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

RichHTML

14

PDF

220

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"

[1] BARELLI L, MOONJELY S, BEHIE S W, BIDOCHKA M J. Fungi with multifunctional lifestyles: Endophytic insect pathogenic fungi. Plant Molecular Biology, 2016,90(6):657-664.
[2] JULIYA R F. Biocontrol potential and genetic diversity of Metarhizium anisopliae lineage in agricultural habitats. Journal of Applied Microbiology, 2019,127(2):556-564.
[3] CLARKSON J M, CHARNLEY A K. New insights into the mechanisms of fungal pathogenesis in insects. Trends in Microbiology, 1996,4(5):197-203.
[4] BARELLI L, PADILLA-GUERRERO I E, BIDOCHKA M J. Differential expression of insect and plant specific adhesin genes, MAD1 and MAD2, in Metarhizium robertsii. Fungal Biology, 2011,115(11):1174-1185.
[5] WANG C, ST LEGER R J. The MAD1 adhesin of Metarhizium anisopliae links adhesion with blastospore production and virulence to insects, and the MAD2 adhesin enables attachment to plants. Eukaryotic Cell, 2007,6(5):808-816.
[6] 赵宇, 张佳诗, 刘艳微, 王佳江, 徐文静, 张冬娜, 赵慧, 张正坤, 李启云. 创制转Mad1基因球孢白僵菌工程菌株提高对玉米螟毒力. 玉米科学, 2019,27(5):39-44, 51.
ZHAO Y, ZHANG J S, LIU Y W, WANG J J, XU W J, ZHANG D N, ZHAO H, ZHANG Z K, LI Q Y. Construction of Mad1 gene transgenic Beauveria bassiana strain to promote the toxicity against Ostrinia furnacalis. Journal of Maize Sciences, 2019,27(5):39-44, 51. (in Chinese)
[7] ABRO N A, WANG G J, ULLAH H, GUO L L, HAO K, NONG X Q, CAI N, TU X B, ZHANG Z H. Influence of Metarhizium anisopliae (IMI330189) and Mad1 protein on enzymatic activities and Toll- related genes of migratory locust. Environmental Science and Pollution Research International, 2019,26(17):17797-17808.
[8] SASAN R K, BIDOCHKA M J. The insect-pathogenic fungus Metarhizium robertsii (Clavicipitaceae) is also an endophyte that stimulates plant root development. American Journal of Botany, 2012,99(1):101-107.
[9] GARCÍA J E, POSADAS J B, PERTICARI A, LECUONA R E. Metarhizium anisopliae (Metschnikoff) Sorokin promotes growth and has endophytic activity in tomato plants. Advances in Biological Research, 2011,5(1):22-27.
[10] CAI N, WANG F, NONG X Q, WANG G J, MCNEILL M, CAO G C, HAO K, LIU S F, ZHANG Z H. Visualising confirmation of the endophytic relationship of Metarhizium anisopliae with maize roots using molecular tools and fluorescent labelling. Biocontrol Science and Technology, 2019,29(11):1023-1036.
[11] TANG D, WANG G, ZHOU J M. Receptor kinases in plant-pathogen interactions: More than pattern recognition. The Plant Cell, 2017,29(4):618-637.
[12] JONES J D, DANGL J L. The plant immune system. Nature, 2006,444(7117):323-329.
[13] WOLF S. Plant cell wall signalling and receptor-like kinases. The Biochemical Journal, 2017,474(4):471-492.
[14] ZHU H Y, RIELY B K, BURNS N J, ANÉ J M. Tracing nonlegume orthologs of legume genes required for nodulation and arbuscular mycorrhizal symbioses. Genetics, 2006,172(4):2491-2499.
[15] BROTMAN Y, LANDAU U, CUADROS-INOSTROZA Á, TOHGE T, TAKAYUKI T, FERNIE A R, CHET I, VITERBO A, WILLMITZER L. Trichoderma-plant root colonization: Escaping early plant defense responses and activation of the antioxidant machinery for saline stress tolerance. PLoS Pathogens, 2013,9(3):e1003221.
