Revealing a signaling role of phytosphingosine-1-phosphate in yeast

Perturbing metabolic systems of bioactive sphingolipids with genetic approachMultiple types of “omics” data collected from the systemSystems approach for integrating multiple “omics” informationPredicting signal transduction information flow: lipid; TF activation; gene expressio

Induction of androgenesis and production of haploid embryos in anther cultures of borage (Borago officinalis L.)

[EN] Borage (Borago officinalis L.) is an important medicinal plant with different culinary, pharmaceutical and industrial properties. Unfortunately, there are no published reports on the establishment of protocols to produce DHs in this species up to now. In this work, we show for the first time the induction of borage microspores to become embryogenic calli, from which haploid embryos are produced. In addition, we evaluated the effect of using different flower bud sizes, carbon sources, concentrations of 2,4-D and BAP, cold (4 A degrees C) pretreatments and heat shock treatments. Production of total calli, embryogenic calli and callus-derived embryos was differently affected by the different parameters studied. Our results showed that the use of 5-7 mm-long flower buds, a cold (4 A degrees C) pretreatment during 4 days, a 32 A degrees C heat shock for 3 days, and the addition of 3 % maltose and 2 mgl(-1) 2,4-D and 1 mgl(-1) BAP to the culture medium, was beneficial for embryo production. Overall, this work demonstrates that DH technology is possible in borage, and opens the door for future improvements needed to finally obtain borage DH plants.Eshaghi, ZC.; Abdollahi, MR.; Moosavi, SS.; Deljou, A.; Seguí-Simarro, JM. (2015). Induction of androgenesis and production of haploid embryos in anther cultures of borage (Borago officinalis L.). Plant Cell, Tissue and Organ Culture. 122:321-329. doi:10.1007/s11240-015-0768-5S321329122Abdollahi MR, Moieni A, Javaran MJ (2004) Interactive effects of shock and culture density on embryo induction in isolated microspore culture of Brassica napus L. cv. Global Iranian J Biotech 2:97–100Bohanec B, Neskovic M, Vujicic R (1993) Anther culture and androgenetic plant regeneration in buckwheat (Fagopyrum esculentum Moench). Plant Cell Tissue Organ Cult 35:259–266Calleberg E, Johansson L (1996) Effect of gelling agents on anther cultures. In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants, vol 23. Springer, Netherlands, pp 189–203Custers JBM, Cordewener JHG, Nöllen Y, Dons JJ, van Lookeren-Campagne MM (1994) Temperature controls both gametophytic and sporophytic development in microspore cultures of Brassica napus. Plant Cell Rep 13:267–271Ferrie AMR (2013) Advances in microspore culture technology: a biotechnological tool for the improvement of medicinal plants. In: Chandra S et al (eds) Biotechnology for medicinal plants. Springer, Berlin, pp 191–206Ferrie AMR, Caswell KL (2011) Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell Tissue Organ Cult 104:301–309Ferrie AMR, Bethune T, Mykytyshyn M (2011) Microspore embryogenesis in the Apiaceae. Plant Cell Tissue Organ Cult 104:399–406Forster BP, Heberle-Bors E, Kasha KJ, Touraev A (2007) The resurgence of haploids in higher plants. Trends Plant Sci 12:368–375Gamborg OL, Miller RA, Ojiwa K (1968) Nutrient requirements of suspension culture of soybean root callus. Exp Cell Res 50:151–158Guil-Guerrero JL, García-Maroto F, Vilches-Ferrón MA, López-Alonso D (2003) Gamma-linolenic acid from fourteen Boraginaceae species. Ind Crop Prod 18:85–89Horrobin DF (1983) The regulation of prostaglandin biosynthesis by the manipulation of essential fatty acid metabolism. Rev Pure Appl Pharmacol Sci 4:339–383Irikova T, Grozeva S, Rodeva V (2011) Anther culture in pepper (Capsicum annuum L.) in vitro. Acta Physiol Plant 33:1559–1570Lauxen MS, Kaltchuk-Santos E, Hu CY, Callegari-Jacques SM, Bodanese-Zanettini MH (2003) Association between floral bud size and developmental stage in soybean microspores. Braz Arch Biol Technol 46:515–520Leach CR, Mayo O, Bürger R (1990) Quantitatively determined self-incompatibility. Outcrossing in Borago officinalis. Theoret Appl Genetics 79:427–430Lichter R (1982) Induction of haploid plants from isolated pollen of Brassica napus. Z Pflanzenphysiol 105:427–434Maluszynski M, Kasha KJ, Szarejko I (2003) Published doubled haploid protocols in plant species. