Variation In Bombyx Mori Immune Response Against Fungal Pathogen Beauveria Bassiana With Variability In Cell Wall β-1,3-glucan

Bombyx mori is an important model insect to explore the interaction mechanisms between insect and microorganism.Beauveria bassiana has been one of the most studied broad-spectrum entomopathogenic fungi since the development of insect pathogenic fungi reagent.Fungal cell wall is the main structure on the infection of host insects.The analysis of B.mori immune mechanism and B.bassiana cell wall components can provide key information for understanding the mechanism of fungi-hostinteraction.In this study,immune mechanism of B.mori against B.bassiana with different virulence and the effect of cell wall components of B.bassiana on its pathogenicity were analyzed,those results could provide theoretical basis and significance for improving the pathogenicity of entomopathogenic fungi and the immunity of insect with economic value such as B.mori.The results are as follows:1.Selection of B.bassiana strains with different virulence in the hemolymphThrough the growth rate determination,conidial yield capacity and cuticle penetration assays of 20 B.bassiana strains of Bb9 with strong pathogenicity,Bb20 with medium pathogenicity and Bb6 with weak pathogenicity were obtained.2.Transcriptome analysis of B.mori inner immune response against B.bassiana with different virulenceTo delineate the differences in gene expression from B.mori infected with different virulence B.bassiana strains,the genome-wide transcriptome analysis was performed with RNA-seq for B.mori infected with high,medium and low virulence strains Bb9,Bb20 and Bb6（represented by HI,MI and LI,sterile water as the control group C,respectively）.The results showed that B.mori regulated different genes to resist the infection of against B.bassiana strains with virulence variant.Compared to the control group,a large number of immunity-related genes were differential expressed in these groups.Furthermore,numerous immune-related genes were also differential expressed among the three infected silkworm by different B.bassiana strains,including a immune recognition protein BmβGRP4.3.Analysis of BmβGRP gene expression from B.mori To explore the expression profile of genes encoding peptidoglycan recognition proteins（βGRPs）in B.mori against different B.bassiana strains,the gene expression of BmβGRP1,BmβGRP2,BmβGRP3 and BmβGRP4 during infection stages was determined by q RT-PCR.The expression levels of BmβGRP1,BmβGRP2 and BmβGRP3 changed after being infected by the strain,but there was no obvious correlation between the change trend and the virulence of the strain.Importantly,BmβGRP4 were significantly differentially expressed among these samples at 12 h:BmβGRP4 was expressed at high levels with the low-virulence strain,at middle levels with the medium-virulence strain,at low levels with the high-virulence strain,which suggested that BmβGRP4 might participate in silkworm innate immune defense against different virulence B.bassiana strains at 12 h.4.Conidial cell wall composition of B.bassiana strains with virulence variantTo clarify the differences in cell wall components of different pathogenic B.bassiana,contents of the cell wall surface chitin,total chitin,α-1,6-mannan,total dextran,α-1,3-dextran,β-1,3-glucan were examined with immunofluorescent staining.The results showed that the cell wall components were significantly different among those B.bassiana strains.Importantly,β-1,3-glucan from Bb9-H was the least,from Bb20-M was medium,and from Bb6-L was the most,which was consistent with BmβGRP4 expression in B.mori at 12 h infection with those strains.5.Silkworm immune activation with BmβGRP4 recognition ofβ-1,3-glucanTo determine whether the amount ofβ-1,3-glucan induced the differences in BmβGRP4expression and thus activated the B.mori innate immune,the impact of addition ofβ-1,3-glucan and silencing of BmβGRP4 on B.bassiana pathogenicity was assessed.The expression of BmβGRP4 in B.mori was increased sharply with increasing amounts ofβ-1,3-glucan injection,and it was stably expressed until addition of 600 ngβ-1,3-glucan.Analysis of LT₅₀of B.mori showed injection ofβ-1,3-glucan together with B.bassiana strains could reduce the Bb9-H pathogenicity but not Bb20-M and Bb6-L,whereas pathogenicity enhanced by BmβGRP4 silencing for Bb20-M and Bb6-L but not Bb9-H.There was no significant differences among LT₅₀s of B.mori infected by different virulence B.bassiana together withβ-1,3-glucan addition or BmβGRP4 silencing.These results confirmed that B.bassiana pathogenicity was affected by cell wallβ-1,3-glucan contents,which could been recognized by BmβGRP4.