In incompatible interactions growth of C. fulvum is arrested soon after penetration of the leaf of a resistant genotype (Lazarovits and Higgins, 1976a; De Wit, 1977). Fungal growth is restricted to the area of a few mesophyll cells, while the cells contacted by hyphae or in advance of the growing hyphae often die. Fairly accurate measurements of fungal biomass were achieved by inoculating tomato with a transformant of C. fulvum constitutively expressing f3-g1ucuronidase (GUS) (Oliver et al., 1993). By inoculating near-isogenic lines of tomato homozygous or heterozygous for different Cf genes with race 4 of C. fulvum, expressing GUS, it was found that the different Cf genes confer distinct abilities to restrict C. fulvum infection (Hammond-Kosack and Jones, 1994). However, one should keep in mind that the effectiveness of a given Cf gene is also dependent on the amount and stability of the race-specific elicitor produced by the matching Avr gene. In addition, it was found that all Cf genes show incomplete dominance. Significantly less growth of C. fulvum occurred in plants that are homozygous, compared to plants that are heterozygous for a given Cf gene.
1. Callose Deposition and Cell Wall Appositions
One of the early defence responses observed in incompatible interactions involves deposition of callose and cell wall thickening. Callose deposition is predominantly associated with incompatible interactions, but under suboptimal conditions for disease development it also occurs in compatible interactions (Lazarovits and Higgins, 1976a;
De Wit, 1977). Callose is deposited between the plant cell wall and plasma membrane.
Cell wall thickening usually ocurrs at the interface between intercellular hyphae and mesophyll cells. Thickened cell walls contain polyphenolic compounds (Lazarovits and Higgins, 1976a). When conidia of virulent or avirulent races of C. fulvum are injected into the intercellular space of tomato leaves the conidia and the primary germ tube invoke strong cell wall appositions containing predominantly callose. Secondary germ tubes ceased to invoke callose deposition and cell wall appositions in susceptible plants, but continued to do so in resistant plants (Higgins, 1982). Non-specific elicitors which are present in the cell walls of conidia and primary germ tubes possibly induce those responses in both susceptible and resistant plants. However, enzymes of plant origin present in the apoplast might degrade these non-specific elicitors, explaining why secondary hyphae of virulent races no longer induce these responses in susceptible plants while avirulent races continue to induce these responses. An additional plant response associated with incompatible interactions is the enlargement of host cells that are in contact with or slightly in front of the advancing hyphae (Lazarovits and Higgins, 1976a; Hammond-Kosack and Jones, 1994).
2. Phytoalexins
In tomato leaves inoculated with C. fulvum, accumulation of phytoalexins occurs in incompatible interactions (De Wit and Flach, 1979; De Wit and Kodde, 1981a).
The phytoalexins detected in leaves are not sesquiterpenes, the common phytoalexins detected in solanaceous plants, but the poly acetylene phytoalexins, falcarinol and falcarindiol (De Wit and Kodde, 1981a). The latter two phytoalexins accumulate quicker
A Case Study: Cladosporium fulvum-tomato Interaction 59
and usually to a higher level in incompatible interactions compared to compatible ones (De Wit and Flach, 1979). Pericarp tissue of green tomato fruits responds with accumulation of the sequiterpene rishitin, after treatment with conidia of virulent or avirulent races of C. fulvum, or non-specific elicitors isolated from culture filtrates or cell walls of virulent or avirulent races (De Wit and Roseboom, 1980; De Wit and Kodde, 1981b). However, accumulation of rishitin in green tomato fruit tissue of genotypes carrying different Cf genes after treatment with those elicitors, is similar in compatible and inompatible interactions. Thus, the accumulation of polyacetylene phytoalexins in leaves is race-specific, but the accumulation of rishitin in tomato fruits is not (De Wit and Flach, 1979; De Wit and Roseboom, 1980; De Wit and Kodde, 1981a,b)
3. The Hypersensitive Response
In incompatible interactions, after penetrating stomata, hyphae that are in contact with mesophyll cells induce necrosis which is considered a hypersensitive response (HR) (Lazarovits and Higgins, 1976a; De Wit 1977). In incompatible interactions, involving different Cf-genes, necrosis was sometimes observed rather late (Hammond-Kosack and Jones, 1994). AF isolated from leaves that ar~ fully colonized by virulent races of C. fulvum, carrying different genes for avirulence, induced HR after injection in Cflines that contain a matching resistaDce gene. HR induced by race-specific elicitors present in these AFs, will be discussed later. HR that occurs in the natural plant-pathogen interaction is very difficult to quantify. As will be discussed later, the outcome of a particular race-genotype interaction depends on the stability and activity of both the race specific elicitor and the receptor. Plants that are homozygous for a particular Cf gene respond with HR to a two-fold lower race-specific elicitor concentration as compared with with heterozygous plants. This indicates that the receptor concentration might be a limiting factor in inducing HR (Hammond-Kosack and Jones, 1994).
