The Cf-2 gene of tomato confers resistance to strains of the biotrophic pathogenic fungus Cladosporium fulvum carrying avirulence gene Avr2. To allow dissection of the biochemical mechanism of perception of AVR2 by Cf-2, we set out to clone the Avr2 gene. Here, we report the functional cloning of Avr2 cDNA, based on the induction of a hypersensitive response (HR) by the encoded AVR2 protein in Cf2 tomato plants. Analysis of strains of C. fulvum that are virulent on Cf2 tomato lines revealed various independent frameshift mutations in the Avr2 open reading frame (ORF) and a point mutation resulting in a premature stop codon. All modifications result in the production of truncated AVR2 proteins. Interestingly, an additional modification involves the insertion of a LINE-like element, Cfl1, in the Avr2 ORF. Cfl1 is the first LINE-like element identified in C. fulvum and provides the first example of loss of avirulence of a plant pathogen caused by insertion of a retrotransposable element in an Avr gene. Rcr3 represents an additional plant protein that is specifically required for Cf-2-mediated resistance. Analysis of two different rcr3 mutant Cf2 tomato plants revealed that their ability to respond to AVR2 with a HR correlates with their degree of resistance to AVR2-producing strains of C. fulvum. These data support a role for Rcr3 in the perception of AVR2 by Cf-2.
Host specificity in plant–pathogen interactions has been described by the gene-for-gene model (Flor, 1942; 1946). This model postulates that, for every dominant gene determining resistance in the host plant, there is a matching dominant gene conditioning avirulence in the pathogen. The simplest biochemical model for perception of an avirulence (AVR) protein by a resistant host plant involves direct interaction of the AVR protein with the matching resistance (R) gene product (Gabriel and Rolfe, 1990; Keen, 1990). Perception of an avirulence protein by the host plant elicits a hypersensitive response (HR), culminating in resistance. To date, a variety of R and Avr genes have been cloned (reviewed by Takken and Joosten, 2000; Van’t Slot and Knogge, 2002). However, the number of host–pathogen relationships for which a direct interaction between R and Avr gene products has been detected is still very limited (reviewed by Luderer and Joosten, 2001). In fact, for most gene-for-gene relationships studied so far, experimental evidence is more consistent with indirect perception of an AVR protein by an R protein than with a direct physical interaction be-tween these proteins (reviewed by Luderer and Joosten, 2001). Indirect perception of an AVR protein by an R protein implies that at least a third component is required for specific recognition.
The interaction between tomato (Lycopersicon spp.) and the biotrophic fungus Cladosporium fulvum complies with the gene-for-gene model. The tomato R genes Cf-2, Cf-4, Hcr9-4E, Cf-5 and Cf-9 confer resistance to strains of C. fulvum that carry the corresponding Avr gene, Avr2, Avr4, Avr4E, Avr5 and Avr9 respectively (reviewed by Joosten and De Wit, 1999). All Cf genes are predicted to encode a receptor-like protein with an extracellular LRR region, a transmembrane domain and a short cytoplasmic tail with no homology to known signalling domains (reviewed by Joosten and De Wit, 1999).
Growth of C. fulvum is confined to the apoplastic space of tomato leaves (De Wit, 1977). Two race-specific avirulence proteins of C. fulvum, AVR9 and AVR4, were isolated from apoplastic fluid, and the corresponding genes have been cloned (Scholtens-Toma and De Wit, 1988; Van Kan et al., 1991; Van den Ackerveken et al., 1992; Joosten et al., 1994). Consistent with the presence of the AVR proteins in the apoplastic space, both Avr9 and Avr4 encode a protein with a signal peptide for extracellular targeting (Van Kan et al., 1991; Joosten et al., 1994). The Avr9 gene encodes a precursor protein of 63 amino acids (aa), which is further processed by fungal and plant proteases into a 28-aa peptide that contains six cysteine residues (Van Kan et al., 1991; Van den Ackerveken et al., 1993). These cysteine residues were shown to be involved in disulphide bridges (Van den Hooven et al., 2001). In strains of C. fulvum that are virulent on Cf9 tomato plants, the Avr9 gene is absent (Van Kan et al., 1991). Avr4 encodes a 135-aa preproprotein, which upon secretion is N- and C-terminally processed, resulting in an 86-aa mature AVR4 protein (Joosten et al., 1994; 1997). The mature AVR4 protein contains eight cysteine residues, which are thought to be involved in disulphide bridges. Strains of C. fulvum that are virulent on Cf4 tomato plants show various single point mutations in the Avr4 open reading frame (ORF) that result in the production of unstable AVR4 isoforms (Joosten et al., 1997).
