Transcriptional Regulation of Plant Immune Response
TRANSCRIPTIONAL REGULATORY NETWORKS GOUVERNING THE PLANT IMMUNE RESPONSE
The plant innate immune system consists of two interconnected branches termed MTI (MAMP-Triggered Immunity) and ETI (Effector-Triggered Immunity) that initiate massive transcriptional reprogramming. MTI is provoked by microbe associated molecular patterns (MAMPs), molecular signatures ubiquitously decorating various microbes. Several microorganisms secrete effector proteins into host cells that intercept PAMP triggered defense signals and thereby attenuate PTI. Co-evolution of virulent pathogens with their hosts resulted in the establishment of ETI, a manifestation of gene-for-gene resistance. ETI is triggered by plant resistance (R) proteins that provoke highly efficient defense responses upon specific detection of pathogen effectors. The major differences between PTI and ETI appear more quantitative and/or temporal rather than qualitative, suggesting that most pathogens trigger a common/interconnected plant signaling network. The graded transcriptional responses associated with immunity clearly indicate the existence of a complex regulatory circuitry comprised of transcriptional activators and repressors fine-tuning the expression of defense genes. The nuclear end of the signaling cascades remains ill studied. Very little is known about the biochemical signals and how they are relayed and linked to nuclear components and specific transcription factors. Equally fragmentary are our insights into the intricate transcriptional machinery responsible for executing proper temporal and spatial control of immune response genes. Certain members of several transcription factor families are known to modulate the defense transcriptome. In particular, zinc-finger-type WRKY factors appear to play a broad and pivotal role in regulating host defenses. WRKY factors exert their functions predominantly by binding to a cis-regulatory DNA element termed, W box (TTGACT/C). WRKY genes are functionally connected forming a transcriptional network composed of positive and negative feedback loops and feed-forward modules.
1. WRKY transcription factors act as positive and negative regulators of plant immunity
a) WRKY18 and WRKY40 act as negative regulators of plant immunity
WRKY18 and WRKY40 act as negative regulators of MTI in a partly redundant manner. Loss-of-WRKY18/40 functions render plants highly resistant to the powdery mildew fungus Golovinomyces orontii.
b) WRKY33 transcription factor acts as a positive regulator of plant immunity
The role of WRKY33 in mediating resistant towards necrotrophic pathogens in being investigated. Loss-of-WRKY33 function leads to susceptibility of Arabidopsis plants towards the fungus Botrytis cinerea.
In both cases detailed comparative global expression arrays have been performed to define gene networks controlled by these WRKY factors. Transgenic lines expressing functional epitope-tagged versions of the respective WRKY transcription factors have been generated. Chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) are currently being used to define direct in vivo target genes of these two WRKY factors on a genome-wide level. Moreover, the interplay between different defense signalling pathways and how they are influenced during the infection process are being studied using molecular, biochemical and genetic approaches. In addition, identification of nuclear components interacting with these WRKY proteins is being pursued.
2. Search for additional transcription factors modulating defense gene expression
Previously, we have functionally identified several cis-acting DNA elements originating from numerous MAMP-responsive gene promoters (Rushton et al., 2002). In transgenic Arabidopsis plants, these elements are both necessary and sufficient to confer pathogen/MAMP-responsiveness on a reporter gene.
Novel pathogen-responsive cis-regulatory DNA elements.
This project aims at identifying novel cis-acting regulatory DNA elements responsive to abiotic and biotic stimuli based on their in vivo activity. We have plant-adapted the use of the RNA Pol II (HaploChIP) procedure followed by deep second-generation sequencing (ChIP-Seq). The methodology involves the use of a random-sequence oligonucleotide library as an unbiased source of DNA elements, transformation of such libraries (as reporter constructs) into plant protoplasts, two to three rounds of enrichments of the actively transcribed DNA constructs by chromatin immunoprecipitation (ChIP) employing a specific RNA polymerase II antibody, and subsequent deep sequencing (Solexa, Illumina) of the enriched DNA pools. Bioinformatic analyses are helping to identify candidate elements, which subsequently are being rigorously tested with respect to functionality.
Future research plans
It is obvious that WRKY factors are just one of several key transcriptional regulators implicated in plant immuntiy. Moreover, dynamic stimulus-dependent changes in chromatin structure and organization also contribute to proper transcriptional outputs. Thus, in the future we would like to delineate the majority of early MAMP-dependent changes across the genome with respect to DNA-binding-site occupancies by various transcription factors, determine their gene recruitment kinetics to define potential cooperative interactions, and monitor for chromatin modifications. This will allow us to establish a hierarchical network of transcriptional regulators and to define the regulatory program underlying early stages of PTI. Such a broad global mapping of protein-DNA interactions is becoming feasible thanks to the advent of second-generation sequencing technology in combination with classical molecular methods By quickly adapting such newly developed methodologies we hope to extensively broaden but also refine our current knowledge of the plant defense transcriptome.