AVR9 Peptide Research Articles

CITRX thioredoxin is a putative adaptor protein connecting Cf-9 and the ACIK1 protein kinase during the Cf-9/Avr9- induced defence response

Tomato Cf- 9, a receptor-like protein (RLP), confers resistance to races of the fungal pathogen Cladosporium fulvum that express the Avr9 avirulence gene. CITRX ( Cf-9- interacting thio redo xin) was previously identified in a yeast two-hybrid screen as a protein interacting with the cytoplasmic domain of Cf-9 and shown to be a negative regulator of the cell death induced after Cf-9/Avr9 interaction. ACIK1 is a Ser/Thr protein kinase that is specifically required for the Cf- 9 and Cf- 4 dependent defence response in tomato. In this paper we present data suggesting that CITRX may act as an adaptor recruiting the ACIK1 kinase to the cytoplasmic domain of Cf-9 upon elicitation with the Avr9 peptide. Interestingly, the catalytic activities of both CITRX and ACIK1 are not required for their interaction.

The E3 Ubiquitin Ligase Activity of Arabidopsis PLANT U-BOX17 and Its Functional Tobacco Homolog ACRE276 Are Required for Cell Death and Defense

Previous analysis of transcriptional changes after elicitation of Cf-9 transgenic tobacco (Nicotiana tabacum) by Avr9 peptide revealed a rapidly upregulated gene, ACRE276. We show that ACRE276 is transiently induced in wounded leaves within 15 min, but upon Avr9 elicitor treatment, this upregulation is enhanced and maintained until cell death onset in Cf-9 tobacco. ACRE276 RNA interference (RNAi) silencing in tobacco results in loss of hypersensitive response (HR) specified by Cf resistance genes. ACRE276 RNAi plants are also compromised for HR mediated by the tobacco mosaic virus defense elicitor p50. Silencing tomato (Lycopersicon esculentum) ACRE276 leads to breakdown of Cf-9-specified resistance against Cladosporium fulvum leaf mold. We confirmed that tobacco ACRE276 is an E3 ubiquitin ligase requiring an intact U-box domain. Bioinformatic analyses revealed Arabidopsis thaliana PLANT U-BOX17 (PUB17) and Brassica napus ARC1 as the closest homologs of tobacco ACRE276. Transiently expressing PUB17 in Cf-9 tobacco silenced for ACRE276 restores HR, while mutant PUB17 lacking E3 ligase activity fails to do so, demonstrating that PUB17 ligase activity is crucial for defense signaling. Arabidopsis PUB17 knockout plants are compromised in RPM1- and RPS4-mediated resistance against Pseudomonas syringae pv tomato containing avirulence genes AvrB and AvrRPS4, respectively. We identify a conserved class of U-box ARMADILLO repeat E3 ligases that are positive regulators of cell death and defense across the Solanaceae and Brassicaceae.

P-Coumaroylnoradrenaline, a Novel Plant Metabolite Implicated in Tomato Defense against Pathogens

The Avr9 peptide elicitor from the fungus Cladosporium fulvum, the bacterial pathogen Pseudomonas syringae pathovar tomato carrying the avirulence gene avrPto (Pst (avrPto)), and the organophosphorous insecticide fenitrothion induce resistance-related responses in tomato lines carrying the Cf-9, Pto, and Fen genes, respectively. These responses were associated with synthesis of p-coumaroyloctopamine and p-coumaroylnoradrenaline, a novel compound for plants. In susceptible near isogenic tomato lines (Cf-0, pto, fen) and wounded tomato leaves, the levels of these compounds were reduced or undetectable. The elevated levels of p-coumaroyloctopamine and p-coumaroylnoradrenaline were accompanied by elevated mRNA levels of genes encoding phenylalanine ammonia lyase, p-coumarate CoA ligase, and hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT), enzymes that are involved in the hydroxycinnamic acid amide biosynthesis. Southern hybridization indicated that THT is encoded by a multigene family in tomato. Four different THT full-length cDNAs were derived by reverse transcriptase-PCR using degenerate primers based on potato and tobacco THT sequences. Transcripts for all four homologs were present in unchallenged tomato leaves, but only tomTHT1-3 was highly expressed following challenge with Pst (avrPto). Furthermore, tomTHT1-3 showed a more substantial and rapid induction in the incompatible interaction than in the compatible interaction. The cDNAs tomTHT1-3, tomTHT7-1, and tomTHT7-8 encoded proteins with a high degree of amino acid sequence homology, although the recombinant proteins had different preferences for octopamine and noradrenaline. The fourth cDNA, tomTHT1-4, directed synthesis of a truncated enzymatically inactive protein due to the presence of a premature stop codon.

