Bendahmane Abdelhafid, Farnham Garry, Moffett Peter, Baulcombe David C
The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK.
Plant J. 2002 Oct;32(2):195-204. doi: 10.1046/j.1365-313x.2002.01413.x.
Rx in potato encodes a protein with a nucleotide binding site (NBS) and leucine-rich repeats (LRR) that confers resistance against Potato virus X. The NBS and LRR domains in Rx are present in many disease resistance proteins in plants and in regulators of apoptosis in animals. To investigate structure-function relationships of NBS-LRR proteins we exploited the potential of Rx to mediate a cell death response. With wild-type Rx cell death is elicited only in the presence of the viral coat protein. However, following random mutagenesis of Rx, we identified mutants in which cell death is activated in the absence of viral coat protein. Out of 2500 Rx clones tested there were seven constitutive gain-of-function mutants carrying eight independent mutations. The mutations encoded changes in the LRR or in conserved RNBS-D and MHD motifs of the NBS. Based on these findings we propose that there are inhibitory domains in the NBS and LRR. The constitutive gain-of-function phenotypes would be due to deletion or modification of these inhibitory domains. However activation of Rx is not simply release of negative regulation by the LRR and adjacent sequence because deleted forms of Rx that lack constitutive gain of function mutations are not active unless the protein is overexpressed.
马铃薯中的Rx基因编码一种具有核苷酸结合位点（NBS）和富含亮氨酸重复序列（LRR）的蛋白质，该蛋白质赋予马铃薯对X病毒的抗性。Rx中的NBS和LRR结构域存在于植物中的许多抗病蛋白以及动物的细胞凋亡调节因子中。为了研究NBS-LRR蛋白的结构-功能关系，我们利用Rx介导细胞死亡反应的潜力。野生型Rx只有在病毒外壳蛋白存在时才引发细胞死亡。然而，在对Rx进行随机诱变后，我们鉴定出了在没有病毒外壳蛋白的情况下激活细胞死亡的突变体。在测试的2500个Rx克隆中，有7个组成型功能获得突变体携带8个独立突变。这些突变编码了LRR或NBS保守的RNBS-D和MHD基序中的变化。基于这些发现，我们提出在NBS和LRR中存在抑制结构域。组成型功能获得表型可能是由于这些抑制结构域的缺失或修饰。然而，Rx的激活不仅仅是通过LRR和相邻序列释放负调控，因为缺乏组成型功能获得突变的Rx缺失形式没有活性，除非该蛋白质过表达。
