Pectin-modifying enzymes in plant development and interactions with fungi

植物发育中的果胶修饰酶及其与真菌的相互作用

• 批准号: RGPIN-2020-05147
• 负责人: Deyholos, Michael
• 金额: $ 3.22万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740412
• 关键词: Pectin; modifying; enzymes; plant; development

The plant cell wall plays a critical role in processes such as morphogenesis and fungal interactions. Pectins (e.g. rhamnogalacturonan I, RG-I) are a particularly dynamic part of the cell wall environment, especially within the middle lamella that forms the interface between adjoining cells. As such, pectins are critical to adhesion and separation of cells (among their other functions). Pectins are remodeled dynamically, including the breakage and rejoining of pectin molecules. In plants, cleavage of the RG-I portion of pectins is believed to mediated by enzymes called RG lyases (RGLs). Our objective is to characterize plant RGL enzyme families, and their role in plant development and interactions with the environment. We will use computational tools to explore the diversity of putative RGLs, by analysis of the genomes and transcriptomes currently available from over 1,200 plant species. This will provide insight into the diversity and evolution of these genes. We will also conduct meta-analyses of existing RNASeq data from a wide range of species to make inferences about RGL functions, including exploring co-expression with other regulators or cell wall; enzymes. Based on the bioinformatic analyses described above, we expect to make new inferences about the evolution of the pectin-modifying genes and regulators, and their roles in plant physiology. Despite their annotations as RGLs, no plant RGL has yet been empirically proven to have RGL activity.  We will characterize the enzymatic activity of selected RGLs in vitro, by purifying them from appropriate hosts, and analyzing their effects on diverse pectic substrates, primarily through a novel capillary-electrophoresis based approach.   To complement the other analyses proposed above, we will also conduct mutational analysis, with a focus on cell adhesion/separation in morphogenesis and plant-microbe interactions. We will use Arabidopsis thaliana and flax (Linum usitatissimum) for these mutational studies. The advantages of arabidopsis as a model system are already well known, and flax offers other benefits because it demonstrates intrusive growth (i.e. of phloem fibers), and associations with both arbuscular mycorrhizal fungi (AMF) and pathogens. We have already established many resources relevant to these studies in both flax and arabidopsis. Loss-of-function RGL mutants will be compared to wild-type using a range of phenotypic assays including: immunohistochemistry, analytical chemistry, and growth and hormone assays, and fungal colonization assays. This research program will provide key insights into the role of one of the least-understood enzymes families in cell wall biology. It will also provide training opportunities for HQP in bioinformatics, gene editing, plant genetics, and analytical chemistry. Finally, the information learned in this research will also facilitate the development of crops (especially flax) with modified pectins for specific industrial end uses.

植物细胞壁在形态发生和真菌相互作用等过程中起着至关重要的作用。果胶(如鼠李半乳糖苷I、RG-I)是细胞壁环境中一个特别动态的部分，特别是在形成相邻细胞之间界面的中层。因此，果胶对细胞的黏附和分离(以及它们的其他功能)至关重要。果胶是动态重塑的，包括果胶分子的断裂和重新连接。在植物中，果胶的RG-I部分的裂解被认为是由称为RG裂解酶(RGL)的酶介导的。我们的目标是确定植物RGL酶家族的特征，以及它们在植物发育和与环境相互作用中的作用。我们将使用计算工具，通过分析目前从1,200多种植物中获得的基因组和转录本，来探索推测的RGL的多样性。这将提供对这些基因的多样性和进化的洞察。我们还将对来自广泛物种的现有RNAseq数据进行荟萃分析，以推断RGL的功能，包括探索与其他调控因子或细胞壁、酶的共表达。基于上述生物信息学分析，我们期望对果胶修饰基因和调控因子的进化及其在植物生理中的作用做出新的推断。尽管它们被注释为RGL，但还没有植物RGL被经验证明具有RGL活性。我们将通过从合适的宿主中纯化RGL，并分析它们对不同果胶底物的影响，主要是通过一种新的基于毛细管电泳法的方法，在体外表征它们的酶活性。为了补充上面提出的其他分析，我们还将进行突变分析，重点是形态发生和植物-微生物相互作用中的细胞黏附/分离。我们将利用拟南芥和亚麻(Linum Usitatisimum)进行这些突变研究。拟南芥作为模式系统的优势已经广为人知，而亚麻也提供了其他好处，因为它表现出侵入性生长(即韧皮部纤维)，并与丛枝菌根真菌(AMF)和病原体相关联。我们已经在亚麻和拟南芥中建立了许多与这些研究相关的资源。功能丧失的Rgl突变体将使用一系列表型分析方法与野生型进行比较，包括：免疫组织化学、分析化学、生长和激素分析以及真菌定植分析。这项研究计划将提供对细胞壁生物学中最不被了解的酶家族之一的作用的关键见解。它还将为HQP提供生物信息学、基因编辑、植物遗传学和分析化学方面的培训机会。最后，从这项研究中了解到的信息也将有助于开发用于特定工业终端用途的改性果胶作物(特别是亚麻)。