iTRAQ analysis of the tobacco leaf proteome reveals that RNA-directed DNA methylation (RdDM) has important roles in defense against geminivirus-betasatellite infection.

Geminiviruses have caused serious losses in crop production. To investigate the mechanisms underlying host defenses against geminiviruses, an isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomic approach was used to explore the expression profiles of proteins in Nicotiana benthamiana (N. benthamiana) leaves in response to tomato yellow leaf curl China virus (TYLCCNV) with its betasatellite (TYLCCNB) at an early phase. In total, 4155 proteins were identified and 272 proteins were changed differentially in response to TYLCCNV/TYLCCNB infection. Bioinformatics analysis indicated that S-adenosyl-l-methionine cycle II was the most significantly up-regulated biochemical process during TYLCCNV/TYLCCNB infection. The mRNA levels of three proteins in S-adenosyl-l-methionine cycle II were further analyzed by qPCR, each was found significantly up-regulated in TYLCCNV/TYLCCNB-infected N. benthamiana. This result suggested a strong promotion of the biosynthesis of available methyl groups during geminivirus infections. We further tested the potential role of RdDM in N. benthamiana by virus-induced gene silencing (VIGS) and found that a disruption in RdDM resulted in more severe infectious symptoms and higher accumulation of viral DNA after TYLCCNV/TYLCCNB infection. Although the precise functions of these proteins still need to be determined, our proteomic results enhance the understanding of plant antiviral mechanisms. BIOLOGICAL SIGNIFICANCE: One of the major limitations to crop growth in the worldwide is the prevalence of geminiviruses. They are able to infect food and cash crops and cause serious crop failures and economic losses worldwide, especially in Africa and Asia. Tomato yellow leaf curl China virus (TYLCCNV), which causes severe viral diseases in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). However, the mechanisms underlying the TYLCCNV/TYLCCNB defense in plants are still not fully understood at the molecular level. In this study, the combined proteomic, bioinformatic and VIGS analyses revealed that TYLCCNV/TYLCCNB invasion caused complex proteomic alterations in the leaves of N. benthamiana involving the processes of stress and defense, energy production, photosynthesis, protein homeostasis, metabolism, cell structure, signal transduction, transcription, transportation, and cell growth/division. Promotion of available methyl groups via the S-adenosyl-l-methionine cycle II pathway in N. benthamiana appeared crucial for antiviral responses. These findings enhance our understanding in the proteomic aspects of host antiviral defenses against geminiviruses, and also demonstrate that the combination of proteomics with bioinformatics and VIGS analysis is an effective approach to investigate systemic plant responses to geminiviruses and to shed light on plant-virus interactions.