[16] CAO Y R, HALANE M K, GASSMANN W, STACEY G. The role of plant innate immunity in the legume-rhizobium symbiosis. Annual Review of Plant Biology, 2017,68:535-561.
[17] HAO K, WANG F, NONG X Q, MCNEILL M R, LIU S F, WANG G J, CAO G C, ZHANG Z H. Response of peanut Arachis hypogaea roots to the presence of beneficial and pathogenic fungi by transcriptome analysis. Scientific Reports, 2017,7(1):964.
[18] MICHÉ L, BALANDREAU J. Effects of rice seed surface sterilization with hypochlorite on inoculated Burkholderia vietnamiensis. Applied and Environmental Microbiology, 2001,67(7):3046-3052.
[19] PFAFFL M W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, 2001,29(9):e45.
[20] KAPTEYN J C, HOYER L L, HECHT J E, MÜLLER W H, ANDEL A, VERKLEIJ A J, MAKAROW M, VAN DEN ENDE H, KLIS F M. The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants. Molecular Microbiology, 2000,35(3):601-611.
[21] RICHARD M L, PLAINE A. Comprehensive analysis of glycosylphosphatidylinositol-anchored proteins in Candida albicans. Eukaryotic Cell, 2007,6(2):119-133.
[22] KULKARNI R D, KELKAR H S, DEAN R A. An eight-cysteine- containing CFEM domain unique to a group of fungal membrane proteins. Trends in Biochemical Sciences, 2003,28(3):118-121.
[23] 张丽勍, 段可, 邹小花, 何成勇, 高清华. 草莓胶孢炭疽菌CFEM候选效应子的生物信息学鉴定及其侵染过程中的转录分析. 植物保护, 2017,43(5):43-51.
ZHANG L Q, DUAN K, ZOU X H, HE C Y, GAO Q H. Bioinformatic identification and transcriptional analysis of Colletotrichum gloeosporioides candidate CFEM effector proteins. Plant Protection, 2017,43(5):43-51. (in Chinese)
[24] DEZWAAN T M, CARROLL A M, VALENT B, SWEIGARD J A. Magnaporthe grisea pth11p is a novel plasma membrane protein that mediates appressorium differentiation in response to inductive substrate cues. The Plant Cell, 1999,11(10):2013-2030.
[25] DESAKI Y, KOHARI M, SHIBUYA N, KAKU H. MAMP-triggered plant immunity mediated by the LysM-receptor kinase CERK1. Journal of General Plant Pathology, 2019,85:1-11.
[26] WANG C, WANG G, ZHANG C, ZHU P K, DAI H L, YU N, HE Z H, XU L, WANG E. OsCERK1-mediated chitin perception and immune signaling requires receptor-like cytoplasmic kinase 185 to activate an MAPK cascade in rice. Molecular Plant, 2017,10(4):619-633.
[27] YAMADA K, YAMAGUCHI K, YOSHIMURA S, TERAUCHI A, KAWASAKI T. Conservation of chitin-induced MAPK signaling pathways in rice and Arabidopsis. Plant and Cell Physiology, 2017,58(6):993-1002.
[28] OSAKABE Y, MARUYAMA K, SEKI M, SATOU M, SHINOZAKI K, YAMAGUCHI-SHINOZAKIA K. Leucine-rich repeat receptor- like kinase1 is a key membrane-bound regulator of abscisic acid early signaling in Arabidopsis. The Plant Cell, 2005,17(4):1105-1119.
[29] OSAKABE Y, MIZUNO S, TANAKA H, MARUYAMA K, OSAKABE K, TODAKA D, FUJITA Y, KOBAYASHI M, SHINOZAKI K, YAMAGUCHI-SHINOZAKI K. Overproduction of the membrane-bound receptor-like protein kinase 1, RPK1, enhances abiotic stress tolerance in Arabidopsis. The Journal of Biological Chemistry, 2010,285(12):9190-9201.
