Warm temperature-triggered developmental reprogramming requires VIL1-mediated, genome-wide H3K27me3 accumulation in Arabidopsis.

Changes in ambient temperature immensely affect developmental programs in many species. Plants adapt to high ambient growth temperature in part by vegetative and reproductive developmental reprogramming, known as thermo-morphogenesis. Thermo-morphogenesis is accompanied by massive changes in the transcriptome upon temperature change. Here, we show that transcriptome changes induced by warm ambient temperature require VERNALIZATION INSENSITIVE 3-LIKE 1 (VIL1), a facultative component of the Polycomb repressive complex PRC2, in Arabidopsis. Warm growth temperature elicits genome-wide accumulation of H3K27me3 and VIL1 is necessary for the warm temperature-mediated accumulation of H3K27me3. Consistent with its role as a mediator of thermo-morphogenesis, loss of function of VIL1 results in hypo-responsiveness to warm ambient temperature. Our results show that VIL1 is a major chromatin regulator in responses to high ambient temperature.

Abscisic acid negatively regulates the Polycomb-mediated H3K27me3 through the PHD-finger protein, VIL1.

Polycomb dictates developmental programs in higher eukaryotes, including flowering plants. A phytohormone, abscisic acid (ABA), plays a pivotal role in seed and seedling development and mediates responses to multiple environmental stresses, such as salinity and drought. In this study, we show that ABA affects the Polycomb Repressive Complex 2 (PRC2)-mediated Histone H3 Lys 27 trimethylation (H3K27me3) through VIN3-LIKE1/VERNALIZATION 5 (VIL1/VRN5) to fine-tune the timely repression of ABSCISIC ACID INSENSITIVE 3 (ABI3) and ABSCISIC ACID INSENSITIVE 4 (ABI4) in Arabidopsis thaliana. vil1 mutants exhibit hypersensitivity to ABA during early seed germination and show enhanced drought tolerance. Our study revealed that the ABA signaling pathway utilizes a facultative component of the chromatin remodeling complex to demarcate the level of expression of ABA-responsive genes.

Orthogonal control of gene expression in plants using synthetic promoters and CRISPR-based transcription factors.

BACKGROUND: The construction and application of synthetic genetic circuits is frequently improved if gene expression can be orthogonally controlled, relative to the host. In plants, orthogonality can be achieved via the use of CRISPR-based transcription factors that are programmed to act on natural or synthetic promoters. The construction of complex gene circuits can require multiple, orthogonal regulatory interactions, and this in turn requires that the full programmability of CRISPR elements be adapted to non-natural and non-standard promoters that have few constraints on their design. Therefore, we have developed synthetic promoter elements in which regions upstream of the minimal 35S CaMV promoter are designed from scratch to interact via programmed gRNAs with dCas9 fusions that allow activation of gene expression. RESULTS: A panel of three, mutually orthogonal promoters that can be acted on by artificial gRNAs bound by CRISPR regulators were designed. Guide RNA expression targeting these promoters was in turn controlled by either Pol III (U6) or ethylene-inducible Pol II promoters, implementing for the first time a fully artificial Orthogonal Control System (OCS). Following demonstration of the complete orthogonality of the designs, the OCS was tied to cellular metabolism by putting gRNA expression under the control of an endogenous plant signaling molecule, ethylene. The ability to form complex circuitry was demonstrated via the ethylene-driven, ratiometric expression of fluorescent proteins in single plants. CONCLUSIONS: The design of synthetic promoters is highly generalizable to large tracts of sequence space, allowing Orthogonal Control Systems of increasing complexity to potentially be generated at will. The ability to tie in several different basal features of plant molecular biology (Pol II and Pol III promoters, ethylene regulation) to the OCS demonstrates multiple opportunities for engineering at the system level. Moreover, given the fungibility of the core 35S CaMV promoter elements, the derived synthetic promoters can potentially be utilized across a variety of plant species.

Phytochrome B triggers light-dependent chromatin remodelling through the PRC2-associated PHD finger protein VIL1.

To compensate for a sessile nature, plants have developed sophisticated mechanisms to sense varying environmental conditions. Phytochromes (phys) are light and temperature sensors that regulate downstream genes to render plants responsive to environmental stimuli1-4. Here, we show that phyB directly triggers the formation of a repressive chromatin loop by physically interacting with VERNALIZATION INSENSITIVE 3-LIKE1/VERNALIZATION 5 (VIL1/VRN5), a component of Polycomb Repressive Complex 2 (PRC2)5,6, in a light-dependent manner. VIL1 and phyB cooperatively contribute to the repression of growth-promoting genes through the enrichment of Histone H3 Lys27 trimethylation (H3K27me3), a repressive histone modification. In addition, phyB and VIL1 mediate the formation of a chromatin loop to facilitate the repression of ATHB2. Our findings show that phyB directly utilizes chromatin remodelling to regulate the expression of target genes in a light-dependent manner.

