Polyamines and linear DNA mediate bacterial threat assessment of bacteriophage infection.

Monitoring the extracellular environment for danger signals is a critical aspect of cellular survival. However, the danger signals released by dying bacteria and the mechanisms bacteria use for threat assessment remain largely unexplored. Here, we show that lysis of Pseudomonas aeruginosa cells releases polyamines that are subsequently taken up by surviving cells via a mechanism that relies on Gac/Rsm signaling. While intracellular polyamines spike in surviving cells, the duration of this spike varies according to the infection status of the cell. In bacteriophage-infected cells, intracellular polyamines are maintained at high levels, which inhibits replication of the bacteriophage genome. Many bacteriophages package linear DNA genomes and linear DNA is sufficient to trigger intracellular polyamine accumulation, suggesting that linear DNA is sensed as a second danger signal. Collectively, these results demonstrate how polyamines released by dying cells together with linear DNA allow P. aeruginosa to make threat assessments of cellular injury.

A Filamentous Bacteriophage Protein Inhibits Type IV Pili To Prevent Superinfection of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in a variety of settings. Many P. aeruginosa isolates are infected by filamentous Pf bacteriophage integrated into the bacterial chromosome as a prophage. Pf virions can be produced without lysing P. aeruginosa. However, cell lysis can occur during superinfection, which occurs when Pf virions successfully infect a host lysogenized by a Pf prophage. Temperate phages typically encode superinfection exclusion mechanisms to prevent host lysis by virions of the same or similar species. In this study, we sought to elucidate the superinfection exclusion mechanism of Pf phage. Initially, we observed that P. aeruginosa that survive Pf superinfection are transiently resistant to Pf-induced plaquing and are deficient in twitching motility, which is mediated by type IV pili (T4P). Pf utilize T4P as a cell surface receptor, suggesting that T4P are suppressed in bacteria that survive superinfection. We tested the hypothesis that a Pf-encoded protein suppresses T4P to mediate superinfection exclusion by expressing Pf proteins in P. aeruginosa and measuring plaquing and twitching motility. We found that the Pf protein PA0721, which we termed Pf superinfection exclusion (PfsE), promoted resistance to Pf infection and suppressed twitching motility by binding the T4P protein PilC. Because T4P play key roles in biofilm formation and virulence, the ability of Pf phage to modulate T4P via PfsE has implications in the ability of P. aeruginosa to persist at sites of infection. IMPORTANCE Pf bacteriophage (phage) are filamentous viruses that infect Pseudomonas aeruginosa and enhance its virulence potential. Pf virions can lyse and kill P. aeruginosa through superinfection, which occurs when an already infected cell is infected by the same or similar phage. Here, we show that a small, highly conserved Pf phage protein (PA0721, PfsE) provides resistance to superinfection by phages that use the type IV pilus as a cell surface receptor. PfsE does this by inhibiting assembly of the type IV pilus via an interaction with PilC. As the type IV pilus plays important roles in virulence, the ability of Pf phage to modulate its assembly has implications for P. aeruginosa pathogenesis.

Apple ALMT9 Requires a Conserved C-Terminal Domain for Malate Transport Underlying Fruit Acidity.

Malate accumulation in the vacuole largely determines apple (Malus domestica) fruit acidity, and low fruit acidity is strongly associated with truncation of Ma1, an ortholog of ALUMINUM-ACTIVATED MALATE TRANSPORTER9 (ALMT9) in Arabidopsis (Arabidopsis thaliana). A mutation at base 1,455 in the open reading frame of Ma1 leads to a premature stop codon that truncates the protein by 84 amino acids at its C-terminal end. Here, we report that both the full-length protein, Ma1, and its naturally occurring truncated protein, ma1, localize to the tonoplast; when expressed in Xenopus laevis oocytes and Nicotiana benthamiana cells, Ma1 mediates a malate-dependent inward-rectifying current, whereas the ma1-mediated transmembrane current is much weaker, indicating that ma1 has significantly lower malate transport activity than Ma1. RNA interference suppression of Ma1 expression in 'McIntosh' apple leaves, 'Empire' apple fruit, and 'Orin' apple calli results in a significant decrease in malate level. Genotyping and phenotyping of 186 apple accessions from a diverse genetic background of 17 Malus species combined with the functional analyses described above indicate that Ma1 plays a key role in determining fruit acidity and that the truncation of Ma1 to ma1 is genetically responsible for low fruit acidity in apple. Furthermore, we identified a C-terminal domain conserved in all tonoplast-localized ALMTs essential for Ma1 function; protein truncations into this conserved domain significantly lower Ma1 transport activity. We conclude that the truncation of Ma1 to ma1 reduces its malate transport function by removing a conserved C-terminal domain, leading to low fruit acidity in apple.

