Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase

Abstract

SummarySalicylic acid (SA) plays a central role as a signalling molecule involved in plant defense against microbial attack. Genetic manipulation of SA biosynthesis may therefore help to generate plants that are more disease-resistant. By fusing the two bacterial genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, respectively, we have engineered a novel hybrid enzyme with salicylate synthase (SAS) activity. The pchB-A fusion was expressed in Arabidopsis thaliana under the control of the constitutive cauli¯ower mosaic virus (CaMV) 35S promoter, with targeting of the gene product either to the cytosol (c-SAS plants) or to the chloroplast (p-SAS plants). In p-SAS plants, the amount of free and conjugated SA was increased more than 20-fold above wild type (WT) level, indicating that SAS is functional in Arabidopsis. P-SAS plants showed a strongly dwarfed phenotype and produced very few seeds. Dwar®sm could be caused by the high SA levels per se or, perhaps more likely, by a depletion of the chorismate or isochorismate pools of the chloroplast. Targeting of SAS to the cytosol caused a slight increase in free SA and a signi®cant threefold increase in conjugated SA, probably re¯ecting limited chorismate availability in this compartment. Although this modest increase in total SA content did not strongly induce the resistance marker PR-1, it resulted nevertheless in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c-SAS lines was paralleled with reduced seed production. Taken together, these results illustrate that SAS is a potent tool for the manipulation of SA levels in plants.

Selective Interaction Between Chloroplast β-ATPase and TGB1L88 Retards Severe Symptoms Caused by Alternanthera mosaic virus Infection

“…Interestingly, proteolytic fragments of γ-ATPase functioned to induce salicylic acid (SA), jasmonic acid (JA) and ethylene causing defense response against insect attack (Schmelz et al, 2006, 2007). It is also notable that SA and JA are formed from amino acid biosynthetic precursors generated primarily in the chloroplast (Mauch et al, 2001; Schmid and Amrhein, 1995; Wasternack et al, 2006), which may suggest that disabling chloroplast function impairs host defense signaling (Dardick, 2007). Except for these recent reports and our own data we were unable to find information linking chloroplast ATPase and virus infection.…”

Arabidopsis Isochorismate Synthase Functional in Pathogen-induced Salicylate Biosynthesis Exhibits Properties Consistent with a Role in Diverse Stress Responses

“…38). Furthermore, constitutive expression of a PchBA SAS fusion protein in Arabidopsis resulted both in a Ͼ20-fold increase in SA levels and in severe dwarfism/infertility (20). The severity of the dwarfism/infertility phenotype compared with mutants constitutively overexpressing SA is most likely due to the channeling of chorismate to SA at the expense of other essential chorismate-and IC-derived products (e.g.…”

“…First, studies with transgenic tobacco plants overexpressing bacterial monofunctional ICS and IPL enzymes targeted to either the plastid or cytosol singly or in combination found that only transgenic plants in which both ICS and IPL were targeted to the plastid exhibited an SA overexpression functional phenotype: SAG levels similar to local tobacco mosaic virus-infected leaves, constitutive expression of pathogenesis-related genes, and a reduction in tobacco mosaic virus-induced lesion size (56). Second, in Arabidopsis, an SA synthase PchBA fusion protein was constitutively expressed in either the plastid or the cytosol (20). Here too, a true SA overexpression phenotype (SAG ϳ20 g/g fresh weight, PR1 expression) was only observed when the fusion protein was targeted to the plastid, implying that SA synthesis occurs in the plastid in Arabidopsis.…”

“…chorismate is synthesized in the plastid and largely localized to the plastid (1,20,21); however, experimental evidence supporting these predictions has been lacking. Past biochemical characterizations of plant ICS enzymes utilized ICS (partially) purified from elicited cell suspension cultures of Galium mollugo L (22,23), Rubia tinctorum (24), or Catharanthus roseus (19), because these systems provided sufficient activity, protein, and product.…”

Nuclear localization of NPR1 is required for regulation of salicylate tolerance, isochorismate synthase 1 expression and salicylate accumulation in Arabidopsis

“…This pathway contains two reactions, which are catalyzed by isochorismate synthase 1 (ICS1) and isochorismate pyruvate lyase (IPL), respectively (Serino et al, 1995). Mutants that lack ICS1 are deficient in activation of SAR (Nawrath and M etraux, 1999;Wildermuth et al, 2001), whereas expression of bacterial ICS and IPL in plants causes SA accumulation and constitutive SAR (Verberne et al, 2000;Mauch et al, 2001). Exogenous application of SA also induces SAR (White, 1979).…”