Salicylic acid is part of the Mi-1-mediated defense response to root-knot nematode in tomato

The Mi-1 gene of tomato confers resistance against three species of root-knot nematode in tomato (Lycopersicon esculentum). Transformation of tomato carrying Mi-1 with a construct expressing NahG, which encodes salicylate hydroxylase, a bacterial enzyme that degrades salicylic acid (SA) to catechol, results in partial loss of resistance to root-knot nematodes. Exogenous SA was toxic to roots expressing NahG but not to control roots. This toxicity is most likely due to the production of catechol from SA, and we report here that 100 microM catechol is toxic to tomato roots. Benzothiadiazole, a SA analog, completely restores nematode resistance in Mi-1 roots transformed with NahG but does not confer resistance to susceptible tomato roots. The localized cell death produced by transient expression in Nicotiana benthamiana of Mi-DS4, a constitutively lethal chimera of Mi-1 with one of its homologs, was prevented by coexpression of NahG. These results indicate that SA is an important component of the signaling that leads to nematode resistance and the associated hypersensitive response.     

Mi-1-Mediated aphid resistance involves salicylic acid and mitogen-activated protein kinase signaling cascades

The tomato Mi-1 gene confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphids (Macrosiphum eluphorbiae), and whiteflies (Bemisia tabaci and B. tabaci biotype B). Resistance to potato aphid is developmentally regulated and is not associated with induction of a hypersensitive response. The NahG transgene that eliminates endogenous salicylic acid (SA) was used to test the role of the SA signaling pathway in the resistance mediated by Mi-1 to potato aphids. Aphids survived longer on NahG tomato plants than on wild type. However, aphid reproduction was not affected on NahG tomato. Aphid resistance in Mi-1 NahG plants was completely abolished and the phenotype was successfully rescued by application of BTH (benzo(1,2,3)-thiaiazole-7-carbothioic acid S-methyl ester), indicating that the SA signaling pathway is an important component of Mi-1-mediated aphid resistance. Using virus-induced gene silencing, one or more mitogen-activated protein kinase (MAPK) cascades required for Mi-1-mediated aphid resistance were identified. Silencing plants for MAPK kinase (LeMKK2) and MAPKs (LeMPK2 and LeMPK1, or LeMPK3) resulted in attenuation of Mi-1-mediated aphid resistance. These results further demonstrate that resistance gene-mediated signaling events against piercing-sucking insects are similar to those against other plant pathogens.     

Probenazole induces systemic acquired resistance in Arabidopsis with a novel type of action

Probenazole (PBZ; 3-allyloxy-1,2-benzisothiazole-1,1-dioxide), which is the active ingredient in Oryzemate, has been used widely in Asia to protect rice plants against the rice blast fungus Magnaporthe grisea. To study PBZ's mode of action, we analyzed its ability, as well as that of its active metabolite 1, 2-benzisothiazol-3 (2H)-one 1,1-dioxide (BIT) to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild-type Arabidopsis treated with PBZ or BIT exhibited increased expression of several pathogenesis-related genes, increased levels of total salicylic acid (SA), and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC 3000 and the oomycete pathogen Peronospora parasitica Emco5. The role of several defense signaling hormones, such as SA, ethylene and jasmonic acid (JA), in activating resistance following PBZ or BIT treatment was analyzed using NahG transgenic plants and etr1-1 and coi1-1 mutant plants, respectively. In addition, the involvement of NPR1, a key component in the SA signaling pathway leading to defense responses, was assessed. PBZ or BIT treatment did not induce disease resistance or PR-1 expression in NahG transgenic or npr1 mutant plants, but it did activate these phenomena in etr1-1 and coi 1-1 mutant plants. Thus SA and NPR1 appear to be required for PBZ- and BIT-mediated activation of defense responses, while ethylene and JA are not. Furthermore, our data suggest that PBZ and BIT comprise a novel class of defense activators that stimulate the SA/NPR1-mediated defense signaling pathway upstream of SA.     

