Avirulence Genes in Magnaporthe oryzae 007: Cloning, Identification and Expression Analysis

doi:10.11924/j.issn.1000-6850.casb20190400047

Abstract

Abstract:

To clarify the avirulence genes contained in the international standard strain Magnaporthe oryzae race 007, and the possible mainly effective avirulence genes, we designed five common avirulence gene-specific primers for cloning and identification. And the expression of five avirulence genes in Magnaporthe oryzae race 007 strain were detected separately in the liquid culture condition and the process of infecting rice. The results showed that Magnaporthe oryzae race 007 strain contained five avirulence genes including PWL2, AvrPiz-t, Avr-pik, Avr-pita and ACE1. The result of cloning and identification indicated that the five avirulent genes did not change much. Compared with that under the liquid culture condition, in the process of infecting rice, the expression of PWL2 was up-regulated, and the expressions of ACE1, AvrPiz-t and Avr-Pik were basically unchanged, while the expression of Avr-Pita gene was down-regulated. Thus, the expression level of PWL2 gene was significantly changed. We further analyzed promoter region sequences of PWL2 gene, and the results showed that there were some cis-acting elements in the promoter region of PWL2 gene to affect the expression of PWL2 gene. This study show that Magnaporthe oryzae race 007 strain contains five common avirulent genes with little sequence variation and the expression of PWL2 gene is regulated and it may play a major pathogenic role in Magnaporthe oryzae race 007.

Key words: Magnaporthe oryzae, avirulence gene, cloning, expression analysis

CLC Number:

S432.1

Xiong Tiantian, Han Haojie, Lu Yanmei, Zhang Ning, Wang Chuntai, Liu Xingqiong, Cheng Gang. Avirulence Genes in Magnaporthe oryzae 007: Cloning, Identification and Expression Analysis[J]. Chinese Agricultural Science Bulletin, 2020, 36(18): 124-129.

