Salicylic Acid Biosynthetic Genes Research Articles

Rapeseed NTL4 transcription factor positively regulates leaf senescence and resistance against bacterial pathogen via controlling transcription of ROS and SA biosynthetic genes

Rapeseed (Brassica napus L.) is widely planted for both edible oil and biodiesel. However, its growth is frequently affected by adverse environmental factors such as pathogens. Moreover, rapeseed shows significant falling of leaves during the whole growth period, making it important to control leaf senescence for better nutrient utilization efficiency and improved growth. Membrane-tethered transcription factors (MTTF) constitute a special group of proteins, which facilitate rapid response to adverse conditions. However, the role of MTTFs in rapeseed has seldomly been reported. Here BnaNTL4 was functionally characterized to transcriptionally regulate leaf senescence and resistance towards a bacterial pathogen in rapeseed. BnaNTL4 encodes a member-associated transcription factor and its expression was induced by several stimuli including salicylic acid and hydrogen peroxide. Both stable and transient expression demonstrated BnaNTL4 could induce reactive oxygen species (ROS) production and leaf chlorosis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay of differentially expressed genes in BnaNTL4 overexpression lines and control plants uncovered that several genes related to programmed cell death (γVPE, BFN1), ROS production (RbohF), salicylic acid (SA) synthesis (PBS3) and senescence (SEN4) were induced by BnaNTL4 overexpression. Further, a dual luciferase reporter assay revealed BnaNTL4 transactivated the activities of the promoters of putative target genes. At last, an electrophoretic mobility shift assay discovered that BnaNTL4 directly bound to promoters of BnaRbohF, BnaPBS3 and BnaSEN4. Overexpression of BnaNTL4 also enhances resistance towards a bacterial pathogen Pseudomonas syringae DC3000. Thus, the results uncovered a dual role of BnaNTL4 in leaf senescence and disease resistance in rapeseed.

Preliminary analysis reveals that RCF1 confers resistance to Pseudomonas syringae pv. tomato DC3000 but impairs Botrytis cinerea infection

The adaptation process is a way of life for plants, and they must contend with biotic and abiotic stresses for their chance of survival. These stresses can lead to disastrous crop production and limited distribution of crops geographically. The use of chemicals to address those stresses in agriculture is limited, while management strategies often fail to meet the needs of the industry. Therefore, novel techniques are needed to control plant diseases and to maintain food security. RCF1 has been found to encode an RNA helicase containing DEAD-box domains and loss-of-function mutation of this gene increases the sensitivity of plants to cold stress. In this study, a single mutant generated in RCF1 was used to investigate the function of the RCF1 gene in pathogen infection stress (i.e., Pst DC3000 and B. cinerea). RFC1 was found to confer resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) but negatively affect Botrytis cinerea (B. cinerea) infection by repressing SNI1 pathways and JA signaling while activating SA pathways and its receptor NPR1. The generated rcf1 allelic mutant (name: 407) showed sensitivities to bacteria pathogen (Pst DC3000) but tolerance to fungus infection (B. cinerea), and SNI1 was highly upregulated in it. Free and conjugated Salicylic Acid (SA) contents were much lower in the allelic mutant 407 than in complementary materials and is sensitive to Pst DC3000 infection, and NPR1 depending on SA biosynthetic genes were down-regulated in it. Our preliminary results reveal that RCF1 confers resistance to Pst DC3000 by SA and NPR1 dependence manner but blocks SNI1 and JA pathways.

Histone acetyltransferase HAM1 interacts with molecular chaperone DNAJA2 and confers immune responses through salicylic acid biosynthetic genes in cassava.

Cassava bacterial blight (CBB) is one of the most serious diseases in cassava production, so it is essential to explore the underlying mechanism of immune responses. Histone acetylation is an important epigenetic modification, however, its relationship with cassava disease resistance remains unclear. Here, we identified 10 histone acetyltransferases in cassava and found that the transcript of MeHAM1 showed the highest induction to CBB. Functional analysis showed that MeHAM1 positively regulated disease resistance to CBB through modulation of salicylic acid (SA) accumulation. Further investigation revealed that MeHAM1 directly activated SA biosynthetic genes' expression via promoting lysine 9 of histone 3 (H3K9) acetylation and lysine 5 of histone 4 (H4K5) acetylation of these genes. In addition, molecular chaperone MeDNAJA2 physically interacted with MeHAM1, and MeDNAJA2 also regulated plant immune responses and SA biosynthetic genes. In conclusion, this study illustrates that MeHAM1 and MeDNAJA2 confer immune responses through transcriptional programming of SA biosynthetic genes via histone acetylation. The MeHAM1 & MeDNAJA2-SA biosynthesis module not only constructs the direct relationship between histone acetylation and cassava disease resistance, but also provides gene network with potential value for genetic improvement of cassava disease resistance.

