Rapid induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glycine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis

The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1–2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.Abbreviations cDNA copy DNA - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

Biosynthesis of avenanthramides in suspension cultures of oat (<Emphasis Type="Italic">Avena sativa</Emphasis>)

Oats produce a group of secondary metabolites termed avenanthramides (avn). These compounds are biosynthesized through the action of the enzyme hydroxycinnamoyl CoA: hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) which catalyzes the condensation of one of several cinnamate CoA thioesters with the amine functionality of anthranilic acid, 4-hydroxy- or 5-hydroxy-anthranilic acid. In oat leaf tissue the biosynthesis of avenanthramides appears to result from elicitation by fungal infection. Here we demonstrate the biosynthesis of several avenanthramides in suspension cultures of oat apical meristem callus tissue. This phenomenon appears as a generalized pathogen response, evidenced by the production of PR-1 mRNA, in response to elicitation with chitin (poly-N-acetyl glucosamine). The suspension cultures also produce relatively large quantities of avnA and G in response to chitin elicitation. Under certain culture conditions avnB and C are also produced as well as three additional metabolites tentatively identified as avnH, O and R. These findings portend the utility of oat suspension culture as a tool for more detailed investigation of the mechanisms triggering their biosynthesis as well as the factors dictating the particular types of avenanthramides biosynthesized.     

Co-ordinated synthesis of phytoalexin biosynthetic enzymes in biologically-stressed cells of bean (Phaseolus vulgaris L.)

Changes in the rates of synthesis of three enzymes of phenyl-propanoid biosynthesis in Phaseolus vulgaris L. (dwarf French bean) have been investigated by immunoprecipitation of [³⁵S]methionine-labeled enzyme subunits with mono-specific antisera. Elicitor causes marked, rapid but transient co-ordinated increases in the rate of synthesis of phenyl-alanine ammonia-lyase, chalcone synthase and chalcone isomerase concomitant with the phase of rapid increase in enzyme activity at the onset of accumulation of phenyl-propanoid-derived phytoalexin antibiotics in suspension cultures of P. vulgaris. Co-ordinate induction of enzyme synthesis is also observed in hypocotyl tissue during race:cultivar-specific interactions with Colletotrichum lindemuthianum, causal agent of anthracnose. In an incompatible interaction (host resistant) there are early increases apparently localized to the initial site of infection prior to the onset of phytoalexin accumulation and expression of hypersensitive resistance. In contrast, in a compatible interaction (host susceptible) there is no induction of synthesis in the early stages of infection, but a delayed widespread response at the onset of lesion formation associated with attempted lesion limitation. It is concluded that expression of the phytoalexin defense response in biologically stressed cells of P. vulgaris characteristically involves co-ordinate induction of synthesis of phytoalexin biosynthetic enzymes.     

In situ localization of PR-1 mRNA and PR-1 protein in compatible and incompatible interactions of pepper stems withPhytophthora capsici

Summary In situ hybridization and immunogold labeling were performed to examine the temporal and spatial expression pattern of pathogenesis-related protein 1 (CABPR1) mRNA and PR-1 protein in pepper (Capsicum annuum L.) stem tissues infected by virulent and avirulent isolates ofPhytophthora capsici. CABPR1 mRNA accumulation was confirmed in the infected pepper stem tissue by Northern blot analysis and in situ hybridization. Northern blot analysis showed that the temporal expression ofCABPR1 mRNA varied greatly between compatible and incompatible interactions. An earlier expression of theCABPR1 gene, 6 h after inoculation, was observed in the incompatible interaction. In situ hybridization results revealed thatCABPR1 mRNA was expressed in the phloem areas of vascular bundles in infected pepper stem tissues, but especially strongly in the incompatible interaction. PR-1 protein was predominantly found in the intercellular spaces of pepper stem cells in the compatible and incompatible interactions 24 h after inoculation. Strikingly, the immunogold labeling was associated with fibrillar and electron-dense material localized in the intercellular space. Dense labeling of PR-1 protein was also seen at the interface of the pathogen and the host cell wall, whereas few gold particles were detected over the host cytoplasm. However, PR-1 protein was not detected over the fungal cell wall in either interaction.     

Phytoalexins and polar metabolites from the oilseeds canola and rapeseed: differential metabolic responses to the biotroph Albugo candida and to abiotic stress

