Principal investigator:

El Kasmi, Farid, Dr.,
Universität Tübingen

Zentrum für Molekularbiologie der Pflanzen (ZMBP)

Pflanzenphysiologie

Auf der Morgenstelle 32

72076 Tübingen

Phone:

E-mail: elkasmispam prevention@email.unc.edu

Summary:

Plants have evolved nucleotide binding leucine-rich repeat receptor proteins (NLRs) to detect the presence/activity of pathogen-derived effector proteins, thus ensuring activation of effector-triggered immunity (ETI). Canonical NLR activity depends on a functional P-loop motif important for nucleotide binding. Recent work demonstrates that a few NLRs function in genetically linked pairs to initiate immunity, i.e. the NLR pairs RGA4/RGA5 in rice or RPS4/RRS1 in Arabidopsis, whereas others require a certain NLR subclade, termed “helper”-NLRs (hNLRs) for their activity during an immune response or in autoimmunity. The Arabidopsis genome encodes 5 full-length hNLR genes grouped in two families, the ADRs (ADR1, ADR1-L1, and ADR1-L2) and NRGs (NRG1.1, NRG1.2). hNLR function, at least for ADR1-L2, was demonstrated to be P-loop independent (non-canonically). ADR1-L2, however, also has a P-loop dependent canonical function in cell death regulation. Although an important role of the ADRs and in particular ADR1-L2 in (auto-) immunity is described, we still lack a clear mechanistic understanding of hNLR function and activation (canonical vs. non-canonical). What the molecular and cellular components associated with hNLR activity are and whether they have specific or redundant functions in immunity needs to be investigated so that we better understand the regulation of NLR-mediated immunity, and thus how plants fight microbial infections. This proposal aims to determine the unique and overlapping functions of hNLRs in immunity, by studying combinatorial mutants of the 5 hNLR genes, created using the CRISPR/Cas9 gene editing technique, and their requirement in responses to a broad range of biotic stresses. We want to elucidate whether hNLRs self-associate or form complexes with other NLRs or with immune-signaling components and whether these interactions are required for their function. We propose to dissect the molecular mechanism of hNLR activation, by functionally characterizing mutations in the ADR1-L2 protein that we have identified in a suppressor screen, in which the constitutive pathogen response-like phenotype of an auto-activated hNLR (ADR1-L2^D484V-HA) is reduced or eliminated. We have isolated 209 candidate suppressor mutants in this screen, likely including intragenic suppressor mutations, which will provide us with a toolbox to test canonical and non-canonical functions, and to test for interaction capability with ADR1-L2 partners/regulators (e.g. EDS1). Extragenic suppressors will aid in the construction of a genetic network required for NLR (auto-) activity and candidate genes will be tested for their requirement for canonical or non-canonical functions of ADR1-L2 and other hNLRs. The proposed project advances our understanding of the wide modes of NLR function, by illuminating core principles of hNLR activity and regulation.