Fast-track applications: The potential for direct delivery of proteins and nucleic acids to plant cells for the discovery of gene function

In animal systems, several methods exist for the direct delivery of nucleic acids and proteins into cells for functional analysis. Until recently, these methods have not been applied to plant systems. Now, however, several preliminary reports suggest that both nucleic acids and proteins can also be delivered into plant cells by very simple, direct application. This promises to open the way for high-throughput screening for gene function in a range of plant species.     

Cell type-specific characterization of nuclear DNA contents within complex tissues and organs

Background  

Eukaryotic organisms are defined by the presence of a nucleus, which encloses the chromosomal DNA, and is characterized by its DNA content (C-value). Complex eukaryotic organisms contain organs and tissues that comprise interspersions of different cell types, within which polysomaty, endoreduplication, and cell cycle arrest is frequently observed. Little is known about the distribution of C-values across different cell types within these organs and tissues.     

Preliminary results on detection of maize treated with electrons for seed dressing and according decontamination and infestation

Saatgut und Getreide kann mit niederenergetischen Elektronen (<300 kev) oder=" hochenergetischen=" elektronen=" (1–10 mev)=" wirksam=" behandelt=" werden,=" um=" mikroorganismen=" und=" insekten=" abzutöten.=" in=" dieser=" vorläufigen=" studie=" wurde=" mais=" mit=" niederenergetischen=" (125 kev)=" und=" hochenergetischen=" elektronen=" (10 mev)=" behandelt.=" um=" diese=" elektronenbehandlung=" nachzuweisen,=" wurden=" verschiedene=" verfahren=" eingesetzt:=" photostimulierte=" lumineszenz=" (psl),=" thermolumineszenz=" (tl)=" und=" dna-kometentest.=" für=" diese=" drei=" nachweismethoden=" existieren=" bereits=" europäische=" normen=" und=" sie=" sind=" als=">Allgemeine Codex Methoden zum Nachweis bestrahlter Lebensmittel etabliert. Die Ergebnisse zeigen, dass PSL und TL geeignete Verfahren sind, um sowohl eine Behandlung von Mais mit niederenergetischen als auch mit hochenergetischen Elektronen zu erkennen. Der DNA-Kometentest erwies sich als weniger geeignet: die Behandlung mit niederenergetischen Elektronen konnte—wie erwartet—nicht nachgewiesen werden. Die Behandlung mit hochenergetischen Elektronen konnte bei einer Maissorte erkannt werden, jedoch nicht bei einer anderen Sorte.     

Maceration of plant tissue by fungi is inhibited by recombinant antipectinase antibodies

Polyclonal antiserum from mice immunized with extracellular proteins from Rhizoctonia solani inhibited pectinase and cellulase activities in cell free culture supernatants of Rhizoctonia solani. Spleen mRNA from these mice was used to construct a cDNA library from which antipectinase ScFv antibodies were isolated using phage display techniques. Soluble ScFv antibodies produced by individual clones in Escherichia coli inhibited polygalacturonase in the culture supernatants of a range of fungal pathogens, including ascomycetes, basidiomycetes, and oomycetes. The soluble antibodies also inhibited maceration of potato tissue by these pathogens.     

Epitope identification and <Emphasis Type="Italic">in silico</Emphasis> prediction of the specificity of antibodies binding to the coat proteins of <Emphasis Type="Italic">Potato Virus Y</Emphasis> strains

A phage library containing 2.7 × 10⁹ randomly expressed peptides was used to determine the epitopes of three monoclonal antibodies that bind to the coat protein of potato virus Y. Construction of the consensus sequences for the peptides obtained after three selection rounds indicated that each antibody recognized a different epitope located within the first 50 N-terminal amino acids of the coat protein. The location of the epitopes was confirmed by heterologous expression of the N-terminal part of the coat protein in Escherichia coli, and, subsequently, by performing an immunological test with the three antibodies. The accuracy of the phage library was demonstrated by predicting in silico the cross-reactivity of the three antibodies with other potyvirus family members. ELISA and in silico predictions revealed the same results in almost every case. The potential of peptide phage libraries to optimize the use of antibodies in plant virology is discussed.     