[30] MENG X, ZHANG S. MAPK cascades in plant disease resistance signaling. Annual Review of Phytopathology, 2013,51:245-266.
[31] YU X, FENG B M, HE P, SHAN L. From chaos to harmony: Responses and signaling upon microbial pattern recognition. Annual Review of Phytopathology, 2017,55:109-137.
[32] COUTO D, ZIPFEL C. Regulation of pattern recognition receptor signalling in plants. Nature Reviews Immunology, 2016,16(9):537-552.
[33] 武志刚, 武舒佳, 王迎春, 郑琳琳. 植物中钙依赖蛋白激酶(CDPK)的研究进展. 草业学报, 2018,27(1):204-214.
WU Z G, WU S J, WANG Y C, ZHENG L L. Advances in studies of calcium-dependent protein kinase (CDPK) in plants. Acta Prataculturae Sinica, 2018,27(1):204-214. (in Chinese)
[34] LIU J Z, HORSTMAN H D, BRAUN E, GRAHAM M A, ZHANG C Q, NAVARRE D, QIU W L, LEE Y, NETTLETON D, HILL J H, WHITHAM S A. Soybean homologs of MPK4 negatively regulate defense responses and positively regulate growth and development. Plant Physiology, 2011,157(3):1363-1378.
[35] GLYAN’KO A K, ISCHENKO A A. Immunity of a leguminous plant infected by nodular bacteria Rhizobium spp. F. Applied Biochemistry and Microbiology, 2017,53(2):140-148.
[36] LIANG Y, CAO Y, TANAKA K, THIBIVILLIERS S, WAN J, CHOI J, KANG C, QIU J, STACEY G. Nonlegumes respond to rhizobial nod factors by suppressing the innate immune response. Science, 2013,341(6152):1384-1387.
[37] TELLSTRM V, USADEL B, THIMM O, STITT M, KÜSTER H, KARSTEN H, NIEHAUS K. The lipopolysaccharide of Sinorhizobium meliloti suppresses defense-associated gene expression in cell cultures of the host plant Medicago truncatula. Plant Physiology, 2007,143(2):825-837.
[1] WU Wei,XU HuiLi,WANG ZhengLiang,YU XiaoPing. Cloning and Function Analysis of a Serine Protease Inhibitor Gene Nlserpin2 in Nilaparvata lugens [J]. Scientia Agricultura Sinica, 2022, 55(12): 2338-2346.
[2] DU Yu,FAN XiaoXue,JIANG HaiBin,WANG Jie,FENG RuiRong,ZHANG WenDe,YU KeJun,LONG Qi,CAI ZongBing,XIONG CuiLing,ZHENG YanZhen,CHEN DaFu,FU ZhongMin,XU GuoJun,GUO Rui. MicroRNA-Mediated Cross-Kingdom Regulation of Apis mellifera ligustica Worker to Nosema ceranae [J]. Scientia Agricultura Sinica, 2021, 54(8): 1805-1820.
[3] HanXi LIU,Hao LÜ,GuangYu GUO,DongXu LIU,Yan SHI,ZhiJun SUN,ZeXin ZHANG,YanJiao ZHANG,YingNan WEN,JieQi WANG,ChunYan LIU,QingShan CHEN,DaWei XIN,JinHui WANG. Effect of rhcN Gene Mutation on Nodulation Ability of Soybean Rhizobium HH103 [J]. Scientia Agricultura Sinica, 2021, 54(6): 1104-1111.
[4] CAI Ni,YAN DuoZi,NONG XiangQun,WANG GuangJun,TU XiongBing,ZHANG ZeHua. Adhesin Gene mad2 Knockout and Functional Effects on Biological Characteristics and Inducing Plant Responses in Metarhizium anisopliae [J]. Scientia Agricultura Sinica, 2021, 54(22): 4800-4812.
