Rebecca Schwab

Control of leaf morphogenesis by microRNAs

Plants with altered microRNA metabolism have pleiotropic developmental defects, but direct evidence for microRNAs regulating specific aspects of plant morphogenesis has been lacking. In a genetic screen, we identified the JAW locus, which produces a microRNA that can guide messenger RNA cleavage of several TCP genes controlling leaf development. MicroRNA-guided cleavage of TCP4 mRNA is necessary to prevent aberrant activity of the TCP4 gene expressed from its native promoter. In addition, overexpression of wild-type and microRNA-resistant TCP variants demonstrates that mRNA cleavage is largely sufficient to restrict TCP function to its normal domain of activity. TCP genes with microRNA target sequences are found in a wide range of species, indicating that microRNA-mediated control of leaf morphogenesis is conserved in plants with very different leaf forms.

Highly Specific Gene Silencing by Artificial MicroRNAs in Arabidopsis

Plant microRNAs (miRNAs) affect only a small number of targets with high sequence complementarity, while animal miRNAs usually have hundreds of targets with limited complementarity. We used artificial miRNAs (amiRNAs) to determine whether the narrow action spectrum of natural plant miRNAs reflects only intrinsic properties of the plant miRNA machinery or whether it is also due to past selection against natural miRNAs with broader specificity. amiRNAs were designed to target individual genes or groups of endogenous genes. Like natural miRNAs, they had varying numbers of target mismatches. Previously determined parameters of target selection for natural miRNAs could accurately predict direct targets of amiRNAs. The specificity of amiRNAs, as deduced from genome-wide expression profiling, was as high as that of natural plant miRNAs, supporting the notion that extensive base pairing with targets is required for plant miRNA function. amiRNAs make an effective tool for specific gene silencing in plants, especially when several related, but not identical, target genes need to be downregulated. We demonstrate that amiRNAs are also active when expressed under tissue-specific or inducible promoters, with limited nonautonomous effects. The design principles for amiRNAs have been generalized and integrated into a Web-based tool (http://wmd.weigelworld.org).

Antagonistic regulation of PIN phosphorylation by PP2A and PINOID directs auxin flux

In plants, cell polarity and tissue patterning are connected by intercellular flow of the phytohormone auxin, whose directional signaling depends on polar subcellular localization of PIN auxin transport proteins. The mechanism of polar targeting of PINs or other cargos in plants is largely unidentified, with the PINOID kinase being the only known molecular component. Here, we identify PP2A phosphatase as an important regulator of PIN apical-basal targeting and auxin distribution. Genetic analysis, localization, and phosphorylation studies demonstrate that PP2A and PINOID both partially colocalize with PINs and act antagonistically on the phosphorylation state of their central hydrophilic loop, hence mediating PIN apical-basal polar targeting. Thus, in plants, polar sorting by the reversible phosphorylation of cargos allows for their conditional delivery to specific intracellular destinations. In the case of PIN proteins, this mechanism enables switches in the direction of intercellular auxin fluxes, which mediate differential growth, tissue patterning, and organogenesis.

Gene silencing in plants using artificial microRNAs and other small RNAs

Comprehensive analysis of gene function requires the detailed examination of mutant alleles. In Arabidopsis thaliana, large collections of sequence-indexed insertion and chemical mutants provide potential loss-of-function alleles for most annotated genes. However, limitations for phenotypic analysis include gametophytic or early sporophytic lethality, and the ability to recombine mutant alleles in closely linked genes, especially those present as tandem duplications. Transgene-mediated gene silencing can overcome some of these shortcomings through tissue-specific, inducible and partial gene inactivation, or simultaneous targeting of several, sequence-related genes. In addition, gene silencing is a convenient approach in species or varieties for which exhaustive mutant collections are not yet available. Typically, gene function is reduced post-transcriptionally, effected by small RNAs that act in a sequence-specific manner by base pairing to complementary mRNA molecules. A recently introduced approach is the use of artificial microRNAs (amiRNAs). Here, we review various strategies for small RNA-based gene silencing, and describe in detail the design and application of amiRNAs in many plant species.

Dual Effects of miR156-Targeted SPL Genes and CYP78A5/KLUH on Plastochron Length and Organ Size in Arabidopsis thaliana

Leaves of flowering plants are produced from the shoot apical meristem at regular intervals, with the time that elapses between the formation of two successive leaf primordia defining the plastochron. We have identified two genetic axes affecting plastochron length in Arabidopsis thaliana. One involves microRNA156 (miR156), which targets a series of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. In situ hybridization studies and misexpression experiments demonstrate that miR156 is a quantitative, rather than spatial, modulator of SPL expression in leaf primordia and that SPL activity nonautonomously inhibits initiation of new leaves at the shoot apical meristem. The second axis is exemplified by a redundantly acting pair of cytochrome P450 genes, CYP78A5/KLUH and CYP78A7, which are likely orthologs of PLASTOCHRON1 of rice (Oryza sativa). Inactivation of CYP78A5, which is expressed at the periphery of the shoot apical meristem, accelerates the leaf initiation rate, whereas cyp78a5 cyp78a7 double mutants often die as embryos with supernumerary cotyledon primordia. The effects of both miR156-targeted SPL genes and CYP78A5 on organ size are correlated with changes in plastochron length, suggesting a potential compensatory mechanism that links the rate at which leaves are produced to final leaf size.

