The Effects Of Pepper Mild Mottle Virus Infection On Small RNA Expression Of Peppers And The Mechanism Of PMMoV-induced Autophagy

Pepper is an important horticultural crop with a planting area of 1.5 million to 1.6 million hm2,accounting for about 10% of the total vegetable planting area in China.Pepper virus disease is an important disease in pepper production.At present,nearly 40 types of viruses have been found to infect peppers in the world.Pepper mild mottle virus(PMMo V)belongs to the genus Tobamovirus,which can infect different varieties of peppers.PMMo V infection initially causes mild foliar mosaic symptoms followed by mottling and malformation of leaves and fruits,resulting in significant losses of pepper yield.This study gave a further research on the interaction between PMMo V and host plants.We preliminarily explored the functions of vsi RNA,mi RNA and related host factors in the interaction between PMMo V and host plants,and revealed that PMMo V infection-induced autophagy is an important host antiviral mechanism.The main results are as follows:In this paper,an efficient RT-RPA assay was established for rapid detection of PMMo V and Chi VMV that infect peppers.This assay required only a pair of specific primers and could be amplified at 38 ? for 30 minutes with high specificity and sensitivity.It had no cross-reaction with other related viruses and is about 10-fold sensitive than the conventional RT-PCR.The RT-RPA method was further verified by field-collected samples.In this paper,high-throughput sequencing was used to study the characteristics of vsi RNAs after PMMo V infection in peppers.The important function of small RNAs in the interaction between PMMo V and pepper was explored.The results showed that the length of vsi RNAs was mainly 21-nt and 22-nt,and U was the most abundant nucleotide at the 5'-terminal.vsi RNAs were derived from the sense and antisense strands of the PMMo V genome with heterogeneous distribution.There were more hotspots in the sense strand of PMMo V than that in the antisense strand,and the regions corresponding to Rd Rp and CP contained more hotspots.Target predictions for vsi RNAs indicated that 42 specific vsi RNAs were predicted to target 66 annotated genes in pepper.Under certain conditions,most vsi RNAs in the sense strand had multiple targets,while most antisense vsi RNAs had only one target.GO analysis of these targets showed that many annotated targets were involved in physiological pathways related to stress response,cell regulation,and metabolism process.In addition,PMMo V infection induced significant up-regulation of Ca AGO1a/1b/2,Ca DCL2 and Ca RDR1 in pepper plants.Further analysis of the sequencing results showed that PMMo V infection affected the expression of endogenous small RNA in pepper plants.In this study,a total of 88 known mi RNAs in pepper plants were identified,including 20 specific mi RNAs,and 198 novel mi RNAs were predicted.We used bioinformatics software to predict mi RNA targets and analyze their functions.GO analysis showed that most of the targets played important roles in the development and defense signaling process of pepper plants after PMMo V infection,which laid the foundation for search on the impact of virus infection on mi RNA regulatory network in pepper plants.In order to further understand the interaction between pepper plants and PMMo V,the transcriptomic profiles of Mock-infected and PMMo V-infected pepper plants were compared by high-throughput sequencing technology.A total of 24,227 genes were obtained from the different samples.We identified a total of 197 differentially expressed genes(DEGs),of which 172 genes were significantly up-regulated and 25 genes significantly down-regulated compared with the control group.By GO and KEGG pathway enrichment analysis of these DEGs,53 significant enrichment GO terms and 6 significant enrichment KEGG pathways were obtained.Additionally,the expressions of several autophagy-related genes(ATGs)were affected by PMMo V infection in pepper plants,suggesting that autophagy may play an important role in the process of PMMo V infection.Recently,the direct interaction between autophagy and plant viruses has also become the research hotspots.In this study,based on the model plant Nicotiana benthamiana,we detected the formation of autophagic structure and the expression characteristics of autophagy-related genes Nb VPS15,Nb ATG3,Nb ATG5,Nb Beclin1,Nb ATG7,Nb ATG8 a,Nb ATG9 in tobacco leaves after PMMo V infection by transmission electron microscopy,autophagy marker GFP-ATG8 a fluorescence,immunoblot,and real-time RT-PCR,respectively.We demonstrated that PMMo V infection could cause host autophagy.Silencing of key autophagy genes,such as Nb ATG3,Nb ATG5,Nb Beclin1,Nb ATG7 and Nb TG8 a by virus-induced gene silencing(VIGS),could increase PMMo V accumulation and accelerate the production of disease symptoms.Moreover,treatments with autophagy inhibitors 3-MA and E64 d could also increase PMMo V accumulation.The yeast two-hybrid results showed that P126 encoded by PMMo V could interact with Nb ATG8 f,in which the helicase region Hel was the key domain of the interaction,suggesting that P126 was the target of autophagic degradation by binding to Nb ATG8 f.These results demonstrated that autophagy plays an antiviral role during PMMo V infection.