The metabolites produced in leaves of the oilseeds canola and rapeseed (Brassica rapa L.) inoculated with either different races of the biotroph Albugo candida or sprayed with CuCl(2) were determined. This investigation established consistent phytoalexin (spirobrassinin, cyclobrassinin, and rutalexin) and phytoanticipin (indolyl-3-acetonitrile, arvelexin, caulilexin C, and 4-methoxyglucobrassicin) production in canola and rapeseed in response to both biotic and abiotic elicitation. In addition, a wide number of polar metabolites were isolated from infected leaves, including six new phenylpropanoids and two new flavonoids. The extractable chemical components of zoosporangia of A. candida and the anti-oomycete activity of phytoalexins were determined as well. Overall, the results suggest that during the initial stage of the interaction, leaves of B. rapa have a similar response to virulent and avirulent races of A. candida, with respect to the accumulation of chemical defenses. After this stage, despite the higher phytoalexin concentration, the "compatible" races could overcome the plant defense system for further infection, but growth of the "incompatible" races was inhibited. Since results of bioassays showed that cyclobrassinin and brassilexin were more inhibitory to A. candida than rutalexin, the apparent redirection of the phytoalexin pathway towards rutalexin, avoiding cyclobrassinin and brassilexin accumulation might be caused by the pathogen. Alternatively, A. candida might be able to detoxify both cyclobrassinin and brassilexin, similar to necrotrophic plant pathogens. Overall, the correlation between phytoalexin production in infected or stressed leaves and the outcome of the plant-pathogen interaction suggested that A. candida was able to elude the plant defense mechanisms by, for example, redirecting the phytoalexin biosynthetic pathway.     

Cloning,sequence and characterization of a sunflower (Helianthus annuus L.) pathogen-induced gene showing sequence homology with auxin-induced genes from plants

The establishment of a plant-pathogen interaction involves changes in gene expressions in both organisms. To isolate Helianthus annuus genes whose expression is induced during processes of resistance to Plasmopara halstedii, a comparison of the expression pattern of healthy sunflowers was made with sunflowers infected with 2 races of P. halstedii, either virulent or avirulent, using differential display of mRNA. A full-length cDNA, HaAC1, representing a sunflower gene whose expression is enhanced during early stages of the incompatible interaction, was isolated. Different timing of RNA accumulation is observed between compatible and incompatible combinations. Sequence analysis and database search revealed significant homology with auxin-induced genes from plants. The expression of this gene, is also induced after treatment with 2,4-dichlorophenoxyacetic acid (2,4-D), salicylic acid (SA) and wounding.     

A cytochrome P450 gene is differentially expressed in compatible and incompatible interactions between pepper (Capsicum annuum) and the anthracnose fungus, Colletotrichum gloeosporioides

The anthracnose fungus, Colletotrichum gloeosporioides, was previously shown to have an incompatible interaction with ripe-red fruit of pepper (Capsicum annuum). However, the fungus had a compatible interaction with unripe-mature-green fruit. Using mRNA differential display, we isolated and characterized a PepCYP gene expressed in the incompatible interaction. The PepCYP gene encodes a protein homologous to cytochrome P450 proteins containing a heme-binding domain. The expression level of PepCYP is higher in the incompatible interaction than in the compatible interaction, and then remains elevated in the incompatible interaction. In the compatible interaction, the expression of PepCYP is transient. The induction of PepCYP gene is up-regulated by wounding or jasmonic acid treatment during ripening. Analysis of PepCYP expression by in situ hybridization shows that the accumulation of PepCYP mRNA is localized in the epidermal cell layers, but not in the cortical cell layers. An examination of transverse sections of the fruits inoculated with the fungus shows that the fungus invades and colonizes the epidermal cell layers of the unripe fruit at 24 and 72 h after inoculation, respectively, but not those of the ripe fruit. These results suggest that the PepCYP gene product plays a role in the defense mechanism when the fungus invades and colonizes the epidermal cells of fruits in the incompatible interaction during the early fungal infection process.     

Further investigations of race:cultivar-specific induction of enzymes related to phytoalexin biosynthesis in soybean roots following infection with Phytophthora megasperma f.sp. glycinea

The activities of the following enzymes in soybean roots were determined at early times after infection of the roots with zoospores of an incompatible or a compatible race of Phytophthora megasperma f.sp. glycinea: dimethylallyl-diphosphate : 3,6a,9-trihydroxypterocarpan dimethylallyltransferase (prenyltransferase), an enzyme specific for glyceollin biosynthesis; NADPH-cytochrome reductase and hydroxymethylglutaryl-CoA reductase, enzymes related to the glyceollin pathway; and isocitrate dehydrogenase. Already at 4 h after infection there was a higher activity of the prenyltransferase in the incompatible interaction than in the compatible interaction, and enzyme activity in the incompatible interaction increased considerably between 4 and 8 h after infection. In the compatible interaction prenyltransferase activity was only slightly higher than in uninfected roots. The activity of the other enzymes in infected roots was not significantly different from that in the uninfected roots. No qualitative differences could be detected between the two-dimensional patterns of unlabelled proteins or proteins labelled with L-[35S]methionine of infected and uninfected roots at early times after infection. We conclude from these and earlier results (A. Bonhoff et al. (1986) Arch. Biochem. Biophys. 246, 149-154) that infection of the soybean roots with an incompatible race of the fungus leads to selective induction of the phytoalexin pathway and presumably to induction of other as yet unknown defense mechanisms.     

Quantitative Localization of the Phytoalexin Glyceollin I in Relation to Fungal Hyphae in Soybean Roots Infected with Phytophthora megasperma f. sp. glycinea

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 (EC₉₀in 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.     

