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According to their lifestyle, plant pathogens are divided into biotrophic and necrotrophic organisms. Biotrophic pathogens exclusively nourish living host cells, whereas necrotrophic pathogens rapidly kill host cells and nourish cell walls and cell contents. To this end, the necrotrophic fungus Botrytis cinerea secretes large amounts of phytotoxic proteins and cell wall-degrading enzymes. However, the precise role of these proteins during infection is unknown. Here, we report on the identification and characterization of the previously unknown toxic protein hypersensitive response-inducing protein 1 (Hip1), which induces plant cell death. We found the adoption of a structurally conserved folded Alternaria alternata Alt a 1 protein structure to be a prerequisite for Hip1 to exert its necrosis-inducing activity in a host-specific manner. Localization and the induction of typical plant defense responses by Hip1 indicate recognition as a pathogen-associated molecular pattern at the plant plasma membrane. In contrast to other secreted toxic Botrytis proteins, the activity of Hip1 does not depend on the presence of the receptor-associated kinases BRI1-associated kinase 1 and suppressor of BIR1-1. Our results demonstrate that recognition of Hip1, even in the absence of obvious enzymatic or pore-forming activity, induces strong plant defense reactions eventually leading to plant cell death. Botrytis hip1 overexpression strains generated by CRISPR/Cas9 displayed enhanced infection, indicating the virulence-promoting potential of Hip1. Taken together, Hip1 induces a noncanonical defense response which might be a common feature of structurally conserved fungal proteins from the Alt a 1 family., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Levels of the C6-polyol mannitol were observed to rise dramatically in the biotrophic interaction of the rust fungus Uromyces fabae and its host plant Vicia faba. Mannitol was found in millimolar concentrations in extracts and apoplastic fluids of infected leaves and also in extracts of spores. We suggest that this polyol might have at least a dual function: first, as a carbohydrate storage compound, and second, as a scavenger of reactive oxygen species. Mannitol accumulation is accompanied by high expression of a mannitol dehydrogenase (MAD1) in haustoria. While MAD1 transcripts were detected in haustoria only, immunolocalization studies show that the gene product is also present in spores. Kinetic and thermodynamic analyses of the MAD1p catalyzed reactions indicate that the enzyme might be responsible for the production of mannitol in haustoria and for the utilization of mannitol in spores. Since V. faba is normally unable to synthesize or utilize polyols, the multipurpose usage of mannitol seems an ideal strategy for the fungal pathogen.

Low-temperature stress was shown to cause a rapid increase in steady-state levels of alcohol dehydrogenase-1 message (Adh1) and protein activity (ADH1) in maize (Zea mays) (B37N, A188) and rice (Oryza sativa) (Taipei 309, Calmochi 101) seedlings. Maize roots and rice shoots and roots from 7-day seedlings shifted to low temperature (10 degrees C) contained as much as 15-fold more Adh1 mRNA and 8-fold more ADH1 protein activity than the corresponding tissues from untreated seedlings. Time-course studies showed that these tissues accumulated Adh1 mRNA and ADH1 activity severalfold within 4- to 8-hour, levels plateaued within 20 to 24 hours, and remained elevated at 4 days of cold treatment. Within 24 hours of returning cold-stressed seedlings to ambient temperature, Adh1 mRNA and ADH1 activity decreased to pretreatment levels. Histochemical staining of maize and rice tissue imprints showed that ADH activity was enhanced along the lengths of cold-stressed maize primary roots and rice roots, and along the stems and leaves of rice shoots. Our observations suggest that short-term cold stress induces Adh1 gene expression in certain plant tissues, which, reminiscient of the anaerobic response, may reflect a fundamental shift in energy metabolism to ensure tissue survival during the stress period.

An elicitor of phytoalexin accumulation (endogenous elicitor) is solubilized from purified cell walls of soybean (Glycine max [L.] Merr., cv. Wayne) by extracting the walls with hot water or by subjecting the walls to partial acid hydrolysis. The endogenous elicitor obtained from soybean cell walls binds to an anion exchange resin. The elicitor-active material released from the resin contains oligosaccharides rich in galacturonic acid; small amounts of rhamnose and xylose are also present. The preponderance of galacturonic acid in the elicitor-active fragments suggests that the elicitor is, in fact, a fragment of a pectic polysaccharide. This possibility is supported by the observation that treatment of the wall fragments with a highly purified endopolygalacturonase destroys their ability to elicit phytoalexin accumulation. This observation, together with other evidence presented in this paper, suggests that galacturonic acid is an essential constituent of the elicitor-active wall fragments. Endogenous elicitors were also solubilized by partial hydrolysis from cell walls of suspension-cultured tobacco, sycamore, and wheat cells.