The Raf-like Kinase ILK1 and the High Affinity K+ Transporter HAK5 Are Required for Innate Immunity and Abiotic Stress Response.

Plant perception of pathogen-associated molecular patterns (PAMPs) and other environmental stresses trigger transient ion fluxes at the plasma membrane. Apart from the role of Ca(2+) uptake in signaling, the regulation and significance of PAMP-induced ion fluxes in immunity remain unknown. We characterized the functions of INTEGRIN-LINKED KINASE1 (ILK1) that encodes a Raf-like MAP2K kinase with functions insufficiently understood in plants. Analysis of ILK1 mutants impaired in the expression or kinase activity revealed that ILK1 contributes to plant defense to bacterial pathogens, osmotic stress sensitivity, and cellular responses and total ion accumulation in the plant upon treatment with a bacterial-derived PAMP, flg22. The calmodulin-like protein CML9, a negative modulator of flg22-triggered immunity, interacted with, and suppressed ILK1 kinase activity. ILK1 interacted with and promoted the accumulation of HAK5, a putative (H(+))/K(+) symporter that mediates a high-affinity uptake during K(+) deficiency. ILK1 or HAK5 expression was required for several flg22 responses including gene induction, growth arrest, and plasma membrane depolarization. Furthermore, flg22 treatment induced a rapid K(+) efflux at both the plant and cellular levels in wild type, while mutants with impaired ILK1 or HAK5 expression exhibited a comparatively increased K(+) loss. Taken together, our results position ILK1 as a link between plant defense pathways and K(+) homeostasis.

Sea urchin akt activity is Runx-dependent and required for post-cleavage stage cell division.

In animal development following the initial cleavage stage of embryogenesis, the cell cycle becomes dependent on intercellular signaling and controlled by the genomically encoded ontogenetic program. Runx transcription factors are critical regulators of metazoan developmental signaling, and we have shown that the sea urchin Runx gene runt-1, which is globally expressed during early embryogenesis, functions in support of blastula stage cell proliferation and expression of the mitogenic genes pkc1, cyclinD, and several wnts. To obtain a more comprehensive list of early runt-1 regulatory targets, we screened a Strongylocentrotus purpuratus microarray to identify genes mis-expressed in mid-blastula stage runt-1 morphants. This analysis showed that loss of Runx function perturbs the expression of multiple genes involved in cell division, including the pro-growth and survival kinase Akt (PKB), which is significantly underexpressed in runt-1 morphants. Further genomic analysis revealed that Akt is encoded by two genes in the S. purpuratus genome, akt-1 and akt-2, both of which contain numerous canonical Runx target sequences. The transcripts of both genes accumulate several fold during blastula stage, contingent on runt-1 expression. Inhibiting Akt expression or activity causes blastula stage cell cycle arrest, whereas overexpression of akt-1 mRNA rescues cell proliferation in runt-1 morphants. These results indicate that post-cleavage stage cell division requires Runx-dependent expression of akt.

Aluminum tolerance in maize is associated with higher MATE1 gene copy number.

Genome structure variation, including copy number variation and presence/absence variation, comprises a large extent of maize genetic diversity; however, its effect on phenotypes remains largely unexplored. Here, we describe how copy number variation underlies a rare allele that contributes to maize aluminum (Al) tolerance. Al toxicity is the primary limitation for crop production on acid soils, which make up 50% of the world's potentially arable lands. In a recombinant inbred line mapping population, copy number variation of the Al tolerance gene multidrug and toxic compound extrusion 1 (MATE1) is the basis for the quantitative trait locus of largest effect on phenotypic variation. This expansion in MATE1 copy number is associated with higher MATE1 expression, which in turn results in superior Al tolerance. The three MATE1 copies are identical and are part of a tandem triplication. Only three maize inbred lines carrying the three-copy allele were identified from maize and teosinte diversity panels, indicating that copy number variation for MATE1 is a rare, and quite likely recent, event. These maize lines with higher MATE1 copy number are also Al-tolerant, have high MATE1 expression, and originate from regions of highly acidic soils. Our findings show a role for copy number variation in the adaptation of maize to acidic soils in the tropics and suggest that genome structural changes may be a rapid evolutionary response to new environments.

