The first two authors contributed equally to this work. SummaryLipid peroxidation, often associated with hypersensitive cell death, may be initiated either by active oxygen species (AOS) or lipoxygenases (LOX). Here we report a detailed analysis of this oxidative process in both incompatible and compatible interactions between the cotton cultivar Reba B50 and Xanthomonas campestris pv. malvacearum (Xcm). The hypersensitive reaction (HR) was characterized by a massive production of polyunsaturated fatty acid (PUFA) hydroperoxides together with typical tissue dehydration. Among these, isomers peroxidized on carbon 9, largely predominant, were chiral, showing an excess in the S enantiomer. The HR process was accompanied by an increase in 9S-LOX activity and preceded by transcription of a LOX gene (GhKLox1). These results showed that: (i) AOS produced during the oxidative burst were not involved in PUFA peroxidation during HR; and (ii) as previously described in elicited leaves of tobacco, the massive enzymatic lipid peroxidation was closely associated with hypersensitive cell death. During disease development in this cotton cultivar, the 9-lipoxygenation of PUFAs was late, weak, preceded by a faint accumulation of GhKLox1 transcripts, and associated with chlorosis but not with necrosis. Consequently, the main difference between incompatible and compatible interactions was in the precocity and intensity of the oxidative process, rather than in its nature. These data provide the evidence for a correlation between lipid peroxidation and hypersensitive cell death induced by pathogens.

Interactions between Gossypium spp. and the bacterial pathogen Xanthomonas campestris pv. malvacearum are understood in the context of the gene-for-gene concept. Reviewed here are the genetic basis for cotton resistance, with reference to resistance genes, resistance gene analogs, and bacterial avirulence genes, together with the physiological mechanisms involved in the hypersensitive response to the pathogen, including production of signaling hormones, synthesis of antimicrobial molecules and alteration of host cell structures. This host-pathogen interaction represents the most complex resistance gene/avr gene system yet known and is one of the few in which phytoalexins are known to be specifically localized in HR cells at anti-microbial concentrations.

We analyzed the production of reactive oxygen species, the accumulation of salicylic acid (SA), and peroxidase activity during the incompatible interaction between cotyledons of the cotton (Gossypium hirsutum) cv Reba B50/Xanthomonas campestris pv malvacearum (Xcm) race 18. SA was detected in petioles of cotyledons 6 h after infection and 24 h post inoculation in cotyledons and untreated leaves. The first peak of SA occurred 3 h after generation of superoxide (O 2 ⅐؊ ), and was inhibited by infiltration of catalase. Peroxidase activity and accumulation of SA increased in petioles of cotyledons and leaves following H 2 O 2 infiltration of cotyledons from 0.85 to 1 mM. Infiltration of 2 mM SA increased peroxidase activity in treated cotyledons and in the first leaves, but most of the infiltrated SA was rapidly conjugated within the cotyledons. When increasing concentrations of SA were infiltrated 2.5 h post inoculation at the beginning of the oxidative burst, the activity of the apoplastic cationic O 2 ⅐؊ -generating peroxidase decreased in a dose-dependent manner. We have shown that during the cotton hypersensitive response to Xcm, H 2 O 2 is required for local and systemic accumulation of SA, which may locally control the generation of O 2 ⅐؊ . Detaching cotyledons at intervals after inoculation demonstrated that the signal leading to systemic accumulation of SA was emitted around 3 h post inoculation, and was associated with the oxidative burst. SA produced 6 h post infection at HR sites was not the primary mobile signal diffusing systemically from infected cotyledons.

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Cotton cotyledons displayed a hypersensitive reaction (HR) in the cultivar Réba B50 after infiltration with the avirulent race 18 from Xanthomonas campestris pv. malvacearum. Two sets of peroxidases were associated with the HR time course. Early but transient accumulation of peroxidase in material encapsulating the bacteria in intercellular areas was observed by immunocytochemistry at 3 h postinfection and coincided with the oxidative burst. Total guaiacol-peroxidase activity was highly increased in cells undergoing HR, from 12 h after treatment. Molecular characterization of seven cloned peroxidase genes revealed highly conserved B, D, and F domains, with similarities to plant class III peroxidases. Analysis of gene expression showed variation in transcript accumulation during both compatible (race 20) and incompatible interactions for four of these genes: pod2, pod3, pod4, and pod6. Pod4 and pod6 were more intensely up-regulated during resistance than during disease and in the control, while pod3 was specifically down-regulated during the HR after the oxidative burst. Pod2 was induced by pathogen infection and weakly stimulated in the control. These data suggest that cotton peroxidases may have various functions in the defense response to Xanthomonas infections.