4. Pathogenesis-Related Protein Accumulation
Early accumulation of several host-encoded pathogenesis-related (PR) proteins in the apop!ast is characteristic for incompatible interactions (De Wit and Van der Meer, 1986; De Wit et al., 1986; Joosten and De Wit, 1989). Biochemical characterization revealed that many of these proteins are 1,3-J3-glucanases and chitinases which are hydrolytic enzymes potentially able to degrade hyphal walls that contain 1,3-J3-glucans and chitin (Joosten and De Wit, 1989; Bol et al., 1990). The cDNAs encoding the various basic and acidic 1,3-J3-glucanases and chitinases have been cloned (Van Kan et aI., 1992; Danhash et aI., 1993) and the expression of the genes (Van Kan et al., 1992; Danhash et aI., 1993; Wubben et aI., 1994a) and localization of the transcripts and the encoded proteins have been studied (Wubben et al., 1992; Wubben et al., 1994b). Although the early accumulation of hydrolytic enzymes in the incompatible interaction coincides with the expression of HR and arrest of fungal growth, it is not clear whether the induced PR proteins play a decisive role in resistance of tomato against C. fulvum (Joosten et al., 1995).
III. Non-Specific and Race-specific Elicitors of Cladosporium fulvum A. NON-SPECIFIC ELICITORS
The search for race-specific elicitors in the 1970s was mainly carried out with C. Julvum grown in vitro. Virulent and avirulent races were grown in stationary or shake cultures and culture filtrates and cell wall fractions were analysed for the presence of race-specific elicitors based on different bio-assays. Assays were based on electrolyte leakage, necrosis and callose deposition (Van Dijkman and Kaars Sijpesteijn, 1973;
Dow and Callow, 1979a,b; Lazarovits and Higgins, 1979; Lazarovits et ai., 1979) or phytoalexin accumulation (De Wit and Roseboom, 1980; De Wit and Kodde, 1981a,b).
In all cases elicitor activity was observed, but this activity was never race-specific.
The elicitors appeared to be glycoproteins of different sizes containing glucose, galactose and mannose. They were called proteoglucogalactomannans (De Wit and Kodde, 1981 b) and occur in culture filtrates as well as cell walls. Their presence and significance during growth of C. fulvum in planta is unclear. If produced during the infection process the glycoproteins are probably quickly degraded by enzymes of plant origin present in the apoplast. It was found that AF obtained from healthy plants could inactivate the necrosis-inducing activity of these glycoprotein elicitors (Higgins, 1982;
Peever and Higgins, 1989a). So far, the biological relevance of non-specific elicitors of C. fulvum can be questioned as it is unknown whether they occur in infected plants.
If they playa role, it is probably only during the late stages of infection when tomato leaves become necrotic.
