Maize 16-kD γ-zein forms very unusual disulfide-bonded polymers in the endoplasmic reticulum: implications for prolamin evolution.

In the lumen of the endoplasmic reticulum (ER), prolamin storage proteins of cereal seeds form very large, ordered heteropolymers termed protein bodies (PBs), which are insoluble unless treated with alcohol or reducing agents. In maize PBs, 16-kD γ-zein locates at the interface between a core of alcohol-soluble α-zeins and the outermost layer mainly composed of the reduced-soluble 27-kD γ-zein. 16-kD γ-zein originates from 27-kD γ-zein upon whole-genome duplication and is mainly characterized by deletions in the N-terminal domain that eliminate most Pro-rich repeats and part of the Cys residues involved in inter-chain bonds. 27-kD γ-zein also forms insoluble PBs when expressed in transgenic vegetative tissues. We show that in Arabidopsis leaves, 16-kD γ-zein assembles into disulfide-linked polymers that fail to efficiently become insoluble. Instead of forming PBs, these polymers accumulate as very unusual threads that markedly enlarge the ER lumen, resembling amyloid-like fibers. Domain-swapping between the two γ-zeins indicates that the N-terminal region of 16-kD γ-zein has a dominant effect in preventing full insolubilization. Therefore, a newly evolved prolamin has lost the ability to form homotypic PBs, and has acquired a new function in the assembly of natural, heteropolymeric PBs.

Construction of a synthetic infectious cDNA clone of Grapevine Algerian latent virus (GALV-Nf) and its biological activity in Nicotiana benthamiana and grapevine plants.

BACKGROUND: Grapevine Algerian latent virus (GALV) is a tombusvirus first isolated in 1989 from an Algerian grapevine (Vitis spp.) plant and more recently from water samples and commercial nipplefruit and statice plants. No further reports of natural GALV infections in grapevine have been published in the last two decades, and artificial inoculations of grapevine plants have not been reported. We developed and tested a synthetic GALV construct for the inoculation of Nicotiana benthamiana plants and different grapevine genotypes to investigate the ability of this virus to infect and spread systemically in different hosts. METHODS: We carried out a phylogenetic analysis of all known GALV sequences and an epidemiological survey of grapevine samples to detect the virus. A GALV-Nf clone under the control of the T7 promoter was chemically synthesized based on the full-length sequence of the nipplefruit isolate GALV-Nf, the only available sequence at the time the project was conceived, and the infectious transcripts were tested in N. benthamiana plants. A GALV-Nf-based binary vector was then developed for the agroinoculation of N. benthamiana and grapevine plants. Infections were confirmed by serological and molecular analysis and the resulting ultrastructural changes were investigated in both species. RESULTS: Sequence analysis showed that the GALV coat protein is highly conserved among diverse isolates. The first epidemiological survey of cDNAs collected from 152 grapevine plants with virus-like symptoms did not reveal the presence of GALV in any of the samples. The agroinoculation of N. benthamiana and grapevine plants with the GALV-Nf binary vector promoted efficient infections, as revealed by serological and molecular analysis. The GALV-Nf infection of grapevine plants was characterized in more detail by inoculating different cultivars, revealing distinct patterns of symptom development. Ultrastructural changes induced by GALV-Nf in N. benthamiana were similar to those induced by tombusviruses in other hosts, but the cytopathological alterations in grapevine plants were less severe. CONCLUSIONS: This is the first report describing the development of a synthetic GALV-Nf cDNA clone, its artificial transmission to grapevine plants and the resulting symptoms and cytopathological alterations.

Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells.

Lupin seed γ-Conglutin is a protein capable of reducing glycaemia in mammalians and increasing glucose uptake by model cells. This work investigated whether γ-Conglutin is internalised into the target cells and undergoes any covalent change during the process, as a first step to understanding its mechanism of action. To this purpose, γ-Conglutin-treated and untreated HepG2 cells were submitted to confocal and transmission electron microscopy. Immune-revelation of γ-Conglutin at various intervals revealed its accumulation inside the cytosol. In parallel, 2D-electrophoresis of the cell lysates and antibody reaction of the blotted maps showed the presence of the protein intact subunits inside the treated cells, whilest no trace of the protein was found in the control cells. However, γ-Conglutin-related spots with an unexpectedly low pI were also observed in the maps. These spots were excised, trypsin-treated and submitted to MS/MS spectrometric analysis. The presence of phosphorylated amino acids was detected. These findings, by showing that γ-Conglutin is internalised by HepG2 cells in an intact form and is modified by multiple phosphorylation, open the way to the understanding of the lupin γ-Conglutin insulin-mimetic activity.

Phenotypic and histochemical traits of the interaction between Plasmopara viticola and resistant or susceptible grapevine varieties.

