The Cladosporium fulvum - tomato interaction : physiological and molecular aspects of pathogenesis

In this thesis research on the physiological and molecular aspects of pathogenesis in the interaction between tomato and <em>Cladosporium fulvum</em> Cooke (syn. <em>Fulvia fulva</em> [Cooke] Cif) is described. This plant-fungus interaction is envisaged to be based on a gene-for-gene relationship. Incompatible interactions (plants are resistant) between certain races of <em>C. fulvum</em> and tomato are thought to result from a specific interaction between products of fungal avirulence genes (racespecific elicitors) and products of corresponding resistance genes (cultivar-specific receptors) that are present in the host. After the elicitor has bound to the receptor, host defense genes are activated. A major feature of the activation of host defense is the accumulation of several pathogenesis-related (PR) proteins. Generally these proteins, which also accumulate in several other plant species, are of low molecular weight, accumulate in the apoplast, are highly resistant to proteolytic cleavage and have extreme iso-electric points. In compatible interactions (plants are susceptible) presumably no molecular recognition of the fungus occurs, resulting in colonization of the apoplastic space between the leaf mesophyll cells.In chapter 2 the purification of a fungal protein (designated P1, molecular mass 14 kD), specific for compatible <em>C. fulvum</em> -tomato interactions is described. Polyclonal antibodies were raised and the protein was shown to be only present in apoplastic fluid isolated from compatible <em>C. fulvum</em> -tomato interactions. Immunolocalization experiments revealed that in compatible interactions the protein was present in the electron-dense matrix between the walls of leaf mesophyll cells and fungal hyphae (chapter 6). Probably P1 plays a role in the establishment or maintenance of basic compatibility and can be regarded as a basic pathogenicity factor.In compatible interactions the fungus is able to hydrolyze the translocation sugar sucrose to glucose and fructose, which in turn are converted into the polyol mannitol by mannitol dehydrogenase (MTLDH) (chapter 3). During the colonization process of the intercellular spaces of the tomato leaves, increasing amounts of mannitol present in the apoplastic fluid coincided with increasing levels of MTLDH activity. The fungal metabolite mannitol cannot be utilized by the plant and possibly functions as a carbohydrate reserve for the fungus. In incompatible interactions no functional nutritional relationship between host and fungus is established and consequently no mannitol accumulation was observed.Chapter 4 describes the partial purification of a race-specific elicitor, the putative product of avirulence gene 4 ( <em>avr4</em> ) of <em>C. fulvum.</em> The race-specific elicitor precipitated in 60% (v/v) acetone, migrated on high pH, native gels and bound to an anion-exchange column at pH 9.0. The elicitor preparation induced a hypersensitive response and accumulation of PR proteins in near-isogenic line Cf4 of tomato (carrying resistance gene 4), indicating that active host defense is triggered by recognition of a race-specific elicitor by the plant.In incompatible interactions between tomato and <em>C. fulvum</em> the inhibition of fungal growth coincides with a substantial accumulation of PR proteins in the apoplast of the tomato leaf (chapter 5). Two abundantly occurring PR proteins of 35 kD and 26 kD in molecular mass were purified and were shown to have 1,3-β-glucanase and chitinase activity, respectively. Fungal walls that partly consist of 1,3-β-glucans and chitin, can be affected by these hydrolytic enzymes. With polyclonal antibodies that were raised against the purified enzymes one additional 1,3-β-glucanase (33 kD) and three additional chitinases (27, 30 and 32 kD) were detected in apoplastic fluids or homogenates of tomato leaves after inoculation with <em>C.</em><em>fulvum.</em> Upon inoculation with <em>C.</em><em>fulvum</em> apoplastic chitinase and 1,3-β-glucanase activities increased more rapidly in incompatible interactions than in compatible ones, indicating that these hydrolytic enzymes might play a role in active host defense.Immunolocalization experiments revealed that in incompatible tomato- <em>C.</em><em>fulvum</em> interactions 1,3-β-glucanases and chitinases accumulated in intercellular spaces, cytoplasm and electron-dense material that was present in the vacuoles of leaf mesophyll cells (chapter 6). Often 1,3-β-glucanases and chitinases were found to be associated with the electron-dense outer layer of the fungal cell wall. In compatible interactions no localized accumulation of 1,3-β-glucanases and chitinases was observed.In addition to the rapid induction and accumulation of 1,3-β-glucanases and chitinases in incompatible tomato- <em>C.</em><em>fulvum</em> interactions, a substantial accumulation of PR proteins of about 15 kD in molecular mass occurred. It was shown that in apoplastic fluids isolated from induced tomato leaves three basic PR proteins are present that migrate similarly to the earlier characterized tomato PR protein P14 on SDS-polyacrylamide gels (chapter 7). Two proteins, designated P4 and P6, molecular mass 15.5 kD, isoelectric points (pI) 10.9 and 10.7, respectively, appeared to be serologically related to each other and to the tobacco PR-1 proteins. The third protein, designated P2, molecular mass 15 kD, pI 10.4, was found to be serologically related to PR-R from tobacco. The biological function of P2, P4 and P6 is still unknown.In chapter 8 the characterization of messenger RNA (mRNA) for P6, the most abundant isomer of P14, is described. The mRNA contains an open reading frame of 477 nucleotides, encoding a protein of 159 amino acids, with an N-terminal signal peptide of 24 amino acids. Synthesis of P6 is regulated at the transcriptional level. In the incompatible interaction Cf4/race 5 there was a much faster accumulation of the P6 mRNA than in the compatible one (Cf5/race 5). There are probably two to four genes present in the genome of tomato that encode P14-like proteins.