The role of pectate lyase and the jasmonic acid defense response in Pseudomonas viridiflava virulence

Pseudomonas viridiflava is a common pathogen of Arabidopsis thaliana in wild populations, yet very little is known about mechanisms of resistance and virulence in this interaction. We examined the induced defense response of A. thaliana to several strains of P. viridiflava collected from this host by quantifying the expression of PR-1 and LOX2/PDF1.2, which serve as markers for induction of the salicylic and jasmonic acid (JA) pathways, respectively. Growth of these strains then was assessed on Col-0, the fad3/7/8 and coil-1 mutants deficient in JA- and ethylene (ET)-induced defense responses, and the sid2-1 mutant deficient in salicylic acid-induced defense responses. All strains of P. viridiflava induced high expression of LOX2 and PDF1.2 on Col-0. In contrast, PR-1 expression was delayed and reduced relative to PDF1.2 expression. Additionally, three of four P. viridiflava strains were more virulent on fad3/7/8 relative to Col-0, whereas all strains were more virulent on coil-1 relative to Col-0, indicating that P. viridiflava generally may be suppressed by JA/ET-mediated defense responses. In contrast, no increase in the growth of P. viridiflava strains was observed in the sid2-1 mutant relative to Col-0. Parallel experiments were performed with the closely related P. syringae pv. tomato for comparative purposes. In addition, we assessed the role of pectate lyase and the alternative sigma factor HrpL in P. viridiflava virulence on A. thaliana and found that pectate lyase activity is correlated with virulence, whereas the removal of pectate lyase or HrpL significantly reduced virulence.     

Analysis of the plant bos1 mutant highlights necrosis as an efficient defence mechanism during D. dadantii/Arabidospis thaliana interaction

Dickeya dadantii is a broad host range phytopathogenic bacterium provoking soft rot disease on many plants including Arabidopsis. We showed that, after D. dadantii infection, the expression of the Arabidopsis BOS1 gene was specifically induced by the production of the bacterial PelB/C pectinases able to degrade pectin. This prompted us to analyze the interaction between the bos1 mutant and D. dadantii. The phenotype of the infected bos1 mutant is complex. Indeed, maceration symptoms occurred more rapidly in the bos1 mutant than in the wild type parent but at a later stage of infection, a necrosis developed around the inoculation site that provoked a halt in the progression of the maceration. This necrosis became systemic and spread throughout the whole plant, a phenotype reminiscent of that observed in some lesion mimic mutants. In accordance with the progression of maceration symptoms, bacterial population began to grow more rapidly in the bos1 mutant than in the wild type plant but, when necrosis appeared in the bos1 mutant, a reduction in bacterial population was observed. From the plant side, this complex interaction between D. dadantii and its host includes an early plant defence response that comprises reactive oxygen species (ROS) production accompanied by the reinforcement of the plant cell wall by protein cross-linking. At later timepoints, another plant defence is raised by the death of the plant cells surrounding the inoculation site. This plant cell death appears to constitute an efficient defence mechanism induced by D. dadantii during Arabidopsis infection.     

An Arabidopsis homologue of human seven-in-absentia-interacting protein is involved in pathogen resistance

Human seven-in-absentia (SIAH)-interacting protein (SIP) is a component of the E3 ligase complex targeting beta-catenin for destruction. Arabidopsis has one SIP protein (AtSIP) with 32% amino acid sequence identity to SIP. To investigate the functions of AtSIP, we isolated an atsip knockout mutant, and generated transgenic plants overexpressing AtSIP. The growth rates and morphologies of the atsip and transgenic plants were indistinguishable from those of wild type. However, atsip plants were more susceptible to Pseudomonas syringae infection, and the transgenic plants overexpressing AtSIP were more resistant. Consistent with this, RNA blot analysis showed that the AtSIP gene is strongly induced by wounding and hydrogen peroxide treatment. In addition, when plants were infected with P. syringae, AtSIP was transiently induced prior to PR-1 induction. These observations show that Arabidopsis AtSIP plays a role in resistance to pathogenic infection.     

