RESUMO
Co-agroinjection of Nicotiana benthamiana leaves with the pectin methylesterase (proPME) gene and the TMV:GFP vector resulted in a stimulation of virus-induced RNA silencing (inhibition of GFP production, virus RNA degradation, stimulation of siRNAs production). Conversely, co-expression of TMV:GFP with either antisense PME construct or with enzymatically inactive proPME restored synthesis of viral RNA. Furthermore, expression of proPME enhanced the GFP transgene-induced gene silencing accompanied by relocation of the DCL1 protein from nucleus to the cytoplasm and activation of siRNAs and miRNAs production. It was hypothesized that DCL1 relocated to the cytoplasm may use as substrates both miRNA precursor and viral RNA. The capacity for enhancing the RNA silencing is a novel function for the polyfunctional PME.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Nicotiana/enzimologia , Interferência de RNA , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Precursores Enzimáticos , Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , MicroRNAs/biossíntese , Epiderme Vegetal/citologia , Folhas de Planta/citologia , Folhas de Planta/microbiologia , Proteínas de Plantas/metabolismo , Transporte Proteico , Estabilidade de RNA/genética , RNA Interferente Pequeno/biossíntese , RNA Viral/metabolismo , Rhizobium/genética , Vírus do Mosaico do Tabaco/fisiologia , TransgenesRESUMO
We report that unprocessed tobacco pectin methylesterase (PME) contains N-terminal pro-sequence including the transmembrane (TM) domain and spacer segment preceding the mature PME. The mature portion of PME was replaced by green fluorescent protein (GFP) gene and various deletion mutants of pro-sequence fused to GFP were cloned into binary vectors and agroinjected in Nicotiana benthamiana leaves. The PME pro-sequence delivered GFP to the cell wall (CW). We showed that a transient binding of PME TM domain to endoplasmic reticulum membranes occurs upon its transport to CW. The CW targeting was abolished by various deletions in the TM domain, i.e., anchor domain was essential for secretion of GFP to CW. By contrast, even entire deletion of the spacer segment had no influence on GFP targeting.