Phenotypic analysis of Phytophthora parasitica by using high throughput phenotypic microarray.

OBJECTIVE: We studied the phenotypic characterization of Phytophthora parasitica Dastur var. nicotianae. METHODS: Phenotypic characterization of the pathogen was studied to provide information for disease management program by using BIOLOG phenotype MicroArray (PM ). Using PM plates 1 to 10, 950 different phenotypic characterizations were tested. RESULTS: P. parasitica was able to metabolize 74% of tested carbon sources, 96% of nitrogen sources, 100% of sulfur sources, and 98% of phosphorus sources. Most informative utilization patterns for carbon sources of P. parasitica were organic acids and carbohydrates, and for nitrogen were various amino acids. The pathogen presented 285 different nitrogen pathways. It had wide range adaptabilities in osmolytes with up to 1% sodium chloride, up to 3% potassium chloride, up to 5% sodium sulfate, up to 20% ethylene glycol, up to 2% sodium formate, up to 5% urea, and up to 2% sodium lactate. It also exhibited active metabolism under pH values between 3.5 and 10, with optimal pH of around 7.0. The pathogen showed both decarboxylase and deaminase activities in the presence of various amino acids. CONCLUSION: These phenotypic characterizations of P. parasitica provided the theoretical basis for the next study of the pathogen in physiology and metabolism, and provided potential new way for tobacco black shank management.

General and hybrid correlation nuclear magnetic resonance analysis of phosphorus in Phytophthora palmivora.

Generalized two-dimensional (Gen2D) correlation analysis and hybrid correlation analysis have been applied to a series of dynamic (31)P nuclear magnetic resonance (NMR) spectra to monitor the in vivo metabolic changes of the plant pathogen Phytophthora palmivora in the presence and absence of phosphonate over an 18-h period. Results indicate that phosphonate exposure causes cleavage in organism polyphosphate chains as well as an increase in total sugar phosphates. In the presence of phosphonate, the NMR resonances attributed to terminal polyphosphate phosphorus reduced at a lower rate than those of middle polyphosphate phosphorus, indicating a change in average chain length and suggesting cleavage in the middle of the chain as well as at the ends. The correlation analysis techniques serve to identify and confirm spectral regions undergoing major change in the time-series data and facilitate the analysis of these dynamic changes.

Use of the cryptogein gene to stimulate the accumulation of Bacopa saponins in transgenic Bacopa monnieri plants.

Genetic transformation of the Indian medicinal plant, Bacopa monnieri, using a gene encoding cryptogein, a proteinaceous elicitor, via Ri and Ti plasmids, were established and induced bioproduction of bacopa saponins in crypt-transgenic plants were obtained. Transformed roots obtained with A. rhizogenes strain LBA 9402 crypt on selection medium containing kanamycin (100 mg l(-1)) dedifferentiated forming callus and redifferentiated to roots which, spontaneously showed shoot bud induction. Ri crypt-transformed plants thus obtained showed integration and expression of rol genes as well as crypt gene. Ti crypt-transformed B. monnieri plants were established following transformation with disarmed A. tumefaciens strain harboring crypt. Transgenic plants showed significant enhancement in growth and bacopa saponin content. Bacopasaponin D (1.4-1.69 %) was maximally enhanced in transgenic plants containing crypt. In comparison to Ri-transformed plants, Ri crypt-transformed plants showed significantly (p ≤ 0.05) enhanced accumulation of bacoside A(3), bacopasaponin D, bacopaside II, bacopaside III and bacopaside V. Produced transgenic lines can be used for further research on elicitation in crypt-transgenic plants as well as for large scale production of saponins. Key message The cryptogein gene, which encodes a proteinaceous elicitor is associated with increase in secondary metabolite accumulation-either alone or in addition to the increases associated with transformation by A. rhizogenes.

[Effects of elicitors on accumulation of phenolic acids and tanshinones in Salvia miltiorrhiza hairy root].

