RESUMO
SUMMARY: In contrast to the accumulated data on nuclear transport mechanisms of macromolecules, little is known concerning the regulated release of nuclear-exported complexes and their subsequent trans-cytoplasmic movement. The bipartite begomovirus nuclear shuttle protein (NSP) facilitates the nuclear export of viral DNA and cooperates with the movement protein (MP) to transport viral DNA across the plant cell wall. Here, we identified a cellular NSP-interacting GTPase (NIG) with biochemical properties consistent with a nucleocytoplasmic transport role. We show that NIG is a cytosolic GTP-binding protein that accumulates around the nuclear envelope and possesses intrinsic GTPase activity. NIG interacts with NSP in vitro and in vivo (under transient expression), and redirects the viral protein from the nucleus to the cytoplasm. We propose that NIG acts as a positive contributor to geminivirus infection by modulating NSP nucleocytoplasmic shuttling and hence facilitating MP-NSP interaction in the cortical cytoplasm. In support of this, overexpression of NIG in Arabidopsis enhances susceptibility to geminivirus infection. In addition to highlighting the relevance of NIG as a cellular co-factor for NSP function, our findings also have implications for general nucleocytoplasmic trafficking of cellular macromolecules.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Begomovirus/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas do Movimento Viral em Plantas/metabolismo , Transporte Ativo do Núcleo Celular , Arabidopsis/metabolismo , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Begomovirus/metabolismo , Núcleo Celular/metabolismo , DNA Viral/genética , Microscopia Confocal , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Doenças das Plantas/virologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas do Movimento Viral em Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plasmídeos , Protoplastos/metabolismo , RNA de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Nicotiana/genética , Nicotiana/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
All geminiviruses found in Brazil belong to the Begomovirus genus with a bipartite genome that is split between two genomic components, DNA-A and DNA-B. The DNA-A of the bipartite begomovirus ToCMoV-[MG-Bt] (Tomato chlorotic mottle virus), however, possesses as a peculiar characteristic the capacity to systemically infect Nicotiana benthamiana. Here we further characterize this variant DNA-A and show that it also infects Solanum lycopersicum and other host plants, in the absence of DNA-B. The ToCMoV-[MG-Bt]-DNA-A encodes an additional ORF, designated AC5, but otherwise its genome organization is similar to other DNA-A from Western Hemisphere begomoviruses. We showed that this AC5 putative ORF is not essential for infection, as disruption of its coding capacity caused no effect on ToCMoV-[MG-Bt]-DNA-A-mediated infection process. Likewise, the ToCMoV-[MG-Bt]-DNA-A ac4 mutant was indistinguishable from its wild type counterpart in all hosts tested. In contrast, an av1 (coat protein) mutant was unable to infect systemically N. benthamiana and Chenopodium quinoa in the absence of DNA-B. However, inclusion of DNA-B in the infection assay fully rescued the movement defect of the ToCMoV-[MG-Bt]-DNA-A av1 mutant. These results suggest that at suboptimal conditions for infection the coat protein is required for ToCMoV-[MG-Bt] systemic movement.
Assuntos
Begomovirus/genética , DNA Viral/genética , Sequência de Aminoácidos , Sequência de Bases , Begomovirus/classificação , Begomovirus/patogenicidade , Chenopodium quinoa , Primers do DNA/genética , Variação Genética , Genoma Viral , Solanum lycopersicum/virologia , Dados de Sequência Molecular , Recombinação Genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , NicotianaRESUMO
The nuclear shuttle protein (NSP) from bipartite geminiviruses facilitates the intracellular transport of viral DNA from the nucleus to the cytoplasm and acts in concert with the movement protein (MP) to promote the cell-to-cell spread of the viral DNA. A proline-rich extensin-like receptor protein kinase (PERK) was found to interact specifically with NSP of Cabbage leaf curl virus (CaLCuV) and of tomato-infecting geminiviruses through a yeast two-hybrid screening. The PERK-like protein, which we designated NsAK (for NSP-associated kinase), is structurally organized into a proline-rich N-terminal domain, followed by a transmembrane segment and a C-terminal serine/threonine kinase domain. The viral protein interacted stably with defective versions of the NsAK kinase domain, but not with the potentially active enzyme, in an in vitro binding assay. In vitro-translated NsAK enhanced the phosphorylation level of NSP, indicating that NSP functions as a substrate for NsAK. These results demonstrate that NsAK is an authentic serine/threonine kinase and suggest a functional link for NSP-NsAK complex formation. This interpretation was corroborated by in vivo infectivity assays showing that loss of NsAK function reduces the efficiency of CaLCuV infection and attenuates symptom development. Our data implicate NsAK as a positive contributor to geminivirus infection and suggest it may regulate NSP function.