FEBS Letters
Volume 582, Issue 4 , Pages 473-478, 20 February 2008

Salicylic acid production in response to biotic and abiotic stress depends on isochorismate in Nicotiana benthamiana

Edited by Michael R. Sussman

Department of Biology, University of Fribourg, Ch. du Musée 10, 1700 Fribourg, Switzerland

Received 19 November 2007; received in revised form 9 December 2007; accepted 21 December 2007. published online 15 January 2008.

Article Outline

Abstract 

Salicylic acid (SA) is an important signal involved in the activation of defence responses against abiotic and biotic stress. In tobacco, benzoic acid or glucosyl benzoate were proposed to be precursors of SA. This is in sharp contrast with studies in Arabidopsis thaliana, where SA derives from isochorismate. We have determined the importance of isochorismate for SA biosynthesis in Nicotiana benthamiana using virus-induced gene silencing of the isochorismate synthase (ICS) gene. Plants with silenced ICS expression do not accumulate SA after exposure to UV or to pathogen stress. Plants with silenced ICS expression also exhibit strongly decreased levels of phylloquinone, a product of isochorismate. Our data provide evidence for an isochorismate-derived synthesis of SA in N. benthamiana

Keywords: Isochorismate synthase, Pathogen stress, Phylloquinone, Salicylic acid, UV stress, Nicotiana

 

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1. Introduction 

The phytohormone salicylic acid (SA) has been associated with various physiological processes including flowering, thermogenesis, stomatal closure and responses to biotic and abiotic stress (reviewed in [1]). It has been most intensely studied in relation to the response of plants to pathogens where it acts as a signalling molecule in the activation of defence responses against microbial pathogens (review in [2]). The importance of SA in the induction of defence responses has been well documented by many studies with mutants and transgenic plants that exhibit altered levels of SA (reviewed in [3]). SA is integrated in a signalling network together with two other phytohormones, jasmonic acid and ethylene. This network exhibits the typical hallmarks found in integrated systems including negative and positive crosstalk interactions and specific responses to combinations of stimuli [4].

Based on previous studies, SA is synthesized from phenylalanine and, depending on the plant species, either from free benzoic acid, benzoyl glucose or o-coumaric acid as direct precursors [5]. Most of these findings were supported by results obtained in tobacco, rice, cucumber and potato that showed an accumulation of radiolabelled 14C-SA in tissues incubated with 14C-phenylalanine [5]. Suppression of the expression of one phenylalanine ammonia-lyase (PAL) gene in transgenic tobacco leads to a decrease in SA accumulation [6]. The identification of SA-deficient Arabidopsis thaliana mutant sid2 [7] and the subsequent localization of this mutation in a gene encoding a functional isochorismate synthase (ICS) shed some new light on the biosynthetic pathway of SA [8], [9]. Only minimal levels of SA could be detected in sid2 mutants after infection, UV or ozone exposure [7] providing a strong support for isochorismate as a precursor of stress-induced SA accumulation in A. thaliana. The biosynthetic pathway for SA in A. thaliana is therefore related to that described in bacteria where SA is synthesized from chorismate via the rate-limiting enzyme ICS and isochorismate pyruvate lyase (IPL) (reviewed in [10]). An increase in SA levels and heightened resistance to tobacco mosaic virus was shown to take place in transgenic tobacco plants transformed with bacterial ICS and IPL, indicating that this pathway can operate in plants [11]. The discrepancy in the biosynthetic pathway of SA proposed for A. thaliana and other plants was often assumed to reflect differences among species. To clarify the biosynthesis of SA in tobacco we have cloned the ICS gene from Nicotiana benthamiana (NbICS) and used virus-induced gene silencing (VIGS) to suppress the expression of NbICS in N. benthamiana. This led to an effective disruption of the accumulation of SA and of phylloquinone (PHQ) in plants undergoing a biotic and abiotic stress indicating that the ICS enzyme is also required for SA biosynthesis in N. benthamiana.

