RESUMEN
Group-II introns are self-splicing mobile genetic elements consisting of catalytic intron-RNA and its related intron-encoded splicing maturase protein cofactor. Group-II sequences are particularly plentiful within the mitochondria of land plants, where they reside within many critical gene loci. During evolution, the plant organellar introns have degenerated, such as they lack regions that are are required for splicing, and also lost their evolutionary related maturase proteins. Instead, for their splicing the organellar introns in plants rely on different host-acting protein cofactors, which may also provide a means to link cellular signals with respiratory functions. The nuclear genome of Arabidopsis thaliana encodes four maturase-related factors. Previously, we showed that three of the maturases, nMAT1, nMAT2 and nMAT4, function in the excision of different group-II introns in Arabidopsis mitochondria. The function of nMAT3 (encoded by the At5g04050 gene locus) was found to be essential during early embryogenesis. Using a modified embryo-rescue method, we show that nMAT3-knockout plants are strongly affected in the splicing of nad1 introns 1, 3 and 4 in Arabidopsis mitochondria, resulting in complex-I biogenesis defects and altered respiratory activities. Functional complementation of nMAT3 restored the organellar defects and embryo-arrested phenotypes associated with the nmat3 mutant line. Notably, nMAT3 and nMA4 were found to act on the same RNA targets but have no redundant functions in the splicing of nad1 transcripts. The two maturases, nMAT3 and nMAT4 are likely to cooperate together in the maturation of nad1 pre-RNAs. Our results provide important insights into the roles of maturases in mitochondria gene expression and the biogenesis of the respiratory system during early plant life.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Desoxirribonucleasas/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Proteínas Mitocondriales/metabolismo , Arabidopsis/embriología , Proteínas de Arabidopsis/genética , Núcleo Celular/genética , Desoxirribonucleasas/genética , Intrones/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Fenotipo , Empalme del ARN , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismoRESUMEN
Tomato yellow leaf curl virus (TYLCV), a major tomato pathogen causing extensive crop losses, is a whitefly-transmitted geminivirus. V2 mutants of TYLCV-Is and related viruses tend to induce symptomless infection with attenuated viral DNA levels, while accumulating close to wild-type DNA levels in protoplasts, suggesting V2 as a movement protein. The discovery of plant-silencing mechanisms and viral silencing suppressors, V2 included, led us to reconsider V2׳s involvement in viral movement. We studied two mutant versions of the virus, one impaired in V2 silencing-suppression activity, and another carrying a non-translatable V2. While both mutant viruses spread in the infected plant to newly emerged leaves at the same rate as the wild-type virus, their DNA-accumulation levels were tenfold lower than in the wild-type virus. Thus, we suggest that the setback in virus proliferation, previously ascribed to a movement impediment, is due to lack of silencing-suppression activity.
Asunto(s)
Begomovirus/fisiología , Proteínas de Movimiento Viral en Plantas/metabolismo , Replicación Viral , Begomovirus/genética , Técnicas de Inactivación de Genes , Proteínas de Movimiento Viral en Plantas/genética , Nicotiana/virologíaRESUMEN
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.
Asunto(s)
Begomovirus/fisiología , Interacciones Huésped-Patógeno , Enfermedades de las Plantas/virología , Proteínas Virales/metabolismo , Replicación Viral , Begomovirus/genética , Codón sin Sentido , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Nicotiana/virología , Proteínas Virales/genéticaRESUMEN
Bhendi yellow vein mosaic disease is caused by a complex consisting of a monopartite begomovirus associated with a ß-satellite. The C2 protein of bhendi yellow vein mosaic virus (BYVMV) is a suppressor of post-transcriptional gene silencing and also functions as a transcriptional activator. To explore the molecular mechanisms of its nuclear trafficking and self-interaction, fusion proteins of fluorescent proteins with wild-type or mutated constructs of BYVMV C2 were expressed in tobacco protoplasts. Analyses revealed that the BYVMV C2 nuclear localization signal (NLS) was located in the N terminus of the protein, comprising aa 17-31 of C2. NLSs are recognized by a class of soluble transport receptors termed karyopherins α and ß. The BYVMV C2 NLS was found to be necessary for this protein's interaction with its nuclear import mediator, karyopherin α, ensuring its nuclear localization. Nevertheless, when deleted, C2 was found in both the cytoplasm and the nucleus, suggesting NLS-independent nuclear import of this protein. Homotypic interaction of BYVMV C2 was also found, which correlates with the nuclear localization needed for efficient activation of transcription.