RESUMO
Viroids are single-stranded, circular, noncoding RNAs that infect plants, causing devastating diseases. In this work, we employed 2D DIGE, followed by MS identification, to analyze the response of tomato plants infected by Citrus exocortis viroid (CEVd). Among the differentially expressed proteins detected, 45 were successfully identified and classified into different functional categories. Validation results by RT-PCR allowed us to classify the proteins into two expression groups. First group included genes with changes at the transcriptional level upon CEVd infection, such as an endochitinase, a ß-glucanase, and pathogenesis-related proteins, PR10 and P69G. All these defense proteins were also induced by gentisic acid, a pathogen-induced signal in compatible interactions. The second group of proteins showed no changes at the transcriptional level and included several ribosomal proteins and translation factors, such as the elongation factors 1 and 2 and the translation initiation factor 5-alpha. These results were validated by 2D Western blot, and possible PTMs caused by CEVd infection were detected. Moreover, an interaction between eukaryotic elongation factor 1 and CEVd was observed by 2D Northwestern. The present study provides new protein-related information on the mechanisms of plant resistance to pathogens.
Assuntos
Regulação da Expressão Gênica de Plantas/fisiologia , Solanum lycopersicum/fisiologia , Viroides/fisiologia , Western Blotting , Eletroforese em Gel Bidimensional , Fator de Iniciação 1 em Eucariotos/química , Fator de Iniciação 1 em Eucariotos/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Gentisatos/farmacologia , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/virologia , Doenças das Plantas , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase , Ligação Proteica , Modificação Traducional de Proteínas/efeitos dos fármacos , Modificação Traducional de Proteínas/fisiologia , Proteoma/efeitos dos fármacos , Proteoma/fisiologia , RNA Viral/química , RNA Viral/metabolismo , Reprodutibilidade dos Testes , Ácido Salicílico/farmacologiaRESUMO
We have observed that treatments with salicylic acid (SA) or gentisic acid (GA) induced resistance to RNA pathogens such as ToMV and CEVd in tomato and Gynura auriantiaca, respectively. Accumulation of SA and GA has been found to occur in plants infected by these pathogens, thus pointing out a possible defence role of both molecules. To study the molecular basis of the observed induced resistance to RNA pathogens the induction of silencing-related genes by SA and GA was considered. For that purpose, we searched for tomato genes which were orthologous to those described in Arabidopsis thaliana, such as AtDCL1, AtDCL2, AtDCL4, AtRDR1, AtRDR2 and AtRDR6, and we tracked their induction in tomato along virus and viroid infections. We observed that CEVd significantly induced all these genes in tomato, with the exception of ToRDR6, being the induction of ToDCL4 the most outstanding. Regarding the ToMV asymptomatic infection, with the exception of ToRDR2, we observed a significant induction of all the indicated silencing-related genes, being ToDCL2 the most induced gene. Subsequently, we analyzed their transcriptional activation by SA and at the time when ToMV was inoculated on plants. ToDCL2, ToRDR1 and ToRDR2 were significantly induced by both SA and GA, whereas ToDCL1 was only induced by SA. Such an induction resulted more effective by SA treatment, which is in agreement with the stronger SA-induced resistance observed. Our results suggest that the observed delay in the RNA pathogen accumulation could be due to the pre-induction of RNA silencing-related genes by SA or GA.