Pathogen‐induced expression of plant ATP:citrate lyase¹

Abstract

Hypersensitive response (HR) in plants is generally characterized by a rapid and localized cell death, cell wall strengthening and synthesis of phytoalexins and PR proteins [1]. In order to understand the molecular and cellular defense mechanisms better during HR that results from the interaction between plant pathogen and its non-host, we inoculated hot pepper (Capsicum annuum cv. Pukang) leaves with soybean pustule pathogen, Xanthomonas campestris pv. glycines8ra. A pool of genes induced or repressed by infection of the pathogen has been isolated by using a di¡erential displaypolymerase chain reaction (DD-PCR) technique (Yi et al., unpublished data). Approximately one half of the isolated genes had no sequence similarity in existing databases and the major portion of identi¢ed DNA fragments consisted of enzymes involved in primary or secondary metabolic pathways. One of the fragments was found to have signi¢cant sequence homology with ATP:citrate lyase (ACL) known from diverse organisms including alga, fungi, and mammals.ACL is known to catalyze the following reaction: citrate+ CoA+ATPCoxaloacetate+ADP+Pi+acetyl-CoA. In the cytosol of animals, oleaginous yeast, and fungi, acetyl-CoA produced by the action of ACL is the major precursor for fatty acid and sterol biosynthesis [2]. However, in plants, the biosynthetic origin of cytosolic acetyl-CoA that is needed for biosynthesis of phytochemicals is not established. In sweet potato, ACL activity has been observed to increase 10-fold in root tissues response to infection by the fungal pathogen, Ceratocystis ¢mbriata, and to correspond to the accumulation of phytoalexin ipomeamarone [3], but the molecular characterization of a plant ACL has not yet been reported.In order to isolate a full-length cDNA clone encoding ACL, a cDNA library was constructed from hot pepper leaves inoculated with X. campestris pv. glycines8ra. After screening of the cDNA library by using a cDNA fragment of ACL produced by DD-PCR as a probe, a cDNA clone with an open reading frame containing 608 amino acids was ¢nally isolated and designated as Ca-ACL1 (GenBank accession number: AF290958). The deduced amino acid of Ca-ACL1 gene has signi¢cant sequence homology with the C-terminal part of known animal ACLs. Calculated molecular mass of hot pepper ACL polypeptide was 66 kDa, which is only half of the animal ACLs. The ACL catalytic center, GHAGA, and the histidine residue that is autophosphorylated by ATP during catalysis are highly conserved in Ca-ACL1, but none of the three additional phosphorylation sites (Tyr446, Ser450, Ser454) that are involved in the regulation of rat ACL activity exists [2].To investigate the expression kinetics of the Ca-ACL1 transcripts during inoculation with X. campestris pv. glycines8ra, total RNA was isolated from hot pepper leaves 0, 4, 8, and 12 h after inoculation, and Northern blot analysis was performed by using 32 P-labeled Ca-ACL1 as a probe. HR cell death on pepper leaves appeared at approximately 15 h post-in¢ltra-tion, whereas no visible symptom was de...

Molecular Characterization of a Heteromeric ATP-Citrate Lyase That Generates Cytosolic Acetyl-Coenzyme A in Arabidopsis,

“…More recently, EST cDNA clones that share sequence similarity with the animal ACL have further supported this supposition (Wurtele et al, 1998;Suh et al, 2001). The data presented herein formally establish the occurrence of ACL in plants.…”

“…In transgenic maize cells, an ACL-like mRNA is one of many mRNAs induced by ectopic expression of the transcription factors C1/R and P, which induce flavonoid synthesis (Bruce et al, 2000). Fungal infection of peppers (Capsicum annuum) induces the accumulation of an ACLB homolog (Suh et al, 2001), possibly associated with the production of the sesquiterpene phytoalexin, capsidiol (Back et al, 1998).…”

CaAlaAT1 catalyzes the alanine: 2-oxoglutarate aminotransferase reaction during the resistance response against Tobacco mosaic virus in hot pepper

“…The induction of CaAlaAT1 in defense response would be related to the abnormal condition of hot pepper in need of much energy by any stress (Urban et al 1999;Suh et al 2001). Pyruvate can be utilized as a carbon and energy source (de Vos et al 1998).…”

“…Although the energy-yielding steps of glycolysis generate low amount of ATP while reducing NAD + to NADH, much more energy can be extracted aerobically by means of the citric acid cycle and the electron transport chain. The entry point to this oxidative pathway is acetyl coenzyme A (acetyl CoA), which is needed for biosynthesis of phytochemicals, important components in defense (Suh et al 2001). When infected by any pathogen, the plant needs high amount of ATP from pyruvate.…”

Implication of a pepper h-type thioredoxin in type I- and II-nonhost resistance to Xanthomonas axonopodis

“…For the pathogen challenge, the incompatible bacterial pathogen Xanthomonas axonopodis pv. glycines 8ra and its type III-secretion-system null mutant 8-13 (Xag; of OD 600 = 0.4 in 10 mM MgCl 2 ) were pressure-infiltrated into pepper leaves with a needle-less syringe, as described previously (Park and Hwang 1999;Suh et al 2001). For the salicylic acid (SA) treatment, 5 mM of SA was applied by spraying whole plants.…”