A linear RNA replicon from the oomycete Phytophthora infestans

An unusual RNA element was discovered in an isolate of the oomyceteous fungus Phytophthora infestans. The RNA exists predominantly as single-stranded molecules of about 625 nucleotides with complementary strands present at a ratio of approximately 130:1. Gel mobility and PCR assays indicated that the element was linear. The RNA appeared to be an autonomous element, since P. infestans DNA did not contain cross-hybridizing sequences. Standard methods for virus purification yielded no evidence for encapsidation of the RNA, or for other virus particles in the isolate bearing the replicon. The replicon contained polyU and polyA tracts at its 5′ and 3′ termini, respectively, with a central region that had a GC content of 47%, and lacked obvious ORFs. Two-thirds of the replicon co-purified with nuclei, at about 200 copies per nucleus, while one-third resided in a cytoplasmic but non-mitochondrial location. Maternal inheritance was observed in sexual crosses, with a few exceptions. The replicon was not widely distributed throughout the species and had little effect on growth or pathogenicity. The data suggest that the RNA is best characterized as a novel linear RNA plasmid. Received: 3 September 1999 / Accepted: 12 December 1999     

Cloning and characterization of a theta class glutathione transferase from the potato pathogen Phytophthora infestans

A glutathione transferase (GST) related to the theta (T) class of enzymes found in plants and animals has been cloned from the potato pathogen Phytophthora infestans. The cDNA encoded a 25kDa polypeptide termed PiGSTT1 which was expressed in E. coli as the native protein. The purified recombinant enzyme behaved as a dimer (PiGSTT1-1) and while being unable to catalyse the glutathione conjugation of 1-chloro-2,4-dintrobenzene, was highly active as a glutathione peroxidase with organic hydroperoxide substrates. In addition to reducing the synthetic substrate cumene hydroperoxide, PiGSTT1-1 was shown to be highly active toward 9(S)-hydroperoxy-(10E,12Z,15Z)-octadecatrienoic acid=9(S)-HPOT, which is formed in potato plants during infection by P. infestans as a precursor of the antifungal oxylipin colnelenic acid. An antiserum was raised to PiGSTT1-1 and used to demonstrate that the respective enzyme was abundantly expressed in P. infestans both cultured on pea agar and during the infection of potato plants.     

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.     

Dominance and recessiveness at loci for virulence against potato and tomato in Phytophthora infestans

Summary In this study we investigated the genetic control of virulence in the diploid fungal pathogen, Phytophthora infestans, against host resistance genes R1, R2, R3, and R4 (potato) and Ph1 (tomato). For four of these virulence traits, the presence or absence of segregation indicated conclusively which phenotype was dominant. We observed a 31 (virulentavirulent) segregation on R2 in the progeny of parents which were both virulent, suggesting that virulence is dominant and both parents are heterozygous. In a cross in which one parent was virulent and the other avirulent on potato gene R3, all progeny tested were avirulent, so avirulence against R3 is dominant. The same virulent parent crossed with a different avirulent parent produced virulent and avirulent progeny in a 13 ratio, indicating that a second locus may be involved. The progeny of two parents virulent on R4 segregated for virulence and avirulence, so virulence against R4 is dominant. For Ph1, a 13 segregation in the progeny of two avirulent parents showed that the avirulent phenotype is dominant, and a 31 ration in a second cross suggested the involvement of a second locus. The segregations for virulence against R1 did not indicate which phenotype was dominant, but did suggest singlelocus control.     

Inhibitory effect of a defensin gene from the Andean crop maca (Lepidium meyenii) against Phytophthora infestans

In this study, we report the isolation of a defensin gene, lm-def, isolated from the Andean crop 'maca' (Lepidium meyenii) with activity against the pathogen Phytophthora infestans responsible of late blight disease of the potato and tomato crops. The lm-def gene has been isolated by polymerase chain reaction (PCR) using degenerate primers corresponding to conserved regions of 13 plant defensin genes of the Brassicaceae family assuming that defensin genes are highly conserved among cruciferous species. The lm-def gene belongs to a small multigene family of at least 10 members possibly including pseudogenes as assessed by genomic hybridization and nucleotide sequence analyses. The deduced mature Lm-Def peptide is 51 amino acids in length and has 74-94% sequence identity with other plant defensins of the Brassicaceae family. The Lm-Def peptide was produced as a fusion protein using the pET-44a expression vector and purified using an immobilized metal ion affinity chromatography. The recombinant protein (NusA:Lm-Def) exhibited in vitro activity against P. infestans. The NusA:Lm-Def protein caused growth inhibition and hyphal damage at concentration not greater than 0.4 microM. In contrast, the NusA protein alone expressed and purified similarly did not show any activity against P. infestans. Therefore, these results indicate that the lm-def gene isolated from maca belong to the plant defensin family with activity against P. infestans. Its expression in potato, as a transgene, might help to control the late blight disease caused by P. infestans with the advantage of being of plant origin.     