Peroxisomal Acyl-CoA oxidase 4 activity differs between Arabidopsis accessions

In plants, peroxisomes are the primary site of fatty acid β-oxidation. Following substrate activation, fatty acids are oxidized by Acyl-CoA Oxidase (ACX) enzymes. Arabidopsis has six ACX genes, although ACX6 is not expressed. Biochemical characterization has revealed that each ACX enzyme acts on specific chain-length targets, but in a partially overlapping manner, indicating a degree of functional redundancy. Genetic analysis of acx single and double mutants in the Columbia (Col-0) accession revealed only minor phenotypes, but an acx3acx4 double mutant from Wassileskija (Ws) is embryo lethal. In this study, we show that acx3acx4(Col) and acx1acx3acx4(Col) mutants are viable and that enzyme activity in these mutants is significantly reduced on a range of substrates compared to wild type. However, the triple mutant displays only minor defects in seed-storage mobilization, seedling development, and adult growth. Although the triple mutant is defective in the three most active and highly-expressed ACX proteins, increases in ACX2 expression may support partial β-oxidation activity. Comparison of acx mutant alleles in the Col-0 and Ws accessions reveals independent phenotypes; the Ws acx4 mutant uniquely shows increased sensitivity to propionate, whereas the Col-0 acx4 allele has sucrose-dependent growth in the light. To dissect the issues between Col-0 and Ws, we generated mixed background mutants. Although alleles with the Col-0 acx4 mutant were viable, we were unable to isolate an acx3acx4 line using the Ws acx4 allele. Reducing ACX4 expression in several Arabidopsis backgrounds showed a split response, suggesting that the ACX4 gene and/or protein functions differently in Arabidopsis accessions.     

Salicylate accumulation inhibits growth at chilling temperature in Arabidopsis

The growth of Arabidopsis plants in chilling conditions could be related to their levels of salicylic acid (SA). Plants with the SA hydroxylase NahG transgene grew at similar rates to Col-0 wild types at 23 degrees C, and growth of both genotypes was slowed by transfer to 5 degrees C. However, at 5 degrees C, NahG plants displayed relative growth rates about one-third greater than Col-0, so that by 2 months NahG plants were typically 2.7-fold larger. This resulted primarily from greater cell expansion in NahG rosette leaves. Specific leaf areas and leaf area ratios remained similar in both genotypes. Net assimilation rates were similar in both genotypes at 23 degrees C, but higher in NahG at 5 degrees C. Chlorophyll fluorescence measurements revealed no PSII photodamage in chilled leaves of either genotype. Col-0 shoots at 5 degrees C accumulated SA, particularly in glucosylated form. SA in NahG shoots showed similar tendencies at 5 degrees C, but at greatly depleted levels. Catechol was not detected as a metabolite of the NahG transgene product. We also examined growth and SA levels in SA signaling and metabolism mutants at 5 degrees C. The partially SA-insensitive npr1 mutant displayed growth intermediate between NahG and Col-0, while the SA-deficient eds5 mutant behaved like NahG. In contrast, the cpr1 mutant at 5 degrees C accumulated very high levels of SA and its growth was much more inhibited than wild type. At both temperatures, cpr1 was the only SA-responsive genotype in which oxidative damage (measured as thiobarbituric acid-reactive substances) was significantly different from wild type.     

The Pseudomonas syringae type III effector AvrRpm1 induces significant defenses by activating the Arabidopsis nucleotide-binding leucine-rich repeat protein RPS2

Plant disease resistance (R) proteins recognize potential pathogens expressing corresponding avirulence (Avr) proteins through 'gene-for-gene' interactions. RPM1 is an Arabidopsis R-protein that triggers a robust defense response upon recognizing the Pseudomonas syringae effector AvrRpm1. Avr-proteins of phytopathogenic bacteria include type III effector proteins that are often capable of enhancing virulence when not recognized by an R-protein. In rpm1 plants, AvrRpm1 suppresses basal defenses induced by microbe-associated molecular patterns. Here, we show that expression of AvrRpm1 in rpm1 plants induced PR-1, a classical defense marker, and symptoms including chlorosis and necrosis. PR-1 expression and symptoms were reduced in plants with mutations in defense signaling genes ( pad4 , sid2 , npr1 , rar1 , and ndr1 ) and were strongly reduced in rpm1 rps2 plants, indicating that AvrRpm1 elicits defense signaling through the Arabidopsis R-protein, RPS2. Bacteria expressing AvrRpm1 grew more on rpm1 rps2 than on rpm1 plants. Thus, independent of its classical 'gene-for-gene' activation of RPM1, AvrRpm1 also induces functionally relevant defenses that are dependent on RPS2. Finally, AvrRpm1 suppressed host defenses and promoted the growth of type III secretion mutant bacteria equally well in rps2 and RPS2 plants, indicating that virulence activity of over-expressed AvrRpm1 predominates over defenses induced by weak activation of RPS2.     

