Lesion mimic mutants: keys for deciphering cell death and defense pathways in plants?

The identification of several lesion mimic mutants (LMM) that misregulate cell death constitutes a powerful tool to unravel programmed cell death (PCD) pathways in plants, particularly the hypersensitive response (HR), a form of PCD associated with resistance to pathogens. Recently, the characterization of novel LMM has enabled genes that might regulate cell death programmes to be identified as well as the dissection of defense signaling pathways and of crosstalk between multiple pathways in ways that might not be possible by studying the responses of wild-type plants to pathogens.     

Pepper pathogenesis‐related protein 4c is a plasma membrane‐localized cysteine protease inhibitor that is required for plant cell death and defense signaling

Xanthomonas campestris pv. vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis‐related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT‐triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease‐inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H₂O₂ and significant induction of some defense‐response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT‐triggered resistance responses, and enabled Xcv proliferation in infected leaves. H₂O₂ accumulation, cell‐death induction, and defense‐response gene expression were distinctly reduced in CaPR4c‐silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling.     

Preexisting systemic acquired resistance suppresses hypersensitive response-associated cell death in Arabidopsis hrl1 mutant

The hypersensitive response (HR) displayed by resistant plants against invading pathogens is a prominent feature of plant-pathogen interactions. The Arabidopsis hypersensitive response like lesions1 (hrl1) mutant is characterized by heightened defense responses that make it more resistant to virulent pathogens. However, hrl1 suppresses avirulent pathogen-induced HR cell death. Furthermore, the high PR-1 expression observed in hrl1 remains unaltered after avirulent and virulent pathogen infections. The suppressed HR phenotype in hrl1 is observed even when an elicitor is expressed endogenously from an inducible promoter, suggesting that an impaired transfer of avirulent factors is not the reason. Interestingly, the lack of HR phenotype in hrl1 is reversed if the constitutive defense responses are compromised either by a mutation in NON EXPRESSOR OF PR-1 (NPR1) or by depleting salicylic acid due to the expression of the nahG gene. The rescue of HR cell death in hrl1 npr1 and in hrl1 nahG depends on the extent to which the constitutive systemic acquired response (SAR) is compromised. Pretreating Arabidopsis wild-type plants with SAR-inducers, before pathogen infection resulted in a significant decrease in HR cell death. Together, these results demonstrate that the preexisting SAR may serve as one form of negative feedback loop to regulate HR-associated cell death in hrl1 mutant and in the wild-type plants.     

Alerted Defense System Attenuates Hypersensitive Response-Associated Cell Death in <Emphasis Type="BoldItalic">Arabidopsis siz1</Emphasis> Mutant

Plants defend themselves by inducing sophisticated multilevel defense responses against pathogenic attack. The first line of defense against microbial pathogens is the process of nonself-recognition, which mediates the activation of the necessary defense repertoire. The hypersensitive response (HR), a macroscopic collapse of plant leaves in primary infection site, is one of such plant resistance responses. Subsequently, the HR triggers a general resistance mechanism called systemic acquired resistance (SAR), rendering uninfected parts of the plants less sensitive to further pathogenic attacks. Here, we show that SIZ1 mutation-mediated preexisting SAR attenuates HR-associated cell death in Arabidopsis thaliana. In siz1 mutant, the amount of PR1 and PR5 stayed high level, and the growth of pathogenic bacteria Pseudomonas syringae pv. maculicola (Pma) strain M6CΔE was reduced. Early callose deposition, spontaneous formation of microscopic cell death, and reactive oxygen species (ROS) were also observed in siz1 mutant.     