A quantitative Real Time PCR based method for the detection of Phytophthora infestans causing Late blight of potato,in infested soil

A fast and simple polymerase chain reaction method has been developed for detection of Phytophthora infestans oospores, the causal agent of Late blight of Potato in soil. The method involves the disruption of oospores by grinding dry soil, using abrasive properties, in the presence of glass powder and skimmed milk powder within a short time. The latter prevents loss of DNA by adsorption to soil particles or by degradation and reduces the co-extraction of PCR inhibitors with the DNA. After phenol/chloroform extraction; the DNA is suitable for direct PCR amplification without a precipitation step. This amplification leads to detection of pathogen in infested soils before planting of crop. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. infestans detection to confirm positive inoculum level in potato seeds and elsewhere. With increasing amounts of standard DNA templates, the respective threshold cycle (Ct) values were determined and a linear relationship was established between these Ct values and the logarithm of initial template amounts. The method is rapid, cost efficient, and when combined with suitable internal controls can be applied to the detection and quantification of P. infestans oospores on a large-scale basis.     

Beta-xylosidase activity of a GH3 glucosidase/xylosidase from yak rumen metagenome promotes the enzymatic degradation of hemicellulosic xylans

Aims: To characterize the duel activities of a glycosyl hydrolase family 3 β‐glucosidase/xylosidase from rumen bacterial metagenome and to investigate the capabilities of its β‐d ‐xylosidase activities for saccharification of hemicellulosic xylans. Methods and Results: A β‐glucosidase/xylosidase gene RuBGX1 was cloned from yak (Bos grunniens) rumen using the metagenomic technology. Recombinant RuBGX1, expressed in Escherichia coli, demonstrated high hydrolytic activities on both p‐nitrophenyl‐β‐d ‐glucopyranoside (pNP‐Glc) and p‐nitrophenyl‐β‐d ‐xylopyranoside (pNP‐Xyl) substrates. Analysis of the kinetic properties indicated that RuBGX1 had a lower affinity for pNP‐Glc substrate as the K_m was 0·164 mmol l^?1 for pNP‐Glc and 0·03 mmol l^?1 for pNP‐Xyl at pH 6·0 and 50°C, respectively. The capabilities of RuBGX1 β‐xylosidase for hydrolysis of xylooligosaccharide substrates were further investigated using an endoxylanase‐coupled assay. Hydrolysis time courses illustrated that a significant increase (about 50%) in the reducing sugars, including xylobiose, xylotriose and xylotetraose, was achieved by supplementing endoxylanase with RuBGX1. Enzymatic product analysis using high‐performance anion‐exchange chromatography‐pulsed amperometric detection showed that RuBGX1 could release xyloses from intermediate xylooligosaccharides produced by endoxylanase. Conclusions: The RuBGX1 shows β‐glucosidase activity in hydrolysis of cello‐oligosaccharides; meanwhile, it has β‐xylosidase activity and functions synergistically with endoxylanase to promote the degradation of hemicellulosic xylans. Significance and Impact of the study: This was the first to report the β‐xylosidase activity of family 3 β‐glucosidase/xylosidase functioned in the degradation of hemicellulosic xylans. The bifunctional β‐glucosidase/xylosidase property of RuBGX1 can be used in simultaneous saccharification of cellulose and xylan into fermentable glucose and xylose.     

General resistance to late blight of Solanum tuberosum plants regenerated from callus resistant to culture filtrates of Phytophthora infestans

Summary Resistance of potato leaflets to culture filtrates of Phytophthora infestans is correlated with lower growth of the congenial parasite but not with lower sporulation.     

