Yang Yong, Liu Jun, Yin Chuanchun, de Souza Vespoli Luciano, Ge Dongdong, Huang Yanyan, Feng Baomin, Xu Guangyuan, Manhães Ana Marcia E de A, Dou Shijuan, Criswell Cameron, Shan Libo, Wang Xiaofeng, He Ping
State Key Laboratory of Crop Stress Biology in Arid Areas, College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, China.
Department of Plant Pathology and Microbiology, Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, Texas 77843.
Plant Physiol. 2020 May;183(1):331-344. doi: 10.1104/pp.19.01555. Epub 2020 Mar 12.
A wide variety of intrinsic and extrinsic cues lead to cell death with unclear mechanisms. The infertility of some death mutants often hurdles the classical suppressor screens for death regulators. We have developed a transient RNA interference (RNAi)-based screen using a virus-induced gene silencing approach to understand diverse cell death pathways in Arabidopsis (). One death pathway is due to the depletion of a MAP kinase (MAPK) cascade, consisting of MAPK kinase kinase 1 (MEKK1), MKK1/2, and MPK4, which depends on a nucleotide-binding site Leu-rich repeat (NLR) protein SUMM2. Silencing of by virus-induced gene silencing resembles the mutant with autoimmunity and defense activation. The RNAi-based screen toward Arabidopsis T-DNA insertion lines identified SUMM2, MEKK2, and Calmodulin-binding receptor-like cytoplasmic kinase 3 (CRCK3) to be vital regulators of RNAi -induced cell death, consistent with the reports of their requirement in the death pathway. Similar with , overexpression of caused dosage- and SUMM2-dependent cell death, and the transcripts of were up-regulated in , , and MEKK2-induced cell death depends on CRCK3. Interestingly, CRCK3-induced cell death also depends on MEKK2, consistent with the biochemical data that MEKK2 complexes with CRCK3. Furthermore, the kinase activity of CRCK3 is essential, whereas the kinase activity of MEKK2 is dispensable, for triggering cell death. Our studies suggest that MEKK2 and CRCK3 exert concerted functions in the control of NLR SUMM2 activation and MEKK2 may play a structural role, rather than function as a kinase, in regulating CRCK3 protein stability.
多种内在和外在信号会导致细胞死亡，但其机制尚不清楚。一些死亡突变体的不育性常常阻碍对死亡调节因子进行经典的抑制子筛选。我们开发了一种基于瞬时RNA干扰（RNAi）的筛选方法，利用病毒诱导的基因沉默方法来了解拟南芥中的多种细胞死亡途径。一种死亡途径是由于丝裂原活化蛋白激酶（MAPK）级联反应的缺失，该级联反应由MAPK激酶激酶1（MEKK1）、MKK1/2和MPK4组成，它依赖于一个核苷酸结合位点富含亮氨酸重复序列（NLR）的蛋白SUMM2。通过病毒诱导的基因沉默使SUMM2沉默类似于具有自身免疫和防御激活的SUMM2突变体。对拟南芥T-DNA插入系进行基于RNAi的筛选，确定SUMM2、MEKK2和钙调蛋白结合受体样胞质激酶3（CRCK3）是RNAi诱导的细胞死亡的重要调节因子，这与它们在SUMM2死亡途径中的需求报道一致。与SUMM2类似，SUMM2的过表达导致剂量和SUMM2依赖性细胞死亡，并且SUMM2的转录本在MEKK2、CRCK3和MEKK2诱导的细胞死亡中上调。有趣的是，CRCK3诱导的细胞死亡也依赖于MEKK2，这与MEKK2与CRCK整合的生化数据一致。此外，CRCK3的激酶活性对于触发细胞死亡是必不可少的，而MEKK2的激酶活性则是可有可无的。我们的研究表明，MEKK2和CRCK3在控制NLR SUMM2激活中发挥协同作用，并且MEKK2在调节CRCK3蛋白稳定性方面可能起结构作用，而不是作为激酶发挥作用。
