Cloning of tobacco genes that elicit the hypersensitive response
Cloning of tobacco genes that elicit the hypersensitive response
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DOI：
10.1023/a:1005949304445
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发表时间：
1998-03-01
影响因子：
5.1
通讯作者：
Holt, CA
中科院分区：
文献类型：
--
作者：
Karrer, EE;Beachy, RN;Holt, CA
采用功能筛选的方法分离出植物病原防御过程中引发过敏反应（hypersensitive response， HR）通路的基因。以烟草花叶病毒（TMV）为载体，克隆了烟草花叶病毒基因文库。产生了侵染转录本，并将其用于接种缺乏抗氮基因（基因型Xanthi N）的烟草植株。大约1/1000的感染转录本产生局部病变，因此可能引发HR。利用RT-PCR方法从50个病变形成克隆中提取cDNA插入片段，并对12个独特克隆进行测序。与蛋白质数据库的比较显示其同源性(a)泛素，(b)烟草肿瘤相关蛋白，类似于kunitz型胰蛋白酶抑制剂和(c)核糖体蛋白S14。剩下的9个克隆与已知的蛋白质没有同源性，因此被认为是新的。5个克隆能够诱导烟草HR特异性激活基因PR2的表达。对编码泛素的克隆感染叶片的Northern和western blot分析强烈表明，感染产生了共抑制反应；染病叶片中泛素mRNA和蛋白的内源水平比健康叶片低50%左右。这一观察结果支持了先前关于泛素系统参与烟草HR[2]的报道，并验证了功能性克隆方法的实用性。
We used a functional screening method to isolate genes whose products elicit the hypersensitive response (HR) pathway of defense against plant pathogens. A cDNA library derived from tobacco leaves undergoing the HR was cloned into a tobacco mosaic virus (TMV)-based expression vector. Infectious transcripts were generated and used to inoculate tobacco plants lacking the N resistance gene (genotype Xanthi nn). Approximately 1/1000 of the infectious transcripts produced local lesions, and may thus elicit the HR. The cDNA inserts from 50 lesion-forming clones were recovered by RT-PCR, and 12 unique clones were sequenced. Comparisons with protein databases revealed homologies to (a) ubiquitin, (b) tobacco tumor-related protein, similar to Kunitz-type trypsin inhibitors and (c) ribosomal protein S14. The remaining nine clones revealed no homology to known proteins and are thus considered novel. Five clones were able to induce the expression of PR2, a gene which is specifically activated in the tobacco HR. Northern and western blot analyses of leaves infected by the clone encoding ubiquitin strongly suggest that the infection produced a co-suppression response; the endogenous levels of ubiquitin mRNA and protein in infected leaves are ca. 50% less than those found in healthy leaves. This observation supports a previous report on the involvement of the ubiquitin system in the tobacco HR [2], and validates the utility of the functional cloning method.