[5] ZHENG FengSheng,WANG HaiHua,WU QingTao,SHEN Quan,TIAN JianHong,PENG XiXu,TANG XinKe. Genome-Wide Identification of VQ Gene Family in Fagopyrum tataricum and Its Expression Profiles in Response to Leaf Spot Pathogens [J]. Scientia Agricultura Sinica, 2021, 54(19): 4048-4060.
[6] XU HuanHuan,LI Yi,GAO Wei,WANG YongQin,LIU LeCheng. Cloning and Identification of γ-Glutamyl Transpeptidase AcGGT Gene from Onion (Allium cepa) [J]. Scientia Agricultura Sinica, 2021, 54(19): 4169-4178.
[7] YU ZhengWang,ZHOU ZhongXin. Functions of Antibacterial and Inducing Defense Peptide Expression of Medium-Chain Fatty Acid and Its Application in Piglet Feeds [J]. Scientia Agricultura Sinica, 2021, 54(13): 2895-2905.
[8] ZHANG YuFei,CAO ManYuan,WANG LiYing,ZHAO WeiGang,LI XiaoXia,CHANG Tong,XU BaoZeng. Eukaryotic Expression, Purification and Biological Activity of Recombinant Cervus Nippon Activin A Protein [J]. Scientia Agricultura Sinica, 2020, 53(5): 1058-1070.
[9] QI Yue,LÜ JunYuan,ZHANG Yue,WEI Jie,ZHANG Na,YANG WenXiang,LIU DaQun. Puccinia triticina Effector Protein Pt18906 Triggered Two-Layer Defense Reaction in TcLr27+31 [J]. Scientia Agricultura Sinica, 2020, 53(12): 2371-2384.
[10] AN JunXia,ZHAO Yu,ZHANG ZhengKun,SHI HaiPeng,JI DongMing,CAO HongYi,DU Qian,LI QiYun. Induction of Cold Tolerance in Rice at the Breeding Stage by Gongzhulingmycin [J]. Scientia Agricultura Sinica, 2020, 53(11): 2195-2206.
[11] LI YongHua, CHE LuPing, QIU XuSheng, TAN Lei, SUN YingJie, LIU WeiWei, SONG CuiPing, LIAO Ying, DING Chan, WANG JinQuan, MENG ChunChun. Construction of Chicken TIGAR Gene Eukaryotic Expression Plasmid and Evaluation of Its Anti-Apoptotic Function [J]. Scientia Agricultura Sinica, 2019, 52(6): 1102-1109.
[12] TIAN ZhiLong,TANG JiShun,SUN Qing,WANG YuQin,ZHANG XiaoSheng,ZHANG JinLong,CHU MingXing. Tissue Expression and Polymorphism of Sheep SMAD1 Gene and Their Association with Litter Size [J]. Scientia Agricultura Sinica, 2019, 52(4): 755-766.
[13] NING Yue,MI Xue,CHEN XingYi,SHAO JianHang,ZAN LinSen. Silencing and Overexpressing SMAD Family Member 1 (SMAD1) Gene and Its Effect on Myogenesis in Primary Myoblast of Qinchuan Cattle (Bos taurus) [J]. Scientia Agricultura Sinica, 2019, 52(10): 1818-1829.
[14] HE YuJuan, JU Di, WANG Yue, YANG XueQing, WANG XiaoQi . Compositive and Inductive Expression Patterns of Protease Inhibitor Genes OsLTPL164 and OsLTPL151 in Rice (Oryza sativa) [J]. Scientia Agricultura Sinica, 2018, 51(12): 2311-2321.
[15] Feng YANG, ChuanWu CHEN, QiJun FAN, ChunMei SHI, ZongZhou XIE, DaYong GUO, JiHong LIU. Influence of Temperature and Polyamines on Occurrence of Citrus Canker Disease and Underlying Mechanisms [J]. Scientia Agricultura Sinica, 2018, 51(10): 1899-1907.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd