Molecular aspects of avirulence and pathogenicity of the tomato pathogen Cladosporium fulvum

The molecular understanding of host-pathogen interactions and particularly of specificity forms the basis for studying plant resistance. Understanding why a certain plant species or cultivar is susceptible and why other species or cultivars are resistant is of great importance in order to design new strategies for future crop protection by molecular plant breeding.In this thesis molecular aspects of avirulence and pathogenicity of the tomato pathogen Cladosporium fulvum are described. The interaction C. fulvum - tomato is an excellent model system to study fungus - plant specificity as the communication between pathogen and plant is confined to the apoplast (intercellular space). The ability to obtain intercellular fluid from C. fulvum -infected tomato leaves enabled the isolation and characterization of plant and fungal compounds that might play an important role in pathogenesis and/or the induction of resistance. The purification and characterization of a race-specific peptide elicitor provides the basis for most of the experiments described in this thesis. This peptide was thought to be produced only by races of C. fulvum avirulent on tomato genotypes carrying the resistance gene Cf9 , on which the elicitor induced necrosis.Molecular aspects of avirulence of C. fulvum were first studied by the isolation and characterization of the cDNA encoding the AVR9 race-specific peptide elicitor (Chapter 2). The peptide was shown to be indeed produced by C. fulvum . Races virulent on tomato genotype Cf9 , lack the avr9 gene and do not produce the peptide elicitor thereby evading recognition by tomato genotypes carrying the corresponding resistance gene Cf9. To prove that the avr9 gene is a genuine avirulence gene, races virulent on tomato genotype Cf9 , were transformed with the cloned avr9 gene (Chapter 3). The cultivar-specificity of the transformants was changed from virulent to avirulent on tomato genotype Cf9. The avr9 gene can therefore be considered to be a genuine avirulence gene, the first fungal avirulence gene cloned. Additional proof for the role of the avirulence gene avr9 in specificity was provided by the disruption of avr9 in two races avirulent on tomato genotype Cf9 , by gene replacement, resulting in transformants virulent on tomato genotype Cf9 , (Chapter 4).The avr9 gene encodes a 63 amino acids precursor protein. Removal of a signal peptide results in an extracellular peptide of 40 amino acids. Proteases of C. fulvum are involved in further processing this extracellular peptide by removal of N-terminal amino acids resulting in peptides of 32, 33 and 34 amino acids. Plant factors are responsible for further processing, resulting in the stable peptide elicitor of 28 amino acids (Chapter 5).The avirulence gene avr9 is highly expressed in C. fulvum while growing inside the tomato leaf. The expression of avr9 is induced in C. fulvum grown in vitro under conditions of nitrogen limitation. The high expression of avr9 in C. fulvum growing inside the tomato leaf might be caused by nitrogen limiting conditions in the apoplast (Chapter 6).Pathogenicity of C. fulvum was studied at the molecular level by the isolation of two genes encoding extracellular proteins (ECPs). The ecp1 and ecp2 genes were isolated via the amino acid sequence of ECP1, and polyclonal antibodies raised against ECP2, respectively (Chapter 7). The expression of the ecp genes is highly induced in planta as compared to the in vitro situation. The availability of the cloned ecp genes now enables us to study the role and importance of these genes during pathogenesis by reporter gene analysis and gene disruption.Two models describing the C. fulvum -tomato interaction are presented, dealing with basic compatibility and race-specific incompatibility, respectively (Chapter 8). The improved understanding of pathogen recognition can be exploited in future research to elucidate the role of putative receptors in the resistant plant involved in perception of elicitors and induction of active plant defence

Molecular aspects of avirulence and pathogenicity of the tomato pathogen Cladosporium fulvum

