ta-siRNA Pathway Research Articles

Discriminating Mutations of HC-Pro ofZucchini yellow mosaic viruswith Differential Effects on Small RNA Pathways Involved in Viral Pathogenicity and Symptom Development

Helper component-proteinase (HC-Pro), the gene-silencing suppressor of Potyvirus spp., interferes with microRNA (miRNA) and short-interfering RNA (siRNA) pathways. Our previous studies showed that three mutations of highly conserved amino acids of HC-Pro, R(180)I (mutation A), F(205)L (B), and E(396)N (C), of Zucchini yellow mosaic virus (ZYMV) affect symptom severity and viral pathogenicity. The mutant ZYMV GAC (ZGAC) with double mutations, R(180)I/E(396)N, induces transient leaf mottling in host plants followed by recovery. This mutant confers complete cross protection against subsequent infection by the parental ZYMV (ZG) strain. Here, we sought to obtain molecular evidence on the roles of the three highly conserved amino acids of HC-Pro in miRNA and siRNA pathways using transgenic Arabidopsis plants expressing comparable levels of wild-type and mutant HC-Pro proteins. We demonstrated that amino acid residues 180, 205, and 396 of HC-Pro are critical for suppression of miRNA, trans-acting siRNA (ta-siRNA), and virus-induced gene silencing (VIGS) pathways but not for sense-post transcriptional gene silencing (s-PTGS). Because the HC-Pro double mutant (R(180)I/E(396)N) does not interfere with miRNA and ta-siRNA pathways, the ZGAC mutant virus elicits only attenuated symptoms. Furthermore, the recovery seen on ZGAC-infected plants likely results from the weak VIGS suppression by the HC-Pro double AC mutant. Thus, through manipulating these three conserved amino acids on HC-Pro, symptom severity of diseases caused by Potyvirus spp. can be modulated to generate useful cross protectants for field application. Although some of our mutated HC-Pro proteins do not interfere with miRNA and ta-siRNA pathways, they still retain the ability to suppress s-PTGS.

TheREDUCED LEAFLETGenes Encode Key Components of the trans-Acting Small Interfering RNA Pathway and Regulate Compound Leaf and Flower Development inLotus japonicus

The endogenous trans-acting small interfering RNA (ta-siRNA) pathway plays a conserved role in adaxial-abaxial patterning of lateral organs in simple-leafed plant species. However, its function in compound-leafed species is largely unknown. Using the compound-leafed species Lotus japonicus, we identified and characterized two independent mutants, reduced leaflet1 (rel1) and rel3, whose most conspicuous defects in compound leaves are abaxialized leaflets and reduction in leaflet number. Concurrent mutations in REL genes also compromise flower development and result in radial symmetric floral organs. Positional cloning revealed that REL1 and REL3 encode the homologs of Arabidopsis (Arabidopsis thaliana) SUPPRESSOR OF GENE SILENCING3 and ARGONAUTE7/ZIPPY, respectively, which are key components of the ta-siRNA pathway. These observations, together with the expression and functional data, demonstrated that the ta-siRNA pathway plays conserved yet distinct roles in the control of compound leaf and flower development in L. japonicus. Moreover, the phenotypic alterations of lateral organs in ta-siRNA-deficient mutants and the regulation of downstream targets by the ta-siRNA pathway in L. japonicus were similar to those in the monocots but different from Arabidopsis, indicating many parallels between L. japonicus and the monocots in the control of lateral organ development by the ta-siRNA pathway.

Regulation of Small RNA Accumulation in the Maize Shoot Apex

MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) are essential to the establishment of adaxial–abaxial (dorsoventral) leaf polarity. Tas3-derived ta-siRNAs define the adaxial side of the leaf by restricting the expression domain of miRNA miR166, which in turn demarcates the abaxial side of leaves by restricting the expression of adaxial determinants. To investigate the regulatory mechanisms that allow for the precise spatiotemporal accumulation of these polarizing small RNAs, we used laser-microdissection coupled to RT-PCR to determine the expression profiles of their precursor transcripts within the maize shoot apex. Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity. We show that miR390, an upstream determinant in leaf polarity whose activity triggers tas3 ta-siRNA biogenesis, accumulates adaxially in leaves. The polar expression of miR390 is established and maintained independent of the ta-siRNA pathway. The comparison of small RNA localization data with the expression profiles of precursor transcripts suggests that miR166 and miR390 accumulation is also regulated at the level of biogenesis and/or stability. Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well. Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs.

AGO1-miR173 complex initiates phased siRNA formation in plants

MicroRNA (miRNA)-guided cleavage initiates entry of primary transcripts into the transacting siRNA (tasiRNA) biogenesis pathway involving RNA-DEPENDENT RNA POLYMERASE6, DICER-LIKE4, and SUPPRESSOR OF GENE SILENCING3. Arabidopsis thaliana TAS1 and TAS2 families yield tasiRNA that form through miR173-guided initiation–cleavage of primary transcripts and target several transcripts encoding pentatricopeptide repeat proteins and proteins of unknown function. Here, the TAS1c locus was modified to produce synthetic (syn) tasiRNA to target an endogenous transcript encoding PHYTOENE DESATURASE and used to analyze the role of miR173 in routing of transcripts through the tasiRNA pathway. miR173 was unique from other miRNAs in its ability to initiate TAS1c -based syn-tasiRNA formation. A single miR173 target site was sufficient to route non- TAS transcripts into the pathway to yield phased siRNA. We also show that miR173 functions in association with ARGONAUTE 1 (AGO1) during TAS1 and TAS2 tasiRNA formation, and we provide data indicating that the miR173–AGO1 complex possesses unique functionality that many other miRNA–AGO1 complexes lack.

