Plant genome modification by homologous recombination

The mechanisms and frequencies of various types of homologous recombination (HR) have been studied in plants for several years. However, the application of techniques involving HR for precise genome modification is still not routine. The low frequency of HR remains the major obstacle but recent progress in gene targeting in Arabidopsis and rice, as well as accumulating knowledge on the regulation of recombination levels, is an encouraging sign of the further development of HR-based approaches for genome engineering in plants.     

Physiological responses and antioxidant enzyme changes in <Emphasis Type="Italic">Sulla coronaria</Emphasis> inoculated by cadmium resistant bacteria

Plant growth promoting bacteria (PGPB) may help to reduce the toxicity of heavy metals on plants growing in polluted soils. In this work, Sulla coronaria inoculated with four Cd resistant bacteria (two Pseudomonas spp. and two Rhizobium sullae) were cultivated in hydroponic conditions treated by Cd; long time treatment 50 µM CdCl₂ for 30 days and short time treatment; 100 µM CdCl₂ for 7 days. Results showed that inoculation with Cd resistant PGPB enhanced plant biomass, thus shoot and root dry weights of control plants were enhanced by 148 and 35% respectively after 7 days. Co-inoculation of plants treated with 50 and 100 µM Cd increased plant biomasses as compared to Cd-treated and uninoculated plants. Cadmium treatment induced lipid peroxidation in plant tissues measured through MDA content in short 7 days 100 µM treatment. Antioxidant enzyme studies showed that inoculation of control plants enhanced APX, SOD and CAT activities after 30 days in shoots and SOD, APX, SOD, GPOX in roots. Application of 50 µM CdCl₂ stimulated all enzymes in shoots and decreased SOD and CAT activities in roots. Moreover, 100 µM of CdCl₂ increased SOD, APX, CAT and GPOX activities in shoots and increased significantly CAT activity in roots. Metal accumulation depended on Cd concentration, plant organ and time of treatment. Furthermore, the inoculation enhanced Cd uptake in roots by 20% in all treatments. The cultivation of this symbiosis in Cd contaminated soil or in heavy metal hydroponically treated medium, showed that inoculation improved plant biomass and increased Cd uptake especially in roots. Therefore, the present study established that co-inoculation of S. coronaria by a specific consortium of heavy metal resistant PGPB formed a symbiotic system useful for soil phytostabilization.     

Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Late Embryogenesis Abundant (LEA) proteins are associated with tolerance to water-related stress. A wheat (Triticum durum) group 2 LEA proteins, known also as dehydrin (DHN-5), has been previously shown to be induced by salt and abscisic acid (ABA). In this report, we analyze the effect of ectopic expression of Dhn-5 cDNA in Arabidopsis thaliana plants and their response to salt and osmotic stress. When compared to wild type plants, the Dhn-5 transgenic plants exhibited stronger growth under high concentrations of NaCl or under water deprivation, and showed a faster recovery from mannitol treatment. Leaf area and seed germination rate decreased much more in wild type than in transgenic plants subjected to salt stress. Moreover, the water potential was more negative in transgenic than in wild type plants. In addition, the transgenic plants have higher proline contents and lower water loss rate under water stress. Also, Na⁺ and K⁺ accumulate to higher contents in the leaves of the transgenic plants. Our data strongly support the hypothesis that Dhn-5, by its protective role, contributes to an improved tolerance to salt and drought stress through osmotic adjustment.     

Phosphate,phytate and phytases in plants: from fundamental knowledge gained in Arabidopsis to potential biotechnological applications in wheat

Phosphorus (P) is an essential macronutrient for all living organisms. In plants, P is taken up from the rhizosphere by the roots mainly as inorganic phosphate (Pi), which is required in large and sufficient quantities to maximize crop yields. In today’s agricultural society, crop yield is mostly ensured by the excessive use of Pi fertilizers, a costly practice neither eco-friendly or sustainable. Therefore, generating plants with improved P use efficiency (PUE) is of major interest. Among the various strategies employed to date, attempts to engineer genetically modified crops with improved capacity to utilize phytate (PA), the largest soil P form and unfortunately not taken up by plants, remains a key challenge. To meet these challenges, we need a better understanding of the mechanisms regulating Pi sensing, signaling, transport and storage in plants. In this review, we summarize the current knowledge on these aspects, which are mainly gained from investigations conducted in Arabidopsis thaliana, and we extended it to those available on an economically important crop, wheat. Strategies to enhance the PA use, through the use of bacterial or fungal phytases and other attempts of reducing seed PA levels, are also discussed. We critically review these data in terms of their potential for use as a technology for genetic manipulation of PUE in wheat, which would be both economically and environmentally beneficial.     

