Tissue-adapted invasion strategies of the rice blast fungus Magnaporthe oryzae.

Magnaporthe oryzae causes rice blast, the most serious foliar fungal disease of cultivated rice (Oryza sativa). During hemibiotrophic leaf infection, the pathogen simultaneously combines biotrophic and necrotrophic growth. Here, we provide cytological and molecular evidence that, in contrast to leaf tissue infection, the fungus adopts a uniquely biotrophic infection strategy in roots for a prolonged period and spreads without causing a loss of host cell viability. Consistent with a biotrophic lifestyle, intracellularly growing hyphae of M. oryzae are surrounded by a plant-derived membrane. Global, temporal gene expression analysis used to monitor rice responses to progressive root infection revealed a rapid but transient induction of basal defense-related gene transcripts, indicating perception of the pathogen by the rice root. Early defense gene induction was followed by suppression at the onset of intracellular fungal growth, consistent with the biotrophic nature of root invasion. By contrast, during foliar infection, the vast majority of these transcripts continued to accumulate or increased in abundance. Furthermore, induction of necrotrophy-associated genes during early tissue penetration, previously observed in infected leaves, was not seen in roots. Collectively, our results not only report a global characterization of transcriptional root responses to a biotrophic fungal pathogen but also provide initial evidence for tissue-adapted fungal infection strategies.

Nonspecific PCR amplification by high-fidelity polymerases: implications for next-generation sequencing of AFLP markers.

High-fidelity 'proofreading' polymerases are often used in library construction for next-generation sequencing projects, in an effort to minimize errors in the resulting sequence data. The increased template fidelity of these polymerases can come at the cost of reduced template specificity, and library preparation methods based on the AFLP technique may be particularly susceptible. Here, we compare AFLP profiles generated with standard Taq and two versions of a high-fidelity polymerase. We find that Taq produces fewer and brighter peaks than high-fidelity polymerase, suggesting that Taq performs better at selectively amplifying templates that exactly match the primer sequences. Because the higher accuracy of proofreading polymerases remains important for sequencing applications, we suggest that it may be more effective to use alternative library preparation methods.

Analysis of bioavailable arsenic in rice with whole cell living bioreporter bacteria.

A multiwell plate bioassay was developed using genetically modified bacteria (bioreporter cells) to detect inorganic arsenic extracted from rice. The bacterial cells expressed luciferase upon exposure to arsenite, the activity of which was detected by measurement of cellular bioluminescence. The bioreporter cells detected arsenic in all rice varieties tested, with averages of 0.02-0.15 microg of arsenite equivalent per gram of dry weight and a method detection limit of 6 ng of arsenite per gram of dry rice. This amounted to between approximately 20 and 90% of the total As content reported by chemical methods for the same sample and suggested that a major proportion of arsenic in rice is in the inorganic form. Calibrations of the bioassay with pure inorganic and organic arsenic forms showed that the bacterial cells react to arsenite with highest affinity, followed by arsenate (with 25% response relative to an equivalent arsenite concentration) and trimethylarsine oxide (at 10% relative response). A method for biocompatible arsenic extraction was elaborated, which most optimally consisted of (i) grinding rice to powder, (ii) mixing with an aqueous solution containing pancreatic enzymes, (iii) mechanical shearing, (iv) extraction in mild acid conditions and moderate heat, and (v) centrifugation and pH neutralization. Detection of mainly inorganic arsenic by the bacterial cells may have important advantages for toxicity assessment of rice consumption and would form a good complement to total chemical arsenic determination.

The half-size ABC transporters STR1 and STR2 are indispensable for mycorrhizal arbuscule formation in rice.

