Mycorrhizal diversity,seed germination and long‐term changes in population size across nine populations of the terrestrial orchid Neottia ovata

In plant species that rely on mycorrhizal symbioses for germination and seedling establishment, seedling recruitment and temporal changes in abundance can be expected to depend on fungal community composition and local environmental conditions. However, disentangling the precise factors that determine recruitment success in species that critically rely on mycorrhizal fungi represents a major challenge. In this study, we used seed germination experiments, 454 amplicon pyrosequencing and assessment of soil conditions to investigate the factors driving changes in local abundance in 28 populations of the orchid Neottia ovata. Comparison of population sizes measured in 2003 and 2013 showed that nearly 60% of the studied populations had declined in size (average growth rate across all populations: ?0.01). Investigation of the mycorrhizal fungi in both the roots and soil revealed a total of 68 species of putatively mycorrhizal fungi, 21 of which occurred exclusively in roots, 25 that occurred solely in soil and 22 that were observed in both the soil and roots. Seed germination was limited and significantly and positively related to soil moisture content and soil pH, but not to fungal community composition. Large populations or populations with high population growth rates showed significantly higher germination than small populations or populations declining in size, but no significant relationships were found between population size or growth and mycorrhizal diversity. Overall, these results indicate that temporal changes in abundance were related to the ability of seeds to germinate, but at the same time they provided limited evidence that variation in fungal communities played an important role in determining population dynamics.     

Insulin-mediated down-regulation of apolipoprotein A5 gene expression through the phosphatidylinositol 3-kinase pathway: role of upstream stimulatory factor

The apolipoprotein A5 gene (APOA5) has been repeatedly implicated in lowering plasma triglyceride levels. Since several studies have demonstrated that hyperinsulinemia is associated with hypertriglyceridemia, we sought to determine whether APOA5 is regulated by insulin. Here, we show that cell lines and mice treated with insulin down-regulate APOA5 expression in a dose-dependent manner. Furthermore, we found that insulin decreases human APOA5 promoter activity, and subsequent deletion and mutation analyses uncovered a functional E box in the promoter. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that this APOA5 E box binds upstream stimulatory factors (USFs). Moreover, in transfection studies, USF1 stimulates APOA5 promoter activity, and the treatment with insulin reduced the binding of USF1/USF2 to the APOA5 promoter. The inhibition of the phosphatidylinositol 3-kinase (PI3K) pathway abolished insulin's effect on APOA5 gene expression, while the inhibition of the P70 S6 kinase pathway with rapamycin reversed its effect and increased APOA5 gene expression. Using an oligonucleotide precipitation assay for USF from nuclear extracts, we demonstrate that phosphorylated USF1 fails to bind to the APOA5 promoter. Taken together, these data indicate that insulin-mediated APOA5 gene transrepression could involve a phosphorylation of USFs through the PI3K and P70 S6 kinase pathways that modulate their binding to the APOA5 E box and results in APOA5 down-regulation. The effect of exogenous hyperinsulinemia in men showed a decrease in the plasma ApoAV level. These results suggest a potential contribution of the APOA5 gene in hypertriglyceridemia associated with hyperinsulinemia.     

Clonality and α-a recombination in the Australian Cryptococcus gattii VGII population--an emerging outbreak in Australia

Background

Cryptococcus gattii is a basidiomycetous yeast that causes life-threatening disease in humans and animals. Within C. gattii, four molecular types are recognized (VGI to VGIV). The Australian VGII population has been in the spotlight since 2005, when it was suggested as the possible origin for the ongoing outbreak at Vancouver Island (British Columbia, Canada), with same-sex mating being suggested as the driving force behind the emergence of this outbreak, and is nowadays hypothesized as a widespread phenomenon in C. gattii. However, an in-depth characterization of the Australian VGII population is still lacking. The present work aimed to define the genetic variability within the Australian VGII population and determine processes shaping its population structure.

Methodology/Principal Findings

A total of 54 clinical, veterinary and environmental VGII isolates from different parts of the Australian continent were studied. To place the Australian population in a global context, 17 isolates from North America, Europe, Asia and South America were included. Genetic variability was assessed using the newly adopted international consensus multi-locus sequence typing (MLST) scheme, including seven genetic loci: CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and IGS1. Despite the overall clonality observed, the presence of MAT a VGII isolates in Australia was demonstrated for the first time in association with recombination in MATα-MAT a populations. Our results also support the hypothesis of a “smouldering” outbreak throughout the Australian continent, involving a limited number of VGII genotypes, which is possibly caused by a founder effect followed by a clonal expansion.

Conclusions/Significance

The detection of sexual recombination in MATα-MAT a population in Australia is in accordance with the natural life cycle of C. gattii involving opposite mating types and presents an alternative to the same-sex mating strategy suggested elsewhere. The potential for an Australian wide outbreak highlights the crucial issue to develop active surveillance procedures.     

Fatty Acid Production from Amino Acids and α-Keto Acids by Brevibacterium linens BL2

Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and α-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of α-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and α-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from α-keto acids only. BL2 also converted α-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of B. linens ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and α-keto acids and that carbon metabolism is important in regulating this event.     

