Genome-Wide Identification of the Invertase Gene Family in Populus

Invertase plays a crucial role in carbohydrate partitioning and plant development as it catalyses the irreversible hydrolysis of sucrose into glucose and fructose. The invertase family in plants is composed of two sub-families: acid invertases, which are targeted to the cell wall and vacuole; and neutral/alkaline invertases, which function in the cytosol. In this study, 5 cell wall invertase genes (PtCWINV1-5), 3 vacuolar invertase genes (PtVINV1-3) and 16 neutral/alkaline invertase genes (PtNINV1-16) were identified in the Populus genome and found to be distributed on 14 chromosomes. A comprehensive analysis of poplar invertase genes was performed, including structures, chromosome location, phylogeny, evolutionary pattern and expression profiles. Phylogenetic analysis indicated that the two sub-families were both divided into two clades. Segmental duplication is contributed to neutral/alkaline sub-family expansion. Furthermore, the Populus invertase genes displayed differential expression in roots, stems, leaves, leaf buds and in response to salt/cold stress and pathogen infection. In addition, the analysis of enzyme activity and sugar content revealed that invertase genes play key roles in the sucrose metabolism of various tissues and organs in poplar. This work lays the foundation for future functional analysis of the invertase genes in Populus and other woody perennials.     

A Role for MORE AXILLARY GROWTH1 (MAX1) in Evolutionary Diversity in Strigolactone Signaling Upstream of MAX2

