菌根诱导的两个SlGH3基因在番茄丛枝菌根共生中的功能和响应机制研究

Arbuscular mycorrhizal (AM) symbiosis between plant roots and members of an ancient phylum of fungi, the Glomeromycota, is one of the important evolutionary mechanisms of terrestrial plants to increase fitness to environmental stresses. It has been well documented that the establishment of AM symbiosis is regulated by a number of genes, of which the expression were commonly inducible in mycorrhizal roots. In our previous work, we have identified two mycorrhiza-induced genes, SlGH3.3 and SlGH3.4, from the tomato GH3 gene family.Expression and functional analysis revealed that the two genes are responsive to the exogenous IAA signal and their encoding proteins could in vitro catalyze IAA conjugate to different amino acids. Therefore, we arised a hypothesis that the two genes, SlGH3.3 and SlGH3.4, could modulate the establishment of AM symbiosis, probably by mean of regulating the IAA signal pathway. To testify the hypothesis, in this study, we will functionally characterize the two mycorrhiza-induced GH3 genes, by enhancing or silencing their expression levels in transgenic plants, regarding to their physiological roles in regulating mycorrhizal symbiosis and IAA signal pathway. The transgenic plants carrying mutated promoters (a series of truncations and deletions) fusing GUS reporter gene will also be employed to explore the putative cis-elements in response to AM symbiosis in the promoters of the two mycorrhiza-induced GH3 genes. In additon, EMSA (Electrophoretic Mobility Shift Assay) and yeast one hybridization will also be performed to screen and testify the potential transcription factors recognizing the AM-responsive cis-elements. Accomplishing these studies, it will allow us to elucidate the molecular mechanisms underlying the response regulation and physiological roles of GH3 genes during the establishment of the AM symbiosis.

丛枝菌根是土壤中的丛枝菌根真菌与植物根系间建立形成的互惠共生体。研究表明在丛枝菌根中很多基因能够被诱导表达，这些基因往往又能调控丛枝菌根的共生过程。前期我们从番茄GH3基因家族中鉴定到两个菌根诱导表达的成员SlGH3.3和SlGH3.4，并初步证明这两个基因能够响应外源IAA信号，其蛋白能够催化IAA与不同氨基酸联接。为此我们提出并将验证如下假说：SlGH3.3和SlGH3.4能够参与调控番茄丛枝菌根的共生，其调控过程可能与IAA信号途径密切相关。本项目将利用SlGH3.3和SlGH3.4沉默、超表达的转基因材料，研究增强或抑制这两个基因的表达对番茄丛枝菌根共生和IAA信号途径的影响。本项目还将利用报告基因、EMSA和酵母单杂交系统筛选两个基因启动子中响应菌根共生信号的调控元件以及与之互作的转录因子，从而揭示SlGH3.3和SlGH3.4在响应和可能调控番茄丛枝菌根共生中的功能和作用机制。

植物作为定居固着生物需要进化出更为精巧的适应性机制以提高它们抵御不同环境胁迫（如病菌侵入、养分缺乏等）的能力。植物对这些环境挑战的进化适应机制之一是与丛枝菌根真菌（Arbuscular mycorrhizal fungi; 简称AM真菌）形成互惠共生。AM真菌能够促进宿主植株从土壤中吸收养分，尤其是磷素；作为回报，宿主植株给AM真菌提供其生长发育所必需的碳水化合物。丛枝菌根共生体的形成需要AM真菌与宿主之间进行持续的信号交流，其过程受到很多基因程序化表达调控。生长素作为一种植物激素被证实能够调控植物与微生物互作。一些参与生长素合成和代谢相关基因也被发现在丛枝菌根中被诱导，暗示生长素信号参与了菌根共生过程。然而目前对生长素介导的菌根共生机制并没有很好的解析。植物GH3家族基因能够调节植物体内激素稳态，参与调控植物生长发育，其第二亚家族成员主要编码生长素氨基合成酶，能够催化吲哚乙酸与氨基酸结合，调节植物体内生长素动态平衡。在项目中我们发现3个GH3第二亚家族成员在丛枝菌根中被诱导上调表达，其中SlGH3.4的表达水平在接种AM真菌处理的植株中被强烈特异诱导。组织化学染色显示SlGH3.4启动子可以驱动GUS报告基因在烟草、大豆和水稻菌根中强烈表达，且表达部位主要集中在丛枝化的皮层细胞中。对启动子区功能分析发现，一个生长素响应元件和两个新发现的作用元件MYCS1和MYCS2参与了SlGH3.4响应生长素和菌根共生的表达调控。在番茄和水稻中组成型超表达SlGH3.4显著降低植株体内游离态IAA浓度和总的菌根侵染率，并导致丛枝发育异常。通过CRISPR-Cas9系统和RNAi 技术敲除或抑制SlGH3.4表达能够显著增加番茄植株体内游离态IAA含量，但对总侵染率和丛枝发育没有显著影响。与对照相比，超表达SlGH3.4显著抑制生长素受体基因TIR1表达，以及细胞壁扩张蛋白SlEXLB4和SlEXLB2表达。相反，敲除/沉默SlGH3.4显著增强SlEXLB4、SlEXLB2、SlEXPA9和SlEXPA3表达。综上所述，SlGH3.4能够通过控制细胞内生长素稳态和生长素信号途径以及细胞壁扩张蛋白基因表达，调节丛枝菌根共生和丛枝发育过程。