Syringolin A, a new plant elicitor from the phytopathogenic bacterium Pseudomonas syringae pv. syringae, inhibits the proliferation of neuroblastoma and ovarian cancer cells and induces apoptosis.

Syringolin A is a new plant elicitor produced by the plant pathogen Pseudomonas syringae pv. syringae. The goal of this study was to investigate whether syringolin A exhibits anti-proliferative properties in cancer cells. The treatment of human neuroblastoma (NB) cells (SK-N-SH and LAN-1) and human ovarian cancer cells (SKOV3) with syringolin A (0-100 microm) inhibited cell proliferation in a dose-dependent manner. The IC(50) (50% inhibition) for each cell line ranged between 20 microm and 25 microm. In SK-N-SH cells, the treatment with 20 microm syringolin A led to a rapid (24 h) increase of the apoptosis-associated tumour suppressor protein p53. In addition, we found that the treatment of SK-N-SH cells caused severe morphological changes after 48 h such as rounding of cells and loss of adherence, both conditions observed during apoptosis. The induction of apoptosis by syringolin A was confirmed by both poly (ADP-ribose) polymerase (PARP) cleavage and annexin V assay. Taken together, we show for the first time that the natural product syringolin A exhibits anti-proliferative activity and induces apoptosis. Syringolin A and structurally modified syringolin A derivatives may serve as new lead compounds for the development of novel anticancer drugs.

Syringolin reprograms wheat to undergo hypersensitive cell death in a compatible interaction with powdery mildew.

We had previously isolated and characterized syringolin A, one of the molecular determinants secreted by Pseudomonas syringae pv syringae that is perceived by nonhost plant species such as rice. Here, we show that syringolin A is recognized by wheat and that it induces the accumulation of gene transcripts and increases protection against powdery mildew when applied before inoculation. Moreover, syringolin A essentially eradicates powdery mildew from infected wheat if applied after inoculation. This curative effect is accompanied by the induction of cell death and the reactivation of pathogenesis-related genes whose transcript levels initially accumulate after powdery mildew inoculation but then decline during the later course of infection. Because syringolin A has no fungicidal activity against a variety of fungi and its action on wheat cannot be mimicked by the fungicide cyprodinil, syringolin A is hypothesized to counteract the suppression of host defense reactions imposed by the pathogen on the colonized cells.

Characterization of RCI-1, a chloroplastic rice lipoxygenase whose synthesis is induced by chemical plant resistance activators.

A full-length lipoxygenase cDNA (RCI-1) has been cloned from rice (Oryza sativa) whose corresponding transcripts accumulate in response to treatment of the plants with chemical inducers of acquired resistance such as benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), 2,6-dichloroisonicotinic acid (INA), and probenazole. In contrast, RCI-1 transcript levels did not increase after inoculation with compatible and incompatible races of the rice blast fungus Magnaporthe grisea and the nonhost pathogen Pseudomonas syringae pv. syringae. RCI-1 transcript levels also increased after exogenous application of jasmonic acid, but not upon wounding. Dose-response and time course experiments revealed a similar pattern of transcript accumulation and lipoxygenase activity in BTH-treated rice leaves. Enzymatic analysis of recombinant RCI-1 protein produced in Escherichia coli revealed that 13-hydroperoxy-octadecanoic acids were the predominant reaction products when either linoleic or linolenic acid used as a substrate. The RCI-1 sequence features a putative chloroplast targeting sequence at its N-terminus. Indeed, a protein consisting of the putative chloroplast transit peptide fused to green fluorescent protein was exclusively localized in chloroplasts, indicating that RCI-1 is a chloroplastic enzyme.

Interfacial self-assembly of fungal hydrophobins of the lichen-forming ascomycetes Xanthoria parietina and X. ectaneoides.