NAC transcription factor TgNAP promotes tulip petal senescence.

Petal senescence is a crucial determinant for ornamental quality and economic value of floral crops. Salicylic acid (SA) and reactive oxygen species (ROS) are two prominent factors involved in plant senescence regulation. In this study, tulip TgNAP (NAC-like, activated by APETALA3/PISTILLATA) was characterized as positively regulating tulip petal senescence through dually regulating SA biosynthesis and ROS detoxification pathways. TgNAP was upregulated in senescing petals of tulip while exogenous SA and H2O2 treatments substantially promoted petal senescence in tulip. Silencing of TgNAP by VIGS assay delayed SA and H2O2-induced petal senescence in tulip, whereas overexpression of TgNAP promoted the senescence process in Arabidopsis (Arabidopsis thaliana) plants. Additionally, inhibition of SA biosynthesis prolonged the lifespan of TgNAP-silenced petal discs. Further evidence indicated that TgNAP activates the transcriptions of two key SA biosynthetic genes ISOCHORISMATE SYNTHASE 1 (TgICS1) and PHENYLALANINE AMMONIA-LYASE 1 (TgPAL1) through directly binding to their promoter regions. Meanwhile, TgNAP repressed ROS scavenging by directly inhibiting PEROXIDASE 12 (POD12) and POD17 expression. Taken together, these results indicate that TgNAP enhances SA biosynthesis and ROS accumulation to positively regulate petal senescence in tulip.

Senescence and Defense Pathways Contribute to Heterosis.

Hybrids are used extensively in agriculture due to their superior performance in seed yield and plant growth, yet the molecular mechanisms underpinning hybrid performance are not well understood. Recent evidence has suggested that a decrease in basal defense response gene expression regulated by reduced levels of salicylic acid (SA) may be important for vigor in certain hybrid combinations. Decreasing levels of SA in the Arabidopsis (Arabidopsis thaliana) accession C24 through the introduction of the SA catabolic enzyme salicylate1 hydroxylase (NahG) increases plant size, phenocopying the large-sized C24/Landsberg erecta (Ler) F1 hybrids. C24♀ × Ler♂ F1 hybrids and C24 NahG lines shared differentially expressed genes and pathways associated with plant defense and leaf senescence including decreased expression of SA biosynthetic genes and SA response genes. The expression of TL1 BINDING TRANSCRIPTION FACTOR1, a key regulator in resource allocation between growth and defense, was decreased in both the F1 hybrid and the C24 NahG lines, which may promote growth. Both C24 NahG lines and the F1 hybrids showed decreased expression of the key senescence-associated transcription factors WRKY53, NAC-CONTAINING PROTEIN29, and ORESARA1 with a delayed onset of senescence compared to C24 plants. The delay in senescence resulted in an extension of the photosynthetic period in the leaves of F1 hybrids compared to the parental lines, potentially allowing each leaf to contribute more resources toward growth.

Common mycorrhizal networks activate salicylic acid defense responses of trifoliate orange (Poncirus trifoliata).

Citrus canker, caused by Xanthomonas axonopodis pv. citri ('Xac'), is an important quarantine disease in citrus crops. Arbuscular mycorrhizal fungi (AMF) form symbiotic interactions with host plants and further affect their disease resistance, possibly by modulating the activity of salicylic acid (SA), a key phytohormone in disease resistance. Common mycorrhizal networks (CMNs) can interconnect plants, but it is not yet clear whether CMNs promote resistance to citrus canker and, if so, whether SA signaling is involved in this process. To test this possibility, we used a two-chambered rootbox to establish CMNs between trifoliate orange (Poncirus trifoliata) seedlings in chambers inoculated (treated) or not (neighboring) with the AMF, Paraglomus occultum. A subset of the AMF-inoculated seedlings were also inoculated with Xac (+AMF+Xac). At 2 d post-inoculation (dpi), compared with the +AMF-Xac treatment, neighboring seedlings in +AMF+Xac treatment had lower expression levels of the SA biosynthetic genes, PtPAL, PtEPS1, and PtPBS3, but higher SA levels, which attributed to the upregulation of PtPAL and PtPBS3 in treated seedlings and the transfer of SA, via CMNs, to the neighboring seedlings. At 4 dpi, the pathogenesis-related (PR) protein genes, PtPR1, PtPR4, and PtPR5, and the transcriptional regulatory factor gene, PtNPR1, were activated in neighboring seedlings of +AMF+Xac treatment. At 9 dpi, root phenylalanine ammonia-lyase activity and total soluble phenol and lignin concentrations increased in neighboring seedlings of +AMF+Xac treatment, likely due to the linkage and signal transfer, via CMNs. These findings support the hypothesis that CMNs transfer the SA signal from infected to neighboring healthy seedlings, to activate defense responses and affording protection to neighboring plants against citrus canker infection.