Loss of function of FATTY ACID DESATURASE7 in tomato enhances basal aphid resistance in a salicylate-dependent manner

We report here that disruption of function of the ω-3 FATTY ACID DESATURASE7 (FAD7) enhances plant defenses against aphids. The suppressor of prosystemin-mediated responses2 (spr2) mutation in tomato (Solanum lycopersicum), which eliminates the function of FAD7, reduces the settling behavior, survival, and fecundity of the potato aphid (Macrosiphum euphorbiae). Likewise, the antisense suppression of LeFAD7 expression in wild-type tomato plants reduces aphid infestations. Aphid resistance in the spr2 mutant is associated with enhanced levels of salicylic acid (SA) and mRNA encoding the pathogenesis-related protein P4. Introduction of the Naphthalene/salicylate hydroxylase transgene, which suppresses SA accumulation, restores wild-type levels of aphid susceptibility to spr2. Resistance in spr2 is also lost when we utilize virus-induced gene silencing to suppress the expression of NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1), a positive regulator of many SA-dependent defenses. These results indicate that FAD7 suppresses defenses against aphids that are mediated through SA and NPR1. Although loss of function of FAD7 also inhibits the synthesis of jasmonate (JA), the effects of this desaturase on aphid resistance are not dependent on JA; other mutants impaired in JA synthesis (acx1) or perception (jai1-1) show wild-type levels of aphid susceptibility, and spr2 retains aphid resistance when treated with methyl jasmonate. Thus, FAD7 may influence JA-dependent defenses against chewing insects and SA-dependent defenses against aphids through independent effects on JA synthesis and SA signaling. The Arabidopsis (Arabidopsis thaliana) mutants Atfad7-2 and Atfad7-1fad8 also show enhanced resistance to the green peach aphid (Myzus persicae) compared with wild-type controls, indicating that FAD7 influences plant-aphid interactions in at least two plant families.     

The jasmonate signaling pathway in tomato regulates susceptibility to a toxin-dependent necrotrophic pathogen

The plant hormone, jasmonic acid (JA), is known to have a critical role in both resistance and susceptibility against bacterial and fungal pathogen attack. However, little is known about the involvement of JA in the interactions between plants and toxigenic necrotrophic fungal pathogens. Using the tomato pathotype of Alternaria alternata (Aa) and its AAL-toxin/tomato interaction as a model system, we demonstrate a possible role for JA in susceptibility of plants against pathogens, which utilize host-specific toxins as virulence effectors. Disease development and in planta growth of the tomato pathotype of Aa were decreased in the def1 mutant, defective in biosynthesis of JA, compared with the wild-type (WT) cultivar. Exogenous methyl jasmonate (MeJA) application restored pathogen disease symptoms to the def1 mutant and led to increased disease in the WT. On the other hand, necrotic cell death was similarly induced by AAL-toxin both on def1 and WT, and MeJA application to the tomatoes did not affect the degree of cell death by the toxin. These results indicate that the JA-dependent signaling pathway is not involved in host basal defense responses against the tomato pathotype of Aa, but rather might affect pathogen acceptability via a toxin-independent manner. Data further suggest that JA has a promotional effect on susceptibility of tomato to toxigenic and necrotrophic pathogens, such that pathogens might utilize the JA signaling pathway for successful infection.     

The plant growth-promoting fungus Penicillium simplicissimum GP17-2 induces resistance in Arabidopsis thaliana by activation of multiple defense signals

Arabidopsis thaliana grown in soil amended with barley grain inocula of Penicillium simplicissimum GP17-2 or receiving root treatment with its culture filtrate (CF) exhibited clear resistance to Pseudomonas syringae pv. tomato DC3000 (Pst). To assess the contribution of different defense pathways, Arabidopsis genotypes implicated in salicylic acid (SA) signaling expressing the NahG transgene or carrying disruption in NPR1 (npr1), jasmonic acid (JA) signaling (jar1) and ethylene (ET) signaling (ein2) were tested. All genotypes screened were protected by GP17-2 or its CF. However, the level of protection was significantly lower in NahG and npr1 plants than it was in similarly treated wild-type plants, indicating that the SA signaling pathway makes a minor contribution to the GP17-2-mediated resistance and is insufficient for a full response. Examination of local and systemic gene expression revealed that GP17-2 and its CF modulate the expression of genes involved in both the SA and JA/ET signaling pathways. Subsequent challenge of GP17-2-colonized plants with Pst was accompanied by direct activation of SA-inducible PR-2 and PR-5 genes as well as potentiated expression of the JA-inducible Vsp gene. In contrast, CF-treated plants infected with Pst exhibited elevated expression of most defense-related genes (PR-1, PR-2, PR-5, PDF1.2 and Hel) studied. Moreover, an initial elevation of SA responses was followed by late induction of JA responses during Pst infection of induced systemic resistance (ISR)-expressing plants. In conclusion, we hypothesize the involvement of multiple defense mechanisms leading to an ISR of Arabidopsis by GP17-2.     