Extensive binding studies have been performed to detect whether direct interaction occurs between avirulence protein AVR9 and resistance protein Cf-9. Although various experimental approaches were followed, no binding between AVR9 and Cf-9 was detected (Luderer et al., 2001). These results indicate that perception of AVR9 by Cf-9 is indirect, which implies that at least a third component is required. The high-affinity binding site (HABS) for AVR9 that has been identified in several solanaceous species (Kooman-Gersmann et al., 1996) might be the third component that is required for perception of AVR9 by Cf-9. The binding affinity of mutant AVR9 peptides for the HABS was shown to correlate with their ability to induce a HR in tomato lines carrying Cf-9 (Kooman-Gersmann et al., 1998). Thus far, however, attempts to purify the HABS to clone its encoding gene by reversed genetics have not been successful (Van der Hoorn, 2001). Currently, binding studies are performed with AVR4 and Cf-4 to unravel whether a direct interaction between these proteins takes place (N. Westerink, unpublished data).
Recently, the Rcr3 gene (required for C. fulvumresistance 3), which is required for Cf-2-mediated resistance, was identified (Dixon et al., 2000). Rcr3 is specifically required for Cf-2-mediated resistance but not for Cf-5- and Cf-9-mediated resistance (Dixon et al., 2000). The Rcr3 gene has been isolated, and the predicted gene product shows homology to secreted cysteine proteases (J. Krueger, C. M. Thomas, C. Golstein, M. S. Dixon and J. D. G. Jones, personal communication). Cf-2 and Cf-5 are thought to activate the same defence signalling pathway upon perception of the matching AVR protein, and Rcr3 has therefore been proposed to play a role upstream of this common defence pathway and might represent the third component that is required for perception of AVR2 by Cf-2 (Dixon et al., 1996; 1998; 2000). The predicted extracellular localization of Rcr3 is con-sistent with this hypothesis. To allow dissection of the biochemical mechanism of perception of AVR2 by Cf-2, we set out to clone Avr2. Several attempts to purify the AVR2 protein from intercellular fluid obtained from a compatible interaction between tomato and an AVR2-producing strain of C. fulvum were unsuccessful (P. Vossen and M. Joosten, unpublished results). Therefore, we set out to clone Avr2 cDNA using a functional screen based on the HR-inducing activity of the AVR2 protein in Cf2 tomato plants (Takken et al., 2000).
Here, we report the functional cloning of Avr2 cDNA using a potato virus X (PVX)-based binary expression vector. By transformation of C. fulvum with a genomic clone of Avr2, we show that production of AVR2 confers avirulence of the fungus on Cf2 tomato plants. Strains of C. fulvum that are virulent on Cf2 tomato plants contain various modifications in the Avr2 ORF. These modifications all result in the production of truncated AVR2 proteins. Interestingly, one of the modifications involves the insertion of a LINE-like element [also known as non-long terminal repeat (non-LTR) retrotransposable elements]. Analysis of two different rcr3 mutant Cf2 tomato plants revealed that their ability to respond to AVR2 correlates with their degree of resistance to AVR2-producing strains of C. fulvum. The putative role of Rcr3 in the perception of AVR2 by Cf-2 will be discussed.
Functional cloning of Avr2 cDNA
To clone Avr2, a binary PVX-based cDNA library obtained from C. fulvum grown in vitro (Takken et al., 2000) was expressed in Cf2 tomato plants. The strain of C. fulvum that was used to create this library (strain 5a; Takken et al., 2000) is avirulent on Cf2 tomato plants, which indicates that it carries avirulence gene Avr2. The 9600 Agrobacterium tumefaciens colonies comprising the cDNA library were toothpick inoculated on leaflets of Cf2 tomato plants and Cf9 tomato plants were inoculated in parallel as a negative control. Upon inoculation of the plant, A. tumefaciens transfers the PVX expression vector to the plant cells and, subsequently, PVX particles are formed that spread around the inoculation site (Takken et al., 2000). When the virus expresses a C. fulvum cDNA that encodes the AVR2 protein, a spreading HR is ex-pected to be induced specifically in leaflets of Cf2 tomato plants. After a prudent selection, 61 putative positive clones that induced a HR on Cf2 tomato plants were identified in the first screen. Repeated screening revealed that two of these clones reproducibly induced a HR specifically upon expression in Cf2 plants. Sequencing revealed that both clones contained an identical ORF of 237 bp, which we tentatively designated Avr2. The clones were independent, as their 3′ ends were different. The sequence of the largest cDNA clone has been filed in the EMBL database under accession number AJ421629.
