Stereoisomerism in plant disease resistance: induction and isolation of the 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone oxidoreductase, an enzyme introducing chirality during synthesis of isoflavonoid phytoalexins in pea (Pisum sativum L).

Treatment of pea seedlings with CuCl2 induced the activity of the enzyme NADPH:7,2'-dihydroxy-4',5'-methylenedioxyisoflavone oxidoreductase (DMIRase) that introduces (+) stereoisomerism in pisatin. DMIRase was purified approximately 7000 fold from CuCl2-treated pea seedlings to apparent homogeneity by a six-step process. The purification sequence included (NH4)2SO4 fractionation, gel filtration on AcA 44, chromatography on DEAE-Bio-Gel,phenyl-Sepharose CL-4B, and Reactive Red 120-agarose, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration and denaturing electrophoresis showed that the enzyme consisted of a single polypeptide chain with an Mr of 37,500. The pH optimum of DMIRase was determined to be 7.8. The enzyme showed apparent Michaelis constants of 20 microM for 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone and 58 microM for NADPH. The reaction product of the enzyme, sophorol, gave a distinct negative Cotton effect in the region 300-360 nm, which indicated 3S configuration of the molecule. Antibodies against the enzyme were raised in rabbits and characterized for specificity.

One enzyme makes a fungal pathogen, but not a saprophyte, virulent on a new host plant.

Certain genes of Nectria haematococca, a fingal pathogen of pea (Pisum sativum), encode pisatin demethylase (pda), a cytochrome P-450 monoxygenase that detoxifies the phytoalexin pisatin. Because pda is required by N.haematococca for pathogenicity on pea, pisatin helps defend pea against N. haematococca. The possibility that pisatin is a general defense factormicrothat is, that pda can confer pathogenicity to fungi not normally pathogenic on peamicrowas investigated. Genes encoding pda were transformed into and highly expressed in Cochliobolus heterostrophus, a fungal pathogen of maize but not of pea, and in Aspergillus nidulans, a saprophytic fungus, neither of which produces a significant amount of pda. Transformants contained at least as much pda as did wild-type N. haematococca. Recombinant C. heterostrophus was normally virulent on maize, but it also caused symptoms on pea, whereas recombinant A. nidulans did not affect pea. Thus, phytoalexins can function in nonspecific resistance of plants to microbes; saprophytes appear to lack genes for basic pathogenicity.

Detoxification of the phytoalexin pisatin by a fungal cytochrome P-450.

The fungus Nectria haematococca, a pathogen of garden pea (Pisum sativum), can demethylate pisatin, an antimicrobial compound synthesized by infected pea tissue. The phenolic product is less toxic than pisatin to many microorganisms. Cell extracts catalyzing pisatin demethylation were obtained from N. haematococca, and the properties of the reaction were examined. The enzyme activity was greatest in the high-speed pellet fraction, in which rates up to 20 nmol/min/mg protein were observed. The Km for pisatin was relatively low, less than 5 microM. The reaction was dependent on NADPH, which could not be replaced by any other cofactor tested. However, in the presence of NADPH, NADH increased the rate of demethylation. Oxygen uptake by the enzyme was stimulated by addition of pisatin, the increment of oxygen utilization being approximately equimolar with pisatin added. Formaldehyde was a product of the reaction. The effects of various inhibitors were tested to determine whether this reaction is mediated by cytochrome P-450. The respiratory inhibitors KCN (1 mM) and antimycin A strongly inhibited the demethylation of pisatin by intact cells of the fungus, but not by the NADPH-supplemented enzyme. The cytochrome P-450 inhibitors SKF 525-A and 1-(2-isopropylphenyl)imidazole inhibited demethylation both in whole cells and in the enzyme preparation, though the latter compound was effective only at high concentrations. Most other cytochrome P-450 inhibitors tested had little effect. However the reaction was quite sensitive to CO, and this inhibition was readily reversed by light at wavelengths near 450 nm. It is concluded that pisatin demethylase is a cytochrome P-450 monooxygenase.

Susceptibility to Enzymatic Degradation of Cell Walls From Bean Plants Resistant and Susceptible to Rhizoctonia solani Kuhn.

Enzymes in culture filtrates of Rhizoctonia solani Kuhn grown using 4-day old or 20-day old bean (Phaseolus vulgaris L.) hypocotyl cell walls as a carbon source degraded xylan, galactan, galactomannan, araban, polygalacturonic acid, and carboxymethylcellulose. Extracts of lesions from R. solani infected plants, but not healthy plants, contained similar enzymatic activities. These enzyme sources readily solubilized cell wall constituents containing arabinose, galactose, and glucose from 4-day old, but not from 20-day old, bean cell walls. Analysis of cell walls prepared from infected plants revealed that the alterations in cell wall composition in the diseased host were limited largely to the immediate lesion areas and occurred during the early phases of pathogenesis. The cell walls of young susceptible bean seedlings could be degraded by R. solani enzymes, but the cell walls of older plants which are resistant to this pathogen were not susceptible to enzymatic destruction by the same enzyme preparation.