Parasitism by Cuscuta pentagona attenuates host plant defenses against insect herbivores

Considerable research has examined plant responses to concurrent attack by herbivores and pathogens, but the effects of attack by parasitic plants, another important class of plant-feeding organisms, on plant defenses against other enemies has not been explored. We investigated how attack by the parasitic plant Cuscuta pentagona impacted tomato (Solanum lycopersicum) defenses against the chewing insect beet armyworm (Spodoptera exigua; BAW). In response to insect feeding, C. pentagona-infested (parasitized) tomato plants produced only one-third of the antiherbivore phytohormone jasmonic acid (JA) produced by unparasitized plants. Similarly, parasitized tomato, in contrast to unparasitized plants, failed to emit herbivore-induced volatiles after 3 d of BAW feeding. Although parasitism impaired antiherbivore defenses, BAW growth was slower on parasitized tomato leaves. Vines of C. pentagona did not translocate JA from BAW-infested plants: amounts of JA in parasite vines grown on caterpillar-fed and control plants were similar. Parasitized plants generally contained more salicylic acid (SA), which can inhibit JA in some systems. Parasitized mutant (NahG) tomato plants deficient in SA produced more JA in response to insect feeding than parasitized wild-type plants, further suggesting cross talk between the SA and JA defense signaling pathways. However, JA induction by BAW was still reduced in parasitized compared to unparasitized NahG, implying that other factors must be involved. We found that parasitized plants were capable of producing induced volatiles when experimentally treated with JA, indicating that resource depletion by the parasite does not fully explain the observed attenuation of volatile response to herbivore feeding. Collectively, these findings show that parasitic plants can have important consequences for host plant defense against herbivores.     

Multiple hormones act sequentially to mediate a susceptible tomato pathogen defense response

Phytohormones regulate plant responses to a wide range of biotic and abiotic stresses. How a limited number of hormones differentially mediate individual stress responses is not understood. We have used one such response, the compatible interaction of tomato (Lycopersicon esculentum) and Xanthomonas campestris pv vesicatoria (Xcv), to examine the interactions of jasmonic acid (JA), ethylene, and salicylic acid (SA). The role of JA was assessed using an antisense allene oxide cyclase transgenic line and the def1 mutant to suppress Xcv-induced biosynthesis of jasmonates. Xcv growth was limited in these lines as was subsequent disease symptom development. No increase in JA was detected before the onset of terminal necrosis. The lack of a detectable increase in JA may indicate that an oxylipin other than JA regulates basal resistance and symptom proliferation. Alternatively, there may be an increase in sensitivity to JA or related compounds following infection. Hormone measurements showed that the oxylipin signal must precede subsequent increases in ethylene and SA accumulation. Tomato thus actively regulates the Xcv-induced disease response via the sequential action of at least three hormones, promoting expansive cell death of its own tissue. This sequential action of jasmonate, ethylene, and SA in disease symptom development is different from the hormone interactions observed in many other plant-pathogen interactions.     

The wound response mutant suppressor of prosystemin-mediated responses6 (spr6) is a weak allele of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1)

The systemic defense response of tomato plant in response to insect attack and wounding is regulated by the 18 amino acid peptide systemin and the phytohormone jasmonic acid (JA). Recent genetic analyses based mainly on spr (suppressors of prosystemin-mediated responses) mutant screens have led to the hypothesis that systemin acts at, or near, the site of wounding to amplify the production of JA, which in turn functions as a mobile signal to promote the systemic defense response. In order to identify more components involved in the systemin/JA-signaled defense response, we carried out a larger scale screen for new spr mutants in tomato. Here we describe the characterization of spr6, a mutant impaired in wound- and systemin-induced defense gene expression. Using a candidate gene approach based on genetic linkage, we demonstrate that spr6 is allelic to jai1-1, which is a loss-of-function allele of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. We show several aspects of the spr6 mutant phenotype distinct from that of jai1-1. First, the responsiveness of spr6 plants to exogenous JA shows a dosage dependency, i.e. it is more sensitive to JA than jai1-1 while less sensitive to JA than the wild-type. Secondly, unlike the sterile jai1-1, the spr6 plant displays normal fertility and seed set and thus can be maintained as a pure line and does not require selection. Therefore, spr6 provides a valuable tool, which can complement the limitations of jai1-1, to study JA signaling in tomato. The gene identification process of Spr6 we described herein represents an example showing the convenience of a candidate gene approach, based on genetic linkage, to identify gene functions of genetic loci defined by tomato wound response mutants.     