OBJECTIVE: To observe the effects of a biotic elicitor fungal hyphae extract, an abiotic elicitor methyl jasmonate and their synergistic action on the accumulation of phenolic acids and tanshinones in Salvia miltiorrhiza hairy root. METHOD: Different elicitors were added to S. miltiorrhiza hairy root, which was subcultured for 21 days, the dry weight and contents of phenolic acids and tanshinones were determined at different harvest-time. RESULT: S. miltiorrhiza hairy root growth was significantly inhibited by all three treatments and the accumulation of cryptotanshinone and dihydrotanshinone were promoted by each elicition. As for the accumulation of phenolic acids, there were differences between fungal elicitor and methyl jasmonate treatments, they were promoted by methyl jasmonate while inhibited in a certain extent by fungal hyphae extract. CONCLUSION: Fungal elicitor, methyl jasmonate and their synergistic action have significant influence on accumulation of components in S. miltiorrhiza hairy root, and the effect varies between phenolic acids and tanshinones. There is no correlation between production of water-soluble ingredients and fat-soluble components on the whole under three different treatments.

Alpha- and beta-tubulin from Phytophthora capsici KACC 40483: molecular cloning, biochemical characterization, and antimicrotubule screening.

Internal fragments of alpha- and beta-tubulin genes were generated using reverse transcription polymerase chain reaction (RT-PCR), and the termini were isolated using 5'- and 3'-rapid amplification of cDNA ends. Phytophthora capsici alpha- and beta-tubulin specific primers were then used to generate full-length cDNA by RT-PCR. The recombinant alpha- and beta-tubulin genes were expressed in Escherichia coli BL21 (DE3), purified under denaturing conditions, and average yields were 3.38-4.5 mg of alpha-tubulin and 2.89-4.0 mg of beta-tubulin, each from 1-l culture. Optimum conditions were obtained for formation of microtubule-like structures. A value of 0.12 mg/ml was obtained as the critical concentration of polymerization of P. capsici tubulin. Benomyl inhibited polymerization with half-maximal inhibition (IC(50)) = 468 +/- 20 microM. Approximately 18.66 +/- 0.13 cysteine residues per tubulin dimer were accessible to 5,5'-dithiobis-(2-nitrobenzoic acid), a quantification reagent of sulfhydryl and 12.43 +/- 0.12 residues were accessible in the presence of 200 microM benomyl. The order of preference for accessibility to cysteines was benomyl > colchicine > GTP > taxol, and cysteine accessibility changes conformed that binding sites of these ligands in tubulin were folding correctly. Fluorescence resonance energy transfer technique was used for high throughput screening of chemical library in search of antimitotic agent. There was significant difference in relative fluorescence by 210-O-2 and 210-O-14 as compared to colchicine.

A translocation signal for delivery of oomycete effector proteins into host plant cells.

Bacterial, oomycete and fungal plant pathogens establish disease by translocation of effector proteins into host cells, where they may directly manipulate host innate immunity. In bacteria, translocation is through the type III secretion system, but analogous processes for effector delivery are uncharacterized in fungi and oomycetes. Here we report functional analyses of two motifs, RXLR and EER, present in translocated oomycete effectors. We use the Phytophthora infestans RXLR-EER-containing protein Avr3a as a reporter for translocation because it triggers RXLR-EER-independent hypersensitive cell death following recognition within plant cells that contain the R3a resistance protein. We show that Avr3a, with or without RXLR-EER motifs, is secreted from P. infestans biotrophic structures called haustoria, demonstrating that these motifs are not required for targeting to haustoria or for secretion. However, following replacement of Avr3a RXLR-EER motifs with alanine residues, singly or in combination, or with residues KMIK-DDK--representing a change that conserves physicochemical properties of the protein--P. infestans fails to deliver Avr3a or an Avr3a-GUS fusion protein into plant cells, demonstrating that these motifs are required for translocation. We show that RXLR-EER-encoding genes are transcriptionally upregulated during infection. Bioinformatic analysis identifies 425 potential genes encoding secreted RXLR-EER class proteins in the P. infestans genome. Identification of this class of proteins provides unparalleled opportunities to determine how oomycetes manipulate hosts to establish infection.