The cDNA microarray analysis using an Arabidopsis pad3 mutant reveals the expression profiles and classification of genes induced by Alternaria brassicicola attack

The hypersensitive response (HR) was induced in a wild-type Arabidopsis thaliana plant (Columbia) (Col-wt) by inoculation with Alternaria brassicicola that causes the development of small brown necrotic lesions on the leaves. By contrast, pad3-1 mutants challenged with A. brassicicola produced spreading lesions. The cell death in pad3-1 mutants could not inhibit the pathogen growth and development, although both production of H(2)O(2) and localized cell death were similar in Col-wt and pad3-1 plants after the inoculation. The difference between Col-wt and pad3-1 plants is defense responses after the occurrence of cell death. In other words, PAD3 is necessary for defense response to A. brassicicola. Therefore, we examined the changes in the expression patterns of ca. 7,000 genes by cDNA microarray analysis after inoculation with A. brassicicola. The cDNA microarrays were also done to analyze Arabidopsis responses after treatment with signal molecules, reactive oxygen species (ROS)-inducing compounds and UV-C. The results suggested that the pad3-1 mutation altered not only the accumulation of camalexin but also the timing of expression of many defense-related genes in response to the challenge with A. brassicicola. Furthermore, the plants integrate two or more signals that act together for promoting the induction of multiple defense pathways.     

HopPtoN is a Pseudomonas syringae Hrp (type III secretion system) cysteine protease effector that suppresses pathogen-induced necrosis associated with both compatible and incompatible plant interactions

Pseudomonas syringae pv. tomato DC3000 causes bacterial speck disease in tomato, and it elicits the hypersensitive response (HR) in non-host plants such as Nicotiana tabacum and Nicotiana benthamiana. The compatible and incompatible interactions of DC3000 with tomato and Nicotiana spp., respectively, result in plant cell death, but the HR cell death occurs more rapidly and is associated with effective plant defense. Both interactions require the Hrp (HR and pathogenicity) type III secretion system (TTSS), which injects Hop (Hrp outer protein) effectors into plant cells. Here, we demonstrate that HopPtoN is translocated into tomato cells via the Hrp TTSS. A hopPtoN mutant produced eightfold more necrotic 'speck' lesions on tomato leaves than did DC3000, but the mutant and the wild-type strain grew to the same level in infected leaves. In non-host N. tabacum leaves, the hopPtoN mutant produced more cell death, whereas a DC3000 strain overexpressing HopPtoN produced less cell death and associated electrolyte leakage in comparison with wild-type DC3000. Transient expression of HopPtoN via infection with a PVX viral vector enabled tomato and N. benthamiana plants to tolerate, with reduced disease lesions, challenge infections with DC3000 and P. syringae pv. tabaci 11528, respectively. HopPtoN showed cysteine protease activity in vitro, and hopPtoN mutants altered in the predicted cysteine protease catalytic triad (C172S, H283A and D299A) lost HR suppression activity. These observations reveal that HopPtoN is a TTSS effector that can suppress plant cell death events in both compatible and incompatible interactions.     

Ozone-induced ethylene production is dependent on salicylic acid,and both salicylic acid and ethylene act in concert to regulate ozone-induced cell death

Ethylene is known to influence plant defense responses including cell death in response to both biotic and abiotic stress factors. However, whether ethylene acts alone or in conjunction with other signaling pathways is not clearly understood. Ethylene overproducer mutants, eto1 and eto3, produced high levels of ethylene and developed necrotic lesions in response to an acute O3 exposure that does not induce lesions in O3-tolerant wild-type Col-0 plants. Treatment of plants with ethylene inhibitors completely blocked O3-induced ethylene production and partially attenuated O3-induced cell death. Analyses of the responses of molecular markers of specific signaling pathways indicated a relationship between salicylic acid (SA)- and ethylene-signaling pathways and O3 sensitivity. Both eto1 and eto3 plants constitutively accumulated threefold higher levels of total SA and exhibited a rapid increase in free SA and ethylene levels prior to lesion formation in response to O3 exposure. SA pre-treatments increased O3 sensitivity of Col-0, suggesting that constitutive high SA levels prime leaf tissue to exhibit increased magnitude of O3-induced cell death. NahG and npr1 plants compromised in SA signaling failed to produce ethylene in response to O3 and other stress factors suggesting that SA is required for stress-induced ethylene production. Furthermore, NahG expression in the dominant eto3 mutant attenuated ethylene-dependent PR4 expression and rescued the O3-induced HR (hypersensitive response) cell death phenotype exhibited by eto3 plants. Our results suggest that both SA and ethylene act in concert to influence cell death in O3-sensitive genotypes, and that O3-induced ethylene production is dependent on SA.     