Low pH, aluminum, and phosphorus coordinately regulate malate exudation through GmALMT1 to improve soybean adaptation to acid soils.

Low pH, aluminum (Al) toxicity, and low phosphorus (P) often coexist and are heterogeneously distributed in acid soils. To date, the underlying mechanisms of crop adaptation to these multiple factors on acid soils remain poorly understood. In this study, we found that P addition to acid soils could stimulate Al tolerance, especially for the P-efficient genotype HN89. Subsequent hydroponic studies demonstrated that solution pH, Al, and P levels coordinately altered soybean (Glycine max) root growth and malate exudation. Interestingly, HN89 released more malate under conditions mimicking acid soils (low pH, +P, and +Al), suggesting that root malate exudation might be critical for soybean adaptation to both Al toxicity and P deficiency on acid soils. GmALMT1, a soybean malate transporter gene, was cloned from the Al-treated root tips of HN89. Like root malate exudation, GmALMT1 expression was also pH dependent, being suppressed by low pH but enhanced by Al plus P addition in roots of HN89. Quantitative real-time PCR, transient expression of a GmALMT1-yellow fluorescent protein chimera in Arabidopsis protoplasts, and electrophysiological analysis of Xenopus laevis oocytes expressing GmALMT1 demonstrated that GmALMT1 encodes a root cell plasma membrane transporter that mediates malate efflux in an extracellular pH-dependent and Al-independent manner. Overexpression of GmALMT1 in transgenic Arabidopsis, as well as overexpression and knockdown of GmALMT1 in transgenic soybean hairy roots, indicated that GmALMT1-mediated root malate efflux does underlie soybean Al tolerance. Taken together, our results suggest that malate exudation is an important component of soybean adaptation to acid soils and is coordinately regulated by three factors, pH, Al, and P, through the regulation of GmALMT1 expression and GmALMT1 function.

A screen for spore wall permeability mutants identifies a secreted protease required for proper spore wall assembly.

The ascospores of Saccharomyces cerevisiae are surrounded by a complex wall that protects the spores from environmental stresses. The outermost layer of the spore wall is composed of a polymer that contains the cross-linked amino acid dityrosine. This dityrosine layer is important for stress resistance of the spore. This work reports that the dityrosine layer acts as a barrier blocking the diffusion of soluble proteins out of the spore wall into the cytoplasm of the ascus. Diffusion of a fluorescent protein out of the spore wall was used as an assay to screen for mutants affecting spore wall permeability. One of the genes identified in this screen, OSW3 (RRT12/YCR045c), encodes a subtilisin-family protease localized to the spore wall. Mutation of the active site serine of Osw3 results in spores with permeable walls, indicating that the catalytic activity of Osw3 is necessary for proper construction of the dityrosine layer. These results indicate that dityrosine promotes stress resistance by acting as a protective shell around the spore. OSW3 and other OSW genes identified in this screen are strong candidates to encode enzymes involved in assembly of this protective dityrosine coat.

Oral-aboral axis specification in the sea urchin embryo III. Role of mitochondrial redox signaling via H2O2.