The Avr2 ORF encodes a predicted protein of 78 aa, which contains eight cysteine residues (Fig. 1). The AVR2 protein was analysed with the SIGNALP program (Nielsen et al., 1997), which predicted a 20-aa N-terminal signal sequence for extracellular targeting. The Avr2 cDNA se-quence does not have significant homology with se-quences present in databases or with other Avr genes of C. fulvum. Database searches with the predicted AVR2 protein did not reveal significant homology either.
Avr2 cDNA was used to screen a genomic library of C. fulvum (see Experimental procedures ). This screen resulted in the identification of eight independent clones ranging from about 12 kb to over 20 kb that all contained the complete Avr2 ORF. Partial sequencing of two of these clones and comparison of the sequence with the cDNA sequence of Avr2 revealed that the Avr2 gene contains one intron of 54 bp which is positioned after bp 159 of the Avr2 ORF. The genomic sequence of Avr2 was determined from 663 bp upstream to 652 bp downstream of the ORF. This sequence has been filed in the EMBL database under accession number AJ421628.
Expression of Avr2 results in a Cf-2-specific HR
The Avr2 cDNA was cloned from a library derived from an in vitro culture of C. fulvum. To examine the expression of the gene during growth of the fungus in planta, a strain of C. fulvum that is avirulent on Cf2 tomato plants was inoculated onto susceptible Cf0 tomato plants, which do not harbour any known functional Cf resistance gene. At several days after inoculation, infected leaves were harvested for RNA isolation. Figure 2 shows that the Avr2 gene of C. fulvum is expressed during growth of the fungus on tomato. The increase in the transcript level of the fungal actin gene over time indicates successful colonization of the leaves by C. fulvum.
To confirm further the specific HR-inducing activity of AVR2 upon its production in Cf2 tomato plants, the Avr2 ORF was amplified from the cDNA and cloned in a PVX-based expression vector, resulting in PVX::Avr2 (see Experimental procedures). Figure 3 shows that PVX-mediated expression of Avr2 results in severe systemic necrosis in Cf2 tomato plants, whereas inoculation of Cf2 tomato plants with PVX without an Avr2 insert (PVX::-) only results in typical systemic mosaic symptoms caused by the viral infection. Tomato plants carrying resistance gene Cf-4 and Hcr9-4E, Cf-5, Cf-9, Cf-ECP2 (Laugéet al., 1998) or Cf-ECP5 (Haanstra et al., 2000) that were inoculated with PVX::Avr2 or PVX::- only developed systemic mosaic symptoms (results not shown). Together, these data show that expression of Avr2 in tomato induces a Cf-2-specific HR.
Avr2 is the genuine avirulence gene matching Cf-2
To prove that Avr2 confers avirulence of C. fulvum on Cf2 tomato plants, a strain of C. fulvum that is virulent on Cf2 tomato plants (designated #31, producing all known AVR proteins except for AVR2, see below) was co-transformed with a genomic clone of Avr2 and a hygromycin selection marker (see Experimental procedures). Twenty-four hygromycin-resistant C. fulvum transformants were se-lected and inoculated onto Cf0 and Cf2 tomato plants. All transformants were still able to colonize Cf0 tomato plants, indicating that the transformation procedure did not affect their pathogenicity. In contrast to the recipient strain, however, 10 out of the 24 hygromycin-resistant transformants had become avirulent on Cf2 plants (see Fig. 4 for representative symptoms). Standard polymerase chain reaction (PCR) analysis (see Experimental procedures) revealed a strict correlation between the presence of the Avr2 transgene and the inability to colonize Cf2 tomato lines (results not shown). Transformants that were still virulent on Cf2 tomato plants only contained the hygromycin selection marker and lacked the Avr2 transgene. These results demonstrate that Avr2 confers avirulence of C. fulvum on Cf2 tomato plants, thereby rendering Avr2 a genuine avirulence gene.