Ozone-induced cell death occurs via two distinct mechanisms in Arabidopsis: the role of salicylic acid

Previous studies suggest that salicylic acid (SA) plays an important role in influencing plant resistance to ozone (O3). To further define the role of SA in O3-induced responses, we compared the responses of two Arabidopsis genotypes that accumulate different amounts of SA in response to O3 and a SA-deficient transgenic Col-0 line expressing salicylate hydroxylase (NahG). The differences observed in O3-induced changes in SA levels, the accumulation of active oxygen species, defense gene expression, and the kinetics and severity of lesion formation indicate that SA influences O3 tolerance via two distinct mechanisms. Detailed analyses indicated that features associated with a hypersensitive response (HR) were significantly greater in O3-exposed Cvi-0 than in Col-0, and that NahG plants failed to exhibit these HR-like responses. Furthermore, O3-induced antioxidant defenses, including the redox state of glutathione, were greatly reduced in NahG plants compared to Col-0 and Cvi-0. This suggests that O3-induced cell death in NahG plants is due to the loss of SA-mediated potentiation of antioxidant defenses, while O3-induced cell death in Cvi-0 is due to activation of a HR. This hypothesis is supported by the observation that inhibition of NADPH-oxidases reduced O3-induced H2O2 levels and the O3-induced cell death in Cvi-0, while no major changes were observed in NahG plants. We conclude that although SA is required to maintain the cellular redox state and potentiate defense responses in O3 exposed plants, high levels of SA also potentiate activation of an oxidative burst and a cell death pathway that results in apparent O3 sensitivity.     

Tobacco plants that lack expression of functional nitrate reductase in roots show changes in growth rates and metabolite accumulation.

When tobacco is provided with a high nitrate supply, only a small amount of the nitrate taken up by the roots is immediately assimilated inside the roots, while the majority is transported to the leaves where it is reduced to ammonium. To elucidate the importance of root nitrate assimilation, tobacco plants have been engineered that showed no detectable nitrate reductase activity in the roots. These plants expressed the nitrate reductase structural gene nia2 under control of the leaf-specific potato promoter ST-LS1 in the nitrate reductase-mutant Nia30 of Nicotiana tabacum. Homozygous T2-transformants grown in sand or hydroponics with 5.1 mM nitrate had approximately 55-70% of wild-type nitrate reductase acivity in leaves, but lacked nitrate reductase acivity in roots. These plants showed a retarded growth as compared with wild-type plants. The activation state of nitrate reductase was unchanged; however, diurnal variation of nitrate reductase acivity was not as pronounced as in wild-type plants. The transformants had higher levels of nitrate in the leaves and reduced amounts of glutamine both in leaves and roots, while roots showed higher levels of hexoses (3-fold) and sucrose (10-fold). It may be concluded that the loss of nitrate reductase acivity in the roots changes the allocation of reduced nitrogen compounds and sugars in the plant. These plants will be a useful tool for laboratories studying nitrate assimilation and its interactions with carbon metabolism.     

Fusarium oxysporum hijacks COI1-mediated jasmonate signaling to promote disease development in Arabidopsis