Figures/Tables 7

References 29

[1]	Schneider D R, Saraiva A M, Azzoni A R, et al. Overexpression and purification of PWL2D, a mutant of the effector protein PWL2 from Magnaporthe grisea.[J]. Protein Expression & Purification, 2010,74(1):24-31. doi: 10.1016/j.pep.2010.04.020 URL pmid: 20438845
[2]	梁华兵, 王晓婉, 胡晓岚, 等. 水稻稻瘟病抗病基因Pi63顺式作用元件的载体构建[J]. 中国农学通报, 2015,31(30):220-224.
[3]	Couch B C, Kohn L M. A multilocus gene genealogy concordant with host preference indicates segregation of a new species, Magnaporthe oryzae, from M. grisea[J]. Mycologia, 2002,94(4):683-693. doi: 10.1080/15572536.2003.11833196 URL pmid: 21156541
[4]	He Y, Zhu M, Huang J, et al. Biocontrol potential of a Bacillus subtilis strain BJ-1 against the rice blast fungus Magnaporthe oryzae[J]. Canadian Journal of Plant Pathology, 2019,41(1):47-59. doi: 10.1080/07060661.2018.1564792 URL
[5]	Tang W, Jiang H, Zheng Q, et al. Isopropylmalate isomerase MoLeu1 orchestrates leucine biosynjournal, fungal development, and pathogenicity in Magnaporthe oryzae[J]. Applied Microbiology and Biotechnology, 2018,103(1):327-337. doi: 10.1007/s00253-018-9456-9 URL pmid: 30357439
[6]	Yuemin P, Rui P, Leyong T, et al. Pleiotropic roles of O-mannosyltransferase MoPmt4 in development and pathogenicity of Magnaporthe oryzae[J]. Current Genetics, 2018. doi: 10.1007/s00294-020-01073-z URL pmid: 32249353
[7]	Talbot N J. On the Trail of a Cereal Killer: Exploring the Biology of Magnaporthe grisea[J]. Annual Review of Microbiology, 2003,57(1):177-202. doi: 10.1146/annurev.micro.57.030502.090957 URL
[8]	Jones K, Khang C H. Vacuole Dynamics in Rice Cells Invaded by the Blast Fungus Magnaporthe oryzae[J]. Plant Vacuolar Trafficking, 2018,1789:195-203.
[9]	Shao Z, Li Z, Fu Y, et al. Induction of defense responses against Magnaporthe oryzae in rice seedling by a new potential biocontrol agent Streptomyces JD211.[J]. Journal of Basic Microbiology, 2018,58(8):686-697. doi: 10.1002/jobm.201800100 URL pmid: 29901825
[10]	Gladieux P, Condon A B, Ravel B S, et al. Gene Flow between Divergent Cereal- and Grass-Specific Lineages of the Rice Blast Fungus Magnaporthe oryzae[J]. mBio, 2018,9(1):e01219-17. doi: 10.1128/mBio.01219-17 URL pmid: 29487238
[11]	Liang Y, Zhao J, Wang C, et al. Infection with blast fungus (Magnaporthe orzyae), leads to increased expression of an arabinogalactan-protein epitope in both susceptible and resistant rice cultivars[J]. Physiological and Molecular Plant Pathology, 2018: S088557651730334X.
[12]	Allen R L, Bittner-Eddy P D, Grenville-Briggs L J, et al. Host-parasite coevolutionary conflict between Arabidopsis and downy mildew[J]. Science, 2004,306(5703):1957-1960. doi: 10.1126/science.1104022 URL pmid: 15591208
[13]	Zheng Y, Zheng W, Lin F, et al. AVR1-CO39 is a predominant locus governing the broad avirulence of Magnaporthe oryzae 2539 on cultivated rice (Oryza sativa L.)[J]. Mol Plant Microbe Interact, 2011,24(1):13-17. doi: 10.1094/MPMI-10-09-0240 URL pmid: 20879839
[14]	徐鑫, 郭旭梦, 刘松青, 等. 稻瘟病抗性基因Pi63启动子4个构建体的表达及抗性分析[J]. 中南民族大学学报:自然科学版, 2018,37(2):36-40.
[15]	田红刚. 黑龙江省稻瘟病菌致病性和品种抗性改良研究[D]. 沈阳:沈阳农业大学, 2016.
[16]	Liu J L, Wang X J, Mitchell T, et al. Recent progress and understanding of the molecular mechanisms of the rice-Magnaporthe oryzae interaction[J]. Molecular Plant Pathology, 2010,11(3):419-427. doi: 10.1111/j.1364-3703.2009.00607.x URL pmid: 20447289
[17]	姜华, 余欢, 王艳丽, 等. 稻瘟病菌无毒基因序列变异研究进展[J]. 浙江农业学报, 2015,27(3):512-520.
[18]	Orbach M J, Farrall L, Sweigard J A, et al. A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta[J]. The Plant Cell, 2000,12(11):2019-2032. doi: 10.1105/tpc.12.11.2019 URL pmid: 11090206
[19]	Heidi U Böhnert, Fudal I, Dioh W, et al. A Putative Polyketide Synthase/Peptide Synthetase from Magnaporthe grisea Signals Pathogen Attack to Resistant Rice[J]. The Plant Cell, 2004,16(9):2499-2513. doi: 10.1105/tpc.104.022715 URL pmid: 15319478
[20]	Li W, Wang B, Wu J, et al. The Magnaporthe oryzae avirulence gene AvrPiz-t encodes a predicted secreted protein that triggers the immunity in rice mediated by the blast resistance gene Piz-t[J]. Mol Plant Microbe Interact, 2009,22(4):411-420. doi: 10.1094/MPMI-22-4-0411 URL pmid: 19271956
[21]	Yoshida K, Saitoh H, Fujisawa S, et al. Association Genetics Reveals Three Novel Avirulence Genes from the Rice Blast Fungal Pathogen Magnaporthe oryzae[J]. The plant cell online, 2009,21(5):1573-1591. doi: 10.1105/tpc.109.066324 URL
[22]	龙子文, 周杰, 王春台, 等. 鄂西地区稻瘟病菌生理小种文库的构建及初步鉴定[J]. 安徽农业科学, 2012,40(24):12065-12067.
[23]	李泌, 王春台, 周杰, 等. 2011年鄂西地区水稻稻瘟病菌遗传多样性的分析[J]. 安徽农业科学, 2014,42(14):4249-4251.
[24]	田珂, 杨武, 李泌, 等. 鄂西南地区2012-2014年稻瘟病菌致病性变化分析[J]. 华中农业大学学报, 2017,36(05):10-14.
[25]	Xin X, Wu Y, Ke T, et al. Genetic diversity and pathogenicity dynamics of Magnaporthe oryzae in the Wuling Mountain area of China[J]. European Journal of Plant Pathology, 2019,153(3):731-742. doi: 10.1007/s10658-018-1587-4 URL
[26]	Song M Y, Kim C Y, Han M, et al. Differential requirement of Oryza sativa RAR1 in immune receptor-mediated resistance of rice to Magnaporthe oryzae[J]. Molecules and Cells, 2013,35(4):327-334. doi: 10.1007/s10059-013-2317-6 URL
[27]	Chang-Jie J, Xiao-Long L, Xin-Qiong L, et al. Stunted Growth Caused by Blast Disease in Rice Seedlings Is Associated with Changes in Phytohormone Signaling Pathways[J]. Frontiers in Plant Science, 2017,8:1558. doi: 10.3389/fpls.2017.01558 URL pmid: 28932234
[28]	Wan J, Wenli Z, Ron A, et al. A method for determining zygosity of transgenic zebrafish by TaqMan real-time PCR[J]. Analytical Biochemistry, 2005,344(2):240-246. doi: 10.1016/j.ab.2005.06.046 URL pmid: 16061191
[29]	Livak . Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C (T)) Method[J]. Methods, 2001,25(4):402-408. doi: 10.1006/meth.2001.1262 URL pmid: 11846609