Recent advances in the study of tomato Cf resistance genes

Recent advances in the study of tomato <i>Cf</i> resistance genes

Retracted: The Cf-9 Disease Resistance Protein Is Present in an ∼420-Kilodalton Heteromultimeric Membrane-Associated Complex at One Molecule per Complex

The tomato Cf-9 gene confers race-specific resistance to the fungal pathogen Cladosporium fulvum expressing the corresponding avirulence gene Avr9. In tobacco, Cf-9 confers a hypersensitive response to the Avr9 peptide. To investigate Cf-9 protein function in initiating defense signaling, we engineered a functional C-terminal fusion of the Cf-9 gene with the TAP (Tandem Affinity Purification) tag. In addition, we established a transient expression assay in Nicotiana benthamiana leaves for the production of functional Cf-9:myc and Cf-9:TAP. Transiently expressed Cf-9:myc and Cf-9:TAP proteins induced an Avr9-dependent hypersensitive response, consistent with previous results with stably transformed tobacco plants and derived cell suspension cultures expressing c-myc–tagged Cf-9. Gel filtration of microsomal fractions solubilized with octylglucoside revealed that the Cf-9 protein, either as c-myc or TAP fusions, migrated at a molecular mass of 350 to 475 kD. By using blue native gel electrophoresis, the molecular size was confirmed to be ∼420 kD. Our results suggest that only one Cf-9 protein molecule is present in the Cf-9 complex and that Cf-9 is part of a membrane complex consisting of an additional glycoprotein partner(s). The high structural similarity between Cf proteins and Clavata2 (CLV2) of Arabidopsis, together with the similarity of molecular mass between Cf-9 and CLV complexes (420 and 450 kD, respectively), led us to investigate whether Cf-9 is integrated into membrane-associated protein complexes like those formed by CLV1 and CLV2. Unlike CLV2, the Cf-9 protein did not form disulfide-linked heterodimers, no ligand (Avr9)-dependent shift in the molecular mass of the Cf-9 complex was detected, and no Rho-GTPase–related proteins were found associated with Cf-9 under the conditions tested. Thus, Cf-9–dependent defense signaling and CLV2-dependent regulation of meristem development seem to be accomplished via distinct mechanisms, despite the structural similarity of their key components Cf-9 and CLV2.

Disulfide bond structure of the AVR9 elicitor of the fungal tomato pathogen Cladosporium fulvum: evidence for a cystine knot.

Disease resistance in plants is commonly activated by the product of an avirulence (Avr) gene of a pathogen after interaction with the product of a matching resistance (R) gene in the host. In susceptible plants, Avr products might function as virulence or pathogenicity factors. The AVR9 elicitor from the fungus Cladosporium fulvum induces defense responses in tomato plants carrying the Cf-9 resistance gene. This 28-residue beta-sheet AVR9 peptide contains three disulfide bridges, which were identified in this study as Cys2-Cys16, Cys6-Cys19, and Cys12-Cys26. For this purpose, AVR9 was partially reduced, and the thiol groups of newly formed cysteines were modified to prevent reactions with disulfides. After HPLC purification, the partially reduced peptides were sequenced to determine the positions of the modified cysteines, which originated from the reduced disulfide bridge(s). All steps involving molecules with free thiol groups were performed at low pH to suppress disulfide scrambling. For that reason, cysteine modification by N-ethylmaleimide was preferred over modification by iodoacetamide. Upon (partial) reduction of native AVR9, the Cys2-Cys16 bridge opened selectively. The resulting molecule was further reduced to two one-bridge intermediates, which were subsequently completely reduced. The (partially) reduced cysteine-modified AVR9 species showed little or no necrosis-inducing activity, demonstrating the importance of the disulfide bridges for biological activity. Based on peptide length and cysteine spacing, it was previously suggested that AVR9 isa cystine-knotted peptide. Now, we have proven that the bridging pattern of AVR9 is indeed identical to that of cystine-knotted peptides. Moreover, NMR data obtained for AVR9 show that it is structurally closely related to the cystine-knotted carboxypeptidase inhibitor. However, AVR9 does not show any carboxypeptidase inhibiting activity, indicating that the cystine-knot fold is a commonly occurring motif with varying biological functions.