Antagonistic rhizoplane bacteria induce diverse morphological alterations in Peronosporomycete hyphae during <Emphasis Type="Italic">in vitro</Emphasis> interaction

A total of 150 bacteria were isolated from rhizoplanes of the host and non-host plants of a phytopathogenic Peronosporomycete Aphanomyces cochlioides. Upon screening, 5% of the isolates were evaluated as antagonists as they inhibited radial growth of A. cochlioides AC-5 hyphae in a dual culture assay. In addition, those antagonistic bacteria also induced characteristic morphological alterations in the A. cochlioides AC-5 hyphae that grew towards bacterial colonies. Hyphal morphological alterations observed in AC-5 and other tested strains of Peronosporomycetes included excessive branching, curly growth, unusually longer and pointed tip formation and swelling; all of these were comparable to the alterations induced by known antimicrobial compounds. Among the antagonistic bacteria, Pseudomonas sp. strain EC-S101 induced a unique branching pattern (tree-like) in AC-5 hyphae by continuous apical bifurcation of successive hyphae, where increases in number of branches and hyphal area were linearly correlated with time up to 10 h. Our observations suggested that the pathogen might have lost its ability of normal branch production; however maintained the capability of self-branching. Soluble extracts from the culture fluids of Pseudomonas sp. strain EC-S101 and Stenotrophomonas maltophilia EC-S105 induced similar excessive branching and curly growth in A. cochlioides hyphae as the respective bacterium. These results revealed that bacterial metabolites appeared to be responsible for induction of morphological alterations. Interestingly, the antagonistic bacteria that induced hyphal morphological alterations, also efficiently suppressed in vivo damping-off disease caused by AC-5. We suggest that antagonistic rhizoplane bacteria have the capability to induce diverse morphological alterations in Peronosporomycetes hyphae during in vitro interactions. Hyphal morphological alterations associated with growth inhibition and the induction of characteristic morphological changes indicate antagonistic activity against the Peronosporomycete.     

Hydrogen peroxide localization and antioxidant status in the recovery of apricot plants from European Stone Fruit Yellows

Hydrogen peroxide (H₂O₂) localization and roles of peroxidases, malondialdehyde and reduced glutathione were compared in leaves of apricot (Prunus armeniaca) plants asymptomatic, European Stone Fruits Yellows (ESFY)-symptomatic and recovered. Nested PCR analysis revealed that Candidatus Phytoplasma prunorum, is present in asymptomatic, symptomatic and recovered apricot trees, confirming previous observations on this species, in which recovery does not seem to be related to the disappearance of phytoplasma from the plant.H₂O₂was detected cytochemically by its reaction with cerium chloride, which produces electron-dense deposits of cerium perhydroxides. H₂O₂was present in the plasmalemma of the phloem cells of recovered apricot plant leaves, but not in the asymptomatic or symptomatic material. Furthermore, by labelling apricot leaf tissues with diaminobenzidine DAB, no differences were found in the localization of peroxidases.Protein content in asymptomatic, symptomatic and recovered leaves was not significantly different from one another. In contrast, guaiacol peroxidase activity had the following trend: symptomatic > recovered > asymptomatic, whereas reduced glutathione content followed the opposite trend: asymptomatic > recovered > symptomatic. Moreover, no differences were observed in malondialdehyde concentrations between asymptomatic, symptomatic and recovered leaves. The overall results suggest that H₂O₂ and related metabolites and enzymes appear to be involved in lessening both pathogen virulence and disease symptom expression in ESFY-infected apricot plants.     

Luteoforol,a flavan 4-ol,is induced in pome fruits by prohexadione-calciumand shows phytoalexin-like properties against <Emphasis Type="Italic">Erwinia amylovora</Emphasis>and other plant pathogens

Treatments with prohexadione-calcium led to lowered incidence of fire blight, scab and other diseases in pome fruit trees and other crop plants. In addition to acting as a growth regulator, prohexadione-calcium interferes with flavonoid metabolism and induces the accumulation of the 3-deoxycatechin luteoliflavan in shoots of pome fruit trees. Luteoliflavan does not possess any remarkable antimicrobial activity. Therefore luteoforol, its unstable and highly reactive precursor, has been tested in vitro for its bactericidal and fungicidal activities. Luteoforol was found to be highly active against different strains of Erwinia amylovora, the causal agent of fire blight, and all other bacterial and fungal organisms tested. Phytotoxic effects were also observed in pear plantlets. The results obtained indicate that prohexadione-calcium induces luteoforol as an active principle with non-specific biocidal properties. It is proposed that luteoforol is released upon pathogen attack from its cellular compartment and inhibits further disease development by destroying pathogen cells as well as by inducing a hypersensitive-like reaction in the host plant tissue. This mechanism would be closely analogous to the one known for structurally related phytoalexins in sorghum.