Study On The Infection Mechanism Of Ustilaginoidea Virens

Rice false smut caused by Ustilaginoidea virens has developed as one of the three major diseases of rice in China.It not only causes yield losses,but also produces toxins,causing serious threat to food security and food safety in China.With the improvement of artificial inoculation technology of U.virens,studies on the infection mechanism of U.virens were carried out around the world,which greatly improved the knowledge about the infection of U.virens.In this study,a GFP labeled strain of U.virens was used to inoculate the sensitive cultivar Wanxian 98.After inoculation,the infection process of the pathogen and the development of rice flower organs were investigated to analyze the infection mechanisms of U.virens.At the same time,3 U.virens effectors interacted with the rice promoter OsSWEET5 were obtained and verified by yeast one hybrid experiment.Finally,the control effect of seed treatment with fungicides was evaluated.The main results were as follows:(1)A GFP labeled strain of U.virens was used to inoculate the sensitive cultivar Wanxian 98,after inoculation,the infection process of pathogen and development of rice flower organs were investigated daily,the results revealed that the infection of U.virens in rice is a stepwise infection pattern.By using stereofluorescence microscope,laser confocal microscope,scanning electron microscope and transmission electron microscope,it was found that the hyphae extended to the interior of spikelets through the gap between lemma and palea before the flower organs were mature,initial infection occurred on the filaments,and then the pathogen invaded the stigmas and styles,occasionally the ovaries through styles.During the infection,no infection structures such as penetration pegs or haustoria were observed,and hyphae only grew in the intercellular space of host.KI-I2 and DAPI staining experiments showed that almost all the infected rice pollens were not mature,and the ovaries were no longer developed and could not be stained by KI-I2,indicating that the pollination of ovary was blocked by lacking of the mature pollens.Real-time PCR showed that some genes associated with grain filling including b ZIP transcription factor,starch metabolism related genes and seed storage protein genes were induced by U.virens infection,the similar expression pattern with the fertilization of ovaries,suggesting that the infection of stigmas and styles could mimick the fertilization process.In addition,the TGMS line Hua9 s was inoculated with G2,and then the inoculated panicles were pollinated with pollens from cultivar Wanxian 98 at 3 dpi,to evaluate whether the fungus can compete with the fertilized ovary.The results showed that the majority of ovaries developed into young seeds and the ovaries were unaffected even filaments were colonized by the pathogen.The results suggested that the U.virens could not compete successfully with fertilized rice ovaries,and it might be the reason why the U.virens has to attack the filaments first to suppress the formation of mature pollens.The stepwise infection of flower organs and mimicry of ovary fertilization unveiled in this study guided the rice plant into supplying nutrients for false smut ball development,which represented a new and unique biological process of host pathogen interactions.(2)The samples inoculated with U.virens and the PSB as control at 1,5 and 9 dpi,a total of 6 samples were obtained to perform the transcriptome sequencing and analysis.There were 89,308 and 3545 differentially expressed genes(DEGs)identified in three infection stages,and the DEGs were further analyzed by GO enrichment analysis and KEGG Pathway enrichment analysis.The GO enrichment analysis showed that the up-regulated genes were enriched in the carbohydrate metabolism process and some regulation processes at 9 dpi.The KEGG Pathway enrichment analysis showed that the DEGs were mainly concentrated in the two major categories of carbohydrate metabolism and energy metabolism,among them,the DEGs involved in glycolysis and gluconeogenesis pathway were significantly up-regulated at 5 dpi and 9 dpi.In addition,most of the OsSWEET genes were up-regulated after infection of U.virens.Especially at 9 dpi,OsSWEET1 a,OsSWEET1 b,OsSWEET4,OsSWEET5,OsSWEET6 a,OsSWEET14,OsSWEET16 and OsSWEET11-like were significantly up-regulated.OsSWEET5 was selected to do the futher research,using yeast one hybrid technology,three effectors of U.virens named Uv33,Uv46 and Uv48 were identified to be interacting with OsSWEET5 promoter.Expression analysis indicated that Uv33,Uv46 and Uv48 were highly expressed during infection.In addition,yeast one hybrid method was taken to verify the results of screening,and the results showed that effectors Uv33,Uv46 and Uv48 could interact with OsSWEET5 promoter in vivo.In order to verify whether these candidate effectors had secretory function,the p SUC2 yeast secretion system was used to verify the secretory function of signal peptides.The results indicated that the signal peptides of effector Uv33,Uv46 and Uv48 had secretory function.(3)In order to evaluate if seeds polluted by U.virens could be a major primaiy infection sources,three fungicides carbendazim,triadimefon,thiram,and lime were used to treat rice seeds that were contaminated by chlamydospores of U.virens in 2014,2015 and 2017.Samples of rice tissue were taken for detection of U.virens by using specific PCR method at seedling,tillering and booting stage respectively,in addition,the occurrence of rice false smut was investigated and analysed at maturation stage.At seedling stage,rice shoots and roots were taken for detection,and the results showed that the treatment groups had significant reduction of detection rates of U.virens compared with the control group.However,the PCR detection of leaves,stems and roots at the tillering stage,and detection of leaves,stems,panicles and roots at booting stage did not show significant differences between the fungicide treatments and control.Finally investigation of rice false smut balls at maturation stage also did not find significant differences among different treatments including control.The results in this study suggested that seeds polluted by U.virens could not be the major primaiy infection sources of false smut in fields.