Domain swapping and gene shuffling identify sequences required for induction of an Avr-dependent hypersensitive response by the tomato Cf-4 and Cf-9 proteins.

The tomato Cf-4 and Cf-9 genes confer resistance to infection by the biotrophic leaf mold pathogen Cladosporium. Their protein products induce a hypersensitive response (HR) upon recognition of the fungus-encoded Avr4 and Avr9 peptides. Cf-4 and Cf-9 share >91% sequence identity and are distinguished by sequences in their N-terminal domains A and B, their N-terminal leucine-rich repeats (LRRs) in domain C1, and their LRR copy number (25 and 27 LRRs, respectively). Analysis of Cf-4/Cf-9 chimeras, using several different bioassays, has identified sequences in Cf-4 and Cf-9 that are required for the Avr-dependent HR in tobacco and tomato. A 10-amino acid deletion within Cf-4 domain B relative to Cf-9 was required for full Avr4-dependent induction of an HR in most chimeras analyzed. Additional sequences required for Cf-4 function are located in LRRs 11 and 12, a region that contains only eight of the 67 amino acids that distinguish it from Cf-9. One chimera, with 25 LRRs that retained LRR 11 of Cf-4, induced an attenuated Avr4-dependent HR. The substitution of Cf-9 N-terminal LRRs 1 to 9 with the corresponding sequences from Cf-4 resulted in attenuation of the Avr9-induced HR, as did substitution of amino acid A433 in LRR 15. The amino acids L457 and K511 in Cf-9 LRRs 16 and 18 are essential for induction of the Avr9-dependent HR. Therefore, important sequence determinants of Cf-9 function are located in LRRs 10 to 18. This region contains 15 of the 67 amino acids that distinguish it from Cf-4, in addition to two extra LRRs. Our results demonstrate that sequence variation within the central LRRs of domain C1 and variation in LRR copy number in Cf-4 and Cf-9 play a major role in determining recognition specificity in these proteins.

Early defence responses induced by AVR9 and mutant analogues in tobacco cell suspensions expressing the Cf-9 resistance gene

The interaction between the fungal leaf pathogen Cladosporium fulvum and its only host, tomato, fits the gene-for-gene model. In tomato, the Cf-9 resistance gene product mediates specific recognition of the fungal avirulence gene product AVR9, resulting in a hypersensitive response and resistance. Cf9 tomato leaves respond with necrosis after injection with AVR9, whereas Cf9 tomato cell suspensions do not show defence responses after treatment with AVR9. Here we report on early defence responses induced in Cf-9 transgenic tobacco leaves and Cf-9 transgenic tobacco cell suspensions after treatment with synthesized AVR9 and mutant analogues R08K, F10A and F21A. The necrosis-inducing activity of the AVR9 peptides increased in the order F21A, F10A, AVR9, R08K. An oxidative burst was induced at a much lower AVR9 peptide concentration as compared to medium alkalization and necrosis. Interestingly, the mutant peptide F21A failed to induce necrosis and medium alkalization but did induce an oxidative burst. In all assays, the relative differential activities of the AVR9 peptides were similar to those observed in Cf9 tomato leaves. Both AVR9 and F21A activated a MAP kinase in Cf-9 transgenic tobacco cell suspensions. AVR9 also induced specific cell death in these suspensions. The relation between the induction of early defence responses and necrosis is discussed.

Functional, c-myc-tagged Cf-9 resistance gene products are plasma-membrane localized and glycosylated.

