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

Abstract

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.

Maize Homologs of CCoAOMT and HCT, Two Key Enzymes in Lignin Biosynthesis, Form Complexes with the NLR Rp1 Protein to Modulate the Defense Response

“…We have shown that CC D21 , but not the NB-ARC or LRR domain, conferred autoactive HR when fused with EGFP (Wang et al, 2015d). We have also shown that HCT1806/4918 interact with CC D21 to suppress CC D21 :EGFP-induced HR (Wang et al, 2015c).…”

“…However, the effector that elicits the response associated with Rp1-D has not been identified. We previously derived models to elucidate the self-activation/repression of Rp1 proteins (Wang et al, 2015c(Wang et al, , 2015d. In a suppressed state, Rp1 proteins are held in an inactive state through the intramolecular interaction of different domains and the interaction between different Rp1 proteins (Wang et al, 2015d).…”

“…This system has been widely used for the functional analysis of HR mediated by NLRs from both monocots and dicots (Leister et al, 2005;van Ooijen et al, 2008;Slootweg et al, 2010;Tameling et al, 2010;Bai et al, 2012;Qi et al, 2012). We have recently shown that this is an appropriate system for the functional analysis of HR modulated by Rp1-D21 and its regulators (Wang and Balint-Kurti, 2015;Wang et al, 2015cWang et al, , 2015d. When Rp1-D21:HA (Rp1-D21 fused with a 33 hemagglutinin tag at the C terminus) was transiently coexpressed with GUS:EGFP (a GUS gene fused with enhanced GFP at the C terminus), an obvious HR phenotype was observed at 3 d post infiltration, similar to our previous observation (Wang and Balint-Kurti, 2015;Wang et al, 2015cWang et al, , 2015d.…”

“…We have recently shown that this is an appropriate system for the functional analysis of HR modulated by Rp1-D21 and its regulators (Wang and Balint-Kurti, 2015;Wang et al, 2015cWang et al, , 2015d. When Rp1-D21:HA (Rp1-D21 fused with a 33 hemagglutinin tag at the C terminus) was transiently coexpressed with GUS:EGFP (a GUS gene fused with enhanced GFP at the C terminus), an obvious HR phenotype was observed at 3 d post infiltration, similar to our previous observation (Wang and Balint-Kurti, 2015;Wang et al, 2015cWang et al, , 2015d. Also as previously observed, no HR was observed when HCT1806:EGFP and Rp1-D21:HA were transiently coexpressed ( Fig.…”

“…We have found that, when fused with HA tag, Rp1-dp2 has higher expression level than Rp1-D21 and Rp1-D (Wang et al, 2015d). Thus, we chose Rp1-dp2:HA to test the interaction between CCoAOMT and Rp1 proteins.…”