In the symbiotic phenotype of the lichen-forming ascomycetes Xanthoria parietina and X. ectaneoides, a conglutinate, hydrophilic cortex surrounds a system of aerial hyphae with hydrophobic wall surfaces. In X. parietina freeze-fracture electron microscopy showed that a rodlet layer covers the fungal and algal wall surfaces. Extracts of hot SDS-insoluble wall residues isolated from both species contained a protein that revealed a rodlet layer upon interfacial self-assembly. The N-terminal sequence of the 10-kDa protein of X. ectaneoides served to clone cDNA fragments of XEH1 (H1 of X. ectaneoides) and XPH1 (H1 of X. parietina) by RT-PCR. Genomic DNA blot analysis with both lichenized species and the aposymbiotically cultured symbionts of X. parietina showed that XPH1 and XEH1 are fungal single copy genes. The deduced amino acid sequences of the two encoded proteins were 96% identical and showed the characteristics of class I hydrophobins.

Constitutive expression of the defense-related Rir1b gene in transgenic rice plants confers enhanced resistance to the rice blast fungus Magnaporthe grisea.

The Rirlb gene of rice (Oryza sativa) is one of a set of putative defense genes whose transcripts accumulate upon inoculation of rice with the non-host pathogen Pseudomonas syringae pv. syringae. It belongs to a family of genes encoding small extracellular proteins so far only identified in cereals. To assess the function of the Rirlb gene in rice blast resistance, it was placed under the control of the CaMV 35S promoter and transferred into rice plants of the japonica variety Taipei 309 by biolistic transformation of immature embryos. Two out of 12 hygromycin-resistant regenerated plants (OE1 and OE3) were fertile. DNA gel blot analysis suggested that these two T0 plants were independent transformants, each of which had stably incorporated one complete copy of the transgene into the genome. In addition, the OE1 plant appeared also a contain a rearranged copy or incomplete copy. T1 plants homozygous for the transgene were identified by DNA gel blot analysis of individual T2 progeny and further propagated. Expression analysis of the transgene showed that the transgene was active both in T1 plants and homozygous decendants. Challenge inoculation of homozygous transgenic plants with Magnaporthe grisea, the causal agent of rice blast disease, revealed that both independent transgenic lines were more resistant than the untransformed wild type, suggesting that over-expression of the Rirlb gene confers partial resistance against this important pathogen.

A role for ectophosphatase in xenobiotic resistance.

Xenobiotic resistance in animals, plants, yeast, and bacteria is known to involve ATP binding cassette transporters that efflux invading toxins. We present data from yeast and a higher plant indicating that xenobiotic resistance also involves extracellular ATP degradation. Transgenic upregulation of ecto-ATPase alone confers resistance to organisms that have had no previous exposure to toxins. Similarly, cells that are deficient in extracellular ATPase activity are more sensitive to xenobiotics. On the basis of these and other supporting data, we hypothesize that the hydrolysis of extracellular ATP by phosphatases and ATPases may be necessary for the resistance conferred by P-glycoprotein.

Syringolin-mediated activation of the Pir7b esterase gene in rice cells is suppressed by phosphatase inhibitors.

Inoculation of rice plants (Oryza sativa) with the nonhost pathogen Pseudomonas syringae pv. syringae leads to the activation of defense-related genes and ultimately to induced resistance against the rice blast fungus Pyricularia oryzae. One of the molecular determinants of P. syringae pv. syringae that is recognized by the plant cells and evokes these defense responses is syringolin A, an elicitor that is secreted by the bacteria under appropriate conditions. In order to investigate signal transduction events elicited by syringolin A, the response of cultured rice cells to syringolin A application was analyzed. Cultured rice cells were able to sense syringolin A at concentrations in the nanomolar range as observed by the transient accumulation of Pir7b esterase transcripts. Syringolin A-mediated Pir7b transcript accumulation was inhibited by cycloheximide, indicating that de novo protein synthesis was required. Calyculin and okadaic acid, two protein phosphatase inhibitors, blocked Pir7b gene induction, whereas the serine/threonine protein kinase inhibitors staurosporine and K-252a had no effect on Pir7b transcript levels. Actin transcript levels were essentially not affected by inhibitor treatments over the experimental time span. These results imply that dephosphorylation of a phosphoprotein is an important step in the syringolin A-triggered signal transduction pathway.