引物名称	引物序列(5’-3’)			Tm值/℃
PWL2	CATCCCTTACTCCGCCACTT			61
PWL2	CTCTTCTCGCTGTTCACGGT			61
AvrPiz-t	GCGACTTTCTTTCTTTGTCTCCT			55
AvrPiz-t	GACAGTGACCATGTTTTATACCG			55
Avr-pik	TCCTGCTGCTAACTCCATTC			60
Avr-pik	TCAACCAAGCGTAAACCTCG			60
Avr-Pita		CGACCCGTTTCCGCCTTTATT	56.5
Avr-Pita		TCCCTCCATTCCAACACTAACG	56.5
ACE1		CCCAGAGTTGGCGATGATGC	64
ACE1		ATGTGGCGGTGACAGAGGAC	64

引物名称	引物序列(5’-3’)			Tm值/℃
PWL2	CATCCCTTACTCCGCCACTT			61
PWL2	CTCTTCTCGCTGTTCACGGT			61
AvrPiz-t	GCGACTTTCTTTCTTTGTCTCCT			55
AvrPiz-t	GACAGTGACCATGTTTTATACCG			55
Avr-pik	TCCTGCTGCTAACTCCATTC			60
Avr-pik	TCAACCAAGCGTAAACCTCG			60
Avr-Pita		CGACCCGTTTCCGCCTTTATT	56.5
Avr-Pita		TCCCTCCATTCCAACACTAACG	56.5
ACE1		CCCAGAGTTGGCGATGATGC	64
ACE1		ATGTGGCGGTGACAGAGGAC	64

引物名称	引物序列(5’-3’)	Tm值/℃
actin	ACTCCTGCTTCGAGATCCACATC	61
actin	TCGACGTCCGAAAGGATCTGT	61
PWL2	TCCTCCCTTTTGCTTTGGTC	57
PWL2	CGGATGTTGCCGTTGTTTT	57
AvrPiz-t	GACACTGGGGCACGATAAGG	62
AvrPiz-t	CGGAGGAGAGAACATCAGTGG	62
Avr-pik	GTCGAGAGCGAGACCCTAAC	62
Avr-pik	CAACCTGGAGGGAAGTCGC	62
Avr-Pita	CAAGGAATGTAACAGGGACGC	60
Avr-Pita	CCTGCTGTTTACGGGGTTGT	60
ACE1	TGCGTGACGAGATGTGGAAT	57.8
ACE1	TACGAGTGGCGAACATTGCT	57.8

引物名称	引物序列(5’-3’)	Tm值/℃
actin	ACTCCTGCTTCGAGATCCACATC	61
actin	TCGACGTCCGAAAGGATCTGT	61
PWL2	TCCTCCCTTTTGCTTTGGTC	57
PWL2	CGGATGTTGCCGTTGTTTT	57
AvrPiz-t	GACACTGGGGCACGATAAGG	62
AvrPiz-t	CGGAGGAGAGAACATCAGTGG	62
Avr-pik	GTCGAGAGCGAGACCCTAAC	62
Avr-pik	CAACCTGGAGGGAAGTCGC	62
Avr-Pita	CAAGGAATGTAACAGGGACGC	60
Avr-Pita	CCTGCTGTTTACGGGGTTGT	60
ACE1	TGCGTGACGAGATGTGGAAT	57.8
ACE1	TACGAGTGGCGAACATTGCT	57.8