ACR11 modulates levels of reactive oxygen species and salicylic acid-associated defense response in Arabidopsis

The ACT domain (aspartate kinase, chorismate mutase and TyrA), an allosteric effector binding domain, is commonly found in amino acid metabolic enzymes. In addition to ACT domain-containing enzymes, plants have a novel family of ACT domain repeat (ACR) proteins, which do not contain any recognizable catalytic domain. Arabidopsis has 12 ACR proteins, whose functions are largely unknown. To study the functions of Arabidopsis ACR11, we have characterized two independent T-DNA insertion mutants, acr11-2 and acr11-3. RNA gel-blot analysis revealed that the expression of wild-type ACR11 transcripts was not detectable in the acr11 mutants. Interestingly, a lesion-mimic phenotype occurs in some rosette leaves of the acr11 mutants. In addition, high levels of reactive oxygen species (ROS), salicylic acid (SA), and callose accumulate in the mutant leaves when grown under normal conditions. The expression of several SA marker genes and the key SA biosynthetic gene ISOCHORISMATE SYNTHASE1 is up-regulated in the acr11 mutants. Furthermore, the acr11 mutants are more resistant to the infection of bacterial pathogen Pseudomonas syringae pathovar tomato DC3000. These results suggest that ACR11 may be directly or indirectly involved in the regulation of ROS and SA accumulation, which in turn modulates SA-associated defense responses and disease resistance in Arabidopsis.

Salicylic Acid Regulates Systemic Defense Signaling in Chickpea During Fusarium oxysporum f. sp. ciceri Race 1 Infection

Annual loss of productivity of the important crop legume chickpea has received prime scientific concern at recent times. Vascular wilt caused by fungal pathogen Fusarium oxysporum f. sp. ciceris race 1 (Foc1) accounts for major share of yield loss of chickpea. Control of this disease remains a challenge due to the lack of appropriate breeding programs to manage fast pathogen mutability. Previous studies with this pathogen have highlighted the role of reactive oxygen species (ROS) as chemical signal in enkindling downstream systemic resistance response instead of activating site specific defense. But the role of salicylic acid in modulating resistance is still unexplored. Present study explains the probable function of salicylic acid (SA) in coordination with ROS. The external SA application reveals the restoration of relative water content of infected susceptible chickpea plants. The qRT-PCR based expression study of key SA biosynthetic genes indicate that the SA biogenesis takes place by the activity of phenylalanine ammonia lyase (PAL) that activates other SA responsive genes and TGA transcription factors to induce an active defense against Foc1. Finally, detection of SA by LC MS/MS along with the accumulation of transcripts of SA marker genes, PR1 and PR5, strengthens the involvement of SA in translocation of distant systemic signals in chickpea-Foc1 interaction.

Arabidopsis thaliana GLUTATHIONE-S-TRANSFERASE THETA 2 interacts with RSI1/FLD to activate systemic acquired resistance.

A partly infected plant develops systemic acquired resistance (SAR) and shows heightened resistance during subsequent infections. The infected parts generate certain mobile signals that travel to the distal tissues and help to activate SAR. SAR is associated with epigenetic modifications of several defence-related genes. However, the mechanisms by which mobile signals contribute to epigenetic changes are little known. Previously, we have shown that the Arabidopsis REDUCED SYSTEMIC IMMUNITY 1 (RSI1, alias FLOWERING LOCUS D; FLD), which codes for a putative histone demethylase, is required for the activation of SAR. Here, we report the identification of GLUTATHIONE-S-TRANSFERASE THETA 2 (GSTT2) as an interacting factor of FLD. GSTT2 expression increases in pathogen-inoculated as well as pathogen-free distal tissues. The loss-of-function mutant of GSTT2 is compromised for SAR, but activates normal local resistance. Complementation lines of GSTT2 support its role in SAR activation. The distal tissues of gstt2 mutant plants accumulate significantly less salicylic acid (SA) and express a reduced level of the SA biosynthetic gene PAL1. In agreement with the established histone modification activity of FLD, gstt2 mutant plants accumulate an enhanced level of methylated and acetylated histones in the promoters of WRKY6 and WRKY29 genes. Together, these results demonstrate that GSTT2 is an interactor of FLD, which is required for SAR and SAR-associated epigenetic modifications.

