利用R基因RRS1/RPS4探索水稻原生质体瞬时表达转化体系的研究

doi:10.16036/j.issn.1000-2650.2021.03.004

摘要/Abstract

摘要： 【目的】将拟南芥TNL型R基因RRS1/IRPS4转入水稻原生质体中,探索瞬时表达转化体系的最佳条件,并分析AtRRS1/AtRPS4对水稻内源ETI下游抗病基因表达的影响,为揭示水稻EDS1是否参与ETI反应途径提供实验依据。【方法】以日本晴两叶一心期幼苗为植物材料,设计不同梯度的PEG浓度(30%、35%、40%)、转化时的质粒浓度(0.5、1、2μg/μL)以及PEG介导的转化温度(0、18、37℃),利用Box- Benhnken设计三因素三水平来分析不同参数组合条件下对转化效果的影响,并对原生质体转化效率进行预测和试验验证。随后,利用该瞬时系统表达拟南芥TNL型R基因RRS1/IRPS4,并通过检测水稻内源抗病标志基因的表达水平来揭示其对水稻抗病能力的影响。【结果】经过试验验证得到水稻原生质体转化最佳条件:PEG浓度36.16%、质粒浓度1.72 μg/μL转化温度37℃,该组合条件下转化效率可达75%;利用该优化体系开展的瞬转研究发现拟南芥TNL型R基因/基因对RRS1/RPS4能显著提高水稻原生质体中抗病相关基因的表达。【结论】构建了高效的水稻原生质体瞬时转化系统,并以此为平台,研究揭示了拟南芥TNL型R基因RRS1/RPS4可能通过水稻内源的EDS1去诱导ETI下游抗病免疫反应的发生。

关键词: 水稻, 原生质体, 瞬时表达, R基因, 转化体系

Abstract: 【Objective】 The Arabidopsis TNL type R gene RRS1/RPS4 was transferred into rice protoplasts to explore the optimal conditions for transient expression transformation system,and the effect of these R genes on the expression of endogenous ETI downstream disease resistance genes in rice was analyzed to provide experimental basis for revealing whether OsEDS1 is involved in ETI response pathway. 【Method】 Seedlings in two leaf and one heart stage of Nipponbare were used as plant materials to explore the optimal combination of conditions for PEG-mediated transformation. By designing different gradients of PEG concentration（30%,35%,40%）,and PEG-mediated transformation temperature(0,18,37℃),Box-Benhnken was used to design three factors and three levels to analyze the effect of different parameter combinations on the transformation effect,and the protoplast transformation efficiency was predicted and experimentally verified. Subsequently,the Arabidopsis TNL type R gene RRS1/RPS4 was single/cotransformed into the rice protoplast transient expression system,and their influence on rice disease resistance was revealed by detecting the expression level of rice endogenous disease resistance marker genes.【Result】 The optimal conditions verified by experiments for rice protoplast transformation were as follows：PEG concentration 36.16%,plasmid concentration 1.72μg/μL,transformation temperature 37℃,and the conversion efficiency can reach 75% under the combined conditions. Transient studies using this optimized system found that the Arabidopsis TNL type R gene/gene pair RRS1/RPS4 can significantly increase the expression of disease resistance-related genes in rice protoplasts.【Conclusion】 In thisstudy,an efficient protoplast transient transformation system was established and used as a platform,this study revealed that the Arabidopsis TNL type R gene RRS1/RPS4 may induce downstream immune response through the endogenous EDS1 in rice.

Key words: rice, protoplast, transient expression, R gene, transformation system

中图分类号:

S511

李丽, 赵彦丰, 吴琪琦, 黄燕. 利用R基因RRS1/RPS4探索水稻原生质体瞬时表达转化体系的研究[J]. 四川农业大学学报, 2021, 39(3): 297-306.

LLi, ZHAO Yanfeng, WU Qiqi, HUANG Yan. Exploring the Transient Expression and Transformation System of Rice Protoplasts by Using R Gene RRSl/RPS4[J]. Journal of Sichuan Agricultural University, 2021, 39(3): 297-306.

