State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, East Campus, 510006 Guangzhou, China.
Plant Physiol. 2022 Aug 1;189(4):2382-2395. doi: 10.1093/plphys/kiac205.
Type III protein secretion systems of nitrogen-fixing rhizobia deliver effector proteins into leguminous host cells to promote or inhibit the nodule symbiosis. However, mechanisms underlying effector-triggered inhibition of nodulation remain largely unknown. Nodulation outer protein T (NopT) of Sinorhizobium sp. NGR234 is an effector protease related to the Pseudomonas effector Avirulence protein Pseudomonas phaseolicola B (AvrPphB). Here, we constructed NGR234 mutants producing different NopT variants and found that protease activity of NopT negatively affects nodulation of smooth crotalaria (Crotalaria pallida). NopT variants lacking residues required for autocleavage and subsequent lipidation showed reduced symbiotic effects and were not targeted to the plasma membrane. We further noticed that Sinorhizobium fredii strains possess a mutated nopT gene. Sinorhizobium fredii USDA257 expressing nopT of NGR234 induced considerably fewer nodules in soybean (Glycine max) cv. Nenfeng 15 but not in other cultivars. Effector perception was further examined in NopT-expressing leaves of Arabidopsis (Arabidopsis thaliana) and found to be dependent on the protein kinase Arabidopsis AvrPphB Susceptible 1 (AtPBS1) and the associated resistance protein Arabidopsis Resistance to Pseudomonas syringae 5 (AtRPS5). Experiments with Nicotiana benthamiana plants indicated that the soybean homolog GmPBS1-1 associated with AtRPS5 can perceive NopT. Further analysis showed that NopT cleaves AtPBS1 and GmPBS1-1 and thus can activate these target proteins. Insertion of a DKM motif at the cleavage site of GmPBS1-1 resulted in increased proteolysis. Nodulation tests with soybeans expressing an autoactive GmPBS1-1 variant indicated that activation of a GmPBS1-1-mediated resistance pathway impairs nodule formation in cv. Nenfeng 15. Our findings suggest that legumes face an evolutionary dilemma of either developing effector-triggered immunity against pathogenic bacteria or establishing symbiosis with suboptimally adapted rhizobia producing pathogen-like effectors.
固氮根瘤菌的 III 型蛋白分泌系统将效应蛋白输送到豆科宿主细胞中，以促进或抑制根瘤共生。然而，效应蛋白触发的结瘤抑制机制在很大程度上仍然未知。NGR234 中的结瘤外蛋白 T（NopT）是一种与假单胞菌效应因子 Pseudomonas phaseolicola B（AvrPphB）相关的效应蛋白酶。在这里，我们构建了产生不同 NopT 变体的 NGR234 突变体，并发现 NopT 的蛋白酶活性对光滑野豌豆（Crotalaria pallida）的结瘤有负面影响。缺乏自身切割和随后脂化所需残基的 NopT 变体显示出降低的共生效应，并且不靶向质膜。我们进一步注意到，根瘤菌 fredii 菌株具有突变的 nopT 基因。表达 NGR234 nopT 的根瘤菌 fredii USDA257 在大豆（Glycine max）品种 Nenfeng 15 中诱导的结瘤数量明显减少，但在其他品种中没有。进一步在拟南芥（Arabidopsis thaliana）表达 NopT 的叶片中进行了效应物感知实验，发现该实验依赖于蛋白激酶 Arabidopsis AvrPphB Susceptible 1（AtPBS1）和相关的抗性蛋白 Arabidopsis Resistance to Pseudomonas syringae 5（AtRPS5）。与 Nicotiana benthamiana 植物的实验表明，与 AtRPS5 相关的大豆同源物 GmPBS1-1 可以感知 NopT。进一步分析表明，NopT 切割 AtPBS1 和 GmPBS1-1，从而可以激活这些靶蛋白。在 GmPBS1-1 的切割位点插入 DKM 基序会导致蛋白水解增加。在表达自动激活 GmPBS1-1 变体的大豆中进行的结瘤试验表明，激活 GmPBS1-1 介导的抗性途径会损害 Nenfeng 15 品种的结瘤形成。我们的研究结果表明，豆科植物面临着一个进化困境，要么针对病原菌产生效应蛋白触发的免疫，要么与产生类似病原菌效应蛋白的适应性较差的根瘤菌建立共生关系。
