Conserved Threonine Residues within the A-Loop of the Receptor NIK Differentially Regulate the Kinase Function Required for Antiviral Signaling

NSP-interacting kinase (NIK1) is a receptor-like kinase identified as a virulence target of the begomovirus nuclear shuttle protein (NSP). We found that NIK1 undergoes a stepwise pattern of phosphorylation within its activation-loop domain (A-loop) with distinct roles for different threonine residues. Mutations at Thr-474 or Thr-468 impaired autophosphorylation and were defective for kinase activation. In contrast, a mutation at Thr-469 did not impact autophosphorylation and increased substrate phosphorylation, suggesting an inhibitory role for Thr-469 in kinase function. To dissect the functional significance of these results, we used NSP-expressing virus infection as a mechanism to interfere with wild type and mutant NIK1 action in plants. The NIK1 knockout mutant shows enhanced susceptibility to virus infections, a phenotype that could be complemented with ectopic expression of a 35S-NIK1 or 35S-T469A NIK1 transgenes. However, ectopic expression of an inactive kinase or the 35S-T474A NIK1 mutant did not reverse the enhanced susceptibility phenotype of knockout lines, demonstrating that Thr-474 autophosphorylation was needed to transduce a defense response to geminiviruses. Furthermore, mutations at Thr-474 and Thr-469 residues antagonistically affected NIK-mediated nuclear relocation of the downstream effector rpL10. These results establish that NIK1 functions as an authentic defense receptor as it requires activation to elicit a defense response. Our data also suggest a model whereby phosphorylation-dependent activation of a plant receptor-like kinase enables the A-loop to control differentially auto- and substrate phosphorylation

Functional analysis of the naturally recombinant DNA-A of the bipartite begomovirus tomato chlorotic mottle virus

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

A PERK-Like Receptor Kinase Interacts with the Geminivirus Nuclear Shuttle Protein and Potentiates Viral Infection

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

Chocó bio-innovador y sustentable

La formulación del Plan Estratégico Regional en Ciencia, Tecnología e Innovación del departamento del Chocó (PERCTI) tiene como propósito central señalar el camino para fortalecer el desarrollo propio e intercambio de mejores prácticas en sectores claves; reconocer la formación de expertos en áreas temáticas priorizadas; fortalecer la capacitación del talento humano mediante el aprovechamiento de capacidades instaladas en el territorio; coadyuvar a la transferencia de tecnologías necesarias para impulsar el desarrollo endógeno sustentable y, por supuesto, incentivar la incorporación de conocimiento científico y tecnológico en la cotidianidad de las personas para mejorar su calidad de vida