Biological Function Of Chitin Deacetylase Family Proteins In Beauveria Bassiana

Beauveria bassiana,as a typical entomogenous fungus,plays an important role in the field of biological control of agricultural and forestry pests.The first site and barrier of fungal interaction with the host are performed by the insect cuticle and fungal cell wall.Chitin is one of the main component of the cell wall in fungi and in the exoskeleton of insects,which provides the necessary rigidity and mechanical strength for fungal cell walls and the cuticle of arthropods.As an important chitin modifying enzyme,Although the roles of chitin deacetylase（CDA）in other pathogenic fungi have been studied,the deacetylation of chitin in the entomopathogenic fungus B.bassiana has not been reported.Therefore,we identified three members containing conserved domains of carbohydrate esterase 4（CE-4）family in B.bassiana,and constructed three gene knockout mutant（CDAs）strainsΔCDA1,ΔCDA2 andΔCDA3 and their gene complement strains（CDA1^C,CDA2^C and CDA3^C）.By comparing with the phenotypic difference of wild type（WT）strain of B.bassiana,the different functions of CDA family proteins in growth and development,vegetative growth and infection virulence of B.bassiana were revealed.1.Sequence similarity analysis showed that the sequence homology of CDA1,CDA2 and CDA3 was 21.8%and 19.7%,the sequence identity of CDA2 and CDA3 was33.8%,respectively.The 3D structure of the CDAs modeled by the I-TASSER server exhibited a（β/α）₈ barrel fold.2.Through the quantitative determination of various strains of conidial found that the destruction of three CDAs genes led to different results,and the conidial production ofΔCDA1 andΔCDA2 decreased by 25.81%to 47.68%compared with WT at day 5 to day 7,and the conidial production ofΔCDA3 was almost equal to that of the wild type.On day 3,the sporulation yield ofΔCDA1,ΔCDA2 andΔCDA3 was 20.23%,23.21%and27%lower than that of WT,respectively.In addition,50%conidial germination time(GT₅₀)ofΔCDA1,ΔCDA2 andΔCDA3 was 10.16%-12.66%longer than that of WT.The half-lethal time(LT₅₀)of conidia inΔCDA3 was 15.85%lower than that in WT.This indicates that CDAs affect spore quantity and quality of B.bassiana.3.The colony sizes ofΔCDA1 on SDAY,1/4 SDAY,CZA and CZA-N were similar to the colony sizes of the control strains.However,compared with those of WT,the colony sizes ofΔCDA1 decreased by 12.54%and 10.62%on CZA-C and CZA-CN,respectively.ΔCDA1 showed the same growth features as the control strains when the C source was replaced with N-Glc NAc.However,the colony size ofΔCDA1 was 25%less than that of WT on medium with chitin as the single C source.In addition,ΔCDA2 andΔCDA3 were not significantly different from WT in the above medium.These results suggest that CDA1 may perform a vital function in the utilization of C sources（especially chitin）.4.The absence of CDAs affects the virulence of B.bassiana to the host.The larvae of 3-day old Galleria mellonella were used as the host for virulence test.The infection virulence of B.bassiana against the insect host was determined by means of cuticle infection or hemocoel infection.In the cuticle infection test,the half-lethal time(LT₅₀)ofΔCDA1 andΔCDA3 was 29.33%and 23.34%longer than that in WT,respectively.All the gene disruption strains showed results similar to those of the control strains in the hemocoel infection pathway,the CDA family may not contribute to hemocoel infection virulence.However,the surface of the insects infected by the three gene knockout mutant strains were covered by less mycelium.The larvae were continue cultivated,the result was showed that the yield of newly produced conidia from the dead larvae which killed by the three gene deletion mutants was 68.45%,63.84%and 56.65%lower than that of WT,respectively,indicating that the absence of CDAs affected the secondary sporulation capacity of B.bassiana.5.The absence of CDAs affects the penetration and deacetylation of B.bassiana.In the cicada wing penetration experiment.Although all the strains were successfully penetrated the cicadas wing and continued to grow on the medium below the wing,The colony size of three gene deleted mutant strains were reduced by 47.76%、29.12%and33.29%,compared with the WT respectively.These results suggest that CDAs mutants have reduced penetration compared to WT.The enzyme activity ofΔCDA1 andΔCDA3decreased by 84.76%and 83.04%compared with WT,respectively,the enzyme activity ofΔCDA2 was not significantly different from that of WT,indicating that CDA1 and CDA3 may play a more important role in the deacetylation of chitin.In short,members of the CDA protein family plays an significant role in the growth,quantity and quality of spores,virulence,life history of B.bassiana.A novel theoretical basis for the study of biological functions of fungal CDA family proteins was provided in our study,and it also provided a new target for improving insecticidal efficacy of biological fungi control.