Wladimir I. L. Tameling

Mutations in the NB-ARC Domain of I-2 That Impair ATP Hydrolysis Cause Autoactivation

Resistance (R) proteins in plants confer specificity to the innate immune system. Most R proteins have a centrally located NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain. For two tomato (Lycopersicon esculentum) R proteins, I-2 and Mi-1, we have previously shown that this domain acts as an ATPase module that can hydrolyze ATP in vitro. To investigate the role of nucleotide binding and hydrolysis for the function of I-2 in planta, specific mutations were introduced in conserved motifs of the NB-ARC domain. Two mutations resulted in autoactivating proteins that induce a pathogen-independent hypersensitive response upon expression in planta. These mutant forms of I-2 were found to be impaired in ATP hydrolysis, but not in ATP binding, suggesting that the ATP- rather than the ADP-bound state of I-2 is the active form that triggers defense signaling. In addition, upon ADP binding, the protein displayed an increased affinity for ADP suggestive of a change of conformation. Based on these data, we propose that the NB-ARC domain of I-2, and likely of related R proteins, functions as a molecular switch whose state (on/off) depends on the nucleotide bound (ATP/ADP).

To Nibble at Plant Resistance Proteins

To intercept invading microbes that threaten growth and reproduction, plants evolved a sophisticated innate immune system. Recognition of specialized pathogens is mediated by resistance proteins that function as molecular switches. Pathogen perception by these multidomain proteins seems to trigger a series of conformational changes dependent on nucleotide exchange. The activated resistance protein switches on host defenses, often culminating in the death of infected cells. Given their control over life and death, activity of these proteins requires tight regulation that involves intramolecular interactions between the various domains.

Nucleocytoplasmic distribution is required for activation of resistance by the potato NB-LRR receptor Rx1 and is balanced by its functional domains.

The resistance protein Rx1 exists in cytoplasmic and nuclear pools in the cell. Both subcellular pools are necessary for full PVX resistance, and the cytoplasmic compartment could be linked to PVX recognition. A functional phosphate binding loop and the presence of SGT1 are required to sustain the nuclear pool. Functional domains of Rx1 were shown to have opposing roles in Rx1 localization. The Rx1 protein, as many resistance proteins of the nucleotide binding–leucine-rich repeat (NB-LRR) class, is predicted to be cytoplasmic because it lacks discernable nuclear targeting signals. Here, we demonstrate that Rx1, which confers extreme resistance to Potato virus X, is located both in the nucleus and cytoplasm. Manipulating the nucleocytoplasmic distribution of Rx1 or its elicitor revealed that Rx1 is activated in the cytoplasm and cannot be activated in the nucleus. The coiled coil (CC) domain was found to be required for accumulation of Rx1 in the nucleus, whereas the LRR domain promoted the localization in the cytoplasm. Analyses of structural subdomains of the CC domain revealed no autonomous signals responsible for active nuclear import. Fluorescence recovery after photobleaching and nuclear fractionation indicated that the CC domain binds transiently to large complexes in the nucleus. Disruption of the Rx1 resistance function and protein conformation by mutating the ATP binding phosphate binding loop in the NB domain, or by silencing the cochaperone SGT1, impaired the accumulation of Rx1 protein in the nucleus, while Rx1 versions lacking the LRR domain were not affected in this respect. Our results support a model in which interdomain interactions and folding states determine the nucleocytoplasmic distribution of Rx1.

RanGAP2 Mediates Nucleocytoplasmic Partitioning of the NB-LRR Immune Receptor Rx in the Solanaceae, Thereby Dictating Rx Function