Abscisic acid deficiency increases defence responses against Myzus persicae in Arabidopsis

Comparison of Arabidopsis thaliana (Arabidopsis) gene expression induced by Myzus persicae (green peach aphid) feeding, aphid saliva infiltration and abscisic acid (ABA) treatment showed a significant positive correlation. In particular, ABA‐regulated genes are over‐represented among genes that are induced by M. persicae saliva infiltration into Arabidopsis leaves. This suggests that the induction of ABA‐related gene expression could be an important component of the Arabidopsis–aphid interaction. Consistent with this hypothesis, M. persicae populations induced ABA production in wild‐type plants. Furthermore, aphid populations were smaller on Arabidopsis aba1‐1 mutants, which cannot synthesize ABA, and showed a significant preference for wild‐type plants compared with the mutant. Total free amino acids, which play an important role in aphid nutrition, were not altered in the aba1‐1 mutant line, but the levels of isoleucine (Ile) and tryptophan (Trp) were differentially affected by aphids in wild‐type and mutant plants. Recently, indole glucosinolates have been shown to promote aphid resistance in Arabidopsis. In this study, 4‐methoxyindol‐3‐ylmethylglucosinolate was more abundant in the aba1‐1 mutant than in wild‐type Arabidopsis, suggesting that the induction of ABA signals that decrease the accumulation of defence compounds may be beneficial for aphids.     

Plant growth-promoting rhizobacteria systemically protect Arabidopsis thaliana against Cucumber mosaic virus by a salicylic acid and NPR1-independent and jasmonic acid-dependent signaling pathway

Arabidopsis thaliana ecotype Columbia plants (Col-0) treated with plant growth-promoting rhizobacteria (PGPR) Serattia marcescens strain 90-166 and Bacillus pumilus strain SE34 had significantly reduced symptom severity by Cucumber mosaic virus (CMV). In some cases, CMV accumulation was also significantly reduced in systemically infected leaves. The signal transduction pathway(s) associated with induced resistance against CMV by strain 90-166 was determined using mutant strains and transgenic and mutant Arabidopsis lines. NahG plants treated with strains 90-166 and SE34 had reduced symptom severity indicating that the resistance did not require salicylic acid (SA). Strain 90-166 naturally produces SA under iron-limited conditions. Col-0 and NahG plants treated with the SA-deficient mutant, 90-166-1441, had significantly reduced CMV symptom severity with reduced virus accumulation in Col-0 plants. Another PGPR mutant, 90-166-2882, caused reduced disease severity in Col-0 and NahG plants. In a time course study, strain 90-166 reduced virus accumulation at 7 but not at 14 and 21 days post-inoculation (dpi) on the non-inoculated leaves of Col-0 plants. NahG and npr1-1 plants treated with strain 90-166 had reduced amounts of virus at 7 and 14 dpi but not at 21 dpi. In contrast, no decrease in CMV accumulation occurred in strain 90-166-treated fad3-2 fad7-2 fad8 plants. These data indicate that the protection of Arabidopsis against CMV by strain 90-166 follows a signaling pathway for virus protection that is independent of SA and NPR1, but dependent on jasmonic acid.     

The plant growth-promoting rhizobacterium Bacillus cereus AR156 induces systemic resistance in Arabidopsis thaliana by simultaneously activating salicylate- and jasmonate/ethylene-dependent signaling pathways