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants. A manual. Kluwer, Dordrecht, pp 309–335Maraschin SF, de Priester W, Spaink HP, Wang M (2005) Androgenic switch: an example of plant embryogenesis from the male gametophyte perspective. J Exp Bot 56:1711–1726McDonald BE, Fitzpatrick K (1998) Designer Vegetable Oils. In: Mazza G (ed) Functional foods, biochemical and processing aspects. Technomic Publ Co. Inc, Lancaster, pp 265–291Ozkum D, Tipirdamaz R (2002) The effects of cold treatment and charcoal on the in vitro androgenesis of pepper (Capsicum annuum L.). Turk J Bot 26:131–139Parra-Vega V, González-García B, Seguí-Simarro JM (2013a) Morphological markers to correlate bud and anther development with microsporogenesis and microgametogenesis in pepper (Capsicum annuum L.). Acta Physiol Plant 35:627–633Parra-Vega V, Renau-Morata B, Sifres A, Seguí-Simarro JM (2013b) Stress treatments and in vitro culture conditions influence microspore embryogenesis and growth of callus from anther walls of sweet pepper (Capsicum annuum L.). Plant Cell Tissue Organ Cult 112:353–360Raquin C (1983) Utilization of different sugars as carbon sources for in vitro cultures of Petuina. Z Pflanzenphysol 111:453–457Salas P, Rivas-Sendra A, Prohens J, Seguí-Simarro JM (2012) Influence of the stage for anther excision and heterostyly in embryogenesis induction from eggplant anther cultures. Euphytica 184:235–250Seguí-Simarro JM (2010) Androgenesis revisited. Bot Rev 76:377–404Seguí-Simarro JM, Nuez F (2006) Androgenesis induction from tomato anther cultures: callus characterization. Acta Hort 725:855–861Seguí-Simarro JM, Corral-Martínez P, Parra-Vega V, González-García B (2011) Androgenesis in recalcitrant solanaceous crops. Plant Cell Rep 30:765–778Shariatpanahi ME, Bal U, Heberle-Bors E, Touraev A (2006) Stresses applied for the reprogramming of plant microspores towards in vitro embryogenesis. Physiol Plant 127:519–534Simon JE, Chadwick AF, Craker LE (1984) Herbs: an indexed bibliography. 1971–1980. The scientific literature on selected herbs, and aromatic and medicinal plants of the temperate zone. Archon Books, Hamden, CTSkrzypek E, Czyczyło-Mysza I, Marcińska I, Wędzony M (2008) Prospects of androgenetic induction in Lupinus spp. Plant Cell Tissue Organ Cult 94(2):131–137Snape JW (1989) Doubled haploid breeding: theoretical basis and practical applications. In: Mujeeb-Kazi A, Sitch LA (eds) Review of advances in plant biotechnology, 1985–1988: 2nd international symposium genetic manipulation in crops. Mexico and Manila, CIMMYT and IRRI, pp 19–30Tipirdamaz R, Ellialtioğlu Ş (1998) The effects of cold treatments and activated charcoal on ABA contents of anthers and in vitro androgenesis in eggplant (Solanum melongena L.). In: Tsekos I, Moustakas M (eds) Progress in botanical research, Proceedings of the 1st Balkan botanical congress. Kluwer Academic Publishers, The NetherlandsVagera J, Havranek P (1985) In vitro induction of androgenesis in Capsicum annuum L. and its genetic aspests. Biol Plant 27(1):10–21Zur I, Dubas E, Golemiec E, Szechynska-Hebda M, Golebiowska G, Wedzony M (2009) Stress-related variation in antioxidative enzymes activity and cell metabolism efficiency associated with embryogenesis induction in isolated microspore culture of triticale (×Triticosecale Wittm.). Plant Cell Rep 28:1279–128

Rational Diversification of a Promoter Providing Fine-Tuned Expression and Orthogonal Regulation for Synthetic Biology

