Development And Application Of Novel RNAi Biologics Based On Transboundary Sly-miR398a

Tomato is one of the most important vegetable crops in the world,because of its rich nutritional value,widely planted in the world.Tomato wilt disease,caused by Fusarium oxysporum f.sp.Lycopersici(Fol),is one of the most serious fungal diseases in tomato production,and also known as "tomato cancer",is a soil borne fungal disease that causes huge economic losses to the global tomato industry.At present,the common control methods for tomato fusarium wilt include agricultural control,physical control,chemical control,biological control.However,ordinary prevention and control measures often have slow effects,pollution to humans,animals and environment,resistance of tomato.Therefore,further exploration of the pathogenic mechanism of Fusarium oxysporum f.sp.Lycopersici(Fol)can provide new ideas for the control of tomato wilt disease.MicroRNA(miRNA)is a type of small RNA molecule with a length of approximately 20-24 nucleotides.miRNA participates in the regulation and expression of cellular gene transcription.With in-depth research on miRNA,it has been found playing an important regulatory role in plant growth and development,signal transduction,and response to stress.RNA interference(RNAi)is a specific gene silencing phenomenon mediated by double stranded RNA(dsRNA)and involved in specific enzymes which regulates the expression of specific genes at the transcriptional,post transcriptional,and translational levels.Current researches have shown that miRNAs can inhibit the function of target genes in different species through cross-border transportation playing an important role in the interaction between plants and pathogens.Based on the above theory,here,we used Fol treating tomato susceptible cultivar Moneymaker(MM)and resistant cultivar Motelle(Mot).Fol was isolated and cultured from MM stem for a second time,and then total RNA of the next generation colony was extracted to construct a library.We adopted to explore the molecular mechanism of tomato host active defense against disease invasion,and to develop environmentally friendly and specific control technology of tomato wilt.Our results are as follows:(1)High-throughput sequencing revealed that cross-kingdom tomato endogenous miRNA:Sly-miR398a.(2)Sly-miR398a has been shown to be stable and persistent in several next-generation Fol strains.(3)By using bioinformatic analysis,Sly-miR398a was predicted five potential target genes in the Fol genome,and six potential target genes in tomato genome.Fol_AAXH01000165 and Fol_AAXH01000191 in Fol,Solyc01g067740,Solyc08g079830,and Solyc05g005460 in tomato were verified as true target genes of Sly-miR398a using tobacco co-expression system.(4)To further investigate the biological function of target genes in Fol,homologous recombination was used to construct target gene knockout transformants.The results showed that the deletion of Fol_AAXH01000165 and Fol_AAXH01000191 resulted in impairing the sporophyte size and the porosity of the mycelium of Fol.On the other hand,deletion of these two genes didn’t impact the response to abiotic stress.Further,the deletion of Fol_AAXH01000165 and Fol_AAXH01000191 leaded to reduced infection ability of Fol.(5)In order to explore the functions of Sly-miR398a and its target genes in tomato host,transgenic plants of overexpressing and knock outing Sly-miR398a were constructed using Agrobacterium tumefaciens mediated tomato genetic transformation,as well as the confirmed target genes.After obtaining transgenic materials,further research on resistance to tomato wilt disease will be performed.(6)By using nanomaterials to encapsulate genes resistant to tomato wilt disease,RNAi formulations were sprayed on the surface of tomato plants pre-treated with Fol.The results showed an enhanced prevention and control to tomato wilt disease.In summary,we developed a technical and theoretical basis for further exploring the cross-kingdom involvement of miRNAs in plant fungal pathogen interactions,as well as analyzing the function of target genes in tomato resistance to wilt disease.Our results also provided a potential environmentally friendly green strategy to control the tomato wilt disease.