Chiyoko Machida

βC1, the pathogenicity factor of TYLCCNV, interacts with AS1 to alter leaf development and suppress selective jasmonic acid responses

Viruses induce pathogenic symptoms on plants but the molecular basis is poorly understood. Here, we show that transgenic Arabidopsis expressing the pathogenesis protein betaC1 of Tomato yellow leaf curl China virus (TYLCCNV), a geminivirus, can phenocopy to a large extent disease symptoms of virus-infected tobacco plants in having upward curled leaves, radialized leaves with outgrowth tissues from abaxial surfaces, and sterile flowers. These morphological changes are paralleled by a reduction in miR165/166 levels and an increase in PHB and PHV transcript levels. Two factors, ASYMMETRIC LEAVES 1 (AS1) and ASYMMETRIC LEAVES 2 (AS2), are known to regulate leaf development as AS1/AS2 complex. Strikingly, betaC1 plants phenocopy plants overexpressing AS2 at the morphological and molecular level and betaC1 is able to partially complement as2 mutation. betaC1 binds directly to AS1, elicits morphological and gene expression changes dependent on AS1 but not AS2, and attenuates expression of selective jasmonic acid (JA)-responsive gene. Our results show that betaC1 forms a complex with AS1 to execute its pathogenic functions and to suppress a subset of JA responses.

Histone Deacetylases and ASYMMETRIC LEAVES2 Are Involved in the Establishment of Polarity in Leaves of Arabidopsis

We show that two Arabidopsis thaliana genes for histone deacetylases (HDACs), HDT1/HD2A and HDT2/HD2B, are required to establish leaf polarity in the presence of mutant ASYMMETRIC LEAVES2 (AS2) or AS1. Treatment of as1 or as2 plants with inhibitors of HDACs resulted in abaxialized filamentous leaves and aberrant distribution of microRNA165 and/or microRNA166 (miR165/166) in leaves. Knockdown mutations of these two HDACs by RNA interference resulted in phenotypes like those observed in the as2 background. Nuclear localization of overproduced AS2 resulted in decreased levels of mature miR165/166 in leaves. This abnormality was abolished by HDAC inhibitors, suggesting that HDACs are required for AS2 action. A loss-of-function mutation in HASTY, encoding a positive regulator of miRNA levels, and a gain-of-function mutation in PHABULOSA, encoding a determinant of adaxialization, suppressed the generation of abaxialized filamentous leaves by inhibition of HDACs in the as1 or as2 background. AS2 and AS1 were colocalized in subnuclear bodies adjacent to the nucleolus where HDT1/HD2A and HDT2/HD2B were also found. Our results suggest that these HDACs and both AS2 and AS1 act independently to control levels and/or patterns of miR165/166 distribution and the development of adaxial-abaxial leaf polarity and that there may be interactions between HDACs and AS2 (AS1) in the generation of those miRNAs.

The AtNACK1/HINKEL and STUD/TETRASPORE/AtNACK2 genes, which encode functionally redundant kinesins, are essential for cytokinesis in Arabidopsis

Cytokinesis is the critical step during which daughter cells are separated. We showed previously that a protein complex that consists of NACK1 (and NACK2) kinesin‐like protein and NPK1 MAPKKK and its substrate NQK1 MAPKK are required for progression of cytokinesis in Nicotiana tabacum. The genome of Arabidopsis thaliana encodes homologues of NACK1 and NACK2, namely, AtNACK1/HINKEL and STUD/TETRASPORE/AtNACK2, respectively. Loss‐of‐function mutations in AtNACK1/HINKEL and STUD/TETRASPORE/AtNACK2 result in the occasional failure of somatic and male‐meiotic cytokinesis, respectively. However, it is likely that these genes function redundantly to some extent in somatic tissues and female gametogenesis. We describe the phenotypes of Arabidopsis plants that have mutations in both the AtNACK1/HINKEL and STUD/TETRASPORE/AtNACK2 genes. These phenotypes suggest that the two genes are essential during both male and female gametogenesis. Female gametes with atnack1 atnack2 double mutations failed to cellularize and to generate a central cell, synergids and the egg cells. Male gametes with atnack1 atnack2 mutations were also not transmitted to the next generation. The AtNACK1/HINKEL and STUD/TETRASPORE/AtNACK2 genes for kinesin‐like proteins have overlapping functions that are essential for gametogenetic cytokinesis. They appear to be essential components of a MAP kinase cascade that promotes cytokinesis of plant cells in both gametophytic (haploid) and sporophytic (diploid) proliferation.