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2. Materials and methods 

2.1. Plant growth 

N. benthamiana plants were grown in pots in potting soil at 25°C in a growth chamber under 16h light/8h dark cycle.

2.2. Plasmid constructions 

pTRV1 and pTRV2 VIGS vectors and derivatives have been described previously [12]. pTRV2-attB2-NbICS-attB1 harboured a 700bp fragment of N. benthamiana ICS cDNA containing the attB1 and attB2 sequences and was obtained by polymerase chain reaction (PCR) using the primers 5′-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTG CTG CAA CTA TTG CAT GGG-3′ and 5′-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CCC ACA AAC TGC TGG AGT AGG-3′. This NbICS PCR product was recombined into pDONR-207 (Invitrogen, Basel, Switzerland) containing the attP1 and attP2 recombination sites using the BP CLONASE, the pTRV2-attR1-attR2 destination vector and the LR CLONASE. The product pTRV2-attB2-NbICS-attB1 was transformed into DH10B cells and selected on kanamycin-containing LB plates. pTRV2-attB2-AtPDS-attB1 contained a 442bp fragment corresponding to the eleventh intron of the A. thaliana phytoene desaturase gene (PDS) (At4g14210) was amplified from genomic DNA with the primers: 5′-CAC CTA TAG AAT TCA AAG GTA CTT TGA TTG GTC-3′ and 5′-TAT AAA GCT TAG CTA TCT GGA GGAAGA C-3′. This PCR product was cloned into the pENTR vector using the TOPO cloning kit (Invitrogen) and pTRV2-attR1-attR2 as destination vector and the LR CLONASE. The final plasmid pTRV2-attB2-AtPDS-attB1 was transformed into DH10B cells and selected on kanamycin-containing LB plates.

For the VIGS assay, pTRV1 or pTRV2 and their derivatives were introduced into Agrobacterium strain GV3101 by electroporation. A 3-ml culture was grown overnight at 28°C in 50mg/l rifampicin and 50mg/l kanamycin. Agrobacterium cells were harvested and resuspended in infiltration media (10mM MgCl2, 10mM MES, 150μM acetosyringone), adjusted to an OD of 0.4 and left at room temperature for 2h. For VIGS, transformed Agrobacterium was injected into the two primary leaves of a plant at the four-leaves stage using a needle-less 1ml syringe. Plants infiltrated with the co-culture of Agrobacterium containing pTRV1 and pTRV2-attB2-NbICS-attB1 plasmids were named TRV-NbICS plants. Plants infiltrated with the co-culture of Agrobacterium containing pTRV1 and pTRV2-attB2-AtPDS-attB1 plasmids were named TRV-INTRON plants. The effectiveness of VIGS was tested using the PDS gene as described in [13].

2.3. Southern blot 

Genomic DNA of leaves was isolated using cetyldimethylethylammonium bromide and was digested with BglII and HindIII, separated and blotted according to Sambrook et al. [14] A PCR-amplified NbICS fragment was radiolabelled with P32-dCTP using the prime-a-gene-labelling kit (Promega, Basel, Switzerland) was used as the probe for hybridization.

2.4. Race PCR 

The full-length NbICS cDNA sequence was isolated using a RLM-RACE cDNA amplification kit (Ambion, Austin, USA), following the manufacturer’s instructions. Degenerated primers were first designed based on an alignment of the three ICS genes of A. thaliana, Catharanthus roseus and Lycopersicon pennelii. Using these primers a sequence of NbICS was obtained corresponding to that of NtICS. Other primers were then designed using the NbICS sequence to perform the RACE. For the 5′cDNA-end amplification, the primers used were 5′ RACE inner: 5′-ACG GAG GAA ACC TGC CAT ATT G-3′ and 5′ RACE outer: 5′-TGC GAG TAG ATG TGA ACG AGA T-3′. For the 3′cDNA-end amplification the primers used were 3′ RACE outer: 5′-TTG CTC GTA GCT CCA GAG TTG T-3′ and 3′ RACE inner: 5′-CCA GTC AGC GGC ATT CAT T-3′.

