大豆MEKK1调控细胞死亡与免疫反应分子机理的研究

It has been established in model plant Arabidopsis that MAPK signaling pathway plays a critical role in regulating cell death and immune responses. However, the studies of this signaling pathway in crops are still at rudimentary stage. Through virus-induced gene silencing in combination with transient expression mediated by agro-infiltration, we have identified a soybean MAP3K, designated GmMEKK1 hereafter, that plays both positive and negative roles in regulating cell death. In addition, our in vivo kinase assay revealed that the phosphorylation activity of an unidentified protein was GmMEKK1-dependent, implying a possibility that this protein functions downstream of GmMEKK1. Based on the preliminary results, we shall use the proposed strategies and methodologies to 1) identify the GmMAP2K and GmMAPK that function downstream of GmMEKK1 to establish GmMEKK1-GmMKKx-GmMPKx signaling module; 2) identify both the target proteins that function downstream of GmMEKK1-GmMKKx-GmMPKx signaling module and the biological processes in which the target proteins are involved; 3) explore the relationship between the identified target proteins and cell death; 4) reveal the identity of the protein whose phosphorylation activity is dependent upon GmMEKK1 and explore its role in cell death; 5) examine whether the cell death induced by silencing GmMEKK1 and the cell death induced by overexpressing GmMEKK1share the same mechanism? The ultimate goal of this proposal is to gain insight into the molecular mechanism of GmMEKK1 participated signaling pathways in regulation of cell death and immunity.

在模式植物拟南芥中已证实MAPK信号转导途径在植物细胞死亡和免疫反应中起着重要作用，但在粮食作物中的研究尚处于起步阶段。我们前期通过病毒诱导的基因沉默(VIGS)及农杆菌介导的瞬时表达鉴定出一个对细胞死亡兼起正、负调控作用的大豆MAP3K， GmMEKK1；并通过活体激酶分析发现一个磷酸化活性依赖于GmMEKK1、身份尚未被鉴定的蛋白。本申请拟在前期工作的基础上经所提议的研究手段及策略1)鉴定出作用于大豆GmMEKK1下游的GmMAP2K及GmMAPK; 2)鉴定出受该激酶途径调控的靶蛋白及其所参与的生物学过程; 3) 探明靶蛋白及其所参与的生物学过程与细胞死亡间的关系；4）查明磷酸化活性依赖于GmMEKK1未知蛋白的身份并探明其在细胞死亡中的作用；5)比较沉默与过表达GmMEKK1诱导的细胞死亡是否享有相同的机制。通过以上研究期望揭示大豆GmMEKK1调控细胞死亡与免疫反应的分子机理。

MAPK信号通路在植物免疫中起着重要作用。我们前期发现大豆GmMPK4是细胞死亡与免疫反应的负调控因子, 而大豆GmMPK6在细胞死亡与免疫反应中兼具正、负调控作用。 但作用于GmMPK4及GmMPK6上游的GmM2K（GmMAPKK）及GmM3K(GmMAPKKK)尚未鉴定出。利用菜豆豆荚斑驳病毒 (Bean pod mottle virus, BPMV)介导的基因沉默技术，通过沉默大量GmM2K及GmM3K编码基因，我们发现沉默一个GmM3K基因，GmMEKK1，可导致沉默植株接种叶片及系统叶片上发生超敏性细胞死亡 (Hypersensitive responses, HR)，说明GmMEKK1负调控HR。GmMEKK1沉默株中过量积累水杨酸 (SA)与过氧化氢 (H2O2),说明GmMEKK1通过负调控SA与H2O2而参与HR的调控。RNAseq结果表明沉默株中参与初级代谢、转录与翻译、光合作用及生长发育相关的基因与对照株相比显著下调； 而参与防御反应及逆境胁迫的基因的表达则显著上调, 说明防御反应的激活是以牺牲生长发育为代价的。与防御反应激活的表型相一致，沉默株对大豆花叶病毒 (Soybean mosaic virus, SMV)及白粉病的抗性显著增强。我们还发现，沉默GmMEKK1降低了GmMPK6对flg22的应答激活，但增强了GmMPK3对flg22的应答激活，说明GmMEKK1正调控GmMPK6的激活，但负调控GmMPK3的激活。出乎意外的是，与拟南芥中不同，flg22、SA及细菌、病毒侵染均不能激活大豆中的GmMPK4。有趣的是在烟草叶片中通过农杆菌注射瞬时表达GmMEKK1可诱导细胞死亡，说明其也可正调控HR，亦即GmMEKK1兼具正负调控HR的效应。综述所述，GmMEKK1可能是通过促进GmMPK6的活性及抑制GmMPK3的活性而参与HR与免疫反应的调控。GmMPK4在大豆HR及免疫反应中的作用还有待进一步研究。