Isolation and Characterization of an Elicitor of Necrosis Isolated from Intercellular Fluids of Compatible Interactions of Cladosporium fulvum (Syn. Fulvia fulva) and Tomato

Intercellular fluids of compatible race-cultivar interactions of Cladosporium fulvum and tomato contain specific elicitors of necrosis. These elicitors which are of fungal origin induce chlorosis and necrosis in resistant but not in susceptible plants. With the tomato cultivar Sonatine (carrying resistance gene Cf9, resistant to the fungal races 0, 4, 5, 2, 2.4, and 2.4.5 but susceptible to race 2.4.5.9) as the test plant for assaying necrosis-inducing activity, we isolated and partially characterized an elicitor of necrosis on this cultivar. The elicitor bound to CM-Sephadex but not to DEAE-Sephadex; it was stable to heat (10 minutes at 100°C), HCl (0.01 normal), NaOH (0.01 normal), and NaIO₄ (0.02 molar), sensitive to pronase and protease (from Bacillus polymyxa) but not to other proteases such as α-chymotrypsin and trypsin. After electrophoresis of partially purified elicitor preparations under low pH conditions, the necrosis-inducing activity was association with a peptide with an apparent molecular weight of 5500. Races 0, 4, 5, 2.4, and 2.4.5 but not race 2.4.5.9 produced this elicitor in high yields. The elicitor is probably a product of avirulence gene A9 which is present in all races except in race 2.4.5.9 and induces necrosis in cultivars carrying resistance gene Cf9.     

Electrolyte Leakage, Lipoxygenase, and Lipid Peroxidation Induced in Tomato Leaf Tissue by Specific and Nonspecific Elicitors from Cladosporium fulvum

Glycoprotein nonspecific elicitor (NSE) and a specific elicitor preparation from intercellular fluids (SE) of tomato (Lycopersicon esculentum Mill. cv Bonny Best or Potentate) infected with race 2.4.5 of Cladosporium fulvum Cooke [syn. Fulvia fulva (Cooke) Ciferri] were injected into cv Sonatine (resistant to race 2.4.5) to compare electrolyte leakage, lipoxygenase activity, and lipid peroxidation induced in response to these elicitors. Increased electrolyte leakage was induced by NSE or SE; the leakage due to NSE but not to SE was inhibited by the nonsteroidal antiinflammatory drug (NSAID) piroxicam. Under normal photoperiod conditions, higher levels of lipoxygenase activity were detected 6 hours after injection with either elicitor. This activity peaked by 12 hours with both elicitors and declined to control levels by 24 hours when visible necrosis could be detected. Both NSE and SE-induced lipoxygenase was inhibited by piroxicam in vitro. Lipid peroxidation in elicitor-treated tissue was also assayed at 6, 12, and 24 hours after injection using the TBA test for malonaldehyde. Increased peroxidation was detected in response to NSE or SE at 12 hours with similar values obtained at 24 hours. With plants incubated in the dark, lipoxygenase, and lipid peroxidation were similarly induced in SE-injected tissue whereas necrosis induction by SE was light dependent.     

Identification of G proteins mediating fungal elicitor-induced dephosphorylation of host plasma membrane H+ -ATPase

Tomato cells (with the Cf-5 resistance gene) were treated withelicitor preparations containing the avr5 gene product fromtwo Cf-5 incompatible races of the fungal pathogen Cladosporiumfulvum (race 2.3 and race 4), or with elicitor preparationscontaining no avr5 gene product from two Cf-5 compatible races(race 5 and race 2.4.5.9 [EC] .11). Elicitor preparations from race2.3 or race 4 caused dephosphorylation of host plasma membraneH⁺ -ATPase in isolated plasma membranes, while the preparationsfrom race 5 or race 2.4.5.9 [EC] .11 did not. GTP()S, AlF₄^–and cholera toxin (CTX) each induced similar dephosphorylationin the absence of active elicitors. The elicitor-induced dephosphorylationof the H⁺ -ATPase was blocked by preincubation of membraneswith an antibody raised against a stimulatory G protein -subunit(anti-G_s This antibody cross-reacted with a 42 kDa polypeptidefrom tomato plasma membranes. A 42 kDa polypeptide was alsoADP-ribosylated by CTX. When plasma membranes were treated withelicitor preparations from race 4 and separated on non-dissociatingPAGE, two proteins were detected on Western blots with the antibodyraised against the -subunit, suggesting the dissociation ofthe trimeric complex. No dissociation of the complex was detectedwith antibodies raised against either the - or ß-subunitswhen the plasma membranes were treated with elicitor preparationsfrom race 5. The results provide evidence for the activationof a stimulatory subtype of trimeric G proteins in the stimulationof elicitor-induced host defences to fungal pathogens. Key words: G protein, dephosphorylation, H⁺ -ATPase, fungal elicitor, tomato     