Strigolactones (SLs) are carotenoid-derived phytohormones with diverse roles. They are secreted from roots as attractants for arbuscular mycorrhizal fungi and have a wide range of endogenous functions, such as regulation of root and shoot system architecture. To date, six genes associated with SL synthesis and signaling have been molecularly identified using the shoot-branching mutants more axillary growth (max) of Arabidopsis (Arabidopsis thaliana) and dwarf (d) of rice (Oryza sativa). Here, we present a phylogenetic analysis of the MAX/D genes to clarify the relationships of each gene with its wider family and to allow the correlation of events in the evolution of the genes with the evolution of SL function. Our analysis suggests that the notion of a distinct SL pathway is inappropriate. Instead, there may be a diversity of SL-like compounds, the response to which requires a D14/D14-like protein. This ancestral system could have been refined toward distinct ligand-specific pathways channeled through MAX2, the most downstream known component of SL signaling. MAX2 is tightly conserved among land plants and is more diverged from its nearest sister clade than any other SL-related gene, suggesting a pivotal role in the evolution of SL signaling. By contrast, the evidence suggests much greater flexibility upstream of MAX2. The MAX1 gene is a particularly strong candidate for contributing to diversification of inputs upstream of MAX2. Our functional analysis of the MAX1 family demonstrates the early origin of its catalytic function and both redundancy and functional diversification associated with its duplication in angiosperm lineages.Strigolactones (SLs) are carotenoid-derived terpenoid lactones, which have been identified as signaling molecules in several areas of plant biology. SLs were first identified as germination stimulants for seeds of plants in the genus Striga (Cook et al., 1966). Striga spp. and related Orobanchaceae are parasitic weeds that germinate in response to host plant root exudates and develop haustoria to penetrate the host tissue and draw nutrients. Striga spp. are major agricultural pests across much of tropical and subtropical Asia and are present in two-thirds of arable land in Africa, where they are the greatest biological cause of crop damage (Humphrey and Beale, 2006). The secretion of SLs by roots, despite its exploitation by Striga spp., has been preserved because it also serves to recruit arbuscular mycorrhizal (AM) fungi (Akiyama et al., 2005). AM fungi form symbiotic associations with most land plants, whereby the plant gains access to mineral nutrients, particularly phosphate, absorbed by the fungal hyphae, and in exchange the fungus gains fixed carbon from the plant. In several flowering plant species, SL production is correspondingly increased when phosphate availability is limiting, thereby presumably increasing fungal recruitment (Yoneyama et al., 2007, 2012).AM symbioses can be traced back to the origin of land plants, between 360 to 450 million years ago, and are thought to have facilitated plant colonization of the terrestrial environment (Simon et al., 1993). Although AM symbiosis has been lost from some lineages, such as Brassicaceae, it is still widespread, with 80% of land plants able to form associations with AM fungi (Schüssler et al., 2001). In support of a similarly ancient origin for SL secretion, the liverwort Marchantia polymorpha and the moss Physcomitrella patens, both basal land plant groups, have been shown to produce SLs (Proust et al., 2011; Delaux et al., 2012). Furthermore, the presence of SLs in charophyte algae indicates that SL production may predate the emergence of land plants (Delaux et al., 2012), and Chara corallina responds to SL treatment by producing longer rhizoids (Delaux et al., 2012). In P. patens, SLs appear to act as intercolony coordination signals, regulating colony growth and competition by controlling flexible developmental processes such as protonemal branching (Proust et al., 2011; Delaux et al., 2012). In flowering plants, SLs have also been implicated in development, including several processes regulated in response to phosphate limitation (Kohlen et al., 2011; Ruyter-Spira et al., 2011). In particular, SLs play important roles in the regulation of shoot branching in higher plants (Gomez-Roldan et al., 2008; Umehara et al., 2008). It is through work on their effects on shoot branching that some of the genes in the SL pathway were first identified.Arabidopsis (Arabidopsis thaliana) MORE AXILLARY GROWTH (MAX) mutants show increased branching and reduced stature relative to wild-type plants, and analogous phenotypes have been identified in pea (Pisum sativum; RAMOSUS [RMS]), petunia (Petunia hybrida; DECREASED APICAL DOMINANCE [DAD]), and rice (Oryza sativa; DWARF [D] or HIGH TILLERING DWARF) mutants. So far, six MAX/RMS/DAD/D genes have been identified, with roles in SL biosynthesis or signaling. MAX3/RMS5/HIGH TILLERING DWARF1/D17 (Booker et al., 2004; Johnson et al., 2006; Zou et al., 2006) and MAX4/RMS1/DAD1/D10 (Sorefan et al., 2003; Snowden et al., 2005; Arite et al., 2007) encode carotenoid cleavage dioxygenases (CCD7 and CCD8, respectively). These enzymes are capable of sequentially cleaving the carotenoid 9-cis-β-carotene to produce a novel compound, carlactone, a putative strigolactone intermediate (Alder et al., 2012). Another biosynthetic gene, D27, was originally mutationally defined in rice (Lin et al., 2009), and reverse genetic approaches in Arabidopsis indicate a similar function in this species (Waters et al., 2012a). D27 is an iron-containing protein with isomerase activity that can produce the 9-cis-β-carotene substrate for MAX3 from all-trans-β-carotene (Alder et al., 2012). The fourth gene known to be involved in SL biosynthesis, MAX1, encodes a cytochrome p450 monooxygenase belonging to the CYP711 clan (Booker et al., 2005). Mutant phenotypes associated with this gene have so far only been identified in one species, Arabidopsis, although the gene is present in all tracheophytes (Nelson et al., 2008). The excessive-branching phenotypes associated with mutations in all of these genes can be rescued by exogenous application of SL, while mutants in the two remaining genes in the pathway are SL insensitive. D14 encodes an α/β hydrolase, which is proposed to act in signaling or in the hydrolysis of SLs to an active compound and provides specificity to signaling via MAX2/RMS4/D3, an F-box protein that mediates both SL signaling and signaling of karrikins (Stirnberg et al., 2002, 2007; Ishikawa et al., 2005; Johnson et al., 2006; Arite et al., 2009; Hamiaux et al., 2012; Waters et al., 2012b). Karrikins are compounds structurally related to SLs that are found in smoke and act as germination stimulants for plants that colonize ground cleared by forest fires (Nelson et al., 2010; Waters et al., 2012b).Homology searches described in the original publications for each of the MAX/D genes revealed two general patterns. MAX1, MAX3, and MAX4 are members of widespread gene families and are more closely related to nonplant sequences than to other plant genes (Sorefan et al., 2003; Booker et al., 2005). By contrast, MAX2, D14, and D27 are members of plant-specific gene families (Stirnberg et al., 2002; Arite et al., 2009; Lin et al., 2009). These contrasting patterns of SL pathway gene ancestry and the diverse biological roles of SLs present interesting evolutionary questions. The identification of SLs and SL responses in charophyte algae demonstrate their early evolution, but these species lack many of the genes required for SL synthesis and signaling in angiosperms. In an attempt to trace the evolution of the angiosperm SL pathway, we conducted a phylogenetic analysis of the known SL biosynthesis and signaling genes, allowing the correlation of events in the evolution of the genes with the evolution of SL function. Our analysis suggests that the notion of a distinct SL pathway is inappropriate. Instead, the angiosperm pathway seems to have been defined by the rapid evolution of MAX2 in early land plants. Upstream of MAX2, there appears to be much greater flexibility, especially in the requirements for the synthesis of SLs. We present evidence for the contribution of MAX1 to this flexibility. Our functional analysis of MAX1 orthologs from phylogenetically diverse species demonstrates the early origin of its catalytic activity and both redundancy and functional diversification associated with its duplication in angiosperm lineages.     

