| Beauveria bassiana is an important entomopathogenic fungus,which the pathogenic process to insects can be divided into three stages: epidermis penetration,colonization and growth in the blood of insects,the hyphae re-penetration of the host surface,and reproduction from in vivo to in vitro.So far,a large number of related genes have been identified in each infection stage of B.bassiana,and the main goal is to improve the virulence of fungi and biological control.In the process of infection,B.bassiana can infect the host,whose range is wide,through the insect epidermis.However,B.bassiana isolated from a specific host shows significant differences in pathogenicity when infecting different host insects,the molecular mechanism of the pathogenicity difference and how to adapt to the host environment of different insects for B.bassiana are still unclear.The main purpose of this study is to find the genes related to the parasitic adaptive function of B.bassiana in the process of infecting different hosts.We analyzed the gene expression characteristics of B.bassiana in response to different hosts by comparative transcriptomics method,uncovered the molecular mechanism of different pathogenicity of the specific B.bassiana strain to different insect hosts in this study,and found some special and differential expression genes of B.bassiana adapting to different hosts,such as the allergen gene（Bb-f2,BBA05395）,Class III chitinase Chi A2 gene（Bbchi-17,BBA 05353）and other genes,the expression of allergen protein Bb-f2 of highly virulent strain（GXsk1011）isolated from Bombyx mori,in particular,was significantly up-regulated in response to the epidermis of other hosts.The biological functions and effects of the Bb-f2 gene werestudied by gene knockout and overexpression methods.The expression characteristics of pattern recognition receptors PRRS,Toll-like receptors TLR,cytoplasmic signal transduction factor and antimicrobial peptide genes in the Toll pathway of silkworm were detected by heterologous eukaryotic cells expressing Bb-f2 protein and injecting the protein into silkworms,the mechanism of Bb-f2 protein in the process of B.bassiana infecting insects were analyzed and explored.The main results are as follows:1.Pathogenicity of B.bassiana GXsk1011 to Bombyx mori,Helicoverpa armigera and Clanis bilineataWhen B.bassiana GXsk1011 was used to infect three lepidopteran insects,fifth instar of B.mori,H.armigera and C.bilineata at a concentration of 1 × 108 conidia /ml,the mortality rates of B.bassiana to B.mori,H.armigera and C.bilineata were 100 %,52.50 % and 8.88 % respectively;the LC50 of B.mori,H.armigera and C.bilineata were successively 1.25 × 106,4.67 × 107 and 5.25 × 1010 conidia /ml.The results showed that the virulence of B.bassiana to the originally isolated（susceptible）host B.mori was significantly different from that of the two non-originally isolated hosts.2.Differentially expressed genes in response of B.bassiana to three lepidopteran insectsTo reveal the reasons for the differences in pathogenicity,the transcriptome data of B.bassiana responding to three insect epidermis were analyzed.Due to the low biomass of B.bassiana in the early infection stage at 24 h,and to avoid the digestion of RNase in living tissues and the influence of other microorganisms,in vitro simulation infection test and RNA-seq were used,and the transcriptome data were registered by the laboratory in the NCBI database（registration number: SRR3036777,SRR 3043073 and SRR 3043097）.The analysis of transcriptome data showed that,through pairwise comparison,there were about 600 to 900 genes of up-regulated expression and 400 to 800 genes of down-regulated expression.Through the annotation of differential genes,the differentially expressed genes mainly involving the biological processes and pathways of metabolism,catalytic activity,transporter activity and localization of strains,some key virulence genes closely related to pathogenicity differences and gene expression patterns of strains responding to different host epidermis were identified.The analysis of the top 20 differentially expressed genes with the highest multiple of difference（Top 20）showed that the key factors involved in spore wall adhesion,cuticleprotein degradation and some growth