Responses of Cultured Tobacco Cells to Cryptogein, a Proteinaceous Elicitor from Phytophthora cryptogea

Abstract

In culture, the phytopathogenic fungus Phytophthora cryptogea secretes a protein which elicits hypersensitive-like necroses and protects tobacco plants against invasion by the pathogen Phytophthora parasitca var. nicotianae. This protein, named cryptogein, has been purified and its amino acid sequence determined. In this work, we studied the effect of cryptogein on tobacco cell suspension cultures. Cryptogein was lethal at about 0.10 micromolar. When added at sublethal doses, it elicited the production of ethylene and phytoalexins. It also induced a rapid increase in pH and conductivity of the extracellular medium without affecting the integrity of the plasma membrane. Cryptogein reduced the fusicoccin-induced acidification of the extracellular medium. The concentration which inhibited the fusicoccin response by 50% was 0.8 nanomolar, while 1 micromolar erythrosine B, an ATPase inhibitor, was needed to produce the same inhibition. However, cryptogein did not inhibit the activity of a purified plasma membrane ATPase. Results of binding studies with whole cells suggested the presence of elicitor-binding sites with a high affinity for cryptogein. The involvement of the plasma membrane during the initial interaction between elicitor and cells is discussed.

Involvement of lipid-protein complexes in plant-microorganism interactions

“…This is also supported by cryptogein-effect reversion with fusicoccin, a well-known activator of H + -ATPase [20,22], according to similar observations reported from tomato cells treated with systemin [45]. At the same time, a strong and rapid alkalization of the extracellular medium and a concomitant acidification of the cytosol are observed [20,22]. Few minutes later, a transient oxidative burst is noticed [22,23].…”

“…Cryptogein-treated tobacco cells were also used to describe the early changes in gene expression [28][29][30][31][32]. All the responses described above are likely to depend on elicitin recognition by specific high affinity binding sites [20], and by protein phosphorylation events [33]. …”

Mediation of Elicitin Activity on Tobacco Is Assumed by Elicitin-Sterol Complexes

“…Iodination of elicitins was performed as previously described (Blein et al, 1991). Specific radioactivity of labeled ligand was ϳ200 Ci/ mmol.…”

“…In tobacco plants, these elicitors induce both a hypersensitive response (leaf necrosis) and nonspecific systemic acquired resistance (SAR; Bonnet et al, 1996;Keller et al, 1996). In cell suspension cultures, classical mechanisms of elicitation have been reported, such as calcium influx (Tavernier et al, 1995), alkalization of the extracellular medium (Blein et al, 1991), production of active oxygen species (Rustérucci et al, 1996;Simon-Plas et al, 1997), and cell wall modifications (Kieffer et al, 2000). The presence of a single family of high-affinity sites for elicitins has been reported (Blein et al, 1991).…”

“…In cell suspension cultures, classical mechanisms of elicitation have been reported, such as calcium influx (Tavernier et al, 1995), alkalization of the extracellular medium (Blein et al, 1991), production of active oxygen species (Rustérucci et al, 1996;Simon-Plas et al, 1997), and cell wall modifications (Kieffer et al, 2000). The presence of a single family of high-affinity sites for elicitins has been reported (Blein et al, 1991). They are located on the plasma membrane, represent 220 fmol of sites/mg of protein (K d ϭ 2 nM), and exhibit a sharp optimum pH near 7.0 .…”