Subcellular localization of fungal and plant proteins in the Cladosporium fulvum - tomato interaction

Abstract

In this thesis data are presented on the localization of gene expression and accumulation of differentially accumulating proteins in the interaction between the plant pathogenic fungus Cladosporium fulvum and tomato. Differential accumulation of proteins in the apoplast of C. fulvum - infected leaves in compatible and incompatible interactions initiated detailed studies on isolation and characterization of these proteins in order to establish their role in the infection process. On the one hand, plant proteins accumulating in the apoplast of C. fulvum infected tomato leaves had been suggested to be involved in host defence responses. On the other hand, fungal proteins present in the apoplast of C. fulvum -infected tomato leaves had been suggested to play a role in establishment and maintenance of basic compatibility or in specific recognition events resulting in compatible or incompatible interactions.Accumulation of pathogenesis-related (PR) plant proteins has been observed to occur 2-4 days earlier in incompatible interactions between C. fulvum and tomato than in compatible interactions. A significant role in plant defence against fungal pathogens was proposed for chitinases and 1,3-β-glucanases. These PR proteins were identified as potential antifungal proteins due to their ability to hydrolyse chitin and 1,3-β-glucan polymers present in fungal cell walls. Indeed it has been shown that these proteins could inhibit fungal growth in vitro.In this thesis the localized accumulation of chitinases and 1,3-β-glucanases. in C. fulvum -infected tomato leaves has been described. Immunogold cytochemistry revealed PR protein accumulation near stomatal guard cells in the lower epidermis of C. fulvum-infected tomato leaves. Differential PR protein accumulation near stomata has not been observed between compatible and incompatible interactions. Accumulation of chitinases and 1,3-β-glucanases near stomata can be regarded as a general defence response of plants against pathogens, which is induced to a similar extent in compatible and incompatible interactions.More detailed localization studies of chitinases and 1,3-β-glucanases in C. fulvum infected tomato leaves have been carried out by electron microscope immunogold labelling. Specific accumulation of chitinases and 1,3-β-glucanases. has been observed in vacuoles of mesophyll cells and in the intercellular space in extracellular material present around fungal hyphae and between tomato mesophyll cells. Specific association of chitinases and 1,3-β- glucanases with cell walls of C. fulvum has not been observed.In addition to accumulation of the hydrolytic PR proteins, chitinases and 1,3-β-glucanases the spatial distribution of PR-1 protein accumulation in C. fulvum -infected tomato leaves has also been described. These proteins accumulate in the apoplast of C. fulvum infected tomato leaves, primarily in extracellular material around fungal hyphae. Spatial differences in PR-1 accumulation between compatible and incompatible interactions have not been observed. A role for PR-1 proteins involved in cell wall modifications, possibly leading to resistance against pathogens, as has been reported previously, could not be supported in this study. Apart from the localized accumulation near fungal hyphae, we did not obtain further information on a function for PR-1 proteins in the interaction between C. fulvum and tomato.Cell wall modifications, potentially resulting from the activity of hydrolytic enzymes in the apoplast of C. fulvum -infected tomato leaves, have been examined indirectly by determining the distribution of substrates for these enzymes in cell walls of either the plant or the fungus using immunocytochemical labelling. In these studies probes specific for pectin compounds in plant cell walls or for chitin residues in fungal cell walls were used. Alterations in the distribution of unesterified pectin and methylesterified pectin in the plant extracellular matrix between C. fulvum -infected and uninfected tomato leaf tissue were not observed. However, some accumulation of unesterified pectin was observed in extracellular material near fungal hyphae. This accumulation might result from cell wall degrading enzymes of plant or fungal origin. Furthermore, differences in chitin distribution in cell walls of C. fulvum grown in vitro or in planta has not been observed. Chitin in cell walls of C. fulvum , is probably embedded in a matrix of amorphous material and therefore not accessible for chitinases that accumulate in the intercellular space of C. fulvum-infected tomato leaves.Results on the spatial distribution of PR proteins in C. fulvum -infected tomato leaves, did provide further information on a role in defence of tomato against C. fulvum . In addition, northern analyses, revealed accumulation of transcripts encoding extracellular PR proteins, 2-4 days earlier in incompatible interactions than in compatible ones. Expression of genes encoding vacuolar PR proteins was induced to a similar extent in compatible and incompatible interactions. Spatial distribution of PR gene expression in tomato upon infection by C. fulvum was studied by in situ hybridization. Expression of the extracellular, acidic isoforms of chitinase and 1,3-β-glucanase occurred near vascular tissue and near epidermal cells, whereas expression of intracellular, basic isoforms was less restricted to particular tissue. No preferential accumulation of transcripts has been observed near penetrating fungal hyphae. Temporal differences in accumulation of gene transcripts between compatible and incompatible interactions could only be observed for the acidic PR proteins, thus confirming the northern analyses carried out before.Induced gene expression of acidic chitinases and 1,3-β-glucanases has been observed in a resistance gene-dependent manner upon injection of purified race-specific elicitors, AVR4 and AVR9, in tomato genotypes, Cf4 and Cf9, respectively. Induced gene expression, primarily observed in resistant tomato genotypes, correlates with preferential induction of chitinase and 1,3- β-glucanase gene expression in incompatible interactions as previously observed after inoculation by C. fulvum.An significant role for hydrolytic PR proteins in active defence of tomato against C. fulvum could not be proven. Only temporal differences were observed in accumulation of acidic isoforms of chitinase and 1, 3-β-glucanase which, however, have been shown to possess only limited in vitro antifungal properties. Furthermore, non of the purified chitinases and 1,3-β-glucanases. did inhibit growth of C. fulvumin vitro. Therefore, it is unlikely that they play an important role in direct defence of tomato against C. fulvum .Two extracellular fungal proteins, ECP1 and ECP2, have been described in the last section of this thesis. These proteins are produced by C. fulvum primarily during growth on tomato. Immunogold localization experiments revealed that ECP1 and ECP2 accumulated abundantly in extracellular material near fungal and host cell walls. Expression studies, using the β- glucuronidase reporter gene, revealed induction of ecp 1 and ecp 2 expression only during growth of C. fulvum in intercellular spaces of tomato leaves. Fungal hyphae growing near vascular tissue in infected tomato leaves, showed high ecp gene expression. No Ecp gene expression was observed in conidia used for inoculation nor in newly formed conidia on infected plants. A function for ECP1 and ECP2 in pathogenesis of C. fulvum on tomato is not clear. However, the accumulation near extracellular material associated with fungal and host cell walls, and the induced expression of ecp 1 and ecp 2 in planta suggest a role for both genes during intercellular growth of C. fulvum in tomato