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.

Defense related phytohormones regulation in arbuscular mycorrhizal symbioses depends on the partner genotypes.

Arbuscular mycorrhizal (AM) symbioses are mutualistic associations between soil fungi and most vascular plants. Modulation of the hormonal and transcriptional profiles, including changes related to defense signalling, has been reported in many host plants during AM symbioses. These changes have been often related to the improved stress tolerance common in mycorrhizal plants. However, results on the alterations in phytohormones content and their role on the symbiosis are controversial. Here, an integrative analysis of the response of phylogenetically diverse plants (i.e., tomato, soybean, and maize) to two mycorrhizal fungi -Funneliformis mosseae and Rhizophagus irregularis- was performed. The analysis of the defense-related hormones salicylic acid, abscisic acid, and jasmonates, and the expression of marker genes of the pathways they regulate, revealed significant changes in the roots of mycorrhizal plants. These changes depended on both the plant and the AM fungus (AMF) involved. However, general trends can be identified: roots associated with the most effective colonizer R. irregularis showed fewer changes in these defense-related traits, while the colonization by F. mosseae led to significant modifications in all plants tested. The up-regulation of the jasmonate pathway by F. mosseae was found to be highly conserved among the different plant species, suggesting an important role of jasmonates during this AM interaction. Our study evidences a strong influence of the AMF genotype on the modulation of host defense signalling, and offers hints on the role of these changes in the symbiosis.

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont.

• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

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.

Laparoscopic appendectomy for complicated acute appendicitis.

The role of laparoscopic appendectomy in complicated appendicitis is still not widely accepted. The authors report their retrospective study performed to evaluate the effectiveness of the laparoscopic approach in the management of complicated appendicitis. From January 2003 to October 2008, 552 patients underwent appendectomy in our surgical department. Among these, 358 were not complicated appendicitis while 194 were complicated. Of the 194 cases of complicated appendicitis, 121 patients underwent laparoscopic appendectomy while the remaining 73 cases were treated by conventional open surgery. The average length of hospital stay was 5.7 days, with a range from 4 to 13 days. Post-operative complications were observed in a total 11 patients (9.1%), including 3 cases of intra abdominal abscess (2.5%), 2 cases of umbilical wound infection (1.6%) and 6 cases of prolonged ileus (4.9%). Our experience suggests that the laparoscopic procedure is a valid, safe and feasible option to manage acute complicated appendicitis.

[Radiological study of anastomotic leakages following colorectal surgery]. / Il controllo radiologico delle deiscenze anastomotiche in chirurgia colo-rettale.

The anastomotic leakage is one of the most serious complications following colorectal surgery. The incidence rate is between 3% and 21% considering the different experiences, pathology and surgical techniques. Our aim is to verify the role of radiological study in 45 patients with clinical and subclinical colorectal anastomotic leakage total anastomoses = 252). In 31 patients at risk, the operation was concluded with a loop ileostomy. The radiological study gastrografin enema was performed in all patients (26 symptomatic and 19 asymptomatic patients with loop ileostomy). The dehiscence incidence resulted 5.5%: 14 of 252 patients. In our experience the radiological study of selected colorectal anastomoses allowed to show the site and the flow of the leakage and to plan the proper management. In asymptomatic patients the study allowed to programme a specific follow up in patients with higher risk of postinflammatory stenosis or perhaps neoplastic relapse.

The impact of tillage practices on arbuscular mycorrhizal fungal diversity in subtropical crops.

