MGOS: A resource for studying Magnaporthe grisea and Oryza sativa interactions

The MGOS (Magnaporthe grisea Oryza sativa) web-based database contains data from Oryza sativa and Magnaporthe grisea interaction experiments in which M. grisea is the fungal pathogen that causes the rice blast disease. In order to study the interactions, a consortium of fungal and rice geneticists was formed to construct a comprehensive set of experiments that would elucidate information about the gene expression of both rice and M. grisea during the infection cycle. These experiments included constructing and sequencing cDNA and robust long-serial analysis gene expression libraries from both host and pathogen during different stages of infection in both resistant and susceptible interactions, generating >50,000 M. grisea mutants and applying them to susceptible rice strains to test for pathogenicity, and constructing a dual O. sativa-M. grisea microarray. MGOS was developed as a central web-based repository for all the experimental data along with the rice and M. grisea genomic sequence. Community-based annotation is available for the M. grisea genes to aid in the study of the interactions.     

Vision impairs the abilities of bats to avoid colliding with stationary obstacles

Background

Free-flying insectivorous bats occasionally collide with stationary objects they should easily detect by echolocation and avoid. Collisions often occur with lighted objects, suggesting ambient light may deleteriously affect obstacle avoidance capabilities. We tested the hypothesis that free-flying bats may orient by vision when they collide with some obstacles. We additionally tested whether acoustic distractions, such as “distress calls” of other bats, contributed to probabilities of collision.

Methodology/Principal Findings

To investigate the role of visual cues in the collisions of free-flying little brown bats (Myotis lucifugus) with stationary objects, we set up obstacles in an area of high bat traffic during swarming. We used combinations of light intensities and visually dissimilar obstacles to verify that bats orient by vision. In early August, bats collided more often in the light than the dark, and probabilities of collision varied with the visibility of obstacles. However, the probabilities of collisions altered in mid to late August, coincident with the start of behavioural, hormonal, and physiological changes occurring during swarming and mating. Distress calls did not distract bats and increase the incidence of collisions.

Conclusions/Significance

Our findings indicate that visual cues are more important for free-flying bats than previously recognized, suggesting integration of multi-sensory modalities during orientation. Furthermore, our study highlights differences between responses of captive and wild bats, indicating a need for more field experiments.     

A critical role for Syk protein tyrosine kinase in Fc receptor-mediated antigen presentation and induction of dendritic cell maturation

Dendritic cells (DCs) are the only APCs capable of initiating adaptive immune responses. The initiation of immune responses requires that DCs 1) internalize and present Ags; and 2) undergo a differentiation process, called "maturation", which transforms DCs into efficient APCs. DC maturation may be initiated by the engagement of different surface receptors, including certain cytokine receptors (such as TNFR), Toll-like receptors, CD40, and FcRs. The early activation events that link receptor engagement and DC maturation are not well characterized. We found that FcR engagement by immune complexes induced the phosphorylation of Syk, a protein tyrosine kinase acting immediately downstream of FcRs. Syk was dispensable for DC differentiation in vitro and in vivo, but was strictly required for immune complexes internalization and subsequent Ag presentation to T lymphocytes. Importantly, Syk was also required for the induction of DC maturation and IL-12 production after FcR engagement, but not after engagement of other surface receptors, such as TNFR or Toll-like receptors. Therefore, protein tyrosine phosphorylation by Syk represents a novel pathway for the induction of DC maturation.     

