ARGONAUTE (AGO) RNA-binding proteins are involved in RNA silencing. They bind to short interfering RNAs (siRNAs) and microRNAs (miRNAs) through a conserved PAZ domain, and, in animals, they assemble into a multisubunit RNA-induced silencing complex (RISC). The mammalian AGO2, termed Slicer, directs siRNA-and miRNA-mediated cleavage of a target RNA. In Arabidopsis, there are 10 members of the AGO family, and the AGO1 protein is potentially the Slicer component in different RNAsilencing pathways. Here, we show that AGO1 selectively recruits certain classes of short silencing-related RNA. AGO1 is physically associated with miRNAs, transacting siRNAs, and transgenederived siRNAs but excludes virus-derived siRNAs and 24-nt siRNAs involved in chromatin silencing. We also show that AGO1 has Slicer activity. It mediates the in vitro cleavage of a mir165 target RNA in a manner that depends on the sequence identity of amino acid residues in the PIWI domain that are predicted by homology with animal Slicer-competent AGO proteins to constitute the RNase catalytic center. However, unlike animals, we find no evidence that AGO1 Slicer is in a high molecular weight RNA-induced silencing complex. The Slicer activity fractionates as a complex of Ϸ150 kDa that likely constitutes the AGO1 protein and associated RNA without any other proteins. Based on sequence similarity, we predict that other Arabidopsis AGOs might have a similar catalytic activity but recruit different subsets of siRNAs or miRNAs.posttranscriptional regulation ͉ ribonuclease ͉ viral RNA ͉ silencing A RGONAUTE (AGO) proteins are implicated in RNAsilencing processes that also involve 21-to 26-nt short RNAs (sRNAs) (1) cleaved from double-stranded or partially doublestranded (ds) RNAs by the RNase III enzyme Dicer. There are several types of RNA-silencing mechanisms, including RNA interference (RNAi), the micro RNA (miRNA) pathway, and RNAdirected chromatin silencing (1). RNAi is a type of RNA silencing in which the Dicer substrate is fully double stranded, the sRNA cleavage product is short interfering RNA (siRNA), and the outcome is targeted destruction of siRNA-complementary RNAs. The miRNA pathway is similar except that the Dicer substrate is an inverted repeat RNA with a partially ds structure, the sRNA is referred to as a miRNA, and the target RNAs can be suppressed at the translational level or degraded as in RNAi, depending on the degree of complementarity between the sRNA and its target. Plants possess an additional class of degradative sRNAs called transacting siRNAs (ta-siRNAs) whose formation depends on the miRNAmediated cleavage of their precursor and its conversion into a dsRNA by RDR6 (2-4). The last pathway, RNA-directed chromatin silencing, is similar to RNAi, but the siRNA targets are either DNA or chromatin-associated RNAs and the outcome is DNA methylation or histone modification at the target locus.In the best understood of these RNA-silencing mechanisms, the duplex siRNAs or miRNAs produced by Dicer are unwound in an ATP-dependent process. One ...

Plant and animal viruses encode suppressor proteins of an adaptive immunity mechanism in which viral double-stranded RNA is processed into 21-25 nt short interfering (si)RNAs. The siRNAs guide ARGONAUTE (AGO) proteins so that they target viral RNA. Most viral suppressors bind long dsRNA or siRNAs and thereby prevent production of siRNA or binding of siRNA to AGO. The one exception is the 2b suppressor of Cucumoviruses that binds to and inhibits AGO1. Here we describe a novel suppressor mechanism in which a Polerovirus-encoded F box protein (P0) targets the PAZ motif and its adjacent upstream sequence in AGO1 and mediates its degradation. F box proteins are components of E3 ubiquitin ligase complexes that add polyubiquitin tracts on selected lysine residues and thereby mark a protein for proteasome-mediated degradation. With P0, however, the targeted degradation of AGO is insensitive to inhibition of the proteasome, indicating that the proteasome is not involved. We also show that P0 does not block a mobile signal of silencing, indicating that the signal molecule does not have AGO protein components. The ability of P0 to block silencing without affecting signal movement may contribute to the phloem restriction of viruses in the Polerovirus group.

