of Bbstr1 and Bbstr2,respectively.The functional analysis is as follows:1)Growth on various nitrogen sources.The colony growth of?Bbstr1 was slightly slower than wild-type strain?WT?in PDA,1/2 SDAY,CZM,tryptone,?NH₄?₂SO₄,NH₄NO₃ media.However,the growth of?Bbstr1 was significantly slower than WT in NaNO₂ and Urea media.While,the growth of?Bbstr2 in these nitrogen media was significantly slower than WT.These results suggested that Bbstr1 and Bbstr2 could regulate nitrogen utilization and metabolism of B.bassiana.2)Bbstr1 and Bbstr2 affect conidial resistance to H₂O₂ and Congo red.In the H₂O₂ medium,the growth of?Bbstr1 was faster than WT,and the edge of mycelium was denser than WT.The growth inhibition rate of?Bbstr1 mutant significantly reduced by25%.In Congo red medium,the growth of?Bbstr1 was significantly slower and the growth inhibition rate increased by 49%.This showed that deletion of Bbstr1 increased the tolerance of conidia to H₂O₂,but reduced the tolerance to Congo Red.Nevertheless,in the H₂O₂ medium and Congo red,the growth of?Bbstr2 strain was significantly slower and growth inhibition rates were 38%and 10%higher than those in the wild type,respectively.The result showed that Bbstr2 could regulate the tolerance of conidia to H₂O₂ and Congo red stress.3)Bbstr1 and Bbstr2 affect the virulence of B.bassiana.We examined the role of Bbstr1 and Bbstr2 in B.bassiana during infection of the 3rd-instar larvae of G.mellonella.Compared to wild type,deletion of Bbstr1 or Bbstr2 increased fungal virulence,decreasing mean lethal time(LT₅₀)by 12.87%and 12.32%,respectively.4)Bbstr1 and Bbstr2 influenced the conidial production of B.bassiana.Compared to wild type,knockout of Bbstr1 in B.bassiana reduced the conidial production on CZM,PDA and 1/4SDAY,by 21.6%,16.2%,and 25.6%,respectively.Additionally,deletion of Bbstr2 also led to the reduction in conidia production.These results indicated the two zinc finger protein,Bbstr1 and Bbstr2,play crucial roles in colony growth,conidiation,virulence,and stress responses of B.bassiana.