Development of genetic engineering technology and molecular characterization of plant defense responses have provided strategies for controlling plant diseases additional to those based on chemical control or classical breeding programs. Most of these alternative strategies are based on the overproduction of one component of the plant's own defense response. Some strategies exploit the hypersensitive response, a rapid, localized death of tissue surrounding the infection site, which is observed in many resistant plants upon unsuccessful pathogen attack. Most approaches to increase resistance to fungi have been described to be successful under laboratory conditions. Incorporation of these successful, alternative strategies in resistance breeding programs of agriculturally important crops will depend on the results obtained from field experiments.

The interaction between the biotrophic fungal pathogen Cladosporium fulvum and tomato complies with the genefor-gene model. Resistance, expressed as a hypersensitive response (HR) followed by other defence responses, is based on recognition of products of avirulence genes from C. fulvum (race-specific elicitors) by receptors (putative products of resistance genes) in the host plant tomato. The AVR9 elicitor is a 28 amino acid (aa) peptide and the AVR4 elicitor a 106 aa peptide which both induce HR in tomato plants carrying the complementary resistance genes Cf9 and Cf4, respectively. The 3-D structure of the AVR9 peptide, as determined by 1H NMR, revealed that AVR9 belongs to a family of peptides with a cystine knot motif. This motif occurs in channel blockers, peptidase inhibitors and growth factors. The Cf9 resistance gene encodes a membrane-anchored extracellular glycoprotein which contains leucine-rich repeats (LRRs). 125I labeled AVR9 peptide shows the same affinity for plasma membranes of Cf9+ and Cf9- tomato leaves. Membranes of solanaceous plants tested so far all contain homologs of the Cf9 gene and show similar affinities for AVR9. It is assumed that for induction of HR, at least two plant proteins (presumably CF9 and one of his homologs) interact directly or indirectly with the AVR9 peptide which possibly initiates modulation and dimerisation of the receptor, and activation of various other proteins involved in downstream events eventually leading to HR. We have created several mutants of the Avr9 gene, expressed them in the potato virus X (PVX) expression system and tested their biological activity on Cf9 genotypes of tomato. A positive correlation was observed between the biological activity of the mutant AVR9 peptides and their affinity for tomato plasma membranes. Recent results on structure and biological activity of AVR4 peptides encoded by avirulent and virulent alleles of the Avr4 gene (based on expression studies in PVX) are also discussed as well as early defence responses induced by elicitors in tomato leaves and tomato cell suspensions.

The race-specific Cladosporium fulvum peptide elicitor AVRQ, which specifically induces a hypersensitive response in tomato genotypes carrying the Cf-9 resistance gene, was labeled with iodine-125 at the N-terminal tyrosine residue and used in binding studies. 1251-AVR9 showed specific, saturable, and reversible binding to plasma membranes isolated from leaves of tomato cultivar Moneymaker without Cf resistance genes (MM-CfO) or from a near-isogenic genotype with the Cf.9 resistance gene (MM-CR). The dissociation constant was found to be 0.07 nM, and the receptor concentration was 0.8 pmollmg microsomal protein. Binding was highly influenced by pH and the ionic strength of the binding buffer and by temperature, indicating the involvement of both electrostatic and hydmphobic interactions. Binding kinetics and bind- ing capacity were similar for membranes of the MM-CfO and MM-CR genotypes. In all solanaceous plant species tested, an AVRS binding site was present, whereas in the nonsolanaceous species that were analyzed, such a binding site could not be identified. The ability of membranes isolated from different solanaceous plant species to bind AVRS seems to correlate with the presence of members of the Cf-9 gene family, but whether this correlation is functional remains to be determined.

Three constructs were used to study the expression of the avirulence gene 9 from the fungal tomato pathogen in plants. They include pAVIR1, pAVIR2 and pAVIR21, encoding the wild-type AVR9 protein and two hybrid AVR9 proteins containing the signal sequences of the pathogenesis-related proteins PR-S and PR-1a, respectively. Transgenic tobacco plants obtained with the three constructs showed a normal phenotype and produced AVR9 elicitor with the same specific necrosis-inducing activity as the wild-type AVR9 elicitor produced by isolates of containing the 9 gene. Level of expression was not correlated with number of T-DNA integrations, but plants homozygous for the 9 gene produced more elicitor protein than heterozygous plants. The amino acid sequence of the processed AVR9 peptide present in apoplastic fluid (AF) of pAVIR1 transformed plants producing the wild-type AVR9 elicitor was identical to that of the wild-type AVR9 peptide isolated from -infected tomato leaves. Transgenic Cf0 genotypes of tomato, obtained by transformation with construct pAVIR21, showed a normal phenotype. However, transgenic F1 plants expressing the 9 gene, obtained from crossing transgenic Cf0 genotypes with wild-type Cf9 genotypes, showed delayed growth, necrosis and complete plant death indicating that the AVR9 peptide produced in plants carrying the gene is deleterious. The necrotic defence response observed in Cf9 genotypes expressing the 9 gene support the potential to apply avirulence genes in molecular resistance breeding.

