Grapevine leafroll-associated virus 3 (GLRaV-3) is a formidable threat to the stability of the global grape and wine industries. It is the primary etiological agent of grapevine leafroll disease (GLD) and significantly impairs vine health, fruit quality, and yield. GLRaV-3 is a member of the genus Ampelovirus, Closteroviridae family. Viral genes within the 3′ proximal unique gene blocks (UGB) remain highly variable and poorly understood. The UGBs of Closteroviridae viruses include diverse open reading frames (ORFs) that have been shown to contribute to viral functions such as the suppression of the host RNA silencing defense response and systemic viral spread. This study investigates the role of GLRaV-3 ORF8, ORF9, and ORF10, which encode the proteins p21, p20A, and p20B, respectively. These genes represent largely unexplored facets of the GLRaV-3 genome. Here, we visualize the subcellular localization of wildtype and mutagenized GLRaV-3 ORFs 8, 9, and 10, transiently expressed in Nicotiana benthamiana. Our results indicate that p21 localizes to the cytosol, p20A associates with microtubules, and p20B is trafficked into the nucleus to carry out the suppression of host RNA silencing. The findings presented herein provide a foundation for future research aimed at the characterization of the functions of these ORFs. In the long run, it would also facilitate the development of innovative strategies to understand GLRaV-3, mitigate its spread, and impacts on grapevines and the global wine industry.

Coffee is a perennial plant that exhibits asynchronous flowering while maintaining concomitant vegetative growth. This growth dichotomy affects fruit development and maturation time. To better understand flowering in coffee, we characterized a phosphatidylethanolamine binding protein (PEBP) homolog with high similarity to Arabidopsis thaliana TERMINAL FLOWER1 (TFL1). The interaction of TFL1 with floral regulator bZIP transcription factor, FD, forms a floral repressor complex that maintains inflorescence meristems in an indeterminate state. Arabidopsis TFL1 is expressed only in shoot apical meristems, yet CaTFL1a transcripts were detected exclusively in coffee leaves. Moreover, leaf-derived CaTFL1a transcript retains an intron, which has not been reported for TFL1 orthologs in other species. The ectopic expression of CaTFL1a in Arabidopsis causes extreme late flowering or prevents flowering altogether. Notably, the most severe floral repressive activity occurred in transgenic plants that spliced out the extra intron from CaTFL1. Yeast Two-Hybrid assays show that full-length CaTFL1a protein (fl-CaTFL1a) encoded by the fully spliced mRNA interacts with FD and Arabidopsis 14-3-3 protein AtGRF3, whereas truncated protein (tr-CaTFL1a) encoded by transcript that retains an intron does not interact. This evidence suggests that CaTFL1a may affect flowering in coffee by acting as a leaf-derived, long-distance floral repressor whose activity is controlled by alternative splicing.