Posttranslational Modifications of the Master Transcriptional Regulator NPR1 Enable Dynamic but Tight Control of Plant Immune Responses.

NPR1, a master regulator of basal and systemic acquired resistance in plants, confers immunity through a transcriptional cascade, which includes transcription activators (e.g., TGA3) and repressors (e.g., WRKY70), leading to the massive induction of antimicrobial genes. How this single protein orchestrates genome-wide transcriptional reprogramming in response to immune stimulus remains a major question. Paradoxically, while NPR1 is essential for defense gene induction, its turnover appears to be required for this function, suggesting that NPR1 activity and degradation are dynamically regulated. Here we show that sumoylation of NPR1 by SUMO3 activates defense gene expression by switching NPR1's association with the WRKY transcription repressors to TGA transcription activators. Sumoylation also triggers NPR1 degradation, rendering the immune induction transient. SUMO modification of NPR1 is inhibited by phosphorylation at Ser55/Ser59, which keeps NPR1 stable and quiescent. Thus, posttranslational modifications enable dynamic but tight and precise control of plant immune responses.

Salicylic acid activates DNA damage responses to potentiate plant immunity.

DNA damage is normally detrimental to living organisms. Here we show that it can also serve as a signal to promote immune responses in plants. We found that the plant immune hormone salicylic acid (SA) can trigger DNA damage in the absence of a genotoxic agent. The DNA damage sensor proteins RAD17 and ATR are required for effective immune responses. These sensor proteins are negatively regulated by a key immune regulator, SNI1 (suppressor of npr1-1, inducible 1), which we found is a subunit of the structural maintenance of chromosome (SMC) 5/6 complex required for controlling DNA damage. Elevated DNA damage caused by the sni1 mutation or treatment with a DNA-damaging agent markedly enhances SA-mediated defense gene expression. Our study suggests that activation of DNA damage responses is an intrinsic component of the plant immune responses.

NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants.

Salicylic acid (SA) is a plant immune signal produced after pathogen challenge to induce systemic acquired resistance. It is the only major plant hormone for which the receptor has not been firmly identified. Systemic acquired resistance in Arabidopsis requires the transcription cofactor nonexpresser of PR genes 1 (NPR1), the degradation of which acts as a molecular switch. Here we show that the NPR1 paralogues NPR3 and NPR4 are SA receptors that bind SA with different affinities. NPR3 and NPR4 function as adaptors of the Cullin 3 ubiquitin E3 ligase to mediate NPR1 degradation in an SA-regulated manner. Accordingly, the Arabidopsis npr3 npr4 double mutant accumulates higher levels of NPR1, and is insensitive to induction of systemic acquired resistance. Moreover, this mutant is defective in pathogen effector-triggered programmed cell death and immunity. Our study reveals the mechanism of SA perception in determining cell death and survival in response to pathogen challenge.

The Arabidopsis chromatin modifier ATX1, the myotubularin-like AtMTM and the response to drought.

Plants respond to environmental stresses by altering transcription of genes involved in the response. The chromatin modifier ATX1 regulates expression of a large number of genes; consequently, factors that affect ATX1 activity would also influence expression from ATX1-regulated genes. Here, we demonstrate that dehydration is such a factor implicating ATX1 in the plant's response to drought. In addition, we report that a hitherto unknown Arabidopsis gene, At3g10550, encodes a phosphoinositide 3'-phosphatase related to the animal myotubularins (AtMTM1). Myotubularin activities in plants have not been described and herein, we identify an overlapping set of genes co-regulated by ATX1 and AtMTM under drought conditions. We propose that these shared genes represent the ultimate targets of partially overlapping branches of the pathways of the nuclear ATX1 and the cytoplasmic AtMTM1. Our analyses offer first genome-wide insights into the relationship of an epigenetic factor and a lipid phosphatase from the other end of a shared drought responding pathway in Arabidopsis.

