RESUMEN
Tsip1, a Zn finger protein that was isolated as a direct interactor with tobacco stress-induced 1 (Tsi1), plays an important role in both biotic and abiotic stress signaling. To further understand Tsip1 function, we searched for more Tsip1-interacting proteins by yeast two-hybrid screening using a tobacco cDNA library. Screening identified a new Tsip1-interacting protein, Nicotiana tabacum Tsip1-interacting ferredoxin 1 (NtTfd1), and binding specificity was confirmed both in vitro and in vivo. The four repeats of a cysteine-rich motif (CXXCXGXG) of Tsip1 proved important for binding to NtTfd1. Virus-induced gene silencing of NtTfd1, Tsip1, and NtTfd1/Tsip1 rendered plants more susceptible to salinity stress compared with TRV2 control plants. NtTfd1- and Tsip1-silenced tobacco plants were more susceptible to infection by Cucumber mosaic virus compared with control plants. These results suggest that NtTfd1 might be involved in the regulation of biotic and abiotic stresses in chloroplasts by interaction with Tsip1.
Asunto(s)
Ferredoxinas/fisiología , Nicotiana/fisiología , Proteínas de Plantas/fisiología , Estrés Fisiológico , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Clorofila/metabolismo , Cucumovirus/fisiología , Resistencia a la Enfermedad/genética , Ferredoxinas/genética , Ferredoxinas/metabolismo , Regulación de la Expresión Génica de las Plantas , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Datos de Secuencia Molecular , Fenotipo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Tolerancia a la Sal/genética , Nicotiana/genética , Nicotiana/metabolismo , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Tobacco stress-induced1 (Tsi1) is an ethylene-responsive-element binding protein/APETALA2-type transcription factor that plays an important role in both biotic and abiotic stress signaling pathways. We show that Tsi1-interacting protein1 (Tsip1), a DnaJ-type Zn finger protein, interacts with Tsi1 in vitro and in yeast (Saccharomyces cerevisiae). The transcript level of Tsip1 in tobacco (Nicotiana tabacum) increased upon treatment with salicylic acid (SA), ethylene, gibberellic acid, NaCl, and virus challenge. Tsip1 appeared to be physically associated with the chloroplast surface but dissociated from it after SA treatment. Tsip1 colocalized and coimmunoprecipitated with Tsi1 in plant cells following SA treatment. Tsip1 expression increased Tsi1-mediated transcription and was able to functionally compensate for loss of the Tsi1 transcriptional activation domain through a direct interaction with Tsi1. Transgenic plants simultaneously coexpressing Tsi1 and Tsip1 displayed stronger pathogen resistance and salt tolerance than did transgenic plants expressing either Tsi1 or Tsip1 alone. Concurrent with this, the expression of a subset of stress-related genes was induced in a cooperative manner in Tsi1/Tsip1 transgenic plants. These results together implied that Tsi1 recruits Tsip1 to the promoters of stress-related genes to potentiate Tsi1-mediated transcriptional activation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Nicotiana/genética , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Secuencia de Bases , Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Datos de Secuencia Molecular , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Plantas Modificadas Genéticamente/efectos de los fármacos , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/virología , Protoplastos/metabolismo , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Ácido Salicílico/farmacología , Nicotiana/efectos de los fármacos , Nicotiana/metabolismo , Factores de Transcripción/fisiología , Activación Transcripcional , Dedos de Zinc/fisiologíaRESUMEN
Using a yeast two-hybrid system, we identified a plant cellular factor that interacts with the proteins of the Cucumber mosaic virus (CMV). Initially 14 candidate genes were isolated from Nicotiana tabacum, using a full-length CMV 1a gene as bait. Among the candidate genes, two were encoding thaumatin-like proteins (TLP), and were designated as Nicotiana tabacum thaumatin-like protein 1 (NtTLP1). Consistent with this observation, recombinant GST-NtTLP1 protein, which was expressed and purified in E. coli, bound tightly to CMV 1a in vitro. In planta interaction was also verified via co-immunoprecipitation. Additionally, NtTLP1 specifically interacted with the CMV movement-related proteins, movement protein and coat protein, in yeast. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the expression of NtTLP1 increased as the result of CMV inoculation.
