Genetic evidence that expression of NahG modifies defence pathways independent of salicylic acid biosynthesis in the Arabidopsis-Pseudomonas syringae pv. tomato interaction

The salicylic acid (SA)-induction deficient (sid) mutants of Arabidopsis, eds5 and sid2 accumulate normal amounts of camalexin after inoculation with Pseudomonas syringae pv. tomato (Pst), while transgenic NahG plants expressing an SA hydroxylase that degrades SA have reduced levels of camalexin and exhibit a higher susceptibility to different pathogens compared to the sid mutants. SID2 encodes an isochorismate synthase necessary for the synthesis of SA. NahG was shown to act epistatically to the sid mutant phenotype regarding accumulation of camalexin after inoculation with Pst in eds5NahG and sid2NahG plants. The effect of the pad4 mutation on the sid mutant phenotype was furthermore tested in eds5pad4 and sid2pad4 double mutants, and it was demonstrated that PAD4 acts epistatically to EDS5 and SID2 regarding the production of camalexin after inoculation with Pst. NahG plants and pad4 mutants were also found to produce less ethylene (ET) after infection with Pst in comparison to the wild type (WT) and sid mutants. Both PAD4 and NahG acted epistatically to SID regarding the Pst-dependent production of ET that was found to be necessary for the accumulation of camalexin. Early production of jasmonic acid (JA) 12 h after inoculation with Pst/avrRpt2 was absent in all plants expressing NahG compared to the other mutants tested here. These genetic studies unravel pleiotropic changes in defence signalling of NahG plants that are unlikely to result from their low SA content. This adds unexpected difficulties in the interpretation of earlier findings based solely on NahG plants.     

Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation.

In Arabidopsis, systemic acquired resistance against pathogens has been associated with the accumulation of salicylic acid (SA) and the expression of the pathogenesis-related proteins PR-1, PR-2, and PR-5. We report here the isolation of two nonallelic mutants impaired in the pathway leading to SA biosynthesis. These SA induction-deficient (sid) mutants do not accumulate SA after pathogen inoculation and are more susceptible to both virulent and avirulent forms of Pseudomonas syringae and Peronospora parasitica. However, sid mutants are not as susceptible to these pathogens as are transgenic plants expressing the nahG gene encoding an SA hydroxylase that degrades SA to catechol. In contrast to NahG plants, only the expression of PR-1 is strongly reduced in sid mutants, whereas PR-2 and PR-5 are still expressed after pathogen attack. Furthermore, the accumulation of the phytoalexin camalexin is normal. These results indicate that SA-independent compensation pathways that do not operate in NahG plants are active in sid mutants. One of the mutants is allelic to eds5 (for enhanced disease susceptibility), whereas the other mutant has not been described previously.     

Arabidopsis local resistance to Botrytis cinerea involves salicylic acid and camalexin and requires EDS4 and PAD2, but not SID2, EDS5 or PAD4

Salicylic acid (SA) is an important regulator of plant defense responses, and a variety of Arabidopsis mutants impaired in resistance against bacterial and fungal pathogens show defects in SA accumulation, perception, or signal transduction. Nevertheless, the role of SA-dependent defense responses against necrotrophic fungi is currently unclear. We determined the susceptibility of a set of previously identified Arabidopsis mutants impaired in defense responses to the necrotrophic fungal pathogen Botrytis cinerea. The rate of development of B. cinerea disease symptoms on primary infected leaves was affected by responses mediated by the genes EIN2, JAR1, EDS4, PAD2, and PAD3, but was largely independent of EDS5, SID2/ICS1, and PAD4. Furthermore, plants expressing a nahG transgene or treated with a phenylalanine ammonia lyase (PAL) inhibitor showed enhanced symptoms, suggesting that SA synthesized via PAL, and not via isochorismate synthase (ICS), mediates lesion development. In addition, the degree of lesion development did not correlate with defensin or PR1 expression, although it was partially dependent upon camalexin accumulation. Although npr1 mutant leaves were normally susceptible to B. cinerea infection, a double ein2 npr1 mutant was significantly more susceptible than ein2 plants, and exogenous application of SA decreased B. cinerea lesion size through an NPR1-dependent mechanism that could be mimicked by the cpr1 mutation. These data indicate that local resistance to B. cinerea requires ethylene-, jasmonate-, and SA-mediated signaling, that the SA affecting this resistance does not require ICS1 and is likely synthesized via PAL, and that camalexin limits lesion development.     

Constitutive salicylic acid-dependent signaling in cpr1 and cpr6 mutants requires PAD4

Salicylic acid (SA)-dependent signaling controls activation of a set of plant defense mechanisms that are important for resistance to a variety of microbial pathogens. Many Arabidopsis mutants that display altered SA-dependent signaling have been isolated. We used double mutant analysis to determine the relative positions of the pad4, cpr1, cpr5, cpr6, dnd1 and dnd2 mutations in the signal transduction network leading to SA-dependent activation of defense gene expression and disease resistance. The pad4 mutation causes failure of SA accumulation in response to infection by certain pathogens, while the other mutations cause constitutively high levels of SA, defense gene expression and resistance. The cpr1 pad4, cpr5 pad4, cpr6 pad4, dnd1 pad4 and dnd2 pad4 double mutants were constructed and assayed for stature, presence of spontaneous lesions, resistance to Pseudomonas syringae and Peronospora parasitica, SA levels, expression of PAD4, PR-1 and PDF1.2, and accumulation of camalexin. We found that the effects of the cpr1 and cpr6 mutations on SA-dependent gene expression are completely dependent on PAD4 function. In contrast, SA accumulation in the lesion-mimic mutant cpr5 is partially PAD4-independent, while in dnd1 and dnd2 mutants it is completely PAD4-independent. A model describing a possible arrangement of activities in the signal transduction network is presented.     

Characterisation of an Arabidopsis-Leptosphaeria maculans pathosystem: resistance partially requires camalexin biosynthesis and is independent of salicylic acid, ethylene and jasmonic acid signalling

Out of 168 Arabidopsis accessions screened with isolates of Leptosphaeria maculans, one (An-1) showed clear disease symptoms. In order to identify additional components involved in containment of L. maculans in Arabidopsis, a screen for L. maculans-susceptible (lms) mutants was performed. Eleven lms mutants were isolated, which displayed differential susceptibility responses to L. maculans. lms1 was crossed with Columbia (Col-0) and Ws-0, and mapping data for both populations showed the highest linkage to a region on chromosome 2. Reduced levels of PR-1 and PDF1.2 expression were found in lms1 compared to wild-type plants 48 h after pathogen inoculation. In contrast, the lms1 mutant displayed upregulation of either marker gene upon chemical treatment, possibly as an effect of an altered ethylene (ET) response. To assess the contribution of different defence pathways, genotypes implicated in salicylic acid (SA) signalling plants expressing the bacterial salicylate hydroxylase (nahG) gene, non-expressor of PR1 (npr1)-1 and phytoalexin-deficient (pad4-1), jasmonic acid (JA) signalling (coronatine insensitive (coi)1-16, enhanced disease susceptibility (eds)8-1 and jasmonic acid resistant (jar)1-1) and ET signalling (eds4-1, ethylene insensitive (ein)2, ein3-1 and ethylene resistant (etr)1-1) were screened. All the genotypes screened were as resistant as wild-type plants, demonstrating the dispensability of the pathways in L. maculans resistance. When mutants implicated in cell death responses were assayed, responsive to antagonist 1 (ran1)-1 exhibited a weak susceptible phenotype, whereas accelerated cell death (acd)1-20 showed a rapid lesion development. Camalexin is only partially responsible for L. maculans containment in Arabidopsis, as pad3-1 and enhanced susceptibility to Alternaria (esa)1 clearly showed a susceptible response while wild-type levels of camalexin were present in An-1 and lms1. The data presented point to the existence of multiple defence mechanisms controlling the containment of L. maculans in Arabidopsis.     