Transfer of chimeric plasmids among Salmonella typhimurium strains by P22 transduction

Salmonella typhimurium bacteriophage P22 transduced plasmids having P22 sequences inserted in the vector pBR322 with high frequency. Analysis of the structure of the transducing particle DNA and the transduced plasmids indicates that this plasmid transduction involves two homologous recombination events. In the donor cell, a single recombination between the phage and the homologous sequences on the plasmid inserted the plasmid into the phage chromosome, which was then packaged by headfuls into P22 particles. The transducing particle DNA contained duplications of the region of homology flanking the integrated plasmid vector sequences and lacked some phage genes. When these defective phage genomes containing the inserted plasmid infected a recipient cell, recombination between the duplicated regions regenerated the plasmid. A useful consequence of this sequence of events was that genetic markers in the region of homology were readily transferred from phage to plasmid. Plasmid transduction required homology between the phage and the plasmid, but did not depend on the presence of any specific P22 sequence in the plasmid. When the infecting P22 carried a DNA sequence homologous to the ampicillin resistance region of pBR322, the vector plasmid having no P22 insert could be transduced. P22-mediated transduction is a useful way to transfer chimeric plasmids, since most S. typhimurium strains are poorly transformed by plasmid DNA.     

Mapping of avirulence genes in the rice blast fungus, Magnaporthe grisea, with RFLP and RAPD markers

Three genetically independent avirulence genes, AVR1-Irat7, AVRI-MedNoi; and AVR1-Ku86, were identified in a cross involving isolates Guy11 and 2/0/3 of the rice blast fungus, Magnaporthe grisea. Using 76 random progeny, we constructed a partial genetic map with restriction fragment length polymorphism (RFLP) markers revealed by probes such as the repeated sequences MGL/MGR583 and Pot3/MGR586, cosmids from the M. grisea genetic map, and a telomere sequence oligonucleotide. Avirulence genes AVR1-MedNoi and AVR1-Ku86 were closely linked to telomere RFLPs such as marker TelG (6 cM from AVR1-MedNoi) and TelF (4.5 cM from AVR1-Ku86). Avirulence gene AVR1-Irat7 was linked to a cosmid RFLP located on chromosome 1 and mapped at 20 cM from the avirulence gene AVR1-CO39. Using bulked segregant analysis, we identified 11 random amplified polymorphic DNA (RAPD) markers closely linked (0 to 10 cM) to the avirulence genes segregating in this cross. Most of these RAPD markers corresponded to junction fragments between known or new transposons and a single-copy sequence. Such junctions or the whole sequences of single-copy RAPD markers were frequently absent in one parental isolate. Single-copy sequences from RAPD markers tightly linked to avirulence genes will be used for positional cloning.     

A telomeric avirulence gene determines efficacy for the rice blast resistance gene Pi-ta

Genetic mapping showed that the rice blast avirulence gene AVR-Pita is tightly linked to a telomere on chromosome 3 in the plant pathogenic fungus Magnaporthe grisea. AVR-Pita corresponds in gene-for-gene fashion to the disease resistance (R) gene Pi-ta. Analysis of spontaneous avr-pita(-) mutants indicated that the gene is located in a telomeric 6.5-kb BglII restriction fragment. Cloning and DNA sequencing led to the identification of a candidate gene with features typical of metalloproteases. This gene is located entirely within the most distal 1.5 kb of the chromosome. When introduced into virulent rice pathogens, the cloned gene specifically confers avirulence toward rice cultivars that contain Pi-ta. Frequent spontaneous loss of AVR-Pita appears to be the result of its telomeric location. Diverse mutations in AVR-Pita, including point mutations, insertions, and deletions, permit the fungus to avoid triggering resistance responses mediated by Pi-ta. A point mutation in the protease consensus sequence abolishes the AVR-Pita avirulence function.     

'PACLIMS': A component LIM system for high-throughput functional genomic analysis

Background

Recent advances in sequencing techniques leading to cost reduction have resulted in the generation of a growing number of sequenced eukaryotic genomes. Computational tools greatly assist in defining open reading frames and assigning tentative annotations. However, gene functions cannot be asserted without biological support through, among other things, mutational analysis. In taking a genome-wide approach to functionally annotate an entire organism, in this application the ~11,000 predicted genes in the rice blast fungus (Magnaporthe grisea), an effective platform for tracking and storing both the biological materials created and the data produced across several participating institutions was required.