Characterization of genes in the ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES (AS2/LOB) family in Arabidopsis thaliana, and functional and molecular comparisons between AS2 and other family members.

The ASYMMETRIC LEAVES2 (AS2) gene is required for the generation of the flat and symmetrical shape of the leaf lamina in Arabidopsis. AS2 encodes a plant-specific protein with an AS2/LATERAL ORGAN BOUNDARIES (AS2/LOB) domain that includes a cysteine repeat, a conserved single glycine residue and a leucine-zipper-like sequence in its amino-terminal half. The Arabidopsis genome contains 42 genes, including AS2, that encode proteins with an AS2/LOB domain in their amino-terminal halves, and these genes constitute the AS2/LOB gene family. In the present study, we cloned and characterized cDNAs that covered the putative coding regions of all members of this family, and investigated patterns of transcription systematically in Arabidopsis plants. Comparisons among amino acid sequences that had been deduced from the cloned cDNAs revealed eight groups of genes, with two or three members each, and high degrees of identity among entire amino acid sequences, suggesting that some members of the AS2/LOB family might have redundant function(s). Moreover, no member of the family exhibited significant similarity, in terms of the deduced amino acid sequence of the carboxy-terminal half, to AS2. Results of domain swapping between AS2 and other members of the family showed that the AS2/LOB domain of AS2 cannot be functionally replaced by those of other members of the family, and that only a few dissimilarities among respective amino acid residues of the AS2/LOB domain of AS2 and those of other members are important for the specific functions of AS2.

Novel receptor-like kinase ALE2 controls shoot development by specifying epidermis in Arabidopsis

The epidermis plays crucial roles in the development of various organs and in water retention in both animals and plants. In Arabidopsis thaliana, the subtilase ABNORMAL LEAF SHAPE 1 (ALE1) and the Arabidopsis homolog of the Crinkly4 (ACR4) receptor-like protein kinase (RLK) have been implicated in the intercellular communication that is required for surface functions of the epidermis. We have identified a novel mutant gene in Arabidopsis, ale2, which is associated with various epidermal defects, including disorganization of epidermis-related tissues, defects in the leaf cuticle and the fusion of organs. ALE2 encodes a previously uncharacterized RLK with a cluster of basic amino acid residues followed by a cysteine-containing sequence in the putative extracellular domain. Our genetic investigations suggest that ALE2 and ACR4 function in the same process, whereas ALE1 has a different mode of action, and that these three genes play partially overlapping roles in positively regulating protoderm-specific gene expression and for the formation of leafy organs. We propose that at least two modes of intercellular communication facilitate the specification of epidermis, thereby promoting shoot organogenesis in Arabidopsis.

Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana.

Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic β-glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F1 and F2 progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F1 progeny and most of the F2 progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F2 individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F1 generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.

Dual regulation of ETTIN (ARF3) gene expression by AS1-AS2, which maintains the DNA methylation level, is involved in stabilization of leaf adaxial-abaxial partitioning in Arabidopsis

Leaf primordia are generated at the periphery of the shoot apex, developing into flat symmetric organs with adaxial-abaxial polarity, in which the indeterminate state is repressed. Despite the crucial role of the ASYMMETRIC LEAVES1 (AS1)-AS2 nuclear-protein complex in leaf adaxial-abaxial polarity specification, information on mechanisms controlling their downstream genes has remained elusive. We systematically analyzed transcripts by microarray and chromatin immunoprecipitation assays and performed genetic rescue of as1 and as2 phenotypic abnormalities, which identified a new target gene, ETTIN (ETT)/AUXIN RESPONSE FACTOR3 (ARF3), which encodes an abaxial factor acting downstream of the AS1-AS2 complex. While the AS1-AS2 complex represses ETT by direct binding of AS1 to the ETT promoter, it also indirectly activates miR390- and RDR6-dependent post-transcriptional gene silencing to negatively regulate both ETT and ARF4 activities. Furthermore, AS1-AS2 maintains the status of DNA methylation in the ETT coding region. In agreement, filamentous leaves formed in as1 and as2 plants treated with a DNA methylation inhibitor were rescued by loss of ETT and ARF4 activities. We suggest that negative transcriptional, post-transcriptional and epigenetic regulation of the ARFs by AS1-AS2 is important for stabilizing early leaf partitioning into abaxial and adaxial domains.