B. RACE-SPECIFIC ELICITORS AND THEIR ENCODING GENES 1. Isolation of Race-Specific Elicitors and Cloning of their Encoding Genes A major breakthrough in research on the C. Julvum-tomato interaction was the discovery of race-specific elicitors, the inducers of HR present in AF of C. fulvum-colonized tomato leaves (De Wit and Spikman, 1982; De Wit et ai., 1984, 1985; Higgins and De Wit, 1985). Injection of such AF into healthy leaves of near-isogenic lines containing different Cf genes, resulted in the differential induction of an HR. Proteinaceous compounds which induceq HR on resistant tomato cultivars were correlated with the presence of avirulence genes in the races of C. fulvum used for inoculations.
The first race-specific peptide elicitor purified from AF of C. fulvum-infected tomato leaves was a peptide of28 amino acids (Scholtens-Toma and De Wit, 1988; De Wit, 1992).
The purified peptide elicitor specifically induced HR on tomato genotypes that carried the matching Cf-9 resistance gene. Races that are virulent on tomato genotype Cf-9 do not produce the elicitor (Van Kan et ai., 1991). In a similar way, the race-specific elicitor A VR4, which induces HR on Cf4 tomato genotype, has been isolated (Joosten et ai., 1994). From both proteinaceous elicitors the amino acid sequence has been determined and by reverse genetics the encoding avirulence (A vr) genes A vr9 and A vr4, have been cloned (Van Kan et ai., 1991; Van den Ackerveken et al., 1992; Joosten et ai., 1994). The cloned genes revealed that both elicitors present in C. Julvum-infected tomato plants are post-translationally processed. The Avr9 gene encodes a protein of
A Case Study: Cladosporium fulvum-tomato Interaction 61
63 amino acids including a signal sequence of 23 amino acids. In culture filtrates of transformants of C. fulvum that constitutively produce the A VR9 elicitor, predominantly N-terminally processed peptides of 32, 33 or 34 amino acids are present (Van den Ackerveken et al., 1993a). When these latter peptides were incubated with AF from healthy tomato leaves, they were further processed into the mature elicitor of 28 amino acids, indicating that plant proteases are required for the final processing (Van den Ackerveken et al., 1993a). The A vr4 gene encodes a protein of 135 amino acids, including a signal peptide of 18 amino acids (Joosten et aI., 1994). Here, the mature peptide elicitor is processed at both the N- and C-terminus leaving a mature peptide elicitor of 86 amino acids (Joosten et aI., 1997). Although there is clear evidence for the presence of proteinaceous elicitors in AF of infected leaves that induce HR on Cf-2 and Cf5 genotypes of tomato, the matching A VR2 and A VR5 elicitors have not yet been characterized.
2. Regulation of Avirulence Genes Avr4 and Avr9
In vitro-grown C. fulvum hardly produces A VR4 or A VR9 elicitors. RNA gel blot analysis indicated that expression of the Avr4 and Avr9 genes of C. fulvum is specifically induced in planta. Accumulation o( mRNAs encoding the race-specific elicitors correlates with an increase in fungal biomass in tomato leaves during pathogenesis in compatible interactions (Van Kan et aI., 1991; Joosten et aI., 1994).
Studies on the expression of the Avr4 and Avr9 genes in planta, using transformants of C. fulvum carrying Avr promoter-GUS fusions, showed activation of the promoter in hyphae immediately after penetration of stomata, with expression levels particularly high in mycelia growing in the vicinity of the vascular tissue (Van den Ackerveken et aI., 1994, Joosten et aI., 1997).
The promoter of the Avr4 gene does not contain motifs homologous to sequences known for binding of regulatory proteins. So far, the Avr4 gene can hardly be induced under different growth conditions in vitro. In contrast, the Avr9 gene could be induced under limiting concentrations of nitrogen when grown in liquid medium (Van den Ackerveken et aI., 1994, P.J.G.M De Wit, unpublished; S. Snoeijers, Wageningen Agricultural University, personal communication). Analysis of the Avr9 promoter sequence revealed six copies of the hexanucleotide TAGATA (Van den Ackerveken et aI., 1994), which has been identified as the recognition site of the NIT2 protein, a transcription factor which positively regulates gene expression under nitrogen-limiting conditions ir Neurospora crassa and many other filamentous fungi (Marzluf, 1996).
