We previously identified Arabidopsis thaliana mutants defective in sense transgene posttranscriptional gene silencing (S-PTGS) that defined six loci; here, we describe mutants that define nine additional loci, including HYPER RECOMBINATION1 (HPR1), SILENCING DEFECTIVE3 (SDE3), and SDE5. Our analyses extend previous findings by showing that the requirement for the putative RNA helicase SDE3 is inversely proportional to the strength of the PTGS inducer and that the putative RNA trafficking protein SDE5 is an essential component of the trans-acting small interfering RNA (tasiRNA) pathway and is required for S-PTGS but not inverted repeat transgene-mediated PTGS (IR-PTGS). Our screen also identified HPR1 as a PTGS actor. We show that hpr1 mutations negatively impact S-PTGS, IR-PTGS, and tasiRNA pathways, resulting in increased accumulation of siRNA precursors and decreased accumulation of mature siRNA. In animals, HPR1/THO1 is a member of the conserved RNA trafficking THO/TREX complex, which also includes TEX1/THO3. We show that tex1 mutants, like hpr1 mutants, impact TAS precursor and mature tasiRNA levels, suggesting that a THO/TREX complex exists in plants and that this complex is important for the integrity of the tasiRNA pathway. We propose that both HPR1 and TEX1 participate in the trafficking of siRNA precursors to the ARGONAUTE catalytic center.

The Arabidopsis ARGONAUTE1 (AGO1) and ZWILLE/PINHEAD/AGO10 (ZLL) proteins act in the miRNA and siRNA pathways and are essential for multiple processes in development. Here, we analyze what determines common and specific function of both proteins. Analysis of ago1 mutants with partially compromised AGO1 activity revealed that loss of ZLL function re-establishes both siRNA and miRNA pathways for a subset of AGO1 target genes. Loss of ZLL function in ago1 mutants led to increased AGO1 protein levels, whereas AGO1 mRNA levels were unchanged, implicating ZLL as a negative regulator of AGO1 at the protein level. Since ZLL, unlike AGO1, is not subjected to small RNA-mediated repression itself, this cross regulation has the potential to adjust RNA silencing activity independent of feedback dynamics. Although AGO1 is expressed in a broader pattern than ZLL, expression of AGO1 from the ZLL promoter restored transgene PTGS and most developmental defects of ago1, whereas ZLL rescued only a few AGO1 functions when expressed from the AGO1 promoter, suggesting that the specific functions of AGO1 and ZLL are mainly determined by their protein sequence. Protein domain swapping experiments revealed that the PAZ domain, which in AGO1 is involved in binding small RNAs, is interchangeable between both proteins, suggesting that this common small RNA-binding domain contributes to redundant functions. By contrast, the conserved MID and PIWI domains, which are involved in 5′-end small RNA selectivity and mRNA cleavage, and the non-conserved N-terminal domain, to which no function has been assigned, provide specificity to AGO1 and ZLL protein function.

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SummaryTT8/bHLH042 is a key regulator of anthocyanins and proanthocyanidins (PAs) biosynthesis in Arabidopsis thaliana. TT8 transcriptional activity has been studied extensively, and relies on its ability to form, with several R2R3-MYB and TTG1 (WD-Repeat protein), different MYBbHLH-WDR (MBW) protein complexes. By contrast, little is known on how TT8 expression is itself regulated.Transcriptional regulation of TT8 expression was studied using molecular, genetic and biochemical approaches.Functional dissection of the TT8 promoter revealed its modular structure. Two modules were found to specifically drive TT8 promoter activity in PA-and anthocyanin-accumulating cells, by differentially integrating the signals issued from different regulators, in a spatio-temporal manner. Interestingly, this regulation involves at least six different MBW complexes, and an unpredicted positive feedback regulatory loop between TT8 and TTG2. Moreover, the results suggest that some putative new regulators remain to be discovered. Finally, specific cis-regulatory elements through which TT8 expression is regulated were identified and characterized.Together, these results provide a molecular model consistent with the specific and highly regulated expression of TT8. They shed new light into the transcriptional regulation of flavonoid biosynthesis and provide new clues and tools for further investigation in Arabidopsis and other plant species.