Function Analysis Of NRPSs From Beauveria Bassiana

Beauveria bassiana is an entomopathogenic fungus that can infect more than 200 species of insects from 6 Orders 15 Family.B.bassiana can produce several secondary metabolites,such as beauvericin and oosporein,to disorder metabolism and cause death after it invades insect body.Most studies focused on infection mechanism of B.bassiana,and only a few researches elucidated the biosynthesis and biological function of its secondary metabolites.Non-ribosomal peptides?NRP?metabolites are the main secondary metabolites of fungi.Genomic analysis showed that there are 21 nonribosomal peptide synthase?NRPS?genes in B.bassiana ARSEF 2860,but only 4 genes functions were reported,and the functions of the rest NRPS genes are still unknown.In order to study the functions of the NRPSs in ARSEF 2860,21 NRPS were systematically analyzed by bioinformatics method,and were divided into 3 groups: basic metabolism,pathogenicityand unknown functions,and 3 NRPS genes were identified to be involved in the infection of Plutella xylostella by gene knockout and function test.The detailed results are as follows:1?21 NRPS genes and 3 polyketide synthase-nonribosomal peptide synthase?PKS-NRPS?hybrid genes,were obtained from the ARSEF 2860 genome using secondary metabolite analysis tools such as antiSMASH and hmmsearch.The adenylation domains?A?of the 21 NRPSs were extracted for phylogenetic analysis.The NRPSs of B.bassiana was divided into three groups according their predictive functions: basic metabolism?7 NRPSs?,pathogenicity?12 NRPSs?and unknown functions?2 NRPSs?.2?The NRPSs expressions of ARSEF 2860 were different among laboratory growth condition and infection condition.Reverse transcription PCR is conducted and found BBA₀7611?pathogenicity?and BBA₀3671?pathogenicity?are activated,while transcription level of BBA₀3671?pathogenicity?increase during infection of Diamondback moth.Analysis of real-time PCR shows transcription level of 10 NRPS genes increase,including BBA₀7611,BBA₀3671 and BBA₀3671.These genes are very likely to involve infection.The result of gene fuction prediction is verified by RT-PCR and qRT-PCR,turning out the practicality of NRPS gene functional prediction using phylogeney.3?To verify the predicted function of some NRPSs in B.bassiana,4 NRPS genes were knocked out using Agrobacterium tumefaciens mediated transformation method.These genes include the non-canonical siderophore-synthetase PKS/NRPS hybrid BBA₀4028,the emerice llamide-synthetase-like NRPS BBA₀8222,ChNPS8-like NRPS BBA₀3671,and the B.bassiana specific NRPS BBA₀6727.The growths of wild type and mutants were observed similar when they were cultured on PDA plates,while the infection effects to P.xylostella larvae were much significant difference after 6 days of infection.The survival rates of P.xylostella larvae infected with BBA₀4028,BBA₀8222 and BBA₀3671 mutants were 31.9%,37.3% and 54.7%,respectively,while the survival rate of P.xylostella larvae with wild type infection was 10.6%.The deletion of BBA₀6727 gene did not significantly reduce the pathogenicity of B.bassiana to P.xylostella,and the survival rate of P.xylostella was 10.6%.This study analysis the function of NRPS of B.bassiana,and characterize the function via experiment.The effort builds a relationship between secondary metabolites and pathogenicity,making a theoretical and practical foundation for further clarify the mechanism of infection.