G. Honée

A high-affinity binding site for the AVR9 peptide elicitor of Cladosporium fulvum is present on plasma membranes of tomato and other solanaceous plants.

The race-specific Cladosporium fulvum peptide elicitor AVR9, 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. 125I-AVR9 showed specific, saturable, and reversible binding to plasma membranes isolated from leaves of tomato cultivar Moneymaker without Cf resistance genes (MM-Cf0) or from a near-isogenic genotype with the Cf-9 resistance gene (MM-Cf9). The dissociation constant was found to be 0.07 nM, and the receptor concentration was 0.8 pmol/mg 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 hydrophobic interactions. Binding kinetics and binding capacity were similar for membranes of the MM-Cf0 and MM-Cf9 genotypes. In all solanaceous plant species tested, an AVR9 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 AVR9 seems to correlate with the presence of members of the Cf-9 gene family, but whether this correlation is functional remains to be determined.

The fungal gene Avr9 and the oomycete gene inf1 confer avirulence to potato virus X on tobacco

The AVR9 peptide of the fungal pathogen Cladosporium fulvum and the INF1 protein of the oomycete pathogen Phytophthora infestans elicit the hypersensitive response (HR) on Cf9 tomato or Cf-9 transgenic tobacco and on all cultivars of tobacco, respectively. Expression of either the functional Avr9 or inf1 genes from engineered potato virus X (PVX) genomes resulted in localized HR lesions on tobacco plants responsive to the elicitors and inhibited spread of the recombinant virus. In contrast, PVX derivatives producing mutant forms of AVR9 and INF1 with reduced elicitor activity caused systemic necrotic and/or mosaic symptoms, and were unable to inhibit PVX spread. These results demonstrate that HR is a highly versatile defense mechanism active against unrelated pathogens irrespective of the HR-inducing agent, and that resistance to recombinant PVX in tobacco is correlated with the strength of the transgene-encoded elicitor.

PRODUCTION OF THE AVR9 ELICITOR FROM THE FUNGAL PATHOGEN CLADOSPORIUM FULVUM IN TRANSGENIC TOBACCO AND TOMATO PLANTS

Three constructs were used to study the expression of the avirulence gene Avr9 from the fungal tomato pathogen Cladosporium fulvum 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 in planta by isolates of C. fulvum containing the Avr9 gene. Level of expression was not correlated with number of T-DNA integrations, but plants homozygous for the Avr9 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 C. fulvum-infected tomato leaves. Transgenic Cf0 genotypes of tomato, obtained by transformation with construct pAVIR21, showed a normal phenotype. However, transgenic F1 plants expressing the Avr9 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 Cf9 gene is deleterious. The necrotic defence response observed in Cf9 genotypes expressing the Avr9 gene support the potential to apply avirulence genes in molecular resistance breeding.

MOLECULAR CHARACTERIZATION OF THE INTERACTION BETWEEN THE FUNGAL PATHOGEN CLADOSPORIUM FULVUM AND TOMATO

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.

Molecular and biochemical basis of the interaction between tomato and its fungal pathogen Cladosporium fulvum.

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.

Molecular communication between host plant and the fungal tomato pathogen Cladosporium fulvum.

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.

Avirulence genes of the tomato pathogen Cladosporium fulvum and their exploitation in molecular breeding for disease-resistant plants.

Of the fungal tomato pathogen Cladosporium fulvum a number of avirulence genes and their products have been isolated and characterized. Avirulence genes which encode race-specific elicitors interact with the products of complementary resistance genes in the host plant resulting in a hypersensitive response and other defense responses. Avirulence gene avr9 of C. fulvum is the first fungal avirulence gene that has been cloned. The regulation of this gene has been studied in vitro and in plania. In vitro, the gene is induced under low nitrogen conditions, whereas in planta the gene is highly expressed around the vascular tissue. Avirulent races carrying the avr9 gene become virulent on Cf9 genotypes of tomato after disruption of avr9.

The gene-for-gene concept in plant pathogen interactions: tomato-Cladosporium fulvum.

The phenomenon of host-genotype specificity in interactions between plants and fungal pathogens has intrigued plant pathologists for more than a century. The discovery that resistance to infection by the rust fungus Puccinia graminis f. sp. tritici in wheat is genetically determined (Farrer 1898) and obeys Mendel’s laws (Biffen 1905) provided the possibility to breed for resistance against pathogens. However, as a result of selection pressure, the pathogen is able to develop new variants that regain virulence. Already at the beginning of this century, Stakman and coworkers showed that newly introduced genes for resistance against the stem rust fungus of wheat were rapidly overcome by new “biological forms” of the pathogen (Stakman 1917; Stakman et al. 1918).

Molecular Cloning and Functions of Avirulence and Pathogenicity Genes of the Tomato Pathogen Cladosporium Fulvum

Of the fungal tomato pathogen Cladosporium fulvum a number of in planta induced genes and their products have been isolated and characterized. They include: (i) putative pathogenicity genes of which two genes, (extracellular proteins) ecp1 and ecp2, have been cloned and sequenced; the function of those genes during pathogenesis is not known yet; effects of ecp gene disruption on pathogenicity are being studied, and: (ii) avirulence genes which encode race-specific elicitors interacting with the products of complementary resistance genes in the host plant, resulting in a hypersensitive response and other defense responses. Avirulence gene avr9 of C. fulvum is the first fungal avirulence gene that has been cloned. The regulation of this gene has been studied in vitro and in planta. In vitro, the gene is induced under low nitrogen conditions, whereas in planta the gene is highly expressed around the vascular tissue. Avirulent races carrying the avr9 gene become virulent on Cf9 genotypes of tomato after disruption of avr9.