Editorial: Salicylic Acid Signaling Networks

Editorial: Salicylic Acid Signaling Networks

Effects of volatile organic compound ether on cell responses and gene expressions in Arabidopsis.

Background The volatile organic compound ether is widely used as an industrial solvent and easily released to the environment. Our previous research indicated that ether triggers reactive oxygen species (ROS) production and activates ethylene biosynthetic genes and defense gene expressions in tomato. In the present study, we investigated the effect of ether on cell responses and gene expressions in Arabidopsis and compared the ROS and phytohormones produced in Arabidopsis and tomato plants in response to different air pollutants (O3 vs. ether).ResultsEther induced the sequential production of superoxide anion and hydrogen peroxide in Arabidopsis. Ether also triggered expressions of ethylene, salicylic acid and jasmonic acid biosynthetic genes. The temporal expression patterns of MAP kinase and protein phosphatase genes are in good accordance with those of the ethylene and salicylic acid biosynthetic genes, suggesting that induction of these phytohormone biosynthesis were through signaling pathways including both phosphorylation and/or dephosphorylation. By contrast, expression pattern of protein phosphatase PP2A3&4 coincided well with the expression of jasmonic acid biosynthetic gene LOX4, suggesting that induction of jasmonic acid biosynthesis is through PP2A3&4. However, the production of ROS and temporal expression patterns of phytohormone biosynthetic genes in Arabidopsis in response to ether were different from those to O3 and were different from those in tomato as well.ConclusionsDifferent plants have different strategies to respond to the same abiotic stress, and each plant species possesses its own unique signaling pathways that regulate the responding process.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-015-0112-8) contains supplementary material, which is available to authorized users.

Functional characterization of a Nudix hydrolase AtNUDX8 upon pathogen attack indicates a positive role in plant immune responses.

Nudix hydrolases comprise a large gene family of twenty nine members in Arabidopsis, each containing a conserved motif capable of hydrolyzing specific substrates like ADP-glucose and NADH. Until now only two members of this family, AtNUDX6 and AtNUDX7, have been shown to be involved in plant immunity. RPP4 is a resistance gene from a multigene family that confers resistance to downy mildew. A time course expression profiling after Hyaloperonospora arabidopsidis inoculation in both wild-type (WT) and the rpp4 mutant was carried out to identify differentially expressed genes in RPP4-mediated resistance. AtNUDX8 was one of several differentially expressed, downregulated genes identified. A T-DNA knockout mutant (KO-nudx8) was obtained from a Salk T-DNA insertion collection, which exhibited abolished AtNUDX8 expression. The KO-nudx8 mutant was infected separately from the oomycete pathogen Hpa and the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. The mutant displayed a significantly enhanced disease susceptibility to both pathogens when compared with the WT control. We observed a small, stunted phenotype for KO-nudx8 mutant plants when grown over a 12/12 hour photoperiod but not over a 16/8 hour photoperiod. AtNUDX8 expression peaked at 8 hours after the lights were turned on and this expression was significantly repressed four-fold by salicylic acid (SA). The expression of three pathogen-responsive thioredoxins (TRX-h2, TRX-h3 and TRX-h5) were downregulated at specific time points in the KO-nudx8 mutant when compared with the WT. Furthermore, KO-nudx8 plants like the npr1 mutant, displayed SA hypersensitivity. Expression of a key SA biosynthetic gene ICS1 was repressed at specific time points in the KO-nudx8 mutant suggesting that AtNUDX8 is involved in SA signaling in plants. Similarly, NPR1 and PR1 transcript levels were also downregulated at specific time points in the KO-nudx8 mutant. This study shows that AtNUDX8 is involved in plant immunity as a positive regulator of defense in Arabidopsis.