The central structure of the symbiotic association between plants and arbuscular mycorrhizal (AM) fungi is the fungal arbuscule that delivers minerals to the plant. Our earlier transcriptome analyses identified two half-size ABCG transporters that displayed enhanced mRNA levels in mycorrhizal roots. We now show specific transcript accumulation in arbusculated cells of both genes during symbiosis. Presently, arbuscule-relevant factors from monocotyledons have not been reported. Mutation of either of the Oryza sativa (rice) ABCG transporters blocked arbuscule growth of different AM fungi at a small and stunted stage, recapitulating the phenotype of Medicago truncatula stunted arbuscule 1 and 2 (str1 and str2) mutants that are deficient in homologous ABCG genes. This phenotypic resemblance and phylogenetic analysis suggest functional conservation of STR1 and STR2 across the angiosperms. Malnutrition of the fungus underlying limited arbuscular growth was excluded by the absence of complementation of the str1 phenotype by wild-type nurse plants. Furthermore, plant AM signaling was found to be intact, as arbuscule-induced marker transcript accumulation was not affected in str1 mutants. Strigolactones have previously been hypothesized to operate as intracellular hyphal branching signals and possible substrates of STR1 and STR2. However, full arbuscule development in the strigolactone biosynthesis mutants d10 and d17 suggested strigolactones to be unlikely substrates of STR1/STR2. Interestingly, rice STR1 is associated with a cis-natural antisense transcript (antiSTR1). Analogous to STR1 and STR2, at the root cortex level, the antiSTR1 transcript is specifically detected in arbusculated cells, suggesting unexpected modes of STR1 regulation in rice.

Seed-specific silencing of OsMRP5 reduces seed phytic acid and weight in rice.

Phytic acid (PA) is poorly digested by humans and monogastric animals and negatively affects human/animal nutrition and the environment. Rice mutants with reduced PA content have been developed but are often associated with reduced seed weight and viability, lacking breeding value. In the present study, a new approach was explored to reduce seed PA while attaining competitive yield. The OsMRP5 gene, of which mutations are known to reduce seed PA as well as seed yield and viability, was down-regulated specifically in rice seeds by using an artificial microRNA driven by the rice seed specific promoter Ole18. Seed PA contents were reduced by 35.8-71.9% in brown rice grains of transgenic plants compared to their respective null plants (non-transgenic plants derived from the same event). No consistent significant differences of plant height or number of tillers per plant were observed, but significantly lower seed weights (up to 17.8% reduction) were detected in all transgenic lines compared to null plants, accompanied by reductions of seed germination and seedling emergence. It was observed that the silencing of the OsMRP5 gene increased the inorganic P (Pi) levels (up to 7.5 times) in amounts more than the reduction of PA-P in brown rice. This indicates a reduction in P content in other cellular compounds, such as lipids and nucleic acids, which may affect overall seed development. Put together, the present study demonstrated that seed specific silencing of OsMRP5 could significantly reduce the PA content and increase Pi levels in seeds; however, it also significantly lowers seed weight in rice. Discussions were made regarding future directions towards producing agronomically competitive and nutritionally valuable low PA rice.

Rapid and selective surveillance of Arabidopsis thaliana genome annotations with Centrifuge.

Centrifuge is a user-friendly system to simultaneously access Arabidopsis gene annotations and intra- and inter-organism sequence comparison data. The tool allows rapid retrieval of user-selected data for each annotated Arabidopsis gene providing, in any combination, data on the following features: predicted protein properties such as mass, pI, cellular location and transmembrane domains; SWISS-PROT annotations; Interpro domains; Gene Ontology records; verified transcription; BLAST matches to the proteomes of A.thaliana, Oryza sativa (rice), Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens. The tool lends itself particularly well to the rapid analysis of contigs or of tens or hundreds of genes identified by high-throughput gene expression experiments. In these cases, a summary table of principal predicted protein features for all genes is given followed by more detailed reports for each individual gene. Centrifuge can also be used for single gene analysis or in a word search mode. AVAILABILITY: http://centrifuge.unil.ch/ CONTACT: edward.farmer@unil.ch.

Spatio-temporal transcript profiling of rice roots and shoots in response to phosphate starvation and recovery.

Using rice (Oryza sativa) as a model crop species, we performed an in-depth temporal transcriptome analysis, covering the early and late stages of Pi deprivation as well as Pi recovery in roots and shoots, using next-generation sequencing. Analyses of 126 paired-end RNA sequencing libraries, spanning nine time points, provided a comprehensive overview of the dynamic responses of rice to Pi stress. Differentially expressed genes were grouped into eight sets based on their responses to Pi starvation and recovery, enabling the complex signaling pathways involved in Pi homeostasis to be untangled. A reference annotation-based transcript assembly was also generated, identifying 438 unannotated loci that were differentially expressed under Pi starvation. Several genes also showed induction of unannotated splice isoforms under Pi starvation. Among these, PHOSPHATE2 (PHO2), a key regulator of Pi homeostasis, displayed a Pi starvation-induced isoform, which was associated with increased translation activity. In addition, microRNA (miRNA) expression profiles after long-term Pi starvation in roots and shoots were assessed, identifying 20 miRNA families that were not previously associated with Pi starvation, such as miR6250. In this article, we present a comprehensive spatio-temporal transcriptome analysis of plant responses to Pi stress, revealing a large number of potential key regulators of Pi homeostasis in plants.

