Functional Analysis Of The Nuclear Zinc Finger Transcription Factor Gene MaCrz1in Metarhizium Acridum

As an insect pathogenic fungus, Metarhizium acridum is one of the most popularmodel organisms in recent studies. It has been gradually used in the biological control inrecent years due to its obvious advantages in environmental safety and so forth. Forinstance, it leads to less pest resistance and does not cause any pesticide residues andthereby reducing agricultural pests rampant again effectively. Meanwhile, for piercingsucking insects such as cicadas, M. acridum has particular advantages, but as a new typeof biological pesticides, its deficiencies are also prominent. For example, the lethal timeof M. acridum infected pests is longer, the processes of infection to environment changeare sensitive and the control efficiency is not so stable in comparison to chemicalinsecticides. These disadvantages hold back the more widely application of M. acridumas a biological pesticide. In this study, we analyzed the functions of aCa2+/calmodulin-dependent protein phosphatase related gene Crz1(calcineurinresponsive zinc finger1) in M. acridum to explore its roles in environmental stress andits roles in virulence by genetic engineering method, providing more information andresources for improving the environmental adaptation and enhancing its virulence inpest infection.In this study, the MaCrz1gene function was analyzed using gene knockout andrecovery techniques in the insect pathogenic fungus M. acridum.The main results were asfollows:1）Bioinformatics analysis of the MaCrz1Full genomic DNA sequence of MaCrz1was obtained by amplification. TheMaCrz1gene was predicted to encode a peptide of729amino. Alignment of MaCrz1revealed that it shared conserved secondary structures, the C2H2zinc finger domain, inMaCrz1.2）Disruption of MaCrz1To explore the biological role of the MaCrz1in the development and infectionprocesses of M. acridum, we constructed the ΔMaCrz1disruption mutant throughhomologous recombination of the MaCrz1ORF with a glufosinate-ammonium (GA)resistance gene.3）MaCrz1affect M. acridum susceptible to calcium ionsGrowth of ΔMaCrz1on1/4SDAY supplemented with calcium ions showed that ΔMaCrz1had inhibited growth compared with the wild type and complemented strain,indicating that MaCrz1plays an important role in the regulation of intracellular calciumhomeostasis.4）MaCrz1is important for tolerance to oxidative stress and stress resistanceGrowth of ΔMaCrz1on medium with oxidative stress or cell wall disturbing agentsshowed that ΔMaCrz1mutant had decreased growth compared with the wild type,suggesting that MaCrz1has important functions in stress tolerance.5）MaCrz1is required for M. acridum to tolerance UV-B radiationUV radiation tolerance capability of the wild type, ΔMaCrz1mutant, andcomplemented M. acridum strains were assayed. GT50of ΔMaCrz1mutant decreasedsignificantly, suggesting MaCrz1involved in regulation of the UV tolerance.6）MaCrz1affected M. acridum virulenceThe virulence was significantly decreased in ΔMaCrz1mutant. In topicalinoculation bioassay, the LT50of ΔMaCrz1was significanly longer than WT. However,in injection assay, no difference was found in LT50between WT and ΔMaCrz1.7）MaCrz1affect appressoria formationAppressorium formation was analyzed on locust wing. The ability of appressoriumformation was reduced when MaCrz1was knocked out.8）MaCrz1is important for cell wall integrityTo test the integrity of the cell wall of the ΔMaCrz1mutant, chitn and β-1-3glucanwere visualized by CFW and β-1,3-glucan-specific antibody. ΔMaCrz1mutant hadweaker intensity with two dyes than WT, suggesting that Crz1may participate in cellwall construction.