Calcineurin Catalytic A Subunit Of The Entomopathogenic Fungus Beauveria Bassiana Is Involved In Conidiation, Virulence And Characteristics Associated With Pathogenicity

Entomopathogenic fungal are important natural regulators of insect populations and have attracted much attention as biological control agents for insect pests. Understanding of the molecular mechanism of fungal pathogenesis is essential for commercial development and improvement of the biocontrol fungi.Calcineurin (CN) is a highly conserved Ser/Thr protein phosphatase, which forms a complex with Ca2+ and calmodulin and regulates a variety of biological processes. Calcineurin is considered the center of biological stress response regulatory protein, and involved in fungal spore and mycelium morphology, intracellular ion balance, cell wall synthesis and pathogenicity. And thus, calcineurin-mediated signaling pathways might provide a clue for understanding the molecular mechanism of biological process of fungal pathogens.Here we characterized a calcineurin catalytic subunit gene Bbcna using gene disruption strategy in the entomopathogenic fungus Beauveria bassiana, which has been widely used for biocontrol pest insect as fungal agents. The results showed that Bbcna is involved in regulation of conidiation, virulence and characteristics related to virulence.The major findings obtained are as follows:BbCNA affects colony morphology, conidiation and interaction of hyphae. Analysis of biological characteristics showed that development of aerial hypha of Bbcna disruption mutants was obviously thiner than the wild-type strain on agar plates and conidiation of B. bassiana was significantly decreased by disruption of Bbcna. Conidal yield of Bbcna mutants was decreased by 83-85% on CZM plate and 51-62% on 1/4SDAY plate, compared to the wild-type strain. Hyphae of Bbcna mutants tended to aggregate together to form pellets, suggesting that the calcineurin signal pathway play an important role in regulation of interaction of hyphae.BbCNA regulates sensitivity of B. bassiana to fungicide fludioxonil. Bbcna disruption mutants were more sensitive to than the wild-tipe strain. When exposed to the minimum inhibitory concentration of fludioxonil on B. bassiana (0.1μg/l), obvious inhibition zone was observed in plates inoculated Bbcna mutant. The result suggested that Bbcna is involved in regulation of sensitivity fungicide fludioxonil in B. bassiana.Disruption of BbCNA causes an evident reduction in virulence. Insect bioassay showed that pathogenicity of B. bassiana to Pieris brassicae larva was significantly decreased when Bbcna was disrupted. When inoculated topically host insect at a concentration of 1×107 spores/ml,7 days post inoculation the infection death rate of the insect for Bbcna mutant was reduced by 55.02-61.93%. When inoculated host insect by injecting 3μl conidia suspension directly into the hemocoel at a concentration of 1×107 spores/ml, no significant difference was observed in infection death rates between the wild-type and Bbcna mutant strains. Determination of hyphal bodies in insect hemocoel showed the reproduction number of the fungal cells for the Bbcna mutant was decreased by 37.70% compared to the wild-type strain at 48 h after injection. These results suggested that Bbcna is an important virulent determinant of B. bassiana.BbCNA is involved in conidial hydrophobicity, adhesion and appressorium formation rate. Conidia hydrophobicity of B. bassiana produced on CZM and CZP plates was decreased by 56-82% and 80-87%, respectively, when Bbcna was disrupted. Conidia adhension of Bbcna disruption mutant to insect cuticle was reduced by 17.70-22.36% compared to the wild-type strain. On cicada hind wings, Bbcna mutant form appressoria with normal motphology, whereas appressorium formation rate was decreased by 75.50-76.53%.