Novel Mutations Detected in Avirulence Genes Overcoming Tomato Cf Resistance Genes in Isolates of a Japanese Population of Cladosporium fulvum

Leaf mold of tomato is caused by the biotrophic fungus Cladosporium fulvum which complies with the gene-for-gene system. The disease was first reported in Japan in the 1920s and has since been frequently observed. Initially only race 0 isolates were reported, but since the consecutive introduction of resistance genes Cf-2, Cf-4, Cf-5 and Cf-9 new races have evolved. Here we first determined the virulence spectrum of 133 C. fulvum isolates collected from 22 prefectures in Japan, and subsequently sequenced the avirulence (Avr) genes Avr2, Avr4, Avr4E, Avr5 and Avr9 to determine the molecular basis of overcoming Cf genes. Twelve races of C. fulvum with a different virulence spectrum were identified, of which races 9, 2.9, 4.9, 4.5.9 and 4.9.11 occur only in Japan. The Avr genes in many of these races contain unique mutations not observed in races identified elsewhere in the world including (i) frameshift mutations and (ii) transposon insertions in Avr2, (iii) point mutations in Avr4 and Avr4E, and (iv) deletions of Avr4E, Avr5 and Avr9. New races have developed by selection pressure imposed by consecutive introductions of Cf-2, Cf-4, Cf-5 and Cf-9 genes in commercially grown tomato cultivars. Our study shows that molecular variations to adapt to different Cf genes in an isolated C. fulvum population in Japan are novel but overall follow similar patterns as those observed in populations from other parts of the world. Implications for breeding of more durable C. fulvum resistant varieties are discussed.     

Mapping strategy for resistance genes against Cladosporium fulvum on the short arm of Chromosome 1 of tomato: Cf-ECP5 near the Hcr9 Milky Way cluster

In the past, numerous Lycopersicon accessions have been described that harbor resistance genes to Cladosporium fulvum (Cf genes). Several Cf genes have been isolated, like Cf-4, Cf-4A and Cf-9, which are present on the short arm of Chromosome 1, and Cf-2 and Cf-5, which reside on Chromosome 6. To identify Cf genes linked to the Hcr9 cluster ”Milky Way” on the short arm of Chromosome 1, we test-crossed 66 resistant Lycopersicon accessions to the near-isogenic line Moneymaker-Cf4, and the F₁s were crossed to the susceptible tomato cultivar Moneymaker. Putative linkage between an unknown Cf gene and Cf-4 was concluded based on small-scale allelic tests from an under-representation of susceptible genotypes in the progenies of 24 plants after inoculation with race 0 of C. fulvum. In this way, of the 21 resistant lines tested, 10 harbored a Cf gene that was linked to the Hcr9 Milky Way cluster. Moreover, one of the lines harboring a Cf gene closely linked to Cf-4 specifically recognizes the extracellular protein ECP5 of C. fulvum and was designated Cf-ECP5. Using a testcross population of 338 plants, we mapped Cf-ECP5 more accurately at 4 cM proximal to the Hcr9 Milky Way locus. This report shows that the method of small-scale allelic tests provides a useful tool to rapidly screen for Cf genes on the short arm of Chromosome 1. Further analysis of these Cf genes will elucidate the complex genetic organization of Cf genes on Chromosome 1 of tomato. Received: 23 August 1999 / Accepted: 12 January 2000     

Molecular communication between host plant and the fungal tomato pathogenCladosporium 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.     