2.5. RNA isolation and semi-quantitative RT-PCR analysis 

Total RNA was extracted from control, silenced and non-silenced N. benthamiana plants (two leaves per plant) by using the phenol–guanidine isothiocyanate procedure (Trizol; Invitrogen). First strand cDNA was synthesized using 1μg of total RNA, oligo d(T) primer and omniscript reverse transcriptase (Qiagen, Hombrechtikon, Switzerland). Fragments of ICS cDNAs from N. benthamiana were amplified by PCR from 2-μl aliquots of the cDNA reaction mixture with primers: 5′-ATT TCA TGG TCC CTC AGG TTG-3′ and 5′-TTC CTC GGT CAA ACA TTT CG-3′. As a control, fragments of EF1α were amplified using primers: 5′-TGG TGT CCT CAA GCC TGG TAT GGT TGT-3′ and 5′-ACG CTT GAG ATC CTT AAC CGC AAC ATT CTT-3′. Aliquots of 10μl were taken every 10 PCR cycles until 50 cycles.

2.6. SA analysis 

Leaf material of about 400mg was harvested on ice and SA was extracted and quantified as previously described [15].

2.7. UVc light exposure 

Plants were exposed to UVc light (254nm, 16W) during 1h at a 50cm distance from the lamp (CAMAG, Muttenz, Switzerland) to induce the production of SA. SA was measured at the end of the treatment and three days later.

2.8. Inoculation with Pseudomonas syringae pv. tomato (Pto) DC3000 

Pto DC3000 was plated onto fresh KB medium [16] with the appropriate antibiotics. After growth for 24h at 25°C, 10ml of 10mm MgCl2 were added to the plate. Ten minutes later, the bacterial suspension was washed out of the plates with a pipette, the samples adjusted to an OD600=0.05 and Silwet L-77 was added to a final concentration of 200μll−1. Several 6μl droplets of the bacterial suspension were deposited on each leaf.

2.9. PHQ analysis 

Ground leaves (500mg) were mixed with two volumes of n-heptane at 20°C overnight. After solvent evaporation, extracts were resuspended in 300μl MeOH for HPLC analysis using a Waters 2695 HPLC system (Milford, MA, USA) equipped with a photodiode array detector coupled to a Bruker Daltonics Esquire HCT ESI/MS spectrometer (Bremen, Germany). Separation was performed on a Macherey-Nagel (Düren, Germany) Nucleodur C18 pyramid 5μ column (125×3mm i.d). Gradient elution was performed using a water MeOH gradient starting at 90% of MeOH for 4min, to reach 100% in 10min, MeOH was then maintained at 100% for another 20min. The flow rate was set to 0.2mlmin−1, column temperature at 30°C and detection at 274nm. For negative ionization mode, the pressure of the nebulizer (N2) was set at 50psi, dry gas flow at 9mlmin−1 and the temperature of the drying gas (N2) at 32°C. The voltage of the skimmer lens and the entrance lens in the ion source were automatically optimized by direct inlet of a solution of standard PHQ (Sigma–Aldrich, Buchs, Switzerland) at a concentration of 0.001mgml−1 in MeOH. PHQ was detected on the basis of UV absorption and the m/z of the deprotonated ion [M−H]− m/z 449 in comparison with the standard solution.

