A conditionally dispensable chromosome controls host-specific pathogenicity in the fungal plant pathogen Alternaria alternata.
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The filamentous fungus Alternaria alternata contains seven pathogenic variants (pathotypes), which produce host-specific toxins and cause diseases on different plants. Previously, the gene cluster involved in host-specific AK-toxin biosynthesis of the Japanese pear pathotype was isolated, and four genes, named AKT genes, were identified. The AKT homologs were also found in the strawberry and tangerine pathotypes, which produce AF-toxin and ACT-toxin, respectively. This result is consistent with the fact that the toxins of these pathotypes share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid structural moiety. In this study, three of the AKT homologs (AFT1-1, AFTR-1, and AFT3-1) were isolated on a single cosmid clone from strain NAF8 of the strawberry pathotype. In NAF8, all of the AKT homologs were present in multiple copies on a 1.05-Mb chromosome. Transformation-mediated targeting of AFT1-1 and AFT3-1 in NAF8 produced AF-toxin-minus, nonpathogenic mutants. All of the mutants lacked the 1.05-Mb chromosome encoding the AFT genes. This chromosome was not essential for saprophytic growth of this pathogen. Thus, we propose that a conditionally dispensable chromosome controls host-specific pathogenicity of this pathogen. (+info)
Intestinal absorption enhancement of the ester prodrug tenofovir disoproxil fumarate through modulation of the biochemical barrier by defined ester mixtures.
(2/200)
The effect of discrete esters and ester mixtures on the intestinal stability and absorption of tenofovir disoproxil fumarate (tenofovir DF, an esterase-sensitive prodrug of the antiviral tenofovir) was compared with the effect of strawberry extract, which has been shown to enhance the absorption of the prodrug across Caco-2 monolayers and in rat ileum. In addition, the mechanism of absorption enhancement was investigated. In rat intestinal homogenates, complete inhibition of the conversion of tenofovir DF (as obtained by strawberry extract) could only be obtained at relatively high concentrations of the discrete esters or by using mixtures of esters (e.g., propyl p-hydroxybenzoate 0.02%, octyl acetate 0.02%, ethyl caprylate 0.01%). Coincubation of tenofovir DF with this mixture also resulted in an enhancement of its absorption in the in vitro Caco-2 system as well as in rat ileum. As tenofovir DF is a substrate for P-glycoprotein (P-gp)-related efflux carriers in the Caco-2 model, the modulatory effect of the ester mixtures was studied on the functionality of P-gp using cyclosporin A (CsA) as a model substrate. Strawberry extract as well as the mixture of three esters interfered with the absorptive transport of CsA across Caco-2 monolayers, illustrating that both mixtures interfere with both esterase-activity and P-gp functionality. This concerted barrier was not observed in rat ileum, suggesting differential functional activities of the biochemical barrier toward tenofovir DF in different absorption systems. Overall, our results illustrate that modulation of the biochemical barrier (metabolism and efflux) of tenofovir DF by ester mixtures can be used to increase the intestinal absorption of tenofovir DF in an in vitro and an in situ absorption model; the mechanism of action appears to be a complex interplay of different systems; the differential expression of carriers and enzymes in different systems illustrates the difficulty of extrapolating observations between different systems/species. (+info)
Characterization of a strawberry cDNA clone homologous to calcium-dependent protein kinases that is expressed during fruit ripening and affected by low temperature.
(3/200)
A full-length cDNA clone (FaCDPK1) encoding a calcium-dependent protein kinase (CDPK) has been isolated from a strawberry fruit cDNA library. FaCDPK1 contains the basic features of CDPKs: a catalytic kinase domain linked to a regulatory calmodulin-like domain by a junction sequence that has been shown to act as an autoinhibitory pseudosubstrate. Although the calmodulin-like domain of CDPKs typically contains four EF-hand calcium-binding motifs, FaCDPK1 was predicted to contain only three EF-hand motifs. FaCDPK1 gene expression was observed in roots, stolons, meristems, flowers, and leaves. FaCDPK1 mRNA was not detected in young fruits, but accumulated as fruit turned to white, suggesting a role for this gene in the developing strawberry fruit. In ripe fruit the levels of transcript increased in response to low temperature. (+info)
Isolation and promoter analysis of two genes encoding different endo-beta-1,4-glucanases in the non-climacteric strawberry.
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Two endo-beta-1,4-glucanase (EGase; EC 3.2.1.4.) genes, highly expressed during ripening of the non-climacteric strawberries (Fragariaxananassa Duch. cv. Chandler), were isolated. Serial promoter deletions of both genes (i.e. FaEG1 and FaEG3) fused to GUS were transiently assayed in strawberry fruits by using a technique recently developed in this laboratory. Although differences were observed with the short fragments, GUS activity became comparable with the largest fragments of both promoters. The apparently similar strength of the two largest promoter fragments was in contrast with previous results of Northern analyses which demonstrated different transcripts amounts for the two genes. The inclusion of the 3' flanking region of both genes in the transient assays showed that, in the case of FaEG3, the 3' region had a down-regulating effect on the expression of GUS, and this might account for the lower amount of FaEG3 mRNA usually observed in ripe fruits compared to that of FaEG1. Downstream instability elements might be involved in such down-regulation. (+info)
Cloning and characterization of two ripening-related strawberry (Fragaria x ananassa cv. Chandler) pectate lyase genes.