Wall-modifying genes regulated by the Arabidopsis homolog of trithorax, ATX1: repression of the XTH33 gene as a test case.

The plant cell wall is a dynamic structure playing important roles in the control of plant cell growth and differentiation. These processes involve global reprogramming of the genome driven by dynamic changes in chromatin structure. The chromatin modifier ARABIDOPSIS HOMOLOG OF TRITHORAX (ATX1) methylates lysine residue 4 on histone H3 (H3K4me), acting as an epigenetic mark on associated genes. The remarkable overrepresentation in the ATX1-regulated gene fraction of genes encoding plasma membrane and cell wall-remodeling activities suggested a link between two separate factors affecting growth, development and adaptation in Arabidopsis: the wall-modifying activities regulating cell extension, growth and fate, and the epigenetic mechanisms regulating chromatin structure and gene expression. A co-regulated fraction of specific wall-modifying proteins suggests that they may function together. Here, we study the ATX1-dependent expression of the gene encoding the wall-loosening factor XTH33 as a test case for development- and tissue-specific effects displayed by the chromatin modifier. In addition, we show that XTH33 is, most likely, an integral plasma membrane protein. A putative transmembrane domain is conserved in some, but not all, XTH family members, suggesting that they may be differently positioned when functioning as wall modifiers.

Dynamic and stable histone H3 methylation patterns at the Arabidopsis FLC and AP1 loci.

Mechanisms that chemically modify nucleosomes leading to inheritable activation or repression of pertinent genes are defined as epigenetic. H3K4me3 and H3K27me3 are interpreted as 'activating' and 'silencing' marks, respectively. Here, we demonstrate that even for related genes neither modification, alone, could serve as an indicator of expression status: despite being members of the same gene family selectively activated by ATX1, FLC and AP1 nucleosomes may be similarly decorated but, also, surprisingly different. 'Activating' H3K4me3 and 'silencing' H3K27me3 modifications co-exist at 5'-end nucleosomes of transcriptionally active FLC-gene, while highly transcribed AP1 displays neither of the two marks. The results suggest that distinct mechanisms 'read' and operate at each locus. In a remarkable contrast, H3K4me3-H3K27me3 profiles at downstream FLC and AP1 gene sequences remain unchanged and transmitted as stable marks throughout development. We propose that H3K4me3 and H3K27me3 produce a distinct bi-modular 'syllable' in the histone 'code' conveying different meaning on specific genes. Evidence that certain chromatin modifications might be common for active or non-active genome regions but, also, that the same histone signs might have gene-specific 'meaning', as reported here, might be critically important for large-scale genome analyses. ATX1 and CLF encode enzyme activities involved in establishing the H3K4me3 and H3K27me3 marks, respectively. The potential involvement of ATX1 and CLF in generating the dual H3K4me3 and H3K27me3 marks on FLC and AP1 nucleosomes was investigated.

An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants.

Chromatin immunoprecipitation (ChIP) is a powerful tool for the characterization of covalent histone modifications and DNA-histone interactions in vivo. The procedure includes DNA-histone cross-linking in chromatin, shearing DNA into smaller fragments, immunoprecipitation with antibodies against the histone modifications of interest, followed by PCR identification of associated DNA sequences. In this protocol, we describe a simplified and optimized version of ChIP assay by reducing the number of experimental steps and isolation solutions and shortening preparation times. We include a nuclear isolation step before chromatin shearing, which provides a good yield of high-quality DNA resulting in at least 15 mug of DNA from each immunoprecipitated sample (from 0.2 to 0.4 g of starting tissue material) sufficient to test > or =25 genes of interest. This simpler and cost-efficient protocol has been applied for histone-modification studies of various Arabidopsis thaliana tissues and is easy to adapt for other systems as well.

The highly similar Arabidopsis homologs of trithorax ATX1 and ATX2 encode proteins with divergent biochemical functions.

