UCR

Institute for Integrative Genome Biology



Members


Thomas EulgemThomas Eulgem

Associate Professor of Plant Cell Biology

Mailing Address:

Botany and Plant Sciences
Genomics Building /3234A 
University of California  
Riverside, CA  92521

Phone: (951) 827-7740
Fax: (951) 827-4437
Email: thomas.eulgem@ucr.edu

Degree(s):

PhD 1999 Max-Planck-Institut fuer Zuechtungsforschung; Cologne, Germany

College/Division Affiliation:

College of Natural and Agricultural Sciences

Center/Inst Affiliation(s):

Center for Plant Cell Biology

Areas Of Expertise:

Plant Immune Biology; Defense Signaling; Gene Regulation; Transcriptomics; Functional Genomics

Research Summary:

Transcriptional Reprogramming During Plant Immune Responses
Plant immune responses are associated with extensive transcriptional reprogramming (Figure 1). However, molecular mechanisms that translate recognition of pathogens into defined transcriptional outputs are still poorly understood. The plant defense transcriptome is controlled by a complex signaling network consisting of two interconnected branches termed PTI (PAMP-triggered Immunity) and ETI (Effector-triggered Immunity). PTI is activated by receptor-mediated recognition of pathogen-associated molecular patterns (PAMPs), molecular signatures which are ubiquitously present in certain types of pathogens. Multiple pathogens transfer effector proteins into host cells that intercept PAMP triggered signals and thereby attenuate PTI. The remaining weak immune response, often called basal defense, is typically insufficient to prevent disease. Co-evolution of virulent pathogens with their hosts frequently resulted in the establishment of effector-triggered immunity (ETI). Of key importance for ETI are plant disease resistance (R) genesthat mediate specific recognition of pathogen effectors and trigger strong disease resistance by boosting basal defense reactions and activation of programmed death of plant cells at pathogen infection sites (hypersensitive reaction, HR).
Figure 1: Simplified model of the regulatory network controlling the plant defense transcriptome. Receptor-mediated recognition of PAMPs (pathogen-associated molecular patterns) triggers signaling processes resulting in massive transcriptional reprogramming and activation of defense reactions (PAMP-triggered immunity; PTI). PAMP-dependent signals target transcription factors (TFs) that control the defense transcriptome. Pathogen-derived effector (E) proteins intercept plant defense signals attenuating PTI. R proteins recognize effectors by direct or indirect mechanisms and boost defense reactions resulting in strong effector-triggered immunity (ETI).

CIRRENT PROJECTS

1) Identification of New Regulatory Elements Controlling Defense Gene Clusters

We are using microarray data to define clusters of genes coordinately responding to Peronospora recognition and, hence, likely to be controlled by common regulatory mechanisms (Fig. 2A). Conserved sequence motifs we identified in the promoters of such co-regulated genes may constitute cis-elements responsible for their coordinated activity. We proved that some of these motifs interact with nuclear-localized proteins and act as defense-associated cis-elements in reporter gene assays (Fig. 2B). Furthermore, we identified putative transcription factors (TFs) interacting with these elements by a proteomics-based approach.  Their functional connection to plant immune responses is currently being studied. In addition, we are dissecting the promoters of members of the LURP cluster, a set of tightly co-regulated defense genes, by 5’ deletion analyses using promoter reporter fusions. Short regions within these promoters that bear strong pathogen-response elements have been identified and are currently being further examined (Fig. 2C & D).  Moreover, we found that the transcription factor AtWRKY70 contributes to the regulation of LURP genes (Knoth et al., 2007). Defense promoter elements and their cognate TFs identified in this study will serve as starting points to design new strategies to improve disease resistance in crops.

 

