Study On MicroRNAome Of Nicotiana Benthamiana And Beta Macrocarpa Responding To Beet Necrotic Yellow Vein Virus Infection And Viral Pathogenesis Mechanism

Plant microRNAs(miRNAs)are a class of 20-24 nt non-coding RNAs that play important regulatory roles in plant development,disease defense and symptom formation.The study on microRNAome could increase our knowledge on the pathogenicity of Beet necrotic yellow vein virus(BNYVV)and antivirus mechanism of plants,which might be helpful for future management strategies.BNYW,causing rhizomania disease,is the most destructive and prevalent virus on sugar beet.BNYW is a multiparticle virus with four or five positive-sense,single-stranded RNAs(RNA1-5)packaged into rod-shaped virion individually.BNYVV can infect Nicotiana benthamiana and Beta macrocarpa systemically,and exhibits different pathogenicity not only in different hosts,but also in the same host infected by different combinations of viral RNAs.However,its pathogenicity mechanism remains unclear.The aim of this study was to investigate the causes of BNYVV-induced diseases in N.benthamiana and B.macrocarpa by studying the microRNAome from virus-infected plants.The results were as following:In N.benthamiana,BNYW infection induced not only stunting and leaf curling,but also root and floral development defect.The microarray analysis with 1596 probes and RT-qPCR were performed to explore the differential expression of miRNAs and their targets.8 species-specific miRNAs of N.benthamiana were identified.129 differentially expressed miRNAs were responsive to BN 1234 infection,and their targets were also predicted.The further analysis,combined with the results of the transcriptome sequencing,showed that GA,IAA and JA hormone signaling pathways were inhibited,and Ethylene and SA hormone signaling pathways were induced;GA-GRF1-miR396 pathway was associated with dwarf symptom;IAA-ARFs pathway was related to the development defects of root and flower;the induced Ethylene hormone signaling pathway might take part in the formation of leaf curling;and miR397/LACs cooperated with miR164/NAC21/22 to regulate plant apical meristem growth and function in dwarf symptom,too;Ethylene and JA signaling pathways are benefit to BNYVV accumulation.Our research described changes of miRNAs and hormone pathways induced by BNYW infection,and provided a reference for analyzing pathogenic mechanisms of BNYVV.In B.macrocarpa,BNYW infection led to chlorosis,stunting and abnormal development of axillary bud.B.macrocarpa microRNAome were first investigated by small RNA sequencing.Here,547 known miRNAs,representing 129 miRNA families,and 282 potential novel miRNAs were identified in B.macrocarpa.A phylogenetic analysis was performed,and 8 Beta lineage-specific miRNAs were identified.miRNAs responding to BNYVV infection were identified through a differential expression analysis,and confirmed using a microarray analysis.In total,103 known miRNAs,representing 38 miRNA families,and 45 potential novel miRNAs were differentially regulated with at least a two-fold change in BNYVV-infected plants compared with the mock-inoculated control.Their targets were also predicted.These differentially expressed miRNAs were involved in hormone biosynthesis and signal transduction pathways,and enhanced axillary bud development and plant defenses.The up-regulated miR156 was associated with abnormal development of axillary buds.The microRNAome comparative analysis of plants infected by various plant viruses showed that the up-regulated miR168 and miR398 was the same commonality of differential expression of plant miRNAs when symptoms of viral disease were obvious,and may be related to virus-encoded RNA silencing suppressors,and the similar primary defense response among plant species,respectively.The induced miR398 could enhance plant defense to viruses by increasing the concentration of ROI.p25 encoded by BNYVV RNA3 is the key pathogenic factor of BNYVV in sugar beet.In order to facilitate the reaearch on the pathogenicity of p25,transgenic N.bethamiana with overexpressed p25 was obtained using Agrobacteria-mediated leaf-disk method,and insertion sites of p25 gene were analyzed.The results showed that p25 alone had no obvious pathogenicity to root or leaf of N.benthamiana,and this work has provided a material for further studying the characteristics of p25.In addition,transgenic N.bethamiana with a tandem hairpin RNAs structure(BNYVV-hpRNA-BBSV-hpRNA)was obtained and studied.The bioassay results revealed that transgenic plants were resistant to both BNYVV and BBSV.So this research lays the foundation for the development of virus-resistant sugar beet via genetic engineering.To sum up,this study provides a clearer pattern and effective transgenic plant materials for further research on the pathogenesis of BNYVV.