Suppression of DS1 Phosphatidic Acid Phosphatase Confirms Resistance to Ralstonia solanacearum in Nicotiana benthamiana

Nicotianabenthamiana is susceptible to Ralstonia solanacearum. To analyze molecular mechanisms for disease susceptibility, we screened a gene-silenced plant showing resistance to R. solanacearum, designated as DS1 (Disease suppression 1). The deduced amino acid sequence of DS1 cDNA encoded a phosphatidic acid phosphatase (PAP) 2. DS1 expression was induced by infection with a virulent strain of R. solanacearum in an hrp-gene-dependent manner. DS1 rescued growth defects of the temperature-sensitive ∆lpp1∆dpp1∆pah1 mutant yeast. Recombinant DS1 protein showed Mg2+-independent PAP activity. DS1 plants showed reduced PAP activity and increased phosphatidic acid (PA) content. After inoculation with R. solanacearum, DS1 plants showed accelerated cell death, over-accumulation of reactive oxygen species (ROS), and hyper-induction of PR-4 expression. In contrast, DS1-overexpressing tobacco plants showed reduced PA content, greater susceptibility to R. solanacearum, and reduced ROS production and PR-4 expression. The DS1 phenotype was partially compromised in the plants in which both DS1 and NbCoi1 or DS1 and NbrbohB were silenced. These results show that DS1 PAP may affect plant immune responses related to ROS and JA cascades via regulation of PA levels. Suppression of DS1 function or DS1 expression could rapidly activate plant defenses to achieve effective resistance against Ralstonia solanacearum.

本氏烟草（Nicotiana benthamiana）对青枯雷尔氏菌（Ralstonia solanacearum）易感。为解析病害易感的分子机制，我们筛选得到一株对青枯雷尔氏菌具有抗性的基因沉默植株，将其命名为DS1（病害抑制因子1，Disease suppression 1）。DS1 cDNA推导得到的氨基酸序列编码一种2型磷脂酸磷酸酶（phosphatidic acid phosphatase, PAP）。DS1的表达会被青枯雷尔氏菌强致病菌株以依赖hrp基因的方式诱导。DS1能够恢复温度敏感型∆lpp1∆dpp1∆pah1突变酵母的生长缺陷。重组DS1蛋白表现出不依赖Mg²+的PAP活性。DS1沉默植株的PAP活性降低，且磷脂酸（phosphatidic acid, PA）含量升高。接种青枯雷尔氏菌后，DS1沉默植株表现出细胞死亡加速、活性氧（reactive oxygen species, ROS）过量积累，以及病程相关蛋白4（PR-4）表达的超诱导。与之相反，过表达DS1的烟草植株磷脂酸含量降低，对青枯雷尔氏菌的易感性增强，且活性氧产生量与PR-4的表达水平均显著降低。当同时沉默DS1与NbCoi1，或同时沉默DS1与NbrbohB时，DS1沉默植株的抗性表型会被部分削弱。上述结果表明，DS1编码的PAP可能通过调控磷脂酸水平，影响与活性氧和茉莉酸（JA）信号级联相关的植物免疫应答。抑制DS1的功能或其表达能够快速激活植物防御系统，从而实现对青枯雷尔氏菌的有效抗性。