Foliar treatments with Gaultheria procumbens essential oil induce defense responses and resistance against a fungal pathogen in Arabidopsis.

Essential oil from Gaultheria procumbens is mainly composed of methylsalicylate (MeSA) (>96%), a compound which can be metabolized in plant tissues to salicylic acid, a phytohormone inducing plant immunity against microbial pathogens. The potential use of G. procumbens essential oil as a biocontrol agent was evaluated on the model plant Arabidopsis thaliana. Expression of a selection of defense genes was detected 1, 6, and 24 h after essential oil treatment (0.1 ml/L) using a high-throughput qPCR-based microfluidic technology. Control treatments included methyl jasmonate and a commercialized salicylic acid (SA) analog, benzo(1,2,3)-thiadiazole-7carbothiolic acid (BTH). Strong induction of defense markers known to be regulated by the SA pathway was observed after the treatment with G. procumbens essential oil. Treatment induced the accumulation of total SA in the wild-type Arabidopsis line Col-0 and analysis of the Arabidopsis line sid2, mutated in a SA biosynthetic gene, revealed that approximately 30% of MeSA sprayed on the leaves penetrated inside plant tissues and was demethylated by endogenous esterases. Induction of plant resistance by G. procumbens essential oil was tested following inoculation with a GFP-expressing strain of the Arabidopsis fungal pathogen Colletotrichum higginsianum. Fluorescence measurement of infected tissues revealed that treatments led to a strong reduction (60%) of pathogen development and that the efficacy of the G. procumbens essential oil was similar to the commercial product BION®. Together, these results show that the G. procubens essential oil is a natural source of MeSA which can be formulated to develop new biocontrol products.

Salicylic Acid Biosynthesis and Metabolism

Salicylic acid (SA) has been shown to regulate various aspects of growth and development; it also serves as a critical signal for activating disease resistance in Arabidopsis thaliana and other plant species. This review surveys the mechanisms involved in the biosynthesis and metabolism of this critical plant hormone. While a complete biosynthetic route has yet to be established, stressed Arabidopsis appear to synthesize SA primarily via an isochorismate-utilizing pathway in the chloroplast. A distinct pathway utilizing phenylalanine as the substrate also may contribute to SA accumulation, although to a much lesser extent. Once synthesized, free SA levels can be regulated by a variety of chemical modifications. Many of these modifications inactivate SA; however, some confer novel properties that may aid in long distance SA transport or the activation of stress responses complementary to those induced by free SA. In addition, a number of factors that directly or indirectly regulate the expression of SA biosynthetic genes or that influence the rate of SA catabolism have been identified. An integrated model, encompassing current knowledge of SA metabolism in Arabidopsis, as well as the influence other plant hormones exert on SA metabolism, is presented.

The Cytokinin-Activated Transcription Factor ARR2 Promotes Plant Immunity via TGA3/NPR1-Dependent Salicylic Acid Signaling in Arabidopsis

Cytokinins affect plant immunity to various pathogens; however, the mechanisms coupling plant-derived cytokinins to pathogen responses have been elusive. Here, we found that plant-derived cytokinins promote resistance of Arabidopsis to Pseudomonas syringae pv. tomato DC3000 (Pst). Modulated cytokinin levels or signaling activity in CKX- or IPT-overexpressing plants or in ahk2 ahk3 mutants correlated with altered resistance. In fact, the cytokinin-activated transcription factor ARR2 contributes specifically to Pst resistance. The salicylic acid (SA) response factor TGA3 binds ARR2, and mutation of TGA-binding cis-elements in the Pr1 promoter abolished cytokinin- and ARR2-dependent Pr1 activation. Cytokinin treatment did not increase pathogen resistance in tga3 plants, as the cytokinin-dependent induction of Pr1 was eliminated. Moreover, SA signaling enhanced binding of ARR2/TGA3 to the Pr1 promoter. Taken together, these results show that cytokinins modulate the SA signaling to augment resistance against Pst, a process in which the interaction between TGA3 and ARR2 is important.

