Biomedical subjects

P J De Wit

Publications and source records attributed to P J De Wit.

At least 19 recordsLinked to original sources

PubMed2001

Intragenic recombination generated two distinct Cf genes that mediate AVR9 recognition in the natural population of Lycopersicon pimpinellifolium.

Resistance gene Cf-9 of cultivated tomato (Lycopersicon esculentum) confers recognition of the AVR9 elicitor protein of the fungal pathogen Cladosporium fulvum. The Cf-9 locus, containing Cf-9 and four homologs (Hcr9s), originates from Lycopersicon pimpinellifolium (Lp). We examined naturally occurring polymorphism in Hcr9s that confer AVR9 recognition in the Lp population. AVR9 recognition occurs frequently throughout this population. In addition to Cf-9, we discovered a second gene in Lp, designated 9DC, which also confers AVR9 recognition. Compared with Cf-9, 9DC is more polymorphic, occurs more frequently, and is more widely spread throughout the Lp population, suggesting that 9DC is older than Cf-9. The sequences of Cf-9 and 9DC suggest that Cf-9 evolved from 9DC by intragenic recombination between 9DC and another Hcr9. The fact that the 9DC and Cf-9 proteins differ in 61 aa residues, and both mediate recognition of AVR9, shows that in nature Hcr9 proteins with the same recognitional specificity can vary significantly.

Amino Acid Sequence↗

PubMed2001

Expression of the Avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum is regulated by the global nitrogen response factor NRF1.

Here we describe the role of the Cladosporium fulvum nitrogen response factor 1 (Nrf1) gene in regulation of the expression of avirulence gene Avr9 and virulence on tomato. The Nrf1 gene, which was isolated by a polymerase chain reaction-based strategy, is predicted to encode a protein of 918 amino acid residues. The protein contains a putative zinc finger DNA-binding domain that shares 98% amino acid identity with the zinc finger of the major nitrogen regulatory proteins AREA and NIT2 of Aspergillus nidulans and Neurospora crassa, respectively. Functional equivalence of Nrf1 to areA was demonstrated by complementation of an A. nidulans areA loss-of-function mutant with Nrf1. Nrf1-deficient transformants of C. fulvum obtained by homologous recombination were unable to utilize nitrate and nitrite as a nitrogen source. In contrast to what was observed in the C. fulvum wild-type, the Avr9 gene was no longer induced under nitrogen-starvation conditions in Nrf1-deficient strains. On susceptible tomato plants, the Nrf1-deficient strains were as virulent as wild-type strains of C. fulvum, although the expression of the Avr9 gene was strongly reduced. In addition, Nrf1-deficient strains were still avirulent on tomato plants containing the functional Cf-9 resistance gene, indicating that in planta, apparently sufficient quantities of stable AVR9 elicitor are produced. Our results suggest that the NRF1 protein is a major regulator of the Avr9 gene.

Amino Acid Sequence↗

PubMed2001

No evidence for binding between resistance gene product Cf-9 of tomato and avirulence gene product AVR9 of Cladosporium fulvum.

The gene-for-gene model postulates that for every gene determining resistance in the host plant, there is a corresponding gene conditioning avirulence in the pathogen. On the basis of this relationship, products of resistance (R) genes and matching avirulence (Avr) genes are predicted to interact. Here, we report on binding studies between the R gene product Cf-9 of tomato and the Avr gene product AVR9 of the pathogenic fungus Cladosporium fulvum. Because a high-affinity binding site (HABS) for AVR9 is present in tomato lines, with or without the Cf-9 resistance gene, as well as in other solanaceous plants, the Cf-9 protein was produced in COS and insect cells in order to perform binding studies in the absence of the HABS. Binding studies with radio-labeled AVR9 were performed with Cf-9-producing COS and insect cells and with membrane preparations of such cells. Furthermore, the Cf-9 gene was introduced in tobacco, which is known to be able to produce a functional Cf-9 protein. Binding of AVR9 to Cf-9 protein produced in tobacco was studied employing surface plasmon resonance and surface-enhanced laser desorption and ionization. Specific binding between Cf-9 and AVR9 was not detected with any of the procedures. The implications of this observation are discussed.

