ReDivia - Repositorio Digital de l'Instit Valencià d'Investigacions Agràries
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RinA controls phage-mediated packaging and transfer of virulence genes in Gram-positive bacteria
Get PDFPhage-mediated transfer of microbial genetic elements plays a crucial role in bacterial life style and evolution. In this study, we identify the RinA family of phage-encoded proteins as activators required for transcription of the late operon in a large group of temperate staphylococcal phages. RinA binds to a tightly regulated promoter region, situated upstream of the terS gene, that controls expression of the morphogenetic and lysis modules of the phage, activating their transcription. As expected, rinA deletion eliminated formation of functional phage particles and significantly decreased the transfer of phage and pathogenicity island encoded virulence factors. A genetic analysis of the late promoter region showed that a fragment of 272 bp contains both the promoter and the region necessary for activation by RinA. In addition, we demonstrated that RinA is the only phage-encoded protein required for the activation of this promoter region. This region was shown to be divergent among different phages. Consequently, phages with divergent promoter regions carried allelic variants of the RinA protein, which specifically recognize its own promoter sequence. Finally, most Gram-postive bacteria carry bacteriophages encoding RinA homologue proteins. Characterization of several of these proteins demonstrated that control by RinA of the phage-mediated packaging and transfer of virulence factor is a conserved mechanism regulating horizontal gene transfer
Detection and absolute quantitation of Broad bean wilt virus 1 (BBWV-1) and BBWV-2 by real time RT-PCR
No full textBroad bean wilt virus 1 (BBWV-1) and BBWV-2 are the two most significant viruses in the genus Fabavirus, causing damage to many economically important agricultural crops worldwide. A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using two TaqMan (R) MGB probes was developed for sensitive and specific detection and quantitation of BBWV-1 and BBWV-2. Primers and probes were designed from conserved sequence stretches to detect all isolates of each virus. Standard curves using RNA transcripts identical to both TaqMan (R) MGB probes enabled absolute quantitation, with a wide dynamic range and high sensitivity (10(3)-10(10) RNA molecules). RT-qPCR was assayed with genetically divergent BBWV-1 and BBWV-2 isolates from different plant hosts and countries, and was used to evaluate the temporal accumulation of BBWV-1 RNA in two plant hosts. (C) 2011 Elsevier B.V. All rights reserved
Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs
No full textNon-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca(2+) concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca(2+) concentration contained in the injection medium. in Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3-66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation. (C) 2011 Elsevier Inc. All rights reserved
Soil saturated hydraulic conductivity assessment from expert evaluation of field characteristics using an ordered logistic regression model
Get PDFThe knowledge of the soil saturated hydraulic conductivity (Ks) is essential for irrigation management purposes and for hydrological modelling. Several attempts have been done to estimate Ks in base of a number of soil parameters. However, a reliable enough model for qualitative Ks estimation based on the expert assessment of field characteristics had not been developed up to date. Five field characteristics, namely macroporosity (M), stoniness (S), texture (T), compaction (C) and sealing (L), in addition to tillage (G) were carefully assessed according to three classes each, in 202 sites in an agricultural irrigated area in Eastern Mediterranean Spain. After the evaluation of field characteristics, a single ring infiltrometer was used to determine the Ks value as the solution of the infiltration equation when the steady state was reached. The distribution of the Ks was assessed and five classes with 10-fold separations in class limits were defined accordingly. The relationships among site characteristics and Ks were analyzed through a correspondence analysis (CA). Next, an ordered logistic regression model (OLRM) for the prediction of the Ks class was developed. The CA revealed that, though tightly related, the set of six site characteristics should not be simplified into a smaller set, because each characteristic explains a significantly different aspect of Ks. Consequently, the OLRM was based on the six characteristics, which presented the following order of importance: L > M > G > T > C > S. According to the cross-validation of the OLRM the hit probability for the prediction of the Ks class attained an average value of 50%, which increased to 63% for the highest class of Ks. Moreover, wrong estimation of the Ks class exceeded the ±1 range only in 3% of sites. Therefore, a reliable enough assessment of Ks can be based on the expert assessment of field characteristics in combination with an OLRM. Highlights • The soil saturated hydraulic conductivity (Ks) was measured in 202 sites. • Sealing, macroporosity, tillage, texture, compaction and stoniness were assessed on basis expert criteria. • The six field features are related but explain different parts of Ks variability. • A generalized ordered logit model was developed for prediction of the Ks class. • An average hit probability of 50% was attained in the cross-validation
Identification and genetic characterization of an ethylene-dependent polygalacturonase from apricot fruit
No full textDuring fruit ripening a loss of firmness occurs, which is a key factor limiting postharvest life. In apricot, Prunus armeniaca La wide range of fruit firmness at commercial maturity has been observed in different cultivars. Endopolygalacturonase (endoPG) activity has been reported to be associated with differences in firmness in many fruit species, but never in apricot. In this paper, we reported the identification of an apricot cDNA (PaPG) coding for an endoPG-like protein with 393 amino acids. Protein sequence comparison with known polygalacturonases (PGs) revealed that multiple features as conserved domains and functional residues and a predicted signal peptide were present in PaPG. Moreover, a phylogenetic analysis of this and other plant PGs placed PaPG into a clade containing endoPGs expressed in fruit, abscission and dehiscence zones without a propeptide sequence, very close to PRF5 from peach (Prunus persica L Batsch). PaPG gene expression increased during postharvest storage of the fruit, correlating with fruit softening and ethylene release, and it responded to exogenous ethylene treatments. We localized the PaPG gene in apricot linkage group 4 after genetic mapping based on SNP analysis, in a position apparently syntenic to the PRF5 locus from peach. Results obtained offer genetic evidence supporting the hypothesis that PaPG and PRF5 are orthologous genes, and consequently position PaPG as a gene of interest for studies on fruit softening in apricot, and contribute to the development of molecular tools for breeding apricots with longer shelf life