MYB96‐mediated abscisic acid signals induce pathogen resistance response by promoting salicylic acid biosynthesis in Arabidopsis

*The Arabidopsis MYB96 transcription factor plays a role in abscisic acid (ABA)-mediated drought response. Notably, anthocyanins accumulate in the activation-tagging myb96-1d line, suggesting a role of MYB96 in biotic and abiotic stress responses in plants. Here, we investigate the role of MYB96 in salicylic acid (SA) biosynthesis and plant defense and explore the mechanisms underlying the ABA-SA interaction. *myb96-1d and myb96-1 were subject to pathogen infection assays, and expression of SA biosynthetic and defense genes was examined. myb96-1d was crossed with the NahG transgenic plants to investigate the role of MYB96 in ABA regulation of SA biosynthesis. *Whereas myb96-1d exhibited an enhanced disease resistance, myb96-1 was susceptible to pathogen infection. A subset of pathogenesis-related (PR) genes was up-regulated in myb96-1d. However, PR transcript abundances were reduced in myb96-1d X NahG. Interestingly, a SA biosynthetic gene SALICYLIC ACID INDUCTION DEFICIENT2 (SID2) was up-regulated, and concentrations of SA and SA-beta-glucoside (SAG) were elevated in myb96-1d. In addition, the inductive effects of abiotic stresses on SID2 were reduced in aba3-1. *Our observations indicate that MYB96-mediated ABA signals enhance plant disease resistance by inducing SA biosynthesis. It is envisioned that MYB96 is a molecular link that mediates ABA-SA crosstalks.

Increased SA in NPR1‐silenced plants antagonizes JA and JA‐dependent direct and indirect defenses in herbivore‐attacked Nicotiana attenuata in nature

The phytohormone jasmonic acid (JA) is known to mediate herbivore resistance, while salicylic acid (SA) and non-expressor of PR-1 (NPR1) mediate pathogen resistance in many plants. Herbivore attack on Nicotiana attenuata elicits increases in JA and JA-mediated defenses, but also increases SA levels and Na-NPR1 transcripts from the plant's single genomic copy. SA treatment of wild-type plants increases Na-NPR1 and Na-PR1 transcripts. Plants silenced in NPR1 accumulation by RNAi (ir-npr1) are highly susceptible to herbivore and pathogen attack when planted in their native habitat in Utah. They are also impaired in their ability to attract Geocorus pallens predators, due to their decreased ability to release cis-alpha-bergamotene, a JA-elicited volatile 'alarm call'. In the glasshouse, Spodoptera exigua larvae grew better on ir-npr1 plants, which had low levels of JA, JA-isoleucine/leucine, lipoxygenase-3 (LOX3) transcripts and JA-elicited direct defense metabolites (nicotine, caffeoyl putrescine and rutin), but high levels of SA and isochorismate synthase (ICS) transcripts, suggesting de novo biosynthesis of SA. A microarray analysis revealed downregulation of many JA-elicited genes and upregulation of SA biosynthetic genes. JA treatment restored nicotine levels and resistance to S. exigua in ir-npr1 plants. We conclude that, during herbivore attack, NPR1 negatively regulates SA production, allowing the unfettered elicitation of JA-mediated defenses; when NPR1 is silenced, the elicited increases in SA production antagonize JA and JA-related defenses, making the plants susceptible to herbivores.

Induction of Systemic Resistance Against Bacterial Wilt in Eucalyptus urophylla by Fluorescent Pseudomonas spp

The ability of selected strains of fluorescent Pseudomonas spp. to cause induced systemic resistance (ISR) in Eucalyptus urophylla against bacterial wilt caused by Ralstonia solanacearum was investigated. Four of the five strains used can produce salicylic acid (SA) in vitro and, therefore, chemical SA, that is known to induce resistance in many plant species, was used as a reference treatment. Whereas a soil drench with SA did induce systemic resistance in E. urophylla, infiltration of SA into leaves did not. None of the fluorescent Pseudomonas spp. strains caused ISR against bacterial wilt when applied to the soil, but two strains, P. putida WCS358r and P. fluorescens WCS374r triggered ISR when infiltrated into two lower leaves 3–7 days before challenge inoculation. A mutant of strain WCS358r defective in the biosynthesis of the fluorescent siderophore pseudobactin, did not cause ISR, while the purified siderophore of WCS358r did, suggesting that pseudobactin358 is the ISR determinant of WCS358. A siderophore-minus mutant of WCS374r induced the same level of disease resistance as its parental strain, but the purified siderophore induced resistance as well, indicating that both the siderophore and another, unknown, inducing determinant(s) of WCS374r can trigger ISR in Eucalyptus. A possible role of WCS374r-produced SA remains uncertain. Transformation of a siderophore-minus mutant of WCS358 with the SA biosynthetic gene cluster from WCS374 did not enable this transformant to cause ISR in E. urophylla.