Arbuscular mycorrhizal fungi (AMF) are a main component of soil microbiota in most agrosystems. As obligately mutualistic symbionts, they colonize the roots of the majority of plants, including crop plants. We used molecular techniques to investigate how different tillage systems (moldboard, shred-bedding, subsoil-bedding, and no tillage) can influence the AM fungal community colonizing maize, bean, and sorghum roots in an experimental site located in northern Tamaulipas, Mexico. Roots from 36 plants were analyzed using AM fungal-specific primers to partially amplify the small subunit (SSU) of the ribosomal DNA genes. More than 880 clones were screened for restriction fragment length polymorphism (RFLP) variation, and 173 of these were sequenced. Ten AM fungal types were identified and clustered into three AM fungal families: Gigasporaceae, Glomaceae, and Paraglomaceae. Glomus was the dominating taxon in all the samples. Four of the 10 identified types were distinct from any previously published sequences and could correspond to either known unsequenced species or unknown species. The fungal diversity was low in the four agriculture management systems, but the multidimensional scaling (MDS) analysis and log-linear-saturated model indicated that the composition of the AMF community was significantly affected by the tillage system. In conclusion, since some fungal types were treatment specific, agricultural practices could directly or indirectly influence AM biodiversity.

[Primary duodenal adenocarcinoma: report of three cases, prognostic factors and therapeutic approach]. / Adenocarcinoma primitivo del duodeno: tre casi clinici e considerazioni prognostico-terapeutiche.

Three cases of histologically proven primary non-ampullary adenocarcinoma of the duodenum, observed in our Department from 2001 to 2004, are described. The cases were treated by pancreaticoduodenectomy, duodenal resection and transduodenal excision, respectively. The rarity of this pathology is documented by few retrospective studies and justifies discussion about the main prognostic factors and the best therapeutic approach. We analyze diagnostic, therapeutic and prognostic factors after a revision of literature.

[Covered perforation of solitary cecal diverticulum: case report]. / Perforazione coperta di un diverticolo solitario del cieco: case report.

Acute right lower abdominal pain is often clinically difficult to diagnose. The diagnosis, especially in young patients, is frequently oriented to appendicular disease. Surgical exploration only confirms diagnosis or surprises the surgeon, revealing an unexpected right colon diverticulitis. This emergency condition challenges the surgeon with the dilemma about the best therapeutic choice: conservative or radical treatment? The elective localization of diverticulitis to the right colon is very rare (6.6-14%). The authors report a case of covered perforation of a solitary cecal diverticulum.

[Surgical timing in bleeding liver adenoma: case report]. / Timing chirurgico nell'adenoma epatico sanguinante: case report.

The diagnosis of liver adenoma, which etiopathogenesis most often involves a prolonged assumption of estrogen (90% of adenomas occurs in women after more than 5 years of estrogen therapy), always imposes a surgical resection. The reason depend from neoplasia characteristics like the malignant evolution (4%) and the high risk of abdominal/intratumoral bleeding (30-50%), that increases during pregnancy and postpartum period. Regression of lesion after discontinuation of hormone therapy is rare and does not remove the degeneration and/or haemorrhagic risk. Liver resection should be performed with appropriate selective endovascular embolization, considering that an inept emergency surgery may impose a greater risk ot the liver, exposing the patient to major risk of morbidity and mortality. The correct timing from embolization to elective surgery is not yet standardized in the literature. The surgeon's personal experience and mainly a careful patient follow-up suggest the timing of surgery after embolization. The authors relate their own experience about the therapeutic strategy and surgical timing in a case of bleeding liver adenoma.

Expression profiles of a phosphate transporter gene (GmosPT) from the endomycorrhizal fungus Glomus mosseae.

Arbuscular mycorrhizal (AM) fungi have long been shown to successfully contribute to phosphate uptake by plant roots. The first step of the fungus-mediated uptake is carried out by fungal membrane Pi transporters (PT) that transfer Pi from the soil into the extraradical hyphae. In the present work we report the identification and characterisation of a PT gene from Glomus mosseae, an AM fungus important for natural and agricultural ecosystems. Degenerate primers and rapid amplification of cDNA ends-polymerase chain reaction (PCR) allowed us to obtain a sequence (GmosPT) showing a highly significant similarity with GiPT and GvPT, the only two other PT genes already isolated from AM fungi. Reverse transcriptase-PCR experiments were carried out to study GmosPT expression profiles in structures corresponding to different fungal life stages (quiescent and germinated sporocarps, intraradical and extraradical hyphae) and in extra- and intraradical hyphae exposed to high and low Pi concentrations. GmosPT showed an expression pattern similar to GiPT, the Glomus intraradices PT gene, since its transcript was more abundant in the extraradical mycelium treated with micromolar Pi levels. In addition, the intraradical mycelium also showed a significant GmosPT expression level that was independent from external Pi concentrations. This finding opens new questions about the role and functioning of high-affinity PT in AM fungi.