Characterization of the FKBP12-Encoding Genes in Aspergillus fumigatus

Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the Δfkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn’t localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don’t play an important role in virulence. While the Δfkbp12-2 and Δfkbp12-3 strains didn’t show any discernable phenotype, the Δfkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated “paradoxical growth effect” at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A. fumigatus FKBP12-1 and human FKBP12 will be critical for the generation of fungal-specific FK506 analogs to inhibit fungal calcineurin and treat invasive fungal disease.     

Phylogenetic utility of MORE AXILLARY GROWTH4 (MAX4)-like genes: a case study in Digitalis/Isoplexis (Plantaginaceae)

We present the first assessment of phylogenetic utility of a potential novel low-copy nuclear gene region in flowering plants. A fragment of the MORE AXILLARY GROWTH 4 gene (MAX4, also known as RAMOSUS1 and DECREASED APICAL DOMINANCE1), predicted to span two introns, was isolated from members of Digitalis/Isoplexis. Phylogenetic analyses, under both maximum parsimony and Bayesian inference, were performed and revealed evidence of putative MAX4-like paralogues. The MAX4-like trees were compared with those obtained for Digitalis/Isoplexis using ITS and trnL-F, revealing a high degree of incongruence between these different DNA regions. Network analyses indicate complex patterns of evolution between the MAX4 sequences, which cannot be adequately represented on bifurcating trees. The incidence of paralogy restricts the use of MAX4 in phylogenetic inference within the study group, although MAX4 could potentially be used in combination with other DNA regions for resolving species relationships in cases where paralogues can be clearly identified.     

Cloning and Functional Characterization of Two BTB Genes in the Predatory Mite Metaseiulus occidentalis

Proteins containing the BTB (Bric-à-brac, tramtrack, and Broad Complex) domain typically share low sequence similarities and are involved in a wide range of cellular functions. We previously identified two putative and closely related BTB genes, BTB1 and BTB2, in the genome of the predatory mite Metaseiulus occidentalis. In the current study, full-length BTB1 and BTB2 cDNAs were cloned and sequenced. BTB1 and BTB2 encode proteins of 380 and 401 amino acids, respectively. BTB1 and BTB2 proteins each contain an N-terminal BTB domain and no other identifiable domains. Thus, they belong to a large category of BTB-domain proteins that are widely distributed in eukaryotes, yet with largely unknown function(s). BTB1 and BTB2 gene knockdowns in M. occidentalis females using RNAi reduced their fecundity by approximately 40% and 73%, respectively, whereas knockdown had no impact on their survival or the development of their offspring. These findings suggest these two proteins may be involved in processes related to egg production in this predatory mite, expanding the list of functions attributed to these diverse proteins.     

