Induced Genome-Wide Binding of Three Arabidopsis WRKY Transcription Factors during Early MAMP-Triggered Immunity.

During microbial-associated molecular pattern-triggered immunity (MTI), molecules derived from microbes are perceived by cell surface receptors and upon signaling to the nucleus initiate a massive transcriptional reprogramming critical to mount an appropriate host defense response. WRKY transcription factors play an important role in regulating these transcriptional processes. Here, we determined on a genome-wide scale the flg22-induced in vivo DNA binding dynamics of three of the most prominent WRKY factors, WRKY18, WRKY40, and WRKY33. The three WRKY factors each bound to more than 1000 gene loci predominantly at W-box elements, the known WRKY binding motif. Binding occurred mainly in the 500-bp promoter regions of these genes. Many of the targeted genes are involved in signal perception and transduction not only during MTI but also upon damage-associated molecular pattern-triggered immunity, providing a mechanistic link between these functionally interconnected basal defense pathways. Among the additional targets were genes involved in the production of indolic secondary metabolites and in modulating distinct plant hormone pathways. Importantly, among the targeted genes were numerous transcription factors, encoding predominantly ethylene response factors, active during early MTI, and WRKY factors, supporting the previously hypothesized existence of a WRKY subregulatory network. Transcriptional analysis revealed that WRKY18 and WRKY40 function redundantly as negative regulators of flg22-induced genes often to prevent exaggerated defense responses.

Identification of functional cis-regulatory elements by sequential enrichment from a randomized synthetic DNA library.

BACKGROUND: The identification of endogenous cis-regulatory DNA elements (CREs) responsive to endogenous and environmental cues is important for studying gene regulation and for biotechnological applications but is labor and time intensive. Alternatively, by taking a synthetic biology approach small specific DNA binding sites tailored to the needs of the scientist can be generated and rapidly identified. RESULTS: Here we report a novel approach to identify stimulus-responsive synthetic CREs (SynCREs) from an unbiased random synthetic element (SynE) library. Functional SynCREs were isolated by screening the SynE libray for elements mediating transcriptional activity in plant protoplasts. Responsive elements were chromatin immunoprecipitated by targeting the active Ser-5 phosphorylated RNA polymerase II CTD (Pol II ChIP). Using sequential enrichment, deep sequencing and a bioinformatics pipeline, candidate responsive SynCREs were identified within a pool of constitutively active DNA elements and further validated. These included bonafide biotic/abiotic stress-responsive motifs along with novel SynCREs. We tested several SynCREs in Arabidopsis and confirmed their response to biotic stimuli. CONCLUSIONS: Successful isolation of synthetic stress-responsive elements from our screen illustrates the power of the described methodology. This approach can be applied to any transfectable eukaryotic system since it exploits a universal feature of the eukaryotic Pol II.

Chromatin immunoprecipitation to identify global targets of WRKY transcription factor family members involved in plant immunity.

The completion of the alfalfa, Arabidopsis, papaya, poplar, and rice genome sequences along with ongoing sequencing projects of various crop species, offers an excellent opportunity to study gene expression at the whole genome level and to unravel the complexity of gene networks underlying the reprogramming of plant defense toward pathogen challenge. Gene expression in eukaryotic cells is mainly controlled by regulatory elements that recruit transcription factors (TFs) to modulate transcriptional outputs. Therefore, methods allowing the identification of all cognate TF binding sites (TFBS) within the regulatory regions of target genes on a genome-wide basis are the next obvious step to elucidate the plant defense transcriptome. Chromatin immunoprecipitation (ChIP) is one such powerful technique for analyzing functional cis-regulatory DNA elements. The ChIP assay allows the identification of specific regulatory DNA regions associated with trans-acting regulatory factors in vivo. ChIP assays can provide spatial and temporal snapshots of the regulatory components involved in reprogramming host gene expression upon pathogen ingress. Moreover, the use of ChIP-enriched DNA for hybridization to tiling microarrays (ChIP-chip) or for direct sequencing (ChIP-Seq) by means of massively parallel sequencing technology has expanded this methodology to address global changes in gene expression.

Transcriptional reprogramming regulated by WRKY18 and WRKY40 facilitates powdery mildew infection of Arabidopsis.

