RESUMO
Proteins belonging to the enhancer of RNA interference-1 subfamily of 3'-5' exoribonucleases participate in divergent RNA pathways. They degrade small interfering RNAs (siRNAs), thus suppressing RNA interference, and are involved in the maturation of ribosomal RNAs and the degradation of histone messenger RNAs (mRNAs). Here, we report evidence for the role of the plant homologue of these proteins, which we termed ENHANCED RNA INTERFERENCE-1-LIKE-1 (ERIL1), in chloroplast function. In vitro assays with AtERIL1 proved that the conserved 3'-5' exonuclease activity is shared among all homologues studied. Confocal microscopy revealed that ERL1, a nucleus-encoded protein, is targeted to the chloroplast. To gain insight into its role in plants, we used Nicotiana benthamiana and Arabidopsis thaliana plants that constitutively overexpress or suppress ERIL1. In the mutant lines of both species we observed malfunctions in photosynthetic ability. Molecular analysis showed that ERIL1 participates in the processing of chloroplastic ribosomal RNAs (rRNAs). Lastly, our results suggest that the missexpression of ERIL1 may have an indirect effect on the microRNA (miRNA) pathway. Altogether our data point to an additional piece of the puzzle in the complex RNA metabolism of chloroplasts.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , RNA Ribossômico/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Regulação da Expressão Gênica de Plantas , Interferência de RNA , RNA Ribossômico/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Agroinfiltration is a very fast and powerful method to express in planta any sequences in a transient fashion. Agroinfiltration has proven very useful for the overexpression of proteins in the infiltrated zone when a short-term effect can be informative. However, it has been a real success story in the induction of local and eventually systemic silencing. Here, we describe the use of agroinfiltration for the induction of local silencing of an endogene or a transgene, for the systemic silencing of a transgene and for co-infiltration assays. We also provide protocols for the evaluation of the efficiency of the assay, by detecting the specific siRNAs characteristic of RNA silencing and measuring the effects on the target sequences.