Activation of the TAS3-derived tasiRNA pathway in the root system of Arabidopsis thaliana

Activation of the TAS3-derived tasiRNA pathway in the root system of Arabidopsis thaliana

An Interplay Between Small Regulatory RNAs Patterns Leaves

Adaxial/abaxial (dorsoventral) leaf polarity is a unique example of a developmental process in which early patterning decisions are determined by small regulatory RNAs. In maize, an abaxial gradient of the microRNA miR166 in the incipient leaf defines the localization of HD-ZIPIII genes that promote adaxial identity. Loss of leafbladeless1 (lbl1) function, necessary for the biogenesis of TAS3-derived trans-acting short-interfering RNAs (ta-siRNAs), leads to ectopic accumulation of miR166 throughout the initiating leaf. These findings indicate that the TAS3 ta-siRNA pathway specifies leaf polarity by spatially restricting miR166 to the abaxial side. Here, we briefly discuss possible mechanisms by which the TAS3 ta-siRNA pathway could regulate miR166 expression. Such regulatory mechanisms likely involve AUXIN RESPONSE FACTORS and the phytohormone auxin. The convergence of small RNAs, and perhaps auxin, on key determinants of adaxial/abaxial organ polarity implicates them as candidate mobile signals that act at the plant apex to relay positional information from the meristem to the initiating leaf.

The small interfering RNA production pathway is required for shoot meristem initiation in rice

The shoot apical meristem (SAM) is a group of stem cells that are responsible for plant development. Mutations in rice SHOOTLESS2 (SHL2), SHL4/SHOOT ORGANIZATION2 (SHO2), and SHO1 cause complete deletion or abnormal formation of the SAM. In this study we showed that defects in SAM formation in shl mutants are associated with the loss of expression of the homeodomain-leucine zipper (HD-ZIPIII) family genes. Rice SHL2, SHL4/SHO2, and SHO1 encoded orthologues of Arabidopsis RNA-dependent RNA polymerase 6, ARGONAUTE (AGO) 7, and DICER-like 4, respectively, whose mutations affect leaf development through the trans-acting siRNA (ta-siRNA) pathway. This suggested that the ta-siRNA pathway regulates the critical step of SAM formation during rice embryogenesis. The gain-of-function experiment by the ectopic expression of SHL4 resulted in reduced accumulation of an microRNA, miR166, and partial adaxialization of leaves, supporting a role for the ta-siRNA pathway in the maintenance of leaf polarity as previously reported in maize. Analysis of the spatiotemporal expression patterns of HD-ZIPIII and miR166 in wild-type and shl mutant embryos suggested that the loss of HD-ZIPIII expression in the SAM region of the developing embryo is the result of ectopic expression of miR166. Our analysis of shl mutants demonstrated that HD-ZIPIII expression regulated by miR166 is sensitive to the ta-siRNA pathway during SAM formation in rice embryogenesis.

DRB4-Dependent TAS3 trans-Acting siRNAs Control Leaf Morphology through AGO7

trans-acting siRNAs (ta-siRNAs) are endogenous RNAs that direct the cleavage of complementary mRNA targets . TAS gene transcripts are cleaved by miRNAs; the cleavage products are protected against degradation by SGS3, copied into dsRNA by RDR6, and diced into ta-siRNAs by DCL4 . We describe hypomorphic rdr6 and sgs3 Arabidopsis mutants, which do not exhibit the leaf developmental defects observed in null mutants and which, like null alleles, are impaired in sense-transgene-induced posttranscriptional gene silencing and virus resistance. Null rdr6 and sgs3 mutants lack TAS1, TAS2, and TAS3 ta-siRNAs and overaccumulate ARF3/ETTIN and ARF4 mRNAs, which are TAS3 ta-siRNA targets. A hypomorphic rdr6 mutant accumulates wild-type TAS3 ta-siRNA levels but not TAS1 and TAS2 ta-siRNAs, suggesting that TAS3 is required for proper leaf development. Consistently, tas3 but not tas1 or tas2 mutants exhibits leaf morphology defects, and ago7/zip and drb4 mutants, which exhibit leaf morphology defects, lack TAS3 but not TAS1 and TAS2 ta-siRNAs in leaves. These results indicate that the dsRNA binding protein DRB4 is required for proper ta-siRNA production, presumably by interacting with DCL4, an interaction analogous to that of HYL1 with DCL1 during miRNA production , and that TAS3 ta-siRNAs are required for proper leaf development through the action of AGO7/ZIPPY.

Organ Polarity in Plants Is Specified through the Opposing Activity of Two Distinct Small Regulatory RNAs

Small RNAs and their targets form complex regulatory networks that control cellular and developmental processes in multicellular organisms. In plants, dorsoventral (adaxial/abaxial) patterning provides a unique example of a developmental process in which early patterning decisions are determined by small RNAs. A gradient of microRNA166 on the abaxial/ventral side of the incipient leaf restricts the expression of adaxial/dorsal determinants. Another class of small RNAs, the TAS3-derivated trans-acting short-interfering RNAs (ta-siRNAs), are expressed adaxially and repress the activity of abaxial factors. Loss of maize leafbladeless1 (lbl1) function, a key component of the ta-siRNA biogenesis pathway, leads to misexpression of miR166 throughout the initiating leaf, implicating ta-siRNAs in the spatiotemporal regulation of miR166. The spatial restriction of tasiRNA biogenesis components suggests that this pathway may act non-cell-autonomously in the meristem and possibly contributes to the classic meristem-borne adaxializing Sussex signal. Here, we discuss the key participants in adaxial/abaxial patterning and point out the intriguing possibility that organ polarity in plants is established by the opposing action of specific ta-siRNAs and miRNAs.