Yeast is an ideal organism for the development and application of synthetic biology, yet there remain relatively few well-characterised biological parts suitable for precise engineering of this chassis. In order to address this current need, we present here a strategy that takes a single biological part, a promoter, and re-engineers it to produce a fine-graded output range promoter library and new regulated promoters desirable for orthogonal synthetic biology applications. A highly constitutive Saccharomyces cerevisiae promoter, PFY1p, was identified by bioinformatic approaches, characterised in vivo and diversified at its core sequence to create a 36-member promoter library. TetR regulation was introduced into PFY1p to create a synthetic inducible promoter (iPFY1p) that functions in an inverter device. Orthogonal and scalable regulation of synthetic promoters was then demonstrated for the first time using customisable Transcription Activator-Like Effectors (TALEs) modified and designed to act as orthogonal repressors for specific PFY1-based promoters. The ability to diversify a promoter at its core sequences and then independently target Transcription Activator-Like Orthogonal Repressors (TALORs) to virtually any of these sequences shows great promise toward the design and construction of future synthetic gene networks that encode complex “multi-wire” logic functions

Gene Annotation and Drug Target Discovery in Candida albicans with a Tagged Transposon Mutant Collection

Candida albicans is the most common human fungal pathogen, causing infections that can be lethal in immunocompromised patients. Although Saccharomyces cerevisiae has been used as a model for C. albicans, it lacks C. albicans' diverse morphogenic forms and is primarily non-pathogenic. Comprehensive genetic analyses that have been instrumental for determining gene function in S. cerevisiae are hampered in C. albicans, due in part to limited resources to systematically assay phenotypes of loss-of-function alleles. Here, we constructed and screened a library of 3633 tagged heterozygous transposon disruption mutants, using them in a competitive growth assay to examine nutrient- and drug-dependent haploinsufficiency. We identified 269 genes that were haploinsufficient in four growth conditions, the majority of which were condition-specific. These screens identified two new genes necessary for filamentous growth as well as ten genes that function in essential processes. We also screened 57 chemically diverse compounds that more potently inhibited growth of C. albicans versus S. cerevisiae. For four of these compounds, we examined the genetic basis of this differential inhibition. Notably, Sec7p was identified as the target of brefeldin A in C. albicans screens, while S. cerevisiae screens with this compound failed to identify this target. We also uncovered a new C. albicans-specific target, Tfp1p, for the synthetic compound 0136-0228. These results highlight the value of haploinsufficiency screens directly in this pathogen for gene annotation and drug target identification

The Transcriptomes of Two Heritable Cell Types Illuminate the Circuit Governing Their Differentiation

The differentiation of cells into distinct cell types, each of which is heritable for many generations, underlies many biological phenomena. White and opaque cells of the fungal pathogen Candida albicans are two such heritable cell types, each thought to be adapted to unique niches within their human host. To systematically investigate their differences, we performed strand-specific, massively-parallel sequencing of RNA from C. albicans white and opaque cells. With these data we first annotated the C. albicans transcriptome, finding hundreds of novel differentially-expressed transcripts. Using the new annotation, we compared differences in transcript abundance between the two cell types with the genomic regions bound by a master regulator of the white-opaque switch (Wor1). We found that the revised transcriptional landscape considerably alters our understanding of the circuit governing differentiation. In particular, we can now resolve the poor concordance between binding of a master regulator and the differential expression of adjacent genes, a discrepancy observed in several other studies of cell differentiation. More than one third of the Wor1-bound differentially-expressed transcripts were previously unannotated, which explains the formerly puzzling presence of Wor1 at these positions along the genome. Many of these newly identified Wor1-regulated genes are non-coding and transcribed antisense to coding transcripts. We also find that 5′ and 3′ UTRs of mRNAs in the circuit are unusually long and that 5′ UTRs often differ in length between cell-types, suggesting UTRs encode important regulatory information and that use of alternative promoters is widespread. Further analysis revealed that the revised Wor1 circuit bears several striking similarities to the Oct4 circuit that specifies the pluripotency of mammalian embryonic stem cells. Additional characteristics shared with the Oct4 circuit suggest a set of general hallmarks characteristic of heritable differentiation states in eukaryotes

The Novel Candida albicans Transporter Dur31 Is a Multi-Stage Pathogenicity Factor