Premature leaf senescence modulated by the Arabidopsis PHYTOALEXIN DEFICIENT4 gene is associated with defense against the phloem-feeding green peach aphid

Aphids, which are phloem-feeding insects, cause extensive loss of plant productivity and are vectors of plant viruses. Aphid feeding causes changes in resource allocation in the host, resulting in an increase in flow of nutrients to the insect-infested tissue. We hypothesized that leaf senescence, which is involved in the programmed degradation of cellular components and the export of nutrients out of the senescing leaf, could be utilized by plants to limit aphid growth. Using Arabidopsis (Arabidopsis thaliana) and green peach aphid (GPA; Myzus persicae Sulzer), we found that GPA feeding induced premature chlorosis and cell death, and increased the expression of SENESCENCE ASSOCIATED GENES (SAGs), all hallmarks of leaf senescence. Hypersenescence was accompanied by enhanced resistance against GPA in the Arabidopsis constitutive expresser of PR genes5 and suppressor of SA insensitivity2 mutant plants. In contrast, resistance against GPA was compromised in the phytoalexin deficient4 (pad4) mutant plant. The PAD4 gene, which is expressed at elevated level in response to GPA feeding, modulates the GPA feeding-induced leaf senescence. In comparison to the wild-type plant, GPA feeding-induced chlorophyll loss, cell death, and SAG expression were delayed in the pad4 mutant. Although PAD4 is associated with camalexin synthesis and salicylic acid (SA) signaling, camalexin and SA signaling are not important for restricting GPA growth; growth of GPA on the camalexin-biosynthesis mutant, pad3, and the SA deficient2 and NahG plants and the SA-signaling mutant, nonexpresser of PR genes1, were comparable to that on the wild-type plant. Our results suggest that PAD4 modulates the activation of senescence in the aphid-infested leaves, which contributes to basal resistance to GPA.     

Characterization of the early response of Arabidopsis to Alternaria brassicicola infection using expression profiling

All tested accessions of Arabidopsis are resistant to the fungal pathogen Alternaria brassicicola. Resistance is compromised by pad3 or coi1 mutations, suggesting that it requires the Arabidopsis phytoalexin camalexin and jasmonic acid (JA)-dependent signaling, respectively. This contrasts with most well-studied Arabidopsis pathogens, which are controlled by salicylic acid-dependent responses and do not benefit from absence of camalexin or JA. Here, mutants with defects in camalexin synthesis (pad1, pad2, pad3, and pad5) or in JA signaling (pad1, coi1) were found to be more susceptible than wild type. Mutants with defects in salicylic acid (pad4 and sid2) or ethylene (ein2) signaling remained resistant. Plant responses to A. brassicicola were characterized using expression profiling. Plants showed dramatic gene expression changes within 12 h, persisting at 24 and 36 h. Wild-type and pad3 plants responded similarly, suggesting that pad3 does not have a major effect on signaling. The response of coi1 plants was quite different. Of the 645 genes induced by A. brassicicola in wild-type and pad3 plants, 265 required COI1 for full expression. It is likely that some of the COI1-dependent genes are important for resistance to A. brassicicola. Responses to A. brassicicola were compared with responses to Pseudomonas syringae infection. Despite the fact that these pathogens are limited by different defense responses, approximately 50% of the induced genes were induced in response to both pathogens. Among these, requirements for COI1 were consistent after infection by either pathogen, suggesting that the regulatory effect of COI1 is similar regardless of the initial stimulus.     

PAD4 functions upstream from salicylic acid to control defense responses in Arabidopsis.

The Arabidopsis PAD4 gene was previously shown to be required for synthesis of camalexin in response to infection by the virulent bacterial pathogen Pseudomonas syringae pv maculicola ES4326 but not in response to challenge by the non-host fungal pathogen Cochliobolus carbonum. In this study, we show that pad4 mutants exhibit defects in defense responses, including camalexin synthesis and pathogenesis-related PR-1 gene expression, when infected by P. s. maculicola ES4 326. No such defects were observed in response to infection by an isogenic avirulent strain carrying the avirulence gene avrRpt2. In P. s. maculicola ES4 326-infected pad4 plants, synthesis of salicylic acid (SA) was found to be reduced and delayed when compared with SA synthesis in wild-type plants. Moreover, treatment of pad4 plants with SA partially reversed the camalexin deficiency and PR-1 gene expression phenotypes of P. s. maculicola ES4 326-infected pad4 plants. These findings support the hypothesis that PAD4 acts upstream from SA accumulation in regulating defense response expression in plants infected with P. s. maculicola ES4 326. A working model of the role of PAD4 in governing expression of defense responses is presented.     

