The Catalytic Subunit Of Calcineurin Bbcna Regulates Growth And Development, Cell Wall Integrity And Virulence In The Insect Fungal Pathogen Beauveria Bassiana

Calcineurin is known as the only Ser/Thr phosphatase protein which is regulated by Ca2+, consisting of a CNA catalytic subunit and a regulatory subunit CNB. The catalytic subunit of CNA is mainly involved in fungal spore formation and hyphal morphology, the regulation of intracellular cation balance as well as pathogenicity and synthesis of fungi cell wall. Previous cloning of calcineurin catalytic subunit from the entomopathogenic fungus Beauveria bassiana, found that the Bbcna had an effect on colony morphology, sensitive to fungicide fludioxonil. In addition, the disruption of Bbcna caused an obvious reduction in virulence, conidia hydrophobicity, adhesion to conidia surface and appressorium formation. However, we found that the Bbcna mutant is difficult to preserve, in which suggest that Bbcna maybe related to Beauveria bassiana vigor. Moreover, the mechanism that Bbcna regulate the strain growth and cell surface characteristics are not clear. Facing with these problems, we reconstructed the Bbcna destruction mutant, reply complementary, to further research the Bbcna strain viability, development and differentiation together with cell surface characteristics, virulence and possible mechanisms and the relationship between the structural integrity of the cell wall(CWI) signaling pathway Bbslt2and Bbcna was analyzed via transcriptome.The main results are as following:1. Bbcna affects growth and development. Disruption of Bbcna caused a reduction in colony growth, a significant decrease in conidia yield and conida viability. In1/4SDAY, PDA and CZM culture medium, the growth rate of ABbcna decreased by16.23%,44.12%and28.57%as compared to wild-type strain, respectively. In complete medium1/4SDAY and PDA culture medium, sporulation ABbcna were decreased by43.68%and51.89%than wild-type strain. Conidia germination showed that the destruction of Bbcna delay the spore germination rate, the LT50of conidia germination extended about2.5h than wild-type strain. With the extension of storage time, the activity decreased rapidly. In4℃and26℃for40d, the wild-type strain spore viability were90.35%and92.08%, while ABbcna decreased to33.32%and55.37%. The conidia of reactive oxygen species (ROS) detection results show extend the△Bbcna spores with storage time, intracellular ROS increase. Therefore. Bbcna may participate in the regulation of intracellular reactive oxygen species which influence the spore activity.2. Bbcna regulates cell wall integrity. Bbcna strain has a sensitivity to cell wall synthesis inhibitor Congo Red, the EC50of Congo Red for△Bbcna is2.819 mg/mL. significantly lower than wild-type strain in which EC50is4.642mg/mL. Through Transmission Electron Microscope, found that destruction of ABbcna affected the structure of cell wall. Cell wall components change so much, distruption of Bbcna leads to the total sugar content of cell wall β-1,3and β-1.6glucan increased. while the chitin was decreased significantly. This shows that. ABbcna regulate the cell wall, cell wall integrity.3. Inactivation of Bbcna affects Beauveria bassiana surface characteristics. Disruption of Bbcna resulted in a reduction in conidia hydrophobicity, with41.54%of decrease compared to the wild type. ABbcna conidia adhesion to hydrophobic surface is significant lower than wild-type strain, while in the hydrophilic surface was significant higher than wild-type strain. Inactivation of Bbcna led to a decrease in reaction of conidia with lectins ConA (glucan) and WGA (GlcNAc). while increased the binding ability to the GNL (α-mannose binding). The results demonstrated suggested that Bbcna contribute to conidia hydrophobicity, adhesion and cell surface epitopes.4. Disruption of Bbcna causes a decrease in virulence. Insect bioassay showed that pathogenicity of B.bassiana to Galleria mellonella was singnificantly decreased when Bbcna was disrupted.When inoculated topically host insect with inoculation and injection, the LT50of mutant strain is increased by19.87%and21.3%.5. Comparative gene expression redulated by Bbcna and Bbslt2. When sressed by Congo Red, compared with the wild-type strain, ABbcna has a total of603genes changed significantly in transcription level, including350upregulated genes and253down regulated genes; ABbslt2has a total of102genes changed significantly, including31upregulated genes and71down regulated genes. Compared with the wild-type strain,8genes upregulated both in△Bbslt2and△Bbcna,32genes downregulated, has a percentage of25.81%and45.07%in up regulated of△Bbslt2and down regulated genes. The common up-regulated and down regulated genes of these40differences, involved in cell wall synthesis, cell membrane metabolism, oxidative stress and other biological process. Therefore, the calcineurin signal pathway (Bbcna) and Bbslt2pathways exist obvious interaction in the regulation of Beauveria bassiana cell wall structural integrity process (crosstalking).