This paper describes the effect of a plant-derived polygalacturonase-inhibiting protein (PGIP) on the activity of endopolygalacturonases isolated from fungi. PGIP's effect on endopolygalacturonases is to enhance the production of oligogalacturonides that are active as elicitors of phytoalexin (antibiotic) accumulation and other defense reactions in plants. Only oligogalacturonides with a degree of polymerization higher than nine are able to elicit phytoalexin synthesis in soybean cotyledons. In the absence of PGIP, a 1-minute exposure of polygalacturonic acid to endopolygalacturonase resulted in the production of elicitor-active oligogalacturonides. However, the enzyme depolymerized essentially all of the polygalacturonic acid substrate to elicitor-inactive oligogalacturonides within 15 minutes. When the digestion of polygalacturonic acid was carried out with the same amount of enzyme but in the presence of excess PGIP, the rate of production of elicitor-active oligogalacturonides was dramatically altered. The amount of elicitor-active oligogalacturonide steadily increased for 24 hours. It was only after about 48 hours that the enzyme converted the polygalacturonic acid into short, elicitor-inactive oligomers. PGIP is a specific, reversible, saturable, high-affinity receptor for endopolygalacturonase. Formation of the PGIP-endopolygalacturonase complex results in increased concentrations of oligogalacturonides that activate plant defense responses. The interaction of the plant-derived PGIP with fungal endopolygalacturonases may be a mechanism by which plants convert endopolygalacturonase, a factor important for the virulence of pathogens, into a factor that elicits plant defense mechanisms.

A previously unrecognized phytoalexin has been isolated from soybean cotyledons that had been infected with bacteria or exposed to ultraviolet light. The phytoalexin has been purified to homogeneity by silica gel flash chromatography and high pressure liquid chromatography. It has been structurally characterized by its ultraviolet, circular dichroism and nuclear magnetic resonance spectra, polarimetry, and its mass spectrometric fragmentation pattern. The phytoalexin, (6aS,11aS)-3,6a,9-trihydroxypterocarpan, is a compound that had previously been detected in CuCl(2)-treated soybeans and is structurally related to the previously identified soybean phytoalexins glycerollins I to IV. It is proposed that the trivial name glycinol be used for this phytoalexin. Glycinol is a broad spectrum antibiotic capable of prolonging the lag phase of growth of all six bacteria examined, namely Erwinia carotovora, Pseudomonas glycinea (races 1 and 3), Escherichia coli, Xanthomonas phaseoli, and Bacillus subtilis. Glycinol also inhibits the growth of the fungi Phytophthora megasperma f. sp. glycinea (race 1), Saccharomyces cerevisiae, and Cladosporium cucumerinum. Glycinol is a static agent against the six bacterial species listed above and against S. cerevisiae, and appears to be static against the other fungi examined. As with other phytoalexins, there is no correlation between the pathogenicity of a microorganism and its sensitivity to glycinol.

The effects of cold on protein and RNA metabolism in leaves of rice (Oryza sativa L.) seedlings were investigated. Treatment with a diurnal cycle of 15/10 degrees C or 11/6 degrees C for up to 1 week resulted in progressive changes in the protein synthesis pattern after in vivo labeling of intact rice leaves with [(35)S]methionine. These changes were reversed when the seedlings were returned to normal growth temperatures. While de novo accumulation of several abundant proteins was suppressed, some polypeptides were consistently found to be cold-induced. Synthesis of ribulose 1,5-bisphosphate carboxylase (Rubisco) was drastically reduced after 7 days of cold. Using immunoprecipitation of Rubisco, evidence was obtained that the suppression was greater for the small subunit (over 90%) than for the large subunit (80%), indicating a partial loss of coordination in their synthesis. Preformed Rubisco as well as other cold-suppressed proteins were stable for up to 7 days at 11/6 degrees C. Cold-sensitive rice cultivars responded with similar but more drastic changes in the protein synthesis pattern when compared to cold-tolerant varieties. The suppression of Rubisco synthesis by cold was shown to result from reduced levels of the mRNAs encoding both subunits; their decrease paralleled the lower protein synthesis of each. The levels of other chloroplast-encoded mRNAs, especially psaB, and of the nuclear encoded chlorophyll a/b binding protein, also strongly decreased in the cold, whereas the transcripts of the mitochondrial genes apt9, coxIII, and most nuclear genes analyzed were unaffected or only slightly reduced. These data indicate that some chloroplast functions are disturbed during cold stress. One nuclear gene known to be induced by water stress and ABA (Rab21) was also found to be induced by cold treatment.