Technical advance. Double-stranded RNA interferes with gene function at the single-cell level in cereals.

Double-stranded RNA (dsRNA) has been shown to specifically interfere with gene function in several organisms including tobacco and the model plant Arabidopsis. Here, we report on rapid and sequence-specific interference of dsRNA with gene function in cereals. Delivery of cognate dsRNA into single epidermal cells of maize, barley or wheat by particle bombardment interfered with the function of co-bombarded UidA (GUS) and TaGLP2a:GFP reporter genes. Cognate dsRNA was also found to specifically interfere with the function of the endogenous genes A1 and Ant18 encoding dihydroflavonol-4-reductase in maize and barley, respectively. Dihydroflavonol-4-reductase is an essential enzyme of the anthocyanin biosynthetic pathway in maize and barley. This pathway can be induced by transient expression of the C1- and b-Peru genes that encode transcription factors. In the presence of dsRNA corresponding to the dihydroflavonol-4-reductase gene, C1- and b-Peru-dependent, cell-autonomous accumulation of red anthocyanin pigments in bombarded cells of maize and barley was reduced. dsRNA was also demonstrated to negatively interfere with Mlo, which encodes a negative regulator of race non-specific resistance to the powdery mildew fungus in barley. In the presence of Mlo dsRNA, transformed cells became more resistant, thereby phenocopying plants that carry a heritable loss-of function mlo resistance allele. The results suggest that direct delivery of dsRNA to cereals leads to a rapid and sequence-specific interference with gene function at the single-cell level.

Transient expression of members of the germin-like gene family in epidermal cells of wheat confers disease resistance.

The wheat genome encodes a family of germin-like proteins that differ with respect to regulation and tissue specificity of expression of the corresponding genes. While germin exhibits oxalate oxidase (E.C. 1.2.3.4.) activity, the germin-like proteins (GLPs) have no known enzymatic activity. A role of oxalate oxidase in plant defence has been proposed, based on the capacity of the enzyme to produce H2O2, a reactive oxygen species. The role in defence of germin and other members of the germin-like gene family was functionally assessed in a transient assay system based on particle bombardment of wheat leaves. Transient expression of the pathogen-induced germin gf-2.8 gene, but not of the constitutively expressed HvGLP1 gene, reduced the penetration efficiency of Blumeria (syn. Erysiphe) graminis f.sp. tritici, the causal agent of wheat powdery mildew, on transformed cells. Two engineered germin-gf-2.8 genes and the TaGLP2a gene, which all encoded proteins without oxalate oxidase activity, also reduced the penetration efficiency of the fungus, demonstrating that oxalate oxidase activity is not required for conferring enhanced resistance. Instead, activity tagging experiments showed that in cells transiently expressing the germin gf-2.8 gene, the transgene product became insolubilized at sites of attempted fungal penetration where localised production of H2O2 was observed. Thus, germin and GLPs may play a structural role in cell-wall re-enforcement during pathogen attack.

Involvement of an ABC transporter in a developmental pathway regulating hypocotyl cell elongation in the light

In the dark, plant seedlings follow the skotomorphogenetic developmental program, which results in hypocotyl cell elongation. When the seedlings are exposed to light, a switch to photomorphogenetic development occurs, and hypocotyl cell elongation is inhibited. We have manipulated the expression of the AtPGP1 (for Arabidopsis thaliana P glycoprotein1) gene in transgenic Arabidopsis plants by using sense and antisense constructs. We show that within a certain light fluence rate window, overexpression of the AtPGP1 gene under the control of the cauliflower mosaic virus 35S promoter causes plants to develop longer hypocotyls, whereas expression of the gene in antisense orientation results in hypocotyls shorter than those occurring in the wild type. In the dark, hypocotyls of transgenic and wild-type plants are indistinguishable. Because the AtPGP1 gene encodes a member of the superfamily of ATP binding cassette-containing (ABC) transporters, these results imply that a transport process is involved in a hypocotyl cell elongation pathway active in the light. The AtPGP1 transporter is localized in the plasmalemma, as indicated by immunohistochemical techniques and biochemical membrane separation methods. Analysis of the AtPGP1 expression pattern by using reporter gene constructs and in situ hybridization shows that in wild-type seedlings, AtPGP1 is expressed in both the root and shoot apices.