genes were significantly up-regulated expression in the infection process when B.bassiana responded to the sensitive host B.mori,such as adhesion protein（Mad1）,trypsin-like protease（Pr2）,O-methyltransferase,family 3（BBA09875）,cell surface protein（BBA09174）and so on,besides the Top 20 genes,endothiapepsin-like aspartate protease gene（Bbenp L-1）,which can degrade the epidermis,was also up-regulated expression,due to the high expression of these virulence genes,the strain has high infectivity（100% mortality）to B.mori;however,when B.bassiana responded to the non-original hosts of H.armigera and C.bilineata,the genes significantly up-regulated were allergen protein major allergen Asp f 2-like protein（Bb-f2,BBA05395）,Class III chitinase Chi A2（Bbchi-17,BBA05353）,nonribosomal peptide synthase（BBA08424）and so on,therefore,the pathogenicity to H.armigera and C.bilineata were only about 52 % and 8 % respectively.Additionally,due to the significant differences in mortality between H.armigera and C.bilineata,the further analysis showed that the gene expression characteristics of the strain were mainly related to epidermis degradation and fungal colonization,in response to H.armigera,the expressions of many epidermis degradations and colonization genes were significantly up-regulated,while the expressions of adhesion gene（Mad1）,allergen protein（Bb-f2）,Class III chitinase（Bbchi-17）showed no significant difference in terms of the two aspects.In addition to the significant up-regulation of Bb-f2 expression in B.bassiana responding to non-original hosts,transcriptome analysis also showed that some other allergens related genes rasp f7 allergen and allergen V5 / tpx-1 family protein were also up-regulated（not among the top 20）,it is speculated that the high expressions of Bb-f2 and other genes in the strain may be the result of different host environment stress.This study revealed the molecular mechanism of pathogenicity difference of B.bassiana infection in different hosts through the genomic analysis of differentially expressed genes,and also showed that when a specific strain of B.bassiana infects different insect hosts,its metabolic response and infection strategy will change accordingly to adapt to the new host,and one of the key factors to the infection adaptability is to break through the chitin barrier of different insect epidermis and resist the immune pressure from different hosts.3.Quantitative Real-time PCR detection of Bb-f2 allergen gene in B.bassianaThe Bb-f2 gene was lowly expressed when B.bassiana responded to sensitive hostB.mori and was highly expressed when responded to non-original isolated hosts H.armigera and C.bilineata epidermis,to verify the expression pattern of Bb-f2 gene in other non-original hosts,four additional insects（two Lepidoptera insects,Galleria mellonella and Spodoptera litura,two Coleoptera insects,Tenebrio molitor and Zophobas atratus）were selected to detect the expression of Bb-f2 gene.The pathogenicity of strain GXsk1011 to Galleria mellonella,Spodoptera litura and Tenebrio molitor was different,and the mortality rates were 46.67%,50% and 26.67%,respectively,non-infected with Zophobas atratus.The q RT-PCR results showed that the expression of the Bb-f2 gene,compared with responding to B.mori epidermis,was upregulated when the strains responded to the epidermis of Spodoptera litura,H.armigera,Galleria mellonella,Tenebrio molitor,C.bilineata and Zophobas atratus,it was speculated that Bb-f2 may have a tendency of high expression when B.bassiana respond to non-original host,and high expression of Bb-f2 is a kind of immune response for the strain to better adapt to a new host environment.When the same strain of B.bassiana infects sensitive and non-original hosts,the expression level of Bb-f2 is different,and what is the expression pattern of Bb-f2 when different strains of B.bassiana infect the same host B.mori? This study discussed this situation through q RT-PCR,the Quantitative Real-time results of 5 B.bassiana strains（2 biocontrol strains,3 separated strains from different areas of B.mori,the order of LC50 from small to large was B.mori-separated strains GXsk1011 4.2×105,YNsk1106 4.8×105,SCsk1006 5.4×105 and biocontrol strains GXtr1010 8.9×107,GXtr1009 1.0×108 conidia /m