Functional modulators of the pro-begomoviral protein NIG (NSP-Interacting GTPase)

• Ano de defesa: 2021
• Autor(a) principal: Raimundo, Gabriel Angelo Saraiva
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Universidade Federal de Viçosa; Genética e Melhoramento
• Palavras-chave em Português: Begomovirus; Proteínas; Biologia Molecular
• Link de acesso: https://locus.ufv.br//handle/123456789/31522; https://doi.org/10.47328/ufvbbt.2022.466

The Begomovirus (Geminiviridae family) genome codes for multifunctional proteins responsible for viral replication, viral transport, subverting, and co-opting host functions to favor viral infections. The movement protein NSP (Nuclear Shuttle Protein) binds and escorts vDNA from the nucleus to the cytosol. The Arabidopsis thaliana cytosolic protein NIG (NSP-Interacting GTPase) accessorizes NSP transport from the nucleus to the cytosol. Accordingly, the overexpression of NIG confers enhanced susceptibility to begomovirus, thus placing NIG as a potential pro-begomoviral protein. Among host proteins, NIG associates with the endosomal NISP (NSP-interacting syntaxin domain- containing protein) to help NSP-vDNA traffic through the cytoplasm; and WWP1 (WW domain-containing protein 1), which entraps NIG in nuclear bodies. CSN5A (COP9 Signalosome Subunit 5a) is a potential NIG partner that has been shown to redirect it to the nucleus; however, NIG-CSN5A dynamic has not been elucidated. Since NIG partners influence its nuclear import, we examined whether some physiological stimulus could affect NIG localization. A phytohormone screening by confocal microscopy showed that salicylic acid (SA) could redirect NIG to the nucleus. Nuclear fractionation assays also indicated that SA could alter NIG localization to the nucleus. A WWP1 Knockout line overexpressing NIG displayed the same confocal and nuclear fractionation pattern, indicating that SA-mediated transport was independent of WWP1-mediated nuclear import of NIG. Additionally, SA promoted NIG ubiquitination and degradation, a process prevented by the proteasome inhibitor MG132. To elucidate the underlying mechanism for the proteasome-mediated degradation of NIG, the interaction between NIG and CSN5A was further investigated. CSN5A negatively regulated NIG turnover and interacted with NIG in vivo by co-immunoprecipitation assays. In the absence of stimuli, the NIG-CSN5A complex was predominantly formed in the cytosol, as shown by the BiFC (Bimolecular Fluorescence Complementation) system, yet several lines of evidence were provided indicating that SA-mediated degradation of NIG may occur in the nucleus. First, SA mediated the nuclear relocalization and degradation of NIG. Second, treatment with MG132 increased the SA-induced nuclear pool of NIG but did not alter the cytosolic levels of the protein. Finally, NIG formed a complex with CSN5A that participates in the proteolytic activity of the COP9 signalosome in the nucleus. To approach NIG role via reverse genetics, an NIG knockout line and independent complemented lines were obtained. Col-0, NIG knockout line, and NIG complemented lines were infected with the begomovirus CabLCV (Cabbage Leaf Curl Virus) through biolistics, but these genotypes did not display differences in resistance parameters against the begomovirus. These results complement the current knowledge on NIG functional modulators in the context of begomovirus infections, which may further elucidate the dynamic regarding this GTPase pro-begomoviral function. Keywords: Begomovirus. NIG. NSP.