Analysis Of The Pathogenic Function Of FolRDR1 And The Research And Application Of RNAi In Tomato Wilt Disease

Tomato is widely cultivated worldwide as a nutrient-rich vegetable crop.Tomato wilt disease by Fusarium oxysporum f.sp.Lycopersici（Fol）is one of the most serious fungal diseases in tomato production,causing huge economic losses to tomato agricultural production worldwide.At present,the prevention and control of the disease mainly rely on chemical control,but the long-term use of a large number of chemical agents prone to ecological pollution,pesticide residues and resistance,endangering human health and other problems.Therefore,in-depth investigation of the pathogenic mechanism of Fol and the development of efficient and environmentally friendly biocontrol agents will be a new direction for the prevention and treatment of tomato wilt disease.RNA interference（RNAi）is a highly conserved evolutionary phenomenon induced by double-stranded RNA（dsRNA）and efficiently and specifically degraded by homologous mRNAs,which is an important gene silencing pathway in eukaryotes.Pathogenic organisms produce RNAi effects through the uptake of external dsRNA,which in turn interferes with the normal growth of the pathogen and its pathogenicity to the host plant.This study provides a new strategy for disease prevention and treatment in tomato agricultural production based on RNAi technology to prevent and control the occurrence of tomato wilt through exogenous application of dsRNA.In the early stage of our research,RDR1（RNA-dependent RNA polymerase 1）was discovered in the Fol genome by homologous gene comparison method.In order to further study the function of this gene,the biological function of FolRDR1 and the application of FolRDR1-RNAi in the control of tomato wilt disease were carried out in this study.The findings of the study are as follows:1.The analysis of the conservation and evolutionary relationships between FolRDR1 and other specialized strains of RDR1 showed that the gene is highly conserved among different specialized strains,suggesting that it may have an important biological function.2.Analysis of the biological functions of FolRDR1 in Fol and the response to external abiotic stresses showed that FolRDR1 played a regulatory role in the growth and development of Fol,such as the metamorphosis growth of mycelium,larger conidia,significantly reduced spore production,and significantly regulated the penetration ability of Fol.In addition,FolRDR1 was not involved in the response of Fol to external abiotic stresses such as salt,alkali,and osmotic stress.3.The analysis of the pathogenicity of FolRDR1 knockout strains showed that the deletion of FolRDR1 significantly reduced the infestation of Fol,indicating that FolRDR1 plays an important role in regulating the pathogenicity of Fol.4.Establishment of an efficient system for bacterial prokaryotic expression of dsRNA targeting FolRDR1.5.Based on fluorescent tracing and using RT-PCR,Fol was shown to be able to efficiently take up dsRNA from the external environment and deliver it in its body;Exogenous dsRNA could be transported to roots,stems and leaves with the help of plant vascular tissues after entering plants,and its presence could be detected within 0.5 d-7 d;The detection of dsRNA retention time on leaves showed that naked dsRNA could remain on leaves for 5 d-7 d.6.Detection of the effect on the expression level of FolRDR1 after exogenous application of dsRNA showed that the silencing efficiency of FolRDR1 was best at the working concentration of 150 ng/mL of dsRNA and 1 d of treatment,and the silencing efficiency of FolRDR1-dsRNA1 and FolRDR1-dsRNA2 were 88.84%and 81.34%,respectively.7.Exogenous application of 2 μg of dsRNA on Fol-infested tomato leaves was used to assess the efficacy against tomato wilt.The results showed that after 15 d of treatment,the susceptible line MM showed slight yellowing of the lower leaves and exhibited the initial symptoms of wilt,and with subsequent growth,the plants grew well and showed significant reduction of wilt symptoms after 25 d of treatment.8.FolRDR1 is highly specific without sequence off-target in related plants and animals.In summary,this research clarifies that FolRDR1 is positively correlated with the pathogenicity of Fol and plays a regulatory role in the growth and development of Fol;It is demonstrated that Fol can efficiently take up dsRNA from the external environment and translocate it in its body to cause RNAi effects;The induction system of bacterial prokaryotic expression of dsRNA was further established which indicates that the exogenously applied dsRNA was effective against tomato wilt.The results of the study provide the preliminary technical and scientific support for the next step of perfecting the development of dsRNA-based RNAi biologics,and provide a new strategy for the prevention and control of tomato wilt disease in agricultural production.