The interaction of the immune receptor Rx with RanGAP2 is required for stability and balanced partitioning of Rx between the cytoplasm and nucleus, which is essential for activation of the immune response against Potato virus X. The potato (Solanum tuberosum) nucleotide binding–leucine-rich repeat immune receptor Rx confers resistance to Potato virus X (PVX) and requires Ran GTPase-activating protein 2 (RanGAP2) for effective immune signaling. Although Rx does not contain a discernible nuclear localization signal, the protein localizes to both the cytoplasm and nucleus in Nicotiana benthamiana. Transient coexpression of Rx and cytoplasmically localized RanGAP2 sequesters Rx in the cytoplasm. This relocation of the immune receptor appeared to be mediated by the physical interaction between Rx and RanGAP2 and was independent of the concomitant increased GAP activity. Coexpression with RanGAP2 also potentiates Rx-mediated immune signaling, leading to a hypersensitive response (HR) and enhanced resistance to PVX. Besides sequestration, RanGAP2 also stabilizes Rx, a process that likely contributes to enhanced defense signaling. Strikingly, coexpression of Rx with the Rx-interacting WPP domain of RanGAP2 fused to a nuclear localization signal leads to hyperaccumulation of both the WPP domain and Rx in the nucleus. As a consequence, both Rx-mediated resistance to PVX and the HR induced by auto-active Rx mutants are significantly suppressed. These data show that a balanced nucleocytoplasmic partitioning of Rx is required for proper regulation of defense signaling. Furthermore, our data indicate that RanGAP2 regulates this partitioning by serving as a cytoplasmic retention factor for Rx.

Resistance proteins: scouts of the plant innate immune system

Recognition of non-self in plants is mediated by specialised receptors that upon pathogen perception trigger induction of host defence responses. Primary, or basal, defence is mainly triggered by trans-membrane receptors that recognise conserved molecules released by a variety of (unrelated) microbes. Pathogens can overcome these basal defences by the secretion of specific effectors. Subsequent recognition of these effectors by specialised receptors (called resistance proteins) triggers induction of a second layer of plant defence responses. These responses are qualitatively similar to primary defence responses; however, they are generally faster and stronger. Here we give an overview of the predicted (domain) structures of resistance proteins and their proposed mode of action as molecular switches of plant innate immunity. We also highlight recent advances revealing that some of these proteins act in the plant nucleus as transcriptional co-regulators and that crosstalk can occur between members of different resistance protein families.

Defense activation triggers differential expression of phospholipase-C (PLC) genes and elevated temperature induces phosphatidic acid (PA) accumulation in tomato

Recently, we provided the first genetic evidence for the requirement of tomato PLC4 and PLC6 genes in defense activation and disease resistance. The encoded enzymes were catalytically active as they were able to degrade phosphatidylinositol (PI), thereby producing diacylglycerol (DG). Here we report differential PLC gene expression following the initiation of defense signaling by the interaction between Cladosporium fulvum resistance (R) protein Cf-4 and its matching effector Avr4 in tomato hybrid seedlings that express both Cf-4 and Avr4. Furthermore, we observed that PLC3 and PLC6 gene expression is upregulated by elevated temperature in the control seedlings. This upregulation coincides with an increase in the levels of phosphatidic acid (PA) and a decrease in the levels of PI and phosphatidylinositol phosphate (PIP). The decrease in PI and PIP levels matches with the activation of PLC. In addition, the levels of the structural phospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) declined transiently during recovery after the exposure to elevated temperature., Further studies will be required to explain the mechanism causing the sustained accumulation of PA during recovery, combined with a reduction in the levels of structural phospholipids.

Random mutagenesis of the nucleotide‐binding domain of NRC1 (NB‐LRR Required for Hypersensitive Response‐Associated Cell Death‐1), a downstream signalling nucleotide‐binding, leucine‐rich repeat (NB‐LRR) protein, identifies gain‐of‐function mutations in the nucleotide‐binding pocket

Plant nucleotide-binding, leucine-rich repeat (NB-LRR) proteins confer immunity to pathogens possessing the corresponding avirulence proteins. Activation of NB-LRR proteins is often associated with induction of the hypersensitive response (HR), a form of programmed cell death. NRC1 (NB-LRR Required for HR-Associated Cell Death-1) is a tomato (Solanum lycopersicum) NB-LRR protein that participates in the signalling cascade leading to resistance to the pathogens Cladosporium fulvum and Verticillium dahliae. To identify mutations in NRC1 that cause increased signalling activity, we generated a random library of NRC1 variants mutated in their nucleotide-binding domain and screened them for the ability to induce an elicitor-independent HR in Nicotiana tabacum. Screening of 1920 clones retrieved 11 gain-of-function mutants, with 10 of them caused by a single amino acid substitution. All substitutions are located in or very close to highly conserved motifs within the nucleotide-binding domain, suggesting modulation of the signalling activity of NRC1. Three-dimensional modelling of the nucleotide-binding domain of NRC1 revealed that the targeted residues are centred around the bound nucleotide. Our mutational approach has generated a wide set of novel gain-of-function mutations in NRC1 and provides insight into how the activity of this NB-LRR is regulated.

