State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, 100871, China.
Institute for Advanced Studies, Wuhan University, Wuhan, 430072, China.
J Integr Plant Biol. 2022 Sep;64(9):1789-1802. doi: 10.1111/jipb.13324. Epub 2022 Jul 28.
Nucleotide binding, leucine-rich repeat (NB-LRR) proteins are critical for disease resistance in plants, while we do not know whether S-acylation of these proteins plays a role during bacterial infection. We identified 30 Arabidopsis mutants with mutations in NB-LRR encoding genes from the Nottingham Arabidopsis Stock Center and characterized their contribution to the plant immune response after inoculation with Pseudomonas syringae pv tomato DC3000 (Pst DC3000). Of the five mutants that were hyper-susceptible to the pathogen, three (R5L1, R5L2 and RPS5) proteins contain the conserved S-acylation site in the N-terminal coiled-coil (CC) domain. In wild-type (WT) Arabidopsis plants, R5L1 was transcriptionally activated upon pathogen infection, and R5L1 overexpression lines had enhanced resistance. Independent experiments indicated that R5L1 localized at the plasma membrane (PM) via S-acylation of its N-terminal CC domain, which was mediated by PROTEIN S-ACYL TRANSFERASE 13/16 (PAT13, PAT16). Modification of the S-acylation site reduced its affinity for binding the PM, with a consequent significant reduction in bacterial resistance. PM localization of R5L1 was significantly reduced in pat13 and pat16 mutants, similar to what was found for WT plants treated with 2-bromopalmitate, an S-acylation-blocking agent. Transgenic plants expressing R5L1 in the pat13 pat16 double mutant showed no enhanced disease resistance. Overexpression of R5L1 in WT Arabidopsis resulted in substantial accumulation of reactive oxygen species after inoculation with Pst DC3000; this effect was not observed with a mutant R5L1 carrying a mutated S-acylation site. Our data suggest that PAT13- and PAT16-mediated S-acylation of R5L1 is crucial for its membrane localization to activate the plant defense response.
核苷酸结合富含亮氨酸重复（NB-LRR）蛋白对于植物的抗病性至关重要，然而，我们尚不清楚这些蛋白的 S-酰化在细菌感染过程中是否发挥作用。我们从诺丁汉拟南芥中心（Nottingham Arabidopsis Stock Center）鉴定了 30 个拟南芥突变体，这些突变体的基因编码 NB-LRR 蛋白，并在接种丁香假单胞菌 pv 番茄 DC3000（Pst DC3000）后对其在植物免疫反应中的作用进行了表征。在对病原体超敏感的五个突变体中，三个（R5L1、R5L2 和 RPS5）蛋白在 N 端卷曲螺旋（CC）结构域中含有保守的 S-酰化位点。在野生型（WT）拟南芥植物中，R5L1 在病原体感染后转录激活，并且 R5L1 过表达系具有增强的抗性。独立实验表明，R5L1 通过其 N 端 CC 结构域的 S-酰化定位于质膜（PM），这由蛋白 S-酰基转移酶 13/16（PAT13、PAT16）介导。S-酰化位点的修饰降低了其与 PM 结合的亲和力，导致细菌抗性显著降低。在 pat13 和 pat16 突变体中，R5L1 的 PM 定位显著减少，与用 S-酰化阻断剂 2-溴棕榈酸处理的 WT 植物相似。在 pat13 pat16 双突变体中表达 R5L1 的转基因植物在接种 Pst DC3000 后没有表现出增强的抗病性。在 WT 拟南芥中过表达 R5L1 后，接种 Pst DC3000 后会大量积累活性氧；而在携带突变 S-酰化位点的突变体 R5L1 中未观察到这种效应。我们的数据表明，R5L1 的 PAT13 和 PAT16 介导的 S-酰化对于其膜定位以激活植物防御反应至关重要。
