1.9.32
ENHANCEMENT OF COTTON PHYTOALEXIN POTENCY: PURIFICATION OF DESOXYHEMIGOSSYPOL-O-METHYLTRANSFERASE

RD STIPANOVIC1, JG LIU1, CR BENEDICT2 and AA BELL1

1USDA, ARS, Southern Crops Research Laboratory, College Station, Texas, USA; 2Texas A&M University, Department of Biochemistry, College Station, Texas, USA

Background and objectives
Plants employ a number of strategies to protect themselves from pathogens. These include production of structural barriers, antimicrobial enzymes (beta-1,3-glucanase and chitinase), polymers (tannins, lignins and suberins), and low-molecular-weight antibiotics (either pre-formed or induced, i.e. phytoalexins) [1]. In the case of cotton, the latter are sesquiterpenoids, aldehydes and naphthofurans. These terpenoids appear to be particularly important in protecting the plant from soilborne pathogens. The phytoalexins are synthesized in diseased tissues as part of an active defence reaction. Resistant cotton cultivars respond more quickly and produce higher concentrations of the phytoalexins after infection with Verticullium dahliae as compared with susceptible cultivars.

The phytoalexins desoxyhemigossypol (dHG), desoxyhemigossypol-6-methyl ether (dMHG), hemigossypol (HG), and hemigossypol-6-methyl ether (MHG) are synthesized by the paravascular cells next to the xylem vessels. The phytoalexins are exuded first into the vessels and then into the surrounding intercellular spaces [2]. Cui Y et al. (unpublished data) have shown that resistant cotton cultivars recognize the presence of V. dahiiae as soon as 12 h after inoculation with conidia, while in susceptible cultivars the response is significantly delayed. We believe that increasing the potency of the phytoalexins offers a plausible avenue of research to further increase resistance.

All of the phytoalexins exhibit some degree of toxicity to the pathogenic fungi V dahliae and Fusarium oxysporum f. sp. vasinfectum (Fov) [3]. From studies on the toxicity of these compounds to these pathogens, the following conclusions can be deduced: (i) dHG is the most toxic against all of these pathogens; (ii) methylation of the phenolic group in the C-6 position inevitably reduces the toxicity to the pathogens to one-half or less. We have purified and are attempting to sequence and clone the enzyme dHG-O-methyltransferase (dHG-OMT). Work in our laboratory has shown dHG acts as the unique substrate for this enzyme (i.e. HG does not act as a substrate). Since methylation of the phytoalexins reduces toxicity, expression of antisense constructs derived from DHG-OMT clones should provide a more potent mixture of the phytoalexins and lead to an increase in resistance.

Results and conclusions
A crude protein preparation was prepared from inoculated cotton stem tissue. The homogenate, after centrifugation, was fractionated on an O-sepharose FF column. Pooled active fractions were chromatographed on an Ultragel AcA34 gel filtration column. Three enzymes (DHG-OMT, catechol OMT and coniferyl alcohol dehydrogenase, CAD) co-eluted. DHG-OMT was purified from these co-eluting enzymes utilizing affinity chromatography.

References
1. Lamb CJ, Ryals JA, Ward ER, Dixon RA, 1992. Biotechnology 10, 1436-1445.
2. Mace ME, Stipanovic RD, Bell AA, 1989. New Phytologist 111, 229-232.
3. Zhang J, Mace ME, Stipanovic RD, Bell AA, 1993. Journal of Phytopathology 139, 247-252.