9 research outputs found

    Evaluation of dsRNA delivery methods for targeting macrophage migration inhibitory factor MIF in RNAi-based aphid control

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    Macrophage migration inhibitory factors (MIFs) are multifunctional proteins regulating major processes in mammals, including activation of innate immune responses. In invertebrates, MIF proteins participate in the modulation of host immune responses when secreted by parasitic organisms, such as aphids. In this study, we assessed the possibility to use MIF genes as targets for RNA interference (RNAi)-based control of the grain aphid Sitobion avenae (Sa) on barley (Hordeum vulgare). When nymphs were fed on artificial diet containing double-stranded (ds)RNAs (SaMIF-dsRNAs) that target sequences of the three MIF genes SaMIF1, SaMIF2 and SaMIF3, they showed higher mortality rates and these rates correlated with reduced MIF transcript levels as compared to the aphids feeding on artificial diet containing a control dsRNA (GFP-dsRNA). Comparison of different feeding strategies showed that nymphs' survival was not altered when they fed from barley seedlings sprayed with naked SaMIF-dsRNAs, suggesting they did not effectively take up dsRNA from the sieve tubes of these plants. Furthermore, aphids' survival was also not affected when the nymphs fed on leaves supplied with dsRNA via basal cut ends of barley leaves. Consistent with this finding, the use of sieve tube-specific YFP-labeled Arabidopsis reporter lines confirmed that fluorescent 21 nt dsRNA(Cy3), when supplied via petioles or spraying, co-localized with xylem structures, but not with phloem tissue. Our results suggest that MIF genes are a potential target for insect control and also imply that application of naked dsRNA to plants for aphid control is inefficient. More efforts should be put into the development of effective dsRNA formulations.Peer reviewe

    Functional Sieve Element Protoplasts1[OA]

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    Sieve element (SE) protoplasts were liberated by exposing excised phloem strands of Vicia faba to cell wall-degrading enzyme mixtures. Two types of SE protoplasts were found: simple protoplasts with forisome inclusions and composite twin protoplasts—two protoplasts intermitted by a sieve plate—of which one protoplast often includes a forisome. Forisomes are giant protein inclusions of SEs in Fabaceae. Membrane integrity of SE protoplasts was tested by application of CFDA, which was sequestered in the form of carboxyfluorescein. Further evidence for membrane intactness was provided by swelling of SE protoplasts and forisome dispersion in reaction to abrupt lowering of medium osmolarity. The absence of cell wall remnants as demonstrated by negative Calcofluor White staining allowed patch-clamp studies. At negative membrane voltages, the current-voltage relations of the SE protoplasts were dominated by a weak inward-rectifying potassium channel that was active at physiological membrane voltages of the SE plasma membrane. This channel had electrical properties that are reminiscent of those of the AKT2/3 channel family, localized in phloem cells of Arabidopsis (Arabidopsis thaliana). All in all, SE protoplasts promise to be a powerful tool in studying the membrane biology of SEs with inherent implications for the understanding of long-distance transport and signaling

    Thermodynamic Battle for Photosynthate Acquisition between Sieve Tubes and Adjoining Parenchyma in Transport Phloem

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    In transport phloem, photoassimilates escaping from the sieve tubes are released into the apoplasmic space between sieve element (SE)/companion cell (CC) complexes (SE/CCs) and phloem parenchyma cells (PPCs). For uptake respective retrieval, PPCs and SE/CCs make use of plasma membrane translocators energized by the proton motive force (PMF). Their mutual competitiveness, which essentially determines the amount of photoassimilates translocated through the sieve tubes, therefore depends on the respective PMFs. We measured the components of the PMF, membrane potential and ΔpH, of SE/CCs and PPCs in transport phloem. Membrane potentials of SE/CCs and PPCs in tissue slices as well as in intact plants fell into two categories. In the first group including apoplasmically phloem-loading species (e.g. Vicia, Solanum), the membrane potentials of the SEs are more negative than those of the PPCs. In the second group including symplasmically phloem-loading species (e.g. Cucurbita, Ocimum), membrane potentials of SEs are equal to or slightly more positive than those of PPCs. Pure sieve tube sap collected from cut aphid stylets was measured with H(+)-selective microelectrodes. Under our experimental conditions, pH of the sieve tube saps was around 7.5, which is comparable to the pH of cytoplasmic compartments in parenchymatous cells. In conclusion, only the membrane potential appears to be relevant for the PMF-determined competition between SE/CCs and PPCs. The findings may imply that the axial sinks along the pathway withdraw more photoassimilates from the sieve tubes in symplasmically loading species than in apoplasmically loading species

    Sieve Element Ca2+ Channels as Relay Stations between Remote Stimuli and Sieve Tube Occlusion in Vicia faba[W]

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    Damage induces remote occlusion of sieve tubes in Vicia faba by forisome dispersion, triggered during the passage of an electropotential wave (EPW). This study addresses the role of Ca2+ channels and cytosolic Ca2+ elevation as a link between EPWs and forisome dispersion. Ca2+ channel antagonists affect the initial phase of the EPW as well as the prolonged plateau phase. Resting levels of sieve tube Ca2+ of ∼50 nM were independently estimated using Ca2+-selective electrodes and a Ca2+-sensitive dye. Transient changes in cytosolic Ca2+ were observed in phloem tissue in response to remote stimuli and showed profiles similar to those of EPWs. The measured elevation of Ca2+ in sieve tubes was below the threshold necessary for forisome dispersion. Therefore, forisomes need to be associated with Ca2+ release sites. We found an association between forisomes and endoplasmic reticulum (ER) at sieve plates and pore-plasmodesma units where high-affinity binding of a fluorescent Ca2+ channel blocker mapped an increased density of Ca2+ channels. In conclusion, propagation of EPWs in response to remote stimuli is linked to forisome dispersion through transiently high levels of parietal Ca2+, release of which depends on both plasma membrane and ER Ca2+ channels

    Evolution, polymorphology and multifunctionality of the phloem system

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