miR393 and secondary siRNAs regulate expression of the TIR1/AFB2 auxin receptor clade and auxin-related development of Arabidopsis leaves.

The phytohormone auxin is a key regulator of plant growth and development that exerts its functions through F-box receptors. Arabidopsis (Arabidopsis thaliana) has four partially redundant of these receptors that comprise the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX1 auxin receptor (TAAR) clade. Recent studies have shown that the microRNA miR393 regulates the expression of different sets of TAAR genes following pathogen infection or nitrate treatment. Here we report that miR393 helps regulate auxin-related development of leaves. We found that AtMIR393B is the predominant source for miR393 in all aerial organs and that miR393 down-regulates all four TAAR genes by guiding the cleavage of their mRNAs. A mutant unable to produce miR393 shows developmental abnormalities of leaves and cotyledons reminiscent of enhanced auxin perception by TAARs. Interestingly, miR393 initiates the biogenesis of secondary siRNAs from the transcripts of at least two of the four TAAR genes. Our results indicate that these siRNAs, which we call siTAARs, help regulate the expression of TAAR genes as well as several unrelated genes by guiding the cleavage of their mRNAs. Thus, miR393 and possibly siTAARs regulate auxin perception and certain auxin-related aspects of leaf development.

Massive production of small RNAs from a non-coding region of Cauliflower mosaic virus in plant defense and viral counter-defense.

To successfully infect plants, viruses must counteract small RNA-based host defense responses. During infection of Arabidopsis, Cauliflower mosaic pararetrovirus (CaMV) is transcribed into pregenomic 35S and subgenomic 19S RNAs. The 35S RNA is both reverse transcribed and also used as an mRNA with highly structured 600 nt leader. We found that this leader region is transcribed into long sense- and antisense-RNAs and spawns a massive quantity of 21, 22 and 24 nt viral small RNAs (vsRNAs), comparable to the entire complement of host-encoded small-interfering RNAs and microRNAs. Leader-derived vsRNAs were detected bound to the Argonaute 1 (AGO1) effector protein, unlike vsRNAs from other viral regions. Only negligible amounts of leader-derived vsRNAs were bound to AGO4. Genetic evidence showed that all four Dicer-like (DCL) proteins mediate vsRNA biogenesis, whereas the RNA polymerases Pol IV, Pol V, RDR1, RDR2 and RDR6 are not required for this process. Surprisingly, CaMV titers were not increased in dcl1/2/3/4 quadruple mutants that accumulate only residual amounts of vsRNAs. Ectopic expression of CaMV leader vsRNAs from an attenuated geminivirus led to increased accumulation of this chimeric virus. Thus, massive production of leader-derived vsRNAs does not restrict viral replication but may serve as a decoy diverting the silencing machinery from viral promoter and coding regions.

Transgenerational adaptation of Arabidopsis to stress requires DNA methylation and the function of Dicer-like proteins.

Epigenetic states and certain environmental responses in mammals and seed plants can persist in the next sexual generation. These transgenerational effects have potential adaptative significance as well as medical and agronomic ramifications. Recent evidence suggests that some abiotic and biotic stress responses of plants are transgenerational. For example, viral infection of tobacco plants and exposure of Arabidopsis thaliana plants to UVC and flagellin can induce transgenerational increases in homologous recombination frequency (HRF). Here we show that exposure of Arabidopsis plants to stresses, including salt, UVC, cold, heat and flood, resulted in a higher HRF, increased global genome methylation, and higher tolerance to stress in the untreated progeny. This transgenerational effect did not, however, persist in successive generations. Treatment of the progeny of stressed plants with 5-azacytidine was shown to decrease global genomic methylation and enhance stress tolerance. Dicer-like (DCL) 2 and DCL3 encode Dicer activities important for small RNA-dependent gene silencing. Stress-induced HRF and DNA methylation were impaired in dcl2 and dcl3 deficiency mutants, while in dcl2 mutants, only stress-induced stress tolerance was impaired. Our results are consistent with the hypothesis that stress-induced transgenerational responses in Arabidopsis depend on altered DNA methylation and smRNA silencing pathways.

