Genetic dissection of a putative nucleolar localization signal in the coat protein of ourmia melon virus.

Ourmiaviruses became the object of recent attention for their unusual taxonomic placements among plant viruses. The ourmia melon virus (OuMV) RNA3 encodes a 22-kDa coat protein (CP). Besides its role in virion formation, the OuMV CP facilitates systemic virus spread. In Nicotiana benthamiana, an eGFP-CP fusion protein was localized in the nucleus and preferentially in the nucleolus. By bioinformatics analysis, we identified an arginine- and lysine-rich region at the N-terminus of the CP. Here, we demonstrate by deletion and alanine scanning mutagenesis that this region in the CP is responsible for its preferential accumulation in the nucleolus of host cells.

Multiple exocytotic markers accumulate at the sites of perifungal membrane biogenesis in arbuscular mycorrhizas.

Arbuscular mycorrhizas (AMs) are symbiotic interactions established within the roots of most plants by soil fungi belonging to the Glomeromycota. The extensive accommodation of the fungus in the root tissues largely takes place intracellularly, within a specialized interface compartment surrounded by the so-called perifungal membrane, an extension of the host plasmalemma. By combining live confocal imaging of green fluorescent protein (GFP)-tagged proteins and transmission electron microscopy (TEM), we have investigated the mechanisms leading to the biogenesis of this membrane. Our results show that pre-penetration responses and symbiotic interface construction are associated with extensive membrane dynamics. They involve the main components of the exocytotic machinery, with a major participation of the Golgi apparatus, as revealed by both TEM and in vivo GFP imaging. The labeling of known exocytosis markers, such as v-SNARE proteins of the VAMP72 family and the EXO84b subunit of the exocyst complex, allowed live imaging of the cell components involved in perifungal membrane construction, clarifying how this takes place ahead of the growing intracellular hypha. Lastly, our novel data are used to illustrate a model of membrane dynamics within the pre-penetration apparatus during AM fungal penetration.

Intraradical colonization by arbuscular mycorrhizal fungi triggers induction of a lipochitooligosaccharide receptor.

Functional divergence of paralogs following gene duplication is one of the mechanisms leading to evolution of novel pathways and traits. Here we show that divergence of Lys11 and Nfr5 LysM receptor kinase paralogs of Lotus japonicus has affected their specificity for lipochitooligosaccharides (LCOs) decorations, while the innate capacity to recognize and induce a downstream signalling after perception of rhizobial LCOs (Nod factors) was maintained. Regardless of this conserved ability, Lys11 was found neither expressed, nor essential during nitrogen-fixing symbiosis, providing an explanation for the determinant role of Nfr5 gene during Lotus-rhizobia interaction. Lys11 was expressed in root cortex cells associated with intraradical colonizing arbuscular mycorrhizal fungi. Detailed analyses of lys11 single and nfr1nfr5lys11 triple mutants revealed a functional arbuscular mycorrhizal symbiosis, indicating that Lys11 alone, or its possible shared function with the Nod factor receptors is not essential for the presymbiotic phases of AM symbiosis. Hence, both subfunctionalization and specialization appear to have shaped the function of these paralogs where Lys11 acts as an AM-inducible gene, possibly to fine-tune later stages of this interaction.

Accumulation of cynaropicrin in globe artichoke and localization of enzymes involved in its biosynthesis.

Globe artichoke (Cynara cardunculus var. scolymus) belongs to the Asteraceae family, in which one of the most biologically significant class of secondary metabolites are sesquiterpene lactones (STLs). In globe artichoke the principal STL is the cynaropicrin, which contributes to approximately 80% of its characteristic bitter taste. Cynaropicrin content was assessed in globe artichoke tissues and was observed to accumulate in leaves of different developmental stages. In the receptacle, a progressive decrease was observed during inflorescence development, while the STL could not be detected in the inflorescence bracts. Almost undetectable amounts were found in the roots and inflorescence stems at the commercial stage. Cynaropicrin content was found to correlate with expression of genes encoding CcGAS, CcGAO and CcCOS, which are involved in the STL biosynthesis. A more detailed study of leaf material revealed that cynaropicrin predominantly accumulates in the trichomes, and not in the apoplastic cavity fluids. Analysis of the promoter regions of CcGAO and CcCOS revealed the presence of L1-box motifs, which confers trichome-specific expression in Arabidopsis, suggesting that cynaropicrin is not only stored but also synthesized in trichomes. A transient expression of GFP fusion proteins was performed in Nicotiana benthamiana plants: the CcGAS fluorescence signal was located in the cytoplasm while the CcGAO and CcCOS localized to the endoplasmatic reticulum.

The Lotus japonicus LjSym4 gene is required for the successful symbiotic infection of root epidermal cells.