Although defense responses mediated by the plant oxylipin jasmonic acid (JA) are often necessary for resistance against pathogens with necrotrophic lifestyles, in this report we demonstrate that jasmonate signaling mediated through COI1 in Arabidopsis thaliana is responsible for susceptibility to wilt disease caused by the root-infecting fungal pathogen Fusarium oxysporum . Despite compromised JA-dependent defense responses, the JA perception mutant coronatine insensitive 1 ( coi1 ), but not JA biosynthesis mutants, exhibited a high level of resistance to wilt disease caused by F. oxysporum . This response was independent from salicylic acid-dependent defenses, as coi1/NahG plants showed similar disease resistance to coi1 plants. Inoculation of reciprocal grafts made between coi1 and wild-type plants revealed that coi1 -mediated resistance occurred primarily through the coi1 rootstock tissues. Furthermore, microscopy and quantification of fungal DNA during infection indicated that coi1 -mediated resistance was not associated with reduced fungal penetration and colonization until a late stage of infection, when leaf necrosis was highly developed in wild-type plants. In contrast to wild-type leaves, coi1 leaves showed no necrosis following the application of F. oxysporum culture filtrate, and showed reduced expression of senescence-associated genes during disease development, suggesting that coi1 resistance is most likely achieved through the inhibition of F. oxysporum -incited lesion development and plant senescence. Together, our results indicate that F. oxysporum hijacks non-defensive aspects of the JA-signaling pathway to cause wilt-disease symptoms that lead to plant death in Arabidopsis.     

The Arabidopsis aberrant growth and death2 mutant shows resistance to Pseudomonas syringae and reveals a role for NPR1 in suppressing hypersensitive cell death

A novel Arabidopsis mutant has been identified with constitutive expression of GST1-GUS using plants with a pathogen-responsive reporter transgene containing the beta-glucuronidase (GUS) coding region driven by the GST1 promoter. The recessive mutant, called agd2 (aberrant growth and death2), has salicylic acid (SA)-dependent increased resistance to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae, elevated SA levels, a low level of spontaneous cell death, callose deposition, and enlarged cells in leaves. The enhanced resistance of agd2 to virulent P. syringae requires the SA signaling component NONEXPRESSOR OF PR1 (NPR1). However, agd2 renders the resistance response to P. syringae carrying avrRpt2 NPR1-independent. Thus agd2 affects both an SA- and NPR1-dependent general defense pathway and an SA-dependent, NPR1-independent pathway that is active during the recognition of avirulent P. syringae. agd2 plants also fail to show a hypersensitive cell death response (HR) unless NPR1 is removed. This novel function for NPR1 is also apparent in otherwise wild-type plants: npr1 mutants show a stronger HR, while NPR1-overproducing plants show a weaker HR when infected with P. syringae carrying the avrRpm1 gene. Spontaneous cell death in agd2 is partially suppressed by npr1, indicating that NPR1 can suppress or enhance cell death depending on the cellular context. agd2 plants depleted of SA show a dramatic exacerbation of the cell-growth phenotype and increased callose deposition, suggesting a role for SA in regulating growth and this cell-wall modification. AGD2 may function in cell death and/or growth control as well as the defense response, similarly to what has been described in animals for the functions of NFkappaB.     

Reactive oxygen species generated in chloroplasts contribute to tobacco leaf infection by the necrotrophic fungus Botrytis cinerea

Reactive oxygen species (ROS) play fundamental roles in plant responses to pathogen infection, including modulation of cell death processes and defense‐related gene expression. Cell death triggered as part of the hypersensitive response enhances resistance to biotrophic pathogens, but favors the virulence of necrotrophs. Even though the involvement of ROS in the orchestration of defense responses is well established, the relative contribution of specific subcellular ROS sources to plant resistance against microorganisms with different pathogenesis strategies is not completely known. The aim of this work was to investigate the role of chloroplastic ROS in plant defense against a typical necrotrophic fungus, Botrytis cinerea. For this purpose, we used transgenic Nicotiana tabacum (tobacco) lines expressing a plastid‐targeted cyanobacterial flavodoxin (pfld lines), which accumulate lower chloroplastic ROS in response to different stresses. Tissue damage and fungal growth were significantly reduced in infected leaves of pfld plants, as compared with infected wild‐type (WT) counterparts. ROS build‐up triggered by Botrytis infection and associated with chloroplasts was significantly decreased (70–80%) in pfld leaves relative to the wild type. Phytoalexin accumulation and expression of pathogenesis‐related genes were induced to a lower degree in pfld plants than in WT siblings. The impact of fungal infection on photosynthetic activity was also lower in pfld leaves. The results indicate that chloroplast‐generated ROS play a major role in lesion development during Botrytis infection. This work demonstrates that the modulation of chloroplastic ROS levels by the expression of a heterologous antioxidant protein can provide a significant degree of protection against a canonical necrotrophic fungus.     

