The AVR2-SIX5 gene pair is required to activate I-2-mediated immunity in tomato.

Plant-invading microbes betray their presence to a plant by exposure of antigenic molecules such as small, secreted proteins called 'effectors'. In Fusarium oxysporum f. sp. lycopersici (Fol) we identified a pair of effector gene candidates, AVR2-SIX5, whose expression is controlled by a shared promoter. The pathogenicity of AVR2 and SIX5 Fol knockouts was assessed on susceptible and resistant tomato (Solanum lycopersicum) plants carrying I-2. The I-2 NB-LRR protein confers resistance to Fol races carrying AVR2. Like Avr2, Six5 was found to be required for full virulence on susceptible plants. Unexpectedly, each knockout could breach I-2-mediated disease resistance. So whereas Avr2 is sufficient to induce I-2-mediated cell death, Avr2 and Six5 are both required for resistance. Avr2 and Six5 interact in yeast two-hybrid assays as well as in planta. Six5 and Avr2 accumulate in xylem sap of plants infected with the reciprocal knockouts, showing that lack of I-2 activation is not due to a lack of Avr2 accumulation in the SIX5 mutant. The effector repertoire of a pathogen determines its host specificity and its ability to manipulate plant immunity. Our findings challenge an oversimplified interpretation of the gene-for-gene model by showing requirement of two fungal genes for immunity conferred by one resistance gene.

A nuclear localization for Avr2 from Fusarium oxysporum is required to activate the tomato resistance protein I-2.

Plant pathogens secrete effector proteins to promote host colonization. During infection of tomato xylem vessels, Fusarium oxysporum f. sp. lycopersici (Fol) secretes the Avr2 effector protein. Besides being a virulence factor, Avr2 is recognized intracellularly by the tomato I-2 resistance protein, resulting in the induction of host defenses. Here, we show that AVR2 is highly expressed in root- and xylem-colonizing hyphae three days post inoculation of roots. Co-expression of I-2 with AVR2 deletion constructs using agroinfiltration in Nicotiana benthamiana leaves revealed that, except for the N-terminal 17 amino acids, the entire AVR2 protein is required to trigger I-2-mediated cell death. The truncated Avr2 variants are still able to form homo-dimers, showing that the central region of Avr2 is required for dimerization. Simultaneous production of I-2 and Avr2 chimeras carrying various subcellular localization signals in N. benthamiana leaves revealed that a nuclear localization of Avr2 is required to trigger I-2-dependent cell death. Nuclear exclusion of Avr2 prevented its activation of I-2, suggesting that Avr2 is recognized by I-2 in the nucleus.

Dual regulatory roles of the extended N terminus for activation of the tomato MI-1.2 resistance protein.

Plant resistance (R) proteins mediate race-specific immunity and initiate host defenses that are often accompanied by a localized cell-death response. Most R proteins belong to the nucleotide binding-leucine-rich repeat (NB-LRR) protein family, as they carry a central NB-ARC domain fused to an LRR domain. The coiled-coil (CC) domain at the N terminus of some solanaceous NB-LRR proteins is extended with a solanaceae domain (SD). Tomato Mi-1.2, which confers resistance against nematodes, white flies, psyllids, and aphids, encodes a typical SD-CNL protein. Here, we analyzed the role of the extended N terminus for Mi-1.2 activation. Removal of the first part of the N terminus (Nt1) induced Mi-1.2-mediated cell death that could be suppressed by overexpression of the second half of the N-terminal region. Yet, autoactivating NB-ARC-LRR mutants require in trans coexpression of the N-terminal region to induce cell death, indicating that the N terminus functions both as a negative and as a positive regulator. Based on secondary structure predictions, we could link both activities to three distinct subdomains, a typical CC domain and two novel, structurally-conserved helical subdomains called SD1 and SD2. A negative regulatory function could be assigned to the SD1, whereas SD2 and the CC together function as positive regulators of Mi-1.2-mediated cell death.

The small heat shock protein 20 RSI2 interacts with and is required for stability and function of tomato resistance protein I-2.

