| Entomopathogenic fungi, taking conidium as its main effective insecticidal fungi pesticide unit, have great potential in developing pesticides. To promote the application of insecticidal fungus pesticide, it is quite meaningful to study how to control the entomopathogenic fungi spore production as well as to improve the quality. brlA encodes a C2H2 zinc finger transcription factor, plays a central coordination effect in filamentous fungal spores and hypha growth. brlA-abaA-wet A center pathway controls the expression of specific sporation genes. Like disruption of brlA in Aspergillus nidulans, deficiency of brlA blocked the conidiation. However, the function of Metarhizium acridum brlA gene has not been studied yet. In this work, we identified the M. acridum brlA in conidiation regulation and pathogenicity, and the regulatory mechanisms were elucidated. The main results were as follows:1. Based on the genomic sequence and genome sequence database library of M. acridum, MabrlA full-length cDNA sequence was cloned and the knockout vector was constructed. Building disrupted transformant and complementary transformant of MabrlA were generated and verified by RT-qPCR and Southern blot.2. MabrlA affects the conidiation, stress sensitivity and virulence in M. acridum. Deletion of MabrlA completely blocked asexual development in M. acridum, resulting in white colonies without green conidia formation. The experiment of stress sensitivity showed that MabrlA regulates the thermo- and UV- tolerances of conidia in M. acridum. After heat exposure at 45°C or exposure to UV-B for 2-6 h, the germination rates of conidia declined with exposure time and the conidia germination rates of the mutants appeared to be significantly reduced for each succeeding 2-hour interval when compared with the wild-type strain, while the WT LT50 and CP were conspicuously higher than ?MabrlA.3. We hypothesize a central regulatory pathway involved in MabrlA and MawetA exists in M. acridum. Based on our results, we propose a model of a basal conidiation regulatory pathway: fluG-flbs pathway and Fad A(G?) pathway in M. acridum. This central regulatory pathway controls specific conidiation genes during conidiophore development and spore maturation. Remarkably, specific functions of the central regulatory pathway in conidiogenesis are highly conserved among the fungi in A. nidulans and some other filamentous fungi.4. TEM was performed to obtain the microstructure of the strains and we found that the deficiency of MabrlA has an influence on cell wall content and distribution of M. acridum. By fluorescent staining with antibodies or fluorescent dye, we found that the MabrlA mutant cell wall composition and structure had changed. Component analysis found that MabrlA mutant mannoproteins was only 30% of WT and chitin was increased to three folds than that of WT.5. MabrlA has an influence on cellular and humoral immune response of host, which is the important mechanism of the loss of its virulence. The results indicated that, in contrast to the WT, the ?MabrlA mutant lost its ability to kill insects, ?MabrlA strains of appressorium formation rate was significantly less than the WT, the appressorium turgor pressure is reduced. MabrlA indeced the proteinase Pr1 and Chi in insect appressorium high expression. The melanization of MabrlA disruption in insect hemolymph in vitro became stronger and the PO activity increased than that of WT. MabrlA disruption induced the expression of immune gene Lmspatzle?Lmcactus in insect hemolymph and fat body.Taken together, the results of this study help us to understand the functional of MabrlA in conidiation regulation and control of pathogenicity in insect-pathogenic fungi. It provides new insights into the genetics of conidiation and pathogenicity in filamentous fungi. |
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