RACE-SPECIFIC ELICITORS

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).

A Case Study: Cladosporium fulvum-tomato Interaction 65

Vera-Estrella et al. (1992, 1993) studied biochemical responses in cell cultures of tomato after elicitor treatment. Cell cultures, initiated from callus obtained from tomato genotypes Cf4 and Cf5, retained the specificity of the intact plants from which they originated. Within 10 minutes after treatment with AF containing matching elicitors, a marked extracellular oxidative burst was observed. In addition, the cells showed increased lipid peroxidation followed by increases in extracellular peroxidases and phenolic compounds. Cell cultures of tomato genotype Cf5 treated with AF containing A VR5 elicitor showed also a quick increase in H+-ATPase activity causing acidification of the extracellular medium (Vera-Estrella et aI., 1994a,b). Activation of H+-ATPase in cells and plasma membranes occurred through reversible dephosphorylation (Xing et ai., 1996) The observed acidification of the culture medium contrasts with responses to non-specific elicitors (Vera-Estrella et aI., 1993), which induce a rapid alkalinization of the extracellular medium. Enriched plasma membrane fractions obtained from Cf5 cells also responded to a crude preparation of A VR5 elicitor with a significant increase in NADH oxidase and cytochrome C reductase. Inhibition studies indicated that both phosphatases and G-proteins are involved in these responses (Vera-Estrella et aI., 1994a,b;

Xing et aI., 1996). It was also found that plasma membrane NADPH oxidase was activated in cells after treatment with A VR5 elicitor preparations, while at the same time translocation of components of this enzyme to the plasma membrane was observed (Xing et ai., 1997).

In contrast to cell cultures derived from Cf4 and Cf5 genotypes of tomato, cell cultures and calli of Cf9 genotype treated with A VR9 elicitor did not show Cf-9-dependent defence responses such as generation of an oxidative burst and a pH shift (Honee et aI., 1998). This differs from leaves and shoots of tomato genotype Cf9 treated with A VR9 elicitor which gave strong Cf-9-dependent defence responses (Hammond et ai., 1996;

Honee et aI., 1998). This suggests that AVR9-Cf-9 mediated defence is is not active in undifferentiated tissue such as callus or cells. However, cell cultures of Cf-9 transgenic tobacco do respond with an oxidative burst and extracellular alkalinisation (C.F. De Jong, Wageningen Agricultural University, personal communication). Injection of AVR4 and A VR9 elicitors of C. fuivum into leaves of Cf4 or Cf9 tomato genotypes, respectively, induced expression ofPR protein encoding genes (Wubben et aI., 1996). mRNAs encoding acidic 1,3-[3-glucanase and acidic chitinase were strongly induced. In CfO genotypes the PR mRNAs were hardly induced. The induction pattern of basic chitinase and basic 1,3-[3-glucanase was less clear. In leaves of heterozygous Ct2 and Cf9 plants treated with AF containing AVR2 and AVR9 elicitor, respectively, Ashfield et al. (1994) found levels of induction of acidic and basic 1,3-[3-glucanases comparable to those found by Wubben et al. (1996).

v.

Resistance Genes in Tomato against Cladosporiumfulvum.

Four genes for resistance against C. fuivum (Cf genes) have been mapped at two complex loci. The Cf-9 and Cf-4 genes have been mapped on the short arm of chromosome 1 (Jones et aI., 1993), and the Cf-2 and Cf-5 genes are located on the short arm of chromosome 6 (Dickinson et aI., 1993; Jones et ai., 1993). The Cf-2, Cf-4, Cf-5

and Cf-9 genes have been cloned (Jones et al., 1994; Dixon et aI., 1996; Hammond- Kosack and Jones, 1997; Thomas et aI., 1997). A schematic representation of the four Cf-genes is shown in Figure 2.

Cf.9

IAI B I

C(27 LRRs)

IDMF IGI

⁸⁶³⁸⁸

Cf.4 ~IBI C(25 LRRs)

IDMF IGI

⁸⁰⁶⁸⁸

Cf.2

IAI BI

C (37 LRRs)

JDIEIFIGI

¹¹¹²⁸⁸

Cf.5

IAI BI

C(35 LRRs)

IDIEIFIGI

⁹⁶⁸⁸⁸

Figure 2. Schematic representation of the proteins (number of amino acids is indicated) encoded by the resistance genes Cf-9. Cf-4. Cf-2 and Cf-5. respectively. See text for further details.