Race-specific disease resistance in plants depends on the presence of resistance (R) genes. Most R genes encode NB-ARC-LRR proteins that carry a C-terminal leucine-rich repeat (LRR). Of the few proteins found to interact with the LRR domain, most have proposed (co)chaperone activity. Here, we report the identification of RSI2 (Required for Stability of I-2) as a protein that interacts with the LRR domain of the tomato R protein I-2. RSI2 belongs to the family of small heat shock proteins (sHSPs or HSP20s). HSP20s are ATP-independent chaperones that form oligomeric complexes with client proteins to prevent unfolding and subsequent aggregation. Silencing of RSI2-related HSP20s in Nicotiana benthamiana compromised the hypersensitive response that is normally induced by auto-active variants of I-2 and Mi-1, a second tomato R protein. As many HSP20s have chaperone properties, the involvement of RSI2 and other R protein (co)chaperones in I-2 and Mi-1 protein stability was examined. RSI2 silencing compromised the accumulation of full-length I-2 in planta, but did not affect Mi-1 levels. Silencing of heat shock protein 90 (HSP90) and SGT1 led to an almost complete loss of full-length I-2 accumulation and a reduction in Mi-1 protein levels. In contrast to SGT1 and HSP90, RSI2 silencing led to accumulation of I-2 breakdown products. This difference suggests that RSI2 and HSP90/SGT1 chaperone the I-2 protein using different molecular mechanisms. We conclude that I-2 protein function requires RSI2, either through direct interaction with, and stabilization of I-2 protein or by affecting signalling components involved in initiation of the hypersensitive response.

The effector protein Avr2 of the xylem-colonizing fungus Fusarium oxysporum activates the tomato resistance protein I-2 intracellularly.

To promote host colonization, many plant pathogens secrete effector proteins that either suppress or counteract host defences. However, when these effectors are recognized by the host's innate immune system, they trigger resistance rather than promoting virulence. Effectors are therefore key molecules in determining disease susceptibility or resistance. We show here that Avr2, secreted by the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici (Fol), shows both activities: it is required for full virulence in a susceptible host and also triggers resistance in tomato plants carrying the resistance gene I-2. Point mutations in AVR2, causing single amino acid changes, are associated with I-2-breakingFol strains. These point mutations prevent recognition by I-2, both in tomato and when both genes are co-expressed in leaves of Nicotiana benthamiana. Fol strains carrying the Avr2 variants are equally virulent, showing that virulence and avirulence functions can be uncoupled. Although Avr2 is secreted into the xylem sap when Fol colonizes tomato, the Avr2 protein can be recognized intracellularly by I-2, implying uptake by host cells.

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.

The presence of a virulence locus discriminates Fusarium oxysporum isolates causing tomato wilt from other isolates.

Fusarium oxysporum is an asexual fungus that inhabits soils throughout the world. As a species, F. oxysporum can infect a very broad range of plants and cause wilt or root rot disease. Single isolates of F. oxysporum, however, usually infect one or a few plant species only. They have therefore been grouped into formae speciales (f.sp.) based on host specificity. Isolates able to cause tomato wilt (f.sp. lycopersici) do not have a single common ancestor within the F. oxysporum species complex. Here we show that, despite their polyphyletic origin, isolates belonging to f.sp. lycopersici all contain an identical genomic region of at least 8 kb that is absent in other formae speciales and non-pathogenic isolates, and comprises the genes SIX1, SIX2 and SHH1. In addition, SIX3, which lies elsewhere on the same chromosome, is also unique for f.sp. lycopersici. SIX1 encodes a virulence factor towards tomato, and the Six1, Six2 and Six3 proteins are secreted in xylem during colonization of tomato plants. We speculate that these genes may be part of a larger, dispensable region of the genome that confers the ability to cause tomato wilt and has spread among clonal lines of F. oxysporum through horizontal gene transfer. Our findings also have practical implications for the detection and identification of f.sp. lycopersici.

Structure and function of resistance proteins in solanaceous plants.

Gene-for-gene resistance in plants is based on the presence of a resistance (R) gene in the host and a matching Avirulence (Avr) gene in the pathogen. Many R genes have been cloned over the past two decades, mostly from the Solanaceae. The gene products, called R proteins, display modular domain structures. R protein function has recently been shown to require dynamic interactions between the various domains. In addition to these intramolecular interactions, R proteins interact with other proteins to form signaling complexes that are able to activate an innate immune response that arrests proliferation of the invading pathogen, thereby conferring disease resistance. In this review, we summarize current understanding of R protein structure and function, as well as the molecular mechanisms underlying the activation of defense signaling processes. As well as being a rich source for R genes, Solanaceae are a leading model system in which to study inter- and intramolecular interactions of R proteins.