Changes in the antioxidant status in leaves of Solanum species in response to elicitor from Phytophthora infestans.

Three Solanum genotypes with various polygenic resistance levels to the oomycete pathogen Phytophthora infestans (Mont.) De Bary were studied for their antioxidant response to the pathogen culture filtrate (CF). Detached plant leaves were treated with CF for 6, 18 and 30 h, and assayed for changes in hydrogen peroxide content, total ascorbate and glutathione pools and redox ratios (reduced form to total pool), as well as for changes in activities of ascorbate peroxidase, glutathione reductase and glutathione-S-transferase. In CF treated leaves of non-host resistant S. nigrum var. gigantea and field resistant S. tuberosum cv Bzura, the H(2)O(2) content did not change in comparison to water treated control leaves, whereas in the susceptible S. tuberosum clone H-8105 it decreased below the control level. In CF treated leaves of all genotypes, the total ascorbate pools were relatively unaltered and their redox ratio changed only transiently. In Bzura leaves the total glutathione content increased earlier than in the two other genotypes. The glutathione redox ratio remained rather stable, except for the susceptible clone H-8105, where it decreased transiently by about 42%. The relative increases in activity of all the studied enzymes were the highest in the susceptible clone H-8105. The results are discussed in the light of oxidative processes occurring in CF treated leaves. We conclude that stringent control of pro- and anti-oxidant reactions bringing the H(2)O(2) and/or cellular redox state to the threshold level is decisive for deployment of an effective defense strategy.

Subcellular localization of Strboh proteins and NADPH-dependent O2(-)-generating activity in potato tuber tissues.

Rapid generation of reactive oxygen species (ROS) at the cell surface has been implicated in plant defence responses. Genetic evidence indicates that a plant NADPH oxidase (Rboh; respiratory burst oxidase homologue) is associated with oxidative burst. However, there is not enough physiological evidence of Rboh localization available yet. Isozyme-specific antibodies against potato StrbohA and StrbohB (St; Solanum tuberosum) were prepared to investigate the localization of these proteins. Immunoblot analyses using potato microsomal proteins revealed that StrbohA was expressed constitutively at a low level, whereas the accumulation of StrbohB protein was induced by the cell wall elicitor of the potato pathogen Phytophthora infestans. It is demonstrated here that StrbohA and StrbohB are distributed in plasma membrane fractions which have been separated by sucrose density-gradient centrifugation using their specific antibodies. Green fluorescent protein-tagged Strboh proteins were also located on the plasma membrane by transient expression assay in onion epidermal cells. Additionally, NADPH-dependent O2(-)-generating activities in plasma membrane fractions were diphenylene iodonium-sensitive and NaN3-insensitive. These data suggest that StrbohA and StrbohB are predominantly localized on the plasma membrane and regulate ROS production in defence signalling.

A mating-induced protein of Phytophthora infestans is a member of a family of elicitors with divergent structures and stage-specific patterns of expression.

Five members of an elicitor-like gene family from Phytophthora infestans were examined. The family was identified through the analysis of M81, a mating-induced gene. The predicted M81 product resembled a 42-kDa P. sojae glycoprotein known to elicit defense reactions in plants, including a host of P. infestans, potato. M81 was the most structurally and functionally divergent of the P. infestans genes compared with the P. sojae sequence. M81 lacked elicitor activity, had the lowest protein identity (47%), displayed mating-specific transcription, and had a novel C-terminal domain. The latter contained a 30-residue proline- and threonine-rich motif, which, remarkably, was tandemly repeated 24 to 36 times in different alleles. M81C, M81D, and M81E better resembled the P. sojae protein based on amino acid identity (63 to 75%) and conserved elicitor activity. M81C and M81D mRNA accumulated only during zoosporogenesis, while M81E expression was restricted to hyphae. M81B, an apparent pseudogene, was physically linked to M81. The protein products of each gene were predicted to be extracellular transglutaminases ranging in size from 436 to 1,607 amino acids. Genes with an elicitor, proline- and threonine-rich repeat, and both elicitor and repeat domains were widely distributed throughout Phytophthora infestans. These findings help explain the natural functions of elicitors in pathogen biology and plant-microbe interactions.

