Plants can be induced to develop enhanced resistance to pathogen infection by treatment with a variety of abiotic and biotic inducers. Biotic inducers include infection by necrotizing pathogens and plant-growth-promoting rhizobacteria, and treatment with nonpathogens or cell wall fragments. Abiotic inducers include chemicals which act at various points in the signaling pathways involved in disease resistance, as well as water stress, heat shock, and pH stress. Resistance induced by these agents (resistance elicitors) is broad spectrum and long lasting, but rarely provides complete control of infection, with many resistance elicitors providing between 20 and 85% disease control. There also are many reports of resistance elicitors providing no significant disease control. In the field, expression of induced resistance is likely to be influenced by the environment, genotype, and crop nutrition. Unfortunately, little information is available on the influence of these factors on expression of induced resistance. In order to maximize the efficacy of resistance elicitors, a greater understanding of these interactions is required. It also will be important to determine how induced resistance can best fit into disease control strategies because they are not, and should not be, deployed simply as "safe fungicides". This, in turn, will require information on the interaction of resistance elicitors with crop management practices such as appropriate-dose fungicide use.

A potato gene encoding a putative WRKY protein was isolated from a cDNA library enriched by suppression subtractive hybridization for sequences upregulated 1 h postinoculation with Erwinia carotovora subsp. atroseptica. The cDNA encodes a putative polypeptide of 172 amino acids, containing a single WRKY domain with a zinc finger motif and preceded by a potential nuclear localization site. St-WRKY1 was strongly upregulated in compatible, but only weakly in incompatible, interactions with Phytophthora infestans where, in all cases, it was coregulated with class I endochitinase, associating its expression with a known defense response. Whereas St-WRKY1 was strongly induced by E. carotovora culture filtrate (CF), confirming it to be an elicitor-induced gene, no such induction was detected after treatment with salicylic acid, methyl jasmonate, ethylene, or wounding. St-WRKY1 was upregulated by treatment of potato leaves with CFs from recombinant Escherichia coli containing plasmids expressing E. carotovora pectate lyase genes pelB and pelD, suggesting that either proteins encoded by these genes, or oligogalacturonides generated by their activity, elicit a potato defense pathway associated with St-WRKY1.

Infection of leaves of Arabidopsis thaliana with conidial suspensions of the necrotrophic pathogen Botrytis cinerea resulted in a large decrease in the level of ascorbic acid and increases in intensity of a single-peak free radical and Fe(III) (g=4.27) signals in electron paramagnetic resonance (EPR) spectra. These changes were not confined to the spreading lesions or associated areas of chlorosis, but extended to other apparently healthy tissues in the infected leaves. They are, therefore, consistent with the existence of high levels of oxidative stress being generated as a result of the infection process. The expected accompanying increases in levels of the aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE), were not observed, and in the case of MDA the levels in tissue from infected plants were appreciably lower than in the healthy controls. These last findings are surprising and demonstrate a difference in the response of A. thaliana to infection with B. cinerea compared with tissues from other plant families studied previously.

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SummaryThe application of a variety of structurally different protein phosphatase inhibitors (okadaic acid, acanthifolicin, microcystins, nodularin, tautomycin, calyculin A, cantharidin and endothall) to cut surfaces of soybean cotyledons (Glycine max L.) resulted in the production of isoflavonoid phytoalexins (plant defence compounds). Daidzein was the predominant isoflavonoid produced by soybean cotyledons in response to protein phosphatase inhibitors. In contrast, several isoflavonoid phytoalexins were seen after application of either an elicitor I~-glucan fraction isolated from yeast extract or hepta-(133, l~6)-~-glucoside which is the most potent elicitor-active component isolated from the soybean pathogen Phytophthora megasperma f. sp. glycinea. Isoflavonoid production in response to either protein phosphatase inhibitors or elicitors reached a maximum after 20-24 h. The addition of protein phosphatase inhibitors to a soybean cell suspension culture induced the expression of phenylalanine ammonia-lyase (PAL), the first enzyme in the isoflavonoid biosynthetic pathway. Induction of PAL activity was blocked by protein synthesis inhibitors, cycloheximide or anisomysin, and largely prevented by a protein kinase inhibitor, K252a. Another common response of plant cells to fungal elicitation, alkalinization of the soybean cell culture media, was induced within minutes in response to protein phosphatase inhibitors and was largely prevented by K252a.