The Solanum pimpinellifolium Cf-ECP1 and Cf-ECP4 genes for resistance to Cladosporium fulvum are located at the Milky Way locus on the short arm of chromosome 1

The interaction between tomato and the leaf mould pathogen Cladosporium fulvum is an excellent model to study gene-for-gene interactions and plant disease resistance gene evolution. Most Cf genes were introgressed into cultivated tomato (Solanum lycopersicum) from wild relatives such as S. pimpinellifolium and novel Cf-ECP genes were recently identified in this species. Our objective is to isolate Cf-ECP1, Cf-ECP2, Cf-ECP4 and Cf-ECP5 to increase our understanding of Cf gene evolution, and the molecular basis for recognition specificity in Cf proteins. The map locations of Cf-ECP2 and Cf-ECP5 have been reported previously and we report here that Cf-ECP1 and Cf-ECP4 map to a different locus on the short arm of chromosome 1. The analysis of selected recombinants and allelism tests showed both genes are located at Milky Way together with Cf-9 and Cf-4. Our results emphasise the importance of this locus in generating novel Cf genes for resistance to C. fulvum. Candidate genes for Cf-ECP1 and Cf-ECP4 were also identified by DNA gel blot analysis of bulked segregant pools. In addition, we generated functional cassettes for expression of the C. fulvum ECP1, ECP2, ECP4 and ECP5 proteins using recombinant Potato Virus X, and three ECPs were also expressed in stable transformed plants. Using marker-assisted selection we have also identified recombinants containing Cf-ECP1, Cf-ECP2, Cf-ECP4 or Cf-ECP5 in cis with a linked T-DNA carrying the non-autonomous Zea mays transposon Dissociation. Using these resources it should now be possible to isolate all four Cf-ECPs using transposon tagging, or a candidate gene strategy. Eleni Soumpourou and Michael Iakovidis contributed equally to this work.     

Purification and serological characterization of three basic 15-kilodalton pathogenesis-related proteins from tomato

In tomato (Lycopersicon esculentum) several acidic and basic apoplastic pathogenesis-related (PR) proteins are induced upon inoculation with virulent or avirulent races of Cladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif). One of the most predominant and best characterized tomato PR proteins is P14, a basic protein that shows homology to the tobacco (Nicotiana tabacum) PR-1 protein family. To investigate whether, by analogy with these tobacco PR-1 proteins, P14 also belongs to a family of differently charged isomers, the abundantly occurring PR proteins with molecular masses around 15 kilodaltons (kD) were purified from apoplastic fluids isolated from C. fulvum-infected tomato. Three basic proteins migrating similarly to P14 on sodium dodecyl sulfate polyacrylamide gels were purified to homogeneity by gel filtration followed by high resolution liquid chromatography. Two proteins (15.5 kD, isoelectric point [pl] 10.9 and 10.7 appeared to be serologically related to each other and to the tobacco PR-1 proteins. A third protein (15 kD, pl 10.4) was not serologically related to any other tomato PR protein but was found to be related to PR-R from tobacco.     

Exogenous catechin increases antioxidant enzyme activity and promotes flooding tolerance in tomato (Solanum lycopersicum L.)

We studied the extent to which catechin applied as a soil drench modifies the effects of soil waterlogging on plant growth, the functioning of the free radical scavenging system and on oxidative stress levels. Forty-day-old tomato plants (Solanum lycopersicum L.) were treated with 0 and 2?mM catechin 48 h prior to 5 d waterlogging followed by a 4 d drainage period. Exogenous catechin increased total fresh and dry weight of flooded plants, reduced membrane damage, maintained chlorophyll concentrations, promoted photosynthesis and increased ATP concentration in the leaves, and raised sucrose synthase and alcohol dehydrogenase activities in the roots. Catechin pre-treatment also reduced hydrogen peroxide and superoxide radical concentration and increased various components of the antioxidative system in leaves. Catechin treatment affected superoxide dismutase and catalase activities in close coordination with ascorbate peroxidases and glutathione reductase. Exogenous catechin can markedly reduce the waterlogging injury in leaves and roots of tomato by enhancing free radical scavenging system sufficiently to lower hydrogen peroxide and superoxide concentrations.     