A novel tetratricopeptide repeat-containing J-protein localized in a plasma membrane-bound protein complex of the phytopathogenic oomycete Phytophthora megasperma.

Phytoalexins originating from plant tissues may cause within cells of fungi or oomycetes a change in the localization of actin, tubulin and chaperones. To test the hypothesis in a filamentously growing oomycete, we compared the distribution of cellular markers in the presence and absence of hydroxystilbene phytoalexins. Using cDNA from the phytopathogenic organism Phytophthora megasperma, the causal agent of root rot on soybean and many other plants, and including probes for Hsp70 and Hsp40, we cloned a DnaJ-protein (Jcp) with the capacity of interacting with both a particular Hsp70 isoform via its J-domain and with other proteins via its tetratricopeptide repeat (TPR) domain. Antisera raised against the bacterially expressed protein Jcp allowed the analysis of its intracellular localization during hyphal growth. Following the subfractionation of cell homogenates, we detected virtually all immunoreactive Jcp in the plasma membrane-enriched fraction and as constituent of a membrane-associated protein complex. In agreement with the biochemical findings, immunocytochemical stains of hyphae showed Jcp as part of cortical patches positioned along the plasma membrane similar to the distribution of actin patches. Confocal microscopy, however, revealed that the Jcp-containing patches did not generally co-localize with the patches visualized by the actin stain. The 59-kDa Jcp, characterized by a large 8-fold TPR domain at the N-terminal region and a J-domain close to the C-terminus, is a good candidate for bridging the gap between Hsp70 and Hsp90 by protein-protein interactions. By administration of plant-derived phytoalexins it was shown that the presence of resveratrol or piceatannol significantly reduces the amount of the Jcp-containing patches, but does not lead to a relocalization of intracellular Jcp.

Green fluorescent protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora palmivora.

Transgenic Phytophthora palmivora strains that produce green fluorescent protein (GFP) or beta-glucuronidase (GUS) constitutively were obtained after stable DNA integration using a polyethylene-glycol and CaCl2-based transformation protocol. GFP and GUS production were monitored during several stages of the life cycle of P. palmivora to evaluate their use in molecular and physiological studies. 40% of the GFP transformants produced the GFP to a level detectable by a confocal laser scanning microscope, whereas 75% of the GUS transformants produced GUS. GFP could be visualised readily in swimming zoospores and other developmental stages of P. palmivora cells. For high magnification microscopic studies, GFP is better visualised and was superior to GUS. In contrast, for macroscopic examination, GUS was superior. Our findings indicate that both GFP and GUS can be used successfully as reporter genes in P. palmivora.

Elicitin 172 from an isolate of Phytophthora nicotianae pathogenic to tomato.

Elicitin 172, an acid protein with elicitor activity, has been isolated in true form from culture filtrates of Phytophthora nicotianae, the causal agent of crown and root rot of tomato (Lycopersicon esculentum). The M(r) (10,349 +/- 1) of the purified protein, determined by ES-MS, is identical to that calculated for parasiticein using the mean isotopic composition and assuming the occurrence of three disulfide bridges. The primary structure of elicitin 172, determined using also MALDI-MS experiments, shows complete identity with parasiticein, with elicitin 310 and a cloned elicitin gene from P. parasitica (= P. nicotianae), confirming conservation of the elicitin sequence within a single species. The protein induces necrosis (hypersensitive reaction) on tobacco, but no symptoms on tomato, when applied on the leaves. Tomato pretreated with elicitin 172 was affected by P. nicotianae, as well as by the phytotoxic aggregates, naturally occurring with the elicitin in the non permeated dialysis fraction of culture filtrates. Finally, the elicitin induce protection of capsicum (Capsicum annuum) and vegetable marrow (Cucurbita pepo) from P. capsici.

