RESUMEN
Bhendi yellow vein mosaic virus (BYMV) is a monopartite begomovirus with an associated ß-satellite. ßC1 ORF encoded by the ß-satellite is the symptom determinant and a strong suppressor of post transcriptional gene silencing. To create a virus induced gene silencing vector based upon the ß-satellite associated with BYVMV the ßC1 ORF was replaced with multiple cloning sites. GFP transgene and plant endogenous genes Su, PDS, PCNA and AGO1 were cloned into ß-satellite based VIGS vector. GFP expression was silenced in the GFP expressing transgenic 16c Nicotiana benthamiana plants infiltrated with VIGS vector carrying GFP gene inside. N. benthamiana plants infiltrated with the VIGS vector harboring the endogenous genes Su, PDS, PCNA and AGO1 produced the phenotypic symptoms yellowing of the veins, photobleaching of the veins, stunting of the plant and upward leaf curling, respectively. Real time PCR analyses revealed a reduction in the levels of the corresponding transgene or endogenous target mRNA. The ß-satellite based VIGS vector was able to silence the target genes effectively. Hence, BYVMV ß-satellite based VIGS vector can be used in functional genomics studies.
Asunto(s)
Begomovirus/genética , ADN Satélite/genética , Técnicas de Silenciamiento del Gen/métodos , Vectores Genéticos , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Plantas Modificadas Genéticamente/genética , Nicotiana/genéticaRESUMEN
Bhendi yellow vein mosaic virus (BYVMV) that causes bhendi yellow vein mosaic disease is a monopartite begomovirus with an associated betasatellite. Previous studies have shown that C2 protein of BYVMV acts as a suppressor of post transcriptional gene silencing, activates transcription, localizes to nucleus, and interacts with karyopherin α. To probe the role of C2 in symptom determination and virus replication, the infectious clones of BYVMV containing two stop codons in the C2 ORF were created and used for infection studies. The Nicotiana benthamiana plants infiltrated with the infectious clones harboring stop codons in the C2 ORF did not develop any symptoms unlike plants infiltrated with wild-type BYVMV. Southern blotting and real time PCR analysis revealed that the viral load was reduced drastically in the plants infected with BYVMV containing the nontranslatable version of C2 ORF. However, there was a recovery in viral DNA replication, when co-infiltrated with wild-type betasatellite. Hence we conclude that the C2 protein of BYVMV plays an important role in symptom determination and viral DNA replication.