Roles Of Two Secretory Ribonucleases In Virulence Of Beauveria Bassiana

Ribonucleases(RNases)are ubiquitous in organisms,and are involved in various biological processes such as DNA replication,transcription,RNA cleavege and recycling.RNases are divided into RNase A,RNas T1,and RNase T2 family,according to difference in the hydrolysis of RNA to 3'mononucleotides through 2',3'cyclic nucleotides.RNase A and RNase T1 are basic RNase,with specific hydrolysis of pyrimidine nucleotides and guanosine acids respectively,while RNase T2 is acidic RNase,with no base specificity.Among them,some ribonuclease belongs to secretory proteins,which are independent of intracellular RNA processing and participate in a series of cytological processes.RNase T2 family proteins are usually secreted into extracellular or located in intracellular lysosomes or vacuoles,and are involved in various biological processes,including nucleic acid clearance,nutrient utilization,oxidative stress response,cellular immunity,plant self-incompatibility,leaf senescence,etc.RNase T2 proteins of some viruses show selective cytotoxicity on the mammalian immune cells to interfer with host immune response.However,it is not clear whether other pathogens(bacteria,fungi,etc.)with secretory RNase T2 are involved in the interference of host immune response and pathogenicity.Fungal ribotoxins,mainly produced by filamentous fungi,are extracellular secreted RNases.Similar to RNase T1family proteins,they can specifically lyse guanosinic acid,but they have extra loops,and have a variety of cytotoxic and insecticidal activities.Gene damage of Rib in Beauveria bassiana resulted in attenuated fungal virulence during infection,so it is worth discussing whether overexpression of rib can enhance virulence.In analytic study of spore surface proteins of entomopathogenic fungus,Beauveria bassiana,found that there were two secretory RNase T2 proteins and a ribotoxin,named BbTrv,Bb RNT2 and Bbrib respectively.The discovery contributes to reveal relationship between secretory ribonucleases of pathogenic fungi and virulence.Here in this paper,we explore the role of RNase Trv(BbTrv)and Bbrib in growth and virulence in B.bassiana,through gene expression analysis,gene damage,overexpression and eukaryotic expression.The main results are as follows:1.Sequence structure and phylogenetic analysis of BbTrv and Bb RNT2Through genome search,it was found that there were only two secretory genes encoding RNase T2 family proteins in B.bassiana,BBA?00766 and BBA?00428.Both of them had signal peptide sequences at the N-terminal,two blocks of conserved amino acids(CAS?and CAS?)shared by T2 family proteins,and three histidine residues.BBA?00766 encoded 257 amino acids,with molecular weight of about 27.82 KDa,and isoelectric point of 4.60.The amino acid sequence was 67.7%similar to RNase Trv in Trichoderma viride,which was named BbTrv.BBA?00428 encoded 328 amino acids,with molecular weight of about 36.10 KDa,and isoelectric point of 6.32.The amino acid sequence was only 26.83%similar to RNase Trv,which was named Bb RNT2.Bioinformatics analysis showed that BbTrv and Bb RNT2 had no typical nuclear localization signal.BbTrv had two assumed N-glycosylation sites and two assumed O-glycosylation sites,while there were four N-glycosylation sites and seven O-glycosylation sites in Bb RNT2.Moreover,BbTrv also had B1 and B2 nucleotide binding domains(B1 and B2 nucleotide binding pocket).Phylogenetic tree analysis found that BbTrv and Bb RNT2 clustered into different branches,indicating that BbTrv and Bb RNT2 were different types of RNase T2 in evolution.2.Expression patterns of BbTrv and BbribExpression patterns of BbTrv and Bbrib were analyzed using q RT-PCR and lablling the promoter with GFP.The results showed that expression levels of BbTrv and Bbrib in mycelium morphology were significantly higher than conidia,blastospores and hyphal bodies,and were induced by nutrition starvation,hemolymph and oxidative stress.Therefore,BbTrv and Bbrib may be involved in the nutritional utilization and oxidative stress.3.BbTrv can be secreted into extracellular substrateDistribution of BbTrv was detected by constitution-type expression fusion GFP tag,and it was found that BbTrv with removal of signal peptide was distributed in the cytoplasm,while fusion protein containing signal