Background: R gene-mediated resistance is one of the most effective mechanisms of immunity against pathogens in plants. To date some components that regulate the primary steps of plant immunity have been isolated, however, the molecular dissection of defense signaling downstream of the R proteins remains to be completed. In addition, R genes are known to be highly variable, however, the molecular mechanisms responsible for this variability remain obscure.
Results: To identify novel factors required for R gene-mediated resistance in rice, we used rice insertional mutant lines, induced by the endogenous retrotransposon Tos17, in a forward screen involving the rice blast fungus Magnaporthe oryzae. We inoculated 41,119 mutant lines with the fungus using a high throughput procedure, and identified 86 mutant lines with diminished resistance. A genome analysis revealed that 72 of the 86 lines contained mutations in a gene encoding a nucleotide binding site (NB) and leucine rich repeat (LRR) domain-containing (NLR) protein. A genetic complementation analysis and a pathogenesis assay demonstrated that this NLR gene encodes Pish, which confers resistance against races of M. oryzae containing avrPish. The other 14 lines have intact copies of the Pish gene, suggesting that they may contain mutations in the signaling components downstream of Pish. The genome analysis indicated that Pish and its neighboring three NLR genes are high similar to one another and are tandemly located. An in silico analysis of a Tos17 flanking sequence database revealed that this region is a "hot spot" for insertion. Intriguingly, the insertion sites are not distributed evenly among these four NLR genes, despite their similarity at the sequence and expression levels.
Conclusions: In this work we isolated the R gene Pish, and identified several other mutants involved in the signal transduction required for Pish-mediated resistance. These results indicate that our genetic approach is efficient and useful for unveiling novel aspects of defense signaling in rice. Furthermore, our data provide experimental evidence that R gene clusters have the potential to be highly preferred targets for transposable element insertions in plant genomes. Based on this finding, a possible mechanism underlying the high variability of R genes is discussed.
R基因介導(dǎo)的抗性是植物抵御病原菌的一種最為高效的防衛(wèi)機制。目前已經(jīng)分離到了一些參與調(diào)控植物免疫應(yīng)答主要步驟的組成因子，但對位于R蛋白下游的防衛(wèi)信號的分子機理的研究還不完善。另外，我們知道R基因是高度變異的，但對引起這種變異的分子機制還不是很清楚。為了發(fā)現(xiàn)R基因介導(dǎo)的抗性途徑中的新的因子，作者利用水稻內(nèi)源TOS17反轉(zhuǎn)座子插入突變體，對水稻稻瘟病抗性進行遺傳篩選。利用高通量的方法，作者對41,119個突變體株系接種病原菌，發(fā)現(xiàn)在86個突變株系中抗性被削弱。通過基因組分析，發(fā)現(xiàn)86個株系中的72個，在一個編碼包含核苷酸結(jié)合位點（NBS）和富含亮氨酸重復(fù)序列（LRR）的蛋白（NBS-LRR蛋白）的基因內(nèi)發(fā)生了突變。遺傳互補實驗和病原菌發(fā)生實驗證明了這個NIS-LRR基因編碼Pish，從而產(chǎn)生了對含有avrPish的稻瘟病小種的抗性。在其他14個株系中Pish基因是完整的，表明這些突變發(fā)生在Pish下游的信號組分上。基因組分析顯示Pish和其周圍的3個NBS-LRR基因高度相似并串聯(lián)排列。通過分析數(shù)據(jù)庫中T os17插入位點的旁連序列，表明該區(qū)域是一個插入熱點。有趣的是插入位點在這4個NBS-LRR基因不是均勻分布的，盡管其序列和表達水平都十分相似。綜上所述，本研究分離了Pish這個R基因，并鑒定到一些參與Pish介導(dǎo)的抗性信號傳導(dǎo)途徑中所必需的其他基因的突變體。結(jié)果表明本研究所采用的遺傳策略是有效的，并且能夠發(fā)現(xiàn)一些新的參與水稻防衛(wèi)信號的基因。此外，數(shù)據(jù)還為植物基因組中轉(zhuǎn)座元件可能更偏愛插入R基因簇中提供了實驗證據(jù)。