A structural model for the mechanisms of elicitor release from fungal cell walls by plant beta-1,3-endoglucanase.

The release of elicitor-active carbohydrates from fungal cell walls by beta-1,3-endoglucanase contained in host tissues has been implicated as one of the earliest processes in the interaction between soybean (Glycine max) and the fungal pathogen Phytophthora megasperma f. sp. glycinea leading to host defense responses such as phytoalexin production. The present study was conducted to evaluate the primary structure of the glucanase-released elicitor (RE). Gel-filtration chromatography of carbohydrates released from mycelial walls by purified soybean beta-1,3-endoglucanase resolved them into the four fractions (elicitor-active RE-I, -II, and -III and elicitor-inactive RE-IV). Sugar composition analysis indicated that all of the fractions were composed almost entirely of glucose. 1H- and 13C-nuclear magnetic resonance analysis indicated the presence of both beta-1,3- and beta-1,6-linkages for the elicitor-active RE-I, -II, and -III fractions and only beta-1,3 linkage for the elicitor-inactive RE-IV fraction. Methylation analysis and degradation studies employing beta-1,3-endo- and beta-1,3-exoglucanase further suggested that the basic structure of elicitor-active RE consists of beta-1,6-linked glucan backbone chains of various lengths with frequent side branches composed of beta-1,3-linked one or two glucose moieties. From these structural analyses of RE, a structural model of how RE is originally present in fungal cell walls and released by host beta-1,3-endoglucanase is also proposed.

High affinity binding of a fungal oligopeptide elicitor to parsley plasma membranes triggers multiple defense responses.

An oligopeptide of 13 amino acids (Pep-13) identified within a 42 kDa glycoprotein elicitor from P. mega-sperma was shown to be necessary and sufficient to stimulate a complex defense response in parsley cells comprising H+/Ca2+ influxes, K+/Cl- effluxes, an oxidative burst, defense-related gene activation, and phytoalexin formation. Binding of radiolabeled Pep-13 to parsley microsomes and protoplasts was specific, reversible, and saturable. Identical structural features of Pep-13 were found to be responsible for specific binding and initiation of all plant responses analyzed. The high affinity binding site recognizing the peptide ligand (KD = 2.4 nM) may therefore represent a novel class of receptors in plants, and the rapidly induced ion fluxes may constitute elements of the signal transduction cascade triggering pathogen defense in plants.

Antitumor activity of cell wall beta-1,3/1,6-glucans from Phytophthora species.

From the cell walls of various Phytophthora species (Oomycetes) different water-soluble beta-1,3/1,6-glucans were isolated and characterized. The glucans with relative molecular masses between 13 and 50 kd exhibited varying degrees of branching in the beta-1,3-linked glucan backbone ranging from 14 to 50%. The dose-dependent antitumor activity of the glucans against allogeneic sarcoma-180 was investigated. It could be demonstrated that beta-1,3/1,6-glucans with relative molecular masses below 50 kd can exhibit a prominent antitumor activity (inhibition rate up to 99%), being comparable to that of the high relative molecular mass (450 kd) beta-1,3/1,6-glucans. Biological activity of the glucans could be shown to be correlated with a low degree of branching. The most potent glucan with a degree of branching of 14% also was active against the syngeneic DBA/2-MC.SC-1 fibrosarcoma (inhibition rate up to 90%) and in combination with a suboptimum dose of diethylstilbestrol against Nobel-Nb prostate carcinoma (inhibition rate up to 90%).