Molecular and Functional Analyses of a Maize Autoactive NB-LRR Protein Identify Precise Structural Requirements for Activity

Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR) proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR). Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC), a nucleotide binding (NB-ARC) and a leucine rich repeat (LRR) domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.
Author Summary The plant hypersensitive defense response (HR) is a rapid, localized cell death, usually occurring upon the recognition of specific pathogen-encoded molecules and consequent activation of nucleotide binding-leucine rich repeat (NLR) proteins. Rp1-D21, a naturally-occurring mutant caused by the recombination of two NLR genes, confers a ‘lesion mimic’, HR-like phenotype in the absence of pathogen infection and provides a powerful tool to investigate the molecular mechanisms of NLR regulation. Here we report the results of a genetic screen in maize that identified novel mutations abrogating Rp1-D21-induced HR. To characterize the function of Rp1-D21, we transiently expressed Rp1-D21 and various derivatives in Nicotiana benthamiana to observe the resulting levels of HR. We furthermore examined the protein-protein interactions between and within different Rp1-D21 derivatives. We report novel insights into the precise structural requirements for NLR function and determine the function of a previously undefined motif. These insights enable a better understanding of how NLRs regulate the switch between the resting and the active states.