TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing

SUMMARY: TREE-PUZZLE is a program package for quartet-based maximum-likelihood phylogenetic analysis (formerly PUZZLE, Strimmer and von Haeseler, Mol. Biol. Evol., 13, 964-969, 1996) that provides methods for reconstruction, comparison, and testing of trees and models on DNA as well as protein sequences. To reduce waiting time for larger datasets the tree reconstruction part of the software has been parallelized using message passing that runs on clusters of workstations as well as parallel computers. AVAILABILITY: http://www.tree-puzzle.de. The program is written in ANSI C. TREE-PUZZLE can be run on UNIX, Windows and Mac systems, including Mac OS X. To run the parallel version of PUZZLE, a Message Passing Interface (MPI) library has to be installed on the system. Free MPI implementations are available on the Web (cf. http://www.lam-mpi.org/mpi/implementations/).     

Mutant Allele-Specific Uncoupling of PENETRATION3 Functions Reveals Engagement of the ATP-Binding Cassette Transporter in Distinct Tryptophan Metabolic Pathways

Arabidopsis (Arabidopsis thaliana) PENETRATION (PEN) genes quantitatively contribute to the execution of different forms of plant immunity upon challenge with diverse leaf pathogens. PEN3 encodes a plasma membrane-resident pleiotropic drug resistance-type ATP-binding cassette transporter and is thought to act in a pathogen-inducible and PEN2 myrosinase-dependent metabolic pathway in extracellular defense. This metabolic pathway directs the intracellular biosynthesis and activation of tryptophan-derived indole glucosinolates for subsequent PEN3-mediated efflux across the plasma membrane at pathogen contact sites. However, PEN3 also functions in abiotic stress responses to cadmium and indole-3-butyric acid (IBA)-mediated auxin homeostasis in roots, raising the possibility that PEN3 exports multiple functionally unrelated substrates. Here, we describe the isolation of a pen3 allele, designated pen3-5, that encodes a dysfunctional protein that accumulates in planta like wild-type PEN3. The specific mutation in pen3-5 uncouples PEN3 functions in IBA-stimulated root growth modulation, callose deposition induced with a conserved peptide epitope of bacterial flagellin (flg22), and pathogen-inducible salicylic acid accumulation from PEN3 activity in extracellular defense, indicating the engagement of multiple PEN3 substrates in different PEN3-dependent biological processes. We identified 4-O-β-d-glucosyl-indol-3-yl formamide (4OGlcI3F) as a pathogen-inducible, tryptophan-derived compound that overaccumulates in pen3 leaf tissue and has biosynthesis that is dependent on an intact PEN2 metabolic pathway. We propose that a precursor of 4OGlcI3F is the PEN3 substrate in extracellular pathogen defense. These precursors, the shared indole core present in IBA and 4OGlcI3F, and allele-specific uncoupling of a subset of PEN3 functions suggest that PEN3 transports distinct indole-type metabolites in distinct biological processes.The ATP-binding cassette (ABC) transporters constitute one of the largest protein families in the plant kingdom (Rea, 2007; Verrier et al., 2008; Kang et al., 2011). They share a core structure comprising highly conserved nucleotide-binding domains (NBDs) and transmembrane domains (TMDs). In the model plant Arabidopsis (Arabidopsis thaliana), there are over 120 ABC transporters grouped into 13 subfamilies classified by NBD phylogeny, the length of the protein, and/or the organization of the domains (Verrier et al., 2008). These transmembrane proteins play important roles in plant development, organ formation, and plant response to abiotic and biotic stresses (Kang et al., 2011). Known substrates translocated by characterized ABC transporters cover a range of small molecules, including abscisic acid, auxin and/or auxin precursors (Lin and Wang, 2005; Lewis et al., 2007; Wu et al., 2007), phytochelatin, glutathione and/or glutathione conjugates (Liu et al., 2001; Song et al., 2010), folates and folate homologs (Klein et al., 2004; Raichaudhuri et al., 2009), and many other molecules.Pleiotropic drug resistance (PDR)-type full-size ABC transporters belong to the ABC transporter protein subfamily G (ABCG) and are found exclusively in fungi and plants (Verrier et al., 2008; Kang et al., 2011). The expression of plant genes encoding PDR proteins is often stimulated by microbial infection and defense phytohormones, such as salicylic acid (SA) and jasmonic acid. For example, tobacco (Nicotiana tabacum) PDR1, which is induced by Phytophthora infestans elicitins, flagellin, and methyl jasmonate, directly transports diterpenes in tobacco Bright Yellow-2 suspension cells (Sasabe et al., 2002; Crouzet et al., 2013). NtPDR5 is induced by methyl jasmonate and wounding and plays a role in herbivore resistance (Bienert et al., 2012). Nicotiana plumbaginifolia PDR1 transports diterpene sclareol and is induced by pathogen colonization or jasmonic acid treatment (Stukkens et al., 2005). Wheat (Triticum aestivum) PDR transporter LEAF RUST RESISTANCE34 confers durable, race-nonspecific resistance to multiple fungal pathogens, and the corresponding gene is highly relevant in breeding disease-resistant wheat cultivars (Krattinger et al., 2009). PDR transporters are not only involved in plant defense to pathogenic microorganisms. Petunia hybrida PDR1 is a strigolactone exporter critical for the establishment of symbiotic interactions with arbuscular mycorrhizal fungi (Kretzschmar et al., 2012). Arabidopsis pdr2 plants revealed