Location: Plant Science Research
Project Number: 6070-21220-017-009-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2023
End Date: Sep 30, 2027
Plant disease resistance proteins of the nucleotide binding leucine-rich repeat (NLR) type are activated and induce a strong defense response known as effector-triggered immunity or ETI, upon recognition of specific pathogen-derived effector proteins. The effectiveness of this system depends on its inactivity when the cognate pathogen is not present, rapid induction when a pathogen is recognized and a rapid suppression after induction. The ubiquitin-proteasome pathway, mediated by the sequential actions of E1 (ubiquitin-activating), E2 (ubiquitin-conjugating) and E3 (ubiquitin ligase) enzymes is a major protein modification and degradation pathway found in all eukaryotes. Our preliminary data indicates that maize E3-ligase ZmCER9 mediates degradation of the Rp1-D disease resistance protein specifically after its activation. We have further evidence that ZmCER9 acts in a similar way to degrade other plant resistance proteins once they are activated. This appears to be a previously undescribed mechanism that mediates the deactivation of the ETI defense response after activation. Based on its homology, ZmCER9 is predicted to be a component of the endoplasmic reticulum associated degradation (ERAD), a fundamental eukaryotic quality-control system that degrades incorrectly-folded proteins. In yeast, where it has been best characterized, the ERAD system consists of two membrane protein complexes based around the two E3 ligases Hrd1 and Doa10, the yeast homolog of CER9. ERAD in plants has been poorly characterized and there are no reported substrates of the CER9 complex, meaning that Rp1-D may represent the first known substrate of this branch of the ERAD pathway in plants. We hypothesize that ERAD-Mediated Degradation of Activated NLRs (EMDAN) is a general mechanism for the deactivation of ETI in plants. We will use a range of molecular, genomics and cell biology techniques to: 1.) Determine the importance of EMDAN and related pathways in controlling ETI in plants. 2.) Characterize the role of CER9 in ERAD and identify proteins involved in the plant Doa-10 complex.
1. Elucidate how ubiquitination-related candidate genes control the ETI response mediated by Rp1-D. 2. Determine the conserved molecular mechanisms by which CER9 controls ETI. 3. Characterize ZmCER9 association with the ER and other components of the plant Doa-10 complex We will primarily use two systems for this work. Maize, our most economically valuable crop and a monocot model system, and Arabidopsis, the pre-eminent plant model system. We will use a number of well-established techniques for functional characterization on CER9, NLR proteins and other components of the ubiquitin mediated pathway in plants. These include stable and transient transgenic expression in maize, Arabidopsis and Nicotiana benthamiana, western analysis and assessment of protein-protein interactions by co-immunoprecipitation and using histological techniques. We hope also to use proteomics techniques to identify other components of protein complexes required for modulation of plant defense.