Extreme resistance: The GLK–Rx1 alliance

Abstract

Plants immune surveillance systems depend on nucleotide-binding leucine-rich repeat receptors (NLRs). A subset of NLRs are nuclear-localized, including Rx1, which confers an extreme immunity against potato virus X (PVX). As with many NLRs, the downstream signaling partners of Rx1 are unknown. Townsend et al. identify a Golden-like transcription factor that interacts with Rx1 and mediates antiviral immunity, providing the first insights into the specificity factors that enable the nonspecific DNA-binding Rx1 to confer extreme resistance to PVX.

Introduction

Plants use immune receptors as surveillance systems to perceive pathogenic virulence factors known as effectors and activate a complex defense–signaling web (1, 2). Nucleotide-binding leucine–rich repeat receptors (NLRs)² are immune receptors that detect strain-specific effectors or effector-induced modifications to host target proteins to activate effector-triggered immunity (ETI). ETI activates defense networks causing a reactive oxygen species burst, transcriptional reprogramming, accumulation of anti-microbial compounds, and, in many cases, localized cell death (also known as the hypersensitive response).

NLRs evolve rapidly, adapting to the dynamic pathogen–effector repertoire. As a result, NLR gene families are large and diverse (3). NLRs are considered “molecular immunity switches” (2, 4) because they are maintained in a signaling-competent but auto-inhibited state until perception of their cognate effectors (Fig. 1A). At this time, the NLR binds ATP, conformational changes ensue, and the activated NLR initiates ETI (Fig. 1B). One significant gap in the field is an understanding of the earliest events in ETI activation. With a few exceptions, the identity of the signaling components that act immediately downstream of NLRs have remained elusive. Because ETI promotes transcriptional reprogramming and some NLRs are located within the nucleus, it seems possible that nuclear-localized NLRs interact with transcription factors or transcriptional regulators (5).

Figure 1.

The dynamics of Rx1 interactions and activation of extreme resistance. Rx1 and NbGlk1 binding to DNA segments with and without the GLK cis-acting motif are shown. A, the autoinhibited state (4). RanGAP2 (green) retains Rx1 in the cytoplasm and increases Rx1 protein stability (8). Upon entry into the nucleus, NbGlk (orange triangle) binds the Rx1 CC domain (blue oval). Townsend et al. (10) show that NbGlk1 recruits Rx1 binding to nuclear DNA, and Rx1 prevents NbGLK1 from binding GLK cis-acting elements (red rectangle). B, the activated state. Upon PVX infection, the PVX CP106 binds the LLR domain (striped arc), initiating a conformational change to Rx1's activated form. Within the nucleus, NbGlk1 recruits Rx1 to nuclear DNA. In the Rx1-activated state, NbGlk1 is able to bind to GLK binding sites and activate transcription of genes inducing extreme resistance. As Rx1 overexpression in the presence of CP105 can induce hypersensitive response and NbGlk does not, additional Rx1-interactors are proposed (yellow shapes). PM, plasma membrane; NB, nucleotide binding site.

Whereas many NLRs trigger a hypersensitive response during effector-triggered immunity, the potato NLR Rx1 does not. In response to infection by potato virus X (PVX), Rx1 mediates extreme resistance, a type of immune response in which PVX is perceived, viral replication is rapidly terminated and the plant does not exhibit symptoms like cell death and lesion formation (6). When potato Rx1 is expressed in Nicotiana benthamiana (a relative of tobacco and a model plant for studying plant–pathogen interactions), Rx1's extreme resistance to PVX is recapitulated, indicating that Rx1's downstream signaling components are conserved in these solanaceous plants (6). However, when Rx1 is overexpressed in N. benthamiana, a strong hypersensitive response occurs, indicating that Rx1 levels and activity in planta must be highly controlled.

Rx1 is a coiled-coil (CC) NLR and its cognate effector, the PVX coat protein (CP106), is perceived by the leucine-rich repeat domain (Fig. 1B). Rx1 activation by CP106 occurs in the cytosol, and Rx1 translocation to the nucleus is controlled by the binding of the GTPase RanGAP2 to the Rx1 CC domain (Fig. 1B) (6–8). In planta, CP106-activated Rx1 binds genomic DNA and in vitro Rx1 binds, bends, and melts DNA (9), suggesting a mechanism for Rx1 to report pathogen detection. However, Rx1 binds DNA nonspecifically; thus, Rx1's mechanism for activating extreme resistance must be conferred by other immune components (5). A new study by Townsend et al. (10) identifies a novel Rx1-binding partner from N. benthamiana as a Golden2-like transcription factor (NbGlk1) (Fig. 1). The discovery of NbGlk1 is exciting as it is the first factor to be discovered that confers specificity to a general DNA-binding and -distorting NLR, providing our first insights into the molecular mechanisms that enable Rx1's extreme resistance to PVX.

