Recent advances in the study of tomato Cf resistance genes

Tomato Cf genes confer resistance to the leaf mould pathogen Cladosporium fulvum through recognition of distinct fungal-encoded avirulence (Avr) peptides secreted into the leaf apoplast during infection. These Avr determinants are small cysteine-rich proteins that exhibit no primary sequence homology (Luderer and Joosten, 2001). The tomato–C. fulvum interaction is an attractive model system to study the molecular basis of recognition specificity in plant disease resistance (R) proteins, and R-protein mediated defence responses. Cf proteins are type I transmembrane glycoproteins comprised predominantly of extracytoplasmic leucine-rich repeats (LRRs), a membrane-spanning region, and a short cytoplasmic domain that lacks an obvious signalling function (reviewed in Thomas et al., 1998). These R proteins activate a hypersensitive response (HR) upon recognition of their cognate Avr proteins. It has been proposed that plant R proteins ‘guard’ specific cellular proteins targeted by pathogen-encoded virulence determinants that subsequently trigger the plant defence response (Dangl and Jones, 2001; Dixon et al., 2000). The molecular mechanism for Cf protein-mediated recognition of C. fulvum Avr proteins is not known. However, recent studies suggest that Cf proteins are associated with other membrane proteins which together may constitute a ‘recognition complex’, that has been hypothesized to include the ‘guarded’ protein (Rivas et al., 2002a,b). Heterologous expression of Cf genes in tobacco has also been successfully exploited to investigate Cf-9-mediated changes in gene expression and physiology as well as the molecular basis of recognition specificity in Cf-4 and Cf-9. In this microreview, we summarize recent progress in the functional analysis of tomato Cf proteins and the cellular responses that they activate. Two Cf loci have been extensively studied at the molecular level, the Cf-4/Cf-9 locus on chromosome 1 and the Cf-2/Cf-5 locus on chromosome 6 (reviewed in Thomas et al., 1998). These loci are comprised of tandemly duplicated homologous genes (paralogues). The Cf-4 and Cf-9 haplotypes each contain five paralogous genes. Sequence diversity within these genes is a result of intergenic sequence exchange between paralogues, and mutations in sequences encoding the putative solvent-exposed amino acids of a conserved structural motif in LRR proteins (xxLxLxx, where L = leucine, and x = solvent exposed amino acids). The solvent-exposed residues appear to determine recognition specificity in LRR proteins (Thomas et al., 1998). These studies revealed extensive interspecific sequence polymorphism at Cf loci. Recent analyses suggest Cf loci show considerable intraspecific variation, at least in the wild species Lycopersicon pimpinellifolium (Lauge et al., 1998, 2000). When a limited number of wild accessions were analysed for their response to five C. fulvum extracellular proteins (ECPs), several accessions were identified that induced an ECP-dependent HR. Several of these genes have been mapped to loci containing Cf homologues on the short arm of chromosome 1 (Haanstra et al., 1999, 2000). These studies further illustrate the importance of wild species as novel sources of resistance genes. The molecular basis of recognition specificity has been analysed in Cf-4 and Cf-9. These proteins induce a HR upon recognition of Avr4 and Avr9. Cf-4 and Cf-9 are more than 91% identical at the amino acid level (Thomas et al., 1998) but differ in their LRR copy number (25 LRRs in Cf-4 and 27 in Cf-9). Most of the variant amino acids between Cf-9 and Cf-4 correspond to solvent-exposed amino acids within the xxLxLxx structural motif of their N-terminal LRRs. Sequences required for recognition specificity were identified by functional analysis of Cf-4/Cf-9 chimeras expressed in tobacco and tomato (van der Hoorn et al., 2001a; Wulff et al., 2001). No chimeras that contained 27 LRRs could induce an Avr4-dependent HR and none containing 25 LRRs could induce an Avr9-dependent HR. Only a fraction of the variant amino acids in Cf-4, located in the central LRRs of domain C1 (LRRs 11, 12 and 14), were required to induce an Avr4-dependent HR (van der Hoorn et al., 2001a; Wulff