Functional Mechanism Of Small RNA-Mediated Gene Silencing In Modulating Rice Defense Responses To Rice Sheath Blight And Application Research For Controlling Plant Disease

Plant small RNAs(sRNAs)play an important role in regulating various developmental processes,immune and hormone signaling pathways in rice.Rice blast and rice sheath blight are worldwide rice diseases,which seriously endanger the safety of rice production.At present,small RNA has been studied in the rice-Magnaporthe oryzae interaction system,but the rice-Rhizoctonia solani interaction system is still unknown.To further understand the molecular mechanism of sRNA between rice and R.solani interaction,this study intends to explore the sRNA involved in regulating the immune defense of rice against R.solani through high-throughput sequencing.This study provides a theoretical basis and genetic resources for the prevention and control of sheath blight.In addition,spray-induced gene silencing(SIGS)is an innovative plant protection strategy that uses topical application of double-stranded RNAs(dsRNAs)or small RNAs to target and silence pathogen virulence-related genes to suppress disease.This study also explored the inhibition of spray-induced gene silencing(SIGS)in eukaryotic pathogens and the stability and persistence of dsRNA encapsulated in artificial vesicles were studied,to explore environmentally friendly disease control methods.Specific research contents are as follows:First,siR109944 inhibits the plants immune response to sheath blight by targeting auxin receptor-like proteins We screened rice(Oryza sativa)sRNA expression patterns against Rhizoctonia solani and found that Tourist-miniature inverted-repeat transposable element(MITE)-derived small interfering RNA(siRNA)siR 109944 expression was obviously suppressed upon R.solani infection.One potential target of siR109944 is the F-Box domain and LRR containing protein 55(FBL55),which encodes the transport inhibitor response 1(TIR1)-like protein.We found that rice had significantly enhanced susceptibility when siR109944 was overexpressed,while FBL55 OE plants showed resistance to R.solani challenge.Additionally,multiple agronomic traits of rice,including root length and flag leaf inclination,were affected by siR109944 expression.Auxin metabolism-and signaling pathway-related genes were differentially expressed in the siR109944 OE and FBL55 OE plants compared with wild-type(NPB).Importantly,pretreatment with auxin enhanced sheath blight resistance by affecting endogenous auxin homeostasis in rice.Furthermore,transgenic Arabidopsis overexpressing siR 109944 exhibited early flowering,increased tillers and enhanced susceptibility to R.solani.Our results demonstrate that siR109944 has a conserved function in interfering with plant immunity,growth and development by regulating auxin homeostasis in planta.Thus,siR109944 provides an effective genetic target for plant breeding in the future.Second,Spray-induced gene silencing for disease control is dependent on the efficiency of pathogen RNA uptake Spray-induced gene silencing(SIGS)is the topical application of sRNAs and dsRNAs which target and silence pathogen virulence genes,to achieve the purpose of disease control.It has been successfully utilized as a strategy to control some fungal and viral plant diseases.In this study,to clarify the effect of RNA uptake efficiency on the control of SIGS disease.We first examined the efficiency of RNA uptake in multiple pathogenic and non-pathogenic fungi and an oomycete pathogen.We then chose several pathogens for subsequent analysis of the efficiency of SIGS for disease inhibition.We observed efficient dsRNA uptake in the fungal pathogens Botrytis cinerea,Sclerotinia sclerotiorum,Rhizoctonia solani,Aspergillus niger,and Verticillium dahliae;weak uptake in the beneficial fungus Trichoderma virens;and no uptake in Colletotrichum gloeosporioides.For the oomycete pathogen Phytophthora infestans,RNA uptake varies on different cell types and developmental stages.Topical application of dsRNA targeting virulence-related genes in the pathogens with high RNA uptake efficiency significantly inhibited disease symptoms,whereas the application of dsRNA in pathogens with low RNA uptake did not significantly suppress pathogen infection.Our results revealed that dsRNA uptake efficiencies vary among different types of eukaryotic microbes and in different cell types of a microbe.The success of SIGS for plant protection is largely determined by the RNA uptake efficiency of the pathogens.Third,Artificial vesicles as a carrier can protect RNA from degradation and improve SIGS efficiency Externally applied double-stranded RNA(dsRNA)that target and silence virulence genes can be effectively taken up by fungi to suppress diseases.The instability of dsRNA is the notable drawback that restricts the promotion and application of spray-induced gene silencing(SIGS).Here,we demonstrate that dsRNA can be encapsulated in cationic artificial vesicles(AVs),and the AV-dsRNA complexes can be absorbed by Botrytis cinerea.Applying naked-or AV-dsRNA that targets B.cinerea vesicle-trafficking pathway genes which significantly inhibit gray mold disease on the surface of plants.We found that AVs protect dsRNA from water rinsing and nuclease degradation,as well as limiting fungal growth and pathogenicity for at least 15 days,allowing more than 5-7 days durable and stable protection than that conferred by naked dsRNA.We also provide evidence that naked-and AV-dsRNA were efficiently taken up and systemically transported to inhibit fungal growth in the directly treated and the untreated leaves.Our results indicate that externally applied naked-or AV-dsRNA might able to simultaneously control disease caused by B.cinerea pathogen through targeting pathogen virulence genes,but AVs encapsulated dsRNA treatment provides durable protection than naked dsRNA.Taking the new strategy offers more potential and broader applications in the control of crop fungal diseases through the application of sequence-specific AV-dsRNA.