L）showed that the expression of Bb-f2 gene was the lowest in B.bassiana GXsk1011 with the highest virulence to B.mori,and the highest in B.bassiana GXtr1009 with the lowest virulence to B.mori,the results also suggested that the expression of Bb-f2 gene was related to the immune pressure of the non-original hosts,which can be speculated that the high expression of Bb-f2 gene was beneficial to the protection and survival of the strains themselves.4.Functional research and analysis of Bb-f2 allergen gene of B.bassianaCloning of Bb-f2 gene and Bioinformatics analysis of the Bb-f2 gene by using online website and sequence analysis software,Bb-f2 gene encodes has 260 amino acids,which is a stable protein and contains 22 amino acids in the N-terminal signal peptide and has four N-glycosylation sites,and conserved domain analysis showed that Bb-f2 and Asp f2-like allergen protein have similar conserved domains,belonging toM35-like protein superfamily,however,which contains the conserved motif of HRxx H and generally has no protease activity.The analysis of the conserved domain of Bb-f2 showed that it has a putative zinc ion binding site,which may be speculated to have a zinc ion binding function.Through homologous recombination replacement and strong promoter insertion,the gene knockout（ΔBb-f2）and overexpression mutant（OE-Bb-f2）were successfully obtained.The constructed mutant was cultured on 1/4 Sabouraud’s medium,and the growth of the knockout and overexpression strains were observed and detected,the results showed that the knockout and overexpression of Bb-f2 had no significant effect on the colony growth rate of B.bassiana;overexpression of Bb-f2 gene could significantly increase the mycelia heat tolerance of B.bassiana,and the decrease rate of colony diameter was significantly lower than that of wild-type strain after heat stress for different periods,however,deletion of Bb-f2 gene could also increase the heat tolerance of mycelia,suggesting that deletion of Bb-f2 gene might stimulate other genes to enhance heat tolerance;the results of the sensitivity test for the mutant and wild strain to high permeability（1m Na Cl）,cell wall inhibitor（500 μg / ml Congo red）and metal ions（2 m M Zn2+）showed that: knockout strain Δ Bb-f2 was highly sensitive to Na Cl,Zn Cl2 and Congo red stress,but overexpression strain OE-Bb-f2 was not significantly different from wild strain in colony morphology and diameter,meaning,it was not sensitive.In the pathogenicity test,the pathogenicity of the mutant was tested by cuticle inoculation with B.mori（5th instar）and H.armigera（4th instar）,and the wild strain was used as a control,the overexpression of the Bb-f2 gene enhanced the virulence of the strain to B.mori and H.armigera,however,the knockout of the Bb-f2 gene also significantly enhanced the virulence of the strain to B.mori and H.armigera.Because the lethal rate of the strain to silkworm was 100 %,the virulence of the strain was compared by measuring LT50,the LT50 of overexpression strain OE-Bb-f2 was 4.0 ± 0.02 days,which was lower than the that of the wild type,but the LT50 of ΔBb-f2 strain to B.mori was 3.9 ± 0.02 days,which was also significantly shortened than the LT50 of wild type（4.5 ± 0.03 d）;when the concentration of wild strain,knockout strain and overexpression strain was 1 × 108 conidia /m L,the corrected mortality of H.armigera was 55.3%,67.68 % and 59.77 % respectively,the virulence of knockout strain and overexpression strain was higher than that of wild strain.5.Study on the mechanism of action of Bb-f2 allergen protein of B.bassianaIn this study,expression of Bb-f2 protein in heterologous eukaryotic cell and injecting the protein into B.mori was used to detect the effect on the immune response of B.mori related to Toll pathway,the mechanism of Bb-f2 protein in the process of B.bassiana infecting insects were analyzed and explored.The contents of zinc in the epidermis of B.mori,H.armigera and C.bilineata were determined by the energy spectrum analyzer,the contents of zinc in the epidermis of H.armigera（44.47 μg / g）> B.mori（30.09 μg / g）> C.bilineata（25.21 μg / g）was determined by inductively coupled plasma atomic emission spectrometry（ICP-AES）,which demonstrated that high and low expression levels of Bb-f2 were not