Figure 2: Identification of new promoter elements mediating pathogen-responsive gene expression. A: Clusters of Arabidopsis genes co-regulated after infection with Peronospora. In resistant plants EURP transcripts exhibit an early/transient increase, while LURP transcripts accumulate at later timepoints. B: Analysis of two conserved promoter motifs. Motif 3 (M3) is enriched in the promoters of EURP genes, while motif 6 (M6) is enriched in the promoters of LURP genes. Upper left corner: Electrophoretic Mobility Shift Assays (EMSAs) with 3xM3 (top) and 3xM6 (bottom). 32P-labelled 3xM3 or 3xM6 probes were subject to electrophoresis with nuclear protein extracts from water, Emoy2 or SA-treated Arabidopsis Col-0 seedlings. Emoy2 is a Peronospora isolate recognized by the SA-dependent R gene RPP4.  “-/+”: absence or presence of unlabelled competitor probe. An M3-specific bandshift disappears after Emoy2 or SA treatment, while an M6-specific bandshift is induced by these treatments. Right-hand side: Agrobacterium-mediated transient expression assays in tobacco with trimers of M3 or M6 fused to the -46 CaMV35S core promoter and the GUS coding sequence. “UN”, untreated, “SA24h” SA-treated leaf. Consistent with the EMSA data, M3 acts as a repressor and M6 acts as an enhancer element (Alex Evrard unpublished) C: 5’ deletion analysis with a LURP promoter fused to GUS in stably transformed Arabidopsis lines.  In each well are seedlings of independent T2 lines. The promoter stretch between -333 and -65 harbors a strong pathogen-response element. A permutation of M6 located within this stretch exhibits constitutive and Peronospora (HpEmoy2)-inducible interactions with a nuclear Arabidopsis protein in EMSAs (D) (Colleen Knoth unpublished).

2) Identification of Synthetic Elicitors of Plant Defense Responses and their Targets by Chemical Genomics

EDM2(enhanced downy mildew 2) was identified in a mutant screen for loci required for RPP7 function and isolated by map-based cloning (Eulgem et al., 2007). EDM2 mutations are recessive, block an early defense signaling step, fully abolish RPP7-resistance, and reduce RPP7 transcript levels, strongly suggesting that EDM2 is a positive regulator of RPP7 expression. EDM2 is structurally unrelated to known components of the plant immune system, and bears typical features of transcriptional regulators, but does not belong to any previously characterized classes of TFs. Motifs conserved between EDM2 and EDM2-like proteins (ELPs) from Arabidopsis and rice define a novel plant specific protein family (Fig. 5A) . Our main goal is to understand the molecular roles of EDM2 in disease resistance and its causal connection to RPP7 function. We found that EDM2 is nuclear-localized and activates transcription. Furthermore, we identified by yeast two-hybrid screening multiple chromatin associated proteins and transcriptional regulators that specifically interact with EDM2, suggesting that EDM2 is a component of a transcriptional co-regulator complex. As we found by microarray and RT-PCR analyses, several other R-like genes (besides RPP7) are EDM2-dependent, indicating that EDM2 has additional roles in the plant immune system. Moreover, we identified a putative binding site of EDM2 complexes in the promoters of RPP7 and other EDM2 target genes. Mechanistic details of EDM2-dependent R gene activation, their connection to defense signaling and their significance in disease resistance are being examined. EDM2 appears to be the first TF implicated in controlling R gene activity. We propose that this novel regulator mediates disease resistance by fine-tuning the transcription of RPP7 and additional R genes (Fig. 5B).
Using chemical genomics resources of the CEPCEB we are screening for drug-like organic compounds that activate LURP-promoter/reporter fusions in transgenic Arabidopsis seedlings. The CEPCEB is equipped for high throughput chemical screens, providing libraries representing ~50,000 diverse organic compounds and robotic equipment to deliver them to screening plates.  Chemical genomics offers several advantages over classical genetics and can facilitate the discovery of biological pathway components that cannot be identified by conventional approaches. For example, due to functional redundancy the in vivo roles of many structurally-related proteins are difficult to study by loss-of-function mutations. Chemical genomics can lead to the identification of compounds that target multiple members of protein families and trigger clear phenotypes by simultaneously altering their function. In addition, loss-of-function mutations in genes required for plant development or other essential physiological processes may be lethal, while many gain-of-function mutations have detrimental pleiotropic effects. Bioactive chemicals, however, can be administered in a highly controlled manner (e.g. timing, concentration) minimizing undesired effects. Finally, such compounds can be applied to a wide variety of plant species to examine and manipulate homologous biological mechanisms.

Specific “inducers” of LURP genes are likely to interfere with defense signaling components and can be used instead of pathogens to activate plant immune responses. Our goal is to identify a suite of molecular probes that can be used to target defined nodes and branches of the defense network controlling transcriptional reprogramming. Such synthetic elicitors will be powerful tools for the fine dissection of defense mechanisms, as they are likely to trigger strong, uniform and synchronous defense responses in plants and cell cultures. Protein targets of these synthetic elicitors will be identified by genetic screens for mutants with altered sensitivity to the respective chemical as well as biochemical approaches. In addition, synthetic defense elicitors may allow the development of novel types of pesticides specifically tailored to fight plant diseases by enhancing the plant’s inherent defense capabilities. This project is partially performed in collaboration with the labs of Dr. Thomas Girke and Dr. Isgouhi Kaloshian (both UCR).