The molecular understanding of host-pathogen interactions and particularly of specificity forms the basis for studying plant resistance. Understanding why a certain plant species or cultivar is susceptible and why other species or cultivars are resistant is of great importance in order to design new strategies for future crop protection by molecular plant breeding.In this thesis molecular aspects of avirulence and pathogenicity of the tomato pathogen Cladosporium fulvum are described. The interaction C. fulvum - tomato is an excellent model system to study fungus - plant specificity as the communication between pathogen and plant is confined to the apoplast (intercellular space). The ability to obtain intercellular fluid from C. fulvum -infected tomato leaves enabled the isolation and characterization of plant and fungal compounds that might play an important role in pathogenesis and/or the induction of resistance. The purification and characterization of a race-specific peptide elicitor provides the basis for most of the experiments described in this thesis. This peptide was thought to be produced only by races of C. fulvum avirulent on tomato genotypes carrying the resistance gene Cf9 , on which the elicitor induced necrosis.Molecular aspects of avirulence of C. fulvum were first studied by the isolation and characterization of the cDNA encoding the AVR9 race-specific peptide elicitor (Chapter 2). The peptide was shown to be indeed produced by C. fulvum . Races virulent on tomato genotype Cf9 , lack the avr9 gene and do not produce the peptide elicitor thereby evading recognition by tomato genotypes carrying the corresponding resistance gene Cf9. To prove that the avr9 gene is a genuine avirulence gene, races virulent on tomato genotype Cf9 , were transformed with the cloned avr9 gene (Chapter 3). The cultivar-specificity of the transformants was changed from virulent to avirulent on tomato genotype Cf9. The avr9 gene can therefore be considered to be a genuine avirulence gene, the first fungal avirulence gene cloned. Additional proof for the role of the avirulence gene avr9 in specificity was provided by the disruption of avr9 in two races avirulent on tomato genotype Cf9 , by gene replacement, resulting in transformants virulent on tomato genotype Cf9 , (Chapter 4).The avr9 gene encodes a 63 amino acids precursor protein. Removal of a signal peptide results in an extracellular peptide of 40 amino acids. Proteases of C. fulvum are involved in further processing this extracellular peptide by removal of N-terminal amino acids resulting in peptides of 32, 33 and 34 amino acids. Plant factors are responsible for further processing, resulting in the stable peptide elicitor of 28 amino acids (Chapter 5).The avirulence gene avr9 is highly expressed in C. fulvum while growing inside the tomato leaf. The expression of avr9 is induced in C. fulvum grown in vitro under conditions of nitrogen limitation. The high expression of avr9 in C. fulvum growing inside the tomato leaf might be caused by nitrogen limiting conditions in the apoplast (Chapter 6).Pathogenicity of C. fulvum was studied at the molecular level by the isolation of two genes encoding extracellular proteins (ECPs). The ecp1  and ecp2 genes were isolated via the amino acid sequence of ECP1, and polyclonal antibodies raised against ECP2, respectively (Chapter 7). The expression of the ecp genes is highly induced in planta as compared to the in vitro situation. The availability of the cloned ecp genes now enables us to study the role and importance of these genes during pathogenesis by reporter gene analysis and gene disruption.Two models describing the C. fulvum -tomato interaction are presented, dealing with basic compatibility and race-specific incompatibility, respectively (Chapter 8). The improved understanding of pathogen recognition can be exploited in future research to elucidate the role of putative receptors in the resistant plant involved in perception of elicitors and induction of active plant defence

The in-planta induced ecp2 gene of the tomato pathogen Cladosporium fulvum is not essential for pathogenicity.

During the colonization of tomato leaves, the fungal pathogen Cladosporium fulvum excretes low-molecular-weight proteins in the intercellular spaces of the host tissue. These proteins are encoded by the ecp genes which are highly expressed in C. fulvum while growing in planta but are not, or are only weakly, expressed in C. fulvum grown in vitro. To investigate the function of the putative pathogenicity gene ecp2, encoding the 17-kDa protein ECP2, we performed two successive disruptions of the gene. In the first of these, the ecp2 gene was interrupted by a hygromycin B resistance gene cassette. In the second gene disruption, the ecp2 gene was completely deleted from the genome, and replaced by a phleomycin resistance gene cassette. Both disruption mutants were still pathogenic on tomato seedlings, indicating that the C. fulvum ecp2 gene is not essential for pathogenicity in tomato

The in-planta induced ecp2 gene of the tomato pathogen Cladosporium fulvum is not essential for pathogenicity.

During the colonization of tomato leaves, the fungal pathogen Cladosporium fulvum excretes low-molecular-weight proteins in the intercellular spaces of the host tissue. These proteins are encoded by the ecp genes which are highly expressed in C. fulvum while growing in planta but are not, or are only weakly, expressed in C. fulvum grown in vitro. To investigate the function of the putative pathogenicity gene ecp2, encoding the 17-kDa protein ECP2, we performed two successive disruptions of the gene. In the first of these, the ecp2 gene was interrupted by a hygromycin B resistance gene cassette. In the second gene disruption, the ecp2 gene was completely deleted from the genome, and replaced by a phleomycin resistance gene cassette. Both disruption mutants were still pathogenic on tomato seedlings, indicating that the C. fulvum ecp2 gene is not essential for pathogenicity in tomato