To show that the C. fulvum Avr2 transformants produce AVR2 protein, apoplastic fluid was isolated from Cf0 tomato plants that were colonized by either transformants or the recipient strain. All apoplastic fluids induced necrosis upon injection into leaves of Cf9 plants (Fig. 5), indicating that the apoplastic fluids contain AVR9. The presence of AVR9 confirms that the apoplastic fluids were isolated from leaves that were successfully colonized by strain #31 or derived transformants. Only the apoplastic fluids that were isolated from leaves that were colonized by C. fulvum transformants that contain the Avr2 transgene induced necrosis upon injection in leaves of Cf2 tomato plants (Fig. 5). These results show that only the C. fulvum transformants that contain the Avr2 transgene and had become avirulent on Cf2 tomato plants secrete AVR2 protein in the apoplastic space during colonization of tomato leaves. Altogether, these data show that transformation of C. fulvum with Avr2 results in avirulence of the fungus on Cf2 tomato plants as a result of the production of the AVR2 protein.
Cladosporium fulvum circumvents Cf-2-mediated resistance by various modifications in Avr2
Southern blot analysis of genomic DNA of various strains of C. fulvum revealed that Avr2 is present as a single-copy gene in the genome of strains that are avirulent on Cf2 tomato plants (results not shown). However, a single fragment hybridizing with Avr2 is also present in the genome of strains that are virulent on Cf2 tomato plants, (results not shown). Northern blot analysis of RNA isolated from compatible interactions involving several strains of C. fulvum revealed that, except for two strains (#31 and #41) for which no hybridization was detected (see below), Avr2-hybridizing transcripts are produced by all strains (results not shown). As strains that are virulent on Cf2 tomato plants are not expected to produce a functional AVR2 protein, the Avr2-homologous sequence of all C. fulvum strains present in our collection that are virulent on Cf2 plants was PCR amplified from the genome (using standard conditions; see Experimental procedures) and sequenced. For all strains that are virulent on Cf-2 tomato plants, a mutation was detected in the Avr2 ORF (listed in Table 1). Most modifications are frameshift mutations, resulting from single nucleotide insertions or deletions, which were identified at several sites in the Avr2 ORF. Furthermore, a point mutation was found in four strains, resulting in the introduction of a premature stop codon. All the modifications of the Avr2 ORF that were identified are predicted to result in a truncated AVR2 protein (see Table 1). We therefore conclude that C. fulvum circumvents Cf-2-mediated resistance by producing truncated AVR2 proteins. Compared with the wild-type AVR2 protein, the truncated AVR2 proteins lack at least three cysteine residues (see Table 1) and are therefore expected to be unstable and/or non-functional. The sequence of the Avr2 ORF of 15 strains that are avirulent on Cf2 tomato plants was identical to the sequence that was originally identified. Furthermore, sequencing revealed that, compared with the initially identified genomic sequence of Avr2, some strains lack 3 nucleotides (TGA) at position 27 to 29 of the intron of Avr2. This deletion apparently does not influence the splicing of the intron, as it is present in strains that are virulent on Cf2 plants as well as in strains that are avirulent on Cf2 plants.
Table 1. Mutations in the Avr2 ORF of C. fulvum strains that are virulent of Cf2 tomato plants.
Insertion of a LINE-like element in Avr2 results in circumvention of Cf-2-mediated resistance for two strains of C. fulvum
PCR amplification of the Avr2 gene using standard conditions did not result in a product for the two strains (#31 and #41) for which an Avr2 transcript was not detected by Northern analysis. However, the application of PCR conditions optimized for amplification of long DNA fragments (see Experimental procedures) resulted in a PCR product for these two strains of about 5.3 kb, which is almost 5 kb longer than the product generated from all other strains. The 5.3 kb PCR fragments were initially sequenced partially (see Experimental procedures), and both strains turned out to contain an insert that was located after bp 55 of the Avr2 ORF. The inserts present in both strains were identical for the parts that were sequenced (785 bp of the 5′ end and 550 bp of the 3′ end of the insert). Restriction analysis of the insert with 12 different restriction enzymes with a 6 bp recognition site did not reveal any polymorphisms either. After the insertion, a duplication was identified of 15 bp of the Avr2 ORF preceding the insertion site. From C. fulvum strain #31, the 5 kb insert in the Avr2 ORF was sequenced completely (single stranded), using a primer-walking approach. BLASTX homology searches (Altschul et al., 1997) revealed that the insert sequence contains a region of which the translation product has homology with gag proteins and another region of which the translation product has homology with reverse transcriptases (see Fig. 6 for a schematic representation). The orientation of the protein-encoding regions of the insert is opposite to that of the Avr2 ORF. However, as the insert was only sequenced single stranded, a reliable determination of the exact ORFs is not feasible. The overall features of the insert show resemblance to non-long terminal repeat (non-LTR) retrotransposable elements, which are also called LINE-elements. The insert was therefore named Cfl1, for C. fulvumLINE-like element 1. Translation of nucleotides 1033 to 1112 of Cfl1 reveals the presence of three CysX2CysX4HisX4Cys motifs, of which the second and third motifs are slightly degenerate. This is a typical motif for the first ORF of LINE-like elements (Cambareri et al., 1994). The region of Cfl1 for which the encoded protein shows homology with reverse transcriptases shows the highest homology with a reverse transcriptase of Tad3-2 and Tad1-1, which are LINE-like elements of Neurospora crassa (Cambareri et al., 1994). Moreover, as mentioned above, the insertion site of Cfl1 showed a 15 bp target site duplication. Altogether, these data indicate that Cfl1 represents a novel LINE-like element of C. fulvum. The sequence of Cfl1 has been filed in the EMBL database under accession number AJ421630.
