玉米糖代谢与RBSDV侵染的互作调控研究及葡萄糖防御应答感应子鉴定

Maize rough dwarf disease, caused by Rice black streaked dwarf virus (RBSDV), poses a severe threat to maize production in China. To date, the mechanisms of plant hosts against RBSDV remain largely elusive, and the research on the contribution of plant primary metabolism to plant defense against viruses is also limited. We have found that the sugar metabolism of maize influenced by RBSDV infection, and external applied glucose, in turn, regulated the RBSDV accumulation. To explore the function and underlying mechanism of sugar metabolism against RBSDV, firstly, we will perform an analysis of the accumulation of sugar metabolism-related gene transcripts by RT-qPCR and the soluble sugars by GC-MS（Gas Chromatography-Mass Spectrometer）combined with MEA（microplate enzymatic assays）at different infection stages, in RBSDV-infected maize plants, for revealing the time-course regulation of RBSDV to maize sugar metabolism. Secondly, the effects of external applied glucose with concentration gradient on RBSDV accumulation will be investigated, to characterize the regulation model of glucose metabolism to RBSDV infection. Thirdly, the comparative transcriptome analyses combined with virus-induced gene silencing (VIGS)-dependent gene function analyses will be used to identify the specific sensors responsible for glucose sensing during RBSDV infection. A series of related experiments will be performed, to elucidate the interactive regulation model between sugar metabolism and RBSDV infection, and to reveal the mechanism underlying the regulation of glucose metabolism to RBSDV. Eventually, these efforts will contribute to the durable control of maize rough dwarf disease by genetically regulating primary metabolism-related specific genes. Besides, it will probably expand our current understanding of the interaction between plant primary metabolism and virus infections.

水稻黑条矮缩病毒（RBSDV）引起的玉米粗缩病严重危害我国玉米生产。迄今关于寄主对RBSDV侵染的防御机理及初级代谢在寄主与病毒互作中功能的报道较少。我们前期研究发现RBSDV侵染影响玉米糖代谢，且葡萄糖处理可调控RBSDV积累。为探索糖代谢在RBSDV与玉米互作中的功能及机制，我们拟通过对RBSDV侵染玉米中糖代谢基因表达检测和可溶糖含量的GC-MS/MEA测定，明确RBSDV对玉米糖代谢的时序调控；通过分析不同浓度外源葡萄糖对RBSDV侵染的影响，明确葡萄糖代谢对RBSDV侵染的调控模式；以比较转录组学、生物信息学及基于VIGS系统的基因功能分析，鉴定葡萄糖调控RBSDV侵染的感应子基因。以解析玉米糖代谢与RBSDV侵染的互作模式和葡萄糖调控RBSDV侵染的信号传递机制；为初级代谢路径中抗粗缩病基因挖掘及抗病玉米材料的创制提供理论参考，对于病毒与寄主初级代谢互作理论的拓展也有重要意义。

植物病毒侵染会调控寄主代谢并利用细胞物质以存活和增殖，作为寄主中重要的能量和物质来源，初级代谢路径会被广泛重编程，以维持自身生长发育和胁迫应答等多重需求下的平衡稳态。目前有关病毒与植物寄主初级代谢互作调控的研究较少，且集中于双子叶植物。水稻黑条矮缩病毒（RBSDV）是我国农业生产中的重要病原，可引起玉米粗缩病和水稻黑条矮缩病等病害。我们前期研究发现RBSDV编码p8蛋白与玉米中能量稳态调节器SnRK1复合体AKINβγ亚基直接互作，本项目以RBSDV及其玉米寄主为研究对象，探索玉米糖代谢与RBSDV的互作调控模式及糖感应机制。本研究以RT-qPCR结合MEA法测定RBSDV侵染玉米不同阶段的糖代谢基因表达水平和可溶性糖含量，明确了RBSDV对玉米糖代谢的动态调控过程，调控趋势表现为对蔗糖分解代谢的抑制和对葡萄糖转化的促进，从而导致玉米中蔗糖含量升高，而葡萄糖和果糖含量下降，并明确21 dpi是病毒积累和糖代谢调控的一个关键时间节点。为明确葡萄糖对RBSDV侵染的影响，优化玉米茎干注射和叶面喷施两种外源糖处理方式和适宜浓度，明确0.1 M葡萄糖喷施处理玉米叶面可促进RBSDV症状发展和病毒积累。为鉴定葡萄糖调控RBSDV 侵染的感应子基因，以RT-qPCR法分析葡萄糖处理的玉米中推测的葡萄糖感应基因（HXK1、TOR、SnRK1a和SnRK1b）转录水平，明确SnRK1a基因被下调表达，而TOR基因被上调表达；而RBSDV侵染21 dpi时， SnRK1a下调表达，且沉默SnRK1复合体的ZmAKINβγ亚基促进RBSDV积累。以上结果表明SnRK1a是玉米中感应葡萄糖的重要受体基因，且在RBSDV侵染过程中发挥负调节作用。比较转录组学分析表明0.5 M葡萄糖可诱导玉米中SnRK1复合体调节亚基AKINβ和AKINβγ下调表达及TOR、HXK7和HXK9的上调表达，且影响生长素、赤霉素、乙烯等多条激素路径，进一步证明玉米具有与拟南芥中类似的葡萄糖感应机制且与激素路径存在交互调控。本项目从初级代谢角度解析了RBSDV与玉米互作机制，拓展了对单子叶植物糖代谢与病毒互作模式和信号传递机制的认识；对调控 RBSDV侵染的葡萄糖感应基因的挖掘，有望为通过基因编辑干扰玉米初级代谢以构建长效防控粗缩病的玉米新种质提供候选靶标。