Temporal and spatial patterns of 1, 3-β-glucanase and chitinase induction in potato leaves infected by Phytophthora infestans

Infection of potato (Solarium tuberosum L.) leaves with the fungal pathogen Phytophthora infestans caused a similar, strong and coordinated induction of 1, 3-β-glucanases and chitinases in compatible (plant susceptible) and incompatible (plant resistant) interactions of two selected plant cultivars with appropriate races of the fungus. The temporal and spatial patterns of 1, 3-β-glucanase induction were studied in further detail by immunohistochemical and in-situ hybridization methods. Accumulation of the protein was preceded by progressive activation of the corresponding gene, commencing near infection sites and spreading rapidly throughout the whole infected leaf as well as to adjacent, non-infected leaves. Protein and mRNA distribution patterns were nearly identical in compatible and incompatible interactions. In comparison with 1, 3-β-glucanase mRNA, phenyl-alanine ammonia-lyase mRNA accumulated more rapidly and remained restricted to the vicinity of fungal infection sites, in addition to its constitutive occurrence in the vascular bundles. Even more rapid than any detectable mRNA induction was the accumulation of auto fluorescing material in plant cells immediately surrounding fungal structures, particularly and invariably in incompatible interactions and less frequently in compatible interactions. It is concluded that cultivar-race-specific resistance is established early in the interaction of potato leaves with P. infestans and hence the observed massive accumulation of 1, 3-β-glucanase and chitinase is presumably not involved in determining this specificity.     

Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen Pseudomonas syringae

We performed large-scale mRNA expression profiling using an Affymetrix GeneChip to study Arabidopsis responses to the bacterial pathogen Pseudomonas syringae. The interactions were compatible (virulent bacteria) or incompatible (avirulent bacteria), including a nonhost interaction and interactions mediated by two different avirulence gene-resistance (R) gene combinations. Approximately 2000 of the approximately 8000 genes monitored showed reproducible significant expression level changes in at least one of the interactions. Analysis of biological variation suggested that the system behavior of the plant response in an incompatible interaction was robust but that of a compatible interaction was not. A large part of the difference between incompatible and compatible interactions can be explained quantitatively. Despite high similarity between responses mediated by the R genes RPS2 and RPM1 in wild-type plants, RPS2-mediated responses were strongly suppressed by the ndr1 mutation and the NahG transgene, whereas RPM1-mediated responses were not. This finding is consistent with the resistance phenotypes of these plants. We propose a simple quantitative model with a saturating response curve that approximates the overall behavior of this plant-pathogen system.     

Investigation of the mechanism of glyceollin accumulation in soybean infected by Phytophthora megasperma f. sp. glycinea

Soybean seedlings (Glycine max, cv. Harosoy 63) which had been inoculated in the hypocotyls with mycelium from either race 1 (incompatible) or race 3 (compatible) of Phytophthora megasperma f. sp. glycinea were pulse labeled with ¹⁴CO₂. The time course of accumulation of glyceollin and daidzein and of ¹⁴C incorporation into these compounds was determined. Metabolic rates of glyceollin were measured by pulse-chase experiments. Differences in glyceollin accumulation between the incompatible and compatible interaction were not apparent before about 14 h after inoculation. Subsequently glyceollin accumulated to a higher level in the incompatible interaction. This difference is also reflected in the rate of ¹⁴C incorporation, which declines more rapidly in the compatible interaction. The apparent half-life of glyceollin metabolism was 28 ± 7 h for inoculation with race 1, while no metabolism was observed with race 3. In contrast to a previous report (M. Yoshikawa, K. Yamauchi, and H. Masago (1979)Physiol. Plant Pathol.14, 157–169), our data prove that the higher accumulation of glyceollin in the incompatible interaction is due to a longer duration of synthetic activity and that the level of glyceollin in both the incompatible and compatible interaction is determined predominantly by its rate of synthesis.     

Temporal and subcellular localization of PR-1 proteins in tomato stem tissues infected by virulent and avirulent isolates of Phytophthora capsici

Summary. Immunoblot analysis and immunogold labeling of PR-1 protein (pathogenesis-related protein 1) in tomato (Lycopersicon esculentum Mill.) were performed to examine the temporal and spatial expression patterns of PR-1 protein induced by Phytophthora capsici infection. Soluble proteins with molecular masses of 10, 17, 25, 27 and 75 kDa were induced and accumulated in P. capsici-infected stem tissues during the compatible and incompatible interactions. Western blot analysis revealed that expression of PR-1 protein (17 kDa), at 12 to 24 h after inoculation, occurred earlier in the incompatible than in the compatible interaction. Immunogold labeling of PR-1 proteins occurred over cell walls and cytoplasm of the host and the oomycete pathogen and at the interface between host and oomycete cell walls at 24 h after inoculation in the compatible interaction. In the incompatible interaction, numerous PR-1 proteins accumulated predominantly over oomycete cell walls and at the interface between host and oomycete cell walls. The quantity of PR-1 proteins deposited in both host and oomycete cells was much less in the compatible than the incompatible interaction. Healthy tomato stem tissue was nearly free of immunogold labeling of PR-1 proteins. Received October 9, 2001 Accepted January 18, 2002