ZmACD6和ZmSec61β介导的玉米广谱抗性的分子机制研究

Enhancing the broad spectrum resistance has been a major breeding goal for crop. However, studies on broad spectrum resistance of crops are still lack and the underlying molecular mechanisms remains elusive. ACCELERATED CELL DEATH 6 (ACD6) is a key component of broad spectrum resistance in Arabidopsis that acts in a positive feedback loop with Salicylic acid (SA) to regulate multiple pattern receptors. ACD6 protein contains multiple membrane spanning regions and the efficiency of its biogenesis is induced by SA. During biogenesis of integral membrane protein, protein translocation into endoplasmic reticulum membrane requires Sec61 complex which includes the subunit Sec61β. It has been shown that ZmSec61 β was phosphorylated in response to SA induction. Protein phosphorylation is a reversible posttranslational modification which regulates protein activity, location and stability. Therefore, SA may increase the protein translocation of ZmACD6 and pattern receptors through phosphorylation of ZmSec61 β to enhance maize broad spectrum resistance. We will combine molecular, genetic and biochemical method to discern the molecular mechanism of ZmACD6 and ZmSec61β-mediated broad spectrum resistance. We also screen ZmACD6/ZmSec61β high expression line from a core collection of 290 maize inbred lines in China and check the multiple pathogens resistance in the lines are selected out. Thus, studying on ZmACD6/ZmSec61β not only serves as new entry point for understanding the regulation of the broad spectrum resistance in maize but also has practical applications for maize breeding program with improving broad spectrum resistance.

提高作物广谱抗性是遗传育种的重要目标，但关于作物广谱抗性的研究还很缺乏，其作用机制尚不清楚。ACD6是拟南芥中广谱抗性的关键基因，通过与水杨酸形成正反馈环路，调节了多种模式受体介导的信号。ACD6是跨膜蛋白，其生物合成受水杨酸的调节。跨膜蛋白合成中的蛋白质转运是由 Sec61通道介导的。Sec61β蛋白是Sec61通道的一个亚基，而水杨酸诱导了ZmSec61 β的磷酸化，因此水杨酸可能通过诱导Sec61β磷酸化调节了其活性，提高了ZmACD6及模式受体的转运效率，从而增强了玉米的广谱抗性。本项目将综合利用分子、遗传、生化等手段阐明ZmACD6和ZmSec61β的广谱抗病功能及其分子作用机制。另外，还将从290份玉米核心种质中筛选ZmACD6/ZmSec61β高表达的自交系并鉴定它们的广谱抗病性。项目的开展，不仅为作物广谱抗性的研究找到新的切入点，还将为玉米的抗病分子育种提供基因新资源 。

玉米病害严重影响玉米的产量和品质，危害全球的粮食生产安全。鉴定和利用广谱抗病基因被认为是控制病害发生最有效、最经济的方式。拟南芥质膜蛋白ACD6是水杨酸信号途径的关键调控组分，介导了对多种病原菌的广谱抗性。Sec61β是位于内质网膜上的跨膜复合体Sec61的一个亚基，后者通过为跨膜蛋白插入到细胞膜中提供了通道而调控了膜蛋白生物合成过程中关键性的第一步。已有的报道表明，水杨酸诱导了玉米ZmSec61β蛋白第57位丝氨酸的磷酸化。本课题通过功能分析玉米中ACD6-like基因，揭示ZmANK23为玉米中ACD6的同源基因，并将ZmANK23重新命名为ZmANKACD6。ZmANKACD6表达受水杨酸和病原真菌瘤黑粉病的诱导。利用CRISPR/Cas9介导的基因组编辑技术产生的ZmACD6敲除突变体提高了对瘤黑粉菌的感病性，而ZmACD6高表达的自交系SC-9提高了对瘤黑粉菌抗病性及水杨酸途径标签基因的表达，表明ZmACD6在玉米抗病反应中起重要正向调控作用。其次，本课题还通过筛选拟南芥ACD6基因的功能获得性突变体acd6-1的恢复突变体，获得一株由T-DNA插入AtSec61β启动子而造成功能失活的恢复突变体T-26。T-26部分抑制了acd6-1的自免疫激活表型，提供了Sec61β调控ACD6膜蛋白生物合成的关键遗传学证据。进一步，我们研究了ZmSec61β第57位磷酸化的作用，对ZmSec61β、激酶活性持续激活型ZmSec61βS57D和激酶活性持续抑制型ZmSec61βS57A转基因植株进行接瘤黑粉菌处理，结果表明ZmSec61β第57位磷酸化是发挥其抗病正向调控功能所必需的。重要的，ZmACD6/ZmBAK1在上述转基因植物中的表达与它们的抗病表型正相关。最后，通过转录组测序RNAseq揭示，ZmSec61β57D转基因植株上调了水杨酸信号途径以及赤霉素和生长素途径的核心负向调控因子基因的表达。综上推测，水杨酸诱导的ZmSec61β磷酸化调控了其活性，机制上可能通过促进抗病反应中关键调控膜蛋白如ZmACD6/ZmBAK1的生物合成，上调水杨酸信号途径，抑制赤霉素和生长素途径，从而在植物病原菌入侵时优先防御，更有效激活防卫反应以的抵抗病原菌的入侵。