drastic changes in root exudate profiles, and the composition of root-associated bacterial communities seems to be altered in the mutant plants (Badri et al., 2008, 2009).The Arabidopsis PENETRATION3 (PEN3)/PDR8/ABCG36 PDR-type ABC transporter is unusual, because it has been functionally assigned to several biotic and abiotic stress responses as well as in the transport of the auxin precursor indole-3-butyric acid (IBA). Mutant pen3 plants are defective in extracellular (apoplastic) defense to nonadapted powdery mildew pathogens, including Blumeria graminis and Erysiphe pisi, and the nonadapted oomycete pathogen P. infestans (Stein et al., 2006). Genetic screens for impaired extracellular defense in nonhost resistance to the nonadapted powdery mildew pathogens also identified SYNTAXIN RELATED PROTEIN1 (SYR1), also known as SYP121/PEN1 and PEN2, which encode a plasma membrane-resident syntaxin and a myrosinase, respectively (Leyman et al., 1999; Collins et al., 2003; Lipka et al., 2005; Stein et al., 2006). PEN2 and PEN3 act in the same pathway for extracellular defense, whereas PEN1 functions in a parallel secretory defense pathway (Collins et al., 2003; Kwon et al., 2008; Kim et al., 2014). PEN2 function has been assigned to a glucosinolate metabolic pathway, which comprises biosynthesis of indole glucosinolates (IGs), pathogen-inducible redirection of this biosynthesis through the CYTOCHROME P450 81F2 (CYP81F2) monooxygenase to 4-methoxyindol-3-ylmethylglucosinolate (4MI3G), and 4MI3G activation by PEN2 myrosinase through deglucosylation (Bednarek et al., 2009). Additional metabolized PEN2 products are thought to be exported to the apoplast at pathogen contact sites by plasma membrane-resident PEN3/PDR8/ABCG36 (Stein et al., 2006; Bednarek et al., 2009), but the structures of PEN3 substrates for extracellular defense remain to be identified.PEN genes were originally identified as components of nonhost resistance to nonadapted pathogens. This type of general plant immunity can be triggered upon perception of evolutionary conserved microbe-associated molecular patterns (MAMPs) by membrane-resident pattern recognition receptors (PRRs) or upon activation of intracellular nucleotide-binding and Leu-rich repeat (NLR)-type immune receptors that detect the presence of race-specific pathogen effectors (Schulze-Lefert and Panstruga, 2011). Recently, PEN1, PEN2, and PEN3 were shown to contribute quantitatively to race-specific immune responses against host-adapted bacterial and oomycete pathogens after immune response activation by intracellular NLR-type immune receptors (Johansson et al., 2014). This genetic evidence and known biochemical PEN activities strongly suggest PEN engagements in the execution of plant immune responses triggered by both PRR- and NLR-type immune receptors. Although much is known about plant immune receptors recognizing nonself molecules and subsequent phytohormone-dependent defense signaling, the molecules that execute immune responses to restrict pathogen growth remain largely unknown. In this context, pen mutants are useful tools to identify candidate molecules or compound classes contributing to defense response execution.pen3 null mutant phenotypes include an enhanced disease resistance (edr) to the host-adapted powdery mildew Golovinomyces cichoracearum (formerly Erysiphe cichoracearum), and this infection phenotype is dependent on SA biosynthesis but not dependent on PEN2 (Kobae et al., 2006; Stein et al., 2006). This indicates separable PEN3 functions in extracellular defense to nonadapted powdery mildews and for host colonization by host-adapted G. cichoracearum. PEN2 and PEN3 also act together to limit growth of both host-adapted and nonadapted pathogenic strains of the necrotrophic fungus Plectosphaerella cucumerina. However, pen3 plants are more susceptible to P. cucumerina infection than pen2 plants, suggesting yet another PEN2-independent function of PEN3 in defense of necrotrophic pathogens (Stein et al., 2006; Sanchez-Vallet et al., 2010).The function of PEN3 is not restricted to the innate immune system of Arabidopsis. An excised root tip auxin transport assay showed that root tips of pen3 hyperaccumulate [³H]IBA, suggesting that IBA is a PEN3 substrate. Similarly, a leaf protoplast Cd transport assay has shown that ¹⁰⁹Cd levels are higher in AtPEN3 RNA interference plants and lower in overexpressing plants compared with the wild type, indicating that PEN3 directly transports Cd²⁺ or its conjugates (Kim et al., 2007; Strader and Bartel, 2009; Ruzicka et al., 2010). Together, these data suggest additional PEN3 functions in IBA-mediated auxin homeostasis and cadmium tolerance.In this study, we have isolated and characterized the pen3-5 allele associated with a single-amino acid substitution. The mutant protein, unlike most previously isolated pen3 alleles, accumulates in planta like wild-type PEN3. Using this mutant and all other previously described pen3 alleles, we show an allele-specific uncoupling of a subset of PEN3 functions. We then applied metabolic profiling of pathogen-inoculated wild-type and pen3 plants to identify pathogen-inducible compounds that hyperaccumulate in pen3 leaf tissue. Purification and mass spectrometry of a prominent hyperaccumulating compound identified an indole derivative 4-O-β-d-glucosyl-indol-3-yl formamide (4OGlcI3F), whose biosynthesis is dependent on the PEN2 metabolic pathway. We propose that one or several precursors of this molecule serve as the PEN3 substrate(s) for export into the apoplast during extracellular defense.     

