Small RNA-based Pathogenic Mechanism Of Rice Ragged Stunt Virus In Rice

Rice ragged stunt virus(RRSV),an RNA virus belonging to the genus Oryzavirus in the family Reoviridae,infects rice and induces rice ragged stunt disease.This disease was first discovered in 1976-1977 in Indonesia and the Republic of the Philippines and widely occurred in rice-growing area of most southeast countries and regions including Indonesia,Philippines,Vietnam,and several provinces of China such as Fujian,Guangdong,Jiangxi,Hunan,Zhejiang and Taiwan.This disease damages the production of rice and contributes a major threat to the food safety.Studies on the pathogen characterization,transmission features of vector insects,and molecular biology of RRSV genome has been conducted by researchers since RRSV was discovered.From 2010 to now,progresses have been made on the biological roles of three non-structural proteins including Pns6,Pns7,and Pns10.For instance,Pns6 acts as a movement protein and an RNA silencing suppressor;Pns7 may involve in the spreading process of RRSV in insect vector and in host plants;Pns10 is essential for assembly of the viroplasma and persistent infection of RRSV in its insect vector.However,the pathogenic mechanism of RRSV in rice remains elusive so that no basis can be referred to propose an effective approach to control the rice ragged stunt disease.RRSV infection induces typical symptoms,including stuntedness,excess tillering,dark green leaves,and wider leaves with serrated edges or twisted tips,suggesting that RRSV infection affects the rice growth and development.Considering that small RNAs play crucial roles in plant growth and development including leaf development and morphogenesis,root and floral development etc,thus,we intend to explore the pathogenic mechanism of RRSV from the angle of plant small RNAs.Here,the differential expressed patterns of small RNAs between healthy rice plants(Mock)and RRSV-infected rice plants(RRSV)were first analyzed by small RNA sequencing.The results indicated that RRSV infection affected the expression patterns of rice small RNAs,and the expression of miRNAs closely related to leaf development and morphogenesis such as miR3 19,miR164,and miR156 were significantly induced by RRSV infection.These results were reconfirmed by northern blot assay.The data derived from both small RNA sequencing and northern blot assays indicated that miR319 was affected most significantly by RRSV infection in comparison with miR164 and miR156.Thus,to verify the roles of miR3 19 in RRSV infection,we generated the transgenic rice plants with either miR319a overexpression or miR319b overexpression by using rice genetic transformation system.Positive transgenic lines with miR319 overexpression exhibited disease-like phenotypes such as stuntedness,excess tillering,and dark green and wider leaves with shrink.To assess whether the process of RRSV infection can be affected by miR319 overexpression,both control groups and transgenic rice plants with miR3 19 overexpression were inoculated by RRSV-carrying insects and differences of disease symptoms and morbidity rate were analyzed.The results showed that both transgenic lines were more susceptible to RRSV infection than control groups because the disease symptoms on transgenic lines were more severe than control groups and the morbidity rate of transgenic lines were higher than control groups.The function of miR319 in rice growth and RRSV infection sparks us to check whether miR319-targeted genes play roles in rice growth and RRSV infection.RNAi transgenic rice plants of TCP21,PCF5,PCF6,and PCF8 were obtained through rice genetic transformation system.The results indicated only TCP21 RNAi transgenic lines showed abnormal phenotypes similar to disease-like phenotypes of transgenic lines with miR319 overexpression and disease symptoms of RRSV-infected rice plants,whereas all other three showed normal growth in the vegetative growth stage.To analyze the roles of TCP21,transgenic rice plants with wild-type TCP21 overexpression(TCP21 OE lines)and miR319-resistance TCP21 overexpression(TCP21 Res lines)were generated by using rice genetic transformation system.The differences of disease symptoms and morbidity rate between control groups and TCP21 transgenic lines were compared after RRSV inoculation.The results showed that in comparison with control groups,TCP21 RNAi lines,but not TCP21 OE lines and TCP21 resistance lines,showed enhanced RRSV susceptibility.How do miR319 and TCP21 affect the process of RRSV infection?Based on the research background that jasmonic acid(JA),which can be regulated by miR319-targeted TCPs in Arabidopsis,is vital for host to resist against diverse microbes,we speculated that the increased susceptibility of transgenic rice plants with either miR319 overexpression or TCP21 RNAi may be a result of manipulation of JA biosynthesis and signaling pathway by miR319/TCP21 activity.We found that expression patterns of genes associated with JA biosynthesis and signaling altered in RRSV-infected rice plants by conducting microarray assay and Real-time PCR.Liquid chromatography-mass spectrometry(LC-MS)assay indicated that JA content was reduced in RRSV-infected rice plants.To demonstrate that the JA pathway was manipulated by miR319/TCP21 activity,differential expression of several JA-related genes between control groups and transgenic lines with miR319 overexpression and TCP21 transgenic lines including RNAi,OE,and Res lines were analyzed by Real-time PCR.The JA contents of control groups and transgenic lines were mearsured by LC-MS and the results showed that miR319 and TCP21 regulated the JA accumulation in rice.Additionally,we found that expressions of pathogenesis-related genes were induced and RRSV accumulation was reduced by application of JA in rice.These results suggested the susceptibility of rice to RRSV infection was regulated by miR319/TCP21 activity by the manipulation of JA pathway.Taken together,we demonstrated that miR319 and its target gene TCP21 involve in the RRSV pathogenic process.These results support a scenario in which a plant virus specifically manipulates a miRNA for maximum infection and the manipulation accidentally contributed to symptom induction.With the addition of the findings in wheat-RBSDV system that RBSDV infection significantly induced the miR319 accumulation whereas inhibited the expression of its target gene,we hypothesis that miR319 and its target genes may serve as a common agent in pathogenic process of many rice viruses.