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3. Results 

3.1. NbICS sequence analysis 

In order to clone the N. benthamiana ICS gene, total RNA was isolated from UVc exposed leaves and reverse-transcribed into cDNA. A full-length cDNA designed as NbICSwas isolated using RACE and a sequence of 1296bp long was obtained (available under genBank accession number EU257505). It encodes a protein of 431 amino acids which contains a chorismate-binding domain and a putative plastid-targeting sequence at the N-terminal end. This part of the protein showed 96%, 89%, 87%, 57% and 60% identity with the ICS proteins of N. tabaccum (NtICS), Capsicum annum (CaICS), C. roseus (CaICS) and A. thaliana (AtICS1 and AtICS2), respectively. The chorismate-binding domain is almost identical for all the ICS considered here (Fig. 1A). Southern blot analysis of genomic DNA of N. benthamiana digested with BglII and HindIII revealed two distinct bands indicating the presence of two copies of the ICS gene in the amphidiploı¨d N. benthamiana genome (Fig. 1B).

  • View full-size image.
  • Fig. 1. 

    (A) Clustal alignment of amino acids translated from cDNAs of selected isochorismate synthases. CaICS, Capsicum annuum (AY743431); NtICS, Nicotiana tabacum (AB182580); CrICS, Catharantus roseus (CAA06837); AtICS1, A.thaliana (AY056055); AtICS2, A. thaliana (AAF27094). Color codes: red: identity >90%; blue: >50%, the chorismate-binding domain is shaded in grey. (B) Southern blot analysis of genomic DNA extracted from N. benthamiana using a probe that includes parts of the chorismate-binding domain.

3.2. VIGS of NbICS 

VIGS was performed on N. benthamiana plants for transient suppression of NbICS transcripts. A DNA fragment of 700bp from the coding region of NbICS was cloned into a tobacco rattle virus (TRV)-based vector system. Inoculation of N. benthamiana with both vectors leads to the silencing of the transcripts of the endogenous ICS gene. These plants were referred to as TRV-NbICS. A DNA fragment corresponding to an intron of the PDS gene from A. thaliana was isolated and cloned into the VIGS vectors as a control for VIGS in N. benthamiana. Such plants were termed TRV-INTRON plants. To verify the efficiency of the gene silencing mediated by VIGS, semi-quantitative RT-PCR was performed, and the level of NbICS transcripts in WT, TRV-INTRON and TRV-NbICS plants was determined (Fig. 2). The amount of transcripts of the housekeeping gene EF1α was used to estimate if the same quantity of cDNA of each plant had been used.

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  • Fig. 2. 

    RT-PCR analysis showing the effect of VIGS on transcription of NbICS and EF1α (from N. benthamiana). The first strand cDNA used for PCR was generated from total RNA isolated from upper leaves of WT, TRV-INTRON and TRV-ICS plants (the experiment has been repeated three times, one typical result is presented).

WT and TRV-INTRON plants showed the same quantity of ICS transcript the expression of which is lower than the EF1α transcripts. In TRV-NbICS plants, a band corresponding to the ICS transcripts could be distinguished after 50 PCR cycles in comparison to 30 cycles in WT and TRV-INTRONplants. This difference indicates a reduction in ICS close to an almost complete absence of ICS transcript in the TRV-NbICS plants and is a demonstration of effective silencing by VIGS.

3.3. Effect of ICS silencing on the content of PHQ 

PHQ acts as a photosystem I (PSI) cofactor and is an essential component of the photosynthetic electron transfer chain. In Arabidopsis, ICS was shown to be required for synthesis of PHQ [17] and we verified whether the same process occured in N. benthamiana. PHQ eluted at a retention time (RT) of 21.6min (Fig. 3) when separated by HPLC. Mass spectrometry analysis of this fraction eluting at RT=21.6min presented a mass-to-charge ratio (m/z) of 449.2. In the chromatograms of WT and TRV-INTRON leaves, a peak was observed, eluting at 21.6. This fraction with a m/z ratio of 449.4 (respectively, 449.3) confirms the presence of PHQ in these plants. At the same RT, the chromatogrammes obtained from extracts of TRV-NbICS leaves showed a complete absence of PHQ. No specific peaks corresponding to PHQ could be identified in the mass spectrometry analysis of these plant samples. TRV-NbICS leaves were a paler shade of green than the two other plants and this phenotypic difference might be explained by the quantitative variation in the content of PHQ.