Therefore, the expression of the Avr9 gene is possibly regulated in a similar way, by a C. fulvum-homologue of the NIT2 protein. Indeed deleting a number of the T AGA T A sequences abolished the induction of the A vr9 gene under conditions of low nitrogen concentrations in vitro (PJ.G.M. De Wit, unpublished; S. Snoeijers, Wageningen Agricultural University, personal communication). Two of the six TAGATA sequences are essential for Avr9 regulation under nitrogen-limiting conditions.
3. Avr Alleles in races of C. fulvum Virulent on Tomato Genotypes Cf4 and Cj9 Genomic DNA gel blot analysis, using Avr4 cDNA as a probe, did not reveal any differences between races of C. fulvum avirulent or virulent on tomato genotype Cf4.
All races contain a homologous, single copy gene, not displaying any restnctlOn fragment length polymorphism (Joosten et aI., 1994). Although none of the virulent races produce biologically active A VR4 elicitor, they produce transcripts that hybridize to an Avr4 cDNA probe, proving that those races contain alternative alleles of Avr4 (Joosten et aI., 1994,1997). Sequencing these alleles, showed single base pair mutations in the open reading frame (ORF) encoding the mature A VR4 protein, resulting in single amino acid changes in the AVR4 elicitor (Fig. lB). In one case a frame shift mutation was observed leaving only 13 amino acids at the N-terminus of the A VR4 peptide intact (Joosten et aI., 1994; Joosten et al., 1997). The single amino acid changes in the A VR4 protein cause the peptide to become unstable and probably more sensitive to proteolytic degradation which would prevent it reaching a matching receptor in sufficiently high concentration. Expression of the mutant alleles in the PYX expression system indeed revealed that most still encode an active elicitor molecule which, however, has a much shorter active half life than the peptide elicitor produced by the functional avirulence allele (Joosten et aI., 1997).
Genomic DNA gel blot analysis of races of C. fulvum virulent on tomato genotype Cf9 revealed that in all these races the Avr9 gene is absent (Fig. lA). Thus, there is always a strict correlation between virulence of races on Cf9 genotypes of tomato and absence of the Avr9 gene (Van Kan et aI., 1991).
4. Structure and Function of A VR4 and A VR9 Peptides
Avirulence genes Avr4 and Avr9 both encode relatively small globular peptide elicitors which contain 8 and 6 cysteine residues, respectively. The structure of the AVR9 peptide has been extensively studied by IH-NMR (Vervoort et al., 1997). All cysteines form . disulfide bridges, which are required for elicitor activity. The AVR9 peptide consists of three anti-parallel (-strands forming a rigid region of (-sheet. A VR9 is a member of the family of cystine-knotted peptides, in which the 6 cysteine residues form a typical cystine knot found in several small proteins such as proteinase inhibitors, ion channel blockers and growth factors (Pallaghy et at., 1994). However, structural homology most probably does not represent functional homology.
In contrast to the Avr9 gene, which is absent in all races of C. fulvum virulent on tomato genotype Cf9, the presence of mutated Avr4 alleles in virulent races of C. fulvum might suggest an essential role for its product in virulence (Fig. 1). However, one isolate with a frame-shift mutation in the ORF encoding an A VR4 homologue of only 13 amino acids showed normal virulence, indicating that the A VR4 protein is probably dispensable (Joosten et ai., 1997).