Bacillus cereus AR156 is a plant growth-promoting rhizobacterium that induces resistance against a broad spectrum of pathogens including Pseudomonas syringae pv. tomato DC3000. This study analyzed AR156-induced systemic resistance (ISR) to DC3000 in Arabidopsis ecotype Col-0 plants. Compared with mock-treated plants, AR156-treated ones showed an increase in biomass and reductions in disease severity and pathogen density in the leaves. The defense-related genes PR1, PR2, PR5, and PDF1.2 were concurrently expressed in the leaves of AR156-treated plants, suggesting simultaneous activation of the salicylic acid (SA)- and the jasmonic acid (JA)- and ethylene (ET)-dependent signaling pathways by AR156. The above gene expression was faster and stronger in plants treated with AR156 and inoculated with DC3000 than that in plants only inoculated with DC3000. Moreover, the cellular defense responses hydrogen peroxide accumulation and callose deposition were induced upon challenge inoculation in the leaves of Col-0 plants primed by AR156. Also, pretreatment with AR156 led to a higher level of induced protection against DC3000 in Col-0 than that in the transgenic NahG, the mutant jar1 or etr1, but the protection was absent in the mutant npr1. Therefore, AR156 triggers ISR in Arabidopsis by simultaneously activating the SA- and JA/ET-signaling pathways in an NPR1-dependent manner that leads to an additive effect on the level of induced protection.     

The LeATL6-associated ubiquitin/proteasome system may contribute to fungal elicitor-activated defense response via the jasmonic acid-dependent signaling pathway in tomato

The expression of LeATL6, an ortholog of Arabidopsis ATL6 that encodes a RING-H2 finger protein, was induced in tomato roots treated with a cell wall protein fraction (CWP) elicitor of the biocontrol agent Pythium oligandrum. The LeATL6 protein was expressed as a fusion protein with a maltose-binding protein (MBP) in Escherichia coli, and it catalyzed the transfer of ubiquitin to the MBP moiety on incubation with ubiquitin, the ubiquitin-activating enzyme E1, and the ubiquitin-conjugating enzyme E2; this indicated that LeATL6 represents ubiquitin ligase E3. LeATL6 expression also was induced by elicitor treatment of jail-1 mutant tomato cells in which the jasmonic acid (JA)-mediated signaling pathway was impaired; however, JA-dependent expression of the basic PR-6 and TPI-1 genes that encode proteinase inhibitor II and I, respectively, was not induced in elicitor-treated jail-1 mutants. Furthermore, transient overexpression of LeATL6 under the control of the Cauliflower mosaic virus 35S promoter induced the basic PR6 and TPI-1 expression in wild tomato but not in the jail-1 mutant. In contrast, LeATL6 overexpression did not activate salicylic acid-responsive acidic PR-1 and PR-2 promoters in wild tomato. These results indicated that elicitor-responsive LeATL6 probably regulates JA-dependent basic PR6 and TPI-1 gene expression in tomato. The LeATL6-associated ubiquitin/proteasome system may contribute to elicitor-activated defense responses via a JA-dependent signaling pathway in plants.     

NKS1, Na(+)- and K(+)-sensitive 1, regulates ion homeostasis in an SOS-independent pathway in Arabidopsis

An Arabidopsis thaliana mutant, nks1-1, exhibiting enhanced sensitivity to NaCl was identified in a screen of a T-DNA insertion population in the genetic background of Col-0 gl1sos3-1. Analysis of the genome sequence in the region flanking the T-DNA left border indicated two closely linked mutations in the gene encoded at locus At4g30996. A second allele, nks1-2, was obtained from the Arabidopsis Biological Resource Center. NKS1 mRNA was detected in all parts of wild-type plants but was not detected in plants of either mutant, indicating inactivation by the mutations. Both mutations in NKS1 were associated with increased sensitivity to NaCl and KCl, but not to LiCl or mannitol. NaCl sensitivity was associated with nks1 mutations in Arabidopsis lines expressing either wild type or null alleles of SOS1, SOS2 or SOS3. The NaCl-sensitive phenotype of the nks1-2 mutant was complemented by expression of a full-length NKS1 allele from the CaMV35S promoter. When grown in medium containing NaCl, nks1 mutants accumulated more Na(+) than wild type and K(+)/Na(+) homeostasis was perturbed. It is proposed NKS1, a plant-specific gene encoding a 19kDa endomembrane-localized protein of unknown function, is part of an ion homeostasis regulation pathway that is independent of the SOS pathway.     