Specific recognition of the AVR2 protein by Cf2 tomato and the requirement of Rcr3
The Cf-2 resistance locus contains two functional resist-ance genes, Cf-2.1 and Cf-2.2, which differ by only three aa (Dixon et al., 1996). Because of this feature, it was anticipated that they both confer recognition of the same avirulence protein, AVR2. Thus far, however, it could not be excluded that they each recognize a different AVR protein. For this reason, Cf0 tomato plants, transformed with either Cf-2.1 or Cf-2.2 (Dixon et al., 1996), were inoculated with PVX::Avr2. Figure 7 shows that both transgenic lines developed severe necrotic symptoms, whereas the untransformed Cf0 plant only developed systemic mosaic symptoms typical of the viral infection. These data prove that both Cf-2.1 and Cf-2.2 mediate AVR2 recognition.
The Rcr3 gene is specifically required for Cf-2-mediated resistance (Dixon et al., 2000). Rcr3 mutants have been identified that result in either partial loss of Cf-2-mediated resistance (rcr3-1) or complete loss of Cf-2-mediated resistance (rcr3-3) (Dixon et al., 2000). Inoculation of both rcr3 mutant Cf2 lines with PVX::Avr2 did not result in visible HR symptoms at 14 days after inoculation (results not shown), whereas Cf2 plants with a functional Rcr3 gene show severe necrosis at this time point (see Fig. 3). At 22 days after inoculation, however, chlorosis and slight necrosis was observed in rcr3-1 Cf2 plants inoculated with PVX::Avr2, whereas only typical viral mosaic symptoms were observed in rcr3-3 Cf2 plants (Fig. 8). These experiments show that the ability of rcr3 mutant Cf2 plants to respond to AVR2 with a HR correlates with their degree of resistance to AVR2-producing strains of C. fulvum.
Cloning and characterization of Avr2
Avr2 cDNA was cloned based on the specific HR-inducing activity of the encoded protein in Cf2 tomato plants, according to the method developed by Takken et al. (2000 ). To our knowledge, this is the first novel Avr gene that has been cloned using this strategy. The predicted AVR2 protein is a small, cysteine-rich, secreted protein ( Fig. 1 ). The latter is consistent with the presence of AVR2 protein in apoplastic fluid ( De Wit and Spikman, 1982 ; Fig. 5 ). By analogy with AVR4 and AVR9 ( Van Kan et al., 1991 ; Van den Ackerveken et al., 1993 ; Joosten et al., 1994 ; 1997 ), the AVR2 protein might be processed further by plant and/or fungal proteases after cleavage of the predicted signal peptide. It was not possible, however, to determine the molecular weight of the AVR2 protein present in apoplastic fluid by Western analysis, as attempts to raise antibodies against a synthetic peptide derived from AVR2 were not successful.
Strain #31 of C. fulvum, which is virulent on Cf2 plants because of insertion of a LINE-like element (Cfl1) in the Avr2 ORF, was transformed with a genomic clone of Avr2. The Avr2 ORF containing the Cfl1 insert is too long to be amplified with Avr2-specific primers using standard PCR conditions. As a result, PCR analysis of the transformants using standard conditions only revealed the presence of the Avr2 transgene. Based on previous transformation ex-periments with C. fulvum (Van den Ackerveken et al., 1992; Joosten et al., 1994), the transformants are expected to contain multiple copies of the Avr2 transgene. PCR analysis using an Avr2-specific primer combined with a Cfl1-specific primer revealed that all transformants still contain the Cfl1 insert in the endogenous Avr2 gene (R. Luderer, unpublished results). The latter validates that the transformants have become avirulent on Cf2 plants as a result of incorporation of the Avr2 transgene. These results show that AVR2 is not only inducing a HR in Cf2 tomato plants (Fig. 3), but that expression of Avr2 actually confers avirulence of C. fulvum on Cf2 tomato plants (Fig. 4).