The role of the Lotus japonicus LjSym4 gene during the symbiotic interaction with Mesorhizobium loti and arbuscular mycorrhizal (AM) fungi was analyzed with two mutant alleles conferring phenotypes of different strength. Ljsym4-1 and Ljsym4-2 mutants do not form nodules with M. loti. Normal root hair curling and infection threads are not observed, while a nodC-dependent deformation of root hair tips indicates that nodulation factors are still perceived by Ljsym4 mutants. Fungal infection attempts on the mutants generally abort within the epidermis, but Ljsym4-1 mutants allow rare, successful, infection events, leading to delayed arbuscule formation. On roots of mutants homozygous for the Ljsym4-2 allele, arbuscule formation was never observed upon inoculation with either of the two AM fungi, Glomus intraradices or Gigaspora margarita. The strategy of epidermal penetration by G. margarita was identical for Ljsym4-2 mutants and the parental line, with appressoria, hyphae growing between two epidermal cells, penetration of epidermal cells through their anticlinal wall. These observations define a novel, genetically controlled step in AM colonization. Although rhizobia penetrate the tip of root hairs and AM fungi access an entry site near the base of epidermal cells, the LjSym4 gene is necessary for the appropriate response of this cell type to both microsymbionts. We propose that LjSym4 is required for the initiation or coordinated expression of the host plant cell's accommodation program, allowing the passage of both microsymbionts through the epidermis layer.

Actin versus tubulin configuration in arbuscule-containing cells from mycorrhizal tobacco roots.

The involvement of the cytoskeleton in symbiotic interactions such as arbuscular mycorrhizas has received little attention. In this paper, we examine the organization of actin in tobacco mycorrhizal roots and compare actin and tubulin patterns within arbuscule-containing cells. Our results show drastic reorganization of microfilaments and microtubules upon fungal infection and how those new cytoskeletal patterns relate to the host cytoplasm rearrangement and the intracellular fungal structures. Whereas in uninfected cells a network of cortical and perinuclear actin filaments was observed, in infected cells actin filaments closely follow the fungal branches and envelop the whole arbuscule in a dense coating network. Microtubules are less closely connected with the fungus surface. They run across the whole arbuscule mass, linking branches to each other and to the host cell cortex and nucleus. These major differences between the two cytoskeletal components are used to advance some suggestions concerning their contribution to structural functions in the plant-fungus interactions during the mycorrhizal symbiosis.

Cytoskeleton-related proteins in tobacco mycorrhizal cells: gamma-tubulin and clathrin localisation.

Plant cytoskeletal components respond to the penetration of both pathogenic and symbiotic fungi with a new organization of microtubules and microfilaments. To determine the origin and potential role of microtubule arrays previously observed in tobacco cells colonised by an arbuscular mycorrhizal fungus, we have investigated the patterns of gamma-tubulin and clathrin in uninfected and mycorrhizal cells with immunofluorescence techniques. Antibody against gamma-tubulin revealed microtubule organising centers (MTOC) along the nuclear envelope and along the host membrane that surrounds the plant/fungus interface, while clathrin was observed along the peripheral and perifungal membranes, as well as along a tubular system of endomembranes.

Epidermal cells of a symbiosis-defective mutant of Lotus japonicus show altered cytoskeleton organisation in the presence of a mycorrhizal fungus.

The influence of the mycorrhizal fungus Gigaspora margarita on cytoskeleton organisation in epidermal cells of Lotus japonicus roots was compared between plants of the wild type Gifu and the mutant Ljsym4-2, in which the fungus is confined to the epidermal cells. Immunofluorescence labelling of plant microtubules and microfilaments showed only limited alterations in the peripheral cytoskeleton of epidermal cells during early stages of fungal interaction with the wild type. Later, microtubules and microfilaments enveloped the growing hypha, while the host cell nucleus moved close to the fungus. In contrast, epidermal cells of the mutant responded with disorganisation and disassembly of microtubules and microfilaments before and during fungal penetration attempts. The fungus penetrated only as far as to epidermal cells, whose cytoplasm became devoid of tubulin and actin, suggesting cell death. The close relationship between host cytoskeleton organisation and compatibility with the fungus suggests that a functional Ljsym4 gene is necessary for correct reorganisation of the epidermal cell cytoskeleton in the presence of the fungus and for avoiding hypersensitivity-like reactions.

Vertical transmission of endobacteria in the arbuscular mycorrhizal fungus Gigaspora margarita through generation of vegetative spores.

Arbuscular mycorrhizal (AM) fungi living in symbiotic association with the roots of vascular plants have also been shown to host endocellular rod-shaped bacteria. Based on their ribosomal sequences, these endobacteria have recently been identified as a new taxon, Candidatus Glomeribacter gigasporarum. In order to investigate the cytoplasmic stability of the endobacteria in their fungal host and their transmission during AM fungal reproduction (asexual), a system based on transformed carrot roots and single-spore inocula of Gigaspora margarita was used. Under these in vitro sterile conditions, with no risk of horizontal contamination, the propagation of endobacteria could be monitored, and it was shown, by using primers designed for both 16S and 23S ribosomal DNAs, to occur through several vegetative spore generations (SG0 to SG4). A method of confocal microscopy for quantifying the density of endobacteria in spore cytoplasm was designed and applied; endobacteria were consistently found in all of the spore generations, although their number rapidly decreased from SG0 to SG4. The study demonstrates that a vertical transmission of endobacteria takes place through the fungal vegetative generations (sporulation) of an AM fungus, indicating that active bacterial proliferation occurs in the coenocytic mycelium of the fungus, and suggests that these bacteria are obligate endocellular components of their AM fungal host.