The Roles Of BCNA In Entomppathogenic Fungus, Beauveria Bassiana

Entomopathogenic fungi are important insect mortality agents that have been intensively investigated for application in insect pest control. However, compared to chemical insecticides, fungal agents kill insect slowly and are inconsistent in the field, which has often discouraged the use of fungal formulations in insect pest control. And thus, it is necessary to understand the the molecular mechanism of fungal pathogenicity to explore more effective means to facilitating fungal action at acceptable inoculum levels improvement of pathogens.Calcineurin is a Ca²⁺/Calmodulin-activated protein Phosphatase that is conserved in eukaryotes from yeast to human. Recently, it was demonstrated that calcineurin is as a central regulatory protein in responses to abiotic stress and involved in the fungal colony morphology, cation homeostasis, cell wall synthesis, and pathogenicity, suggesting that the calcineurin-mediated pathway is an important clue to understand the molecular mechanism of corresponding biological process in fungal species. However, the roles of calcineurin in entomopathogenic fungi are still a mystery.To elucidate the roles of calcineurin in the entomopathogenic fungi, a calcineurin catalytic subunit (CNA) encoding gene, designated as BCNA, was cloned from the insect fungal pathogen Beauveria bassiana which is one of the most important entomopathogenic fungi. The roles of BCNA gene in pathogenesis and development of the fungal pathogen were investigated by overexpression and RNA interference technique.1. Cloning and characterization of BCNA geneBased on the conserved amino acid sequence of several filamentous fungus CNAs, a CNA homologous gene, designated as BCNA, was obtained from B. bassiana by degenerate PCR and YADE approachs, and the complete cDNA sequence was cloned using 3'RACE. Sequence analysis demonstrated that the BCNA contained 3 introns, encoding a protein of 525 amino acids with calculated Mr of 60 kDa and pI of 6.04. BLAST analysis indicated that the amino acid sequence of BCNA shared 87%, 87%, 85% and 82% similarity to CNA from Fusarium graminearum, Neurospora crassa, Magnaporthe grisea and Aspergillus nidulans, respectively. BCNA has also been identified as the conserved calcineurin B binding domain, the calmodulin-binding domain, and the autoinhibitory domain. Southern analysis revealed a single copy of BCNA existing in the B. bassiana genome.2. Expression pattern of BCNATo determine the expression pattern of BCNA during growing under different condition, real-time RT-PCR was used to measure the relative expression levels of BCNA in B. bassiana. The result showed that the relative expression level of BCNA was increased by exposure strain to high temperature environment (32℃), high salt stress (0.5 M NaCl), and insect cuticle, suggesting that BCNA was involved in resoponse to acute abiotic stress and pathogenicity in B. bassiana.3. Function analysis of BCNAIn order to invesigate the roles of BCNA in B. bassiana, an antisense vector directed to the BCNA mRNA was introduced in the wild-type strain to deplete BCNA transcript levels. In addition, the transformant overexpressing BCNA was generated by introducing BCNA under the control of fungal constitutive promoter PgpdA in the wild-type strain. Then the biological characteristcs of BCNA overexpression transformants and antisense transformants were invesigated. The main results are as followings:Effect of overexpression of BCNA and transcript depletion of the gene on growth of B. bassiana. The biological characteristics analysis showed that no distinct difference in conidial yield and conidial germination between the wild-type strains and the BCNA antisense or overexpression transformants. Furthermore, under the conidition of high salt (0.8 M NaCl), acidity (pH 5.0), alkalescence (pH 9.0) and high temperature (32℃), there were no distinct difference between wild-type strain and the two transformants. However, when exposed strain to oxidative stress (0.05 % H₂O₂), growth of the transformant overexpressing BCNA was markedly slower than wild-type and BCNA antisense strain, suggesting overexpressing BCNA led to an increase in sensitivity to oxidative stress in B. bassiana.BCNA regulated the sensitivity to fungicide fludioxonil in B. bassiana. Compared to the wild-type strain and the strain overexpressing BCNA, BCNA antisense strain showed a significant resistance to fungicide fludioxonil, which suggested that calcineurin was involved in regulation of sensitivity to fludioxonil in B. bassiana.BCNA was involved in pathogenicity in B. bassiana. The results of bioassay showed that virulence of 5. bassiana to aphids Myzus Persicae was decreased by depletion of BCNA transcript. At a concentration of 1×10⁷ conidia/ml, LT50 of BCNA antisense strain against the aphids was prolonged 7.4 hr compared to the wild-type strain. Whereas, there was no obvious difference between wild-type strain and BCNA overexpression strain in LT50. The result suggested that BCNA is involved in virulence of B. bassiana.BCNA influenced expression of cuticle-degerading enzyme gene and hydrophobin gene in B. bassiana. The result of RT-PCR showed that expressions of cuticle-degrading protease encoding gene Pr1 and hydrophobin encoding gene Hyd2 were reduced in BCNA antisense strain compared to the wild-type strain, suggesting that BCNA partially regulate expression of Pr1 and Hyd2.Taken together, the results demonstrated that BCNA play important roles in the pathogencity, adaptation to oxidative stress, sensitivity to fludioxonil and expression Pr1 and Hyd2.