Characterization of Heterorhabditis Isolates by PCR Amplification of Segments of mtDNA and rDNA Genes

Restriction digests of amplified DNA from the mitochondrial genome and the nuclear ribosomal internally transcribed spacer region have been evaluated as genetic markers for species groups in Heterorhabditis. Six RFLP profiles have been identified. These profiles supported groupings determined by cross-breeding studies and were in agreement with less definitive groupings based on other biochemical and molecular methods. Digestion patterns of both amplification products provided strong evidence for the recognition of species groups, which include Irish, NW European, tropical, and a H. bacteriophora complex. The H. bacteriophora complex could be further resolved into three genotypes represented by H. zealandica, the H. bacteriophora, Brecon (Australian) type isolate for H. bacteriophora, and a grouping composed of isolates NC1, V16, HI82, and HP88. All cultures obtained of the H. megidis isolate were identical to the NW European group. These results could be used to aid monitoring of field release of Heterorhabditis as well as allowing a rapid initial assessment of taxonomic grouping.     

Characterization of Starch-Debranching Enzymes in Pea Embryos

Two distinct types of debranching enzymes have been identified in developing pea (Pisum sativum L.) embryos using native gel analysis and tests of substrate preference on purified or partially purified activities. An isoamylase-like activity capable of hydrolyzing amylopectin and glycogen but not pullulan is present throughout development and is largely or entirely confined to the plastid. Activities capable of hydrolyzing pullulan are present both inside and outside of the plastid, and extraplastidial activity increases relative to the plastidial activity during development. Both types of debranching enzyme are also present in germinating embryos. We argue that debranching enzymes are likely to have a role in starch metabolism in the plastid of the developing embryo and in starch degradation during germination.     

Genome-Wide Identification,Classification, and Expression Analysis of 14-3-3 Gene Family in Populus

Background

In plants, 14-3-3 proteins are encoded by a large multigene family and are involved in signaling pathways to regulate plant development and protection from stress. Although twelve Populus 14-3-3s were identified based on the Populus trichocarpa genome V1.1 in a previous study, no systematic analysis including genome organization, gene structure, duplication relationship, evolutionary analysis and expression compendium has been conducted in Populus based on the latest P. trichocarpa genome V3.0.

Principal Findings

Here, a comprehensive analysis of Populus 14-3-3 family is presented. Two new 14-3-3 genes were identified based on the latest P. trichocarpa genome. In P. trichocarpa, fourteen 14-3-3 genes were grouped into ε and non-ε group. Exon-intron organizations of Populus 14-3-3s are highly conserved within the same group. Genomic organization analysis indicated that purifying selection plays a pivotal role in the retention and maintenance of Populus 14-3-3 family. Protein conformational analysis indicated that Populus 14-3-3 consists of a bundle of nine α-helices (α1-α9); the first four are essential for formation of the dimer, while α3, α5, α7, and α9 form a conserved peptide-binding groove. In addition, α1, α3, α5, α7, and α9 were evolving at a lower rate, while α2, α4, and α6 were evolving at a relatively faster rate. Microarray analyses showed that most Populus 14-3-3s are differentially expressed across tissues and upon exposure to various stresses.

Conclusions

The gene structures and their coding protein structures of Populus 14-3-3s are highly conserved among group members, suggesting that members of the same group might also have conserved functions. Microarray and qRT-PCR analyses showed that most Populus 14-3-3s were differentially expressed in various tissues and were induced by various stresses. Our investigation provided a better understanding of the complexity of the 14-3-3 gene family in poplars.     

