C MARTINEZ¹, E BRESSON¹, K ASSGBETSE¹, JF DANIEL¹, D FERNANDEZ¹, JL MONTILLET¹, JP GEIGER¹ and M NICOLE¹

¹ORSTOM, Department of Plant Pathology, BP 5045, 34032, Montpellier, France; ²CEA, Department of Plant Physiology, 13018, St Paul Lez Durance, France

Background and objectives
Cotton bacterial blight caused by Xanthomonas campestris pv. malvacearum is responsible for considerable yield reduction in most African countries. Interactions between the plant (Gossypium hirsutum) and the pathogen are governed according to the 'gene-for-gene' pattern. In this presentation, we describe some early events involved in the hypersensitive reaction (HR) at infiltration sites of the pathogen.

Results and conclusions
Three hours post-inoculation (h.p.i.) of a resistant cultivar (ev), discs from infiltrated cotyledons reduced exogeneous cytochrome c significantly more than those from a susceptible cv. This reducing activity was inhibited by SOD, suggesting the production of superoxide anions. Peroxidase (Pox) inhibitors, KCN or SHAM, strongly reduced the production of O₂^-, but not the NADPH oxidase inhibitor DPI. NADH oxidase activity detected at this time was inhibited by Pox inhibitors only, demonstrating that NADH Pox was the main source of O₂^- associated with the HR. A strong Pox activity (PoxA) was cytochemically evidenced in cell walls and middle lamellae at 3 h.p.i. Isoelectric focusing (IEF) revealed the increase in cationic PoxA at 3 h.p.i. only. Localization of H₂O₂ by cytochemical staining showed the accumulation of hydrogen peroxide in the apoplast.

Assessment of PoxA by biochemistry (IEF, spectrophotometry, tissue printing) and histochemistry following infection indicated that activity of anionic Pox isozymes dramatically increased at HR sites from 6 h.p.i. A systemic increase in these PoxA was also seen in non-infected leaves, 24 h.p.i. of cotyledons. Northern blot analysis using a Pox clone demonstrated transcription of Pox genes during the HR with detection of accumulation of two peaks of mRNA, 3 and 12 h.p.i.

Salicylic acid (SA) was shown to be produced between 3 and 6 h.p.i. in cells that had undergone the HR. Infiltration of H₂O₂ in cotyledon tissues induced the accumulation of SA. Infiltration of SA triggered anionic PoxA locally at infiltration sites, and systemically in leaves. These experiments suggest that H₂O₂ is likely at the onset of SA accumulation in infected cotton cells and activation of local (LR) and systemic (SR) resistances.

Histochemistry revealed a strong accumulation of flavonoids (9 h.p.i) [1] and sesquiterpenes (48-72 h.p.i.) [2] in Xanthomonas-infiltrated tissues during the incompatible interaction as compared to susceptible infected plants.

References
1. Dai GH, Nicole M, Martinez C et al., 1996. Physiological and Molecular Plant Pathology 49, 285-306.
2. Essenberg M, Pierce M, 1994. In Handbook of Phytoalexin Metabolism and Action, pp. 183-198.