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).

Heat shock protein 90 and its co-chaperone protein phosphatase 5 interact with distinct regions of the tomato I-2 disease resistance protein.

Recent data suggest that plant disease resistance (R) proteins are present in multi-protein complexes. Tomato R protein I-2 confers resistance against the fungal pathogen Fusarium oxysporum. To identify components of the I-2 complex, we performed yeast two-hybrid screens using the I-2 leucine-rich repeat (LRR) domain as bait, and identified protein phosphatase 5 (PP5) as an I-2 interactor. Subsequent screens revealed two members of the cytosolic heat shock protein 90 (HSP90) family as interactors of PP5. By performing in vitro protein-protein interaction analysis using recombinant proteins, we were able to show a direct interaction between I-2 and PP5, and between I-2 and HSP90. The N-terminal part of the LRR domain was found to interact with HSP90, whereas the C-terminal part bound to PP5. The specific binding of HSP90 to the N-terminal region of the I-2 LRR domain was confirmed by co-purifying HSP90 from tomato lysate using recombinant proteins. Similarly, the interaction between PP5 and HSP90 was established. To investigate the role of PP5 and HSP90 for I-2 function, virus-induced gene silencing was performed in Nicotiana benthamiana. Silencing of HSP90 but not of PP5 completely blocked cell death triggered by I-2, showing that HSP90 is required for I-2 function. Together these data suggest that R proteins require, like steroid hormone receptors in animal systems, an HSP90/PP5 complex for their folding and functioning.

A small, cysteine-rich protein secreted by Fusarium oxysporum during colonization of xylem vessels is required for I-3-mediated resistance in tomato.

A 12 kDa cysteine-rich protein is secreted by Fusarium oxysporum f. sp. lycopersici during colonization of tomato xylem vessels. Peptide sequences obtained with mass spectrometry allowed identification of the coding sequence. The gene encodes a 32 kDa protein, designated Six1 for secreted in xylem 1. The central part of Six1 corresponds to the 12 kDa protein found in xylem sap of infected plants. A mutant that had gained virulence on a tomato line with the I-3 resistance gene was found to have lost the SIX1 gene along with neighbouring sequences. Transformation of this mutant with SIX1 restored avirulence on the I-3 line. Conversely, deletion of the SIX1 gene in a wild-type strain results in breaking of I-3-mediated resistance. These results suggest that I-3-mediated resistance is based on recognition of Six1 secreted in xylem vessels.

A tomato xylem sap protein represents a new family of small cysteine-rich proteins with structural similarity to lipid transfer proteins.

The coding sequence of a major xylem sap protein of tomato was identified with the aid of mass spectrometry. The protein, XSP10, represents a novel family of extracellular plant proteins with structural similarity to plant lipid transfer proteins. The XSP10 gene is constitutively expressed in roots and lower stems. The decline of XSP10 protein levels in tomato infected with a fungal vascular pathogen may reflect breakdown or modification by the pathogen.

The tomato R gene products I-2 and MI-1 are functional ATP binding proteins with ATPase activity.

Most plant disease resistance (R) genes known today encode proteins with a central nucleotide binding site (NBS) and a C-terminal Leu-rich repeat (LRR) domain. The NBS contains three ATP/GTP binding motifs known as the kinase-1a or P-loop, kinase-2, and kinase-3a motifs. In this article, we show that the NBS of R proteins forms a functional nucleotide binding pocket. The N-terminal halves of two tomato R proteins, I-2 conferring resistance to Fusarium oxysporum and Mi-1 conferring resistance to root-knot nematodes and potato aphids, were produced as glutathione S-transferase fusions in Escherichia coli. In a filter binding assay, purified I-2 was found to bind ATP rather than other nucleoside triphosphates. ATP binding appeared to be fully dependent on the presence of a divalent cation. A mutant I-2 protein containing a mutation in the P-loop showed a strongly reduced ATP binding capacity. Thin layer chromatography revealed that both I-2 and Mi-1 exerted ATPase activity. Based on the strong conservation of NBS domains in R proteins of the NBS-LRR class, we propose that they all are capable of binding and hydrolyzing ATP.