In Vitro Acquisition of Specific Small Interfering RNAs Inhibits the Expression of Some Target Genes in the Plant Ectoparasite Xiphinema index

Xiphinema index is an important plant parasitic nematode that induces direct damages and specifically transmits the Grapevine fanleaf virus, which is particularly harmful for grapevines. Genomic resources of this nematode species are still limited and no functional gene validation technology is available. RNA interference (RNAi) is a powerful technology to study gene function and here we describe the application of RNAi on several genes in X. index. Soaking the nematodes for 48 h in a suspension containing specific small interfering RNAs resulted in a partial inhibition of the accumulation of some targeted mRNA. However, low reproducible silencing efficiency was observed which could arise from X. index silencing pathway deficiencies. Indeed, essential accustomed proteins for these pathways were not found in the X. index proteome predicted from transcriptomic data. The most reproducible silencing effect was obtained when targeting the piccolo gene potentially involved in endo-exocytosis of synaptic molecules. This represents the first report of gene silencing in a nematode belonging to the Longidoridae family

The GCR2 Gene Family Is Not Required for ABA Control of Seed Germination and Early Seedling Development in Arabidopsis

BACKGROUND: The plant hormone abscisic acid (ABA) regulates diverse processes of plant growth and development. It has recently been proposed that GCR2 functions as a G-protein-coupled receptor (GPCR) for ABA. However, the structural relationships and functionality of GCR2 have been challenged by several independent studies. A central question in this controversy is whether gcr2 mutants are insensitive to ABA, because gcr2 mutants were shown to display reduced sensitivity to ABA under one experimental condition (e.g. 22 degrees C, continuous white light with 150 micromol m(-2) s(-1)) but were shown to display wild-type sensitivity under another slightly different condition (e.g. 23 degrees C, 14/10 hr photoperiod with 120 micromol m(-2) s(-1)). It has been hypothesized that gcr2 appears only weakly insensitive to ABA because two other GCR2-like genes in Arabidopsis, GCL1 and GCL2, compensate for the loss of function of GCR2. PRINCIPAL FINDINGS: In order to test this hypothesis, we isolated a putative loss-of-function allele of GCL2, and then generated all possible combinations of mutations in each member of the GCR2 gene family. We found that all double mutants, including gcr2 gcl1, gcr2 gcl2, gcl1 gcl2, as well as the gcr2 gcl1 gcl2 triple mutant displayed wild-type sensitivity to ABA in seed germination and early seedling development assays, demonstrating that the GCR2 gene family is not required for ABA responses in these processes. CONCLUSION: These results provide compelling genetic evidence that GCR2 is unlikely to act as a receptor for ABA in the context of either seed germination or early seedling development

The transfer and fate of Pb from sewage sludge amended soil in a multi-trophic food chain: a comparison with the labile elements Cd and Zn

The contamination of agroecosystems due to the presence of trace elements in commonly used agricultural materials is a serious issue. The most contaminated material is usually sewage sludge, and the sustainable use of this material within agriculture is a major concern. This study addresses a key issue in this respect, the fate of trace metals applied to soil in food chains. The work particularly addresses the transfer of Pb, which is an understudied element in this respect, and compares the transfer of Pb with two of the most labile metals, Cd and Zn. The transfer of these elements was determined from sludge-amended soils in a food chain consisting of Indian mustard (Brassica juncea), the mustard aphid (Lipaphis erysimi) and a predatory beetle (Coccinella septempunctata). The soil was amended with sludge at rates of 0, 5, 10 and 20 % (w/w). Results showed that Cd was readily transferred through the food chain until the predator trophic level. Zn was the most readily transferred element in the lower trophic levels, but transfer to aphids was effectively restricted by the plant regulating shoot concentration. Pb had the lowest level of transfer from soil to shoot and exhibited particular retention in the roots. Nevertheless, Pb concentrations were significantly increased by sludge amendment in aphids, and Pb was increasingly transferred to ladybirds as levels increased. The potential for Pb to cause secondary toxicity to organisms in higher trophic levels may have therefore been underestimated

Fusicoccin Counteracts the Toxic Effect of Cadmium on the Growth of Maize Coleoptile Segments