The BOS loci of Arabidopsis are required for resistance to Botrytis cinerea infection

Three Botrytis-susceptible mutants bos2, bos3, and bos4 which define independent and novel genetic loci required for Arabidopsis resistance to Botrytis cinerea were isolated. The bos2 mutant is susceptible to B. cinerea but retains wild-type levels of resistance to other pathogens tested, indicative of a defect in a response pathway more specific to B. cinerea. The bos3 and bos4 mutants also show increased susceptibility to Alternaria brassicicola, another necrotrophic pathogen, suggesting a broader role for these loci in resistance. bos4 shows the broadest range of effects on resistance, being more susceptible to avirulent strain of Pseudomonas syringae pv. tomato. Interestingly, bos3 is more resistant than wild-type plants to virulent strains of the biotrophic pathogen Peronospora parasitica and the bacterial pathogen P. syringae pv. tomato. The Pathogenesis Related gene 1 (PR-1), a molecular marker of the salicylic acid (SA)-dependent resistance pathway, shows a wild-type pattern of expression in bos2, while in bos3 this gene was expressed at elevated levels, both constitutively and in response to pathogen challenge. In bos4 plants, PR-1 expression was reduced compared with wild type in response to B. cinerea and SA. In bos3, the mutant most susceptible to B. cinerea and with the highest expression of PR-1, removal of SA resulted in reduced PR-1 expression but no change to the B. cinerea response. Expression of the plant defensin gene PDF1-2 was generally lower in bos mutants compared with wild-type plants, with a particularly strong reduction in bos3. Production of the phytoalexin camalexin is another well-characterized plant defense response. The bos2 and bos4 mutants accumulate reduced levels of camalexin whereas bos3 accumulates significantly higher levels of camalexin than wild-type plants in response to B. cinerea. The BOS2, BOS3, and BOS4 loci may affect camalexin levels and responsiveness to ethylene and jasmonate. The three new mutants appear to mediate disease responses through mechanisms independent of the previously described BOS1 gene. Based on the differences in the phenotypes of the bos mutants, it appears that they affect different points in defense response pathways.     

The Arabidopsis flavin-dependent monooxygenase FMO1 is an essential component of biologically induced systemic acquired resistance

Upon localized attack by necrotizing pathogens, plants gradually develop increased resistance against subsequent infections at the whole-plant level, a phenomenon known as systemic acquired resistance (SAR). To identify genes involved in the establishment of SAR, we pursued a strategy that combined gene expression information from microarray data with pathological characterization of selected Arabidopsis (Arabidopsis thaliana) T-DNA insertion lines. A gene that is up-regulated in Arabidopsis leaves inoculated with avirulent or virulent strains of the bacterial pathogen Pseudomonas syringae pv maculicola (Psm) showed homology to flavin-dependent monooxygenases (FMO) and was designated as FMO1. An Arabidopsis knockout line of FMO1 proved to be fully impaired in the establishment of SAR triggered by avirulent (Psm avrRpm1) or virulent (Psm) bacteria. Loss of SAR in the fmo1 mutants was accompanied by the inability to initiate systemic accumulation of salicylic acid (SA) and systemic expression of diverse defense-related genes. In contrast, responses at the site of pathogen attack, including increases in the levels of the defense signals SA and jasmonic acid, camalexin accumulation, and expression of various defense genes, were induced in a similar manner in both fmo1 mutant and wild-type plants. Consistently, the fmo1 mutation did not significantly affect local disease resistance toward virulent or avirulent bacteria in naive plants. Induction of FMO1 expression at the site of pathogen inoculation is independent of SA signaling, but attenuated in the Arabidopsis eds1 and pad4 defense mutants. Importantly, FMO1 expression is also systemically induced upon localized P. syringae infection. This systemic up-regulation is missing in the SAR-defective SA pathway mutants sid2 and npr1, as well as in the defense mutant ndr1, indicating a close correlation between systemic FMO1 expression and SAR establishment. Our findings suggest that the presence of the FMO1 gene product in systemic tissue is critical for the development of SAR, possibly by synthesis of a metabolite required for the transduction or amplification of a signal during the early phases of SAR establishment in systemic leaves.     