Animals↗

PubMed2001

Identification of distinct specificity determinants in resistance protein Cf-4 allows construction of a Cf-9 mutant that confers recognition of avirulence protein Avr4.

The tomato resistance genes Cf-4 and Cf-9 confer specific, hypersensitive response-associated recognition of Cladosporium carrying the avirulence genes Avr4 and Avr9, respectively. Cf-4 and Cf-9 encode type I transmembrane proteins with extracellular leucine-rich repeats (LRRs). Compared with Cf-9, Cf-4 lacks two LRRs and differs in 78 amino acid residues. To investigate the relevance of these differences for specificity, we exchanged domains between Cf-4 and Cf-9, and mutant constructs were tested for mediating the hypersensitive response by transient coexpression with either Avr4 or Avr9. We show that the number of LRRs is essential for both Cf-4 and Cf-9 function. In addition, Cf-9 specificity resides entirely in the LRR domain and appears to be distributed over several distant LRRs. In contrast, Cf-4 specificity determinants reside in the N-terminal LRR-flanking domain and three amino acid residues in LRRs 13, 14, and 16. These residues are present at putative solvent-exposed positions, and all are required for full Cf-4 function. Finally, we show that Cf-9 carrying the specificity determinants of Cf-4 has recognitional specificity for AVR4. The data indicate that diversifying selection of solvent-exposed residues has been a more important factor in the generation of Cf-4 specificity than has sequence exchange between Cf-4 progenitor genes. The fact that most variant residues in Cf-4 are not essential for Cf-4 specificity indicates that the diverse decoration of R proteins is not fully adapted to confer recognition of a certain avirulence determinant but likely provides a basis for a versatile, adaptive recognition system.

Amino Acid Sequence↗

PubMed2000

Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis.

The avirulence genes Avr9 and Avr4 from the fungal tomato pathogen Cladosporium fulvum encode extracellular proteins that elicit a hypersensitive response when injected into leaves of tomato plants carrying the matching resistance genes, Cf-9 and Cf-4, respectively. We successfully expressed both Avr9 and Avr4 genes in tobacco with the Agrobacterium tumefaciens transient transformation assay (agroinfiltration). In addition, we expressed the matching resistance genes, Cf-9 and Cf-4, through agroinfiltration. By combining transient Cf gene expression with either transgenic plants expressing one of the gene partners, Potato virus X (PVX)-mediated Avr gene expression, or elicitor injections, we demonstrated that agroinfiltration is a reliable and versatile tool to study Avr/Cf-mediated recognition. Significantly, agroinfiltration can be used to quantify and compare Avr/Cf-induced responses. Comparison of different Avr/Cf-interactions within one tobacco leaf showed that Avr9/Cf-9-induced necrosis developed slower than necrosis induced by Avr4/Cf-4. Quantitative analysis demonstrated that this temporal difference was due to a difference in Avr gene activities. Transient expression of matching Avr/Cf gene pairs in a number of plant families indicated that the signal transduction pathway required for Avr/Cf-induced responses is conserved within solanaceous species. Most non-solanaceous species did not develop specific Avr/Cf-induced responses. However, co-expression of the Avr4/Cf-4 gene pair in lettuce resulted in necrosis, providing the first proof that a resistance (R) gene can function in a different plant family.

Agrobacterium tumefaciens↗

PubMed1999

Transcription of the avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum is regulated by a GATA-type transcription factor in Aspergillus nidulans.

The avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum is highly induced during infection of tomato plants. Expression of the Avr9 gene can also be induced in vitro when cells are grown on synthetic liquid medium containing little or no nitrogen. The Avr9 promoter contains six copies of the sequence TAGATA and six additional copies of the core sequence GATA within 0.4 kb upstream of the translation start site. In the filamentous fungi Aspergillus nidulans and Neurospora crassa, these promoter sequences have been identified as the binding sites for a wide-domain GATA-type regulator (AREA in A. nidulans and NIT2 in N. crassa) involved in nitrogen utilization. Quantification of GUS activity of A. nidulans transformants containing a single copy of the fully active Avr9 promoter-uidA (GUS) reporter gene fusion in different areA backgrounds, following starvation for nitrogen, showed that induction of the Avr9 promoter is regulated similarly in A. nidulans and C. fulvum. This suggests that AREA can regulate the Avr9 promoter and that C. fulvum contains an AREA-like regulator that can bind to these specific sequence motifs. Comparison of the induction profiles of Avr9 and niaD showed that Avr9 expression is independent of NIRA, as is niaD expression upon nitrogen starvation. Studies with Avr9 promoter-uidA fusions in which all or most of these sequences had been deleted, showed that Avr9 promoter activity is dependent on the presence of these specific cis-regulatory elements, suggesting that they do indeed function in transcriptional regulation of the Avr9 gene.

Aspergillus nidulans↗

PubMed1998

Successful search for a resistance gene in tomato targeted against a virulence factor of a fungal pathogen.

The interaction between tomato and its fungal pathogen Cladosporium fulvum complies with the gene-for-gene system, in which specific recognition of fungal proteins by plant genotypes with matching resistance genes results in host resistance. Two proteins, ECP1 and ECP2, secreted by C. fulvum during infection, are required for full virulence of the fungus on tomato. We chose the most important virulence factor, ECP2, for a targeted search for hypersensitive response (HR)-based resistance among a collection of tomato genotypes. By screening with recombinant potato virus X that expresses the Ecp2 gene, we identified four lines that respond with HR toward ECP2. The capacity to recognize ECP2 and induce HR is sufficient to confer resistance in tomato against C. fulvum producing ECP2. Resistance is based on a single dominant gene, which we have designated Cf-ECP2, for resistance to C. fulvum through recognition of ECP2. Accordingly, an Ecp2-minus strain created by gene replacement is pathogenic on Cf-ECP2 plants. However, due to lack of ECP2 the mutant strain is only weakly virulent. All strains of a worldwide collection of C. fulvum strains that were tested were found to produce a HR-inducing ECP2 protein. Because the Cf-ECP2 gene operates through recognition of an important virulence factor, we expect it will confer durable resistance against C. fulvum. A similar targeted approach should allow the discovery of new valuable resistance genes in other pathosystems.

Base Sequence↗

PubMed1998

Fungal avirulence genes: structure and possible functions.

Avirulence (Avr) genes exist in many fungi that share a gene-for-gene relationship with their host plant. They represent unique genetic determinants that prevent fungi from causing disease on plants that possess matching resistance (R) genes. Interaction between elicitors (primary or secondary products of Avr genes) and host receptors in resistant plants causes induction of various defense responses often involving a hypersensitive response. Avr genes have been successfully isolated by reverse genetics and positional cloning. Five cultivar-specific Avr genes (Avr4, Avr9, and Ecp2 from Cladosporium fulvum; nip1 from Rhynchosporium secalis; and Avr2-YAMO from Magnaporthe grisea) and three species-specific Avr genes (PWL1 and PWL2 from M. grisea and inf1 from Phytophthora infestans) have been cloned. Isolation of additional Avr genes from these fungi, but also from other fungi such as Uromyces vignae, Melampsora lini, Phytophthora sojae, and Leptosphaeria maculans, is in progress. Molecular analyses of nonfunctional Avr gene alleles show that these originate from deletions or mutations in the open reading frame or the promoter sequence of an Avr gene. Although intrinsic biological functions of most Avr gene products are still unknown, recent studies have shown that two Avr genes, nip1 and Ecp2, encode products that are important pathogenicity factors. All fungal Avr genes cloned so far have been demonstrated or predicted to encode extracellular proteins. Current studies focus on unraveling the mechanisms of perception of avirulence factors by plant receptors. The exploitation of Avr genes and the matching R genes in engineered resistance is also discussed.

Fungi↗

PubMed1998

Solid-phase synthesis, conformational analysis, and biological activity of AVR9 elicitor peptides of the fungal tomato pathogen Cladosporium fulvum.

