Integrated Biological Experiments and Proteomic Analyses of

The plant vascular system is not only a transportation system for delivering nutrients but also a highway transport network for spreading viruses. Tomato spotted wilt orthotospovirus (TSWV) is among the most destructive viruses that cause serious losses in economically important crops worldwide. However, there is minimal information about the long-distance movements of TSWV in the host plant vascular system. In this this study, we confirm that TSWV virions are present in the xylem as observed by transmission electron microscopy (TEM). Further, a quantitative proteomic analysis based on label-free methods was conducted to reveal the uniqueness of protein expression in xylem sap during TSWV infection. Thus, this study identified and quantified 3305 proteins in two groups. Furthermore, TSWV infection induced three viral structural proteins, N, Gn and Gc, and 315 host proteins differentially expressed in xylem (163 up-regulated and 152 down-regulated). GO enrichment analysis showed up-regulated proteins significantly enriched in homeostasis, wounding, defense response, and DNA integration terms, while down-regulated proteins significantly enriched in cell wall biogenesis/xyloglucan metabolic process-related terms. KEGG enrichment analysis showed that the differentially expressed proteins (DEPs) were most strongly associated with plant-pathogen interaction, MAPK signaling pathway, and plant hormone signal transduction. Cluster analysis of DEPs function showed the DEPs can be categorized into cell wall metabolism-related proteins, antioxidant proteins, PCD-related proteins, host defense proteins such as receptor-like kinases (RLKs), salicylic acid binding protein (SABP), pathogenesis related proteins (PR), DNA methylation, and proteinase inhibitor (PI). Finally, parallel reaction monitoring (PRM) validated 20 DEPs, demonstrating that the protein abundances were consistent between label-free and PRM data. Finally, 11 genes were selected for RT-qPCR validation of the DEPs and label-free-based proteomic analysis concordant results. Our results contribute to existing knowledge on the complexity of host plant xylem system response to virus infection and provide a basis for further study of the mechanism underlying TSWV long-distance movement in host plant vascular system.