High-Throughput Targeted Transcriptional Profiling of Defense Genes Using RNA-Mediated Oligonucleotide Annealing, Selection, and Ligation with Next-Generation Sequencing in Arabidopsis.

Tracking RNA transcription has been one of the most powerful tools to gain insight into the biological process. While a wide range of molecular methods such as northern blotting, RNA-seq, and quantitative RT-PCR are available, one of the barriers in transcript analysis is an inability to accommodate a sufficient number of samples to achieve high resolution in dynamic transcriptional changes. RASL-seq (RNA-mediated oligonucleotide Annealing, Selection, and Ligation with next-generation sequencing) is a sequencing-based transcription profiling tool that processes hundreds of samples assessing a set of over a hundred genes with a fraction of the cost of a conventional RNA-seq. We described a RASL-seq protocol for assessing 288 genes mostly including defense genes to capture their dynamic nature. We demonstrated that this transcriptional profiling method produced a highly reliable outcome comparable to a conventional RNA-seq and quantitative RT-PCR.

DEK domain-containing proteins control flowering time in Arabidopsis.

Evolutionarily conserved DEK domain-containing proteins have been implicated in multiple chromatin-related processes, mRNA splicing and transcriptional regulation in eukaryotes. Here, we show that two DEK proteins, DEK3 and DEK4, control the floral transition in Arabidopsis. DEK3 and DEK4 directly associate with chromatin of related flowering repressors, FLOWERING LOCUS C (FLC), and its two homologs, MADS AFFECTING FLOWERING4 (MAF4) and MAF5, to promote their expression. The binding of DEK3 and DEK4 to a histone octamer in vivo affects histone modifications at FLC, MAF4 and MAF5 loci. In addition, DEK3 and DEK4 interact with RNA polymerase II and promote the association of RNA polymerase II with FLC, MAF4 and MAF5 chromatin to promote their expression. Our results show that DEK3 and DEK4 directly interact with chromatin to facilitate the transcription of key flowering repressors and thus prevent precocious flowering in Arabidopsis.

Genome-Wide Analysis of Chromatin Accessibility in Arabidopsis Infected with Pseudomonas syringae.

Changes in chromatin accessibility are an important aspect of the molecular changes that occur in eukaryotic cells responding to stress, and they appear to play a critical role in stress-induced transcriptional activation/reprogramming and epigenetic changes. In plants, pathogen infection has been shown to induce rapid and drastic transcriptional reprogramming; growing evidence suggests that chromatin remodeling plays an essential role in this phenomenon. The recent development of genomic tools to assess chromatin accessibility presents a significant opportunity to investigate the relationship between chromatin dynamicity and gene expression. In this protocol, we have adopted a popular chromatin accessibility assay, DNase-seq, to measure chromatin accessibility in Arabidopsis infected with the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). DNase-seq provides information on chromatin accessibility through the sequencing of DNA fragments generated by DNase I digestion of open chromatin, followed by mapping these sequences on a reference genome. Of the two popular DNase-seq approaches, we based our method on the Stamatoyannopoulos protocol, which involves two DNase cleavages rather than a single cleavage, followed by size fractionation. Please note that this two-cleavage approach is widely accepted and has been used extensively by ENCODE (Encyclopedia of DNA Elements) project, a public research consortium investigating cis- and trans-elements in the transcriptional regulation in animal cells. To enhance the quality of the chromatin accessibility assay, we modified this protocol by including two centrifugation steps for nuclear enrichment and size fractionation and an extra washing step for removal of chloroplasts and Pst. The outcomes obtained by this approach are also discussed.

Pathogen Infection and MORC Proteins Affect Chromatin Accessibility of Transposable Elements and Expression of Their Proximal Genes in Arabidopsis.