Results

The platform designed, named PACLIMS, was built to support our high throughput pipeline for generating 50,000 random insertion mutants of Magnaporthe grisea. To be a useful tool for materials and data tracking and storage, PACLIMS was designed to be simple to use, modifiable to accommodate refinement of research protocols, and cost-efficient. Data entry into PACLIMS was simplified through the use of barcodes and scanners, thus reducing the potential human error, time constraints, and labor. This platform was designed in concert with our experimental protocol so that it leads the researchers through each step of the process from mutant generation through phenotypic assays, thus ensuring that every mutant produced is handled in an identical manner and all necessary data is captured.

Conclusion

Many sequenced eukaryotes have reached the point where computational analyses are no longer sufficient and require biological support for their predicted genes. Consequently, there is an increasing need for platforms that support high throughput genome-wide mutational analyses. While PACLIMS was designed specifically for this project, the source and ideas present in its implementation can be used as a model for other high throughput mutational endeavors.     

Phytoremediation of Heavy and Transition Metals Aided by Legume-Rhizobia Symbiosis

Legumes are important for nitrogen cycling in the environment and agriculture due to the ability of nitrogen fixation by rhizobia. In this review, we introduce an important and potential role of legume-rhizobia symbiosis in aiding phytoremediation of some metal contaminated soils as various legumes have been found to be the dominant plant species in metal contaminated areas. Resistant rhizobia used for phytoremediation could act on metals directly by chelation, precipitation, transformation, biosorption and accumulation. Moreover, the plant growth promoting (PGP) traits of rhizobia including nitrogen fixation, phosphorus solubilization, phytohormone synthesis, siderophore release, and production of ACC deaminase and the volatile compounds of acetoin and 2, 3-butanediol may facilitate legume growth while lessening metal toxicity. The benefits of using legumes inoculated with naturally resistant rhizobia or recombinant rhizobia with enhanced resistance, as well as co-inoculation with other plant growth promoting bacteria (PGPB) are discussed. However, the legume-rhizobia symbiosis appears to be sensitive to metals, and the effect of metal toxicity on the interaction between legumes and rhizobia is not clear. Therefore, to obtain the maximum benefits from legumes assisted by rhizobia for phytoremediation of metals, it is critical to have a good understanding of interactions between PGP traits, the symbiotic plant-rhizobia relationship and metals.     

Synthetic osteogenic extracellular matrix formed by coated silicon dioxide nanosprings

Background

Angiogenesis is widely investigated in conjunction with cancer development, in particular because of the possibility of early stage detection and of new therapeutic strategies. However, such studies are negatively affected by the limitations of imaging techniques in the detection of microscopic blood vessels (diameter 3-5 ??m) grown under angiogenic stress. We report that synchrotron-based X-ray imaging techniques with very high spatial resolution can overcome this obstacle, provided that suitable contrast agents are used.

Results

We tested different contrast agents based on gold nanoparticles (AuNPs) for the detection of cancer-related angiogenesis by synchrotron microradiology, microtomography and high resolution X-ray microscopy. Among them only bare-AuNPs in conjunction with heparin injection provided sufficient contrast to allow in vivo detection of small capillary species (the smallest measured lumen diameters were 3-5 ??m). The detected vessel density was 3-7 times higher than with other nanoparticles. We also found that bare-AuNPs with heparin allows detecting symptoms of local extravascular nanoparticle diffusion in tumor areas where capillary leakage appeared.

Conclusions

Although high-Z AuNPs are natural candidates as radiology contrast agents, their success is not guaranteed, in particular when targeting very small blood vessels in tumor-related angiography. We found that AuNPs injected with heparin produced the contrast level needed to reveal--for the first time by X-ray imaging--tumor microvessels with 3-5 ??m diameter as well as extravascular diffusion due to basal membrane defenestration. These results open the interesting possibility of functional imaging of the tumor microvasculature, of its development and organization, as well as of the effects of anti-angiogenic drugs.