et al., 2001). Sequences required for Cf-9 recognition specificity are distributed over a greater number of LRRs (van der Hoorn et al., 2001a; Wulff et al., 2001), but Wulff et al. (2001) also demonstrated that some essential sequences are located within the central LRRs of domain C1 (Fig. 1). Schematic representation of Cf-4 and Cf-9 resistance proteins. The figure shows their predicted extracellular (B, C1, C2, C3, D and E), transmembrane (F) and cytoplasmic (G) domains (see Thomas et al., 1998 ). The cytoplasmic C-terminal dilysine motif of domain G (KKRY) present in both proteins is also shown. Individual LRRs are depicted as grey boxes. Cf-4 and Cf-9 share more than 91% amino acid identity. The proteins share identical C-terminal domains (‘Conserved’) and sequence variation is confined to their N-terminal LRRs within domain C1 (‘Variable’). These studies demonstrated how variation in LRR copy number of Cf proteins, together with minor sequence variation, can generate novel recognition specificities in R proteins. However, these analyses only addressed the role of variant amino acids and the functional contribution of amino acids conserved between Cf-4 and Cf-9 was not determined. Sequence analysis of a variant L. pimpinellifolium allele of Cf-9 showed that a number of solvent exposed amino acids in the N-terminal LRRs of domain C1 can be substituted without compromising function (van der Hoorn et al., 2001b). Therefore, the full extent of sequences required for recognition specificity in Cf proteins will require further investigation. Mutagenesis of the Cf9 line identified mutants with lesions in Cf-9 (Hammond-Kosack et al., 1994; CM Thomas and JDG Jones, unpublished) and two genes required for its function, Rcr-1 and Rcr-2 (standing for ‘required for Cladosporium resistance’; Hammond-Kosack et al., 1994). However, the rcr-1 and rcr-2 mutants were not amenable to genetic analysis due to the poor penetrance of the mutant phenotypes (C Golstein, CM Thomas and JDG Jones, unpublished results). Mutagenesis of the Cf2 line identified four mutant alleles of Rcr3 that is specifically required for Cf-2 function (Dixon et al., 2000). Rcr3 was shown to encode a secreted cysteine protease homologous to papain (Krüger et al., 2002). It was also shown that the Cf2 line (which contains the Cf-2 gene introgressed from L. pimpinellifolium), contains the L. pimpinellifolium allele of Rcr3 (Rcr3pim), despite the fact that Cf-2 and Rcr3 assort independently (Krüger et al., 2002). Rcr3pim was shown to be allelic to the L. pimpinellifolium gene Ne that is required to suppress the Cf-2-dependent formation of autonecrotic lesions in plants containing the L. esculentum allele of Rcr3 (Rcr3esc or ne). This phenotype can be attributed to a limited number of amino acid sequences that distinguish Rcr3esc from Rcr3pim (Krüger et al., 2002). Rcr3 may function in the processing or maturation of Avr2, and/or it may form part of a ‘recognition complex’ that includes Rcr3, Avr2 and Cf-2 (Dixon et al., 2000). Rcr3 is probably not solely involved in the processing of Avr2 since apoplastic fluids isolated from a compatible tomato–C. fulvum interaction (that presumably contains the ‘mature’ Avr2), do not induce a HR when infiltrated into rcr3 mutants (Dixon et al., 2000). Moreover, it is known that Rcr3esc can induce precocious activation of the Cf-2-dependent defence response (i.e. in the absence of Avr2), which results in autonecrosis (Krüger et al., 2002). These data are consistent with a model where Rcr3 forms part of the ligand that is recognized by Cf-2. In light of the ‘guard’ hypothesis for R protein function, it was proposed that Rcr3 functions as an antifungal protease that is targeted by Avr2. The Rcr3/Avr2 complex formed is recognized by Cf-2 that activates plant defences. The cloning of all three components (Krüger et al., 2002; Luderer and Joosten, 2001) should allow the nature of any protein–protein interactions to be determined. A better understanding of Cf protein function requires the elucidation of their subcellular localization. Both Cf-9 and Cf-4 are highly glycosylated suggesting that they enter a protein secretion pathway (Piedras et al., 2000; Rivas et al., 2002a). Cf-9 and Cf-4 contain a putative