Differential Response of Thor Alfalfa to Meloidogyne chitwoodi Races and M. hapla

Second-stage juveniles (J2) of races 1 and 2 of Meloidogyne chiiwoodi and M. hapla readily penetrated roots of Thor alfalfa and Columbian tomato seedlings; however, few individuals of M. chitwoodi race 1 were able to establish feeding sites and mature on alfalfa. Histopathological studies indicate that J2 of race 1 either failed to initiate feeding sites or they caused cell enlargement without typical cell wall thickening. The protoplasm of these cells coagulated, and juveniles of race 1 did not develop beyond the swollen J2 stage. A few females of race 1 fed on small giant cells and deposited a few eggs at least 20 and 30 days later than M. chitwoodi race 2 and M. hapla, respectively. Failure of race 1 to establish feeding sites was related to egression of J2 from the roots. The M. chitwoodi race 1 J2 egression from alfalfa roots was higher than egression of race 2 and M. hapla. Egression of J2 of M. chitwoodi races 1 and 2 from tomato roots was similar and higher than that of M. hapla. Thus egression plays an important role in the host-parasite relationship of M. chitwoodi and alfalfa.     

Induction of Defense-Related Responses in Cf9 Tomato Cells by the AVR9 Elicitor Peptide of Cladosporium fulvum Is Developmentally Regulated

The AVR9 elicitor from the fungal pathogen Cladosporium fulvum induces defense-related responses, including cell death, specifically in tomato (Lycopersicon esculentum Mill.) plants that carry the Cf-9 resistance gene. To study biochemical mechanisms of resistance in detail, suspension cultures of tomato cells that carry the Cf-9 resistance gene were initiated. Treatment of cells with various elicitors, except AVR9, induced an oxidative burst, ion fluxes, and expression of defense-related genes. Agrobacterium tumefaciens-mediated transformation of Cf9 tomato leaf discs with Avr9-containing constructs resulted efficiently in transgenic callus formation. Although transgenic callus tissue showed normal regeneration capacity, transgenic plants expressing both the Cf-9 and the Avr9 genes were never obtained. Transgenic F₁ seedlings that were generated from crosses between tomato plants expressing the Avr9 gene and wild-type Cf9 plants died within a few weeks. However, callus cultures that were initiated on cotyledons from these seedlings could be maintained for at least 3 months and developed similarly to callus cultures that contained only the Cf-9 or the Avr9 gene. It is concluded, therefore, that induction of defense responses in Cf9 tomato cells by the AVR9 elicitor is developmentally regulated and is absent in callus tissue and cell-suspension cultures, which consists of undifferentiated cells. These results are significant for the use of suspension-cultured cells to investigate signal transduction cascades.     

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-β-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.     

Arbuscular mycorrhiza‐mediated resistance in tomato against Cladosporium fulvum‐induced mould disease

Root colonization with arbuscular mycorrhizal fungi (AMF) enhances plant resistance particularly against soil‐borne pathogenic fungi. In this study, mycorrhizal inoculation with Glomus mosseae (Gm) significantly alleviated tomato mould disease caused by the air‐borne fungal pathogen, Cladosporium fulvum (Cf). The disease index (DI) in local leaves (receiving pathogen inoculation) and systemic leaves (just above the local leaf without pathogen inoculation) was 36.4% and 11.7% in mycorrhizal plants, respectively, whereas DI was 59.6% and 36.4% in the corresponding leaves of AMF non‐inoculated plants, after 50 days of Gm inoculation, corresponding to 15 days after Cf inoculation by leaf infiltration. Foliar spray inoculation with Cf also revealed that AMF pre‐inoculated plants had a higher resistance against subsequent pathogen infection, where the DI was 41.3% in mycorrhizal plants vs. 64.4% in AMF non‐inoculated plants. AMF‐inoculated plants showed significantly higher fresh and dry weight than non‐inoculated plants under both control (without pathogen) and pathogen treatments. AMF‐inoculated plants exhibited significant increases in activities of superoxide dismutase and peroxidase, along with decreases in levels of H₂O₂ and malondialdehyde, compared with non‐inoculated plants after pathogen inoculation. AMF inoculation led to increases in total chlorophyll contents and net photosynthesis rate as compared with non‐inoculated plants under control and pathogen infection. Pathogen infection on AMF non‐inoculated plants led to decreases in chlorophyll fluorescence parameters. However, pathogen infection did not affect these parameters in mycorrhizal plants. Taken together, these results indicate that AMF colonization may play an important role in plant resistance against air‐borne pathogen infection by maintaining redox poise and photosynthetic activity.     