Heterochromatic siRNAs and DDM1 independently silence aberrant 5S rDNA transcripts in Arabidopsis.

5S ribosomal RNA gene repeats are arranged in heterochromatic arrays (5S rDNA) situated near the centromeres of Arabidopsis chromosomes. The chromatin remodeling factor DDM1 is known to maintain 5S rDNA methylation patterns while silencing transcription through 5S rDNA intergenic spacers (IGS). We mapped small-interfering RNAs (siRNA) to a composite 5S rDNA repeat, revealing a high density of siRNAs matching silenced IGS transcripts. IGS transcript repression requires proteins of the heterochromatic siRNA pathway, including RNA polymerase IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Using molecular and cytogenetic approaches, we show that the DDM1 and siRNA-dependent silencing effects are genetically independent. DDM1 suppresses production of the siRNAs, however, thereby limiting RNA-directed DNA methylation at 5S rDNA repeats. We conclude that DDM1 and siRNA-dependent silencing are overlapping processes that both repress aberrant 5S rDNA transcription and contribute to the heterochromatic state of 5S rDNA arrays.

Evolution of Arabidopsis MIR genes generates novel microRNA classes.

In Arabidopsis, canonical 21-nt miRNAs are generated by Dicer-like (DCL) 1 from hairpin precursors. We have identified a novel class of functional 23- to 25-nt long-miRNAs that is generated independently from the same miRNA precursors by DCL3. Long-miRNAs are developmentally regulated and in some cases have been conserved during evolution implying that they have biological functions. Plant microRNA genes (MIR) have been proposed to evolve by inverted duplication of the target gene. We found that recently evolved MIR genes consistently give rise to long-miRNAs, while ancient MIR genes give rise predominantly to canonical miRNAs. Transcripts from inverted repeats representing evolving proto-MIR genes were processed by DCL3 into long-miRNAs and also by DCL1, DCL2 or DCL4 depending on hairpin stem length to produce different sizes of miRNAs. Our results suggest that evolution of MIR genes is associated with gradual, overlapping changes in DCL usage resulting in specific size classes of miRNAs.

The CaMV transactivator/viroplasmin interferes with RDR6-dependent trans-acting and secondary siRNA pathways in Arabidopsis.

Several RNA silencing pathways in plants restrict viral infections and are suppressed by distinct viral proteins. Here we show that the endogenous trans-acting (ta)siRNA pathway, which depends on Dicer-like (DCL) 4 and RNA-dependent RNA polymerase (RDR) 6, is suppressed by infection of Arabidopsis with Cauliflower mosaic virus (CaMV). This effect was associated with overaccumulation of unprocessed, RDR6-dependent precursors of tasiRNAs and is due solely to expression of the CaMV transactivator/viroplasmin (TAV) protein. TAV expression also impaired secondary, but not primary, siRNA production from a silenced transgene and increased accumulation of mRNAs normally silenced by the four known tasiRNA families and RDR6-dependent secondary siRNAs. Moreover, TAV expression upregulated DCL4, DRB4 and AGO7 that mediate tasiRNA biogenesis. Our findings suggest that TAV is a general inhibitor of silencing amplification that impairs DCL4-mediated processing of RDR6-dependent double-stranded RNA to siRNAs. The resulting deficiency in tasiRNAs and other RDR6-/DCL4-dependent siRNAs appears to trigger a feedback mechanism that compensates for the inhibitory effects.

MicroRNA-mediated regulation of stomatal development in Arabidopsis.

The proper number and distribution of stomata are essential for the efficient exchange of gases between the atmosphere and the aerial parts of plants. We show that the density and development of stomatal complexes on the epidermis of Arabidopsis thaliana leaves depend, in part, on the microRNA-mediated regulation of Agamous-like16 (AGL16), which is a member of the MADS box protein family. AGL16 mRNA is targeted for sequence-specific degradation by miR824, a recently evolved microRNA conserved in the Brassicaceae and encoded at a single genetic locus. Primary stomatal complexes can give rise to higher-order complexes derived from satellite meristemoids. Expression of a miR824-resistant AGL16 mRNA, but not the wild-type AGL16 mRNA, in transgenic plants increased the incidence of stomata in higher-order complexes. By contrast, reduced expression of AGL16 mRNA in the agl16-1 deficiency mutant and in transgenic lines overexpressing miR824 decreased the incidence of stomata in higher-order complexes. These findings and the nonoverlapping patterns of AGL16 mRNA and miR824 localization led us to propose that the miR824/AGL16 pathway functions in the satellite meristemoid lineage of stomatal development.

Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing.

Like other eukaryotes, plants use DICER-LIKE (DCL) proteins as the central enzymes of RNA silencing, which regulates gene expression and mediates defense against viruses. But why do plants like Arabidopsis express four DCLs, a diversity unmatched by other kingdoms? Here we show that two nuclear DNA viruses (geminivirus CaLCuV and pararetrovirus CaMV) and a cytoplasmic RNA tobamovirus ORMV are differentially targeted by subsets of DCLs. DNA virus-derived small interfering RNAs (siRNAs) of specific size classes (21, 22 and 24 nt) are produced by all four DCLs, including DCL1, known to process microRNA precursors. Specifically, DCL1 generates 21 nt siRNAs from the CaMV leader region. In contrast, RNA virus infection is mainly affected by DCL4. While the four DCLs are partially redundant for CaLCuV-induced mRNA degradation, DCL4 in conjunction with RDR6 and HEN1 specifically facilitates extensive virus-induced silencing in new growth. Additionally, we show that CaMV infection impairs processing of endogenous RDR6-derived double-stranded RNA, while ORMV prevents HEN1-mediated methylation of small RNA duplexes, suggesting two novel viral strategies of silencing suppression. Our work highlights the complexity of virus interaction with host silencing pathways and suggests that DCL multiplicity helps mediate plant responses to diverse viral infections.

Molecular characterization of geminivirus-derived small RNAs in different plant species.

DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs-the hallmarks of silencing-associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5' end phosphorylated, as shown by phosphatase treatments, and methylated at the 3'-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to beta-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interactions.

RNA silencing systems and their relevance to plant development.

RNA silencing refers to a broad range of phenomena sharing the common feature that large, double-stranded RNAs or stem-loop precursors are processed to ca. 21-26 nucleotide small RNAs, which then guide the cleavage of cognate RNAs, block productive translation of these RNAs, or induce methylation of specific target DNAs. Although the core mechanisms are evolutionarily conserved, epigenetic maintenance of silencing by amplification of small RNAs and the elaboration of mobile, RNA-based silencing signals occur predominantly in plants. Plant RNA silencing systems are organized into a network with shared components and overlapping functions. MicroRNAs, and probably trans-acting small RNAs, help regulate development at the posttranscriptional level. Small interfering RNAs associated with transgene- and virus-induced silencing function primarily in defending against foreign nucleic acids. Another system, which is concerned with RNA-directed methylation of DNA repeats, seems to have roles in epigenetic silencing of certain transposable elements and genes under their control.

Altered expression of an ankyrin-repeat protein results in leaf abnormalities, necrotic lesions, and the elaboration of a systemic signal.

The PR-like proteins, class I beta-1,3-glucanase (GLU I) and chitinase (CHN I), are induced as part of a stereotypic response that can provide protection against viral, bacterial, and fungal pathogens. We have identified two Nicotiana plumbaginifolia ankyrin-repeat proteins, designated Glucanohydrolase Binding Proteins (GBP) 1 and 2, that bind GLU I and CHN I both in vitro and when expressed in yeast cells. Sense as well as antisense transformants of tobacco carrying the GBP1 gene elaborated graft-transmissible acropetally moving signals that induced the downward curling of young leaves. This phenotype was associated with reduced starch, sucrose, and fructose accumulation; the formation of necrotic lesions; and, the induction of markers for the hypersensitive response. GBP1/2 are members of a conserved Plant- Specific Ankyrin- repeat (PANK) family that includes proteins implicated in carbohydrate allocation, reactive oxygen metabolism, hypersensitive cell death, rapid elicitor responses, virus pathogenesis, and auxin signaling. The similarity in phenotype of PANK transformants and transformants altered in carbohydrate metabolism leads us to propose that PANK family members are multifunctional proteins involved in linking plant defense responses and carbohydrate metabolism.