Differential location of alpha-expansin proteins during the accommodation of root cells to an arbuscular mycorrhizal fungus.

alpha-Expansins are extracellular proteins that increase plant cell-wall extensibility. We analysed their pattern of expression in cucumber roots in the presence and in the absence of the mycorrhizal fungus, Glomus versiforme. The distribution of alpha-expansins was investigated by use of two polyclonal antibodies (anti-EXPA1 and anti-EXPA2, prepared against two different cucumber alpha-expansins) in immunoblotting, immunofluorescence, and immunogold experiments. Immunoblot results indicate the presence of a 30-kDa band specific for mycorrhizal roots. The two antibodies identify antigens with a different distribution in mycorrhizal roots: anti-EXPA1 labels the interface zone, but the plant cell walls only weakly. By contrast, the anti-EXPA2 labels only the plant cell walls. In order to understand the potential role of alpha-expansins during the accommodation of the fungus inside root cells, we prepared semi-thin sections to measure the size of cortical cells and the thickness of cortical cell walls in mycorrhizal and non-mycorrhizal root. Mycorrhizal cortical cells were significantly larger than non-mycorrhizal cells and had thicker cell walls. In double-labelling experiments with cellobiohydrolase-gold complex, we observed that cellulose was co-localized with alpha-expansins. Taken together, the results demonstrate that alpha-expansins are more abundant in the cucumber cell walls upon mycorrhizal infection; we propose that these wall-loosening proteins are directly involved in the accommodation of the fungus by infected cortical cells.

Isolation, free-living capacities, and genome structure of "Candidatus Glomeribacter gigasporarum," the endocellular bacterium of the mycorrhizal fungus Gigaspora margarita.

"Candidatus Glomeribacter gigasporarum" is an endocellular beta-proteobacterium present in the arbuscular mycorrhizal (AM) fungus Gigaspora margarita. We established a protocol to isolate "Ca. Glomeribacter gigasporarum" from its host which allowed us to carry out morphological, physiological, and genomic investigations on purified bacteria. They are rod shaped, with a cell wall typical of gram-negative bacteria and a cytoplasm rich in ribosomes, and they present no flagella or pili. Isolated bacteria could not be grown in any of the 19 culture media tested, but they could be kept alive for up to 4 weeks. PCR-based investigations of purified DNA from isolated bacteria did not confirm the presence of all genes previously assigned to "Ca. Glomeribacter gigasporarum." In particular, the presence of nif genes could not be detected. Pulsed-field gel electrophoresis analyses allowed us to estimate the genome size of "Ca. Glomeribacter gigasporarum" to approximately 1.4 Mb with a ca. 750-kb chromosome and a 600- to 650-kb plasmid. This is the smallest genome known for a beta-proteobacterium. Such small genome sizes are typically found in endocellular bacteria living permanently in their host. Altogether, our data suggest that "Ca. Glomeribacter gigasporarum" is an ancient obligate endocellular bacterium of the AM fungus G. margarita.

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.

Chlorophyllous and achlorophyllous specimens of Epipactis microphylla,(Neottieae, Orchidaceae) are associated with ectomycorrhizal septomycetes, including truffles.

