Junshi Yazaki

Genome-wide High-Resolution Mapping and Functional Analysis of DNA Methylation in Arabidopsis

Cytosine methylation is important for transposon silencing and epigenetic regulation of endogenous genes, although the extent to which this DNA modification functions to regulate the genome is still unknown. Here we report the first comprehensive DNA methylation map of an entire genome, at 35 base pair resolution, using the flowering plant Arabidopsis thaliana as a model. We find that pericentromeric heterochromatin, repetitive sequences, and regions producing small interfering RNAs are heavily methylated. Unexpectedly, over one-third of expressed genes contain methylation within transcribed regions, whereas only approximately 5% of genes show methylation within promoter regions. Interestingly, genes methylated in transcribed regions are highly expressed and constitutively active, whereas promoter-methylated genes show a greater degree of tissue-specific expression. Whole-genome tiling-array transcriptional profiling of DNA methyltransferase null mutants identified hundreds of genes and intergenic noncoding RNAs with altered expression levels, many of which may be epigenetically controlled by DNA methylation.

Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis.

The gaseous plant hormone ethylene can trigger myriad physiological and morphological responses in plants. While many ethylene signaling pathway components have been identified and characterized, little is known about the function of the integral membrane protein ETHYLENE-INSENSITIVE2 (EIN2), a central regulator of all ethylene responses. Here, we demonstrate that Arabidopsis thaliana EIN2 is a protein with a short half-life that undergoes rapid proteasome-mediated protein turnover. Moreover, EIN2 protein accumulation is positively regulated by ethylene. We identified two F-box proteins, EIN2 TARGETING PROTEIN1 (ETP1) and EIN2 TARGETING PROTEIN2 (ETP2), that interact with the EIN2 C-terminal domain (EIN2-CEND), which is highly conserved and sufficient to activate most ethylene responses. Overexpression of ETP1 or ETP2 disrupts EIN2 protein accumulation, and these plants manifest a strong ethylene-insensitive phenotype. Furthermore, knocking down the levels of both ETP1 and ETP2 mRNAs using an artificial microRNA (amiRNA) leads to accumulation of EIN2 protein, resulting in plants that display constitutive ethylene response phenotypes. Finally, ethylene down-regulates ETP1 and ETP2 proteins, impairing their ability to interact with EIN2. Thus, these studies reveal that a complex interplay between ethylene, the regulation of ETP1/ETP2 F-box proteins, and subsequent targeting and degradation of EIN2 is essential for triggering ethylene responses in plants.

Utilizing tiling microarrays for whole-genome analysis in plants.

The recent explosion in available genome sequence data has ushered in an era in which analysis of a whole genome can be performed in a single experiment. While DNA microarrays have long been the established technology for measuring gene expression levels, standard expression arrays use relatively few probes for each gene and are typically biased toward known and predicted gene structures. Recently, with the availability of complete genome sequences for many organisms, very-high-density oligonucleotide-based microarrays that span the entire genome have emerged as the preferred platform for genomic analysis. Whole-genome tiling microarrays can be employed for a myriad of purposes, including empirical annotation of the transcriptome, chromatin immunoprecipitation-chip studies, analysis of alternative RNA splicing, characterization of the methylation state of cytosine bases throughout a genome (methylome), and DNA polymorphism discovery. Here, we review several applications of whole-genome technology to obtain a variety of genomic-scale information in plants.

An ABA-increased interaction of the PYL6 ABA receptor with MYC2 Transcription Factor: A putative link of ABA and JA signaling

Abscisic acid (ABA) is a plant hormone that mediates abiotic stress tolerance and regulates growth and development. ABA binds to members of the PYL/RCAR ABA receptor family that initiate signal transduction inhibiting type 2C protein phosphatases. Although crosstalk between ABA and the hormone Jasmonic Acid (JA) has been shown, the molecular entities that mediate this interaction have yet to be fully elucidated. We report a link between ABA and JA signaling through a direct interaction of the ABA receptor PYL6 (RCAR9) with the basic helix-loop-helix transcription factor MYC2. PYL6 and MYC2 interact in yeast two hybrid assays and the interaction is enhanced in the presence of ABA. PYL6 and MYC2 interact in planta based on bimolecular fluorescence complementation and co-immunoprecipitation of the proteins. Furthermore, PYL6 was able to modify transcription driven by MYC2 using JAZ6 and JAZ8 DNA promoter elements in yeast one hybrid assays. Finally, pyl6 T-DNA mutant plants show an increased sensitivity to the addition of JA along with ABA in cotyledon expansion experiments. Overall, the present study identifies a direct mechanism for transcriptional modulation mediated by an ABA receptor different from the core ABA signaling pathway, and a putative mechanistic link connecting ABA and JA signaling pathways.

Analysis of expressed sequence tags in apomictic guineagrass (Panicum maximum).