The Cf-9 resistance gene from tomato confers resistance to races of the fungal pathogen Cladosporium fulvum that express the corresponding avirulence gene, Avr9. Avr9 encodes a secreted peptide. To investigate Cf-9 function, we tagged the Cf-9 protein with a triple myc epitope at either the amino- or carboxy-terminus of the mature protein. Tobacco plants carrying these constructs activate a defence response to Avr9 peptide. The Cf-9 sequence predicts a protein of 94 kDa, with 22 glycosylation sites. Using c-myc antibodies, c-myc : Cf-9 protein was detected as a unique band with a molecular size of 160 kDa. The band shifted to approximately 105 kDa after glucosidase treatment, indicating that Cf-9 protein is highly glycosylated. Plasma membranes were isolated using two-phase partitioning, and c-myc : Cf-9 was enriched in these fractions, indicating that Cf-9 is a plasma membrane protein. This was confirmed by silver-enhanced immunogold labelling of tobacco protoplasts carrying the amino-terminal c-myc tag; a higher labelling density was observed on the surface of protoplasts derived from c-myc : Cf-9 tobacco compared to untransformed control. The presence of Cf-9 in the plasma membrane is consistent with its role in conferring recognition of the extracellular Avr9 peptide.

Folding and conformational analysis of AVR9 peptide elicitors of the fungal tomato pathogen Cladosporium fulvum.

The race-specific elicitor AVR9, produced by the phytopathogenic fungus Cladosporium fulvum, is a 28-residue beta-sheet peptide containing three disulfide bridges. The folding of this peptide to its native conformation was examined in the presence of oxidized (GSSG) and reduced (GSH) glutathione at concentrations resembling those present in the endoplasmic reticulum. The concentrations of GSH and GSSG, and the applied temperature strongly affected the folding efficiency. The effect of temperature appeared reversible. The conditions for in vitro folding were optimized and a maximum yield of 60-70% of correctly folded peptide was obtained. In vitro folded AVR9 is equally as active as native fungal AVR9. They both display similar NMR characteristics, indicating that they have the same 3D structure and identical disulfide bridges. Thus, AVR9 can be folded correctly in vitro. This folding can be described by disulfide bridge formation leading to scrambled three-disulfide species, followed by disulfide reshuffling to acquire the native structure. The presence of urea significantly affected the folding of AVR9, indicating that noncovalent interactions play a role in directing correct folding. Protein disulfide isomerase increased the folding rate at least 15-fold, but had no effect on the yield. The folding procedure has also been applied successfully to two mutant AVR9 peptides, (K23A)AVR9 and biotinylated AVR9. We conclude that the 28-residue sequence, without the preprosequence (as present in vivo), contains sufficient information to direct correct folding and disulfide bridge formation in vitro.

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.

Correlation between binding affinity and necrosis-inducing activity of mutant AVR9 peptide elicitors.

The race-specific peptide elicitor AVR9 of the fungus Cladosporium fulvum induces a hypersensitive response only in tomato (Lycopersicon esculentum) plants carrying the complementary resistance gene Cf-9 (MoneyMaker-Cf9). A binding site for AVR9 is present on the plasma membranes of both resistant and susceptible tomato genotypes. We used mutant AVR9 peptides to determine the relationship between elicitor activity of these peptides and their affinity to the binding site in the membranes of tomato. Mutant AVR9 peptides were purified from tobacco (Nicotiana clevelandii) inoculated with recombinant potato virus X expressing the corresponding avirulence gene Avr9. In addition, several AVR9 peptides were synthesized chemically. Physicochemical techniques revealed that the peptides were correctly folded. Most mutant AVR9 peptides purified from potato virus X::Avr9-infected tobacco contain a single N-acetylglucosamine. These glycosylated AVR9 peptides showed a lower affinity to the binding site than the nonglycosylated AVR9 peptides, whereas their necrosis-inducing activity was hardly changed. For both the nonglycosylated and the glycosylated mutant AVR9 peptides, a positive correlation between their affinity to the membrane-localized binding site and their necrosis-inducing activity in MoneyMaker-Cf9 tomato was found. The perception of AVR9 in resistant and susceptible plants is discussed.

Assignment of amino acid residues of the AVR9 peptide of Cladosporium fulvum that determine elicitor activity.