Identification and Characterization of Two Novel bla KLUC Resistance Genes through Large-Scale Resistance Plasmids Sequencing

Plasmids are important antibiotic resistance determinant carriers that can disseminate various drug resistance genes among species or genera. By using a high throughput sequencing approach, two groups of plasmids of Escherichia coli (named E1 and E2, each consisting of 160 clinical E. coli strains isolated from different periods of time) were sequenced and analyzed. A total of 20 million reads were obtained and mapped onto the known resistance gene sequences. As a result, a total of 9 classes, including 36 types of antibiotic resistant genes, were identified. Among these genes, 25 and 27 single nucleotide polymorphisms (SNPs) appeared, of which 9 and 12 SNPs are nonsynonymous substitutions in the E1 and E2 samples. It is interesting to find that a novel genotype of bla _KLUC, whose close relatives, bla _KLUC-1 and bla _KLUC-2, have been previously reported as carried on the Kluyvera cryocrescens chromosome and Enterobacter cloacae plasmid, was identified. It shares 99% and 98% amino acid identities with Kluc-1 and Kluc-2, respectively. Further PCR screening of 608 Enterobacteriaceae family isolates yielded a second variant (named bla _KLUC-4). It was interesting to find that Kluc-3 showed resistance to several cephalosporins including cefotaxime, whereas bla _KLUC-4 did not show any resistance to the antibiotics tested. This may be due to a positively charged residue, Arg, replaced by a neutral residue, Leu, at position 167, which is located within an omega-loop. This work represents large-scale studies on resistance gene distribution, diversification and genetic variation in pooled multi-drug resistance plasmids, and provides insight into the use of high throughput sequencing technology for microbial resistance gene detection.     

Systems Biology of Lignin Biosynthesis in Populus trichocarpa: Heteromeric 4-Coumaric Acid:Coenzyme A Ligase Protein Complex Formation,Regulation, and Numerical Modeling

As a step toward predictive modeling of flux through the pathway of monolignol biosynthesis in stem differentiating xylem of Populus trichocarpa, we discovered that the two 4-coumaric acid:CoA ligase (4CL) isoforms, 4CL3 and 4CL5, interact in vivo and in vitro to form a heterotetrameric protein complex. This conclusion is based on laser microdissection, coimmunoprecipitation, chemical cross-linking, bimolecular fluorescence complementation, and mass spectrometry. The tetramer is composed of three subunits of 4CL3 and one of 4CL5. 4CL5 appears to have a regulatory role. This protein–protein interaction affects the direction and rate of metabolic flux for monolignol biosynthesis in P. trichocarpa. A mathematical model was developed for the behavior of 4CL3 and 4CL5 individually and in mixtures that form the enzyme complex. The model incorporates effects of mixtures of multiple hydroxycinnamic acid substrates, competitive inhibition, uncompetitive inhibition, and self-inhibition, along with characteristic of the substrates, the enzyme isoforms, and the tetrameric complex. Kinetic analysis of different ratios of the enzyme isoforms shows both inhibition and activation components, which are explained by the mathematical model and provide insight into the regulation of metabolic flux for monolignol biosynthesis by protein complex formation.     

Pathogenomic Inference of Virulence-Associated Genes in Leptospira interrogans

Leptospirosis is a globally important, neglected zoonotic infection caused by spirochetes of the genus Leptospira. Since genetic transformation remains technically limited for pathogenic Leptospira, a systems biology pathogenomic approach was used to infer leptospiral virulence genes by whole genome comparison of culture-attenuated Leptospira interrogans serovar Lai with its virulent, isogenic parent. Among the 11 pathogen-specific protein-coding genes in which non-synonymous mutations were found, a putative soluble adenylate cyclase with host cell cAMP-elevating activity, and two members of a previously unstudied ∼15 member paralogous gene family of unknown function were identified. This gene family was also uniquely found in the alpha-proteobacteria Bartonella bacilliformis and Bartonella australis that are geographically restricted to the Andes and Australia, respectively. How the pathogenic Leptospira and these two Bartonella species came to share this expanded gene family remains an evolutionary mystery. In vivo expression analyses demonstrated up-regulation of 10/11 Leptospira genes identified in the attenuation screen, and profound in vivo, tissue-specific up-regulation by members of the paralogous gene family, suggesting a direct role in virulence and host-pathogen interactions. The pathogenomic experimental design here is generalizable as a functional systems biology approach to studying bacterial pathogenesis and virulence and should encourage similar experimental studies of other pathogens.