Project description:Cucurbit chlorotic yellows virus (CCYV) is a cucurbit-infecting crinivirus. RNA silencing can be initiated as a plant defense against viruses. Viruses encode various RNA silencing suppressors to counteract antiviral silencing. P22 protein encoded by RNA1 of CCYV is a silencing suppressor, but its mechanism of action remains unclear. In this study, the cucumber ribosomal-like protein CsRPS21 was found to interact with P22 protein in vitro and in vivo. A conserved CsRPS21 domain was indispensable for its nuclear localization and interaction with P22. Transient expression of CsRPS21 in Nicotiana benthamiana leaves interfered with P22 accumulation and inhibited P22 silencing suppressor activity. CsRPS21 expression in N. benthamiana protoplasts inhibited CCYV accumulation. Increasing numbers of ribosomal proteins are being found to be involved in viral infections of plants. We identified a P22-interacting ribosomal protein, CsRPS21, and uncovered its role in early viral replication and silencing suppressor activity. Our study increases knowledge of the function of ribosomal proteins during viral infection.
Project description:Plants use RNA silencing as a defense against viruses. In response, viruses encode various RNA silencing suppressors to counteract the antiviral silencing. Here, we identified p22 as a silencing suppressor of cucurbit chlorotic yellows crinivirus and showed that p22 interacts with CsSKP1LB1, a Cucumis sativus ortholog of S-phase kinase-associated protein 1 (SKP1). The F-box-like motif of p22 was identified through sequence analysis and found to be necessary for the interaction using a yeast two-hybrid assay. The involvement of the F-box-like motif in p22 silencing suppressor activity was determined. Proteomics analysis of Nicotiana benthamiana leaves expressing p22, and its F-box-like motif deletion mutant showed 228 differentially expressed proteins and five enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways: ABC transporters, sesquiterpenoid and triterpenoid biosynthesis, ubiquitin-mediated proteolysis, riboflavin metabolism, and cysteine and methionine metabolism. Collectively, our results demonstrate the interaction between p22 and CsSKP1LB1 and show that the deletion of F-box-like motif inhibits p22 silencing suppressor activity. The possible pathways regulated by the p22 through the F-box-like motif were identified using proteomics analysis.
Project description:Aster yellows phytoplasma strain Hyd35 (16SrI-B) in micropropagated periwinkle shoots in collection was used to produce infected plants in pots that were separated according to the diverse symptomatology i.e. phyllody and witches’ broom. Small RNA high-throughput sequencing (HTS) was then used to determine the small RNA pattern of these plants. Bioinformatics analysis revealed the presence of expression changes of different miRNA classes and the presence of phytoplasma derived small RNAs. These results could complement previous studies and serve as a starting point for small RNA omics in phytoplasma research
Project description:BACKGROUND:Cucurbit chlorotic yellows virus (CCYV) is a bipartite cucurbit-infecting crinivirus within the family Closteroviridae. The crinivirus genome varies among genera. P4.9 is the first protein encoded by CCYV RNA2. P5, which is encoded by LIYV, is necessary for efficient viral infectivity in plants; however, it remains unknown whether CCYV P4.9 is involved in movement. FINDING:In this study, we used green fluorescent protein (GFP) to examine the intracellular distribution of P4.9-GFP in plant cells, and observed fluorescence in the cytoplasm and nucleus. Transient expression of P4.9 was localized to the plasmodesmata. Co-infiltration of agrobacterium carrying binary plasmids of P4.9 and GFP facilitated GFP diffusion between cells. Besides P4.9 was able to spread by itself to neighboring cells, and co-localized with a marker specific to the endoplasmic reticulum, HDEL-mCherry, but not with the Golgi marker Man49-mCherry. CONCLUSIONS:Together, these results demonstrate that CCYV P4.9 is involved in cell-cell movement.