A radioimmunoassay for glyceollin I, the major phytoalexin produced by soybean (Glycine max [L.] Merr.), has been developed. Antibodies were raised in rabbits against a glyceollin I-bovine serum albumin conjugate. The antisera were used to establish a radioimmunoassay for glyceollin I using [(125)I]glyceollin I as the tracer. A logit plot of a standard concentration series yielded a straight line in the range of 1 to 100 picomoles (0.34-34 nanograms) of glyceollin I. The structurally related pterocarpan phytoalexins, glyceollins II and III, glyceollidin II and glycinol, which also accumulate in infected soybean tissue, show a low cross-reactivity in the radioimmunoassay (0.5-5% at 50% displacement of the tracer). Two related isoflavones present constitutively in soybean tissue, daidzein and genistein, have cross-reactivities of less than 0.84% and 1.1%, respectively. The radioimmunoassay permitted the quantitative determination of glyceollin I in 15-micrometer microtome sections of soybean hypocotyl tissue infected with zoospores of Phytophthora megasperma f. sp. glycinea.

An elicitor of glyceollin accumulation in soybeans (Glycine max L.) has been isolated from a commercially available extract of brewers' yeast. Yeast is not a known pathogen of plants. The elicitor was isolated by precipitation in 80% (v/v) ethanol followed by column chromatography on DEAE-cellulose, sulfopropyl-Sephadex, and concanavalin A-Sepharose. Compositional and structural analysis showed the elicitor to be a glucan containing terminal, 3-, 6-, and 3,6-linked glucosyl residues. The yeast elicitor stimulates the accumulation of glyceollin in the cotyledons and hypocotyls of soybeans when as little as 15 nanograms or 100 nanograms of the elicitor is applied to the respective tissues. The yeast elicitor is very similar in both structure and absolute elicitor activity to an elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae, a pathogen of soybeans. These and other results of this laboratory suggest that plants are able to respond to the presence of a wide range of fungi by recognizing, as foreign to the plant, structural polysaccharides of the mycelial walls of the fungi.

A radioimmunoassay specific for glyceollin I was used to quantitate this phytoalexin in roots of soybean (Glycine max [L.] Merr. cv Harosoy 63) after infection with zoospores of either race 1 (incompatible) or race 3 (compatible) of Phytophthora megasperma Drechs. f. sp. glycinea Kuan and Erwin. The sensitivity of the radioimmunoassay and an inmmunofluorescent stain for hyphae permitted quantitation of phytoalexin and localization of the fungus in alternate serial cryotome sections from the same root. The incompatible interaction was characterized by extensive fungal colonization of the root cortex which was limited to the immediate vicinity of the inoculation site. Glyceollin I was first detected in extracts of whole roots 2 hours after infection, and phytoalexin content rose rapidly thereafter. Significant concentrations of glyceollin I were present at the infection site in cross-sections (42 micrometers thick) of such roots by 5 hours, and exceeded 0.6 micromoles per milliliter (EC(90)in vitro for glyceollin I) by 8 hours after infection. Longitudinal sectioning (14 micrometers thick) showed that glyceollin I accumulated particularly in the epidermal cell layers, but also was present in the root cortex at inhibitory concentrations. No hyphae were observed in advance of detectable levels of the phytoalexin and, in most roots, glyceollin I concentrations dropped sharply at the leading edge of the infection. In contrast, the compatible interaction was characterized by extensive unchecked fungal colonization of the root stele, with lesser growth in the rest of the root. Only small amounts of glyceollin I were detected in whole root extracts during the first 14 hours after infection. Measurable amounts of glyceollin I were detected only in occasional cross-sections of such roots 11 and 14 hours after infection. The phytoalexin was present at inhibitory concentrations in the epidermal cell layers, but the inhibitory zone did not extend appreciably into the cortex. Altogether, these data support the hypothesis that the accumulation of glyceollin I is an important early response of soybean roots to infection by P. megasperma, but may not be solely responsible for inhibition of fungal growth in the resistant response.