Functional Analysis Of C4-dicarboxylate Transporter MaDCT Genes In Metarhizium Acridum

The absorption and utilization of carbon source is the foundation for the successful colonization and expansion of pathogens in the host.When the glucose content is low,pathogens can use the gluconeogenesis pathway to synthesize glucose for metabolism,and the substrate of the gluconeogenesis pathway is mostly tetracarbon dicarboxylic acid.Tetracarbon dicarboxylic acids are important carbon sources and energy substances.The use of tetracarbon dicarboxylic acid in fungal cells is inseparable from the role of the C4-dicarboxylate transporter(DCT).However,the current research on the C4-dicarboxylate transporter is mainly focused on bacteria,and no report about DCT was found in pathogenic fungi.To explore the function of the DCT genesis very important for studying the growth and colonization of entomopathogenic fungi.In this study,the functions of the MaDCT genes were analyzed by disruption through homologous recombination in M.acridum.The main results are as follows:1.Bioinformatics analysis of MaDCT1and MaDCT2Primers were designed based on the MaDCT1 and MaDCT2 genomic sequences in the NCBI database,and the MaDCT1 and MaDCT2 genes were cloned.Sequence alignment and phylogenetic analysis showed that MaDCT1 had low similarity with MaDCT2.The MaDCT1(accession number XP?007807917)encodes a protein of 458amino acids with a calculated molecular weight of 50.36 kDa and an isoelectric point of6.71.The MaDCT2(accession number XP?007818602),encodes a protein of 365amino acids with a calculated molecular weight of 40.17 kDa and an isoelectric point of7.63.The MaDCT2 genomic DNA had two introns.2.MaDCT disruption and complementationIn order to analyze the functions of MaDCT1 and MaDCT2,a homologous recombination method was used to construct a knockout vector for each gene.The disruption vector was transformed into M.acridum mediated by Agrobacterium tumefaciens.Disruption mutants and complementation strains were verified by PCR analysis and Southern blotting.3.MaDCT affects growth and conidiation in M.acridumThe germination rate,conidiation pattern and sporulation in null mutants and wild type strains were observed under microscopy.Results showed that the?MaDCT1 and the??MaDCT had accelerated germination,but disruption of the MaDCT2 gene did not affect germination.On the nutrient-rich 1/4 SDAY medium,the conidiation was delayed and the mycelial spacing was longer in?MaDCT1 and??MaDCT,while the?MaDCT2 had earlier conidiation process compared to WT.On the microcycle conidiation medium SYA,the conidiation pattern of M.acridum was changed from microcycle conidiation to normal conidiation in the?MaDCT1 and the??MaDCT strains,while deletion of the MaDCT2 gene had no effect on microcycle conidiation process.The sporulation in?MaDCT1 was significantly decreased on 1/4 SDAY and SYA medium.4.MaDCT affects stress resistance of M.acridumThermal and UV-B stress tolerances were assayed in fungal strains.Results showed that disruption of the MaDCT1 gene increased the tolerance to UV-B and the osmotic stress agent sorbitol,and weakened the tolerance to the cell wall disruption agent CFW.Disruption of the MaDCT2 gene had no effect on tolerance of these stressors.The??MaDCT strain had similar phenotypes as?MaDCT1.Quantitative transcription analysis of UV repair-related genes revealed that the expression levels of superoxide dismutase genes sod1 and sod2,collagen gene Mcl2,and chitin synthase gene ChsV were significantly increased compared to that of the wild type.These genes might contributeto UV-B tolerance in?MaDCT1.5.MaDCT affected virulence in M.acridumThe virulence was determined by topical application and injection against the 5th instar of Locusta migratoria.Results showed that disruption of the MaDCT1 gene significantly increased virulence in the topical application bioassay,and the mean LT₅₀value was 8.2±0.15 d for WT and 7.5±0.2 d for?MaDCT1,a significant difference(P<0.05).However,the?MaDCT1 mutant and WT had similar virulence(P>0.05)when conidia were directly injected into the insect haemocoel.Disruption of the MaDCT2gene or double-disruption of two DCT genes had no effect on virulence.6.MaDCT1 transcriptome analysisA transcriptome analysis of the RNA samples of?MaDCT1 on SYA medium during conidiation time revealed that there were 319 up-regulated genes and 165down-regulated genes in?MaDCT1 strains.These genes are mainly related to conidiation,growth,stress resistance and virulence.It contains 19 genes related to conidiation,22 genes related to ultraviolet resistance,68 genes related to virulence,28genes related to carbon metabolism and 19 transcription factors.GO function analysis found the differentially expressed genes are mainly enriched in cell and membrane components,catalytic activity and binding.KEGG Pathway enrichment found that differentially expressed genes mainly focused on carbohydrate metabolism,amino acid metabolism,transport and catabolism,and signal transduction.It indicated that MaDCT1 regulates the M.acridum conidiation pattern shift by regulating the expression of genes related to cell metabolism and conidiation.In summary,the MaDCT genes play important roles in the growth,conidiation,stress resistance,virulence and conidiation pattern shift of M.acridum.