Selection of potato callus for resistance to culture filtrates of Phytophthora infestans and regeneration of resistant plants

Summary Dihaploid calli from Solanum tuberosum were selected, which were resistant to the culture filtrate of Phytophthora infestans. Each of the resistant calli was resistant to all four pathotypes of Phytophthora used in these experiments. The resistance was not lost through regeneration and the induction of new callus.     

The elicitor-induced oxidative processes in leaves of Solanum species with differential polygenic resistance to Phytophthora infestans

Previous studies have shown that suspension-cultured cells of Solanum genotypes with various polygenic resistances to Phytophthora infestans differed in activities of early oxidative processes in response to culture filtrate (CF) from this pathogen. These studies have now been extended by analysing production of reactive oxygen species (ROS), lipid peroxidation and Lipoxygenase (LOX, E.C.1.13.11.12) activity induced by CF in detached leaves of S. tuberosum cv Bzura and clone H-8105, polygenically resistant and susceptible, respectively, as well as S. nigrum, nonhost, completely resistant. The relative increase in the ROS production was higher in the susceptible clone H-8105 than in both resistant genotypes. Lipid peroxidation increased only in the nonhost S. nigrum. An increase in lipid peroxidation in S. nigrum leaves coincided with enhanced LOX activity. In both S. tuberosum genotypes, significant increases in LOX activity were delayed and unaccompanied by changes in the level of lipid peroxidation. LOX activity attained a higher level in both of the resistant genotypes than in the susceptible one. The present results suggest that the involvement of both ROS production and LOX activity in the defense strategy in Solanum species/P. infestans interactions.     

Antimicrobial Activities of the Essential Oils of Various Plants against Tomato Late Blight Disease Agent Phytophthora infestans

The aim of this study was to find an alternative to synthetic fungicides currently used in the control of devastating oomycete pathogen Phytophthora infestans, causal agent of late blight disease of tomato. Antifungal activities of essential oils obtained from aerial parts of aromatic plants such as oregano (Origanum syriacum var. bevanii), thyme (Thymbra spicata subsp. spicata), lavender (Lavandula stoechas subsp. stoechas), rosemary (Rosmarinus officinalis), fennel (Foeniculum vulgare), and laurel (Laurus nobilis), were investigated against P. infestans. Both contact and volatile phase effects of different concentrations of the essential oils used were determined by using two in vitro methods. Chemical compositions of the essential oils were also determined by GC-MS analysis. Major compounds found in essential oils of thyme, oregano, rosemary, lavender, fennel and laurel were carvacrol (37.9%), carvacrol (79.8), borneol (20.4%), camphor (20.2%), anethole (82.8%) and 1,8-cineole (35.5%), respectively. All essential oils were found to inhibit the growth of P. infestans in a dose-dependent manner. Volatile phase effect of oregano and thyme oils at 0.3 μg/ml air was found to completely inhibit the growth of P. infestans. Complete growth inhibition of pathogen by essential oil of fennel, rosemary, lavender and laurel was, however, observed at 0.4–2.0 μg/ml air concentrations. For the determination of the contact phase effects of the tested essential oils, oregano, thyme and fennel oils at 6.4 μg/ml were found to inhibit the growth of P. infestans completely. Essential oils of rosemary, lavender and laurel were inhibitory at relatively higher concentrations (12.8, 25.6, 51.2 μg/ml respectively). Volatile phase effects of essential oils were consistently found to be more effective on fungal growth than contact phase effect. Sporangial production was also inhibited by the essential oil tested. Light and scanning electron microscopic (SEM) observation on pathogen hyphae, exposed to both volatile and contact phase of oil, revealed considerable morphological alterations in hyphae such as cytoplasmic coagulation, vacuolations, hyphal shrivelling and protoplast leakage.     

An in planta induced gene of Phytophthora infestans codes for ubiquitin

An in planta induced gene of Phytophthora infestans (the causal organism of potato late blight) was selected from a genomic library by differential hybridization using labelled cDNA derived from poly(A)+ RNA of P. infestans grown in vitro and labelled cDNA made from potato-P. infestans interaction poly(A)+ RNA as probes. Sequence analysis showed that the gene codes for ubiquitin, a highly conserved protein which plays an important role in several cellular processes. The structure of the polyubiquitin gene (designated ubi3 R) is consistent with the structure of other known polyubiquitin genes. It consists of three repeats in a head-to-tail arrangement without intervening sequences, each encoding a ubiquitin unit of 76 amino acids. The last ubiquitin unit is followed by an extra asparagine residue at the carboxy-terminal end. Northern and Southern blot analyses revealed that the polyubiquitin gene is a member of a multigene family, all genes of which show induced expression in planta.     