The two closely related Arabidopsis transcription factors, WRKY18 and WRKY40, play a major and partly redundant role in PAMP-triggered basal defense. We monitored the transcriptional reprogramming induced by the powdery mildew fungus, Golovinomyces orontii, during early stages of infection with respect to the role of WRKY18/40. Expression of >1300 Arabidopsis genes was differentially altered already 8 hours post infection (hpi), indicating rapid pre-penetration signaling between the pathogen and the host. We found that WRKY18/40 negatively affects pre-invasion host defenses and deduced a subset of genes that appear to be under WRKY18/40 control. A mutant lacking the WRKY18/40 repressors executes pathogen-dependent but exaggerated expression of some defense genes leading, for example, to strongly elevated levels of camalexin. This implies that WRKY18/40 act in a feedback repression system controlling basal defense. Moreover, using chromatin immunoprecipitation (ChIP), direct in vivo interactions of WRKY40 to promoter regions containing W box elements of the regulatory gene EDS1, the AP2-type transcription factor gene RRTF1 and to JAZ8, a member of the JA-signaling repressor gene family were demonstrated. Our data support a model in which WRKY18/40 negatively modulate the expression of positive regulators of defense such as CYP71A13, EDS1 and PAD4, but positively modulate the expression of some key JA-signaling genes by partly suppressing the expression of JAZ repressors.

ROSINA (RSI) is part of a CACTA transposable element, TamRSI, and links flower development to transposon activity.

ROSINA (RSI) was isolated as a DNA binding factor able to bind to the CArG-box present in the promoter of the MADS-box gene DEFICIENS of Antirrhinum majus. The mosaic nature of RSI and its multi-copy presence in the A. majus genome indicated that RSI could be a part of a mobile genetic element. Here we show that RSI is a part of a CACTA transposable element system of A. majus, named TamRSI, which has evolved and is still evolving within the terminal inverted repeats (TIRs) of this CACTA transposon. Interestingly, RSI is always found in opposite orientation with respect to the transcription of a second gene present within the CACTA transposon, which encodes a putative TRANSPOSASE (TNP). This structural configuration has not yet been described for any member of the CACTA transposons superfamily. Internal deletion derivatives of the TamRSI produce aberrant RSI transcripts (RSI-ATs) that carry parts of the RSI RNA fused to parts of the TNP RNA. In addition, an intriguing seed phenotype shown by RNAi transgenic lines generated to silence RSI, relate TamRSI to epigenetic mechanisms and associate the control of flower development to transposon activity.

ROSINA (RSI), a novel protein with DNA-binding capacity, acts during floral organ development in Antirrhinum majus.

Petal and stamen identity of the Antirrhinum majus flower is under the genetic control of the floral homeotic gene DEFICIENS (DEF). To isolate factors involved in the regulation of DEF gene activity, a promoter segment of this B-function gene, containing cis-acting regulatory elements, was used to identify the novel trans-acting factor ROSINA (RSI). RSI does not show an extended similarity with any gene product present in the database. Rather RSI constitutes a protein that contains domains similar to known proteins from organisms of different phyla. The capacity of RSI to bind a sequence element of the DEF promoter, its spatial and temporal expression pattern together with the phenotype of RSI-RNAi interference plants as well as RSI over-expression in Arabidopsis thaliana suggest that RSI is a putative regulator of DEF gene activity in A. majus.

GRAMINIFOLIA promotes growth and polarity of Antirrhinum leaves.

The leaves of higher plants develop distinct cell types along their adaxial-abaxial (dorsal-ventral) axes. Interaction between leaf primordium cells with adaxial and abaxial identities is necessary for lateral growth of the developing leaf blade. We show that the growth and asymmetry of leaves in Antirrhinum majus involves the related YABBY transcription factors GRAMINIFOLIA (GRAM) and PROLONGATA (PROL). GRAM is expressed in abaxial margins of organ primordia where it promotes lateral growth and abaxial cell fate. GRAM, however, is not needed for abaxial fate in the absence of adaxial cell specification, suggesting that it promotes abaxial fate by excluding adaxial identity. Although GRAM expression is abaxially restricted, it functions redundantly with its abaxially expressed paralogue, PROL, and with the ubiquitously expressed PHANTASTICA gene to promote adaxial identity via intercellular signalling. This non cell-autonomous behaviour is consistent with the ability of GRAM in only the abaxial most cell layer to direct normal development of more adaxial cells. The contrasting roles of GRAM in promoting and inhibiting adaxial identity might serve to reinforce and maintain the distinction between adaxial and abaxial domains in the growing leaf primordium.