Study On The Spatiotemporal Expression Pattern Of Rice Grain-filling-related Gene AGF1 Activated By Ustilaginoidea Virens

Rice false smut(RFS)is a rice panicle disease caused by Ustilaginoidea virens(Uv).The only visible symptom is that rice grains are wrapped in rice false smut ball.The disease is occurring more and more frequently in various rice fields worldwide.It can not only cause a significant reduction in rice yield,but also affect the quality of rice by some toxins and harm human or animal health.In recent years,RFS has succeeded in replacing rice sheath blight disease as one of the new Three Major Diseases of rice.At the same time,RFS disease also receives more and more attention from researchers.At present,the research on RFS disease is not deep enough to explain the formation process of the rice false smut ball and pathogenesis at molecular level.Therefore,we need to carry out a lot of scientific research work to deeply analyze the disease mechanism of Uv-rice interaction.The preliminary research foundation has created favorable conditions for the smooth development of our experiment.We found that the rice grain filling process was impeded after the invasion of rice by Uv,and the ovary stopped developing,but some grain-filling-related genes were highly induced.These phenomena led us to speculate that Uv may simulate the rice grain filling process,hijack the rice nutrient system and use the nutrition for the formation of the rice false smut ball.Therefore,this thesis focuses on an important grain-filling related gene AGF1(Activated Grain-Filling-Related Gene 1),and makes an in-depth investigation on its role in the formation of the rice false smut ball and the spatiotemporal expression characteristics activated by Uv.The main results are as follows:1.By inoculating different Uv strains into different rice materials,the disease phenotype was evaluated to search for virulent strains on each rice material.Our data showed that JS was virulent on NPB,with the number of RFS ball per panicle reaching 20.PJ52 and JS were virulent on ZH11 with RFS ball number >10 per panicle.PJ52 and HN were virulent on Dongjin with RFS ball number reaching 20 per panicle.As a susceptible cultivar,Pu6 was vulnerable to all tested Uv strains,forming 40-60 RFS balls per panicle on average.These data are valuable for subsequent experiments in this study.2.By analyzing the seed phenotype data of agf1 and its background material Dongjin,it is concluded that agf1 does affect rice grain filling process.The seed setting rate was decreased by 26% and the number of grains per panicle was deceased more than 40 in agf1 than in Dongjin.Moreover,agf1 had remarkably increased chalkiness degree and chalky kernel percentage compared to Dongjin.3.The artificial inoculation was carried out on agf1 and Dongjin with PJ52 and HN,and the number of rice false smut balls was calculated.The results showed that the much less RFS balls were formed in agf1 than in Dongjin.Paricularly,HN formed over three fold less balls in agf1 than in Dongjin.It is indicated that AGF1 plays an important role in the formation of rice false smut ball.4.The GUS staining experiment on OsAGF1pro-RFP-GUS transgenic rice plants showed that promoter of AGF1 was activated mainly at 5 days after inoculation(dai),most strongly at 9 dai.The activated tissue was mainly in the lodicules of rice flowers.RNA in situ analysis further confirmed the results.Moreover,GUS analysis on promoter-reporter transgenic plants of AGF2,the downstream gene of AGF1,showed that AGF2 promoter was also activated in the lodicules at 9 dai.5.Pu6 was inoculated with the GFP-tagged Uv strain P4,and the proportion of each infected floral organ at different infection stages was calculated.It was found that Uv began to invade into the inner space of rice spikelet at 5 dai.The organ with the highest infection rate was stamen filament and the second was the lodicule,which indicates that the lodicule as well as stamen filament also plays an important role in the infection process of Uv.In summary,AGF1 is a key gene regulating the formation of the RFS ball.AGF1 is activated by Uv infection starting from 5 dai,and the activation reaches a summit at 9 dai.The activation site is mainly in lodicule.As a key site in response to Uv,lodicule may be required for successful colonization of Uv.Therefore,this thesis has preliminarily revealed a new mechanism of Uv infecting rice flower,which is of great significance to the understanding and control of the disease.