Functional Analysis Of Mycorrhizal-induced Gene EXO70I And Lipase3 From Citrus And Their Homolog Genes In Medicago

Arbuscular mycorrhizal fungi(AMF) is a kind of soil microbe widely distributed on the earth, which could form mutually beneficial symbioses with host plant. AMF play a critical role in plant development and growth since it could promote nutrient uptake in root. In general conditions, citrus has a poor root system with rare or no root hair, which results in limited absorption of mineral nutrients through its own root system. It is well known that AMF could not only promote nutrient uptake, but also enhance resistance to biotic and abiotic stresses in citrus plants. Though the physiological effect of AMF on citrus growth and development has been widely reported, little is known about the molecular basis underlying the AM symbiosis in citrus. In this study, we aim to inves tigate the roles of two mycorrhizal- induced genes(EXO70I and Lipase3) in AM symbiosis by using the medicago transient genetic transformation, which might provide basis for further investigation of the function of these genes in citrus root symbiosis.The main contents and results of this thesis are as follows:1. Our previous transcriptomic study identified a set of genes that is induced by AMF colonization, including those encoding EXO70I(PtrEXO70I, Cs3g16120.1), protein plastid movement impaired 2 isoform X1(PtrPMI2, Cs6g18300.1), chitinase 2(PtrChit2, Cs1g05710.1), and triglyceride lipase(PtrLipase3, Cs2g28830.1). Here, I constructed promoter-GUS vectors to analyze the promoters of these four genes. The result from GUS staining experiment showed that all of the four genes were induced by AMF colonization and accumulated in arbuscular cells as stained by WGA488. Particularly, the PtrEXO70 I promoter is highly and exclusively expressed in root cortical cel s containing arbuscules.2. EXO70 subunit plays a key role in exocyst complex in plants. A maximum likelihood phylogenetic tree was generated by using the EXO70 gene members from citrus and other plant species. Phylogenetically, plant EXO70 proteins can be divided into ten subclades(EXO70A to EXO70J). The PtrEXO70 I of citrus was claded together with those from rice, medicago and soybean under the EXO70 I subclade. We then investigated the possible role of EXO70 I in AM symbiosis through RNAi of the EXO70 I gene in medicago(Medtr1g017910.1). Quantitative RT-PCR detection evidenced that gene expression levels of Mt EXO70 I and the AM marker gene MtPT4 are both significantly lower in the RNAi lines than those in the empty vector controls, indicating the RN Ai efficiency is high. Phenotype observation imaged by WGA488 staining showed that the AMF colonization rates are normal in RN Ai lines, however, the arbuscule abundance was significantly lower in RNAi lines than in the controls and large septum was observed in the abnormal arbuscules. The result indicated that the EXO70 I is critical for arbuscule development. Further subcellular localization analysis revealed that the EXO70 I accumulated in spatially restricted zones adjacent to the PAM around the arbuscule hyphal tips, suggesting that EXO70 I may affect arbuscule development by regulating hyphae extension and branching during symbiosis.3. In addition, we also analyzed the possible role of another mycorrhizal- induced gene Lipase3 in AM symbiosis by medicago transient transformation. However, no difference was observed between its RN Ai lines and controls for either AMF colonization rate or arbuscular abundance.