Characterization of the rice pathogen-related protein Rir1a and regulation of the corresponding gene.

In rice (Oryza sativa L.), local acquired resistance against Pyricularia oryzae (Cav.), the causal agent of rice blast, can be induced by a preinoculation with the non-host pathogen Pseudomonas syringae pv. syringae. We have cloned a cDNA (Rir1a) and a closely related gene (Rir1b) corresponding to transcripts that accumulate in leaf tissue upon inoculation with P. syringae pv. syringae. The cDNA encodes a putative 107 amino acid protein, Rir1a, that exhibits a putative signal peptide cleavage site in its hydrophobic N-terminal part and a C-terminal part that is relatively rich in glycine and proline. The Rir1b gene contains a Tourist and a Wanderer miniature transposable element in its single intron and encodes a nearly identical protein. Rir1a is similar in sequence (ca. 35% identical and ca. 60% conservatively changed amino acids) to the putative Wir1 family of proteins that are encoded by pathogen-induced transcripts in wheat. Using antibodies raised against a Rir1a-fusion protein we show that Rir1a is secreted from rice protoplasts transiently expressing a 35S::Rir1a construct and that the protein accumulates in the cell wall compartment of rice leaves upon inoculation with P. syringae pv. syringae. Possible roles of Rir1a in pathogen defense are discussed.

The defense-related rice gene Pir7b encodes an alpha/beta hydrolase fold protein exhibiting esterase activity towards naphthol AS-esters.

Acquired resistance of rice to Pyricularia oryzae, the causing agent of rice blast, can be induced by inoculation with the non-host pathogen Pseudomonas syringae pv. syringae. We have previously cloned a cDNA and a corresponding gene (Pir7b) whose transcripts accumulate upon infiltration with the resistance-inducing bacteria. The putative encoded product Pir7b exhibits significant sequence similarity to two recently cloned hydroxynitrile lyases from Manihot esculenta (cassava) and Hevea brasisliensis, enzymes involved in the release of hydrogen cyanide from cyanogenic glycosides. As rice does not contain cyanogenic glycosides, a similar function of Pir7b appears unplausible. In order to functionally characterize the protein, recombinant Pir7b was produced in Escherichia coli and Saccharomyces cerevisiae. We show that recombinant Pir7b does not have hydroxynitrile lyase activity, but exhibits esterase activity towards naphthol AS-acetate. Using Pir7b-specific antibodies, we show that the protein accumulates in rice leaves inoculated with P. syringae pv. syringae. Both the recombinant and the authentic proteins have an apparent molecular mass of 32 kDa (28.8 kDa calculated) and seem to be active as monomers. Pir7b esterase also exhibits sequence similarity to several expressed sequence tags of Arabidopsis thaliana, indicating that it belongs to a family of proteins widely occuring in plants.

Mechanosensitive expression of a lipoxygenase gene in wheat.

Touch stimulation of wheat (Triticum aestivum L.) seedlings led to a strong and dose-dependent increase in the level of lipoxygenase mRNA transcripts. The touch-induced response occurred within 1 h and was transient. A similar response was observed after wind treatment and wounding. The mechanical strain-regulated lipoxygenase might translate mechanical strain into lipoxygenase pathway-dependent growth responses.

Expression and antisense inhibition of transgenes in Phytophthora infestans is modulated by choice of promoter and position effects.