A Signal peptide B Cysteine-rich region

C Leucine-rich repeat (LRR) region; the number of LRRs is indicated

o Region without specific properties E Acidic region

F Transmembrane region G Intracellular basic region

The Cf-9 gene encodes a 863-amino acid membrane-anchored, predominantly extracytoplasmic glycoprotein containing 27 leucine-rich repeats (LRRs) with an average length of 24 amino acids. These motifs occur in many plant resistance genes (Jones and Jones, 1996; Hammond-Kosack and Jones, 1997). In the Cf-9 protein seven domains (A to G) have been designated. The N-terminal domain A of 23 amino acids is consistent with a signal peptide; domain B of 68 amino acids is cysteine-rich; domain C contains 27 imperfect LRRs; domain D contains 28 amino acids; domain E contains 18 amino acids and is very acidic; domain F contains 37 amino acids and is the presumed trans-membrane domain; C-terminal domain G contains 21 amino acids, is very basic and concludes with the amino acids KKRY. Twenty-two potential N-glycosylation sites are distributed between domains B, C and D. Thus the Cf-9 gene encodes a LRR protein of which the major part (A-F) is extracellular and the C-terminal part (G) is cytoplasmic.

The LRR domain C in the Cf-9 protein is interrupted by a short region originally designated as LRR 24, which has only minimal LRR homology. This domain, now designated C2 is also present in the Cf-2, Cf-4, and Cf-5 genes (Jones and Jones, 1996;

Hammond-Kosack and Jones, 1997). As a result domain C has now been divided in domains Cl (the N-terminal LRRs), C2 (with minor LRR consensus) and domain C3 (the C-terminal LRRs) (Jones and Jones, 1996; Hammond-Kosack and Jones, 1997).

A Case Study: Cladosporium fulvum-tomato Interaction 67

The LRRs match the extracytoplasmatic LRR consensus LxxLxxLxxLxLxxNxLxGxIPxx (Jones and Jones, 1996). LRR regions might be involved in various types of protein- protein interaction (Kobe and Deisenhofer, 1993) as will be discussed later.

The Cf-4 gene is very homologous to the Cf-9 gene. The proteins have >91 ^% identical amino acids (Thomas et al., 1997). The Cf-4 gene encodes an 806-amino acid protein with 25 LRRs. In Cf-4 two complete LRRs are deleted relative to the Cf-9 gene.

DNA sequence analysis suggests that Cf-4 and Cf-9 are derived from a common gene.

The amino acids that distinguish Cf-4 from Cf-9 are located at the N-terminal half of the protein. The C-terminal halves of both genes are almost identical.

The Cf-2 locus contains two functional genes that each independently confer resistance to races of C. fulvum carrying the A vr2 gene (Dixon et al., 1996). Each gene encodes a nearly identical 1112-amino acid protein (three amino acids are different) that is structurally very similar to the Cf-4 and Cf-9 proteins. The Cf-2 protein possesses 37 LRRs. The LRRs of Cf-2 are nearly all 24 amino acids in length and are also interrupted by a short C2 domain which divides the LRRs in a C-terminal block of 33 LRRs and a N-terminal block of 4 LRRs. The highest homology between the Cf-2, and the Cf-4 and Cf-9 proteins resides in the C-terminal part (Hammond-Kosack and Jones, 1997; Thomas et al., 1997).

The Cf-5 gene is closely linked to the Cf2 gene and encodes a 968-amino acid protein that is very similar to the protein encoded by Cf-2 and contains 31 LRRs (Hammond- Kosack and Jones, 1997). The Cf-5 and Cf-2 proteins differ by an exact deletion of six LRRs in Cf-5. The C-terminal part of Cf-2 and Cf-5 is also very conserved.

A. LRR MOTIFS IN CF PROTEINS AND THEIR POTENTIAL FUNCTION