Host Suitability and Response of Asparagus Cultivars to Meloidogyne Species and Races

The host-parasite relationships of asparagus and Meloidogyne spp. were examined under greenhouse and microplot conditions. Meloidogyne species and races differed greatly in their ability to reproduce on asparagus seedlings. Meloidogyne hapla generally failed to reproduce, and M. javanica, M. arenaria race 1, and M. incognita race 3 reproduced poorly, with a reproduction factor (Rf = final population/initial population) usually < 1.0. Only M. arenaria race 2 and M. incognita races 1 and 4 reproduced consistently on all asparagus cultivars tested (Rf typically 1-11). No effect of M. incognita race 4 on host growth was detected. Meloidogyne arenaria race 2 and M. incognita race 1 had slight negative effects (5-10%) on plant and root growth.     

Identification of Several Pathogenesis-Related Proteins in Tomato Leaves Inoculated with Cladosporium fulvum (syn. Fulvia fulva) as 1,3-beta-Glucanases and Chitinases

Inoculation of tomato (Lycopersicon esculentum) leaves with Cladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif) results in a marked accumulation of several pathogenesis-related (PR) proteins in the apoplast. Two predominant PR proteins were purified from apoplastic fluid by ion exchange chromatography followed by chromatofocusing. One protein (molecular mass [M_r] 35 kilodaltons [kD], isoelectric point [pI] ~6.4) showed 1,3-β-glucanase activity, while the other one (M_r26 kD, pI ~6.1) showed chitinase activity. Identification of the products that were released upon incubation of the purified enzymes with laminarin or regenerated chitin revealed that both enzymes showed endo-activity. Using antisera raised against these purified enzymes from tomato and against chitinases and 1,3-β-glucanases isolated from other plant species, one additional 1,3-β-glucanase (M_r33 kD) and three additional chitinases (M_r 27, 30, and 32 kD) could be detected in apoplastic fluids or homogenates of tomato leaves inoculated with C. fulvum. Upon inoculation with C. fulvum, chitinase and 1,3-β-glucanase activity in apoplastic fluids increased more rapidly in incompatible interactions than in compatible ones. The role of these hydrolytic enzymes, potentially capable of degrading hyphal walls of C. fulvum, is discussed in relation to active plant defense.     

RFLP linkage analysis of the Cf-4 and Cf-9 genes for resistance toCladosporium fulvum in tomato

Four different populations segregating for one of the two closely linked (possibly allelic) tomato disease resistance genes to the fungusCladosporium fulvum,Cf-4 andCf-9, were generated and analysed for recombination frequencies between theCf-genes and restriction fragment length polymorphism (RFLP) loci. The population consisting of F₂ progeny from the interspecific crossLycopersicon esculentum carryingCf-9 ×L. pennellii was identified as the most useful for RFLP mapping of theCf-4/9 locus and an RFLP map around this locus was constructed mainly using this population. The two closest markers identified were CP46, 2.6 cM distal, and a group of 11 markers including TG236, 3.7 cM proximal toCf-4/9. A polymerase chain reaction (PCR)-based procedure for the rapid identification of recombination events between these two markers was developed. The regions of foreign DNA introgression surroundingCf-4 andCf-9 in near-isogenic lines were delimited.     