peptide was observed to be fluorescent only on surface of aerial hypha and conidia,while no fluorescence was observed on surface of liquid hyphae,blastospore and hyphal body.It suggested that BbTrv was secreted into extracellular matrix.Western blotting(GFP antibody)detection revealed presence of a fusion protein of 63 k Da and a protein with a molecular weight of 40 k Da in the culture medium,suggesting that the protein was a fusion protein with different degrees of degradation.Thus,BbTrv can be distributed on cell surface and secreted into extracellular matrix.4.BbTrv has typical N-glycosylation sites and RNA hydrolysis activitiesBbTrv was successfully expressed by Pichia pastoris in vitro.The analysis SDS-PAGE and Western blotting revealed that molecular weight of BbTrv was about37 k Da,which was greater than theoretical molecular weight of 27.82 k Da.After the treatment with N-glycosylase,there was a protein migration band of about 30 k Da,which confirmed existence of N-glycosylation modification of BbTrv.The detection of enzyme activity showed that BbTrv had activity of degrading RNA,but only weakly degrade single-stranded DNA,and not double-stranded DNA.The results further proved that BbTrv had ribonuclease activity.5.Overexpression of BbTrv and Bbrib affect development and oxidative stress responseIn order to explore role of BbTrv in growth of strains,gene destruction,recovery complementation and overexpression strains were obtained respectively.The results showed that gene damage did not affect growth of the strain,while overexpression significantly increased growth rate.However,overexpression of gene resulted in abnormal structure of sporulation,and yeild of conidia and blastospore was significantly reduced.Further testing revealed that overexpression of BbTrv significantly down-regulated sporulation-associated genes.Thus,BbTrv is involved in nutrient utilization,regulates transcription of sporulation related genes,and affects sporulation.There was no significant difference in response of?BbTrv to oxidative stress(H₂O₂ and MND),while oxidative tolerance of overexpressed BbTrv strain was significantly enhanced.BbTrv was found to have activity of degrading H₂O₂ in vitro.The results showed that BbTrv had activity of decomposing reactive oxygen species and was involved in oxidative stress response.Overexpression of Bbrib also increased growth rate and tolerance to oxidative stress,but weakened sporulation ability.6.BbTrv and Bbrib are associated with virulence and escape from host immune responseThe bioassay results showed that gene damage of BbTrv did not affect virulence,while the ability of overexpression strain to penetrate insect cuticle was obviously delayed and virulence was reduced.However,after injection of conidia,it was found that although proliferation rate of overexpressed strain in the insect was obviously lagging behind that of wild type,it could cause rapid death of insect.When recombinant protein expressed by yeast was injected with wild strain,this also promoted rapid insect death.The number of hemocytes inoculated with OE-BbTrv was significantly lower than that infected with WT,suggesting that BbTrv was cytotoxic to insect hemocytes.Enzyme activity of phenoloxidase in insects injected with OE-BbTrv was significantly reduced,and the level of reactive oxygen species(H₂O₂)was significantly lower than that of the inoculated wild type.Recombinant protein was injected into insect bodies,and it was found that BbTrv caused significant agglutination of insect blood cells and reduced the number of hemocytes.Therefore,BbTrv secreted by B.bassiana is toxic to insect hemocytes and inhibits host immune response.The microinjection of overexpressed Bbrib strain into insect bodies also led to rapid death of insect,which was accompanied by a significant reduction in the activity of insect phenoloxidase and reactive oxygen species(H₂O₂).The results showed that overexpression of Bbrib could improve virulence of the strain.The results of this study showed that BbTrv,a secretory RNase T2 family protein,affected sporulation of B.bassiana,participated in oxidative stress reaction,had cytotoxicity,interfered with or destroyed immune response of insects,and affected virulence.Overexpression of ribotoxin Bbrib interfered with insect immune response and enhanced virulence.