endoplasmic reticulum (ER)-retention/retrieval signal (KKRY) at their C-terminus. In mammals and yeast the C-terminal dilysine motif (KKXX) signals retrieval of type I membrane proteins from the Golgi apparatus to the ER. Two conflicting studies on the localization of Cf-9 have been published: Cf-9 was first reported to be located predominantly in the plasma membrane (Piedras et al., 2000) but in another study the KKRY sequence was reported to localize Cf-9 to the ER (Benghezal et al., 2000). However, in the latter case, the green fluorescent protein was fused to the transmembrane and cytosolic domains of Cf-9 and expressed in yeast, Arabidopsis and tobacco cells. The functionality of these chimeric proteins could not be tested. More recently it was shown that a free C-terminal dylisine motif is not required for Cf-9 or Cf-4 function (Rivas et al., 2002a,b; Van der Hoorn et al., 2001c), providing an additional argument against localization of Cf-9 and Cf-4 in the ER. A plausible explanation for this observation is that additional proteins mask the KKRY sequence in Cf-9. Indeed Cf-9 has been shown to be a component of a membrane-associated complex (see below). It has been hypothesized that masking of the C-terminal dilysine motif may discriminate between correctly and incorrectly assembled Cf-9 complexes. This control at the ER would only allow correctly assembled complexes to be targeted to the plasma membrane where they would be functional (Van der Hoorn et al., 2001c). Cf-4 and Cf-9 have identical C-terminal halves (Fig. 1) and it is tempting to speculate that they are targeted to the same subcellular location, however, no data regarding the subcellular location of Cf-4 have been reported and the precise location of both proteins remains controversial. The structure of Cf-4 and Cf-9 suggests they function as extracellular receptors for their cognate Avr proteins that are secreted into the leaf apoplast. However, in several studies no direct interaction between Cf-9 and Avr9 could be detected (Luderer et al., 2001). One study identified a high-affinity binding site (HABS) for Avr9 in the plasma membrane fractions isolated from a number of solanaceous species (Kooman-Gersmann et al., 1996). This binding occurs irrespective of the presence or absence of Cf-9. Significantly, the binding affinity of variant Avr9 peptides for the HABS correlated with their ability to induce a Cf-9-dependent HR (Kooman-Gersmann et al., 1998). It has been suggested that Cf-9 may ‘guard’ the HABS and trigger a plant defence response upon Avr9 binding (Fig. 2). Model for Avr9 recognition by the Cf-9 ‘complex’ and subsequent early signalling events. Cf-9 is present in a membrane-associated ∼420 kDa protein complex ( Rivas et al., 2002b ). The ‘guard’ model is consistent with at least two possible molecular mechanisms. The association of Cf-9 with its ‘guarded’ protein might be induced upon Avr9 binding to a pathogen target. Alternatively, Cf-9 might be constitutively associated with this protein and, upon Avr binding, a conformational change is produced that activates downstream signalling components ( Dangl and Jones, 2001 ). In the case of Cf-9, the guarded protein may be the previously described high-affinity binding site (HABS) for Avr9 ( Kooman-Gersmann et al., 1996 ) or a different protein. The early Cf-mediated cellular responses upon perception of Avr9 are shown and discussed in the text. Activation or inhibition of ion channels and H + -ATPase are represented by green or red arrows, respectively. Activation of protein kinases and induction of ACRE genes are thought to play a central role in the regulation of the defence response. However, it remains possible that additional factors are directly or indirectly involved in this regulation. Reported signalling events are shown with a solid arrow, whereas dotted arrows represent plausible signalling pathways. Cf proteins lack an obvious signal transduction domain suggesting that additional proteins are involved in activating plant defences. A paradigm for Cf protein function has been proposed (Dixon et al., 2000; Joosten and De Wit, 1999; Luderer et al., 2001) based on a model proposed for three proteins that control shoot apical meristem differentiation in Arabidopsis (Jeong et al., 1999; Trotochaud et al., 1999). The Arabidopsis Clavata2 (CLV2) protein is structurally similar to Cf proteins and forms an inactive disulphide-linked heterodimer of 185 kDa with CLV1, an extracellular LRR-receptor kinase. As in the case of Cf proteins, the putative ligand for the Clavata complex is a small secreted peptide (CLV3). Upon recognition of CLV3, and phosphorylation of the CLV1 kinase domains, an active 450 kDa complex is formed, containing the protein phosphatase KAPP and a Rho GTPase-related protein (Trotochaud et al., 1999). The possibility that Cf proteins signal through a similar molecular mechanism has been investigated by analysing microsomal fractions from tobacco plants and cell cultures (Rivas et al., 2002a,b). The observed monomeric size of c-myc-tagged Cf-9 and Cf-4 proteins is 160 and 145 KDa, respectively (Piedras et al., 2000; Rivas et al., 2002a). Gel filtration analysis of microsomal fractions solubilized with octylglucoside revealed that epitope-tagged Cf-9 and Cf-4 proteins migrate at a molecular mass of 350–475 kDa (Rivas et al., 2002a,b). Using blue native gel electrophoresis the molecular size was confirmed to be ≈ 420 and ≈ 400 kDa for the Cf-9 and Cf-4 complexes, respectively. Despite this being consistent with more than one Cf protein molecule per complex, results obtained by Rivas et al. (2002a,b) support the notion of a single Cf molecule associated with additional glycoprotein partner(s). The similarity in size observed for the Cf-9 and Cf-4 complexes (≈ 420 and 400 kDa;Rivas et al., 2002a,b) might be coincidental, or alternatively, Cf-4 and Cf-9 may associate with similar partners (i.e. they may ‘guard’ the same cellular proteins), and confer distinct recognition specificities to the 400 and 420 kDa complexes. The sizes of the Cf-4 and Cf-9 complexes are similar to that reported for the CLV complex (450 kDa; Trotochaud et al., 1999). However, unlike the Clavata complex no Avr-dependent shift in the molecular mass of the Cf complex was detected and, in addition, Cf proteins do not form disulphide-linked heterodimers (Rivas et al., 2002a,b). Also, no Rho-GTPase-related proteins were found associated with Cf proteins under the conditions tested (Rivas et al., 2002a,b). Therefore, Cf-dependent defence signalling and the CLV-dependent regulation of meristem development are probably regulated through distinct molecular mechanisms. Cf-9 and Cf-4 can function in tobacco to induce an Avr-dependent HR (Hammond-Kosack et al., 1998; Thomas et al., 2000). This has facilitated the study of Cf-/Avr-dependent biochemical responses and comparison to the previously described signalling events induced by nonspecific pathogen elicitors released during infection, such as oligosaacharides, peptides and proteins. Here we describe the Cf-9/Avr9-dependent early signalling events that have been dissected using tobacco cell cultures, as a model system (see Fig. 2). The rapid production of AOS is characteristic of many R-protein mediated plant defence responses. AOS may play a role in signalling, and possibly direct antimicrobial activity, leading to changes in gene expression (Hammond-Kosack and Jones, 1997). Tobacco cell cultures expressing Cf-9 produce AOS within 5 min of Avr9 addition (Piedras et al., 1998). The synthesis of H2O2 is paralleled by an increase in oxygen uptake (‘oxidative burst’; Fig. 2). Studies with pharmacological inhibitors suggest that uptake of calcium, activation of protein kinases and phospholipase A2 (PLA2) activity are intermediates in the Avr9/Cf-9-dependent signalling pathway that leads to AOS synthesis. The activity of ion channels in Cf-9 tobacco plants after elicitation with Avr9 was investigated in voltage clamp experiments using guard cells from epidermal peels of Cf-9 tobacco (Blatt et al., 1999). After treatment with Avr9, currents of inward- and outward-rectifying K+ channels were measured and both a rapid K+ efflux and a concomitant inhibition of K+ influx were recorded (Fig. 2). These K+ channel responses occurred within 3–5 min in a Cf-9/Avr9-dependent manner. Additionally, an important role for changes in ion channels in early Avr9/Cf-9-dependent signalling was confirmed by the observations that uptake of external Ca2+ is