Figure 3: Screen for LURP inducers. A: Example of a 96-well-plate used for chemical screening with seedlings of a LURP-promoter::GUS line growing in liquid medium. B: Screening plate featuring a “hit” in well C6. C: Pathway targeted by screens for LURP inducers. LURP genes are controlled by a PAD4 and SA-dependent pathway that is utilized by the basal defense system and boosted by RPP4 or RPP7-dependent ETI. A central component of this pathway is the transcription factor WRKY70. We identified synthetic defense elicitors interfering with different hierarchical levels of this pathway (Colleen Knoth & Melinda Salus unpublished; Knoth et al., 2007).

3) Identification of Defense Promoter Elements by Enhancer Trapping

Enhancer trapping is based on random insertions of core-promoter-reporter fusions into genomes. Core promoters are insufficient to mediate gene expression. Only enhancer traps inserted in the vicinity of active enhancers can exhibit reporter gene activity (Fig. 4A). Screening ~11,000 Arabidopsis enhancer trap lines (Campisi et al., 1999, Plant Journal, 17: 699) we identified multiple individuals that exhibit highly localized GUS reporter gene activity in response to Peronospora infections (Fig. 4B). These lines are being analyzed to identify and characterize the respective enhancers. One goal of this project is to identify strictly pathogen-responsive enhancers and to use them to drive defense gene expression in crop plants to improve disease resistance. In addition, we will perform experiments to identify transcription factors interacting with such enhancers and to elucidate their roles in plant defense. 

Figure 4: Enhancer trapping to identify pathogen-responsive enhancers. A: Basic principle of enhancer trapping. Enhancers activate transcription of genes over large distances independent from their orientation. Enhancer trap constructs containing a core promoter fused to a reporter gene can only be transcribed when inserted into the genome close to an active endogenous enhancer. B, C, D: Examples of highly localized Peronospora-induced enhancer trap activity (arrows). These lines were infected with the virulent Peronospora isolate Noco2. GUS-reporter gene activity is detectable in plant tissue areas surrounding Peronospora hyphae (B) or limited to putative spore germination sites (C, D). (Mercedes Schroeder, unpublished).

4) Functional Characterization of EDM2, a Novel Transcriptional Regulator Mediating Disease Resistance in Arabidopsis thaliana

 

Figure 5: Structure and function of EDM2. A: Schematic representation of conserved domains shared by EDM2 and EDM2-like proteins (ELPs) from Arabidopsis and rice. Acidic regions: yellow; PHD finger-like motifs: red; G-protein gamma subunit -like domain: brown; bipartite nuclear localization signals: black; ELP domain: blue; Proline-rich region: cyan. B: Working model of EDM2 function. EDM2 is part of a complex that controls transcription of RPP7 and additional R-like genes (besides other genes). Proper levels and function of the respective R proteins depend on EDM2 (Tokuji Tsuchiya & Linda Wei, unpublished).

Related Press Releases:

UCR Today August 14, 2013: Researchers Discover Beneficial Jumping Gene

UCR June 28, 2006: Plant Cell Biologists, Sociologist Recognized for Supporting Undergraduate Research

 

Selected Publications:

List of publications from PubMed

Lab Personne

Tsuchiya, Tokuji
Postgraduate Researcher —  Identification of EDM2 Complex Components; Analysis of Molecular Functions and Regulation of EDM2
Wei, Yu-Hung (Linda)
Postgraduate Researcher —  Analysis of EDM2/R-promoter Interactions and Roles of EDM2 in RPP7-resistance.
Baig, Ayesha
Graduate Student Researcher —
Rodriguez-Salus, Melinda
Graduate Student Researcher —   Identification of Synthetic Defense Elicitors by Chemical Genomics
Schroeder, Mercedes
Associate —    Enhancer Trap Screens to Identify Pathogen-responsive Enhancers
Bektas, Yasemin
Graduate Student Researcher —

 


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University of California, Riverside
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Riverside, CA 92521
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Institute of Integrative Genome Biology
2150 Batchelor Hall

Tel: (951) 827-7177
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