Induction of H2O2 and related enzymes in tomato roots infected with root knot nematode (M. javanica) by several chemical and microbial elicitors

The effects of chemical and microbial elicitors such as β-aminobutyric acid (BABA), Salicylic acid (SA), and Pseudomonas fluorecens CHAO on hydrogen peroxide generation and activity of the enzymes related to its metabolism, i.e., superoxide dismutase (SOD), guaiacol peroxidase (GPOX), and catalase (CAT) were investigated in tomato roots infected with root-knot nematode (Meloidogyne javanica). Results of this study show that treating the tomato seedlings with the above elicitors significantly reduces the nematode infection level. Among the tested elicitors, BABA has reduced the nematode galls, number of egg masses per plant and number of eggs per individual egg mass more than the others. Additionally, the amount of H₂O₂, a product of oxidative stress, SOD and GPOX specific activities were significantly increased in the elicitor treated plants in comparison to control. Our observation shows that BABA also increases the H₂O₂ accumulation and the SOD and GPOX activities more as compared with the other tested elicitors. Such increases have occurred in two phases and maximum levels of them were observed at 5 days after treatment. In contrast with the increase in SOD and GPOX activities, the CAT activity doesnot show any significant increase in treated plants as compared with the control and other tested elicitors. It can be concluded that BABA, SA, and Pseudomonas fluorescens CHAO induce oxidative stress in tomato roots through generation of reactive oxygen species (ROS) and the enzymes related to their metabolism.     

Salivary signals of European corn borer induce indirect defenses in tomato

Plants can recognize the insect elicitors and activate its defense mechanisms. European Corn Borer (ECB; Ostrinia nubilalis) saliva, produced from the labial salivary glands and released through the spinneret, is responsible for inducing direct defenses in host plants. Glucose oxidase (GOX) present in the ECB saliva induced direct defenses in tomato. By contrast, GOX activity in ECB saliva was insufficient to trigger defenses in maize, suggesting that host-specific salivary elicitors are responsible for inducing direct defenses in host plants. Our current study further examined whether ECB saliva can trigger indirect defenses in tomato. Relative expression levels of TERPENE SYNTHASE5 (TPS5) and HYDROPEROXIDE LYASE (HPL), marker for indirect defenses in host plants, were monitored. Quantitative real-time PCR analysis revealed that ECB saliva can induce the expression of TPS5 and HPL, suggesting that salivary signals can induce indirect defenses in addition to the direct defenses. Further experiments are required to identify different ECB elicitors that are responsible for inducing direct and indirect defenses in host plants.     

Salicylic acid is not required for Cf-2- and Cf-9-dependent resistance of tomato to Cladosporium fulvum

Tomato leaves or cotyledons expressing the Cf-2 or Cf-9 Cladosporium fulvum resistance genes induce salicylic acid (SA) synthesis following infiltration with intercellular washing fluid (IF) containing the fungal peptide elicitors Avr2 and Avr9. We investigated whether SA was required for Cf gene-dependent resistance. Tomato plants expressing the bacterial gene nahG, encoding salicylate hydroxylase, did not accumulate SA in response to IF infiltration but remained fully resistant to C. fulvum. NahG Cf0 plants were as susceptible to C. fulvum as wild-type Cf0. Neither free nor conjugated salicylic acid accumulated in IF-infiltrated Cf2 and Cf9 NahG leaves and cotyledons but conjugated catechol did accumulate. The Cf-9-dependent necrotic response to IF was prevented in NahG plants and replaced by a chlorotic Cf-2-like response. SA also potentiated Cf-9-mediated necrosis in IF-infiltrated wild-type leaves. In contrast, the Cf-2-dependent IF response was retained in NahG leaves and chlorosis was more pronounced than in the wild-type. The distribution of cell death between different cell types was altered in both Cf2 and Cf9 NahG leaves after IF injection. IF-induced accumulation of three SA-inducible defence-related genes was delayed and reduced but not abolished in NahG Cf2 and Cf9 leaves and cotyledons. NahG Tm-22 tomato showed increased hypersensitive response (HR) lesion size upon TMV infection, as observed in TMV-inoculated N gene-containing NahG tobacco plants.