Protein–protein interactions as a proxy to monitor conformational changes and activation states of the tomato resistance protein I-2

Plant resistance proteins (R) are involved in pathogen recognition and subsequent initiation of defence responses. Their activity is regulated by inter- and intramolecular interactions. In a yeast two-hybrid screen two clones (I2I-1 and I2I-2) specifically interacting with I-2, a Fusarium oxysporum f. sp. lycopersici resistance protein of the CC-NB-LRR family, were identified. Sequence analysis revealed that I2I-1 belongs to the Formin gene family (SlFormin) whereas I2I-2 has homology to translin-associated protein X (SlTrax). SlFormin required only the N-terminal CC I-2 domain for binding, whereas SlTrax required both I-2 CC and part of the NB-ARC domain. Tomato plants stably silenced for these interactors were not compromised in I-2-mediated disease resistance. When extended or mutated forms of I-2 were used as baits, distinct and often opposite, interaction patterns with the two interactors were observed. These interaction patterns correlated with the proposed activation state of I-2 implying that active and inactive R proteins adopt distinct conformations. It is concluded that the yeast two hybrid system can be used as a proxy to monitor these different conformational states.

An Outlook on the Localisation and Structure-Function Relationships of R Proteins in Solanum

The co-evolution of plants and plant-pathogens shaped a multi-layered defence system in plants, in which Resistance proteins (R proteins) play a significant role. A fundamental understanding of the functioning of these R proteins and their position in the broader defence system of the plant is essential. Sub-project 3 of the BIOEXPLOIT programme studies how R proteins are activated upon effector recognition and how recognition is conveyed in resistance signalling pathways, using the solanaceous R proteins Rx1 (from S. tuberosum spp. andigena; conferring extreme resistance against Potato Virus X), I-2 (from S. lycopersicon; mediating resistance to Fusarium oxysporum) and Mi-1.2 (from S. lycopersicon; conferring resistance to Meloidogyne incognita) as model systems. The results obtained in this project will serve as a model for other R proteins and will be translated to potential applications or alternative strategies for disease resistance. These include the modification of the recognition specificity of R proteins with the aim to obtain broad spectrum resistance to major pathogens in potato.

Distinct roles of non-overlapping surface regions of the coiled-coil domain in the potato immune receptor Rx1

Mutation of the coiled-coil domain of the intracellular immune receptor Rx1 led to distinct cell death and resistance phenotypes and revealed regions of Rx1 required for interactions between Rx1 and the co-factor RanGAP2. The intracellular immune receptor Rx1 of potato (Solanum tuberosum), which confers effector-triggered immunity to Potato virus X, consists of a central nucleotide-binding domain (NB-ARC) flanked by a carboxyl-terminal leucine-rich repeat (LRR) domain and an amino-terminal coiled-coil (CC) domain. Rx1 activity is strictly regulated by interdomain interactions between the NB-ARC and LRR, but the contribution of the CC domain in regulating Rx1 activity or immune signaling is not fully understood. Therefore, we used a structure-informed approach to investigate the role of the CC domain in Rx1 functionality. Targeted mutagenesis of CC surface residues revealed separate regions required for the intramolecular and intermolecular interaction of the CC with the NB-ARC-LRR and the cofactor Ran GTPase-activating protein2 (RanGAP2), respectively. None of the mutant Rx1 proteins was constitutively active, indicating that the CC does not contribute to the autoinhibition of Rx1 activity. Instead, the CC domain acted as a modulator of downstream responses involved in effector-triggered immunity. Systematic disruption of the hydrophobic interface between the four helices of the CC enabled the uncoupling of cell death and disease resistance responses. Moreover, a strong dominant negative effect on Rx1-mediated resistance and cell death was observed upon coexpression of the CC alone with full-length Rx1 protein, which depended on the RanGAP2-binding surface of the CC. Surprisingly, coexpression of the N-terminal half of the CC enhanced Rx1-mediated resistance, which further indicated that the CC functions as a scaffold for downstream components involved in the modulation of disease resistance or cell death signaling.