Procedures were identified for manipulating the expression of genes in the oomycete fungus, Phytophthora infestans. The activities of five putative promoter sequences, derived from the 5' regions of oomycete genes, were measured in transient assays performed in protoplasts and in stable transformants. The sequences tested were from the ham34 and hsp70 genes of Bremia lactucae, the actin-encoding genes of P. infestans and P. megasperma, and a polyubiquitin-encoding gene of P. infestans. Experiments using the GUS reporter gene (encoding beta-glucuronidase) demonstrated that each 5' fragment had promoter activity, but that their activities varied over a greater than tenfold range. Major variation was revealed in the level of transgene expression in individual transformants containing the same promoter::GUS or promoter::lacZ fusion. The level of expression was not simply related to the number of genes present, suggesting that position effects were also influencing expression. Fusions between the ham34 promoter, and full-length and partial GUS genes in the antisense orientation blocked the expression of GUS in protoplasts and in stable transformants.

Differential induction of distinct glutathione-S-transferases of wheat by xenobiotics and by pathogen attack.

We have previously characterized a pathogen-induced gene from wheat (Triticum aestivum L.) that was named GstA1 based on sequence similarities with glutathione-S-transferases (GSTs) of maize (R. Dudler, C. Hertig, G. Rebmann, J. Bull, F. Mauch [1991] Mol Plant Microbe Interact 4: 14-18). We have constructed a full-length GstA1 cDNA by combinatorial polymerase chain reaction and demonstrate by functional expression of the cDNA in Escherichia coli that the GstA1-encoded protein has GST activity. An antiserum raised against a GstA1 fusion protein specifically recognized a protein with an apparent molecular mass of 29 kD on immunoblots of extracts from bacteria expressing the GstA1 cDNA and extracts from wheat inoculated with Erysiphe graminis. The GstA1-encoded protein was named GST29. RNA and immunoblot analysis showed that GstA1 was only weakly expressed in control plants and was specifically induced by pathogen attack and by the GST substrate glutathione, but not by various xenobiotics. In contrast, a structurally and antigenically unrelated GST with an apparent molecular mass of 25 kD that was detected with an antiserum raised against GSTs of maize was expressed at a high basal level. This GST25 and an additional immunoreactive protein named GST26 were strongly induced by cadmium and by the herbicides atrazine, paraquat, and alachlor, but not by pathogen attack. Compared with the pathogen-induced GST29, GST25 and GST26 showed a high affinity toward glutathione-agarose and were much more active toward the model substrate 1-chloro-2,4-dinitrobenzene. Thus, wheat contains at least two distinct GST classes that are differentially regulated by xenobiotics and by pathogen attack and whose members have different enzymic properties. GST25 and GST26 appear to have a function in xenobiotic metabolism, whereas GST29 is speculated to fulfill a more specific role in defense reactions against pathogens.

Circadian rhythmicity in the expression of a novel light-regulated rice gene.

We have identified and analyzed cDNAs corresponding to a single-copy gene from rice, designated lir1, whose expression exhibits dramatic diurnal fluctuations. The cDNAs encode a putative protein of 128 amino acids with no homology to known proteins. Lir1 mRNA accumulates in the light, reaching maximum and minimum steady-state levels at the end of the light and dark period, respectively. The oscillations of lir1 mRNA abundance persist after the plants have been transferred to continuous light or darkness. Plants germinated in the dark have very low levels of lir1 mRNA, whereas plants germinated in continuous light express lir1 at an intermediate but constant level. These results indicate that lir1 expression is controlled by light and a circadian clock.

Sequence and expression of a wheat gene that encodes a novel protein associated with pathogen defense.

Wheat (Triticum aestivum) exhibits local acquired resistance to the powdery mildew pathogen Erysiphe graminis f. sp. tritici. The resistant state can be induced by a preinoculation with the nonhost pathogen E. g.f. sp. hordei, the barley powdery mildew, and is accompanied by the activation of putative defense genes. Here, we report the sequence of a pathogen-induced gene, WIR1a, and a corresponding cDNA, WIR1, that encode novel defense-related proteins of 88 and 85 amino acids, respectively. Analysis of the primary structure of these proteins predicts them to be integral membrane proteins with extracytoplasmic C-terminal domains rich in proline and glycine, through which the proteins possibly interact with the cell wall.