positively correlated with the cuticle zinc element contents in three insects,it suggested that there may be other host environmental factors affecting the expression of Bb-f2.On the other hand,different concentrations of zinc ions were added to the basic salt medium of B.mori epidermis grown by B.bassiana to create a zinc stress environment,the expression changes of five zinc-related genes and four randomly selected genes were detected,the gene expressions were generally inhibited by Zn2+,at 1 μmol /L Zn2+ concentration,the expression of Bb-f2 gene and zinc finger protein（BBA 05776）of B.bassiana were simultaneously up-regulated,which showed a downward trend with the increase of zinc ion concentration,it is suggested that when at a low concentration of zinc stress,the high expression of Bb-f2 and zinc finger protein might be involved in zinc uptake and zinc metabolism.Through the expression of Bb-f2 protein in eukaryotic Pichia pastoris,together with in vitro zinc ion binding experiment,it was indicated that Bb-f2 has zinc-binding activity and was indicated that Bb-f2 involved in the fungal zinc ions metabolism,it is suggested that zinc can cause the differences of expression,but the differences are not directly determined by the zinc content.q RT-PCR results showed that the expression of zinc finger protein was significantly up-regulated in ΔBb-f2 knockout strain and down-regulated in OE-Bb-f2 overexpression strain.Zinc finger protein gene encodes circular E3 ubiquitin transferase,which determines the specific recognition of target proteins in ubiquitination modification,can degrade target proteins（such as denaturation,overexpression and a great many regulatory proteins）through the ubiquitination pathway.In the Bb-f2 gene knockout strains,the expression of zinc finger protein was significantly up-regulated,which can be speculated that zinc finger protein may play its biological function toregulate the strain to show stronger virulence,and the overexpression strain of Bb-f2 was mainly caused by Bb-f2 itself,besides,compared with the wild type strain,it also showed high pathogenicity to the hosts.By injecting Bb-f2 protein into the B.mori infected with B.bassiana and the B.mori immunized activation with inactivated spores of B.bassiana,compared with PBS injection and BSA injection,the results showed that Bb-f2 protein had a similar effect on the Toll signaling pathway of B.mori infected with B.bassiana and the B.mori immunized activation with inactivated spores of B.bassiana,Bb-f2 protein inhibited the expression of pattern recognition proteins Bmβgrp 2,Bmβgrp 4 and Toll-like receptor Bmtoll 7 in the B.mori,lead to the antimicrobial peptides Bmlebocin 1 and Bmlebocin 2 in the B.mori were significantly down-regulated,which beneficial to the survival and colonization of the strain.After the B.mori was infected with B.bassiana,the injection of Bb-f2 protein through simulation overexpression shortened the half lethal time of B.mori,which indicated that the virulence of strain was enhanced by high-level expression of Bb-f2 gene to adapt to the different host environment.In conclusion,the highly virulent B.bassiana strain GXsk1011 separated from B.mori showed low pathogenicity to H.armigera and C.bilineata,and the analysis of the differentially expressed genes showed that the expressions of typical virulence genes such as adhesion protein Mad1 and protease Pr2 were significantly down-regulated,but the expressions of allergen protein Bb-f2（BBA05395）and type III chitinase（BBA05353）were significantly up-regulated,and the expression of Bb-f2 was upregulated after the expression characteristics of Bb-f2 in the epidermis response of the other four insects were further detected;The study for the function of Bb-f2 gene showed that the gene was involved in heat tolerance of strains,sensitivity of chemical factors and enhancement of virulence;In order to enhance the adaptability of the strain to different hosts,the Bb-f2 protein could affect the Toll signaling pathway of B.mori and inhibit the expression of some antimicrobial peptides in the hosts.This study is of great significance to reveal the pathogenicity difference of B.bassiana infection to different hosts and the molecular mechanism of infection adaptability. |