The fungus Cladosporium fulvum is a biotrophic leaf pathogen of tomato which is the only host it can infect. The infection is initiated by conidia on leaves which germinate, produce runner hyphae and invade stomata without the formation of appressoria, three days post inoculation [1]. Mycelium grows abundantly in the intercellular space between mesophyll cells, where the fungus remains during the main part of its life cycle. Twelve to fourteen days after inoculation, the fungus emerges from the stomata and forms conidiophores producing conidia which may start a new disease cycle. Since the fungus develops in the intercellular space without forming specialized feeding structures such as haustoria, and does not visibly affect the leaf tissue, C.,fulvum co-exists in a highly balanced relationship with tomato. Pathogenicity factors, required to grow in planta and to prevent, or to inactivate, host defence responses, are supposed to play an important role to accomplish and/or maintain basic compatibility. Many different physiologic races of C. fulvum are known, each carrying a different subset of avirulence genes. Resistance genes of tomato have been identified genetically of which several are available in near-isogenic lines of Lycopersicon esculentum cv. MoneyMaker. The outcome of the C. fulvum-tomato interaction can be described by the gene-for-gene model; interaction between a physiologic race of C. fulvum containing an avirulence gene and a tomato cultivar harbouring the complementary resistance gene results in incompatibility. Incompatibility is characterized by appearance of a hypersensitive response (HR), a rapid death of cells surrounding the infection site which prevents further growth of the fungal pathogen and eventually results in resistance. How the fungus accomplishes and maintains compatibility during a successful infection, and which molecular processes determine incompatibility are the main questions to be answered.

The fungus Cladosprium fulvum is a biotrophic leaf pathogen of tomato. The fungus develops in the intercellular space without forming specialized feeding structures and does not affect the leaf tissue. The outcome of the C. fulvum-tomato interaction can be described by the gene-for-gene model. Failure of infection is expressed by a hypersensitive response. Two fungal proteins, ECP1 and ECP2, have been isolated and their corresponding genes have been cloned. In a compatible interaction including many physiological races ECP1 and ECP2 are highly produced and a role in pathogenicity is suggestive. The ecp1 gene shows some homology with tumor necrosis factor receptors (TNFRs) while the ecp2 gene shows no homology with sequences known in data bases. However, disruption of one of the two genes showed no reduced pathogenicity of the fungus. Two race-specific elicitors, AVR4 and AVR9, have been isolated and their corresponding genes have been cloned. The avirulence genes Avr4 and Avr9 are only present in C. fulvum avirulent on Cf-4 and Cf-9 cultivars, respectively. The expression of these two genes is, like the expression of the ecp genes, highly induced when the fungus grows in planta. Disruption of the Avr9 gene in wild type avirulent races leads to virulence on tomato genotypes carrying the complementary resistance gene Cf-9. A single base-pair change in the avirulence gene Avr4 leads to virulence on tomato genotypes carrying the Cf-4 resistance gene. Isolation, characterization and possible function of ECP1, ECP2, AVR4, and AVR9 will be discussed.

Tobacco and tomato plants were generated exhibiting insect resistance due to the introduction of modified cryIA(b) and cryIC genes of Bacillus thuringiensis. Limited modifications at selected regions of the coding sequences of both genes are sufficient to obtain resistance against Spodoptera exigua, Heliothis virescens and Manduca sexta. The criteria used to modify both genes demonstrate that the removal of sequence motifs potentially resulting in premature polyadenylation and transcript instability causes increased insect resistance. The expression of a cryIC-cryIA(b) fusion resulting in protection against S. exigua, H. virescens and M. sexta demonstrates the potential of expressing translational fusions, not only to broaden the insect resistance of transgenic plants, but also to simultaneously employ different gene classes in resistance management strategies.

Host genotype specificity in interactions between biotrophic fungal pathogens and plants in most cases complies with the gene-for-gene model. Success or failure of infection is determined by absence or presence of complementary genes, avirulence and resistance genes, in the pathogen and the host plant, respectively. Resistance, expressed by the induction of a hypersensitive response followed by other defence responses in the host, is envisaged to be based on recognition of the pathogen, mediated through direct interaction between products of avirulence genes of the pathogen (the so-called race-specific elicitors) and receptors in the host plant, the putative products of resistance genes. The interaction between the biothrophic fungusCladosporium fulvum and its only host tomato is a model system to study fungus-plant gene-for-gene relationships. Here we report on isolation, characterization and biological function of putative pathogenicity factors ECP1 and ECP2 and the race-specific elicitors AVR4 and AVR9 ofC. fulvum and cloning and regulation of their encoding genes. Disruption ofecp1 andecp2 genes has no clear effect on pathogenicity ofC. fulvum. Disruption of theavr9 gene, which codes for the race-specific 28 amino acid AVR9 elicitor, in wild type avirulent races, leads to virulence on tomato genotypes carrying the complementary resistance geneCf9. The avirulence geneavr4 encodes a 105 amino acid race-specific elicitor. A single basepair change in the avirulence geneavr4 leads to virulence on tomato genotypes carrying theCf4 resistance gene.

