Research On The Function Of Pathogenic Genes And Disease Control Of Rhizoctonia Solani Using SIGS Technology

Rhizoctonia solani,a soil-borne pathogenic fungus belonging to Agonomycetales,an order of Hyphomycetes,has wide host range,strong saprophytic ability and wide distribution regions,and can harm the plants,more than 260 species and 40 families from Gramineae,Legumes and Solanum,and so on,and cause serious losses.Due to the lack of a complete genetic transformation system,research of the gene function of R.solani is proceeding slowly.Although a few pathogenic genes of R.solani have been studied according to the date of genome,transcriptome,proteome and metabolome,the functions of most pathogenic genes from R solani are unknown.In addition,because of the strong saprophytic properties of R.solani and the occurrence of the diseases caused by R.solain greatly affected by the environment,the control effects of resistant varieties,agricultural measures,physical measures and biological measures are not ideal.To date,the best method to control the diseases caused by R.solani is to use chemical agents.However,excessive and frequent use of chemical agents can easily cause "3R"problems,such as residue,resistance and resurgence,as well as non-target killing.Therefore,developing new methods to research the gene functions and control the diseases caused by R.solani are the main tasks at present.RNA interference(RNAi),a ubiquitous mechanism in eukaryotes,has become an important method to research the functions of genes in pathogenic fungi and the diseases control of crops.However,previous RNAi technologies were all based on genetic modification,and many plants and pathogenic fungi,such as R.solani,can't be genetically manipulated,and countries around the world have low acceptance of transgenic agricultural products,so the safety and applicability of these methods have also been questioned.Spray-induced gene silencing(SIGS)can induce silencing of pathogen target genes through exogenous spraying of dsRNA and does not require to establish genetic transformation systems of pathogens or form transgenic crops.Therefore,it is of great significance in the research of gene function of pathogens and diseases control.On this basis,we studied the functions of some pathogenic genes from R.solani,and have preliminarily obtained dsRNA-nano agents that can be used for controlling the diseases caused by R.solani.The results are as follows:1.Strong fluorescence signal was observed in the mycelia of R.solani,which were co-incubated with the fluorescein-labeled EGFP-dsRNA for 24 hours at room temperature.When the fluorescent mycelia were prepared into protoplasts,the fluorescence signal still can be detected.These results indicated that R.solani can take up dsRNA from the environment.2.According to the genome and transcrip tome data,the polygalacturonase gene RsPGl,the catalase gene RsCAT and the calcineurin-sensitive zinc finger protein gene RsCRZl of R.solani were cloned.The dsRNA targeting three genes were synthesized and co-incubated with R.solani for 24 h.Results of qRT-PCR showed that exogenous dsRNA could silence the target genes,and the silencing effects were from 57.4%to 68.7%.Except the silencing of RsCAT gene affected the formation of sclerotia of R.solani,the silencing of each gene hardly affects the biological characters of R.solani.3.After the dsRNAs targeting RsPG1,RsCAT and RsCRZ1 genes were sprayed on the surface of maize leaves,the hypha blocks of R.solani were inoculated.Three days later,comparing to the control,the expression levels of the three genes of R.solani decrease by 64.5%,59.6%and 68.7%,and the biomass of the strains decreased by 65.5%,59.6%and 68.1%,and the lesion area decreased by67.9%,52.5%and 70.8%in the treated leaves.These results indicated that these genes played a key role in the pathogenic process of R.solani.In addition,the silencing of RsCAT gene in R.solani could lead to aggravated ROS response of the host infected with the strain,while the silencing of RsCRZ1 gene led to the down-regulation of the transcription level of its downstream gene RsNCS in the strain.4.The fluorescein-labeled EGFP-dsRNA was sprayed on the surface of maize leaves.Results of confocal microscopy examination showed that the fluorescent signals were mainly concentrated near the vascular bundles of the leaves,and extended from the spraying sites to the distal end along the vascular bundles.In the injured leaves,the fluorescent signals were much stronger than that of intact leaves,which meant that,as the host crop of R.solani,maize also had the function to take up exogenous dsRNA,and could transport dsRNA from spraying sites to other parts of the leaves through the vascular system.5.If dsRNA was co-incubated with the double-layer hydroxide nanomaterial LDH at mass ratio 1:2,the dsRNA could be fully loaded in 2 hours.While the mass ratio increased to 1:16,the dsRNA could be fully loaded less than half an hour.When the fluorescein-labeled EGFP-dsRNA-LDH was sprayed on the surfaces of maize leaves,the fluorescence signals in the leaves were significantly enhanced compared with the EGFP-dsRNA treatment.After spraying RsCRZ1-dsRNA-LDH on the surface of maize leaves and inoculating R.solani,the lesions significantly reduced comparing to the controls treated with RsCRZl-dsRNA and water.Twenty days past spraying,RsCRZ1-dsRNA still could be detected in the leaves of the RsCRZ1-dsRNA-LDH treatment,while it only can be detected in the leaves of RsCRZ1-dsRNA treatment about 3 days.All above indicated that LDH could load dsRNA to form dsRNA-LDH complex,which could improve the efficiency of dsRNA entering host cells and provide longer protection time.6.Order to obtain more and cheaper dsRNA,we constructed the L4440 vector harbor exogenous dsRNA and transferred it into E.coli HT115,which will lay the foundation for the subsequent application of dsRNA naon fungicides in the field.