Erwinia amylovora Novel Plasmid pEI70: Complete Sequence, Biogeography, and Role in Aggressiveness in the Fire Blight Phytopathogen
Get PDFComparative genomics of several strains of Erwinia amylovora, a plant pathogenic bacterium causal agent of fire blight disease, revealed that its diversity is primarily attributable to the flexible genome comprised of plasmids. We recently identified and sequenced in full a novel 65.8 kb plasmid, called pEI70. Annotation revealed a lack of known virulence-related genes, but found evidence for a unique integrative conjugative element related to that of other plant and human pathogens. Comparative analyses using BLASTN showed that pEI70 is almost entirely included in plasmid pEB102 from E. billingiae, an epiphytic Erwinia of pome fruits, with sequence identities superior to 98%. A duplex PCR assay was developed to survey the prevalence of plasmid pEI70 and also that of pEA29, which had previously been described in several E. amylovora strains. Plasmid pEI70 was found widely dispersed across Europe with frequencies of 5-92%, but it was absent in E. amylovora analyzed populations from outside of Europe. Restriction analysis and hybridization demonstrated that this plasmid was identical in at least 13 strains. Curing E. amylovora strains of pEI70 reduced their aggressiveness on pear, and introducing pEI70 into low-aggressiveness strains lacking this plasmid increased symptoms development in this host. Discovery of this novel plasmid offers new insights into the biogeography, evolution and virulence determinants in E. amylovora
Compatibility and incompatibility in S-RNase-based systems
No full textBackground S-RNase-based self-incompatibility (SI) occurs in the Solanaceae, Rosaceae and Plantaginaceae. In all three families, compatibility is controlled by a polymorphic S-locus encoding at least two genes. S-RNases determine the specificity of pollen rejection in the pistil, and S-locus F-box proteins fulfill this function in pollen. S-RNases are thought to function as S-specific cytotoxins as well as recognition proteins. Thus, incompatibility results from the cytotoxic activity of S-RNase, while compatible pollen tubes evade S-RNase cytotoxicity. Scope The S-specificity determinants are known, but many questions remain. In this review, the genetics of SI are introduced and the characteristics of S-RNases and pollen F-box proteins are briefly described. A variety of modifier genes also required for SI are also reviewed. Mutations affecting compatibility in pollen are especially important for defining models of compatibility and incompatibility. In Solanaceae, pollen-side mutations causing breakdown in SI have been attributed to the heteroallelic pollen effect, but a mutation in Solanum chacoense may be an exception. This has been interpreted to mean that pollen incompatibility is the default condition unless the S-locus F-box protein confers resistance to S-RNase. In Prunus, however, S-locus F-box protein gene mutations clearly cause compatibility. Conclusions Two alternative mechanisms have been proposed to explain compatibility and incompatibility: compatibility is explained either as a result of either degradation of non-self S-RNase or by its compartmentalization so that it does not have access to the pollen tube cytoplasm. These models are not necessarily mutually exclusive, but each makes different predictions about whether pollen compatibility or incompatibility is the default. As more factors required for SI are identified and characterized, it will be possible to determine the role each process plays in S-RNase-based SI
Algunos conceptos para la mejora de la rentabilidad en explotaciones cunícolas
Get PDFLa cunicultura no atraviesa por buenos momentos y la rentabilidad de muchas de las explotaciones españolas está cada vez más comprometida. En este trabajo se intenta conocer y razonar sobre algunas cuestiones generales
que podrían contribuir a la mejora de la rentabilidad de las explotaciones cunícolas. El conocimiento sobre el origen de los ingresos y costes de nuestra explotación tanto fijos como variables es imprescindible a la hora de adoptar medidas adecuadas que contribuyan a mejorar la situación económica de nuestras explotaciones
Terpene Down-Regulation in Orange Reveals the Role of Fruit Aromas in Mediating Interactions with Insect Herbivores and Pathogens
No full textPlants use volatile terpene compounds as odor cues for communicating with the environment. Fleshy fruits are particularly rich in volatiles that deter herbivores and attract seed dispersal agents. We have investigated how terpenes in citrus fruit peels affect the interaction between the plant, insects, and microorganisms. Because limonene represents up to 97% of the total volatiles in orange (Citrus sinensis) fruit peel, we chose to down-regulate the expression of a limonene synthase gene in orange plants by introducing an antisense construct of this gene. Transgenic fruits showed reduced accumulation of limonene in the peel. When these fruits were challenged with either the fungus Penicillium digitatum or with the bacterium Xanthomonas citri subsp. citri, they showed marked resistance against these pathogens that were unable to infect the peel tissues. Moreover, males of the citrus pest medfly (Ceratitis capitata) were less attracted to low limonene-expressing fruits than to control fruits. These results indicate that limonene accumulation in the peel of citrus fruit appears to be involved in the successful trophic interaction between fruits, insects, and microorganisms. Terpene down-regulation might be a strategy to generate broad-spectrum resistance against pests and pathogens in fleshy fruits from economically important crops. In addition, terpene engineering may be important for studying the basic ecological interactions between fruits, herbivores, and pathogens