Nitrate Efflux Is an Essential Component of the Cryptogein Signaling Pathway Leading to Defense Responses and Hypersensitive Cell Death in Tobacco

Abstract

There is much interest in the transduction pathways by which avirulent pathogens or derived elicitors activate plant defense responses. However, little is known about anion channel functions in this process. The aim of this study was to reveal the contribution of anion channels in the defense response triggered in tobacco by the elicitor cryptogein. Cryptogein induced a fast nitrate (NO 3 ؊ ) efflux that was sensitive to anion channel blockers and regulated by phosphorylation events and Ca 2 ؉ influx. Using a pharmacological approach, we provide evidence that NO 3 ؊ efflux acts upstream of the cryptogein-induced oxidative burst and a 40-kD protein kinase whose activation seems to be controlled by the duration and intensity of anion efflux. Moreover, NO 3 ؊ efflux inhibitors reduced and delayed the hypersensitive cell death triggered by cryptogein in tobacco plants. This was accompanied by a delay or a complete suppression of the induction of several defense-related genes, including hsr 203J, a gene whose expression is correlated strongly with programmed cell death in plants. Our results indicate that anion channels are involved intimately in mediating defense responses and hypersensitive cell death.

The Elicitor Cryptogein Blocks Glucose Transport in Tobacco Cells

“…(Bourque et al, 1999), calcium channels (Tavernier et al, 1995b;Lecourieux et al, 2002), potassium and anionic channels (Wendehenne et al, 2002), and an NADPH oxidase responsible for the production of AOS. Channel activation is responsible for a large PM depolarization, and NADPH oxidation leads to the activation of the pentose phosphate pathway, to a decrease in the Glc-6-phosphate content, and to an increase in glycolytic products (Pugin et al, 1997), indicating changes to the plant carbohydrate metabolism.…”

“…Within the first 5 min, cryptogein induces an anion efflux and calcium influx, which give rise to fast and large PM depolarization from Ϫ160 to Ϫ50 mV (Pugin et al, 1997;Wendehenne et al, 2002). This PM depolarization might explain the inhibition of Glc uptake, which is coupled with H ϩ entry and, thus, depends on the transmembrane electrochemical potential difference (⌬⌿; for review, see Delrot et al, 2000).…”

“…Studies on the mode of action of elicitors have revealed that they first activate plasma membrane (PM) proteins involved in recognition (Boller, 1995) and signal transduction. The latter phenomenon involves Ca 2ϩ channels (Zimmermann et al, 1997; Lecourieux et al, 2002), anionic and K ϩ channels (Nü rnberger et al., 1994;Wendehenne et al, 2002), NADPH oxidase (Keller et al, 1998), phospholipases (van der Luit et al., 2000, and probably other proteins that have not yet been identified. When activated, these proteins trigger within a few minutes a complex network of second messengers (free cytosolic calcium increase, cytosolic pH decrease, active oxygen species [AOS], PM depolarization, and changes in metabolism; Batz el al., 1998; Lebrun-Garcia et al, 1999), which in turn triggers defense reactions, as well as the systemic acquired resistance (SAR).…”

“…After its binding to a high-affinity PM N-glycoprotein (Bourque et al, 1999), cryptogein induces a large Ca 2ϩ influx that depends on protein phosphorylation (Tavernier et al, 1995b). This Ca 2ϩ influx initiates many different events, including phosphorylation of at least 18 polypeptides (LecourieuxOuaked et al, 2000), the activation of anionic and K ϩ channels leading to a large PM depolarization (Pugin et al, 1997;Wendehenne et al, 2002), the activation of mitogen-activated protein kinases (MAPKs; LebrunGarcia et al, 1998), NO production (Foissner et al, 2000), microtubule disruption (Binet et al, 2001), and the activation of an NADPH oxidase responsible for AOS production and cytosol acidification. NADPH oxidase activation also leads to a marked decrease in cytosolic Glc-6-phosphate and to the accumulation of glycolytic products (Pugin et al, 1997).…”

The Plant Defense Elicitor Cryptogein Stimulates Clathrin-Mediated Endocytosis Correlated with Reactive Oxygen Species Production in Bright Yellow-2 Tobacco Cells

“…The mode of action of cryptogein begins with the recognition of this elicitor by an unidentified plasma membrane receptor at a high-affinity binding site . This ligand-receptor binding triggers a cascade of events that include phosphorylation processes, rapid calcium influx, ion effluxes, nitric oxide production, extracellular alkalinization, and plasma membrane depolarization both in tobacco plants and cell suspensions (Blein et al, 1991;Tavernier et al, 1995;Wendehenne et al, 2002;Garcia-Brugger et al, 2006). In addition, the activation of a membrane-bound NADPH oxidase (NtrbohD), responsible for reactive oxygen species (ROS) production, has been shown in cryptogein-elicited cells (Simon-Plas et al, 1997, 2002.…”