Bilateral Bi-Cephalic Tdcs with Two Active Electrodes of the Same Polarity Modulates Bilateral Cognitive Processes Differentially

Transcranial direct current stimulation (tDCS) is an innovative method to explore the causal structure-function relationship of brain areas. We investigated the specificity of bilateral bi-cephalic tDCS with two active electrodes of the same polarity (e.g., cathodal on both hemispheres) applied to intraparietal cortices bilaterally using a combined between- and within-task approach. Regarding between-task specificity, we observed that bilateral bi-cephalic tDCS affected a numerical (mental addition) but not a control task (colour word Stroop), indicating a specific influence of tDCS on numerical but not on domain general cognitive processes associated with the bilateral IPS. In particular, the numerical effect of distractor distance was more pronounced under cathodal than under anodal stimulation. Moreover, with respect to within-task specificity we only found the numerical distractor distance effect in mental addition to be modulated by direct current stimulation, whereas the effect of target identity was not affected. This implies a differential influence of bilateral bi-cephalic tDCS on the recruitment of different processing components within the same task (number magnitude processing vs. recognition of familiarity). In sum, this first successful application of bilateral bi-cephalic tDCS with two active electrodes of the same polarity in numerical cognition research corroborates the specific proposition of the Triple Code Model that number magnitude information is represented bilaterally in the intraparietal cortices.     

A general modular framework for gene set enrichment analysis

Background  

Analysis of microarray and other high-throughput data on the basis of gene sets, rather than individual genes, is becoming more important in genomic studies. Correspondingly, a large number of statistical approaches for detecting gene set enrichment have been proposed, but both the interrelations and the relative performance of the various methods are still very much unclear.