Overexpression of a soybean nuclear localized type–III DnaJ domain‐containing HSP40 reveals its roles in cell death and disease resistance

Heat‐shock proteins such as HSP70 and HSP90 are important molecular chaperones that play critical roles in biotic and abiotic stress responses; however, the involvement of their co‐chaperones in stress biology remains largely uninvestigated. In a screen for candidate genes stimulating cell death in Glycine max (soybean), we transiently overexpressed full‐length cDNAs of soybean genes that are highly induced during soybean rust infection in Nicotiana benthamiana leaves. Overexpression of a type‐III DnaJ domain‐containing HSP40 (GmHSP40.1), a co‐chaperone of HSP70, caused hypersensitive response (HR)‐like cell death. The HR‐like cell death was dependent on MAPKKKα and WIPK, because silencing each of these genes suppressed the HR. Consistent with the presence of a nuclear localization signal (NLS) motif within the GmHSP40.1 coding sequence, GFP‐GmHSP40.1 was exclusively present in nuclear bodies or speckles. Nuclear localization of GmHSP40.1 was necessary for its function, because deletion of the NLS or addition of a nuclear export signal abolished its HR‐inducing ability. GmHSP40.1 co‐localized with HcRed‐SE, a protein involved in pri‐miRNA processing, which has been shown to be co‐localized with SR33‐YFP, a protein involved in pre‐mRNA splicing, suggesting a possible role for GmHSP40.1 in mRNA splicing or miRNA processing, and a link between these processes and cell death. Silencing GmHSP40.1 enhanced the susceptibility of soybean plants to Soybean mosaic virus, confirming its positive role in pathogen defense. Together, the results demonstrate a critical role of a nuclear‐localized DnaJ domain‐containing GmHSP40.1 in cell death and disease resistance in soybean.     

The Arabidopsis gain-of-function mutant ssi4 requires RAR1 and SGT1b differentially for defense activation and morphological alterations

A gain-of-function mutation in resistance (R) gene SSI4 causes constitutive activation of defense responses, spontaneous necrotic lesion formation, enhanced resistance against virulent pathogens, and a severe dwarf phenotype. Genetic analysis revealed that ssi4-induced H(2)O(2) accumulation and spontaneous cell death require RAR1, whereas ssi4-mediated stunting is dependent on SGT1b. By contrast, both RAR1 and SGT1b are required in a genetically additive manner for ssi4-induced disease resistance, SA accumulation, and lesion formation after pathogen infection. These data point to cooperative yet distinct functions of RAR1 and SGT1b in responses conditioned by a deregulated nucleotide-binding leucine-rich repeat protein. We also found that RAR1 and SGT1b together contribute to basal resistance because an ssi4 rar1 sgt1b triple mutant exhibited enhanced susceptibility to virulent pathogen infection compared with wild-type SSI4 plants. All ssi4-induced phenotypes were suppressed when plants were grown at 22 degrees C under high relative humidity. However, low temperature (16 degrees C) triggered ssi4-mediated cell death via an RAR1-dependent pathway even in the presence of high humidity. Thus, multiple environmental factors impact on ssi4 signaling, as has been observed for other constitutive defense mutants and R gene-triggered pathways.     

The rice (Oryza sativa) blast lesion mimic mutant, blm, may confer resistance to blast pathogens by triggering multiple defense-associated signaling pathways.