Posttranscriptional gene silencing (PTGS) mediated by siRNAs is an evolutionarily conserved antiviral defense mechanism in higher plants and invertebrates. In this mechanism, viral-derived siRNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation of the corresponding viral RNAs. In Arabidopsis, a key component of RISC is ARGONAUTE1 (AGO1), which not only binds to siRNAs but also carries the RNA slicer activity. At present little is known about posttranslational mechanisms regulating AGO1 turnover. Here we report that the viral suppressor of RNA silencing protein P0 triggers AGO1 degradation by the autophagy pathway. Using a P0-inducible transgenic line, we observed that AGO1 degradation is blocked by inhibition of autophagy. The engineering of a functional AGO1 fluorescent reporter protein further indicated that AGO1 colocalizes with autophagy-related (ATG) protein 8a (ATG8a) positive bodies when degradation is impaired. Moreover, this pathway also degrades AGO1 in a nonviral context, especially when the production of miRNAs is impaired. Our results demonstrate that a selective process such as ubiquitylation can lead to the degradation of a key regulatory protein such as AGO1 by a degradation process generally believed to be unspecific. We anticipate that this mechanism will not only lead to degradation of AGO1 but also of its associated proteins and eventually small RNAs.

In plants, the cell wall is a major determinant of cell morphogenesis. Cell enlargement depends on the tightly regulated expansion of the wall, which surrounds each cell. However, the qualitative and quantitative mechanisms controlling cell wall enlargement are still poorly understood. Here, we report the molecular and functional characterization of LRX1, a new Arabidopsis gene that encodes a chimeric leucine-rich repeat/extensin protein. LRX1 is expressed in root hair cells and the protein is specifically localized in the wall of the hair proper, where it becomes insolubilized during development. lrx1-null mutants, isolated by a reverse-genetic approach, develop root hairs that frequently abort, swell, or branch. Complementation and overexpression experiments using modified LRX1 proteins indicate that the interaction with the cell wall is important for LRX1 function. These results suggest that LRX1 is an extracellular component of a mechanism regulating root hair morphogenesis and elongation by controlling either polarized growth or cell wall formation and assembly.

Gibberellins (GAs) are plant hormones involved in the regulation of plant growth in response to endogenous and environmental signals. GA promotes growth by stimulating the degradation of nuclear growth-repressing DELLA proteins. In Arabidopsis thaliana, DELLAs consist of a small family of five proteins that display distinct but also overlapping functions in repressing GA responses. This study reveals that DELLA RGA-LIKE3 (RGL3) protein is essential to fully enhance the jasmonate (JA)-mediated responses. We show that JA rapidly induces RGL3 expression in a CORONATINE INSENSITIVE1 (COI1)-and JASMONATE INSENSITIVE1 (JIN1/MYC2)-dependent manner. In addition, we demonstrate that MYC2 binds directly to RGL3 promoter. Furthermore, we show that RGL3 (like the other DELLAs) interacts with JA ZIM-domain (JAZ) proteins, key repressors of JA signaling. These findings suggest that JA/MYC2-dependent accumulation of RGL3 represses JAZ activity, which in turn enhances the expression of JA-responsive genes. Accordingly, we show that induction of primary JA-responsive genes is reduced in the rgl3-5 mutant and enhanced in transgenic lines overexpressing RGL3. Hence, RGL3 positively regulates JA-mediated resistance to the necrotroph Botrytis cinerea and susceptibility to the hemibiotroph Pseudomonas syringae. We propose that JA-mediated induction of RGL3 expression is of adaptive significance and might represent a recent functional diversification of the DELLAs.

Regulation of plant height, one of the most important agronomic traits, is the focus of intensive research for improving crop performance. Stem elongation takes place as a result of repeated cell divisions and subsequent elongation of cells produced by apical and intercalary meristems. The gibberellin (GA) phytohormones have long been known to control stem and internodal elongation by stimulating the degradation of nuclear growth-repressing DELLA proteins; however, the mechanism allowing GA-responsive growth is only slowly emerging. Here, we show that DELLAs directly regulate the activity of the plant-specific class I TCP transcription factor family, key regulators of cell proliferation. Our results demonstrate that class I TCP factors directly bind the promoters of core cell-cycle genes in Arabidopsis inflorescence shoot apices while DELLAs block TCP function by binding to their DNA-recognition domain. GAs antagonize such repression by promoting DELLA destruction and therefore cause a concomitant accumulation of TCP factors on promoters of cell-cycle genes. Consistent with this model, the quadruple mutant tcp8 tcp14 tcp15 tcp22 exhibits severe dwarfism and reduced responsiveness to GA action. Altogether, we conclude that GA-regulated DELLA-TCP interactions in inflorescence shoot apex provide a novel mechanism to control plant height.