参考文献

[1] THOMMA B P,NURNBERGER T,JOOSTEN M H.Of PAMPs and effectors:the blurred PTI-ETI dichotomy[J]. The Plant Cell,2011,23(1):4-15.
[2] MAEKAWA T,KUFER T A,SCHULZE-LEFERT P.NLR functions in plant and animal immune systems:so far and yet so close[J]. Nature Immunology,2011,12(9):817-826.
[3] ELLIS J,DODDS P,PRYOR T.Structure,function and evolution of plant disease resistance genes[J]. Current Opinion in Plant Biology,2000,3(4):278-284.
[4] DODDS P N,LAWRENCE G J,CATANZARITI A M,et al.Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes[J]. Proceedings of the National Academy of Sciences of the United States of America,2006,103(23):8888-8893.
[5] KRASILEVA K V,DAHLBECK D,STASKAWICZ B J.Activation of an Arabidopsis resistance protein is specified by the in planta association of its leucine-rich repeat domain with the cognate oomycete effector[J]. The Plant Cell,2010,22(7):2444-2458.
[6] CATANZARITI A M,DODDS P N,VE T,et al.The AvrM effec tor from flax rust has a structured C-terminal domain and interacts directly with the M resistance protein[J]. Molecular Plant-Microbe Interactions,2010,23(1):49-57.
[7] CESARI S,BERNOUX M,MONCUQUET P,et al.A novel conserved mechanism for plant NLR protein pairs:the "integrated decoy" hypothesis[J]. Frontiers in Plant Science,2014,5:606.
[8] SAUCET S B,MA Y,SARRIS P F,et al.Two linked pairs of Arabidopsis TNL resistance genes independently confer recognition of bacterial effector AvrRps4[J]. Nature ommunications,2015,6:6338.
[9] SOHN K H,SEGONZAC C,RALLAPALLI G,et al.The nuclear immune receptor RPS4 is required for RRS1SLH1-dependent constitutive defense activation in Arabidopsis thaliana[J]. PLoS Genetics,2014,10(10):e1004655.
[10] NARUSAKA M,TOYODA K,SHIRAISHI T,et al.Leucine zipper motif in RRS1 is crucial for the regulation of Arabidopsis dual resistance protein complex RPS4/RRS1[J]. Scientific Reports,2016,6:18702.
[11] LE ROUX C,HUET G,JAUNEAU A,et al.A receptor pair with an integrated decoy converts pathogen disabling of transcription factors to immunity[J]. Cell,2015,161(5):1074-1088.
[12] DANGL J L,JONES J D.Plant pathogens and integrated defence responses to infection[J]. Nature,2011,411:826-883
[13] CHISHOLM S T,COAKER G,DAY B,et al.Host-microbe in-teractions:shaping the evolution of the plant immune response[J]. Cell,2006,124:803-814.
[14] WIERMER M,FEYS B J,PARKER J E.Plant immunity:the EDS1 regulatory node[J]. Current Opinion in Plant Biology,2005,8(4):383-389.
[15] ZHOU Z,PANG Z,ZHAO S,et al.Importance of OsRac1 and RAI1 in signalling of nucleotide-binding site leucine-rich repeat protein-mediated resistance to rice blast disease[J]. The New Phytologist,2019,223(2):828-838.
[16] MONOSI B,WISSER R J,PENNILL L,et al.Full-genome analysis of resistance gene homologues in rice[J]. Theoretical and Applied Genetics,2004,109(7):1434-1447.
[17] 刘维,刘浩,董双玉,等. 水稻叶鞘原生质体转化体系的构建及Pik-H4 和AvrPik-H4 蛋白的瞬时表达[J]. 中国农业科学,2017,50(23):4575-4584.
[18] 孙鹤,郎志宏,朱莉,等. 玉米、小麦、水稻原生质体制备条件优化[J]. 生物工程学报,2013,29(2):224-234.
[19] ZHANGY,SU J,DUANS,et al.Ahighly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes[J]. Plant Methods,2011,7(1):30.
[20] SHEEN J.Signal transduction in maize and Arabidopsis mesophyll protoplasts[J]. Plant Physiology,2001,127(4):1466-1475.
[21] 段炼,钱君,郭小雨,等. 一种快速高效的水稻原生质体制备和转化方法的建立[J]. 植物生理学报,2014,50(3):351-357.
[22] 陈越,王玲仙,付坚,等. 云南疣粒野生稻原生质体高效分离培养体系的建立[J]. 西南农业学报,2019,32(10):2265-2272.
[23] KIM N,MOON S J,MIN M K,et al.Functional characterization and reconstitution of ABA signaling components using transient gene expression in rice protoplasts[J]. Frontiers in Plant Science,2015,6:614.
[24] TAKAI R,KANEDA T,ISOGAI A,et al.A new method of defense response analysis using a transient expression system in rice protoplasts[J]. Bioscience Biotechnology and Biochemistry,2007,71(2):590-593.
[25] SHEN J,FU J,MA J,et al.Isolation,culture,and transient transformation of plant protoplasts[J]. Current Protocols in Cell Biology,2014,63:1-17.
[26] 梁大伟. 拟南芥叶肉原生质体分离及瞬时表达体系的建立[D].兰州:兰州大学,2009:19-20.
[27] PONCIANO G,YOSHIKAWA M,LEE J L,et al.Pathogenesisrelated gene expression in rice is correlated with developmentally controlled Xa21-mediated resistance against Xanthomonas oryzae pv. oryzae[J]. Physiological & Molecular Plant Pathology,2006,69(4):131-139.
[28] 谢纯政,刘海燕,李玲,等. 植物病程相关蛋白PR10研究进展[J]. 分子植物育种,2008,6(5):949-953.