Gene duplication followed by functional specialization is a potent force in the evolution of biological diversity. A comparative study of two highly conserved duplicated genes, ARABIDOPSIS TRITHORAX-LIKE PROTEIN1 (ATX1) and ATX2, revealed features of both partial redundancy and of functional divergence. Although structurally similar, their regulatory sequences have diverged, resulting in distinct temporal and spatial patterns of expression of the ATX1 and ATX2 genes. We found that ATX2 methylates only a limited fraction of nucleosomes and that ATX1 and ATX2 influence the expression of largely nonoverlapping gene sets. Even when coregulating shared targets, ATX1 and ATX2 may employ different mechanisms. Most remarkable is the divergence of their biochemical activities: both proteins methylate K4 of histone H3, but while ATX1 trimethylates it, ATX2 dimethylates it. ATX2 and ATX1 provide an example of separated K4 di from K4 trimethyltransferase activity.

The Arabidopsis homologs of trithorax (ATX1) and enhancer of zeste (CLF) establish 'bivalent chromatin marks' at the silent AGAMOUS locus.

Tightly balanced antagonism between the Polycomb group (PcG) and the Trithorax group (TrxG) complexes maintain Hox expression patterns in Drosophila and murine model systems. Factors belonging to the PcG/TrxG complexes control various processes in plants as well but whether they participate in mechanisms that antagonize, balance or maintain each other's effects at a particular gene locus is unknown. CURLY LEAF (CLF), an Arabidopsis homolog of enhancer of zeste (EZ) and the ARABIDOPSIS HOMOLOG OF TRITHORAX (ATX1) control the expression of the flower homeotic gene AGAMOUS (AG). Disrupted ATX1 or CLF function results in misexpression of AG, recognizable phenotypes and loss of H3K4me3 or H3K27me3 histone H3-tail marks, respectively. A novel idea suggested by our results here, is that PcG and TrxG complexes function as a specific pair generating bivalent chromatin marks at the silent AG locus. Simultaneous loss of ATX1 and CLF restored AG repression and normalized leaf phenotypes. At the molecular level, disrupted ATX1 and CLF functions did not lead to erasure of the CLF- and ATX1-generated epigenetic marks, as expected: instead, in the double mutants, H3K27me3 and H3K4me3 tags were partially restored. We demonstrate that ATX1 and CLF physically interact linking mechanistically the observed effects.

Maize DBF1-interactor protein 1 containing an R3H domain is a potential regulator of DBF1 activity in stress responses.

The maize dehydration-responsive element (DRE)-binding factor, DBF1, is a member of the Apetala 2/Ethylene Response Factor transcription factors family and is involved in the regulation of the ABA-responsive gene rab17 through the DRE in an ABA-dependent pathway. In this study we analysed the functionality of DBF1 in abiotic stress responses and found that Arabidopsis plants over-expressing DBF1 were more tolerant to osmotic stress than control plants. In yeast two-hybrid analyses, DBF1 interacted with DBF1-interactor protein 1 (DIP1), a protein containing a conserved R3H single-strand DNA-binding domain. Subcellular localization of DIP1 showed that the protein fusion DIP1-Red Flourescent Protein (RFP) was mainly localized in the cytoplasm. However, after co-transformation of DBF1-GFP and DIP1-RFP, both proteins co-localized in the nucleus. Interestingly, when the N-terminal DBF1-GFP was co-expressed with DIP1-RFP, both proteins co-localized predominantly in the cytoplasmic speckles observed for N-terminal DBF1-GFP fusion protein. These results clearly show in vivo interaction of DBF1 with DIP1 in the cell and that this interaction is necessary for the nuclear localization of DIP1 protein. Analysis of the regulatory effect of the DBF1 and DIP1 interaction on the maize rab17 promoter activity indicated that co-transfection of DBF1 with DIP1 enhances promoter activity in the absence of ABA treatment. We suggest that the regulated association of DBF1 and DIP1 may control the levels of target gene expression during stress conditions.