Structure génétique des cécidomyies des céréales en Tunisie

Genetic structure of Hessian flies in Tunisia. The genetic structure of M. destructor and M. hordei was investigated by sampling 21 fields of cereals in 14 localities of central and southern Tunisia. As previously shown, there was no strict association between the cereal species (wheat, barley and oat) and the Mayetiola species. M. destructor males displayed no heterozygosity at the Pgm3 locus, indicating that they were hemizygous as is the PGM locus in North America. In M. hordei, heterozygous males were observed at all loci, but strong heterozygote deficits were found at two loci (Mdh2 et Hk). Since no such deficit was observed in females, the population structure of M. hordei was studied only in females. Although heterozygosity was two fold higher in M. hordei than in M. destructor, the two species were similar for other genetic characteristics, including a low (Fst < 0.05) but significant (P < 0.05) genetic differentiation, no isolation by distance, and similar rates of gene flow (5.7 ≤ Nm ≤ 9.6). These results are discussed in relation to their consequences in the event of controlling Tunisian Hessian flies using wheat cultivars that are resistant to a M. destructor biotype.     

Co-inoculation Effects of Rhizobium sullae and Pseudomonas sp. on Growth,Antioxidant Status,and Expression Pattern of Genes Associated with Heavy Metal Tolerance and Accumulation of Cadmium in Sulla coronaria

Recently, phytoremediation assisted by soil bacteria has emerged as a potential tool to clean up the metal-contaminated/polluted environment. Three plant-growth-promoting bacteria (PGPBs): Rhizobium sullae, Pseudomonas fluorescens, and Pseudomonas sp. were found to tolerate cadmium (Cd) stress. Sulla coronaria inoculated with these PGPBs, and grown under different Cd concentrations (0, 100, and 200 µM), showed increases in dry biomass and proline content. Notable increases in different gas-exchange characteristics such as photosynthesis rate (A), transpiration rate (E), and water-use efficiency (WUE), as well as increases in nitrogen (N) and Cd accumulations were also recorded in inoculated plants compared to non-inoculated Cd stressed plants. The activities of antioxidant enzymes superoxide dismutase (SOD), guaiacol peroxidase (GPOX), catalase (CAT), and ascorbate peroxidase (APX) in S. coronaria roots increased under Cd stress after PGPB co-inoculation, suggesting that these PGPB species could be used for amelioration of stress tolerance in S. coronaria. The expression patterns of ScPCS, ScMT, ScF-box, ScGR, and ScGST in roots of S. coronaria indicated that these genes are differentially expressed under Cd treatments, suggesting their possible roles in Cd and heavy metal stress responses. The results indicate that co-inoculation with R. sullae and Pseudomonas sp. could alleviate Cd toxicity in S. coronaria. In the present study, the obtained data suggest that the application of PGPBs could be a promising strategy for enhancing the phytostabilization efficiency of Cd-contaminated soils.

     

Comparative evaluation of E-test and CLSI methods for Itraconazole,Fluconazole and Ketoconazole susceptibilities of Microsporum canis strains

The incidence of resistance to antifungal agents for dermatophytes is increasing, but most of the methods currently available to test the antifungal susceptibility of Microsporum canis still require standardization. The aims of this study were: (i) to evaluate the antifungal susceptibility of M. canis strains recovered from animals to ketoconazole (KTZ), fluconazole (FLZ) and itraconazole (ITZ) using a modified CLSI broth microdilution (CLSI M38-A2-BMD) and the E-test® protocols and (ii) to estimate the agreement between the methods. Tentative azole epidemiological cutoff values (ECVs) were also proposed in order to interpret the results of in vitro susceptibility tests and to establish the agreement between the E-test and CLSI BMD methods. A total of forty clinical M. canis strains from animals with skin lesions were tested, and the essential (EA) and categorical agreement (CA) between the two methods were determined. KTZ displayed the lowest MIC values, while ITZ and FLZ the highest. The ECV for KTZ and ITZ were 4 μg/ml, while those of FLZ was 64 μg/ml. Based on ECVs, about 88% of M. canis strains were susceptible to all azoles being a cross-resistance with ITZ-FLZ registered for one strain. A total of five M. canis strains showed MIC?>?ECV for FLZ using CLSI, while one strain showed MIC?>?ECV for ITZ using both tests. KTZ, ITZ and FLZ showed EA ranging from 92.5 to 95%, for all azoles and CA?>?97% except for FLZ (87.5%). The good CA between the E-test and the CLSI BMD provides evidence of the reliability of the former method to test the antifungal susceptibility of M. canis for ITZ and KTZ and not for FLZ.