Here we characterized a rice (Oryza sativa L.) blast lesion mimic (blm) mutant, identified previously in an N-methyl-N-nitrosourea-mutagenized population of the cultivar Hwacheong (wild type). The rice blm displayed spontaneous necrotic lesion formation on the leaves during development under long-day condition and temperature shift from 28 to 24 degrees C in the absence of obvious stress/disease, and provided us with a highly reproducible and convenient experimental system in the growth chamber to study blm. The blm phenotype resembled to the cell death of hypersensitive reaction (HR), and subsequent, two-dimensional gel electrophoresis (2-DGE) revealed induction of many leaf proteins; prominent among them were the three pathogenesis-related (PR) marker proteins of class 5 (one spot) and 10 (two spots). Interestingly, the rice blm manifested HR against all races tested of the rice blast fungus (Magnaporthe grisea), providing high resistance in a non-race specific manner. It was also observed that blm was highly resistant to hydrogen peroxide treatment. Using 2-DGE immunoblotting, we identified the presence of 4 new spots cross-reacting with a superoxide dismutase (SOD) antibody, only in blm, suggesting the expression of potentially new SOD protein (isoforms) during lesion formation. In the leaves of blm, autofluorescent compounds accumulated in and around the site of lesion progression. Moreover, enhanced levels of two major rice phytoalexins, sakuranetin and momilactone A were also observed in the leaves of blm. These results indicate that blm confers broad-spectrum resistance to multiple pathogens, and so, it could be hypothesized that the BLM gene product may control the HR-like cell death and its associated multiple defense signaling pathways, as evidenced by induction of known hallmark features (proteins/metabolites) linked with the defense responses, in rice.     

Overexpression of Pto induces a salicylate-independent cell death but inhibits necrotic lesions caused by salicylate-deficiency in tomato plants

Tomato plants overexpressing the disease resistance gene Pto (35S::Pto) exhibit spontaneous cell death, accumulation of salicylic acid (SA), elevated expression of pathogenesis-related genes, and enhanced resistance to a broad range of pathogens. Because salicylate plays an important role in the cell death and defense activation in many lesion mimic mutants, we investigated the interaction of SA-mediated processes and the 35S::Pto-mediated defense pathway by introducing the nahG transgene that encodes salicylate hydroxylase. Here, we show that SA is not required for the 35S::Pto-activated microscopic cell death and plays a minor role in defense gene activation and general disease resistance in 35S::Pto plants. In contrast, temperature greatly affects the spontaneous cell death and general resistance in 35S::Pto plants, and high temperature inhibits the cell death. The NahG tomato plants develop spontaneous, unconstrained necrotic lesions on leaves. These lesions also are initiated by the inoculation of a virulent strain of Pseudomonas syringae pv. tomato. However, the NahG-dependent necrotic lesions are inhibited in the NahG/35S::Pto plants. This inhibition is most pronounced under conditions favoring the 35S::Pto-mediated spontaneous cell death development. These results indicate that the signaling pathways activated by Pto overexpression suppress the cellular damage that is caused by SA depletion. We also found that ethylene is dispensable for the 35S::Pto-mediated general defense.     

Sucrose-mediated priming of plant defense responses and broad-spectrum disease resistance by overexpression of the maize pathogenesis-related PRms protein in rice plants

Expression of pathogenesis-related (PR) genes is part of the plant's natural defense response against pathogen attack. The PRms gene encodes a fungal-inducible PR protein from maize. Here, we demonstrate that expression of PRms in transgenic rice confers broad-spectrum protection against pathogens, including fungal (Magnaporthe oryzae, Fusarium verticillioides, and Helminthosporium oryzae) and bacterial (Erwinia chrysanthemi) pathogens. The PRms-mediated disease resistance in rice plants is associated with an enhanced capacity to express and activate the natural plant defense mechanisms. Thus, PRms rice plants display a basal level of expression of endogenous defense genes in the absence of the pathogen. PRms plants also exhibit stronger and quicker defense responses during pathogen infection. We also have found that sucrose accumulates at higher levels in leaves of PRms plants. Sucrose responsiveness of rice defense genes correlates with the pathogen-responsive priming of their expression in PRms rice plants. Moreover, pretreatment of rice plants with sucrose enhances resistance to M. oryzae infection. Together, these results support a sucrose-mediated priming of defense responses in PRms rice plants which results in broad-spectrum disease resistance.     

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.