Meiotic transmission of epigenetic changes in the cell-division factor requirement of plant cells.

During the development of tobacco plants, cells undergo epigenetic changes that alter their requirement in culture for the cell-division factor cytokinin. Cultured leaf cells alternate between cytokinin-requiring (C-) and cytokinin-independent (C+) states at extremely high rates of approximately 10-2 per cell generation by a process called pseudodirected variation. Here we show that plants regenerated from most C+ clones express the Habituated leaf (Hl) trait, i.e., leaf tissues exhibit the C+ phenotype rather than the wild-type C- phenotype in culture. This new trait then segregates as a monogenic dominant trait indicating that conversion of C- cells to C+ cells is associated with a meiotically transmissible, genetic modification. Two independent mutants, Hl-2 and Hl-3, derived from C+ variants arising in culture were unstable in planta and reverted gametically at rates roughly comparable to pseudodirected variation in culture. Cells of the Hl-2 mutant, but not of a stable Hl-1 mutant, reverted phenotypically at high rates in culture. This revertant C- phenotype persisted in some plants regenerated from cloned revertant lines, and then showed irregular segregation in two successive sexual generations. These results show for the first time that meiotically transmissible epimutations can occur reversibly and at high rates in culture.

A gene encoding an RNase D exonuclease-like protein is required for post-transcriptional silencing in Arabidopsis.

Post-transcriptional gene silencing (PTGS) and the closely related phenomenon RNA interference (RNAi) result from the initial endonucleolytic cleavage of target mRNAs, which are then presumed to be completely hydrolyzed by exoribonucleases. To date, no plant genes required for PTGS are known to encode exoribonucleases. The Arabidopsis Werner Syndrome-like exonuclease (WEX) gene encodes an RNase D domain most similar to that in human Werner Syndrome protein (WRN), but lacks the RecQ helicase domain. It is also related to Caenorhabditis elegans mut-7, which is essential for RNAi, PTGS, and transposon activity. We isolated a loss-of-function mutant, wex-1, that showed greatly reduced expression of WEX mRNA and early flowering. Although wex-1 did not affect expression of a robust marker for transcriptional gene silencing (TGS), PTGS of a green-fluorescent-protein (GFP) reporter gene was blocked in wex-1 and restored by ectopic expression of WEX, indicating that WEX is required for PTGS but not TGS. Thus, members of the RNase D protein family are required for PTGS in both plants and animals. Interestingly, WEX has been shown to interact with an Arabidopsis RecQ helicase, suggesting that these proteins might comprise a functional equivalent of WRN.

High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants.

Posttranscriptional gene silencing (PTGS) in transgenic plants is an epigenetic form of RNA degradation related to PTGS and RNA interference (RNAi) in fungi and animals. Evidence suggests that transgene loci and RNA viruses can generate double-stranded RNAs similar in sequence to the transcribed region of target genes, which then undergo endonucleolytic cleavage to generate small interfering RNAs (siRNA) that promote degradation of cognate RNAs. The silent state in transgenic plants and in Caenorhabditis elegans can spread systemically, implying that mobile silencing signals exist. Neither the chemical nature of these signals nor their exact source in the PTGS pathway is known. Here, we use a positive marker system and real-time monitoring of green fluorescent protein expression to show that large sense, antisense, and double-stranded RNAs as well as double-stranded siRNAs delivered biolistically into plant cells trigger silencing capable of spreading locally and systemically. Systemically silenced leaves show greatly reduced levels of target RNA and accumulate siRNAs, confirming that RNA can induce systemic PTGS. The induced siRNAs represent parts of the target RNA that are outside of the region of homology with the triggering siRNA. Our results imply that siRNAs themselves or intermediates induced by siRNAs could comprise silencing signals and that these signals induce self-amplifying production of siRNAs.