cell components. A convincing role in pathogenesis, however, has been es­ tablished only for those enzymes that attack the pectic fraction of the plant cell wall. Several fundamental advances in our understanding of these enzymes have been made since the role of pectic enzymes in tissue degradation was reviewed in this series by Bateman & Millar (17) twenty years ago. In the past decade it was demonstrated that highly purified pectic enzymes could macer­ ate and kill plant tissues in a manner similar to that occurring in soft-rot diseases; that the same enzymes could be used to disassemble the dicot primary cell wall, thus revealing the structural importance of pectic polymers in cell-wall architecture; and that a relationship exists between the enzymatic vulnerability of pectic polymers in the cell wall and the regulation of pectic enzyme synthesis in pathogenic fungi. In the present decade it has been demonstrated that fragments released from the cell wall by pectic enzymes can elicit plant defense reactions and that the highly pectolytic bacteria, Erwinia chrysanthemi and E. carotovora, are amenable to a powerful array of molecular genetic manipulations. This paper considers these most recent advances and attempts to project future progress. The following steps occur during the interaction of a pectolytic pathogen and a potential host: (a) The entering pathogen possesses structural genes encoding pectic enzymes with particular physical and catalytic properties. (b)

The Role of Pectic Enzymes in Plant Pathogenesis

Signals and transduction mechanisms for activation of plant defenses against microbial attack.

The plant basal immune system can detect broadly present microbe-associated molecular patterns (MAMPs, also called PAMPs) and induce defenses, but adapted microbes express a suite of effector proteins that often act to suppress these defenses. Plants have evolved other receptors (R proteins) that detect these pathogen effectors and activate strong defenses. Pathogens can subsequently alter or delete their recognized effectors to avoid defense elicitation, at risk of a fitness cost associated with loss of those effectors. Significant research progress is revealing, among other things, mechanisms of MAMP perception, the host defense processes and specific host proteins that pathogen effectors target, the mechanisms of R protein activation, and the ways in which pathogen effector suites and R genes evolve. These findings carry practical ramifications for resistance durability and for future resistance engineering. The present review uses numerous questions to help clarify what we know and to identify areas that are ripe for further investigation.

Elicitors, Effectors, and R Genes: The New Paradigm and a Lifetime Supply of Questions

The biochemical basis for the ability of the pterocarpan phytoalexin glycinol (3,6a,9-trihydroxypterocarpan) to inhibit the growth of bacteria was examined. Glycinol at bacteriostatic concentrations ( e.g. 50 micrograms per milliliter) inhibits the ability of Erwinia carotovora to incorporate [ 3 H]leucine, [ 3 H]thymidine, or [ 3 H]uridine into biopolymers. Exposure of Escherichia coli membrane vesicles to glycinol at 20 micrograms per milliliter results in inhibition of respiration-linked transport of [ 14 C]lactose and [ 14 C]glycine into the vesicles when either d-lactate or succinate is supplied as the energy source. The ability of E. coli membrane vesicles to transport [ 14 C]α-methyl glucoside, a vectorial phosphorylation-mediated process, is also inhibited by glycinol at 20 micrograms per milliliter. Furthermore, exposure of membrane vesicles to glycinol (50 micrograms per milliliter) at 20°C results in the leakage of accumulated [ 14 C]α-methyl glucoside-6-phosphate. The effects of the phytoalexins glyceollin, capsidiol, and coumestrol, and daidzein, a compound structurally related to glycinol but without antibiotic activity, upon the E. coli membrane vesicle respiration-linked transport of [ 14 C]glycine and of [ 14 C]α-methyl glucoside was also examined. Glyceollin and coumestrol (50 micrograms per milliliter), but not daidzein, inhibit both membrane-associated transport processes. These data imply that the antimicrobial activity of glycinol, glyceollin, and coumestrol are due to a general interaction with the bacterial membrane. Capsidiol (50 micrograms per milliliter) inhibits d-lactate-dependent transport of [ 14 C]glycine but not vectorial phosphorylation-mediated transport of [ 14 C]α-methyl glucoside. Thus, capsidiol9s mechanism of antimicrobial action seems to differ from that of the other phytoalexins examined.

/pdf/host-pathogen-interactions-xxiii-the-mechanism-of-the-48fjciu57s.pdf

Host-Pathogen Interactions : XXIII. The Mechanism of the Antibacterial Action of Glycinol, a Pterocarpan Phytoalexin Synthesized by Soybeans.

Suspension cultures of castor bean (Ricinus communis L.) which have been treated with pectic fragment elicitor rapidly accumulate lignin as measured by derivatization with thioglycolic acid. The responsiveness of cultured cells to elicitor is dependent on the stage of culture growth. In 6-day (maximally responsive) cultures, increases in lignin are first evident 3 hours after addition of pectic fragment elicitor with maximal rates of lignin synthesis between 4 and 10 hours. The abundance of lignin in cultures after 12 hours of elicitor treatment is 10- to 20-fold higher than in untreated control cultures and can thereby account for as much as 2% of the dry cell weight. Only intermediate sizes of pectic oligomer are active as elicitors of lignin. Half-maximal accumulation of lignin occurs at 250 to 300 micrograms per milliliter of an optimal elicitor preparation with an average degree of polymerization of seven. We consider the synthesis of lignin in elicited cultures to be a mechanism of plant disease resistance which is induced by the elicitor. Plant peroxidases have been proposed to catalyze the last enzymatic steps in the biosynthesis of both lignin and hydrogen peroxide. Six extracellular isoenzymes of peroxidase (two anionic, designated A1 and A2, and four cationic, designated C2, C3, C4, and C7) are detectable in healthy suspension cultures of castor bean by native gel electrophoresis. Treatment of cultures with elicitor causes substantial changes in the activity of four of these species (A1, C2, C3, and C7). Elicitor treatment also results in the appearance of three new peroxidase isoenzymes that are not readily detectable in healthy cultures (C1, C5, and C6). Increases in the activities of these isoenzymes are concurrent with or slightly precede the accumulation of lignin in elicited 6-day cultures. By 12 hours after addition of elicitor, C1 becomes the most abundant extracellular isoperoxidase. The differential regulation of expression of peroxidase isoenzymes following elicitor treatment suggests that individual isoenzymes of peroxidase may have specific functional roles in the biosynthesis of disease-lignin.