A 3'-end truncated crystal protein gene, derived from Bacillus thuringiensis (Bt) subsp. aizawai 7.21, encoding the toxic fragment of the insecticidal protein cryIA(b), was constructed. The gene was inserted into a transformation vector, also carrying the neomycin phosphotransferase II (nptII) gene and the beta-glucuronidase (gus) gene, and introduced in the oncogenic Agrobacterium tumefaciens strain A281, harbouring the Ti-plasmid pTiBO542. The recombinant Agrobacterium strain was used to transform leaf explants of chrysanthemum (Dendranthema grandiflora) cultivar Parliament. The resulting tumours were kanamycin-resistant, exhibited beta-glucuronidase activity and produced agropine and mannopine. In most tumours, all simultaneously transferred genes were expressed, owing to selection for the presence of both T-DNAs, but no correlation was found between the level of expression of the various genes. A bioassay was developed, in which larvae were fed with tumorous chrysanthemum tissue, in order to detect the effect of the transferred toxin gene on larval development. Using this bioassay with second instar larvae of Heliothis virescens (tobacco budworm), 17 tumour lines were tested. Several of these lines proved to be strongly inhibitory to larval growth. These results indicate that Bt-based insect resistance might be used as a tool in reducing the amount of pesticides used in chrysanthemum culture.

Major drawbacks of the use of chemical insecticides for the control of insect pests are their expense, their persistence in and hazardous effects on the environment, and their escalating rates of application because of decreasing effectiveness. Therefore, over the last two decades efforts have increased to investigate possibilities of biological insect control like the use of predators, parasites, insect viruses or feromones. Likewise, increased utilization of the microbial insect pathogen Bacillusthuringiensis can be regarded as a form of biological insect control. B. thuringiensis is characterized by its ability to produce crystalline inclusions during sporulation which form the major toxic determinant of this bacterium. Most B. thuringiensis strains are active against larvae of lepidopteran species (butterflies), but some show toxicity against larvae of dipteran (flies) or coleopteran (beetles) species. Not only strains, but also different crystal proteins occurring in a single crystal may vary in insecticidal spectra. Sprays based on B. thuringiensis spore/crystal preparations have been used for over thirty years as biological insecticides. Recently, the cloning of crystal protein genes and their expression in transgenic plants and micro-organisms has provided powerful alternative strategies for the protection of crops against insect damage. These additional potential applications resulted in an increased interest in this bacterium and its crystal proteins in recent years. This thesis endeavours to contribute to our understanding of the entomocidal activity of B. thuringiensis, and more in particular of its crystal proteins. Studies are described to identify crystal proteins with new insecticidal spectra, to gain more insight into the mode of action of crystal proteins, and to examine some of their potential applications. Chapter 1 reviews the mode of action of the crystal proteins. It presents a survey of histological and physiological changes observed in the insect and in vitro cultured cells induced by crystal proteins. In addition, investigations using isolated midgut cells, brush border membrane vesicles and artificial phospholipid vesicles are summarized. Finally, conclusions concerning the molecular basis of the mode of action of both Lepidoptera and Diptera specific crystal proteins are discussed. In chapter 2, an analysis of crystal protein genes and their proteins, in particular those occurring in B. thuringiensis serotype entomocidus 60.5, is presented. The nucleotide sequence of a gene isolated from this bacterial strain, the reference type of cryIC , is presented together with its deduced amino acid sequence. Furthermore, several conserved features revealed by an analysis of the amino acid sequences of all crystal proteins are evaluated. In chapter 3, domain-function studies on the crystal proteins are described using hybrid crystal proteins based on CryIA(b) and CryIC constructed in order to improve our understanding of the mode of action of crystal proteins, especially of its molecular basis. Chapter 4 describes the analysis of a translation fusion product of two different insecticidal crystal protein genes. Based on the presented results an alternative strategy for the introduction of more effective insect resistance in transgenic organisms using B. thuringiensis crystal protein genes is proposed. In chapter 5, the generation of insect resistant transgenic plants using the crystal protein genes cryIA(b) and cryIC , is described. Finally, in chapter 6, the three-dimensional structure of one of the crystal proteins, which recently became available, is reviewed as well as alternative strategies for B.thuringiensis applications to prevent crystal protein resistance in insect populations, all in relation to the results presented in this thesis.