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  • Fig. 3. 

    HPLC elution profiles of leaf extracts of WT, TRV-INTRON and TRV-NbICS as well as a standard PHQ solution. Mass spectra of the peaks at the position of PHQ (dashed rectangle) are added on the right for each sample.

3.4. Effect of ICS suppression on SA accumulated after UVc light exposure 

UVc photons are highly energetic and trigger biochemical responses that overlap in part with those induced by necrotizing pathogens [18]. For instance, exposure of tobacco leaves to UVc light induces the accumulation of SA. We have used UVc irradiation as an abiotic stress to increase SA levels in VIGS-plants. Without UVc exposure, WT, TRV-INTRON and TRV-NbICS plants all presented a low level of SA (Fig. 4A). Three days after UVc light treatment, the level of total SA in WT and TRV-INTRON plants had increased more than 10-fold, in accordance with values reported by Yalpani et al. [18] in Nicotiana tabacum L. cv. Xanthi nc. However, the absolute amount of SA measured in N. benthamiana was lower than in N. tabacum, that might indicate a weaker ability to produce SA in N. benthamiana or it might reflect a weaker stimulation by the UVc treatment than in the study cited above. Exposure of TRV-NbICS plants to UVc led to only a fourfold increase in SA, indicating a deficiency in SA production. In all experiments, a very low level of free compared to bound SA was observed (data not shown).

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  • Fig. 4. 

    Total SA accumulation in leaves (1–3) of WT, TRV- INTRON and TRV-NbICS plants: (A) after UVc light exposure for 1h (n=5; ±S.D.), (B) after mock-inoculation or inoculation with Ps DC3000 (black) (n=5; ±S.D.).

3.5. Effect of ICS suppression on SA accumulated after P. syringae pv. tomato (Pto) DC3000 infection 

We have infiltrated the bacteria Pto DC3000 into N. benthamiana leaves as a biotic stress to induce SA (Fig. 4B). Two days after inoculation, TRV-NbICS plants failed to accumulate SA, in contrast to WT and TRV-INTRON plants where the level of SA strongly increased. No accumulation of SA could be detected in WT, TRV-INTRON, TRV-NbICS plants after a mock infiltration (Fig. 4B).

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4. Discussion 

In the earlier studies based to a large extend on radiolabelling, the precursor for SA was postulated to derive from phenylalanine [5]. Recently, genetic and biochemical evidence in A. thaliana demonstrated that SA induced by pathogens or abiotic stress derives from the conversion of isochorismate by ICS [7], [9]. The sequence of several genes encoding ICS has been described in C. roseus [19], Capsicum annuum (accession number AY743431), tobacco [20] as well as in the bacteria Pseudomonas aeruginosa and Escherichia coli [21]. In this article, we describe the sequence of an ICS in N. benthamiana. The chorismate-binding domain shows a strong conservation with other published sequences and was used as a target for VIGS. Suppression of ICS expression by VIGS in N. benthamiana led to substantial decrease in SA accumulation after UVc treatment and to an almost complete lack of SA increase after inoculation with Pto DC3000. These results support a biosynthetic pathway for SA derived from isochorismate and depending on ICS like that described in Arabidopsis [9]. PHQ derives from isochorismate via ICS [17], [22] and did not accumulate in VIGS-plant providing additional experimental support for an efficient suppression of ICS.

After UVc exposure, ICS-suppressed plants still accumulated a residual level of SA above the level observed in untreated plants. An incomplete silencing of NbICS transcripts is unlikely but cannot be ruled out, although VIGS resulted in a strong suppression of ICS (Fig. 2). Alternatively, the residual SA could be explained by the action of another ICS. However, gene silencing was targeted at the conserved chorismate-binding domain (Fig. 1A), so we might reasonably exclude this possibility. The residual SA level could be produced from phenylalanine by PAL [23], [24]. While the PAL pathway did not compensate the lack of SA due to the suppression of NbICS in plants undergoing biotic stress (Fig. 4) the possibility that some SA accumulation is derived from the PAL pathway in response to abiotic stress in N. benthamiana remains a possible explanation.