The AVR9 peptide of Cladosporium fulvum is an elicitor of the hypersensitive response in tomato plants carrying the Cf-9 resistance gene (MM-Cf9). To determine the structure-activity relationship of the AVR9 peptide, amino acids important for AVR9 elicitor activity were identified by independently substituting each amino acid of AVR9 by alanine. In addition, surface-exposed amino acid residues of AVR9 were substituted by other amino acids. Activity of the mutant Avr9 constructs was studied by expressing the constructs in MM-Cf9 tomato plants, using the potato virus X (PVX) expression system and assessing the severity of necrosis induced by each PVX::Avr9 construct. This allowed direct identification of amino acid residues of AVR9 that are essential for elicitor activity. We identified amino acid substitutions that resulted in AVR9 mutants with higher, similar, or lower elicitor activity compared to the wild-type AVR9 peptide. Some mutants had completely lost elicitor activity. A selection of peptides, representing different categories, was isolated and injected into leaves of MM-Cf9 plants. The necrosis-inducing activity of the isolated peptides correlated well with the necrosis induced by the corresponding PVX::Avr9 derivatives. Based on the necrosis-inducing activity of the mutant AVR9 peptides and the global structure of AVR9, we assigned sites in AVR9 that are important for its necrosis-inducing activity. We postulate that the "hydrophobic beta-loop" region of the AVR9 peptide is crucial for necrosis-inducing activity in tomato plants that carry the Cf-9 resistance gene.

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 gene-for-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.

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.

A High-Affinity Binding Site for the AVR9 Peptide Elicitor of Cladosporium fulvum Is Present on Plasma Membranes of Tomato and Other Solanaceous Plants

A High-Affinity Binding Site for the AVR9 Peptide Elicitor of Cladosporium fulvum Is Present on Plasma Membranes of Tomato and Other Solanaceous Plants

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.

Isolation of the tomato Cf-9 gene for resistance to Cladosporium fulvum by transposon tagging.

The tomato Cf-9 gene confers resistance to infection by races of the fungus Cladosporium fulvum that carry the avirulence gene Avr9. The Cf-9 gene was isolated by transposon tagging with the maize transposable element Dissociation. The DNA sequence of Cf-9 encodes a putative membrane-anchored extracytoplasmic glycoprotein. The predicted protein shows homology to the receptor domain of several receptor-like protein kinases in Arabidopsis, to antifungal polygalacturonase-inhibiting proteins in plants, and to other members of the leucine-rich repeat family of proteins. This structure is consistent with that of a receptor that could bind Avr9 peptide and activate plant defense.

Developmentally regulated cell death on expression of the fungal avirulence gene Avr9 in tomato seedlings carrying the disease-resistance gene Cf-9.

Plant defense responses are induced when the products of disease-resistance genes and pathogen avirulence genes interact. We report here the effects of expressing the Cladosporium fulvum avirulence Avr9 gene product in a tomato line containing the Cf-9 disease-resistance gene. A synthetic Avr9 gene was constructed to produce constitutive high-level expression of AVR9 peptide in the plant apoplast. Avr9 expression in Cf-9-containing tomato lines is lethal, but cell death is developmentally regulated, in that necrosis is not visible until 10 days after planting seed. Plant lines lacking Cf-9 and expressing Avr9 remain healthy. The synthetic Avr9 gene exhibited the same specificity of action as the authentic C. fulvum Avr9 gene. Our results have significant implications for strategies using the gene combination Avr9/Cf-9 to engineer plants with enhanced disease resistance.

The AVR9 race-specific elicitor of Cladosporium fulvum is processed by endogenous and plant proteases.

The avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum encodes a race-specific peptide elicitor that induces a hypersensitive response in tomato plants carrying the complementary resistance gene Cf9. The avr9 gene is highly expressed when C. fulvum is growing in the plant and the elicitor accumulates in infected leaves as a 28-amino acid (aa) peptide. In C. fulvum grown in vitro, the peptide elicitor is not produced in detectable amounts. To produce significant amounts of the AVR9 elicitor in vitro, the coding and termination sequences of the avr9 gene were fused to the constitutive gpd-promoter (glyceraldehyde 3-phosphate dehydrogenase) of Aspergillus nidulans. Transformants of C. fulvum were obtained that highly expressed the avr9 gene in vitro and produced active AVR9 peptide elicitors. These peptides were partially sequenced from the N terminus and appeared to consist of 32, 33, and 34 aa's, respectively, and are the precursors of the mature 28-aa AVR9 peptide. We demonstrated that plant factors process the 34-aa peptide into the mature 28-aa peptide. We present a model for the processing of AVR9 involving cleavage of a signal peptide during excretion and further maturation by fungal and plant proteases into the stable 28-aa peptide elicitor.