Frank L. W. Takken

Structure–function analysis of the NB-ARC domain of plant disease resistance proteins

Resistance (R) proteins in plants are involved in pathogen recognition and subsequent activation of innate immune responses. Most resistance proteins contain a central nucleotide-binding domain. This so-called NB-ARC domain consists of three subdomains: NB, ARC1, and ARC2. The NB-ARC domain is a functional ATPase domain, and its nucleotide-binding state is proposed to regulate activity of the R protein. A highly conserved methionine-histidine-aspartate (MHD) motif is present at the carboxy-terminus of ARC2. An extensive mutational analysis of the MHD motif in the R proteins I-2 and Mi-1 is reported. Several novel autoactivating mutations of the MHD invariant histidine and conserved aspartate were identified. The combination of MHD mutants with autoactivating hydrolysis mutants in the NB subdomain showed that the autoactivation phenotypes are not additive. This finding indicates an important regulatory role for the MHD motif in the control of R protein activity. To explain these observations, a three-dimensional model of the NB-ARC domain of I-2 was built, based on the APAF-1 template structure. The model was used to identify residues important for I-2 function. Substitution of the selected residues resulted in the expected distinct phenotypes. Based on the model, it is proposed that the MHD motif fulfils the same function as the sensor II motif found in AAA+ proteins (ATPases associated with diverse cellular activities)-co-ordination of the nucleotide and control of subdomain interactions. The presented 3D model provides a framework for the formulation of hypotheses on how mutations in the NB-ARC exert their effects.

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).

STANDing strong, resistance proteins instigators of plant defence.

Resistance (R) proteins are involved in specific pathogen recognition and subsequent initiation of host defence. Most R proteins are nucleotide binding - leucine rich repeat (NB-LRR) proteins, which form a subgroup within the STAND (signal transduction ATPases with numerous domains) family. Activity of these multi-domain proteins depends on their ability to bind and hydrolyse nucleotides. Since R protein activation often triggers cell-death tight regulation of activation is essential. Autoinhibition, which seems to be accomplished by intramolecular interactions between the various domains, is important to retain R proteins inactive. This review summarizes recent data on intra- and intermolecular interactions that support a model in which pathogen perception triggers a series of conformational changes, allowing the newly exposed NB domain to interact with downstream signalling partners and activate defence signalling.

How to build a pathogen detector: structural basis of NB-LRR function

Many plant disease resistance (R) proteins belong to the family of nucleotide-binding-leucine rich repeat (NB-LRR) proteins. NB-LRRs mediate recognition of pathogen-derived effector molecules and subsequently activate host defence. Their multi-domain structure allows these pathogen detectors to simultaneously act as sensor, switch and response factor. Structure-function analyses and the recent elucidation of the 3D structures of subdomains have provided new insight in how these different functions are combined and what the contribution is of the individual subdomains. Besides interdomain contacts, interactions with chaperones, the proteasome and effector baits are required to keep NB-LRRs in a signalling-competent, yet auto-inhibited state. In this review we explore operational models of NB-LRR functioning based on recent advances in understanding their structure.

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.

Plant resistance genes: their structure, function and evolution.

Plants have developed efficient mechanisms to avoid infection or to mount responses that render them resistant upon attack by a pathogen. One of the best-studied defence mechanisms is based on gene-for-gene resistance through which plants, harbouring specific resistance (R) genes, specifically recognise pathogens carrying matching avirulence (Avr) genes. Here a review of the R genes that have been cloned is given. Although in most cases it is not clear how R gene encoded proteins initiate pathways leading to disease resistance, we will show that there are clear parallels with disease prevention in animal systems. Furthermore, some evolutionary mechanisms acting on R genes to create novel recognitional specificities will be discussed.

Structure-Function Analysis of Barley NLR Immune Receptor MLA10 Reveals Its Cell Compartment Specific Activity in Cell Death and Disease Resistance

Plant intracellular immune receptors comprise a large number of multi-domain proteins resembling animal NOD-like receptors (NLRs). Plant NLRs typically recognize isolate-specific pathogen-derived effectors, encoded by avirulence (AVR) genes, and trigger defense responses often associated with localized host cell death. The barley MLA gene is polymorphic in nature and encodes NLRs of the coiled-coil (CC)-NB-LRR type that each detects a cognate isolate-specific effector of the barley powdery mildew fungus. We report the systematic analyses of MLA10 activity in disease resistance and cell death signaling in barley and Nicotiana benthamiana. MLA10 CC domain-triggered cell death is regulated by highly conserved motifs in the CC and the NB-ARC domains and by the C-terminal LRR of the receptor. Enforced MLA10 subcellular localization, by tagging with a nuclear localization sequence (NLS) or a nuclear export sequence (NES), shows that MLA10 activity in cell death signaling is suppressed in the nucleus but enhanced in the cytoplasm. By contrast, nuclear localized MLA10 is sufficient to mediate disease resistance against powdery mildew fungus. MLA10 retention in the cytoplasm was achieved through attachment of a glucocorticoid receptor hormone-binding domain (GR), by which we reinforced the role of cytoplasmic MLA10 in cell death signaling. Together with our data showing an essential and sufficient nuclear MLA10 activity in disease resistance, this suggests a bifurcation of MLA10-triggered cell death and disease resistance signaling in a compartment-dependent manner.