Candida albicans is the most frequent cause of oral fungal infections. However, the exact pathogenicity mechanisms that this fungus employs are largely unknown and many of the genes expressed during oral infection are uncharacterized. In this study we sought to functionally characterize 12 previously unknown function genes associated with oral candidiasis. We generated homozygous knockout mutants for all 12 genes and analyzed their interaction with human oral epithelium in vitro. Eleven mutants caused significantly less epithelial damage and, of these, deletion of orf19.6656 (DUR31) elicited the strongest reduction in pathogenicity. Interestingly, DUR31 was not only involved in oral epithelial damage, but in multiple stages of candidiasis, including surviving attack by human neutrophils, endothelial damage and virulence in vivo. In silico analysis indicated that DUR31 encodes a sodium/substrate symporter with 13 transmembrane domains and no human homologue. We provide evidence that Dur31 transports histatin 5. This is one of the very first examples of microbial driven import of this highly cytotoxic antimicrobial peptide. Also, in contrast to wild type C. albicans, dur31Δ/Δ was unable to actively increase local environmental pH, suggesting that Dur31 lies in the extracellular alkalinization hyphal auto-induction pathway; and, indeed, DUR31 was required for morphogenesis. In agreement with this observation, dur31Δ/Δ was unable to assimilate the polyamine spermidine

The Functions of Mediator in Candida albicans Support a Role in Shaping Species-Specific Gene Expression

The Mediator complex is an essential co-regulator of RNA polymerase II that is conserved throughout eukaryotes. Here we present the first study of Mediator in the pathogenic fungus Candida albicans. We focused on the Middle domain subunit Med31, the Head domain subunit Med20, and Srb9/Med13 from the Kinase domain. The C. albicans Mediator shares some roles with model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, such as functions in the response to certain stresses and the role of Med31 in the expression of genes regulated by the activator Ace2. The C. albicans Mediator also has additional roles in the transcription of genes associated with virulence, for example genes related to morphogenesis and gene families enriched in pathogens, such as the ALS adhesins. Consistently, Med31, Med20, and Srb9/Med13 contribute to key virulence attributes of C. albicans, filamentation, and biofilm formation; and ALS1 is a biologically relevant target of Med31 for development of biofilms. Furthermore, Med31 affects virulence of C. albicans in the worm infection model. We present evidence that the roles of Med31 and Srb9/Med13 in the expression of the genes encoding cell wall adhesins are different between S. cerevisiae and C. albicans: they are repressors of the FLO genes in S. cerevisiae and are activators of the ALS genes in C. albicans. This suggests that Mediator subunits regulate adhesion in a distinct manner between these two distantly related fungal species

Gene Ontology annotations and resources.

The Gene Ontology (GO) Consortium (GOC, http://www.geneontology.org) is a community-based bioinformatics resource that classifies gene product function through the use of structured, controlled vocabularies. Over the past year, the GOC has implemented several processes to increase the quantity, quality and specificity of GO annotations. First, the number of manual, literature-based annotations has grown at an increasing rate. Second, as a result of a new 'phylogenetic annotation' process, manually reviewed, homology-based annotations are becoming available for a broad range of species. Third, the quality of GO annotations has been improved through a streamlined process for, and automated quality checks of, GO annotations deposited by different annotation groups. Fourth, the consistency and correctness of the ontology itself has increased by using automated reasoning tools. Finally, the GO has been expanded not only to cover new areas of biology through focused interaction with experts, but also to capture greater specificity in all areas of the ontology using tools for adding new combinatorial terms. The GOC works closely with other ontology developers to support integrated use of terminologies. The GOC supports its user community through the use of e-mail lists, social media and web-based resources

The Gene Ontology resource: enriching a GOld mine

The Gene Ontology Consortium (GOC) provides the most comprehensive resource currently available for computable knowledge regarding the functions of genes and gene products. Here, we report the advances of the consortium over the past two years. The new GO-CAM annotation framework was notably improved, and we formalized the model with a computational schema to check and validate the rapidly increasing repository of 2838 GO-CAMs. In addition, we describe the impacts of several collaborations to refine GO and report a 10% increase in the number of GO annotations, a 25% increase in annotated gene products, and over 9,400 new scientific articles annotated. As the project matures, we continue our efforts to review older annotations in light of newer findings, and, to maintain consistency with other ontologies. As a result, 20 000 annotations derived from experimental data were reviewed, corresponding to 2.5% of experimental GO annotations. The website (http://geneontology.org) was redesigned for quick access to documentation, downloads and tools. To maintain an accurate resource and support traceability and reproducibility, we have made available a historical archive covering the past 15 years of GO data with a consistent format and file structure for both the ontology and annotations