     

Alleviation of drought stress in faba bean (Vicia faba L.) by exogenous application of β-aminobutyric acid (BABA)

Physiology and Molecular Biology of Plants - Drought stress is one of the most prevalent environmental factors limiting faba bean (Vicia faba L.) crop productivity. β-aminobutyric acid (BABA)...     

Comparison of disc diffusion, Etest and agar dilution for susceptibility testing of colistin against Enterobacteriaceae

Aims: In this study, we compared different methods of colistin susceptibility testing, disc diffusion, agar dilution and Etest using a set of Enterobacteriaceae isolates that included colistin‐resistant strains. Methods and results: Susceptibility of 200 clinical isolates of Enterobacteriaceae to colistin was tested to compare agar dilution (reference method), disc diffusion (50 and 10 μg) and Etest. MICs (minimum inhibitory concentrations) were interpreted using the criteria established by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Colistin exhibited excellent activity against Escherichia coli and E. cloacae (MIC₉₀ = 0·5 mg l^?1). In contrast, colistin was less active against Klebsiella pneumoniae (MIC₉₀ = 16 mg l^?1). Resistance rates varied from 0% in E. coli to 1·8% in E. cloacae and 13% in K. pneumoniae. High rates of very major errors were observed in the disc diffusion test using either the criteria of the Comité de l’antibiogramme de la Société Française de Microbiologie (CA‐SFM) or the criteria of the Clinical and Laboratory Standards Institute (CLSI), respectively, 3·5 and 2·5%. When the criteria of Gales et al. were applied, the number of very major errors was reduced to one (0·5%). The Etest showed good concordance with agar dilution method. Conclusion: Disc susceptibility testing methods are unreliable on detecting colistin resistance. MIC should be determined to confirm the susceptibility results by disc diffusion. Significance and Impact of the study: We recommend the determination of MIC by Etest for all multidrug‐resistant Enterobacteriaceae when colistin is required for the treatment.     

Structure of the M2 transmembrane segment of GLIC, a prokaryotic Cys loop receptor homologue from Gloeobacter violaceus, probed by substituted cysteine accessibility

GLIC is a homopentameric proton-gated, prokaryotic homologue of the Cys loop receptor family of neurotransmitter-gated ion channels. Recently, crystal structures of GLIC hypothesized to represent an open channel state were published. To explore the channel structure in functional GLIC channels, we tested the ability of p-chloromercuribenzenesulfonate to react with 30 individual cysteine substitution mutants in and flanking the M2 channel-lining segment in the closed state (pH 7.5) and in a submaximally activated state (pH 5.0). Nine mutants did not tolerate cysteine substitution and were not functional. From positions 10' to 27', p-chloromercuribenzenesulfonate significantly modified the currents at pH 7.5 and 5.0 in all mutants except H234C (11'), I235C (12'), V241C (18'), T243C (20'), L245C (22'), and Y250C (27'), which were not functional, except for 12'. Currents for P246C (23') and K247C (24') were only significantly altered at pH 5.0. The reaction rates were all >1000 m(-1) s(-1). The reactive residues were more accessible in the activated than in the resting state. We infer that M2 is tightly associated with the adjacent transmembrane helices at the intracellular end but is more loosely packed from 10' to the extracellular end than the x-ray structures suggest. We infer that the charge selectivity filter is in the cytoplasmic half of the channel. We also show that below pH 5.0, GLIC desensitizes on a time scale of minutes and infer that the crystal structures may represent a desensitized state.