Suppression subtractive hybridization (SSH) was used to generate a cDNA library enriched for sequences induced in a late-blight-resistant potato cultivar undergoing the hypersensitive response (HR). Of 100 partial cDNA sequences submitted to international DNA and protein data bases, 42 showed similarity to 35 genes, of which 31 were from plants. Of these, 13 were previously characterized as either defense-, stress-, or senescence-associated. One sequence matched (75 to 81%) all known serine palmitoyltransferases (SPTs) at the protein level. SPT catalyzes the first committed step in the synthesis of sphingolipids, important signaling molecules involved in cell differentiation and apoptosis. Putative products of other genes identified here may play a role in programmed cell death, including protein degradation, DNA degradation, metal ion chelation, and signal transduction. cDNA-amplified fragment length polymorphism was used to confirm differential expression of sequences isolated by SSH.

ABSTRACrHeat-labile elicitors of phytoalexin accumulation in soybeans (Glycine max L. Merr. cv Wayne) were detected in culture filtrates of Erwinia carotovora grown on a defed medium containing citrus pectin as the sole carbon source. The heat-labile elicitors were highly purified by cation-exchange chromatography on a CM-Sephadex (C-50) column, followed by agarose-affinity chromatography on a Bio-Gel A-05m gel filtration column. The heat-labile elicitor activity co-purified with two a- (14) and from citrus pectin (25).The Gram-negative bacterium E. carotovora has been shown to elicit the accumulation of pterocarpan phytoalexins in wounded soybean cotyledons (33). Ninety-five per cent of this elicitor activity was abolished by killing the bacteria with heat treatments or antibiotics (13). These results suggested that viable E. carotovora cells produce an elicitor when in contact with plant tissue. We report in this paper the purification and characterization of heat-labile elicitors produced by E. carotovora grown on a defined medium containing citrus pectin. These elicitors were found to be PGA2 lyases, pectin-degrading enzymes that have been shown to be secreted by many plant pathogens (3). We also present evidence that the release, by PGA lyase, of oligosaccharides from the pectic polymers of plant cell walls triggered the elicitation of phytoalexin accumulation. This evidence suggests that the release of endogenous elicitors (14, 25) from plant cell walls by pectin-degrading enzymes plays a role in general plant disease resistance to microorganisms. A preliminary report of these results has been published (10).MATERIALS AND METHODS Chemicals. Electrophoresis-grade acrylamide and N,N'-methylenebisacrylamide, low-mol-wt protein standards, silver-stain reagents, dye reagent concentrate for protein assay, and Bio-Gel A-0.5m agarose beads were from Bio-Rad. Sephadex G-15, CMSephadex (C-50), BSA (fraction V), streptomycin sulfate, and Tris were from Sigma. Ion-exchange resin PB 118 and Pharmalyte pH 8-10.5 ampholytes were from Pharmacia. Electrophoresis-grade SDS was from BDH Chemicals Ltd., D-galacturonic acid was from Aldrich, and D-glucose was from Fisher. All other chemicals and solvents were of reagent grade or better.Polysaccharides. Sodium polypectate (grade II), polygalacturonic acid (grade III), and citrus pectin were from Sigma. Cell walls from soybean stems were prepared by M. Woodward and M. Hahn, as described (14). Before use, the cell walls (1 g) were washed with 1 L 5 mm Tris-HCl, 1 mm CaCl2 at pH 8.5, and 1 L deionized H20. The walls were then dried in a vacuum oven. A crude glucan elicitor from Phytophthora megasperma var glycinea was a gift from J. K. Sharp of this laboratory.

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