Elucidating the Subcellular Localization of GLRaV-3 Proteins Encoded by the Unique Gene Block in N. benthamiana Suggests Implications on Plant Host Suppression.

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.

Proximity driven plastid-nucleus relationships are facilitated by tandem plastid-ER dynamics.

Peri-nuclear clustering (PNC) of chloroplasts has largely been described in senescent and pathogen- or ROS- stressed cells. Stromules, tubular plastid extensions are also observed under similar conditions. Coincident observations of PNC and stromules associate the two phenomena in facilitating retrograde signaling between chloroplasts and the nucleus. However, PNC incidence in non-stressed cells under normal growth and developmental conditions, when stromules are usually not observed, remains unclear. Using transgenic Arabidopsis expressing different organelle-targeted fluorescent proteins we show that PNC is a dynamic subcellular phenomenon that continues in the absence of light and is not dependent on stromule formation. PNC is facilitated by tandem plastid-ER dynamics created through membrane contact sites between the two organelles. While PNC increases upon ER-membrane expansion, some plastids may remain in the peri-nuclear region due to their localization in ER-lined nuclear indentions. Moreover, some PNC plastids may sporadically extend stromules into ER-lined nuclear grooves. Our findings strongly suggest that PNC is not an exclusive response to stress caused by pathogens, high light or exogenous-H2O2 treatment and does not require stromule formation. However, morphological and behavioural alterations in ER and concomitant changes in tandem, plastid-ER dynamics play a major role in facilitating the phenomenon.