To assess the role of MORC1 in epigenetics in relation to plant immunity, genome-wide chromatin accessibility was compared between mock- or Pseudomonas syringae pv. tomato-inoculated wild type (WT) Arabidopsis, the morc1/2 double mutant, or both. Most changes in chromatin accessibility, scored by DNase I hypersensitive sites (DHSs), were located in the promoters of genes and transposable elements (TEs). Comparisons between morc1/2 and WT receiving the same treatment revealed differential DHSs (dDHSs) predominantly associated with heterochromatic TEs. By contrast, comparisons between mock- and P. syringae pv. tomato-inoculated plants from the same genotype showed dDHSs associated with biotic and abiotic stress-related genes; a smaller but significant population was in TEs. Moreover, many defense genes, including PR-1, PR-2, and PR-5, were proximal to P. syringae pv. tomato-induced, TE-associated dDHSs. A random subset of these defense genes showed moderately delayed or reduced expression or both in P. syringae pv. tomato-infected morc1/2 as compared with WT. MORC1 was physically bound to chromatin in a P. syringae pv. tomato infection-responsive manner at sites dispersed throughout the genome. Notably, silencing of TE-associated dDHSs proximal to these infection-induced, MORC1-interacting sites led to significant suppression of P. syringae pv. tomato-induced transcription of adjacent defense genes, including PR-1. These results provide evidence that MORC1 is associated with TEs and suggest that a subset of these TEs may help regulate their proximal defense genes.

The compromised recognition of turnip crinkle virus1 subfamily of microrchidia ATPases regulates disease resistance in barley to biotrophic and necrotrophic pathogens.

MORC1 and MORC2, two of the seven members of the Arabidopsis (Arabidopsis thaliana) Compromised Recognition of Turnip Crinkle Virus1 subfamily of microrchidia Gyrase, Heat Shock Protein90, Histidine Kinase, MutL (GHKL) ATPases, were previously shown to be required in multiple layers of plant immunity. Here, we show that the barley (Hordeum vulgare) MORCs also are involved in disease resistance. Genome-wide analyses identified five MORCs that are 37% to 48% identical on the protein level to AtMORC1. Unexpectedly, and in clear contrast to Arabidopsis, RNA interference-mediated knockdown of MORC in barley resulted in enhanced basal resistance and effector-triggered, powdery mildew resistance locus A12-mediated resistance against the biotrophic powdery mildew fungus (Blumeria graminis f. sp. hordei), while MORC overexpression decreased resistance. Moreover, barley knockdown mutants also showed higher resistance to Fusarium graminearum. Barley MORCs, like their Arabidopsis homologs, contain the highly conserved GHKL ATPase and S5 domains, which identify them as members of the MORC superfamily. Like AtMORC1, barley MORC1 (HvMORC1) binds DNA and has Mn2+-dependent endonuclease activities, suggesting that the contrasting function of MORC1 homologs in barley versus Arabidopsis is not due to differences in their enzyme activities. In contrast to AtMORCs, which are involved in silencing of transposons that are largely restricted to pericentromeric regions, barley MORC mutants did not show a loss-of-transposon silencing regardless of their genomic location. Reciprocal overexpression of MORC1 homologs in barley and Arabidopsis showed that AtMORC1 and HvMORC1 could not restore each other's function. Together, these results suggest that MORC proteins function as modulators of immunity, which can act negatively (barley) or positively (Arabidopsis) dependent on the species.

Analysis of segregation distortion and its relationship to hybrid barriers in rice.

BACKGROUND: Segregation distortion (SD) is a frequently observed occurrence in mapping populations generated from crosses involving divergent genotypes. In the present study, ten genetic linkage maps constructed from reciprocal F2 and BC1F1 mapping populations derived from the parents Dasanbyeo (indica) and Ilpumbyeo (japonica) were used to identify the distribution, effect, and magnitude of the genetic factors underlying the mechanisms of SD between the two subspecies. RESULTS: SD loci detected in the present study were affected by male function, female function, and zygotic selection. The most pronounced SD loci were mapped to chromosome 3 (transmitted through male gametes), chromosome 5 (transmitted through male gametes), and chromosome 6 (transmitted through female gametes). The level of SD in BC1F1 populations which defined by chi-square value independence multiple tests was relatively low in comparison to F2 populations. Dasanbyeo alleles were transmitted at a higher frequency in both F2 and BC1F1 populations, suggesting that indica alleles are strongly favored in inter-subspecific crosses in rice. SD loci in the present study corresponded to previously reported loci for reproductive barriers. In addition, new SD loci were detected on chromosomes 2 and 12. CONCLUSION: The identification of the distribution of SD and the effect of genetic factors causing SD in genetic mapping populations provides an opportunity to survey the whole genome for new SD loci and their relationships to reproductive barriers. This provides a basis for future research on the elucidation of the genetic mechanisms underlying SD in rice, and will be useful in molecular breeding programs.