Elicitation of lignin biosynthesis and isoperoxidase activity by pectic fragments in suspension cultures of castor bean.

Heat-labile elicitors of phytoalexin accumulation in soybeans (Glycine max L. Merr. cv Wayne) were detected in culture filtrates of Erwinia carotovora grown on a defined 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-0.5m gel filtration column. The heat-labile elicitor activity co-purified with two α-1,4-endopolygalacturonic acid lyases (EC 4·2·2·2). Endopolygalacturonic acid lyase activity appeared to be necessary for elicitor activity because heat-inactivated enzyme preparations did not elicit phytoalexins. The purified endopolygalacturonic acid lyases elicited pterocarpan phytoalexins at microbial-inhibitory concentrations in the soybean-cotyledon bioassay when applied at a concentration of 55 nanograms per milliliter (1 × 10−9 molar). One of these lyases released heat-stable elicitors from soybean cell walls, citrus pectin, and sodium polypectate. The heat-stable elicitor-active material solubilized from soybean cell walls by the lyase was composed of at least 90% (w/v) uronosyl residues. These results demonstrate that endopolygalacturonic acid lyase elicits phytoalexin accumulation by releasing fragments from pectic polysaccharides in plant cell walls.

/pdf/host-pathogen-interactions-xxv-endopolygalacturonic-acid-3k5ynd137z.pdf

Host-Pathogen Interactions XXV. Endopolygalacturonic Acid Lyase from Erwinia carotovora Elicits Phytoalexin Accumulation by Releasing Plant Cell Wall Fragments

A key regulatory role of complex carbohydrates in the interactions between plants and microbes has been estab— lished. The complex carbohydrates act as regulatory mole— cules or hormones in that the carbohydrates induce de novo protein synthesis in receptive cells. [11 The first complex carbohydrate recognized to possess such regulatory proper— ties is a polysaccharide (PS) present in the walls of fungi (2). Hormonal concentrations of this PS elicit plant cells to accumulate phytoalexins (antibiotics). [2] More recently we have recognized that a PS in the walls of growing plant cells also elicits phytoalexin accumulation; microbes and viruses may cause the release of active fragments of this endogenous elicitor. [3] Another PS in plant cell walls is the Proteinase Inhibitor Inducing Factor (PIIF) (53). This hormone appears to protect plants by inducing synthesis in plants of proteins which specifically inhibit digestive enzymes of insects and bacteria. [4] Glycoproteins secreted by incompatible races (races that do not infect the plant) of a fungal pathogen of soybeans protect seedlings from attack by compatible races. Glycoproteins from compatible races do not protect the seedlings (61). [5] The acidic PS secreted by the nitrogen—fixing rhizobia appear to function in the infection of legumes by the rhizobia. W.D. Bauer and his co—workers have evidence that these PS are required for the development of root hairs capable of being infected by symbiont rhizobia. Current knowledge of the structures of these biologically active complex carbohydrates will be presented.

/pdf/structure-and-function-of-complex-carbohydrates-active-in-1lipull0hy.pdf

Structure and function of complex carbohydrates active in regulating plant-microbe interactions

An extract of frozen and thawed soybean (Glycine max L. Merr. cv. Wayne) stems is active, in wounded soybean cotyledons, as a heat-labile elicitor of phytoalexins. The elicitor activity of the extract is destroyed by heating to 95°C for 10 minutes. The fraction that contains heat-labile elicitor activity releases heat-stable elicitor-active molecules from purified soybean cell walls. Heat-labile elicitor activity voids a Bio-Gel P-6 column and can be absorbed onto and eluted from a DEAE Sephadex ion exchange column. Using the cotyledon phytoalexin elicitor assay, maximum heatlabile elicitor activity was obtained when soybean stems were extracted with acetate buffer at pH 6.0. Addition of 1 millimolar CaCl2 increased apparent heat-labile elicitor activity. The heat-labile elicitor stimulated maximum phytoalexin accumulation when applied to cotyledons immediately after the cotyledons were cut. Partially purified stem extracts lost heat-labile elicitor activity during storage for several days at 3°C. The possible role of a heat-labile elicitor in stimulation of phytoalexin accumulation by both abiotic and biotic elicitors is discussed.

Gary D. Lyon

Papers

Host-Pathogen Interactions XXV. Endopolygalacturonic Acid Lyase from Erwinia carotovora Elicits Phytoalexin Accumulation by Releasing Plant Cell Wall Fragments

Structure and function of complex carbohydrates active in regulating plant-microbe interactions

Host-Pathogen Interactions : XXI. Extraction of a Heat-Labile Elicitor of Phytoalexin Accumulation from Frozen Soybean Stems.