Our results supporting isochorismate-derived SA production in N. benthamiana are in contrast with several other reports in tobacco indicating an exclusive biosynthetic route via phenylalanine in tobacco [20], [23], [24]. These studies demonstrated the conversion of benzoic acid or benzoyl glucose to SA using radiolabelled precursors. While such results certainly reflect a possible source of SA, they do not exclude another pathway of SA production, and are likely to represent only a fraction of total SA produced. A comparison of the specific radioactivities between samples from control and SA-inducing treatments after radiolabelling with a precursor would be needed to answer this question. For example, a decrease in specific radioactivity in the treated sample compared to the control reflects an increase in unlabelled SA and the existence of another source of SA besides the radiolabelled precursor. Unfortunately, such data are not available for SA biosynthesis in plants. Previous studies on tobacco epigenetically suppressed in PAL expression showed a decrease in free SA, associated with a decrease in systemic acquired resistance to tobacco mosaic virus [6]. However, the downregulation of PAL may influence the flux through the isochorismate pathways, as discussed in [25]. In fact, these authors observed an overaccumulation of chlorogenic acid but not of SA in transgenic tobacco over-expressing PAL and there was no clear correlation between the expression of the PAL gene and the accumulation of SA [25]. In N. tabacum plants fumigated with ozone, SA was proposed to be made exclusively via the PAL pathway [20]. This conclusion was based on data showing a weak induction of ICS gene expression and undetectable ICS enzyme activity. However, these observations do not allow a definite conclusion regarding the origin of SA.

Here, we have shown that suppression of ICS expression by VIGS leads to a strong suppression of SA accumulation in N. benthamiana in response to abiotic and pathogen stress demonstrating that the isochorismate pathway for SA biosynthesis is also active at least in one Nicotiana species.

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Acknowledgements 

Support from the Swiss National Science Foundation (Grant no. 3100A0-104224 to J.P.M.) is gratefully acknowledged. Olivier Lamotte and Henk-jan Schoonbeek are thanked for critical reading of the manuscript.