Various modifications were identified in the Avr2 ORF of strains of C. fulvum that are virulent on Cf2 tomato plants. These modifications are all predicted to result in a truncated AVR2 protein (Table 1). The truncated AVR2 proteins all lack at least three cysteine residues compared with the predicted mature AVR2 protein. For AVR4 and AVR9, it has been shown that the substitution of one single cysteine residue severely compromises the HR-inducing activity of the protein (Joosten et al., 1997; Kooman-Gersmann et al., 1997). Moreover, it has been shown that the cysteine residues of AVR9 are involved in disulphide bridges, which are important for the structure of the protein (Van den Hooven et al., 2001). It is therefore not surprising that the truncated AVR2 proteins are not functional as an AVR protein.
Besides their function as avirulence proteins, AVR proteins are thought to contribute to virulence of the pathogen as well (Gabriel, 1999; Kjemtrup et al., 2000; White et al., 2000; Van’t Slot and Knogge, 2002). The truncated AVR2 proteins are probably also compromised in their potential virulence function. Considering the large number of independent, different Avr2 mutations identified in our worldwide C. fulvum collection, the AVR2 protein apparently is not crucial for virulence of C. fulvum. This is consistent with the fact that we did not observe visible differences between growth of C. fulvum Avr2 transformants and the recipient strain on Cf0 tomato plants. The Avr9 gene is absent in strains of C. fulvum that are virulent on Cf9 plants, indicating that AVR9 is dispensable for virulence of C. fulvum as well (Van Kan et al., 1991). This was confirmed under laboratory conditions by disrupting the Avr9 gene in C. fulvum (Marmeisse et al., 1993). It should be taken into account, however, that AVR9 might play a role in virulence of C. fulvum under environmental conditions in the field (Marmeisse et al., 1993). Strains of C. fulvum that are virulent on Cf4 plants contain mutations in the Avr4 ORF (Joosten et al., 1994; 1997). In contrast to the mutations identified in Avr2, the mutations identified in Avr4, except for one, result in single aa substitutions (Joosten et al., 1994; 1997). Although the avirulence function of these AVR4 isoforms is affected, they might still contribute to the virulence of the fungus. From this point of view, the modifications identified in the Avr2 ORF are intrinsically different from most of the modifications identified in the Avr4 ORF. Whether the large number of mutations in Avr2 are the result of the dispensability of AVR2 for virulence of the fungus or whether they are caused by a severe selection pressure by tomato plants carrying the Cf-2 resistance gene cannot be concluded as, for most of the strains in our collection, it is not known under which conditions they have been isolated.
Cfl1, a LINE-like element of C. fulvum
Two strains of C. fulvum present in our collection contain a Cfl1 insertion in their Avr2 ORF (Fig. 6). Cfl1 is the first LINE-like element (also known as non-LTR retrotransposable elements) identified in C. fulvum. Cfl1 is located at the same position in the Avr2 ORF in both strains, with an identical target site duplication. These results indicate that the two strains are related, which is consistent with the fact that they were both isolated on the North American continent. Both strains produce all known elicitor proteins except for AVR2; however, they are different with respect to growth rate and colour of their spores. As a sexual stage has never been observed for C. fulvum, the action of transposable elements is thought to contribute to the genetic variation within the C. fulvum population. Whether the identified Cfl1 element of C. fulvum is still mobile remains to be determined.
To our knowledge, this is the first report of gain of virulence of a plant pathogen towards a specific host genotype as a result of insertion of a retrotransposable element in an Avr gene. As mentioned above, circumvention of recognition of AVR proteins has been reported to occur by absence of the Avr gene, by point mutations in the Avr gene and by frameshift mutations (reviewed by Van’t Slot and Knogge, 2002). Furthermore, insertion of mobile elements that transpose at the DNA level, either in the ORF of an Avr gene or in its promoter, has been reported to result in gain of virulence of plant pathogens towards specific host genotypes (Kearney and Staskawicz, 1990; Kang et al., 2001).
