Project description:We report flg22 regulate the accumulation of AGO1-bound small RNA in arabidopsis. We find that a number of miRNAs are up- or down-regulated by flg22, a well-studied PAMP. Examination of AGO1-bound small RNAs with or without flg22 treatment.
Project description:We report flg22 regulate the accumulation of AGO1-bound small RNA in arabidopsis. We find that a number of miRNAs are up- or down-regulated by flg22, a well-studied PAMP.
Project description:In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role[AQ1] in microRNA (miRNA) and small interfering RNA (siRNA)- mediated silencing and is a key component in antiviral responses. The polerovirus F-box P0 protein triggers AGO1 degradation as a viral counterdefense. Here, we identified a motif in AGO1 that is required for its interaction with the S phase kinase-associated protein1-cullin 1-F-box protein (SCF) P0 (SCFP0) complex and subsequent degradation. The AGO1 P0 degron is conserved and confers P0-mediated degradation to other AGO[AQ2] proteins. Interestingly, the degron motif is localized in the DUF1785 domain of AGO1, in which a single point mutation (ago1-57, obtained by forward genetic screening) compromises recognition by SCFP0. Recapitulating formation of the RNA-induced silencing complex in a cell-free system revealed that this mutation impairs RNA unwinding, leading to stalled forms of AGO1 still bound to double-stranded RNAs. In vivo, the DUF1785 is required for unwinding perfectly matched siRNA duplexes, but is mostly dispensable for unwinding imperfectly matched miRNA duplexes. Consequently, its mutation nearly abolishes phased siRNA production and sense transgene posttranscriptional gene silencing. Overall, our work sheds new light on the mode of AGO1 recognition by P0 and the in vivo function of DUF1785 in RNA silencing.
Project description:ARGONAUTE1 (AGO1) binds directly to small regulatory RNA and is a key effector protein of post-transcriptional gene silencing mediated by microRNA (miRNA) and small interfering RNA (siRNA). The formation of an RNA induced silencing complex (RISC) of AGO1 and small RNA requires the function of the Heat Shock Protein 70/90 chaperone system. Some functions of AGO1 occur in association with endomembranes, in particular the rough endoplasmic reticulum (rER), but proteins interacting with AGO1 in membrane fractions remain unidentified. In this study, we show that the farnesylated Heat Shock Protein 40 homologs, J2 and J3, associate with AGO1 in membrane fractions in a manner that involves protein farnesylation. We also show that three changes in AGO1 function are detectable in mutants in protein farnesylation and J2/J3. First, perturbations of the HSP40/70/90 pathway by mutation of j3, hsp90 and farnesyl transferase affect the amounts of AGO1 associated with membranes. Second, miRNA association with membrane-bound AGO1, and with membrane-bound polysomes is increased in farnesyl transferase and farnesylationdeficient J2/J3 mutants. Third, silencing by non-cell autonomously acting short interfering RNAs (siRNAs) is impaired. These observations highlight the involvement of farnesylated J2/J3 in small RNA-mediated gene regulation, and suggest that the importance of chaperone-AGO1 interaction is not limited to the RISC assembly process.
Project description:Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs in Arabidopsis This study constructed and sequenced two independent small RNA libraries from the upper uninoculated leaves of (i) WT Arabidopsis plants 14 d after mock inoculation and of (ii) rdr1 rdr6 plants 14 d after infection with Fny CMV-Δ2b, and one library each from the upper uninoculated leaves of (iii) WT, (iv) rdr1, and (v) rdr6 plants 14 d after infection with TuMV-GFP. Coimmunoprecipitation with FLAG- and HA-specific antibodies was used to obtain (vi,vii) AGO1 and (viii,ix) AGO2 complexes, respectively, from the FLAG-AGO1/ago1-36 and HA-AGO2 plants 14 d after infection with Fny CMV-Δ2b for extracting total loaded small RNAs for the construction and sequencing of small RNA libraries.