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References 

  1. Raskin I. Role of salicylic-acid in plants. Ann. Rev. Plant Physiol. Plant Molec. Biol. 1992;43:439–463
  2. Nawrath C, Métraux JP, Genoud T. Chemical signals in plant resistance: salicylic acid. In:  Tuzun S,  Bent E editor. Multigenic and Induced Systemic Resistance in Plants. Dordrecht: Kluwer; 2005;p. 143–165
  3. Métraux JP, Durner J. The role of salicylic acid and nitric oxide in programmed cell death and induced resistance. In:  Sandermann H editors. Ecological Studies. vol. 170:Heidelberg, Berlin: Springer-Verlag; 2004;p. 111–150
  4. Genoud T, Santa Cruz MBT, Métraux JP. Numeric simulation of plant signaling networks. Plant Physiol. 2001;126:1430–1437
  5. Garcion C, Métraux JP. Salicylic acid: biosynthesis, metabolism and signal transduction. In:  Hedden P,  Thomas S editor. Plant Hormone Signaling. vol. 24:Oxford: Blackwell Publishing Ltd; 2006;p. 229–255
  6. Pallas JA, Paiva NL, Lamb C, Dixon RA. Tobacco plants epigenetically suppressed in phenylalanine ammonia-lyase expression do not develop systemic acquired resistance in response to infection by tobacco mosaic virus. Plant J. 1996;10:281–293
  7. Nawrath C, Métraux JP. Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation. Plant Cell. 1999;11:1393–1404
  8. Strawn MA, Marr SK, Inoue K, Inada N, Zubieta C, Wildermuth MC. Arabidopsis isochorismate synthase functional in pathogen-induced salicylate biosynthesis exhibits properties consistent with a role in diverse stress responses. J. Biol. Chem. 2007;282:5919–5933
  9. Wildermuth MC, Dewdney J, Wu G, Ausubel FM. Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature. 2001;414:562–565
  10. Verberne MC, Budi Muljono RA, Verpoorte R. Salicylic acid biosynthesis. In:  Libbenga K,  Hall M,  Hooykaas PJJ editor. Biochemistry and Molecular Biology of Plant Hormones. vol. 33:London: Elsevier; 1999;p. 295–312
  11. Verberne MC, Verpoorte R, Bol JF, Mercado-Blanco J, Linthorst HJM. Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistance. Nature Biotech. 2000;18:779–783
  12. Liu YL, Schiff M, Dinesh-Kumar SP. Virus-induced gene silencing in tomato. Plant J. 2002;31:777–786
  13. Liu Y, Schiff M, Marathe R, Dinesh-Kumar SP. Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J. 2002;30:415–429
  14. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1989;
  15. Meuwly P, Métraux JP. Ortho-anisic acid as internal standard for the simultaneous quantitation of salicylic-acid and its putative biosynthetic precursors in cucumber leaves. Anal. Biochem. 1993;214:500–505
  16. King EO, Ward MK, Raney DE. Two simple media for the demonstration of pyocyanin and fluorescin. J. Lab. Clin. Med. 1954;44:301–307
  17. Gross J, et al. A plant locus essential for phylloquinone (vitamin K1) biosynthesis originated from a fusion of four eubacterial genes. J. Biol. Chem. 2006;281:17189–17196
  18. Yalpani N, Enyedi AJ, Leon J, Raskin I. Ultraviolet-light and ozone stimulate accumulation of salicylic-acid, pathogenesis-related proteins and virus-resistance in tobacco. Planta. 1994;193:372–376
  19. van Tegelen LJ, Moreno PR, Croes AF, Verpoorte R, Wullems GJ. Purification and cDNA cloning of isochorismate synthase from elicited cell cultures of Catharanthus roseus. Plant Physiol. 1999;119:705–712
  20. Ogawa D, et al. Salicylic acid accumulation under O3 exposure is regulated by ethylene in tobacco plants. Plant Cell Physiol. 2005;46:1062–1072
  21. Serino L, Reimmann C, Baur H, Beyeler M, Visca P, Haas D. Structural genes for salicylate biosynthesis from chorismate in Pseudomonas aeruginosa. Mol. Gen. Genet. 1995;249:217–228
  22. Verberne MC, Sansuk K, Bol JF, Linthorst HJ, Verpoorte R. Vitamin K1 accumulation in tobacco plants overexpressing bacterial genes involved in the biosynthesis of salicylic acid. J. Biotechnol. 2007;128:72–79
  23. Chong J, Pierrel MA, Atanassova R, Werck-Reithhart D, Fritig B, Saindrenan P. Free and conjugated benzoic acid in tobacco plants and cell cultures induced accumulation upon elicitation of defense responses and role as salicylic acid precursors. Plant Physiol. 2001;125:318–328
  24. Leon J, Yalpani N, Raskin I, Lawton MA. Induction of benzoic acid 2-hydroxylase in virus-inoculated tobacco plant. Plant Physiol. 1993;103:323–328
  25. Shadle GL, Wesley SV, Korth KL, Chen F, Lamb C, Dixon RA. Phenylpropanoid compounds and disease resistance in transgenic tobacco with altered expression of l-phenylalanine ammonia-lyase. Phytochemistry. 2003;64:153–161

PII: S0014-5793(08)00018-5

doi:10.1016/j.febslet.2007.12.039

FEBS Letters
Volume 582, Issue 4 , Pages 473-478, 20 February 2008

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