BACKGROUND: Grapevine downy mildew, caused by Plasmopara viticola, is a very serious disease affecting mainly Vitis vinifera cultivated varieties around the world. Breeding for resistance through the crossing with less susceptible species is one of the possible means to reduce the disease incidence and the application of fungicides. The hybrid Bianca and some of its siblings are considered very promising but their resistance level can vary depending on the pathogen strain. Moreover, virulent strains characterized by high fitness can represent a potential threat to the hybrid cultivation. RESULTS: The host response and the pathogen virulence were quantitatively assessed by artificially inoculating cv Chardonnay, cv Bianca and their siblings with P. viticola isolates derived from single germinating oospores collected in various Italian viticultural areas. The host phenotypes were classified as susceptible, intermediate and resistant, according to the Area Under the Disease Progress Curve caused by the inoculated strain. Host responses in cv Bianca and its siblings significantly varied depending on the P. viticola isolates, which in turn differed in their virulence levels. The fitness of the most virulent strain did not significantly vary on the different hybrids including Bianca in comparison with the susceptible cv Chardonnay, suggesting that no costs are associated with virulence. Among the individual fitness components, only sporangia production was significantly reduced in cv Bianca and in some hybrids. Comparative histological analysis revealed differences between susceptible and resistant plants in the pathogen diffusion and cytology from 48 h after inoculation onwards. Defence mechanisms included callose depositions in the infected stomata, increase in peroxidase activity, synthesis of phenolic compounds and flavonoids and the necrosis of stomata and cells immediately surrounding the point of invasion and determined alterations in the size of the infected areas and in the number of sporangia differentiated. CONCLUSIONS: Some hybrids were able to maintain an intermediate-resistant behaviour even when inoculated with the most virulent strain. Such hybrids should be considered for further field trials.

Uromyces appendiculatus infection in BTH-treated bean plants: ultrastructural details of a lost fight.

The mechanisms of BTH [benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester]-induced resistance against bean rust caused by Uromyces appendiculatus have been explored in Phaseolus vulgaris by light and transmission electron microscopy, following the infection progression in plants challenged 7 days after treatment. While BTH did not affect uredospore germination and fungal penetration in the substomatal cavity, a first impairment to the colonization appeared evident about 48-96 h after inoculation, with alterations of infection hypha structure and reduction in mycelium expansion. No differences were found in this phase regarding the formation and ultrastructure of haustoria in untreated and BTH-treated plants, except for the deposition of electron-opaque material in the extrahaustorial matrix of the latter. A second and decisive impairment in fungal progression was observed at 7-10 days after inoculation when host cell penetrated, or in close contact with the fungal hyphae, were impregnated by phenolic compounds. The same was observed in fungal walls, particularly around haustoria, thus hampering the biotrophic habitus of the fungus and further mycelium spreading. This, in turn, prevented the evasion of fungal reproductive structures, the uredinia, and the appearance of visible symptoms. No particular ultrastructural alterations were observed in most of the penetrated cells, even at late stages of infection, indicating that BTH treatment does not induce host cells to respond with a hypersensitive reaction (HR). A parallel time course of the expression of phenylalanine ammonia lyase (PAL) gene, the key enzyme for the synthesis of phenylpropanoidic phytoalexins and many other phenolics, has shown that PAL mRNA is strongly and persistently transcripted in BTH-treated plants since the 6th h after treatment, though no apparent ultrastructural alterations were detectable up to some days after pathogen challenging. This indicates that BTH, at the employed concentration of 0.3 mM, directly activates the plant's own defences, thus accounting for the observed full protection against bean rust.

Iron availability affects the function of mitochondria in cucumber roots.

* In Strategy-I-plants, iron (Fe) deficiency induces processes leading to increased Fe solubilization in the rhizosphere, including reduction by ferric reductases and active proton extrusion. These processes require active respiration to function. In this work we investigated the effect of Fe deficiency on respiratory activities of cucumber (Cucumis sativus) roots. * We compared oxygen consumption rate and the activities of the respiratory chain complexes on purified mitochondria from roots grown in the presence or absence of Fe using biochemical and molecular approaches. * Oxygen consumption rate in apex roots was increased under Fe deficiency that was mostly resistant to KCN and salycilichydroxamic acid (SHAM) inhibitors, indicating other oxygen-consuming reactions could be present. Indeed, enzyme assays revealed that lack of Fe induced a decrease in the activities of respiratory complexes that was proportional to the number of Fe atoms in each complex. A decrease of cyt c, Rieske and NAD9 proteins was also observed. Transmission electron microscopy (TEM) analysis showed that mitochondria undergo structural changes under Fe deficiency. * Our data show that mitochondria and the electron transport chain are an important target of Fe limitation and that mitochondria modify their function to meet higher demands for organic acids while restricting the activity of enzymes with Fe cofactors.

Three-Dimensional Reconstruction, by TEM Tomography, of the Ultrastructural Modifications Occurring in Cucumis sativus L. Mitochondria under Fe Deficiency.