Sequence and Expression Differences Underlie Functional Specialization of Arabidopsis MicroRNAs miR159 and miR319

Many microRNAs (miRNAs) are encoded by small gene families. In a third of all conserved Arabidopsis miRNA families, members vary at two or more nucleotide positions. We have focused on the related miR159 and miR319 families, which share sequence identity at 17 of 21 nucleotides, yet affect different developmental processes through distinct targets. MiR159 regulates MYB mRNAs, while miR319 predominantly acts on TCP mRNAs. In the case of miR319, MYB targeting plays at most a minor role because miR319 expression levels and domain limit its ability to affect MYB mRNAs. In contrast, in the case of miR159, the miRNA sequence prevents effective TCP targeting. We complement these observations by identifying nucleotide positions relevant for miRNA activity with mutants recovered from a suppressor screen. Together, our findings reveal that functional specialization of miR159 and miR319 is achieved through both expression and sequence differences.

Genome-wide analysis of local chromatin packing in Arabidopsis thaliana

The spatial arrangement of interphase chromosomes in the nucleus is important for gene expression and genome function in animals and in plants. The recently developed Hi-C technology is an efficacious method to investigate genome packing. Here we present a detailed Hi-C map of the three-dimensional genome organization of the plant Arabidopsis thaliana. We find that local chromatin packing differs from the patterns seen in animals, with kilobasepair-sized segments that have much higher intrachromosome interaction rates than neighboring regions, representing a dominant local structural feature of genome conformation in A. thaliana. These regions, which appear as positive strips on two-dimensional representations of chromatin interaction, are enriched in epigenetic marks H3K27me3, H3.1, and H3.3. We also identify more than 400 insulator-like regions. Furthermore, although topologically associating domains (TADs), which are prominent in animals, are not an obvious feature of A. thaliana genome packing, we found more than 1000 regions that have properties of TAD boundaries, and a similar number of regions analogous to the interior of TADs. The insulator-like, TAD-boundary-like, and TAD-interior-like regions are each enriched for distinct epigenetic marks and are each correlated with different gene expression levels. We conclude that epigenetic modifications, gene density, and transcriptional activity combine to shape the local packing of the A. thaliana nuclear genome.

Control of lateral organ development and flowering time by the Arabidopsis thaliana MADS-box Gene AGAMOUS-LIKE6

MADS-domain transcription factors play pivotal roles in various developmental processes. The lack of simple loss-of-function phenotypes provides impediments to understand the biological function of some of the MADS-box transcription factors. Here we have characterized the potential role of the Arabidopsis thaliana AGAMOUS-LIKE6 (AGL6) gene by fusing full-length coding sequence with transcriptional activator and repressor domains and suggest a role for AGL6 in lateral organ development and flowering. Upon photoperiodic induction of flowering, AGL6 becomes expressed in abaxial and proximal regions of cauline leaf primordia, as well as the cryptic bracts subtending flowers. In developing flowers, AGL6 is detected in the proximal regions of all floral organs and in developing ovules. Converting AGL6 into a strong activator through fusion to the VP16 domain triggers bract outgrowth, implicating AGL6 in the development of bractless flowers in Arabidopsis. In addition, ectopic reproductive structures form on both bracts and flowers in gAGL6::VP16 transgenic plants, which is dependent on B and C class homeotic genes, but independent of LEAFY. Overexpression of both AGL6 and its transcriptional repressor form, AGL6::EAR, causes precocious flowering and terminal flower formation, suggesting that AGL6 suppresses the function of a floral repressor.

Directed gene silencing with artificial microRNAs

The characterization of gene function typically includes a detailed analysis of loss-of-function alleles. In model plants, such as Arabidopsis thaliana and rice, sequence-indexed insertion collections provide a large resource of potential null alleles that can often be easily accessed through convenient Web sites (e.g., http://signal.salk.edu ). They are, however, not available for nonmodel species, require stacking for knockout of redundant homologs, and do not easily allow for partial or regulated loss of gene function, which is particularly useful when null alleles are lethal. Transgene approaches that employ directed gene silencing can substitute for null alleles and also enable refined studies of gene function, e.g., by tissue-specific and inducible gene-silencing. This chapter describes the generation and application of artificial microRNAs (amiRNAs) as a gene silencing tool in a wide variety of different plant species.

Activation of a Plant NLR Complex through Heteromeric Association with an Autoimmune Risk Variant of Another NLR

Summary When independently evolved immune receptor variants meet in hybrid plants, they can activate immune signaling in the absence of non-self recognition. Such autoimmune risk alleles have recurrently evolved at the DANGEROUS MIX2 (DM2) nucleotide-binding domain and leucine-rich repeat (NLR)-encoding locus in A. thaliana. One of these activates signaling in the presence of a particular variant encoded at another NLR locus, DM1. We show that the risk variants of DM1 and DM2d NLRs signal through the same pathway that is activated when plant NLRs recognize non-self elicitors. This requires the P loops of each protein and Toll/interleukin-1 receptor (TIR)-domain-mediated heteromeric association of DM1 and DM2d. DM1 and DM2d each resides in a multimeric complex in the absence of signaling, with the DM1 complex shifting to higher molecular weight when heteromerizing DM2 variants are present. The activation of the DM1 complex appears to be sensitive to the conformation of the heteromerizing DM2 variant. Autoimmunity triggered by interaction of this NLR pair thus suggests that activity of heteromeric NLR signaling complexes depends on the sum of activation potentials of partner NLRs.