1.9.18
LOCATION AND TIME OF APPEARANCE OF A PHYTOALEXIN-BIOSYNTHETIC ENZYME RELATIVE TO HYPERSENSITIVE CELL DEATH AT XANTHOMONAS INFECTION SITES IN COTTON COTYLEDONS

CHONG-UK PARK, M ESSENBERG and ML PIERCE

Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078-3035, USA

Background and objectives
Bacterial blight resistance in Gossypium hirsutum is expressed in foliar tissues by a hypersensitive response in which the mesophyll cells closest to single-cell-derived colonies of Xanthomonas campestris pv. malvacearum die and accumulate cadinane phytoalexins, two of which are green-fluorescent. Bacteriostasis occurs 3-5 days post-inoculation. Average cellular concentrations of the phytoalexins at infection sites at the time of bacteriostasis far exceed levels that are inhibitory in vitro [1]. In that study, phytoalexin-containing cells were present at every infection site sooner in the highly resistant cotton line Im216 than in moderately resistant line OK1.2; however, several-fold higher levels of phytoalexins finally accumulated in OK1.2 [1]. The first enzyme of the pathway committed to biosynthesis of cadinanes, (+)-delta-cadinene synthase (CDN1), has been purified [2].

Objectives were: (i) To learn where phytoalexins are synthesized in cotton foliar tissue - in the hypersensitively responding (HR) cells, in healthy neighbouring cells that are not destined to die, or in both cell types? (ii) To test the hypothesis that phytoalexin biosynthesis begins sooner post-inoculation in Im216 than in OK1.2.

Materials and methods
Leafy cotyledons of resistant Im216 and OK1.2 and susceptible Ac44 were infiltrated with X. campestris pv. malvacearum or with sterile infiltration medium. Tissue blocks were excized at intervals, fixed, embedded and sectioned. HR cells were detected by their autofluorescence under UV excitation. Bacteria were detected by immunogold staining with silver enhancement. CDN1 was detected by immunofluorescent staining.

Results and conclusions
(i) Mock-inoculated tissues exhibited no staining for CDN1, and inoculated Ac44 exhibited very little. In the two resistant lines, CDN1 appeared in all HR cells and in a few adjacent living cells during the first 3 days post-inoculation, but by 6 days all CDN1-containing mesophyll cells were dead. Unexpected, intense CDN1 staining was also observed in vascular bundles and in both epidermal cell layers of inoculated resistant cotyledons.
(ii) HR cells and CDN1-containing cells were observed sooner in Im216 than in OK1.2. Observations strongly suggest that the phytoalexins which accumulate in HR cells at bacterial infection sites are principally synthesized by those cells shortly before they die, rather than by surrounding, surviving cells. CDN1 in vascular and epidermal cells may produce either phytoalexins translocated elsewhere, or cadinane products which do not fluoresce in the visible range.

This work was supported by Agreement No. 91-37303-6652 of the NRI Competitive Grants Program/USDA and by the Oklahoma Agricultural Research Station.

References
1. Pierce ML, Cover EC, Richardson PE et al., 1996. Physiological and Molecular Plant Pathology 48, 305-324.
2. Davis EM, Tsuji J, Davis GD et al., 1996. Phytochemistry 41, 1047-1055.