The effects of cadmium (Cd; 0.1–1000 μM) and fusicoccin (FC) on growth, Cd2+ content, and membrane potential (Em) in maize coleoptile segments were studied. In addition, the Em changes and accumulation of Cd and calcium (Ca) in coleoptile segments treated with Cd2+ combined with 1 μM FC or 30 mM tetraethylammonium (TEA) chloride (K+-channel blocker) were also determined. In this study, the effects of Ca2+-channel blockers [lanthanum (La) and verapamil (Ver)] on growth and content of Cd2+ and Ca2+ in coleoptile segments were also investigated. It was found that Cd at high concentrations (100 and 1000 μM) significantly inhibited endogenous growth of coleoptile segments and simultaneously measured proton extrusion. FC combined with Cd2+ counteracted the toxic effect of Cd2+ on endogenous growth and significantly decreased Cd2+ content (not the case for Cd2+ at the highest concentration) in coleoptile segments. Addition of Cd to the control medium caused depolarization of Em, the extent of which was dependent on Cd concentration and time of treatment with Cd2+. Hyperpolarization of Em induced by FC was suppressed in the presence of Cd2+ at 1000 μM but not Cd2+ at 100 μM. It was also found that treatment of maize coleoptile segments with 30 mM TEA chloride caused hyperpolarization of Em and decreased Cd2+ content in coleoptile segments, suggesting that, in the same way as for FC, accumulation of Cd2+ was dependent on plasma membrane (PM) hyperpolarization. Similar to FC, TEA chloride also decreased Ca2+ content in coleoptile segments. La and Ver combined with Cd2+ (100 μM) significantly decreased Cd content in maize coleoptile segments, but only La completely abolished the toxic effect of Cd2+ on endogenous growth and growth in the presence of FC. Taken together, these results suggest that the mechanism by which FC counteracts the toxic effect of Cd2+ (except at 1000 μM Cd2+) on the growth of maize coleoptile segments involves both stimulation of PM H+-ATPase activity by FC as well as Cd2+-permeable, voltage-dependent Ca channels, which are blocked by FC and TEA chloride-induced PM hyperpolarization

The plant multidrug resistance ABC transporter AtMRP5 is involved in guard cell hormonal signalling and water use

Carbon dioxide uptake and water release through stomata, controlling the opening and closure of stomatal pore size in the leaf surface, is critical for optimal plant performance. Stomatal movements are regulated by multiple signalling pathways involving guard cell ion channels. Using reverse genetics, we recently isolated a T-DNA insertion mutant for the Arabidopsis ABC-transporter AtMRP5 (mrp5-1). Guard cells from mrp5-1 mutant plants were found to be insensitive to the sulfonylurea compound glibenclamide, which in the wild type induces stomatal opening in the dark. Here, we report that the knockout in AtMRP5 affects several signalling pathways controlling stomatal movements. Stomatal apertures of mrp5-1 and wild-type Ws-2 were identical in the dark. In contrast, opening of stomata of mrp5-1 plants was reduced in the light. In the light, stomatal closure of mrp5-1 was insensitive to external calcium and abscisic acid, a phytohormone responsible for stomatal closure during drought stress. In contrast to Ws-2, the phytohormone auxin could not stimulate stomatal opening in the mutant in darkness. All stomatal phenotypes were complemented in transgenic mrp5-1 plants transformed with a cauliflower mosaic virus (CaMV) 35S-AtMRP5 construct. Both whole-plant and single-leaf gas exchange measurements demonstrated a reduced transpiration rate of mrp5-1 in the light. Excised leaves of mutant plants exhibited reduced water loss, and water uptake was strongly decreased at the whole-plant level. Finally, if plants were not watered, mrp5-1 plants survived much longer due to reduced water use. Analysis of CO2 uptake and transpiration showed that mrp5-1 plants have increased water use efficiency. Mutant plants overexpressing AtMRP5 under the control of the CaMV 35S promoter again exhibited wild-type characteristics. These results demonstrate that multidrug resistance-associated proteins (MRPs) are important components of guard cell functioning

The plant multidrug resistance ABC transporter AtMRP5 is involved in guard cell hormonal signalling and water use

Carbon dioxide uptake and water release through stomata, controlling the opening and closure of stomatal pore size in the leaf surface, is critical for optimal plant performance. Stomatal movements are regulated by multiple signalling pathways involving guard cell ion channels. Using reverse genetics, we recently isolated a T-DNA insertion mutant for the Arabidopsis ABC-transporter AtMRP5 (mrp5-1). Guard cells from mrp5-1 mutant plants were found to be insensitive to the sulfonylurea compound glibenclamide, which in the wild type induces stomatal opening in the dark. Here, we report that the knockout in AtMRP5 affects several signalling pathways controlling stomatal movements. Stomatal apertures of mrp5-1 and wild-type Ws-2 were identical in the dark. In contrast, opening of stomata of mrp5-1 plants was reduced in the light. In the light, stomatal closure of mrp5-1 was insensitive to external calcium and abscisic acid, a phytohormone responsible for stomatal closure during drought stress. In contrast to Ws-2, the phytohormone auxin could not stimulate stomatal opening in the mutant in darkness. All stomatal phenotypes were complemented in transgenic mrp5-1 plants transformed with a cauliflower mosaic virus (CaMV) 35S-AtMRP5 construct. Both whole-plant and single-leaf gas exchange measurements demonstrated a reduced transpiration rate of mrp5-1 in the light. Excised leaves of mutant plants exhibited reduced water loss, and water uptake was strongly decreased at the whole-plant level. Finally, if plants were not watered, mrp5-1 plants survived much longer due to reduced water use. Analysis of CO2 uptake and transpiration showed that mrp5-1 plants have increased water use efficiency. Mutant plants overexpressing AtMRP5 under the control of the CaMV 35S promoter again exhibited wild-type characteristics. These results demonstrate that multidrug resistance-associated proteins (MRPs) are important components of guard cell functioning. [References: 51] 5

