The Broad-Spectrum Blast Resistance Gene Pi9 Encodes a Nucleotide-Binding Site–Leucine-Rich Repeat Protein and Is a Member of a Multigene Family in Rice

Shaohong Qu, Guifu Liu, Bo Zhou, Maria Bellizzi, Lirong Zeng, Liangying Dai, Bin Han and Guo-Liang Wang
Genetics, 2006, 172(3): 1901-1914  DOI: 10.1534/genetics.105.044891     追溯原文......本站官方QQ群:62473826

The broad-spectrum rice blast resistance gene Pi9 was cloned using a map-based cloning strategy. Sequencing of a 76-kb bacterial artificial chromosome (BAC) contig spanning the Pi9 locus led to identification of six tandemly arranged resistance-like genes with a nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) (Nbs1-Pi9–Nbs6-Pi9). Analysis of selected Pi9 deletion mutants and transformation of a 45-kb fragment from the BAC contig into the susceptible rice cultivar TP309 narrowed down Pi9 to the candidate genes Nbs2-Pi9 and Nbs3-Pi9. Disease evaluation of the transgenic lines carrying the individual candidate genes confirmed that Nbs2-Pi9 is the Pi9 gene. Sequence comparison analysis revealed that the six paralogs at the Pi9 locus belong to four classes and gene duplication might be one of the major evolutionary forces contributing to the formation of the NBS–LRR gene cluster. Semiquantitative reverse transcriptase (RT)–PCR analysis showed that Pi9 was constitutively expressed in the Pi9-resistant plants and was not induced by blast infection. The cloned Pi9 gene provides a starting point to elucidate the molecular basis of the broad-spectrum disease resistance and the evolutionary mechanisms of blast resistance gene clusters in rice.

编码NBS-LRR蛋白的稻瘟病广谱抗性基因Pi9是水稻多基因家族的成员

通过图位克隆的方法克隆了一个水稻稻瘟病广谱抗性基因Pi9。对包含Pi9位点的长76kb的细菌人工染色体重叠群进行测序,鉴定出了6个串联排列的含有核苷酸结合位点(NBS)及富含亮氨酸重复 (LRR)的抗病相关基因(Nbs1-Pi9–Nbs6-Pi9)。对Pi9选择性缺失突变体的分析以及将45kb的BAC重叠群转到感病品种TP309,将Pi9的候选基因缩小为Nbs2-Pi9和Nbs3-Pi9。对转化有单个候选基因的转基因植株进行抗病检测确定了Nbs2-Pi9为Pi9基因。序列比较分析表明位于Pi9位点的这6个旁系同源基因属于4个基因家族,同时基因复制可能是这个NBS–LRR基因簇形成的主要进化选择压力之一。半定量RT-PCR分析表明Pi9在抗病植株中持续表达,不受稻瘟病侵染诱导。Pi9的克隆为阐明广谱抗病性的分子基础以及水稻抗稻瘟病基因簇的进化机制开辟了新的起点。
结果:通过对76kb的基因组片段进行序列分析从Pi9位点鉴定出了一个NBS–LRR基因簇;序列比较和系统发生分析表明这6个Pi9 NBS–LRR基因属于4类旁系同源基因;对Pi9缺失突变体以及大片段DNA插入的分析将Pi9定位在含有Nbs2-Pi9和Nbs3-Pi9的基因组区域中;转化单个候选基因确定Nbs2-Pi9为Pi9基因,同时转化有Pi9的株系与Pi9的供体亲本具有相同的广谱抗病性;Pi9编码一个NBS–LRR蛋白,长1032个氨基酸;Pi9在抗病植株中组成性表达。
讨论:从R基因簇中图位克隆Pi9基因;Pi9基因和蛋白的结构特征;Pi9位点的基因组结构;Pi9对稻瘟病的光谱抗病性的分子机制。


基因列表
  稻瘟病抗性基因 Pi9; Pi2/Piz-5; Pi50; Piz; Pi