Isolation of DNA markers informative in purebred dog families by genomic representational difference analysis (gRDA)

Genomic Representational Difference Analysis (gRDA) is a subtractive DNA method to clone the differences between two related genomes, called tester and driver. We have evaluated this method to obtain polymorphic DNA markers for pedigree dogs. Amplified size-selected genomic restriction fragments (amplicons) of two dog littermates were repeatedly hybridized to each other in order to remove (subtract) those restriction fragments common to both sibs. Already after two rounds of subtractive hybridization, a clear enrichment of presumably tester-specific restriction fragments was observed, which was even more pronounced after the third round of subtraction. A plasmid library of 3000 recombinant clones was constructed of the second round and of the third round difference product. DNA sequence determination of randomly chosen clones of each difference product showed that approximately 1000 unique clones were obtained in the second-round difference product and approximately 500 in the third-round difference product. About half of the clones identified in the second-round difference product were also present in the third-round difference product. Of the second-round difference product, 39 different gRDA fragments could be identified, of which 21 were tester specific. In the third-round difference product, 22 different gRDA fragments were identified, of which 18 were tester specific. There were 13 fragments in common, resulting in a total of 48 different fragments. In order to establish the localization of these markers, we performed mapping using the dog radiation hybrid panel RHDF5000. Of 39 mapped clones, 29 were mapped to 20 existing RH groups, and 10 remained unlinked. It is concluded that gRDA is suitable to generate DNA markers to track disease genes within lines of pedigree dogs. Received: 26 April 2000 / Accepted: 11 May 2000     

Exploiting dendritic cells for cancer immunotherapy: genetic modification of dendritic cells

Dendritic cells (DCs) are pivotal regulators of immune reactivity and immune tolerance. The observation that DCs can recruit naive T cells has invigorated cancer immunology and led to the proposal of DCs as the basis for vaccines designed for the treatment of cancer. Designing effective strategies to load DCs with antigens is a challenging field of research. The successful realization of gene transfer to DCs will be highly dependent on the employed vector system. Here, we review various viral and non-viral gene transfer systems, and discuss their distinct characteristics and possible advantages and disadvantages in respect to their use in DC-based immunotherapy.     

Control of the pattern‐recognition receptor EFR by an ER protein complex in plant immunity

In plant innate immunity, the surface‐exposed leucine‐rich repeat receptor kinases EFR and FLS2 mediate recognition of the bacterial pathogen‐associated molecular patterns EF‐Tu and flagellin, respectively. We identified the Arabidopsis stromal‐derived factor‐2 (SDF2) as being required for EFR function, and to a lesser extent FLS2 function. SDF2 resides in an endoplasmic reticulum (ER) protein complex with the Hsp40 ERdj3B and the Hsp70 BiP, which are components of the ER‐quality control (ER‐QC). Loss of SDF2 results in ER retention and degradation of EFR. The differential requirement for ER‐QC components by EFR and FLS2 could be linked to N‐glycosylation mediated by STT3a, a catalytic subunit of the oligosaccharyltransferase complex involved in co‐translational N‐glycosylation. Our results show that the plasma membrane EFR requires the ER complex SDF2–ERdj3B–BiP for its proper accumulation, and provide a demonstration of a physiological requirement for ER‐QC in transmembrane receptor function in plants. They also provide an unexpected differential requirement for ER‐QC and N‐glycosylation components by two closely related receptors.     

Mycorrhizal specificity does not limit the distribution of an endangered orchid species

What factors determine the distribution of a species is a central question in ecology and conservation biology. In general, the distribution of plant species is assumed to be controlled by dispersal or environmentally controlled recruitment. For plant species which are critically dependent on mycorrhizal symbionts for germination and seedling establishment, specificity in mycorrhizal associations and availability of suitable mycorrhizal fungi can be expected to have a major impact on successful colonization and establishment and thus ultimately on a species distribution. We combined seed germination experiments with soil analyses and fungal assessments using 454 amplicon pyrosequencing to test the relative importance of dispersal limitation, mycorrhizal availability and local growth conditions on the distribution of the orchid species Liparis loeselii, which, despite being widely distributed, is rare and endangered in Europe. We compared local soil conditions, seed germination and mycorrhizal availability in the soil between locations in northern Belgium and France where L. loeselii occurs naturally and locations where conditions appear suitable, but where adults of the species are absent. Our results indicated that mycorrhizal communities associating with L. loeselii varied among sites and plant life cycle stages, but the observed variations did not affect seed germination, which occurred regardless of current L. loeselii presence and was significantly affected by soil moisture content. These results indicate that L. loeselii is a mycorrhizal generalist capable of opportunistically associating with a variety of fungal partners to induce seed germination. They also indicate that availability of fungal associates is not necessarily the determining factor driving the distribution of mycorrhizal plant species.     

Degradation of some phenols and hydroxybenzoates by the imperfect ascomycetous yeastsCandida parapsilosis andArxula adeninivorans: evidence for an operative gentisate pathway

The imperfect ascomycetous yeastsCandida parapsilosis andArxula adeninivorans degraded 3-hydroxybenzoic acid via gentisate which was the cleavage substrate. 4-Hydroxybenzoic acid was metabolized via protocatechuate. No cleavage enzyme for the latter was detected. In stead of this NADH- and NADPH-dependent monooxygenases were present. In cells grown at the expense of hydroquinone and 4-hydroxygenzoic acid, enzymes of the hydroxyhydroquinone variant of the 3-oxoadipate pathway were demonstrated, which also took part in the degradation of 2,4-dihydroxybenzoic acid byC. parapsilosis.Abbreviations HHQ Hydroxyhydroquinone (1,2,4-trihydroxybenzene) - GSH reduced Glutathione