Ribonuclease and proteinase activities in potato leaves immunized against Phytophthora infestans

Local treatment of potato plants with arachidonic acid caused a repeated increase of ribonuclease and proteinase activities in leaves that were directly sprayed with this inducer. Immunization caused no significant systemic changes in the enzyme activity of other potato leaves. However, leaves above the induction zone in the first days after the challenge were found to have a pronounced ribonuclease activity increase in comparison to that gradually rising with disease development in inoculated leaves from plants that were not induced. The proteolytic activity in the leaves of immunized plants after the challenge was decisively on a lower level than that in the leaves of noninduced plants subjected to inoculation.     

New glucosidase activities identified by functional screening of a genomic DNA library from the gut microbiota of the termite Reticulitermes santonensis

β-Glucosidases are widely distributed in living organisms and play a major role in the degradation of wood, hydrolysing cellobiose or cello-oligosaccharides to glucose. Termites are among the rare animals capable of digesting wood, thanks to enzyme activities of their own and to enzymes produced by their gut microbiota. Many bacteria have been identified in the guts of lower termites, some of which possess cellulolytic or/and hemicellulolytic activity, required for digesting wood. Here, having isolated bacterial colonies from the gut of Reticulitermes santonensis, we constructed in Escherichia coli a genomic DNA library corresponding to all of the colonies obtained and screened the library for clones displaying β-glucosidase activity. This screen revealed 8 positive clones. Sequence analysis with the BLASTX program revealed putative enzymes belonging to three glycoside hydrolase families (GH1, GH3 and GH4). Agar-plate tests and enzymatic assays revealed differences between the GH1- and GH3-type enzymes (as regards substrate specificity and regulation) and a difference in substrate specificity within the GH3 group. The substrate specificities and characteristic activities of these enzymes suggest that they may intervene in the depolymerisation of cellulose and hemicellulose.     

The R1 gene conferring race-specific resistance to Phytophthora infestans in potato is located on potato chromosome V.

Summary Late blight in potato is caused by the fungusPhytophthora infestans and can inflict severe damage on the potato crop. Resistance toP. infestans is either based on major dominantR genes conferring vertical, race-specific resistance or on minor genes inducing horizontal, unspecific resistance. A dihaploid potato line was identified which carried theR1 gene, conferring vertical resistance to allP. infestans races, with the exception of those homozygous for the recessive virulence allele of the locusV1. The F₁ progeny of a cross between this resistant parent P(R1) and P(r), a line susceptible to all races, was analysed for segregation ofR1 and of restriction fragment length polymorphism (RFLP) markers distributed on the potato RFLP map comprising more than 300 loci. TheR1 locus was mapped to chromosome V in the interval between RFLP markers GP21 and GP179. The map position ofR1 was found to be very similar to the one ofRx2, a dominant locus inducing extreme resistance to potato virus X.     

Acceptor-dependent regioselectivity of glycosynthase reactions by Streptomyces E383A beta-glucosidase

The nonnucleophilic mutant E383A beta-glucosidase from Streptomyces sp. has proven to be an efficient glycosynthase enzyme, catalyzing the condensation of alpha-glucosyl and alpha-galactosyl fluoride donors to a variety of acceptors. The enzyme has maximal activity at 45 degrees C, and a pH-dependence reflecting general base catalysis with an apparent kinetic pKa of 7.2. The regioselectivity of the new glycosidic linkage depends unexpectedly on the acceptor substrate. With aryl monosaccharide acceptors, beta-(1-->3) disaccharides are obtained in good to excellent yields, thus expanding the synthetic products available with current exo-glycosynthases. With xylopyranosyl acceptor, regioselectivity is poorer and results in the formation of a mixture of beta-(1-->3) and beta-(1-->4) linkages. In contrast, disaccharide acceptors produce exclusively beta-(1-->4) linkages. Therefore, the presence of a glycosyl unit in subsite +II redirects regioselectivity from beta-(1-->3) to beta-(1-->4). To improve operational performance, the E383A mutant was immobilized on a Ni2+-chelating Sepharose resin. Immobilization did not increase stability to pH and organic solvents, but the operational stability and storage stability were clearly enhanced for recycling and scaling-up.