Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (4): 744-753.doi: 10.3864/j.issn.0578-1752.2021.04.007

• PLANT PROTECTION • Previous Articles     Next Articles

Expression in vitro of Metarhizium anisopliae Adhesin MAD1 and Its Effect on Inducing Response in Peanut

YAN DuoZi(),CAI Ni,WANG Feng,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:2020-05-09 Accepted:2020-06-10 Online:2021-02-16 Published:2021-02-16
  • Contact: XiangQun NONG E-mail:yanduozi@163.com;xqnong@sina.com

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Abstract:

【Objective】The fungus Metarhizium anisopliae has the characteristics of insect pathogenicity and plant symbiosis, and its adhesin MAD1 plays an important role in the infection to host insects. The objective of this study is to express MAD1 protein in eukaryotes in vitro, and to clarify the role of MAD1 in symbiosis between M. anisopliae and plants. 【Method】Unigenes of M. anisopliae transcriptome were used as the reference sequence to search and compare the homology in GenBank and design primers. The gene mad1 was cloned from the cDNA template of M. anisopliae IPPM010202. MAD1 protein sequence was translated by DNAMAN software. The online software ProtParam was used to predict the MAD1 protein composition of amino acid and its physicochemical characteristics. The protein structure was analyzed using SMART online program. The recombinant was constructed from eukaryotic expression vector with mad1 and Pichia pastoris transformation. The MAD1 protein was successfully expressed by methanol induction and the purified MAD1 was obtained by Ni-NTA affinity chromatography. The peanut roots were treated with immersion in the purified MAD1 solution (20 μg·mL-1) for 0.5, 6, 12 and 24 h, and then the transcription levels of membrane receptor genes (CERK1, RPK), immune-related cascade related genes (MAPK, MMK1, CDPK) and transcription factor gene (MYB86), cell wall integrator gene (SCW1) as well as defense related genes (PTi1, RML1A) were detected by real-time fluorescence quantitative PCR (qPCR). 【Result】 The adhesive gene mad1 of M. anisopliae was cloned, with a total length of 2 136 bp, encoding 711 amino acids in molecular weight 74.8 kD. Bioinformatics analysis showed that the N-terminal of MAD1 has signal peptide, the C-terminal has a glycosylphosphatidyl inositol (GPI) anchor and contains CFEM functional domain and belongs to hydrophilic protein. The eukaryotic expression system with mad1 was successfully constructed and the active MAD1 protein was efficiently induced, expressed and purified. The results based on qPCR of peanut roots treated with purified MAD1 showed that, for 0.5 h, the root tip cells perceived the protein and activated the expression of membrane recognition receptor gene CERK1. In 0.5-6 h, the transcriptional levels of CERK1 and RPK were up-regulated, the transcription level of membrane-integrated gene SCW1 changed from down-regulation to up-regulation, while the transcriptional levels of MAPK, MMK1, CDPK and MYB86 were temporarily inhibited, and the transcriptional levels of defense genes PTi1 and RML1A were down-regulated, the root immune defense response was inhibited. In 6-12 h, the membrane recognition receptors maintained up-regulation, while the defensive gene RML1A reversed from down-regulation to strong up-regulation. In 12-24 h, CERK1 and RPK, PTi1 and RML1A were all up-regulated, MAPK, MMK1, CDPK and MYB86 appeared slightly up-regulated, the roots initiated immune defense response.【Conclusion】In the early stage of the interaction between M. anisopliae and peanut roots, the adhesin MAD1 protein activates the membrane receptor genes CERK1 and RPK recognition response in peanut at 6 h, while inhibits the expression of peanut immune cascade genes MAPK, CDPK, MMK1 and defense related genes such as PTi1 and RML1A, which is helpful for the colonization of M. anisopliae in peanut root tissue. Subsequently, it induces the up-regulation of expression of cell wall integrin gene SCW1, which participates in the repair and reconstruction of damaged cell wall on the root, and promotes the establishment of symbiotic relationship between M. anisopliae and peanut. Induction of plant immunosuppression and integral cell wall reconstruction may be important steps in the establishment of symbiotic relationship between M. anisopliae and plants.

Key words: MAD1, Metarhizium anisopliae, symbiosis, plant immunity, defense, eukaryotic expression

Table 1

Candidate genes and specific primers"

基因Gene 引物序列Primer sequence 基因Gene 引物序列Primer sequence
CERK1 5′-CTACTCAGCCATTGCCAGTG RPK 5′-AAGGTGGAGTTTGTTATGCC
5′-CAGTCCTAAGAGGGTATGTGATGA 5′-CCTAAGGACTTGAGACCCTG
PTi1 5′-TTTGACCTGGGCACAGAGAG RML1A 5′-CAGTGAGCGTTGCGAGATAC
5′-GACACGGGTGGAATGAAGAC 5′-TTGGGTTTCTTTCAGCGACT
SCW1 5′-GAGTAAGCGATTGCGGACAG CDPK 5′-CAAGACCATTGTTGAGGTTG
5′-TGTTCTCTCCCAAGTTCCCA 5′-CTCCACACATCTGATTCTGG
MMK1 5′-AACTCGGAGACGAATGAGCA MAPK 5′-CTTCCACAATGTCCAAAGCA
5′-TTGGTGAAGGTCAGTGTCCA 5′-CCAGATGAGTTCCTTGATGTC
MYB86 5′-CCACAACAGACAACAGTTTACG RPL7 5′-AGAAGAGGGAGGAGGAATGG
5′-TAGAGCACCATCTCCTACCTC 5′-GGATGCGGATGATAAACAGG

Fig. 1

Amplification of the mad1 full-length gene from M. anisopliae M：DL5000 DNA marker;1：ddH2O;2：绿僵菌cDNA cDNA from M. anisopliae"

Fig. 2

Structure of M. anisopliae adhesin MAD1"

Fig. 3

Examination of positive clones of mad1 transformed yeast by PCR M：DL 5000 DNA marker;N：空载酵母GS115 Empty carrier yeast GS115;1—5：重组单克隆Recombinant clones"

Fig. 4

SDS-PAGE analysis of recombinant yeast induced expression of MAD1 A：发酵液上清Supernatant of fermentation broth;B：菌体破碎提取上清Extract of crushed cell;C：MAD1蛋白纯化Purification of MAD1 protein"

Fig. 5

The relative expression of related genes induced by MAD1 The expression level of the control is 1, indicated by the dotted line. Symbol ‘*’ indicates a significant difference from the control, P＜0.05"

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