Specific recognition of AVR2 by Cf2 plants
Our results demonstrate that AVR2 recognition is mediated by each of the Cf-2 genes (Fig. 7). Furthermore, we revealed a correlation between the ability of two different rcr3 mutant Cf2 tomato lines to respond to AVR2 with a HR and their degree of resistance to AVR2-producing strains of C. fulvum (Fig. 8). The next challenge will be to determine whether the Rcr3 protein actually plays a role in perception of AVR2 by Cf-2 or whether it has another function in AVR2/Cf-2-mediated resistance. We are currently using the yeast Pichia pastoris to produce AVR2 protein that will be used to perform binding studies with Cf-2 and/or Rcr3. These experiments should reveal whether Rcr3 represents the third component that is involved in perception of AVR2 by Cf-2. As suggested by Dixon et al. (2000), Rcr3 might represent the virulence target of AVR2 that is guarded by the Cf-2 protein. Considering its predicted protease activity, Rcr3 might be part of a basic defence mechanism of the plant that is inhibited by AVR2. However, Rcr3 might also be involved in the modification of either AVR2 or Cf-2 to allow the specific recognition of the elicitor to occur. With the cloning of Avr2, all three components have become available to allow the dissection of the biochemical mechanism that is underlying the perception of AVR2 by Cf-2. These experiments can reveal important insights into the perception of AVR proteins by R gene products.
Fungal and plant material
Cladosporium fulvum strains were subcultured on potato-dextrose agar at 22°C. Conidia from 10-day-old cultures were used for plant inoculations as described by De Wit (1977 ). Transformation of C. fulvum was performed as described by Van den Ackerveken et al. (1992 ). C. fulvum was co-transformed with pAN7-1 ( Punt et al., 1987 ), which contains a hygromycin resistance gene, to allow selection of transformants. For DNA isolation, C. fulvum was grown in liquid B5 medium as described by De Wit and Roseboom (1980 ).
Near-isogenic lines of tomato cultivar Moneymaker were grown in the greenhouse in a day–night regime of 16 h, 21°C light and 8 h, 19°C dark at 60% relative humidity. The Moneymaker genotype carrying no known C. fulvum resistance genes is referred to as Cf0 tomato, whereas Cf2 tomato refers to the near-isogenic line carrying the Cf-2 resistance locus. Other Moneymaker genotypes are also designated by the resistance gene they carry.
Functional screening of a cDNA library of C. fulvum
A cDNA library of strain 5a of C. fulvum (Takken et al., 2000), consisting of 9600 Agrobacterium tumefaciens colonies each containing a binary PVX-based vector with a cDNA insert, was toothpick inoculated on Cf2 and Cf9 tomato plants according to the procedure described by Takken et al. (2000). The prudently selected 61 putative positives identified by the first screen were rescreened on various tomato genotypes. From two clones, which reproducibly induced a specific HR upon inoculation onto Cf2 tomato plants, the insert sequence was obtained according to the procedure described by Takken et al. (2000).
For standard PCR analysis, C. fulvum DNA was isolated according to the procedure of Cenis (1992) after growth of the fungus for 5 days in liquid B5 medium. After DNA isolation, the Avr2 ORF was PCR amplified (45 s 94°C, 45 s 60°C, 1 min 72°C, 30 cycles) using AmpliTaq DNA polymerase (Perkin-Elmer) with primers AVR2F (5′-CTCCGCCCAACA TTCGAC-3′) and AVR2R (5′-CTCTTCTCACACTGTCTCC-3′), which flank the Avr2 ORF. The PCR products were sequenced using the AVR2R primer.
For PCR analysis optimized for obtaining long PCR products, C. fulvum DNA was isolated by grinding freeze-dried mycelium obtained from a liquid culture in an Eppendorf tube. After homogenization in 1 ml of extraction buffer (50 mM EDTA, 0.2% SDS, pH 8.5), 10 μl of proteinase K (10 mg ml−1) was added, and the mixture was incubated for 15 min at 70°C. Subsequently, 100 μl of 5 M KAc was added, and the mixture was left on ice for 30 min. After centrifugation (15 min at 15 600 g), the supernatant was treated with RNase (1 μl of 10 mg ml−1), for 30 min at 37°C, extracted twice with phenol–chloroform–isoamyl alcohol (25:24:1) and once with chloroform–isoamyl alcohol (24:1). After extraction, 1/10th volume of 3 M NaAc and 0.5 volume of 7.5 M NH4Ac were added to the upper phase, and the mixture was left on ice for 15 min. After the addition of 1 volume of isopropanol, the mixture was incubated for 5 min at room temperature and centrifuged (15 min at 15 600 g). The precipitated DNA was washed with 70% EtOH and resuspended in TE (10 mM Tris-HCl, pH 8, 1 mM EDTA, pH 8).