Initially identified in a yeast two–hybrid screen, NbGlk1 interactions with Rx1's CC domain were confirmed in planta by size exclusion chromatography and co-immunoprecipitation experiments. NbGlk1 preferentially bound GLK–like DNA motifs. NbGlk1 binding constants for two GLK oligomers were determined by fluorescence anisotropy in the presence and absence of Rx1; the receptor in these experiments was likely in its autoinhibited form, not having been exposed to the viral effector CP106. Surprisingly, when NbGlk binding was assessed in the presence of inactive Rx1, NbGlk1 binding affinities declined >2-fold (Fig. 1). The authors proposed that when the Rx1 CC domain binds NbGlk1, it partially obstructs the NbGlk1 surface that binds the GLK DNA motifs. Alternatively, the Rx1-NbGlk association may alter NbGlk1 conformation making the NbGlk1 DNA-binding surface less accessible. In either case, inactive Rx1 interferes with, but does not abolish, NbGlk1's ability to bind DNA (Fig. 1A). This interaction may keep Rx1's immunity response in a quiescent state until perception of PVX's CP106.

To assess Rx1 and NbGlk interactions with DNA in planta, FRET-FLIM was used to measure the binding of Rx1-GFP and NbGlk1-GFP to genomic DNA. Consistent with previous studies (9), when Rx1-GFP and CP106 were co-infiltrated into N. benthamiana leaves, Rx1 was activated and bound DNA. Unexpectedly, when NbGlk1 and Rx1-GFP were co-infiltrated into N. benthamiana leaves, the CP106 requirement for Rx1-GFP binding DNA was abolished, suggesting that, when overexpressed, NbGlk recruits inactive Rx1-GFP to genomic DNA (Fig. 1A). The reciprocal experiments provided additional insights. NbGlk1-GFP bound DNA only in the presence of Rx1 and CP106, suggesting that activated Rx1 is critical for NbGlk binding (Fig. 1B).

As GLKs are associated with defense signaling in Arabidopsis, Townsend et al. (10) speculated that NbGlk1 should have a role in the two Rx1-mediated immune responses: extreme resistance to PVX and a CP106–promoted cell death response that only occurs when Rx1 is expressed at very high levels (6). Therefore, transient overexpression of Rx1, NbGlk1, and a PVX (expressing a pCP106:GFP reporter gene) in N. benthamiana leaves was used to test NbGlk1's role in immunity. Consistent with previous studies, when Rx1 was expressed at high levels, extensive cell death was observed within the infiltration area. NbGlk1 did not ameliorate or accentuate this hypersensitive response. To assess the role of NbGlk1 in extreme resistance (7), in planta Rx1 levels were controlled by expressing an Rx1 transcript that was translated inefficiently. This prevented the cell death response in the N. benthamiana transient assay and allowed NbGlk's role in extreme resistance to be revealed. Rx1–mediated extreme immunity reported by suppression of PVX–directed GFP expression was observed when Rx1 and PVX were co-infiltrated into leaves. Surprisingly, overexpressed NbGlk1 conferred extreme immunity to PVX in the absence of Rx1. These data indicated that elevated levels of NbGlk1 in this transient assay bypass the requirement for activated Rx1 to trigger extreme resistance to PVX, although it is unlikely that NbGlk levels would reach these levels during PVX infection. Collectively, these data indicate NbGlk is an immune modulator. However, NbGlk overexpression does not phenocopy Rx1 overexpression as a hypersensitive response was not observed within plants overexpressing NbGlk. Therefore, additional immune components are likely important in executing Rx1 functions in vivo when nuclear levels of Rx1 are elevated (Fig. 1B).

Collectively, the data presented in Townsend et al. (10) demonstrate that NbGlk1 is a key regulator and specificity determinant of Rx1-mediated extreme immunity to PVX. In planta, NbGlk and Rx1 have a co-dependence for their association with genomic DNA: NbGlk recruits Rx1, whereas CP106-activated Rx1 promotes NbGlk binding (Fig. 1). In contrast, the in vitro DNA-binding assay suggests that inactive Rx1 antagonizes NbGlk binding to GLK motifs. These data are consistent with the proposal that non-activated Rx1 is in a distinct conformational state (Fig. 1A). Townsend et al. (10) have laid the groundwork for further elucidation of the earliest events in Rx1–mediated extreme resistance. As Rx1 is expected to influence the sites to which NbGlk binds in vivo, it will be of interest to determine NbGlk–binding motifs in the presence and absence of Rx1 using ChIP-seq or DAP-seq. These Rx1-enhanced NbGlk-binding motifs should be correlated with the suite of genes rapidly induced in the cells that participate in Rx1's extreme immunity response to understand the ramifications of the NbGlk–Rx1 alliance.