The plant multidrug resistance ABC transporter AtMRP5 is involved in guard cell hormonal signalling and water use

Carbon dioxide uptake and water release through stomata, controlling the opening and closure of stomatal pore size in the leaf surface, is critical for optimal plant performance. Stomatal movements are regulated by multiple signalling pathways involving guard cell ion channels. Using reverse genetics, we recently isolated a T-DNA insertion mutant for the Arabidopsis ABC-transporter AtMRP5 (mrp5-1). Guard cells from mrp5-1 mutant plants were found to be insensitive to the sulfonylurea compound glibenclamide, which in the wild type induces stomatal opening in the dark. Here, we report that the knockout in AtMRP5 affects several signalling pathways controlling stomatal movements. Stomatal apertures of mrp5-1 and wild-type Ws-2 were identical in the dark. In contrast, opening of stomata of mrp5-1 plants was reduced in the light. In the light, stomatal closure of mrp5-1 was insensitive to external calcium and abscisic acid, a phytohormone responsible for stomatal closure during drought stress. In contrast to Ws-2, the phytohormone auxin could not stimulate stomatal opening in the mutant in darkness. All stomatal phenotypes were complemented in transgenic mrp5-1 plants transformed with a cauliflower mosaic virus (CaMV) 35S-AtMRP5 construct. Both whole-plant and single-leaf gas exchange measurements demonstrated a reduced transpiration rate of mrp5-1 in the light. Excised leaves of mutant plants exhibited reduced water loss, and water uptake was strongly decreased at the whole-plant level. Finally, if plants were not watered, mrp5-1 plants survived much longer due to reduced water use. Analysis of CO2 uptake and transpiration showed that mrp5-1 plants have increased water use efficiency. Mutant plants overexpressing AtMRP5 under the control of the CaMV 35S promoter again exhibited wild-type characteristics. These results demonstrate that multidrug resistance-associated proteins (MRPs) are important components of guard cell functioning. [References: 51] 5

Identification of plant-parasitism genes in nematodes in silico screening and in vivo validation in Meloidogyne incognita.

International audienceRoot-knot nematodes, Meloidogyne spp., are root parasites that annually cause ca. 100 billion Euros losses in crop production worldwide. An initial analysis of the genome of Meloidogyne incognita has allowed the identification of thousands of genes that apparently did not have homolog in model eukaryotes. A proportion of these M. incognita-restricted gene models may be involved in functions linked to plant-parasitism. Our objective was to identify drug target genes to control primarily root-knot nematodes with potential applications to other plant pathogenic and parasitic species. Our hypothesis was, if a gene is involved in a key parasitism process, it might be conserved in plant-parasitic or plant-pathogenic species and absent from non-parasitic species. Our strategy to identify druggable parasitism target genes was to perform a comparative analysis of M. incognita and M. hapla predicted protein coding genes against a selection of fully sequenced genomes and transcriptomes of various eukaryotes with different lifestyles. Our pipeline conserves all sequences shared with parasitic or plant-associated species and eliminates those conserved in species that could be negatively affected by newly developed drugs or control means. In parallel, we performed automatic functional annotation of the identified genes. Those annotations include detection of signal peptide, Pfam domains, assignment of gene ontology (G.O.) terms, and identification of specific motifs. All these features and annotations were stored in a relational database and it is thus possible to produce any combination of feature to filter out genes of interest. Comparison of the relative abundance of G.O. terms assigned to genes restricted to Meloidogyne species versus those assigned to the whole gene sets of M. incognita and M. hapla revealed a number of differences. A second objective was to develop a new RNA interference (RNAi) procedure using siRNA to artificially silence target genes and analyze their role in parasitism. SiRNA are smaller than long dsRNA usually used for silencing in M. incognita. SiRNA might be easier to introduce by soaking in M. incognita. SiRNA are also more specific to a target sequence as they are 21 base-long and their specificity towards target mRNAs can be assessed by BLAST searches in M. incognita genome. SiRNA targeted to various positions on the mRNA secondary structure were tested to optimize siRNA design. Several transfection conditions were tested and validated by quantitative PCR to optimize reproducibility and target mRNA expression inhibition efficiency. To test the effect of gene silencing on nematode infectivity, nematodes treated with optimized siRNA were used for inoculation assays on tomato plants (Solanum esculentum cv. St Pierre). Infectivity was measured by counting gall and egg masse numbers. With this new method we will test the effect of gene silencing for a series of the most promising targets identified during bioinformatics screens

Identification of plant-parasitism genes in nematodes in silico screening and in vivo validation in Meloidogyne incognita.

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