(5/200)
Two genomic clones corresponding to putative pectate lyase genes (plA and plB) were isolated and characterized in strawberry (Fragaria x ananassa cv. Chandler). The corresponding ORFs for the plA and plB genes revealed deduced proteins of 451 and 439 amino acids, respectively, that differ from that of the previously isolated strawberry plC gene. Southern blot analysis has shown that while the plB gene is a single copy gene, the plA gene is probably encoded by a small multigene family. By using specific probes corresponding to the untranslated 3' terminal region of the pl genes, and QRT-PCR methodology, the spatio-temporal expression pattern of both strawberry pl genes have been compared with that of the plC gene. The three transcripts were specifically expressed only in fruit and mainly during the ripening stages. Moreover, the expression of the plA and plB genes was induced in green de-achened fruit, but this increase was reduced by the external application of auxins as was the expression of plC. The expression of both pl genes was also strongly reduced in harvested fruit kept in controlled atmosphere (CA) containing high CO(2) levels. Immunolocalization studies using antibodies raised against the strawberry PL proteins placed the proteins in the cell wall of parenchymatic cells of the fruit receptacle. The role of pl genes in cell-wall disassembly and fruit ripening softening is discussed. (+info)
Plant chitinase as a possible biocontrol agent for use instead of chemical fungicides.
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We investigated whether a plant chitinase can be used as a biocontrol agent instead of chemical fungicides by spraying chitinase E (family 19; class IV) from a yam (Dioscorea opposita Thunb) alone or together with beta-1,3-glucanase directly onto the surface of a powdery mildew infecting strawberry berries and leaves. Results were observed by eye and with a scanning electron microscope. The powdery mildew infecting the strawberries was degraded, mainly by the chitinase, and the disease did not appear again for more than 2 weeks. These results indicated that this kind of plant chitinase might be safe and biodegradable biocontrol agent for use instead of conventional fungicides. (+info)
Strawberry anthocyanins are recovered in urine as glucuro- and sulfoconjugates in humans.
(7/200)
Anthocyanins are phenolic compounds widely distributed in fruits and vegetables. Their consumption has been shown to prevent some chronic diseases. Anthocyanin metabolism, however, is still not fully understood. The aim of this work was to evaluate the bioavailability of anthocyanins in humans consuming a meal containing strawberries and to identify possible metabolites in urine. Six healthy volunteers (three women and three men) consumed a meal containing 200 g strawberries (providing 179 micro mol pelargonidin-3-glucoside). Urine samples were collected before and after the meal and rapidly treated by solid-phase extraction. Identification and quantification of anthocyanin metabolites were carried out by HPLC-ESI-MS-MS and HPLC with UV-visible detection, respectively. In addition to pelargonidin-3-glucoside, five anthocyanin metabolites were identified in urine: three monoglucuronides of pelargonidin, one sulfoconjugate of pelargonidin and pelargonidin itself. Total urinary excretion of strawberry anthocyanin metabolites corresponded to 1.80 +/- 0.29% (mean +/- SEM, n = 6) of pelargonidin-3-glucoside ingested. More than 80% of this excretion was related to a monoglucuronide. Four hours after the meal, more than two-thirds of anthocyanin metabolites had been excreted, although urinary excretion of the metabolites continued until the end of the 24-h experiment. This study demonstrated that anthocyanins were glucuro- and sulfo-conjugated in humans and that the main metabolite of strawberry anthocyanins in human urine was a monoglucuronide of pelargonidin. (+info)
Infectivity of recombinant strawberry vein banding virus DNA.
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Infectivity of the cloned DNA genome of strawberry vein banding virus (SVBV) was demonstrated by particle bombardment of 4-week-old strawberry (Fragaria vesca L. var. UC-5) plants with gold particles coated with the putative full-length 7.9 kb viral DNA. Vein banding symptoms developed on 15 % of inoculated plants 6-7 weeks post-inoculation. An approximate 1.25-mer of the viral DNA was cloned into the binary vector pCGN1547. Particle bombardment of this construct into strawberry plants gave an infection rate of 75 %. The construct was used for transformation of Agrobacterium tumefaciens, and infiltration of these cells into healthy strawberry leaves resulted in development of vein banding symptoms in 100 % of inoculated plants. Gel electrophoresis, Southern blot hybridization with an SVBV probe and sequence analyses of PCR-amplified DNA fragments were used to confirm SVBV infection in symptomatic plants. (+info)