Effect of reduced arginine decarboxylase activity on salt tolerance and on polyamine formation during salt stress in Arabidopsis thaliana

Polyamines have been suggested to play an important role in stress protection. However, attempts to determine the function of polyamines have been complicated by the fact that, dependent on the conditions, polyamine contents increase or decrease during stress. To determine the importance of polyamine formation during salt stress, we analysed polyamine contents and salt tolerance in two Arabidopsis thaliana mutants, spe1-1 and spe2-1 (Watson et al. Plant J 13: 231–239, 1998), with reduced activity of arginine decarboxylase (EC 4.1.1.19), an important enzyme in polyamine synthesis. Polyamines accumulated in wild-type plants (Col-0 and Ler-0) that were pre-treated with 100 m M NaCl before transfer to 125 m M NaCl, but not in plants that were directly transferred to 125 m M NaCl without prior treatment with 100 m M NaCl. This shows that polyamine accumulation depends on acclimation to salinity. The salt treatment that induced polyamine accumulation in wild-type plants did not lead to polyamine accumulation in the spe1-1 and spe2-1 mutants. Decreased fresh weight, chlorophyll content and photosynthetic efficiency indicated that the spe1-1 mutant was more severely affected by salt stress than its wild type, Col-0. In the spe2-1 mutant decreased salt tolerance compared to its wild type, Ler-0, became apparent as bleaching under severe salt stress. The present results demonstrate that decreased polyamine formation due to lower arginine decarboxylase activity leads to reduced salt tolerance.     

Supplementary ultraviolet-B irradiation reveals differences in stress responses between Arabidopsis thaliana ecotypes

Irradiation of Arabidopsis thaliana ecotypes C24, Wassilewskija (Ws) and Columbia-0 (Col-0) with supplementary ultraviolet-A+B (UV-A+B) radiation revealed ecotype-specific differences in expression of the gene for the pathogenesis-related protein PR-5. C24 showed an increased expression level of PR-5 (5- and 20-fold higher compared with Col-0 and Ws, respectively). Expression of other molecular markers such as CHS (encoding chalcone synthase), MEB5.2 [encoding a gene strongly up-regulated by ultraviolet-B (UV-B)] and PYROA [encoding a pyridoxine (Vitamin B6) biosynthesis enzyme] only showed slight differences between ecotypes. Oxidative stress during UVA+B exposure was monitored by staining for H2O2. This analysis also revealed important ecotype-specific differences. 'H2O2 hot spots' were found in C24, whereas an even distribution of H2O2 was found in Ws and Col-0. Necrotic lesions also appeared on C24 leaves after prolonged UV-B exposure. There was a reverse correlation between the H2O2 steady-state concentration and the PR-5 gene expression; Ws showed the highest level of H2O2 accumulation but the lowest expression level of the PR-5 gene. Furthermore, application of paraquat on the rosettes led to similar PR-5 expression and H2O2 accumulation patterns as were found after UV-A+B irradiation. The observed ecotypic differences were also reflected in a statistically significant UV-B-dependent decrease in biomass, rosette size and leaf area for Ws, but not for C24 and Col-0. Our results show that a significant ecotype-specific genetic variability in general UV-B responses in Arabidopsis exists. Moreover, the signal transduction or gene regulation pathway for PR-5 differs from the other molecular markers used in this study.     

Tocopherols modulate leaf vein arrangement and composition without impacting photosynthesis

Photosynthetica - Growth of the tocopherol-deficient vte1 mutant and Col-0 wild type of Arabidopsis thaliana in a sunlit glasshouse revealed both similarities and differences between genotypes....     

Evaluation of the use of the tobacco PR-1a promoter to monitor defense gene expression by the luciferase bioluminescence reporter system

Because of their marked responsiveness to induction signals, genes encoding pathogenesis-related proteins are used as markers to monitor defense gene expression in plants. To develop a non-invasive bioluminescence reporter assay system, we tested acidic PR-1 gene promoters from tobacco and Arabidopsis. These two promoters share common regulatory elements and are believed to show similar responsiveness to various stimuli but the results of transient expression assays by microprojectile bombardment of various plant cells and npr1 mutant Arabidopsis suggest that the tobacco PR-1a promoter is superior to its Arabidopsis counterpart in terms of responsiveness to salicylic acid treatment. Transgenic Arabidopsis seedlings harboring the tobacco PR-1a promoter fused to firefly luciferase showed marked induction in response to treatment with chemicals that induce defense gene expression in plants. These results suggest that the tobacco PR-1a promoter is applicable in monitoring defense-gene expression in various plant species.