The observation that transformants of C. fulvum in which the Avr9 gene was replaced by a selection marker, did not show impaired virulence on non Cj9-containing tomato genotypes, and that in nature avoidance of Cf-9-specific resistance is achieved by complete deletion of the Avr9 gene, suggests that the Avr9 gene is dispensable for growth and virulence of C. fulvum (Marmeisse et ai., 1993). However, the Cf-9 resistance gene, which is present in tomato breeding lines since 1979, still provides good protection toward C. fulvum in commercial tomato crops, indicating that loss of the Avr9 gene is not sufficient to overcome the Cf-9 locus. It was found that isogenic strains of C. fulvum in which the Avr9 gene had been replaced by a selection marker are
A Case Study: Cladosporium fulvum-tomato Interaction 63
A
Avt9
Avirulent
.. I-
Virulent - - - -6. - - - -
B Avr4
Avirulent ~
,. ..
Virulent
"(Cp-+Tyo)
.. _-
Figure 1. The structure of peptides encoded by avirulent and virulent alleles of avirulence genes Avr9 and Avr4. A. Structure of the peptide encoded by avirulence gene Avr9. Single hatched box: signal peptide;
double hatched box: stretch of amino acids removed by proteolytic cleavage; white box: mature elicitor peptide; dotted line: deletion of avirulence gene Avr9 in virulent isolates. B. Structure of the peptide encoded by avirulence gene Avr4. Single hatched box: signal peptide; double hatched box: stretch of amino acids removed by proteolytic cleavage; white box: mature elicitor peptide; shaded box peptide encoded due to frame shift mutation; the various amino acid exchanges in the peptides ecoded by the virulent alleles are indicated.
only weakly virulent on Cf9 genotypes of tomato (Lauge et aI., 1998). This suggests that Cf-9 genotypes of tomato contain functional Cf9 homologs which recognize an elicitor other than AVR9. Infection of Cf-9 genotypes with Avr9-minus strains of C. fulvum was always associated with strong accumulation of PR proteins (Lauge et aI., 1998). most probably homologues of the Cf9 gene provide this protection as will be discussed later.
5. Virulence Factors with Avirulence Properties
As discussed before, C. fulvum secretes avirulence as well as virulence factors into the intercellular space while colonizing tomato leaves (Van den Ackerveken et aI., 1992;
Joosten et ai., 1994; Lauge et ai., 1997). The PYX expression system allows, within the host range of PYX, to search for plants that respond with an HR to virulence factors such as the Ecp2 gene product. A recombinant PYX construct expressing Ecp2 was inoculated onto various lines of species of Lycopersicon. In this way plants were found that responded with HR. All responding plants appeared to originate from the same ancestor. The corresponding resistance gene has been designated Cf-ECP2 (R. Lauge, Wageningen Agricultural University, personal communication). The Cf-ECP2 gene is anticipated to be a durable resistance gene as it recognizes a crucial virulence factor of C. fulvum (R. Lauge, Wageningen Agricultural University, personal communication).
All races of C. fulvum that have been tested so far, contain an Ecp2 gene that induces HR on plants containing the Cf-ECP2 gene. This finding illustrates that tomato has an efficient surveillance system that can recognize not only 'classical' avirulence factors but also crucial virulence factors.
IV. Defence Responses Induced by Non-Specific and Race-Specific Elicitors A. NON-SPECIFIC ELICITORS
The term elicitor was initially coined by Keen (1975) for compounds of pathogen origin which are able to induce the accumulation of phytoalexins, one of the defence responses studied intensively in the seventies. However, presently, the term elicitor is used for all compounds able to induce any defence response including HR, accumulation of phytoalexins, lignification, cell wall thickening, callose deposition or various enzymes involved in defence responses. Non-specific glycoprotein elicitors of C. fulvum induce most of the defence responses reported for other elicitors of biotic origin.
The non-specific glycoprotein elicitors of C. fulvum induce electrolyte leakage, callose deposition, necrosis and accumulation of phytoalexins as discussed before (Van Dijkman and Kaars Sijpesteijn, 1973; Dow and Callow, 1979a,b; Lazarovits and Higgins, 1979; Lazarovits et ai., 1979; De Wit and Roseboom, 1980; De Wit and Kodde, 1981a).