cDNA-AFLP Combined with Functional Analysis Reveals Novel Genes Involved in the Hypersensitive Response

To identify genes required for the hypersensitive response (HR), we performed expression profiling of tomato plants mounting a synchronized HR, followed by functional analysis of differentially expressed genes. By cDNA-AFLP analysis, the expression profile of tomato plants containing both the Cf-4 resistance gene against Cladosporium fulvum and the matching Avr4 avirulence gene of this fungus was compared with that of control plants. About 1% of the transcript-derived fragments (442 out of 50,000) were derived from a differentially expressed gene. Based on their sequence and expression, 192 fragments, referred to as Avr4-responsive tomato (ART) fragments, were selected for VIGS (virus-induced gene silencing) in Cf-4-transgenic Nicotiana benthamiana. Inoculated plants were analyzed for compromised HR by agroinfiltration of either the C. fulvum Avr4 gene or the Inf1 gene of Phytophthora infestans, which invokes a HR in wild-type N. benthamiana. VIGS using 15 of the ART fragments resulted in a compromised HR, whereas VIGS with fragments of ART genes encoding HSP90, a nuclear GTPase, an L19 ribosomal protein, and most interestingly, a nucleotide binding-leucine rich repeat (NB-LRR)-type protein severely suppressed the HR induced both by Avr4 and Inf1. Requirement of an NB-LRR protein (designated NRC1, for NB-LRR protein required for HR-associated cell death 1) for Cf resistance protein function as well as Inf1-mediated HR suggests a convergence of signaling pathways and supports the recent observation that NB-LRR proteins play a role in signal transduction cascades downstream of resistance proteins.

Susceptibility Genes 101: How to Be a Good Host

To confer resistance against pathogens and pests in plants, typically dominant resistance genes are deployed. However, because resistance is based on recognition of a single pathogen-derived molecular pattern, these narrow-spectrum genes are usually readily overcome. Disease arises from a compatible interaction between plant and pathogen. Hence, altering a plant gene that critically facilitates compatibility could provide a more broad-spectrum and durable type of resistance. Here, such susceptibility (S) genes are reviewed with a focus on the mechanisms underlying loss of compatibility. We distinguish three groups of S genes acting during different stages of infection: early pathogen establishment, modulation of host defenses, and pathogen sustenance. The many examples reviewed here show that S genes have the potential to be used in resistance breeding. However, because S genes have a function other than being a compatibility factor for the pathogen, the side effects caused by their mutation demands a one-by-one assessment of their usefulness for application.

Arabidopsis Small Ubiquitin-Like Modifier Paralogs Have Distinct Functions in Development and Defense

This report describes the effect that protein modifications by isoforms of the small ubiquitin-like modifier (SUMO) have on plant development and innate immunity. SUM1 and SUM2 were found to be essential for suppressing defense responses in noninfected plants by preventing accumulation of the defense hormone salicylic acid, whereas SUM3 enhances these defense responses in infected plants. Posttranslational modifications allow dynamic and reversible changes to protein function. In Arabidopsis thaliana, a small gene family encodes paralogs of the small ubiquitin-like posttranslational modifier. We studied the function of these paralogs. Single mutants of the SUM1 and SUM2 paralogs do not exhibit a clear phenotype. However, the corresponding double knockdown mutant revealed that SUM1 and SUM2 are essential for plant development, floral transition, and suppression of salicylic acid (SA)–dependent defense responses. The SUM1 and SUM2 genes are constitutively expressed, but their spatial expression patterns do not overlap. Tight transcriptional regulation of these two SUM genes appears to be important, as overexpression of either wild-type or conjugation-deficient mutants resulted in activation of SA-dependent defense responses, as did the sum1 sum2 knockdown mutant. Interestingly, expression of the paralog SUM3 is strongly and widely induced by SA and by the defense elicitor Flg22, whereas its expression is otherwise low and restricted to a few specific cell types. Loss of SUM3 does not result in an aberrant developmental phenotype except for late flowering, while SUM3 overexpression causes early flowering and activates plant defense. Apparently, SUM3 promotes plant defense downstream of SA, while SUM1 and SUM2 together prevent SA accumulation in noninfected plants.