BACKGROUND: Mitochondria, as recently suggested, might be involved in iron sensing and signalling pathways in plant cells. For a better understanding of the role of these organelles in mediating the Fe deficiency responses in plant cells, it is crucial to provide a full overview of their modifications occurring under Fe-limited conditions. The aim of this work is to characterize the ultrastructural as well as the biochemical changes occurring in leaf mitochondria of cucumber (Cucumis sativus L.) plants grown under Fe deficiency. METHODOLOGY/RESULTS: Mitochondrial ultrastructure was investigated by transmission electron microscopy (TEM) and electron tomography techniques, which allowed a three-dimensional (3D) reconstruction of cellular structures. These analyses reveal that mitochondria isolated from cucumber leaves appear in the cristae junction model conformation and that Fe deficiency strongly alters both the number and the volume of cristae. The ultrastructural changes observed in mitochondria isolated from Fe-deficient leaves reflect a metabolic status characterized by a respiratory chain operating at a lower rate (orthodox-like conformation) with respect to mitochondria from control leaves. CONCLUSIONS: To our knowledge, this is the first report showing a 3D reconstruction of plant mitochondria. Furthermore, these results suggest that a detailed characterization of the link between changes in the ultrastructure and functionality of mitochondria during different nutritional conditions, can provide a successful approach to understand the role of these organelles in the plant response to Fe deficiency.

Cerato-populin and cerato-platanin, two non-catalytic proteins from phytopathogenic fungi, interact with hydrophobic inanimate surfaces and leaves.

Based on sequence homology, several fungal Cys-rich secreted proteins have been grouped in the cerato-platanin (CP) family, which comprises at least 40 proteins involved mainly in eliciting defense-related responses. The core member of this family is cerato-platanin, a moderately hydrophobic protein with a double ψ-ß barrel fold. CP and the recently identified orthologous cerato-populin (Pop1) are involved in host-fungus interaction, and can be considered non-catalytic fungal PAMPs. CP is more active in inducing defense when in an aggregated conformation than in its native form, but little is known about other CP-orthologous proteins. Here, we cloned, expressed, and purified recombinant Pop1, which was used to characterize the protein aggregates. Our results suggest that the unfolded, self-assembled Pop1 is more active in inducing defense, and that the unfolding process can be induced by interaction with hydrophobic inanimate surfaces such as Teflon, treated mica, and gold sheets. In vivo, we found that both CP and Pop1 interact with the hydrophobic cuticle of leaves. Therefore, we propose that the interaction of these proteins with host cuticle waxes could induce unfolding and consequently trigger their PAMP-like activity.

Potato virus X movement in Nicotiana benthamiana: new details revealed by chimeric coat protein variants.

Potato virus X coat protein is necessary for both cell-to-cell and phloem transfer, but it has not been clarified definitively whether it is needed in both movement phases solely as a component of the assembled particles or also of differently structured ribonucleoprotein complexes. To clarify this issue, we studied the infection progression of a mutant carrying an N-terminal deletion of the coat protein, which was used to construct chimeric virus particles displaying peptides selectively affecting phloem transfer or cell-to-cell movement. Nicotiana benthamiana plants inoculated with expression vectors encoding the wild-type, mutant and chimeric viral genomes were examined by microscopy techniques. These experiments showed that coat protein-peptide fusions promoting cell-to-cell transfer only were not competent for virion assembly, whereas long-distance movement was possible only for coat proteins compatible with virus particle formation. Moreover, the ability of the assembled PVX to enter and persist into developing xylem elements was revealed here for the first time.

Histological studies on the mycoparasitism of Cladosporium tenuissimum on urediniospores of Uromyces appendiculatus.

Interactions between the mycoparasite Cladosporium tenuissimum and the bean rust Uromyces appendiculatus were studied through light and electron microscopy in vitro at the host-parasite interface. Urediniospore germination decreased on contact with ungerminated C. tenuissimum conidia, possibly due to antibiosis mechanisms. C. tenuissimum grew towards the bean rust spores and coiled around their germ tubes. Penetration of the urediniospores occurred either enzymatically and/or mechanically, through appressorium or infection cushion structures, from which a thin penetrating hypha was generated. Enzyme production by the mycoparasite was suggested by the loosening of the matricial components of the spore wall, which sometimes left chitin fibrils visible. Mycoparasite hyphae grew within the host spore, emptied its content, and emerged profusely forming conidiophores and conidia. C. tenuissimum was able to grow on media containing laminarin, suggesting the ability of producing glucanases, but not when chitin was used as the sole carbon source. Conidia that had been grown on a sugar-rich medium, filtered, and extracted with organic solvents, were found to contain cladosporol and related compounds. Complete control of the bean rust disease was achieved by application of C. tenuissimum culture filtrates but not by conidial suspensions. This is the first report of parasitism by C. tenuissimum on U. appendiculatus. These investigations provide additional observations on a genus besides Melampsora and Cronartium from which this fungus has been isolated and tested to date. The possible role of environmental factors for the exploitation of this organism as a biocontrol agent is also mentioned.