The race-specific peptide elicitor AVR9 of the fungal pathogen Cladosporium fulvum specifically induces a hypersensitive response in tomato genotypes carrying the complementary resistance gene Cf-9. The total chemical syntheses of this 28-residue AVR9 peptide containing three disulfide bonds, and of three mutant peptides [R8K]AVR9, [F10A]AVR9 and [F21A]AVR9, have been accomplished. The syntheses were carried out using a stepwise solid-phase approach based on tBoc chemistry. The disulfide bridges were formed by air oxidation. The correctness of the chemical structure of all folded synthetic peptides was confirmed by combined NMR and MS analyses. The biological activity and a number of physicochemical properties of folded synthetic AVR9 are identical to those of native fungal 28-residue AVR9. The overall conformations of the folded synthetic mutant peptides were comparable to that of synthetic wild-type AVR9 as demonstrated by NMR spectroscopy. Mutant [R8K]AVR9 showed a threefold higher, and mutant [F10A]AVR9 a threefold lower necrosis-inducing activity when compared to synthetic wild-type AVR9. However, mutant [F21A]AVR9 showed hardly any necrosis-inducing activity. Affinity for polyclonal antibodies raised against native fungal AVR9 is positively correlated with the necrosis-inducing activity of the synthetic AVR9 peptides ([R8K]AVR9 > wild-type AVR9 > [F10A]AVR9 > [F21A]AVR9).

Blotting, Western↗

PubMed1997

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.

Cladosporium↗

PubMed1997

The biotrophic fungus Cladosporium fulvum circumvents Cf-4-mediated resistance by producing unstable AVR4 elicitors.

The avirulence gene Avr4 conditions avirulence of the biotrophic fungus Cladosporium fulvum on tomato genotypes carrying resistance gene Cf-4 (MM-Cf4). Strains of the fungus that circumvent Cf-4-specific resistance show various single point mutations in the coding region of the Avr4 gene. Similar to expression of the Avr4 gene, expression of the various virulent avr4 alleles is specifically induced during pathogenesis. Polyclonal antibodies raised against the AVR4 elicitor, however, did not detect AVR4 isoforms in MM-Cf4 plants infected by the different virulent strains, indicating that these isoforms are unstable. To analyze whether the AVR4 isoforms still possess specific elicitor activity, the avr4 alleles were expressed in MM-Cf4 plants by using the potato virus X (PVX)-based expression system. Inoculation with PVX::Avr4 resulted in the development of spreading lesions, eventually leading to plant death, whereas the various PVX::avr4 derivatives induced symptoms ranging from severe necrosis to no lesions at all. We conclude that instability of the AVR4 isoforms that are produced by virulent strains is a crucial factor in circumvention of Cf-4-mediated resistance.

Alleles↗

PubMed1995

Induction of tomato stress protein mRNAs by ethephon, 2,6-dichloroisonicotinic acid and salicylate.

To study the possible involvement of plant hormones in the synthesis of stress proteins in tomato upon inoculation with Cladosporium fulvum, we investigated the induction of mRNAs encoding PR proteins and ethylene biosynthesis enzymes by ethephon, 2,6-dichloroisonicotinic acid (INA) and salicylic acid (SA) by northern blot analysis. Ethephon slightly induced some but not all mRNAs encoding intra- and extracellular PR proteins. INA induced all PR protein mRNAs analysed, except for intracellular chitinase and extracellular PR-4. SA induced all PR protein mRNAs analyzed, except for intracellular chitinase and osmotin. None of the inducers affected the expression of ACC synthase mRNA, whereas all three induced ethylene-forming enzyme (EFE) mRNA.

Gene Expression↗

PubMed1994

Nitrogen limitation induces expression of the avirulence gene avr9 in the tomato pathogen Cladosporium fulvum.

The avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum encodes a race-specific peptide elicitor that induces the hypersensitive response in tomato plants carrying the complementary resistance gene Cf9. The avr9 gene is not expressed under optimal growth conditions in vitro, but is highly expressed when the fungus grows inside the tomato leaf. In this paper we present evidence for the induction of avr9 gene expression in C. fulvum grown in vitro under conditions of nitrogen limitation. Only growth medium with very low amounts of nitrogen (nitrate, ammonium, glutamate or glutamine) induced the expression of avr9. Limitation of other macronutrients or the addition of plant factors did not induce the expression of avr9. The induced expression of avr9 is possibly mediated by a positive-acting nitrogen regulatory protein, homologous to the Neurospora crassa NIT2 protein, which induces the expression of many genes under conditions of nitrogen limitation. The avr9 promoter contains several putative NIT2 binding sites. The expression of avr9 during the infection process was explored cytologically using transformants of C. fulvum carrying an avr9 promoter-beta-glucuronidase reporter gene fusion. The possibility that expression of avr9 in C. fulvum growing in planta is caused by nitrogen limitation in the apoplast of the tomato leaf is discussed.

Base Sequence↗

PubMed1994

Host resistance to a fungal tomato pathogen lost by a single base-pair change in an avirulence gene.

Host genotype specificity in interactions between biotrophic 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 host plant, respectively. Resistance, expressed by the induction of a hypersensitive response in the host, is envisaged to be based on recognition of the pathogen, mediated through direct interaction between products of pathogen avirulence genes (the so-called race-specific elicitors) and receptors in the host plant, the putative products of resistance genes. The interaction between the biotrophic fungus Cladosporium fulvum and its only host, tomato (Lycopersicon esculentum), is a well-established model system for studying fungus-plant gene-for-gene relationships. Here we report the isolation of race-specific elicitor AVR4 of C. fulvum and the cloning of its encoding avirulence gene. We present evidence that, in nature, a single base-pair change in this avirulence gene leads to virulence of races previously avirulent on tomato genotypes carrying the complementary Cf4 resistance gene.

Amino Acid Sequence↗

PubMed1994

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.

Cladosporium↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1992

Molecular analysis of the avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum fully supports the gene-for-gene hypothesis.

The interaction between the fungal pathogen Cladosporium fulvum and tomato is supposed to have a gene-for-gene basis. Races of C. fulvum which have 'overcome' the resistance gene Cf9 of tomato, lack the avirulence gene avr9 which encodes a race-specific peptide elicitor. Races avirulent on tomato genotypes carrying the resistance gene Cf9 produce the race-specific peptide elicitor, which induces the hypersensitive response (HR) on those genotypes. The causal relationship between the presence of a functional avr9 gene and avirulence on tomato genotype Cf9 was demonstrated by cloning of the avr9 gene and subsequent transformation of C. fulvum. A race virulent on tomato genotype Cf9 was shown to become avirulent by transformation with the cloned avr9 gene. These results clearly demonstrate that the avr9 gene is responsible for cultivar specificity on tomato genotype Cf9 and fully support the gene-for-gene hypothesis. The avr9 gene is the first fungal avirulence gene to be cloned.

Amino Acid Sequence↗

PubMed1990

Subcellular Localization of Chitinase and of Its Potential Substrate in Tomato Root Tissues Infected by Fusarium oxysporum f. sp. radicis-lycopersici.

Antiserum raised against a tomato (Lycopersicon esculentum Mill.) chitinase (molecular mass of 26 kilodaltons) was used as a probe to study the subcellular localization of this enzyme in tomato root tissues infected with Fusarium oxysporum f. sp. radicis-lycopersici. A time-course experiment revealed that chitinase accumulated earlier in the incompatible interaction than in the compatible one. However, in both systems, chitinase deposition was largely correlated with pathogen distribution. The enzyme was found to accumulate in areas where host walls were in close contact with fungal cells. In contrast, the enzyme could not be detected in vacuoles and intracellular spaces. The substantial amount of chitinase found at the fungus cell surface supports the view of an antifungal activity. However, the preferential association of the enzyme with altered fungal wall areas indicates that chitinase activity is either preceded by the hydrolytic action of other enzymes such as beta-1,3-glucanases or coincides with these enzymes. The possibility that fungal glucans released through the action of beta-1,3-glucanases may act as elicitors of chitinase production is discussed.

Journal Article↗