required for AOS production and that addition of Avr9 to Cf-9 cells results in alkalinization of the extracellular medium (Fig. 2). Protein kinases are key intracellular signalling components that are activated in response to extracellular signals. Consistent with the observation that protein kinase activity was needed for changes in ion fluxes and AOS synthesis, activation of protein kinases was observed in Cf-9 tobacco after Avr9 elicitation. Two protein kinases of 46 and 48 kDa were identified that became rapidly and transiently activated in Cf-9 tobacco within 2–5 min of elicitation with Avr9 (Romeis et al., 1999). Using specific antibodies, the two kinases were identified as the previously described mitogen-activated protein (MAP) kinases WIPK (wounding-induced protein kinase; Seo et al., 1999) and SIPK (salicylic acid-induced protein kinase; Zhang and Klessig, 1997). Additionally, Cf-9 tobacco shows an Avr9-dependent accumulation of the WIPK transcript. Studies with pharmacological inhibitors and effectors revealed that Ca2+ uptake and an upstream phosphorylation event are required for MAPK activation (Fig. 2). Neither SIPK nor WIPK are involved in the Avr9-dependent synthesis of AOS. CDPKs comprise a large family of multifunctional serine/threonine protein kinases unique to plants and some protists. A CDPK is induced in Cf-9 tobacco cells 5 min after addition of Avr9 (Fig. 2, Romeis et al., 2000) and in Cf-4 tobacco cells elicited with Avr4 (A Ludwig, S Rivas, JDG Jones and T Romeis, unpublished results). This protein shows a shift in electrophoretic mobility from 68 to 70 kDa and this transition from the ‘nonelicited’ to the ‘elicited’ CDPK form is caused by a trans-phosphorylation event. Interestingly, this elicitation-induced phosphorylation and interconversion of the protein correlates with an increase in enzymatic activity. Studies with pharmacological inhibitors indicate that the CDPK is independent of, or located upstream of, the signalling events leading to AOS production. In a more recent study, two related CDPK cDNAs (NtCDPK2 and NtCDPK3) were isolated from Cf-9 tobacco cell cultures after elicitation with Avr9. The CDPK transcripts are elevated after elicitation and hypo-osmotic stress. NtCDPK2 encodes the previously characterized 68/70 kDa Avr9/Cf-9-dependent CDPK (Romeis et al., 2001). The function of NtCDPK2 in plant defence was investigated using virus-induced gene silencing (VIGS) in Nicotiana benthamiana. NtCDPK2-silenced plants showed a reduced and delayed HR after elicitation in a gene-for-gene interaction that correlated with loss of NtCDPK2 and NtCDPK3 mRNA. cDNA amplified restriction fragment polymorphism (AFLP) analysis was used to identify transcripts whose expression is rapidly altered during the Avr9/Cf-9-mediated defence response in tobacco cell cultures (ACRE, for genes-Avr9/Cf-9 Rapidly Elicited; Durrant et al., 2000). Of 30 000 fragments analysed, 290 showed altered abundance and the majority were induced independently of AOS. A similar pattern of gene induction could be observed when de novo protein synthesis was inhibited suggesting expression of these genes is controlled post-transcriptionally. Analysis of ACRE genes showed they were components of signalling and protein kinases and et al., 2000). ACRE genes showed homology to known sequences such as binding protein which are known to be activated during the plant defence the tobacco resistance a and a protein. The role of these genes in the Cf-dependent HR is being investigated using in benthamiana. progress in understanding of the of Cf gene loci and the molecular basis for Cf gene has been in recent It is also that in understanding of the molecular basis for recognition specificity in this unique of R proteins will increase in the through the analysis of Cf-4 and Cf-9. However, despite the of a number of Cf and their cognate Avr the molecular mechanism of Avr perception by Cf proteins remains a A in the will be to determine how this mechanism to the subsequent signalling events that activate plant defences. This will on and genes that are required for Cf protein function, using or genetic such as in with a biochemical analysis of proteins. are to Jones and all of at the and the of for and for unpublished