Genetic networks regulated by ASYMMETRIC LEAVES1 (AS1) and AS2 in leaf development in Arabidopsis thaliana: KNOX genes control five morphological events

The asymmetric leaves 1 (as1) and as2 mutants of Arabidopsis thaliana exhibit pleiotropic phenotypes. Expression of a number of genes, including three class-1 KNOTTED-like homeobox (KNOX) genes (BP, KNAT2 and KNAT6) and ETTIN/ARF3, is enhanced in these mutants. In the present study, we attempted to identify the phenotypic features of as1 and as2 mutants that were generated by ectopic expression of KNOX genes, using multiple loss-of-function mutations of KNOX genes as well as as1 and as2. Our results revealed that the ectopic expression of class-1 KNOX genes resulted in reductions in the sizes of leaves, reductions in the size of sepals and petals, the formation of a less prominent midvein, the repression of adventitious root formation and late flowering. Our results also revealed that the reduction in leaf size and late flowering were caused by the repression, by KNOX genes, of a gibberellin (GA) pathway in as1 and as2 plants. The formation of a less prominent midvein and the repression of adventitious root formation were not, however, related to the GA pathway. The asymmetric formation of leaf lobes, the lower complexity of higher-ordered veins, and the elevated frequency of adventitious shoot formation on leaves of as1 and as2 plants were not rescued by multiple mutations in KNOX genes. These features must, therefore, be controlled by other genes in which expression is enhanced in the as1 and as2 mutants.

ASYMMETRIC LEAVES2 and Elongator, a Histone Acetyltransferase Complex, Mediate the Establishment of Polarity in Leaves of Arabidopsis thaliana

Leaf primordia are generated around the shoot apical meristem. Mutation of the ASYMMETRIC LEAVES2 (AS2) gene of Arabidopsis thaliana results in defects in repression of the meristematic and indeterminate state, establishment of adaxial-abaxial polarity and left-right symmetry in leaves. AS2 represses transcription of meristem-specific class 1 KNOX homeobox genes and of the abaxial-determinant genes ETTIN/ARF3, KANADI2 and YABBY5. To clarify the role of AS2 in the establishment of leaf polarity, we isolated mutations that enhanced the polarity defects associated with as2. We describe here the enhancer-of-asymmetric-leaves-two1 (east1) mutation, which caused the formation of filamentous leaves with abaxialized epidermis on the as2-1 background. Levels of transcripts of class 1 KNOX and abaxial-determinant genes were markedly higher in as2-1 east1-1 mutant plants than in the wild-type and corresponding single-mutant plants. EAST1 encodes the histone acetyltransferase ELONGATA3 (ELO3), a component of the Elongator complex. Genetic analysis, using mutations in genes involved in the biogenesis of a trans-acting small interfering RNA (ta-siRNA), revealed that ELO3 mediated establishment of leaf polarity independently of AS2 and the ta-siRNA-related pathway. Treatment with an inhibitor of histone deacetylases (HDACs) caused additive polarity defects in as2-1 east1-1 mutant plants, suggesting the operation of an ELO3 pathway, independent of the HDAC pathway, in the determination of polarity. We propose that multiple pathways play important roles in repression of the expression of class 1 KNOX and abaxial-determinant genes in the development of the adaxial domain of leaves and, thus, in the establishment of leaf polarity.

A tobacco protein kinase, NPK2, has a domain homologous to a domain found in activators of mitogen-activated protein kinases (MAPKKs)

A cDNA (cNPK2) that encodes a protein of 518 amino acids was isolated from a library prepared from poly(A)+ RNAs of tobacco cells in suspension culture. The N-terminal half of the predicted NPK2 protein is similar in amino acid sequence to the catalytic domains of kinases that activate mitogen-activated protein kinases (designated here MAPKKs) from various animals and to those of yeast homologs of MAPKKs. The N-terminal domain of NPK2 was produced as a fusion protein in Escherichia coli, and the purified fusion protein was found to be capable of autophosphorylation of threonine and serine residues. These results indicate that the N-terminal domain of NPK2 has activity of a serine/threonine protein kinase. Southern blot analysis showed that genomic DNAs from various plant species, including Arabidopsis thaliana and sweet potato, hybridized strongly with cNPK2, indicating that these plants also have genes that are closely related to the gene for NPK2. The structural similarity between the catalytic domain of NPK2 and those of MAPKKs and their homologs suggests that tobacco NPK2 corresponds to MAPKKs of other organisms. Given the existence of plant homologs of an MAP kinase and tobacco NPK1, which is structurally and functionally homologous to one of the activator kinases of yeast homologs of MAPKK (MAPKKKs), it seems likely that a signal transduction pathway mediated by a protein kinase cascade that is analogous to the MAP kinase cascades proposed in yeasts and animals, is also conserved in plants.