Biological, Transcriptomic, Proteomic Analysis Of Nicotiana Benthamiana And Beta Macrocarpa Infected By Beet Necrotic Yellow Vein Virus

Beet necrotic yellow vein virus (BNYVV), contains either four or five plus-sense single stranded RNAs and is the causal agent of rhizomania disease in sugar beet, which is a widely distributed virus in most regions of the world. The virus exhibits various systemic infectivity and pathogenicity in different hosts. Based on the previous researches, this dissertation was mainly focus on biological, transcriptomic, proteomic analysis of Nicotiana benthamiana and Beta macrocarpa infected by BNYVV with different RNA components.Our previous studies have demonstrated that RNA5was usually eliminated spontaneously in N. benthamiana. To understand the cause of RNA5instability, the maintenance and accumulation of BNYVV RNA5were investigated during systemic infections of N. benthamiana and B. macrocarpa. RT-PCR and Northern blot analysis showed that RNA5had a higher level of accumulation and was maintained more consistently in B. macrocarpa than in N. benthamiana. Comparative analysis of β-glucuroidase (GUS) expression from RNA4and RNA5replicons and recombinants revealed that untranslated regions (UTR) sequence in RNA5had a positive effect on replication in protoplasts and systemic infection of N. benthamiana. These data demonstrate that the low replication efficiency of RNA5depends on the untranslated sequence and regards to systemic infectivity in N. benthamiana.BNYVV can infect N. benthamiana systemically, which causes severe curling and stunting symptoms in the presence of RNA4or mild symptoms in the absence of RNA4. Confocal laser scanning microscopy (CLSM) analysis showed that the RNA4-encoded pathogenicity determinant p31protein fused to the red fluorescent protein (RFP) was localized mainly in the nuclei of N. benthamiana epidermal cells. This suggested that the severe RNA4-induced symptoms might result from p31-dependent modifications of the transcriptome. Therefore, we used next-generation sequencing technologies to analyze the transcriptome profiling of N. benthamiana in the response to infection with different isolates of BNYVV. Comparisons of the transcriptomes of mock, BN3(RNA1+2+3), and BN34(RNA1+2+3+4) infected plants identified3,016differentially expressed transcripts, which provided a list of candidate genes that potentially are elicited as a response to viral infection. Our data indicate that modifications in the expression of genes involved in RNA silencing, ubiquitin-proteasome pathway, cellulose synthesis, and metabolism of the plant hormone gibberellin may contribute to the severe symptoms induced by RNA4from BNYVV. To compare the disease developing mechanism between N. benthamiana and B. macrocarpa, we also characterized the transcriptome of B. macrocarpa and analyzed global gene expression of B. macrocarpa in response to BNYVV infection using Illumina sequencing platform. The overall de novo assembly of cDNA sequence data generated75,917unigenes. Furthermore, comparative analysis of the two transcriptomes revealed that261genes differentially expressed in the infected plants compared to the control, including128up-and133down-regulated genes. GO analysis showed that the changes of the differently expressed genes mainly enriched in response to biotic stimulus and primary metabolic process. Proteins are specific physiological functions executor, the study of protein exrpession changes will directly elucidate the mechanism under physiological and pathological conditions. The research at mRNA level can only reflect the regulation of transcription, but can not reflect the expression of the protein. Proteomics is the large-scale study of proteins, with which we can gain a global understanding of protein expression pattern of samples. Using two-dimesional electrophoresis (2-DE) followed by mass spectrometry and bioinformatics method to analyze the protein expression changes in BN3-and BN34-infected N. benthamiana leaves. The significant expressional changes in N. benthamiana proteins were mostly related to pathogenesis-related proteins, cytoskeletal proteins, stress reponse proteins and proteins involved in photosynthsis, oxidation reduction and metabolism. Thirteen differentially expressed proteins were successfully identified. The study is the first attempt to comapre the complex picture of Nbenthamiana protein expression during BNYVV infection, hence provides large scale valuable protein-related information for better understanding the host immune response to BNYW and potentially mlecular pathogenesis.