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Adhesin Gene mad2 Knockout and Functional Effects on Characteristics of Growth, Virulence, Adhesion and Inducing Plant Responses in Metarhizium anisopliae

CAI Ni, YAN DuoZi, NONG XiangQun, WANG GuangJun, TU XiongBing, ZHANG ZeHua   

  1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193
  • Received:2021-04-14 Accepted:2021-05-12 Published:2021-06-03

Abstract: 【Objective】Metarhizium anisopliae, an entomopathogenic fungus, is found also an endophyte. It was known that MAD2 was one of the two adhesin proteins of M. anisopliae, which play a vital role in adhere and colonize in plants, but its functional mechanism understood little. In this study, we will explore the functional effect of MAD2 protein on the characteristics of growth, virulence, adhesion and inducing plant responses in M. anisopliae by construction of the mad2 mutant strain (Δmad2) of M. anisopliae strain Ma9. 【Method】The genomic DNA sequences of mad2 anteroposterior were obtained from NCBI, and specific primers were designed to amplify mad2 homologous arm genes S1 and S2 by PCR based on genomic DNA template of M. anisopliae strain Ma9. Meanwhile, Hyg-F/R primer pair was designed to amplify hygromycin gene with promoter sequence based on pKH-KO vector DNA template. Then, homologous knockout boxes S1H and S2H of mad2 were constructed by Overlap PCR. Finally, mad2 knockout strains with stable inheritance were obtained by PEG-mediated protoplast transformation of the homologous knockout boxes. By comparing the biological characteristics of the knockout strains (Δmad2) to the wild strains (WT), the effect of MAD2 protein on the characteristics of M. anisopliae growth, virulence, adhesion and inducing the peanut response of symbiosis genes were analyzed.【Result】The result showed that the spore germination rate of mutant strain was significantly decreased and spore semi-germination time was significantly prolonged 5.47 h compare to wild strain (WT), as well as the hypha length of Δmad2 was significantly shorter than WT in 12 h and 14 h incubation, occupied 22.2% and 23.7% respectively. The sporulation in 12-day incubation was reduced by 33.33% compared to WT. The ability of Δmad2 strain adhesion in onion was significantly decreased but showed no differences in adhere to the hind wings of locust. In addition, knockout mad2 did not affect the virulence of M. anisopliae to silkworm. After 12 h inoculating mad2 knockout strain in peanut, the transcription level of peanut symbiosis receptor gene SYMRK, Calcium signal decoding related genes (CAM, CCaMK, DELLA),lipid and nitrogen transfer related genes (LTP1, NRT24, ABCC2) were significantly regulated down compared to the treatment of wild strain. While compared with blank control, Δmad2 still had certain up-regulation of SYMRK, significantly inhibited the transcription levels of CAM, CCAMK and DELLA, but had no effect on the transcription level of ABCC2, LTP1 and NRT24.【Conclusion】In conclusion, M. anisopliae adhesin protein MAD2 affect spore germination, initial growth of hypha, sporulation quantity and plants adhesion, while had no effect on insect adhesion and virulence of strain, and MAD2 was induced the transcription level of symbiosis genes in the initial stage of interaction.

Key words: Metarhizium anisopliae, adhesin protein MAD2, gene knockout, adhesion, virulence, growth trait

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