Mycoheterotrophic species (i.e., achlorophyllous plants obtaining carbon from their mycorrhizal fungi) arose many times in evolution of the Neottieae, an orchid tribe growing in forests. Moreover, chlorophyllous Neottieae species show naturally occurring achlorophyllous individuals. We investigated the fungal associates of such a member of the Neottieae, Epipactis microphylla, to understand whether their mycorrhizal fungi predispose the Neottieae to mycoheterotrophy. Root symbionts were identified by sequencing the fungal ITS of 18 individuals from three orchid populations, including achlorophyllous and young, subterranean individuals. No rhizoctonias (the usual orchid symbionts) were recovered, but 78% of investigated root pieces were colonized by Tuber spp. Other Pezizales and some Basidiomycetes were also found. Using electron microscopy, we demonstrated for the first time that ascomycetes, especially truffles, form typical orchid mycorrhizae. All identified fungi (but one) belonged to taxa forming ectomycorrhizae on tree roots, and four of them were even shown to colonize surrounding trees. This is reminiscent of mycoheterotrophic orchid species that also associate with ectomycorrhizal fungi, although with higher specificity. Subterranean and achlorophyllous E. microphylla individuals thus likely rely on tree photosynthates, and a partial mycoheterotrophy in individuals plants can be predicted. We hypothesize that replacement of rhizoctonias by ectomycorrhizal symbionts in Neottieae entails a predisposition to achlorophylly.

Plants, mycorrhizal fungi and endobacteria: a dialog among cells and genomes.

This review focuses on mycorrhizas, which are associations between fungi and the roots of 90% of terrestrial plants. These are the most common symbioses in the world; they involve about 6000 species of fungi distributed through all the fungal phyla and about 240000 species of plants, including forest and crop plants. Thanks to mycorrhizal symbiosis and nutrient exchanges, regulated by complex molecular signals, the plant improves its vegetative growth, while the fungus accomplishes its life cycle. Molecular and cellular analyses demonstrate that during colonization the cellular organization of the two eukaryotes is completely remodeled. For example, in cortical cells, structural modifications involve both the host and the microbiont. Recent studies revealed that in arbuscular mycorrhizas (AM), system complexity is increased by the presence of a third symbiont: a bacterium living inside the fungus. The presence of this resident genome makes the investigation of the molecular dialogues among the symbiotic partners even more complex. Molecular analysis showed that the bacterium has genes involved in the acquisition of mineral nutrients. The experimental data support the current view that mycorrhizal symbioses are often tripartite associations.

Two genetically related strains of Tuber borchii produce Tilia mycorrhizas with different morphological traits.

Two genetically related strains of Tuber borchii Vittad. (1BO and 43BO) produce mycorrhizas with Tilia platyphyllos Scop. with a different degree of efficiency. The aim of this work was to characterize the morphology of the fungal symbiotic structures in order to examine potential relationships between the anatomical traits of the mycorrhiza, the mycorrhizal capacities of the fungal strains and their effect on the host plants. Some morphological features of mantle hyphae (small size, intense staining, vacuolization, abundance of mitochondria) led to a mantle with morphological features that were isolate-specific. There were unexpected differences, at least under our experimental conditions: 1BO strain mantle cells were larger, less reactive to staining, more highly vacuolated and poorer in mitochondria than those of 43BO. These features were found throughout the mantle in 1BO, while the inner mantle hyphae of 43BO were significantly smaller and more intensely stained than the outer cells. In the 43BO strain there was a positive relation between these features and higher infectivity (evaluated as percentage of mycorrhizal tips) as well as a slightly more effective stimulation of plant growth. These observations suggest that genetically related truffle strains produce mycorrhizas with different morphologies, which may be related to a more efficient response of the host plant to inoculation.

Hydrophobins in ectomycorrhizas: heterologous transcription of the Pisolithus HydPt-1 gene in yeast and Hebeloma cylindrosporum.

Hydrophobins are fungal cell wall proteins involved in aggregation of hyphae. Upon the development of the ectomycorrhizal symbiosis between tree roots and fungal hyphae, the transcripts of hydrophobin genes markedly accumulated. As the precise role of these proteins in symbiosis is not yet known, we develop heterologous expression system of the Pisolithus hydrophobin HYDPt-1. This gene has been introduced in Saccharomyces cerevisiae and in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum. Introns were required for hydPt-1 transcript accumulation in the basidiomycete H. cylindrosporum. Heterologous transcript accumulation did not alter the phenotype of either species. The lack of altered phenotype resulted from the absence of HYDPt-1 polypeptide accumulation in transformed strains.