In fungi and metazoans, the SCF-type Ubiquitin protein ligases (E3s) play a critical role in cell cycle regulation by degrading negative regulators, such as cell cycle-dependent kinase inhibitors (CKIs) at the G1-to-S-phase checkpoint. Here we report that FBL17, an Arabidopsis thaliana F-box protein, is involved in cell cycle regulation during male gametogenesis. FBL17 expression is strongly enhanced in plants co-expressing E2Fa and DPa, transcription factors that promote S-phase entry. FBL17 loss-of-function mutants fail to undergo pollen mitosis II, which generates the two sperm cells in mature A. thaliana pollen. Nonetheless, the single sperm cell-like cell in fbl17 mutants is functional but will exclusively fertilize the egg cell of the female gametophyte, giving rise to an embryo that will later abort, most likely due to the lack of functional endosperm. Seed abortion can, however, be overcome by mutations in FIE, a component of the Polycomb group complex, overall resembling loss-of-function mutations in the A. thaliana cyclin-dependent kinase CDKA;1. Finally we identified ASK11, as an SKP1-like partner protein of FBL17 and discuss a possible mechanism how SCFFBL17 may regulate cell division during male gametogenesis.

We have searched the Arabidopsis and rice (Oryza sativa) genomes for homologs of LRX1, an Arabidopsis gene encoding a novel type of cell wall protein containing a leucine-rich repeat (LRR) and an extensin domain. Eleven and eight LRX (LRR/EXTENSIN) genes have been identified in these two plant species, respectively. The LRX gene family encodes proteins characterized by a short N-terminal domain, a domain with 10 LRRs, a cysteine-rich motif, and a variable C-terminal extensin-like domain. Phylogenetic analysis performed on the conserved domains indicates the existence of two major clades of LRX proteins that arose before the eudicot/monocot divergence and then diversified independently in each lineage. In Arabidopsis, gene expression studies by northern hybridization and promoter::uidA fusions showed that the two phylogenetic clades represent a specialization into "reproductive" and "vegetative" LRXs. The four Arabidopsis genes of the "reproductive" clade are specifically expressed in pollen, whereas the seven "vegetative" genes are predominantly expressed in various sporophytic tissues. This separation into two expression classes is also supported by previous studies on maize (Zea mays) and tomato (Lycopersicon esculentum) LRX homologs and by information on available rice ESTs. The strong conservation of the amino acids responsible for the putative recognition specificity of the LRR domain throughout the family suggests that the LRX proteins interact with similar ligands.With the completion of the Arabidopsis genome sequence, it became clear that many Arabidopsis genes are members of multigene families. Although this had already been suggested by the analysis of expressed sequence tag (EST) databases and by classical gene searches, the availability of the full gene set of a plant provides the unique opportunity to get a complete inventory of all the members of a gene family. Among the 25,500 genes predicted in the Arabidopsis genome, 65% are members of a multigene family and 37% belong to families of more than five members (Arabidopsis Genome Initiative, 2000). Although the predicted total gene number of Arabidopsis is significantly larger than that of other sequenced multicellular eukaryotes such as Caenorhabditis elegans (19,000; C. elegans Sequencing Consortium, 1998) or Drosophila melanogaster (13,600; Adams et al., 2000), the absolute number of gene families and singletons (11,601 in Arabidopsis) is comparable in all these organisms (Arabidopsis Genome Initiative, 2000). This indicates that frequent gene duplications and consequently large gene families are a distinctive feature of the Arabidopsis genome, and possibly of all plant genomes. With the recent publication of a high-quality draft from two different subspecies, rice (Oryza sativa) is the second plant whose genome can be comprehensively investigated. Depending on the stringency applied in gene prediction, the rice genome contains between 32,277 and 61,668 genes (Goff et al., 2002;Yu et al., 2002). With 77% of the genes distributed in about 15,000 m...