The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development

Jasmonic acid (JA) is a fatty acid-derived signaling molecule that regulates a broad range of plant defense responses against herbivores and some microbial pathogens. Molecular genetic studies in Arabidopsis have established that JA also performs a critical role in anther and pollen development but is not essential for other developmental aspects of the plant's life cycle. Here, we describe the phenotypic and molecular characterization of a sterile mutant of tomato (jasmonic acid-insensitive1 [jai1]) that is defective in JA signaling. Although the mutant exhibited reduced pollen viability, sterility was caused by a defect in the maternal control of seed maturation, which was associated with the loss of accumulation of JA-regulated proteinase inhibitor proteins in reproductive tissues. jai1 plants exhibited several defense-related phenotypes, including the inability to express JA-responsive genes, severely compromised resistance to two-spotted spider mites, and abnormal development of glandular trichomes. We demonstrate that these defects are caused by the loss of function of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. These findings indicate that the JA/COI1 signaling pathway regulates distinct developmental processes in different plants and suggest a role for JA in the promotion of glandular trichome-based defenses.     

Components of Arabidopsis defense- and ethylene-signaling pathways regulate susceptibility to Cauliflower mosaic virus by restricting long-distance movement

We analyzed the susceptibility of Arabidopsis mutants with defects in salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) signaling to infection by Cauliflower mosaic virus (CaMV). Mutants cpr1-1 and cpr5-2, in which SA-dependent defense signaling is activated constitutively, were substantially more resistant than the wild type to systemic infection, implicating SA signaling in defense against CaMV. However, SA-deficient NahG, sid2-2, eds5-1, and pad4-1 did not show enhanced susceptibility. A cpr5 eds5 double mutant also was resistant, suggesting that resistance in cpr5 may function partially independently of SA. Treatment of cpr5 and cpr5 eds5, but not cpr1, with salicyl-hydroxamic acid, an inhibitor of alternative oxidase, partially restored susceptibility to wild-type levels. Mutants etr1-1, etr1-3, and ein2-1, and two mutants with lesions in ET/JA-mediated defense, eds4 and eds8, also showed reduced virus susceptibility, demonstrating that ET-dependent responses also play a role in susceptibility. We used a green fluorescent protein (GFP)-expressing CaMV recombinant to monitor virus movement. In mutants with reduced susceptibility, cpr1-1, cpr5-2, and etr1-1, CaMV-GFP formed local lesions similar to the wild type, but systemic spread was almost completely absent in cpr1 and cpr5 and was substantially reduced in etr1-1. Thus, mutations with enhanced systemic acquired resistance or compromised ET signaling show diminished long-distance virus movement.     

The Participation of salicylic and jasmonic acids in genetic and induced resistance of tomato to Meloidogyne incognita (Kofoid and White, 1919)

Salicylic (SA) and jasmonic (JA) acids are the best known mediators of signal systems in plants. In this investigation the participation and character of interactions between SA- and JA-signals under the induced and genetic resistance of plants to nematodes was investigated on the model system tomato (Lycopersicon esculentum) and the root-knot nematode Meloidogyne incognita. This study demonstrates that application of JA and SA to tomato foliage induces systemic effects that suppress root-knot nematode infestation, inhibition of nematode reproduction, and also increased activity of LOX and PAL, the enzymes of biosynthesis of JA and SA. JA treatment did not inhibit Mz-mediated resistance, which suggests a lack of signaling conflicts between these two forms of defense.     

Salicylic acid-dependent expression of host genes in compatible Arabidopsis-virus interactions

Plant viruses elicit the expression of common sets of genes in susceptible hosts. Studies in Arabidopsis (Arabidopsis thaliana) and tomato (Lycopersicon esculentum) indicate that at least one-third of the genes induced in common by viruses have been previously associated with plant defense and stress responses. The genetic and molecular requirements for the induction of these stress and defense-related genes during compatible host-virus interactions were investigated with a panel of Arabidopsis mutant and transgenic plants defective in one or more defense signaling pathways. pad4, eds5, NahG, npr1, jar1, ein2, sid2, eds1, and wild-type Columbia-0 and Wassilewskija-2 plants were infected with two different viruses, cucumber mosaic virus and oilseed rape mosaic virus. Gene expression was assayed by a high-throughput fiber-optic bead array consisting of 388 genes and by RNA gel blots. These analyses demonstrated that, in compatible host-virus interactions, the expression of the majority of defense-related genes is induced by a salicylic acid-dependent, NPR1-independent signaling pathway with a few notable exceptions that did require NPR1. Interestingly, none of the mutant or transgenic plants showed enhanced susceptibility to either cucumber mosaic virus or oilseed rape mosaic virus based on both symptoms and virus accumulation. This observation is in contrast to the enhanced disease susceptibility phenotypes that these mutations or transgenes confer to some bacterial and fungal pathogens. These experimental results suggest that expression of many defense-related genes in compatible host plants might share components of signaling pathways involved in incompatible host-pathogen interactions, but their increased expression has no negative effect on viral infection.     

Proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades

Plants exhibit resistance against incompatible pathogens, via localized and systemic responses as part of an integrated defense mechanism. To study the compatible and incompatible interactions between rice and bacteria, a proteomic approach was applied. Rice cv. Java 14 seedlings were inoculated with compatible (Xo7435) and incompatible (T7174) races of Xanthomonas oryzae pv. oryzae (Xoo). Cytosolic and membrane proteins were fractionated from the leaf blades and separated by 2-D PAGE. From 366 proteins analyzed, 20 were differentially expressed in response to bacterial inoculation. These proteins were categorized into classes related to energy (30%), metabolism (20%), and defense (20%). Among the 20 proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU) was fragmented into two smaller proteins by T7174 and Xo7435 inoculation. Treatment with jasmonic acid (JA), a signaling molecule in plant defense responses, changed the level of protein accumulation for 5 of the 20 proteins. Thaumatin-like protein and probenazole-inducible protein (PBZ) were commonly up-regulated by T7174 and Xo7435 inoculation and JA treatment. These results suggest that synthesis of the defense-related thaumatin-like protein and PBZ are stimulated by JA in the defense response pathway of rice against bacterial blight.     

Erwinia carotovora subsp. carotovora and Erwinia-derived elicitors HrpN and PehA trigger distinct but interacting defense responses and cell death in Arabidopsis

We have used an hrp-positive strain of the soft rot pathogen Erwinia carotovora subsp. carotovora to elucidate plant responses to this bacterial necrotroph. Purified virulence determinants, harpin (HrpN) and polygalacturonase (PehA), were used as tools to facilitate this analysis. We show that HrpN elicits lesion formation in Arabidopsis and tobacco and triggers systemic resistance in Arabidopsis. Establishment of resistance is accompanied by the expression of salicylic acid (SA)-dependent, but also jasmonate/ethylene (JA/ET)-dependent, marker genes PR1 and PDF1.2, respectively, suggesting that both SA-dependent and JA/ET-dependent defense pathways are activated. Use of pathway-specific mutants and transgenic NahG plants show that both pathways are required for the induction of resistance. Arabidopsis plants treated simultaneously with both elictors PehA, known to trigger only JA/ET-dependent defense signaling, and HrpN react with accelerated and enhanced induction of the marker genes PR1 and PDF1.2 both locally and systemically. This mutual amplification of defense gene expression involves both SA-dependent and JA/ET-dependent defense signaling. The two elicitors produced by E. carotovora subsp. carotovora also cooperate in triggering increased production of superoxide and lesion formation.     

Characterization of a novel,defense-related Arabidopsis mutant,cir1, isolated by luciferase imaging

In order to identify components of the defense signaling network engaged following attempted pathogen invasion, we generated a novel PR-1::luciferase (LUC) transgenic line that was deployed in an imaging-based screen to uncover defense-related mutants. The recessive mutant designated cir1 exhibited constitutive expression of salicylic acid (SA), jasmonic acid (JA)/ethylene, and reactive oxygen intermediate-dependent genes. Moreover, this mutation conferred resistance against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and a virulent oomycete pathogen Peronospora parasitica Noco2. Epistasis analyses were undertaken between cir1 and mutants that disrupt the SA (nprl, nahG), JA (jar1), and ethylene (ET) (ein2) signaling pathways. While resistance against both P. syringae pv. tomato DC3000 and Peronospora parasitica Noco2 was partially reduced by npr1, resistance against both of these pathogens was lost in an nahG genetic background. Hence, cirl-mediated resistance is established via NPR1-dependent and -independent signaling pathways and SA accumulation is essential for the function of both pathways. While jar1 and ein2 reduced resistance against P. syringae pv. tomato DC3000, these mutations appeared not to impact cir1-mediated resistance against Peronospora parasitica Noco2. Thus, JA and ET sensitivity are required for cir1-mediated resistance against P. syringae pv. tomato DC3000 but not Peronospora parasitica Noco2. Therefore, the cir1 mutation may define a negative regulator of disease resistance that operates upstream of SA, JA, and ET accumulation.