Histochemical and genetic analysis of host and non-host interactions of Arabidopsis with three Botrytis species: an important role for cell death control

Susceptibility was evaluated of host and non-host plants to three pathogenic Botrytis species: the generalist B. cinerea and the specialists B. elliptica (lily) and B. tulipae (tulip). B. tulipae was, unexpectedly, able to infect plant species other than tulip, and to a similar extent as B. cinerea. To study host and non-host interactions in more detail, the three Botrytis species were inoculated on Arabidopsis wild-types and 23 mutant genotypes. Disease development was monitored macroscopically by quantifying the lesion area and microscopically by bright-field and fluorescence microscopy following histochemical staining. B. cinerea and B. tulipae were very similar in their ability to infect the tested Arabidopsis genotypes, whereas B. elliptica caused disease only on a few Arabidopsis mutant genotypes. Arabidopsis mutants with a delayed or reduced cell death response were generally more resistant to Botrytis infection, whereas mutants in which cell death was accelerated were more susceptible. Differences in susceptibility between genotypes were generally gradual. Only the camalexin-deficient mutant pad 3 was fully susceptible to all three Botrytis species. Cellular changes were monitored during compatible and incompatible interactions. The formation of papillae, the presence of lysosome-like vesicles and the intracellular accumulation of H₂O₂ and nitric oxide were visualized in the infection zones using fluorescent probes. Based on histology and responses of Arabidopsis mutants, a model is proposed in which resistance against Botrytis , besides the production of camalexin, depends on the balance between cell death and survival.     

The glutathione-deficient mutant pad2-1 accumulates lower amounts of glucosinolates and is more susceptible to the insect herbivore Spodoptera littoralis

Plants often respond to pathogen or insect attack by inducing the synthesis of toxic compounds such as phytoalexins and glucosinolates (GS). The Arabidopsis mutant pad2-1 has reduced levels of the phytoalexin camalexin and is known for its increased susceptibility to fungal and bacterial pathogens. We found that pad2-1 is also more susceptible to the generalist insect Spodoptera littoralis but not to the specialist Pieris brassicae . The PAD2 gene encodes a γ-glutamylcysteine synthetase that is involved in glutathione (GSH) synthesis, and consequently the pad2-1 mutant contains about 20% of the GSH found in wild-type plants. Lower GSH levels of pad2-1 were correlated with reduced accumulation of the two major indole and aliphatic GSs of Arabidopsis, indolyl-3-methyl-GS and 4-methylsulfinylbutyl-GS, in response to insect feeding. This effect was specific to GSH, was not complemented by treatment of pad2-1 with the strong reducing agent dithiothreitol, and was not observed with the ascorbate-deficient mutant vtc1-1 . In contrast to the jasmonate-insensitive mutant coi1-1 , expression of insect-regulated and GS biosynthesis genes was not affected in pad2-1 . Our data suggest a crucial role for GSH in GS biosynthesis and insect resistance.     