This DNA (12 ng) was subsequently used for PCR using Expand polymerase mixture (Boehringer Mannheim), according to the protocol of the manufacturer. Amplification [10 cycles of 15 s at 95°C, 45 s at 60°C, 8 min at 68°C, followed by 20 cycles with an addition of 5 s extension (68°C) time per cycle] with AVR2F and AVR2R resulted in a fragment of about 5.3 kb for strains #31 and #41, which was inserted into the pGEM-T easy vector (Promega). Initially, the fragments were sequenced with AVR2F and AVR2R, resulting in a partial sequence. The 5.3 kb fragment derived from strain #31 was sequenced completely following a primer-walking approach (Baseclear).
Screening of a genomic library of C. fulvum
The Avr2 cDNA sequence was amplified with AVR2F and AVR2R using standard PCR conditions. The resulting 334 bp Avr2 fragment was used as a probe to screen a λBlueStar (Novagen) genomic library of C. fulvum. Eight independent clones, ranging from about 12 kb to over 20 kb, were identified that contained a full-length Avr2 ORF. The position of the Avr2 ORF in these clones was estimated by PCR optimized for long PCR products (see above) with vector-specific and Avr2-specific primers. Two clones containing about 10 kb and 11 kb at the 5′ end of the Avr2 ORF and 4.5 kb at the 3′ end of the Avr2 ORF were used for transformation of C. fulvum.
RNA isolation and Northern blotting
Total RNA was isolated from C. fulvum-infected tomato leaves according to the hot-phenol procedure (Extract-A-Plant RNA isolation kit; Clontech). From each sample, 15 μg of glyoxal-denatured RNA was separated on a 1.4% agarose gel and transferred to Hybond-N+ membrane (Amersham). Prehybridization and hybridization with 32P-labelled probes were performed in a phosphate buffer modified after Church and Gilbert (1984) (0.5 M phosphate buffer, pH 7.2, 7% SDS, 1 mM EDTA) at 65°C. Blots were washed (0.5× SSC, 0.5% SDS, 65°C), and X-omat AR film (Kodak) was exposed to the blots at −80°C.
Construction of PVX::Avr2, in vitro transcription and plant inoculation
For transient expression of Avr2 in planta, recombinant potato virus X (PVX) expressing Avr2 (PVX::Avr2) was constructed. The Avr2 cDNA was PCR amplified (1 min at 94°C, 1 min at 50°C, 2 min at 72°C for 30 cycles) with Pfu DNA polymerase (Stratagene), using primers that introduce a ClaI restriction site (underlined) 5′ of the start codon (5′-ATG CAATCGATATGAAGCTCTTCATACTGACC-3′) and 3′ of the stop codon (5′-TACGTATCGATTCATCAACCGCAAAGACC-3′) of the Avr2 ORF. The resulting PCR fragment was digested with ClaI and ligated into ClaI-digested, dephosphorylated PVX plasmid vector (Chapman et al., 1992), 3′ of the duplicated coat protein promoter. The sequence of PVX::Avr2 was verified, and a correct construct was used for in vitro transcription and inoculation of 3- to 4-week-old tomato plants, as described by Kooman-Gersmann et al. (1997).
We thank Xinzhong Cai, Suzan Gabriëls, Renier van der Hoorn, Camiel de Jong, Maarten de Kock, Marco Kruijt, Maita Latijnhouwers and Nienke Westerink for their help with toothpick inoculation of the cDNA library onto tomato plants. Camiel de Jong and Nienke Westerink are also acknowledged for their help with C. fulvum inoculation of tomato plants. We thank Bert Essenstam (Unifarm, Wageningen, The Netherlands) for excellent plant care. W. van Hof (Beeldgroep, Wageningen, The Netherlands) is acknowledged for photographing the plants. Richard Oliver (SABC, Murdoch University, Perth, Australia) kindly provided the genomic library of C. fulvum. We thank the Jones laboratory (Sainsbury Laboratory, Norwich, UK) for making available the Cf-2 transgenic tomato lines and the rcr3 mutant tomato lines. R. Luderer was supported by a grant from the Dutch Foundation for Earth and Life Sciences (ALW), project SLW 805-18.231.