AreB Controls Conidial Germination And Insect Cuticle Penetration In Beauveria Bassiana

In the process of infection and colonization,microbial pathogens feed on their hosts and nutrition acquirement from host is an essential prerequisite for the onset and colonization of an infection by pathogenic microorganisms.B.bassiana can live as saprophytes,plant symbiotic or insect pathogenic fungus,so that it is always used as model fungus to elucidate the relationships between nutrient metabolism regulation and adaptation to niches.Our previous study proved that Are B,a nitrogen metabolism regulatory factor,is a crucial regulator of pathogenesis in B.bassiana.In this work,it aims to explore the mechanism of Are B in regulating the growth and pathogenicity of B.bassiana through gene truncation and complemention,transcriptome analysis and EMSA,combining Co-IP-MS and bioassay.The main results are as follows:1.Are B is a key factor regulateor in conidial polar growth,cuticle penetration and colonization in the insect hemoceolWe change the conidial suspension of inoculate G.mellonella larvae,the mortality caused by wild type(WT)and complemented mutant strain(ΔAre B~C)are both 100%,while the Are B-deleted mutant(ΔAre B)almost lost its virulence and only resulted in1.1%larval death via topical inoculation,and 2.2%larval death via injection.These results indicated that Are B is essential in cuticle penetration and colonization in insect hemoceol in B.bassiana.Deletion Are B impaired the fungal growth on complete medium in B.bassiana,suggesting that the growth defect ofΔAre B has no obvious correlation with nutrients.Further exploration indicated that conidial germination ofΔAre B is also decreased dramatically.Although all conidia are viability.There was no significant difference between WT andΔAre B in the prosess of of conidia swelling by water absorption.These results reveals that ablation of Are B affected the conidial polar growth,which might be one of the important reasons for the decreased virulence.2.Functional analysis of different transcripts of Are BTwo transcripts of Are B were isolated in B.bassiana,previously.In this study,it proved that Are B(a)and Are B(b)possess automatic-activating function to the reporter gene,which imply that both of them has transcriptional activation activity.Further analysis showed that Are B(a)or Are B(b)or their common sequence could restore defects ofΔAre B in nutrition assimilated and conidial germination and the ability of colonization in insect hemoceol,but not in insect cuticle penetration.It means that both Are B(a)and Are B(b)are required for the fungal full virulence,and the sequence shared by Are B(A)and Are B(b)lead to their functional redundancy in codial germination and growth.3.Subcellular localization analysis of Are BTo determine the subcellular localization of Are B,fused gene GFP::Are B and Are B::GFP controlled by its native promoter or constructive promoter Pb3 were constructed and transformed into Are B deletion mutant,respectively.Subcellular localization analysis shows that both of them are localizate in the nucleus of the germinating tube under differnt nutrition condition and the hypha body,this result indicate that neither GFP fused to C-terminus or N-terminus of Are B not affect its localization in nuclear under different nutrient conditions.4.The C-terminus of Are B regulate insect cuticle penetrationPhenotype analysis showed that Are B::GFP could restore the defects ofΔAre B in nutrition assimilation and conidia germination,while GFP::Are B couldn’t restore the defects ofΔAre B in non-preference nutrition assimilation.Bioassay analysis showed that both of them could restore the ability ofΔAre B to colonize insect hemoceol,but only GFP::Are B could fully restore the defects ofΔAre B in pathogenicity.We hypothesized that GFP fused to the C-terminus of Are B might lead to conformational changes.To verify this hypothesis,the smaller tag 13Myc was fused to the C-terminus of Are B and transformed intoΔAre B.Bioassay analysis showed Are B::13Myc showed better performance in restoring the ability of topical infection inΔAre B than Are B::GFP.Furthermore,deletion 150 bp or 75 bp of Are B 3’terminus did not affect growth and conidia germination,but impaired the virulence dramatically.Therefore,the C-terminus of Are B regulate insect cuticle penetration.5.Analysis of differentia expression gene regulated by Are BTo identify the target genes regulated by Are B,we analyzed the differentially expressed genes between WT,ΔAre B and Are B-GFP/ΔAre B under the condition of insect cuticle nutrients through RNA-Seq.It was found that expression of a series of genes encoding PR1 proteases,chitinases and lipases,which contributing to cuticle degradation and penetration,P450 and NRPS involved in secondary metabolism,Nir A family related to nitrogen metabolism were changed both in WT＿vs＿ΔAre B and WT＿vs＿Are B-GFP/ΔAre B.Most promoter regions of the target candidates containging1-8 GATA-binding sequence HGATAR.6.Isolation of Are B interacting proteinsIn this study,GFP tags and Co-IP were used to isolate the proteins interaction with Are B under condition of insect cuticle nutrients.The IP-MS results were predicted by the STRING protein interaction network database,and 16 interaction proteins assumed by Are B were obtained.The Nmr A(BBA＿06457)is one of the candidate protein.our data indicated thatΔNmr A and Are B::GFP/ΔAre B exist similar phenotypes in insect cuticle penetration,tolerance to rapamycin and hust nutrient assimilation.