The ABC transporter BcatrB from Botrytis cinerea exports camalexin and is a virulence factor on Arabidopsis thaliana

Arabidopsis thaliana is known to produce the phytoalexin camalexin in response to abiotic and biotic stress. Here we studied the mechanisms of tolerance to camalexin in the fungus Botrytis cinerea , a necrotrophic pathogen of A. thaliana . Exposure of B. cinerea to camalexin induces expression of BcatrB , an ABC transporter that functions in the efflux of fungitoxic compounds. B. cinerea inoculated on wild-type A. thaliana plants yields smaller lesions than on camalexin-deficient A. thaliana mutants. A B. cinerea strain lacking functional BcatrB is more sensitive to camalexin in vitro and less virulent on wild-type plants, but is still fully virulent on camalexin-deficient mutants. Pre-treatment of A. thaliana with UV-C leads to increased camalexin accumulation and substantial resistance to B. cinerea. UV-C-induced resistance was not seen in the camalexin-deficient mutants cyp79B2/B3 , cyp71A13 , pad3 or pad2 , and was strongly reduced in ups1 . Here we demonstrate that an ABC transporter is a virulence factor that increases tolerance of the pathogen towards a phytoalexin, and the complete restoration of virulence on host plants lacking this phytoalexin.     

CYP71B15 (PAD3) catalyzes the final step in camalexin biosynthesis

Camalexin represents the main phytoalexin in Arabidopsis (Arabidopsis thaliana). The camalexin-deficient phytoalexin deficient 3 (pad3) mutant has been widely used to assess the biological role of camalexin, although the exact substrate of the cytochrome P450 enzyme 71B15 encoded by PAD3 remained elusive. 2-(Indol-3-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid (dihydrocamalexic acid) was identified as likely intermediate in camalexin biosynthesis downstream of indole-3-acetaldoxime, as it accumulated in leaves of silver nitrate-induced pad3 mutant plants and it complemented the camalexin-deficient phenotype of a cyp79b2/cyp79b3 double-knockout mutant. Recombinant CYP71B15 heterologously expressed in yeast catalyzed the conversion of dihydrocamalexic acid to camalexin with preference of the (S)-enantiomer. Arabidopsis microsomes isolated from leaves of CYP71B15-overexpressing and induced wild-type plants were capable of the same reaction but not microsomes from induced leaves of pad3 mutants. In conclusion, CYP71B15 catalyzes the final step in camalexin biosynthesis.     

Signals involved in Arabidopsis resistance to Trichoplusia ni caterpillars induced by virulent and avirulent strains of the phytopathogen Pseudomonas syringae

Plants have evolved different but interconnected strategies to defend themselves against herbivorous insects and microbial pathogens. We used an Arabidopsis/Pseudomonas syringae pathosystem to investigate the impact of pathogen-induced defense responses on cabbage looper (Trichoplusia ni) larval feeding. Arabidopsis mutants [npr1, pad4, eds5, and sid2(eds16)] or transgenic plants (nahG) that are more susceptible to microbial pathogens and are compromised in salicylic acid (SA)-dependent defense responses exhibited reduced levels of feeding by T. ni compared with wild-type plants. Consistent with these results, Arabidopsis mutants that are more resistant to microbial pathogens and have elevated levels of SA (cpr1 and cpr6) exhibited enhanced levels of T. ni feeding. These experiments suggested an inverse relationship between an active SA defense pathway and insect feeding. In contrast to these results, there was increased resistance to T. ni in wild-type Arabidopsis ecotype Columbia plants that were infected with P. syringae pv. maculicola strain ES4326 (Psm ES4326) expressing the avirulence genes avrRpt2 or avrB, which elicit a hypersensitive response, high levels of SA accumulation, and systemic acquired resistance to bacterial infection. Similar results were obtained with other ecotypes, including Landsberg erecta, Cape Verdi Islands, and Shakdara. When infected with Psm ES4326(avrRpt2) or Psm ES4326(avrB), nahG transgenic and npr1 mutant plants (which are more susceptible to virulent and avirulent P. syringae strains) failed to show the increased insect resistance exhibited by wild-type plants. It was surprising that wild-type plants, as well as nahG and npr1 plants, infected with Psm ES4326 not expressing avrRpt2 or avrB, which elicits disease, became more susceptible to T. ni. Our results suggest two potentially novel systemic signaling pathways: a systemic response elicited by HR that leads to enhanced T. ni resistance and overrides the SA-mediated increase in T. ni susceptibility, and a SA-independent systemic response induced by virulent pathogens that leads to enhanced susceptibility to T. ni.