Rice perception of symbiotic arbuscular mycorrhizal fungi requires the karrikin receptor complex.

In terrestrial ecosystems, plants take up phosphate predominantly via association with arbuscular mycorrhizal fungi (AMF). We identified loss of responsiveness to AMF in the rice (Oryza sativa) mutant hebiba, reflected by the absence of physical contact and of characteristic transcriptional responses to fungal signals. Among the 26 genes deleted in hebiba, DWARF 14 LIKE is, the one responsible for loss of symbiosis . It encodes an alpha/beta-fold hydrolase, that is a component of an intracellular receptor complex involved in the detection of the smoke compound karrikin. Our finding reveals an unexpected plant recognition strategy for AMF and a previously unknown signaling link between symbiosis and plant development.

Response of bat species to sylvo-pastoral abandonment

We assessed the effect of abandonment of sylvo-pastoral practices in chestnut orchards (Castanea sativa) on bats in southern Switzerland to determine practical recommendations for bat conservation. We compared bat species richness and foraging activities between traditionally managed and unmanaged chestnut orchards, testing the hypothesis that managed orchards provide better foraging opportunities and harbour more bat species. Echolocation calls of foraging bats were sampled simultaneously at paired sites of managed and unmanaged orchards using custom made recorders. Vegetation structure and aerial insect availability were sampled at the recording sites and used as explanatory variables in the model. In a paired sampling design, we found twice the number of bat species (12) and five times higher total foraging activity in the managed chestnut orchards compared to the unmanaged ones. Bat species with low flight manoeuvrability were 14 times more common in managed orchards, whereas bats with medium to high manoeuvrability were only 5 times more common than in abandoned orchards. The vegetation structure was less dense in managed orchards. However, management did not affect relative insect abundance. Bats primarily visited the most open orchards, free of undergrowth. As a result of restricted access into the overgrown forests, the abandonment of chestnut orchards leads to a decline in bat species richness and foraging activities. Continued management of chestnut orchards to maintain an open structure is important for the conservation of endangered bat species in the southern Swiss Alps.

Identification and characterization of the members of the pho1 gene family in moss and rice

Inorganic phosphate (Pi) is one of the main nutrients limiting plant growth anddevelopment in many agro-ecosystems. In plants, phosphate is acquired from the soil by theroots, and is then transferred to the shoot via the xylem. In the model plant Arabidopsisthaliana, PHO1 was previously identified as being involved in loading Pi into the xylem ofroots. AtPHO1, belongs to a multigenic family composed of 10 additional members, namelyAtPHO1;H1 to AtPHO1;10. In this study, we aimed at further investigating the role of thePHO1 gene family in Pi homeostasis in plants, and to this end we isolated and characterizedthe PHO1 members of two main model plants, the moss Physcomitrella patens and the riceOryza sativa.In the bryophyte P. patens, bioinformatic analyses revealed the presence of seven AtPHO1homologues, highly similar to AtPHO1. The seven moss PHO1 genes, namely PpPHO1;1 toPpPHO1;7 appeared to be differentially regulated, both at the tissue level and in response toPi status. However only PpPHO1;1 and PpPHO1;7 were specifically up-regulated upon Pistarvation, suggesting a potential role in Pi homeostasis. We also characterized the responseof P. patens to Pi starvation, showing that higher and lower plants share some commonstrategies to adapt to Pi-deficiency.In the second part, focusing on the monocotyledon rice, we showed the existence of threePHO1 homologues OsPHO1;1 to OsPHO1;3, with the unique particularity of each havingNatural Antisense Transcripts (NATs). Molecular analyses revealed that both the sense andthe antisense OsPHO1;2 transcripts were by far the most abundantly expressed transcripts ofthe family, preferentially expressed in the roots. The stable expression of OsPHO1;2 in allconditions tested, in opposition with the highly induced antisense transcript upon Pistarvation, suggest a putative role for the antisense in regulating the sense transcript.Moreover, mutant analyses revealed that OsPHO1;2 plays a key role in Pi homeostasis, intransferring Pi from the root to the shoot. Finally, complementing the pho1 mutant inArabidopsis, characterized by low Pi in the shoot and reduced growth, with the riceOsPHO1;2 gene revealed a new role for PHO1 in Pi signaling. Indeed, the complementedplants showed normal growth, with however low Pi content.