Apomixis is an intriguing asexual mode of reproduction, because it produces maternal clones that permit vegetative reproduction through seeds. Guineagrass (Panicum maximum) has both facultative aposporous apomixis and obligate sexual modes of reproduction. Despite the importance of apomixis in guineagrass, expressed sequence tags (ESTs) for this condition have not been studied in this species. We constructed a guineagrass cDNA library from two aposporous strains, Ku5954 and GM64-3A, and utilized them as microarray probes. To find genes uniquely expressed in the immature pistils of apomicts, we performed a microarray analysis using target RNA from another apomict, OKI64. Of the 4608 probes in the microarray, only 394 showed clear gene expression in the immature pistils. Of the 394 expressed probes, 196 were successfully sequenced. Of these, 181 had significant homology with other species, including 10 ESTs with matches in a pistil cDNA library from another aposporous species, Cenchrus ciliaris. Of the remaining ESTs, three showed significant homology only with animal database sequences and the other 12 ESTs showed no homology with any previously registered sequence. In reverse-transcriptase PCR and real-time quantitative PCR, nine ESTs reliably detected ovary-specific gene expression. Of these, three revealed aposporous ovary-specific genes expressed in the early developmental stage, suggesting that these could be apomixis-related genes.

Mapping transcription factor interactome networks using HaloTag protein arrays

Significance Using a newly developed technology, HaloTag nucleic acid programmable protein array (HaloTag-NAPPA), we increase the capacity of in situ protein microarray technology several-fold, such that proteome-scale screening becomes feasible. Many examples of novel protein–protein interactions (PPIs) among plant signaling pathways were observed. With few exceptions, nearly all of these connections are undocumented in the existing literature. This study has resulted in an important new resource for the plant biology community—a plant transcription factor-anchored protein–protein interaction network map. Such transcription factor- and transcriptional regulator-based PPI networks may help in the identification of novel genes for use in the improvement of agronomic traits such as grain quality, disease resistance, and stress tolerance. Protein microarrays enable investigation of diverse biochemical properties for thousands of proteins in a single experiment, an unparalleled capacity. Using a high-density system called HaloTag nucleic acid programmable protein array (HaloTag-NAPPA), we created high-density protein arrays comprising 12,000 Arabidopsis ORFs. We used these arrays to query protein–protein interactions for a set of 38 transcription factors and transcriptional regulators (TFs) that function in diverse plant hormone regulatory pathways. The resulting transcription factor interactome network, TF-NAPPA, contains thousands of novel interactions. Validation in a benchmarked in vitro pull-down assay revealed that a random subset of TF-NAPPA validated at the same rate of 64% as a positive reference set of literature-curated interactions. Moreover, using a bimolecular fluorescence complementation (BiFC) assay, we confirmed in planta several interactions of biological interest and determined the interaction localizations for seven pairs. The application of HaloTag-NAPPA technology to plant hormone signaling pathways allowed the identification of many novel transcription factor–protein interactions and led to the development of a proteome-wide plant hormone TF interactome network.

Structure and expression of two genes encoding secreted acid phosphatases under phosphate-deficient conditions in Pholiota nameko strain N2

Twenty-three polypeptides secreted in response to a deficiency of inorganic phosphate (Pi) were previously found by two-dimensional polyacrylamide gel electrophoresis analysis in mycelia of Pholiota nameko strain N2. In this study, N-terminal sequencing revealed three of them to be identical to known acid phosphatases of P. nameko strain N114. Two cDNAs and the corresponding genomic DNAs of genes PNAP1 and PNAP2 which encode two of the three acid phosphatases were cloned. The deduced amino acid sequences of PNAP1 and PNAP2 showed high similarity to other fungal acid phosphatases and contained a putative catalytic active site of acid phosphatase. PNAP1 and PNAP2 are comprised of five and seven exons interrupted by four and six introns, respectively. Their promoter regions include two cis-acting elements found in Pi deficiency-inducible genes of Saccharomyces cerevisiae, together with several known functional elements such as a TATA box. Northern blot analysis showed that PNAP1 and PNAP2 are expressed in response to a deficiency of Pi.

Profiling Interactome Networks with the HaloTag-NAPPA In Situ Protein Array

Physical interactions between proteins and other molecules can be evaluated at a proteome scale using protein arrays, a type of high-throughput pulldown assay. We developed a modified in situ protein array known as the nucleic acid programmable protein assay (NAPPA) that allows the screening of thousands of open reading frames (ORFs) at a lower cost, with less labor, and in less time than conventional protein arrays. The HaloTag-NAPPA protein array can efficiently capture proteins expressed in situ on a glass slide using the Halo high-affinity capture tag. Here, we describe the fabrication of the array using publicly available resources and detection of protein-protein interactions (PPIs) that can be used to generate a protein interactome map. The Basic Protocol includes procedures for preparing the plasmid DNA spotted on glass slides, in situ protein expression, and PPI detection. The supporting protocols outline the construction of vectors and preparation of ORF clones.