Cannabidiol attenuates cardiac dysfunction, oxidative stress, fibrosis, and inflammatory and cell death signaling pathways in diabetic cardiomyopathy.

Objectives In this study, we have investigated the effects of cannabidiol (CBD) on myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type I diabetic cardiomyopathy and primary human cardiomyocytes exposed to high glucose. Background Cannabidiol, the most abundant nonpsychoactive constituent of Cannabis sativa (marijuana) plant, exerts anti-inflammatory effects in various disease models and alleviates pain and spasticity associated with multiple sclerosis in humans. Methods Left ventricular function was measured by the pressure-volume system. Oxidative stress, cell death, and fibrosis markers were evaluated by molecular biology/biochemical techniques, electron spin resonance spectroscopy, and flow cytometry. Results Diabetic cardiomyopathy was characterized by declined diastolic and systolic myocardial performance associated with increased oxidative-nitrative stress, nuclear factor-kappa B and mitogen-activated protein kinase (c-Jun N-terminal kinase, p-38, p38 alpha) activation, enhanced expression of adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), tumor necrosis factor-alpha, markers of fibrosis (transforming growth factor-beta, connective tissue growth factor, fibronectin, collagen-1, matrix metalloproteinase-2 and -9), enhanced cell death (caspase 3/7 and poly[adenosine diphosphate-ribose] polymerase activity, chromatin fragmentation, and terminal deoxynucleotidyl transferase dUTP nick end labeling), and diminished Akt phosphorylation. Remarkably, CBD attenuated myocardial dysfunction, cardiac fibrosis, oxidative/nitrative stress, inflammation, cell death, and interrelated signaling pathways. Furthermore, CBD also attenuated the high glucose-induced increased reactive oxygen species generation, nuclear factor-kappa B activation, and cell death in primary human cardiomyocytes. Conclusions Collectively, these results coupled with the excellent safety and tolerability profile of CBD in humans, strongly suggest that it may have great therapeutic potential in the treatment of diabetic complications, and perhaps other cardiovascular disorders, by attenuating oxidative/nitrative stress, inflammation, cell death and fibrosis. (J Am Coll Cardiol 2010;56:2115-25) (C) 2010 by the American College of Cardiology Foundation.

Persistence of a biocontrol Pseudomonas inoculant as high populations of culturable and non-culturable cells in 200-cm-deep soil profiles

Little is known about the ecology of soil inoculants used for pathogen biocontrol, biofertilization and bioremediation under field conditions. We investigated the persistence and the physiological states of soil-inoculated Pseudomonas protegens (previously Pseudomonas fluorescens) CHA0 (108 CFU g−1 surface soil) in different soil microbial habitats in a planted ley (Medicago sativa L.) and an uncovered field plot. At 72 days, colony counts of the inoculant were low in surface soil (uncovered plot) and earthworm guts (ley plot), whereas soil above the plow pan (uncovered plot), and the rhizosphere and worm burrows present until 1.2 m depth (ley plot) were survival hot spots (105-106 CFU g−1 soil). Interestingly, strain CHA0 was also detected in the subsoil of both plots, at 102-105 CFU g−1 soil between 1.8 and 2 m depth. However, non-cultured CHA0 cells were also evidenced based on immunofluorescence microscopy. Kogure's direct viable counts of nutrient-responsive cells showed that many more CHA0 cells were in a viable but non-culturable (VBNC) or a non-responsive (dormant) state than in a culturable state, and the proportion of cells in those non-cultured states depended on soil microbial habitat. At the most, cells in a VBNC state amounted to 34% (above the plow pan) and those in a dormant state to 89% (in bulk soil between 0.6 and 2 m) of all CHA0 cells. The results indicate that field-released Pseudomonas inoculants may persist at high cell numbers, even in deeper soil layers, and display a combination of different physiological states whose prevalence fluctuates according to soil microbial habitats.