In sea urchin embryos, specification of the secondary (oral-aboral) axis occurs via nodal, expression of which is entirely zygotic and localized to prospective oral ectoderm at blastula stage. The initial source of this spatial anisotropy is not known. Previous studies have shown that oral-aboral (OA) polarity correlates with a mitochondrial gradient, and that nodal activity is dependent both on mitochondrial respiration and p38 stress-activated protein kinase. Here we show that the spatial pattern of nodal activity also correlates with the mitochondrial gradient, and that the latter correlates with inhomogeneous levels of intracellular reactive oxygen species. To test whether mitochondrial H(2)O(2) functions as a redox signal to activate nodal, zygotes were injected with mRNA encoding either mitochondrially-targeted catalase, which quenches mitochondrial H(2)O(2) and down-regulates p38, or superoxide dismutase, which augments mitochondrial H(2)O(2) and up-regulates p38. Whereas the former treatment inhibits the initial activation of nodal and entrains OA polarity toward aboral when confined to half of the embryo via 2-cell stage blastomere injections, the latter does not produce the opposite effects. We conclude that mitochondrial H(2)O(2) is rate-limiting for the initial activation of nodal, but that additional rate-limiting factors, likely also involving mitochondria, contribute to the asymmetry in nodal expression.

Runx expression is mitogenic and mutually linked to Wnt activity in blastula-stage sea urchin embryos.

BACKGROUND: The Runt homology domain (Runx) defines a metazoan family of sequence-specific transcriptional regulatory proteins that are critical for animal development and causally associated with a variety of mammalian cancers. The sea urchin Runx gene SpRunt-1 is expressed throughout the blastula stage embryo, and is required globally during embryogenesis for cell survival and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Depletion of SpRunt-1 by morpholino antisense-mediated knockdown causes a blastula stage deficit in cell proliferation, as shown by bromodeoxyuridine (BrdU) incorporation and direct cell counts. Reverse transcription coupled polymerase chain reaction (RT-PCR) studies show that the cell proliferation deficit is presaged by a deficit in the expression of several zygotic wnt genes, including wnt8, a key regulator of endomesoderm development. In addition, SpRunt-1-depleted blastulae underexpress cyclinD, an effector of mitogenic Wnt signaling. Blastula stage cell proliferation is also impeded by knockdown of either wnt8 or cyclinD. Chromatin immunoprecipitation (ChIP) indicates that Runx target sites within 5' sequences flanking cyclinD, wnt6 and wnt8 are directly bound by SpRunt-1 protein at late blastula stage. Furthermore, experiments using a green fluorescent protein (GFP) reporter transgene show that the blastula-stage operation of a cis-regulatory module previously shown to be required for wnt8 expression (Minokawa et al., Dev. Biol. 288: 545-558, 2005) is dependent on its direct sequence-specific interaction with SpRunt-1. Finally, inhibitor studies and immunoblot analysis show that SpRunt-1 protein levels are negatively regulated by glycogen synthase kinase (GSK)-3. CONCLUSIONS/SIGNIFICANCE: These results suggest that Runx expression and Wnt signaling are mutually linked in a feedback circuit that controls cell proliferation during development.

The yeast spore wall enables spores to survive passage through the digestive tract of Drosophila.

In nature, yeasts are subject to predation by flies of the genus Drosophila. In response to nutritional starvation Saccharomyces cerevisiae differentiates into a dormant cell type, termed a spore, which is resistant to many types of environmental stress. The stress resistance of the spore is due primarily to a spore wall that is more elaborate than the vegetative cell wall. We report here that S. cerevisiae spores survive passage through the gut of Drosophila melanogaster. Constituents of the spore wall that distinguish it from the vegetative cell wall are necessary for this resistance. Ascospores of the distantly related yeast Schizosaccharomyces pombe also display resistance to digestion by D. melanogaster. These results suggest that the primary function of the yeast ascospore is as a cell type specialized for dispersion by insect vectors.

GAS2 and GAS4, a pair of developmentally regulated genes required for spore wall assembly in Saccharomyces cerevisiae.

The GAS multigene family of Saccharomyces cerevisiae is composed of five paralogs (GAS1 to GAS5). GAS1 is the only one of these genes that has been characterized to date. It encodes a glycosylphosphatidylinositol-anchored protein functioning as a beta(1,3)-glucan elongase and required for proper cell wall assembly during vegetative growth. In this study, we characterize the roles of the GAS2 and GAS4 genes. These genes are expressed exclusively during sporulation. Their mRNA levels showed a peak at 7 h from induction of sporulation and then decreased. Gas2 and Gas4 proteins were detected and reached maximum levels between 8 and 10 h from induction of sporulation, a time roughly coincident with spore wall assembly. The double null gas2 gas4 diploid mutant showed a severe reduction in the efficiency of sporulation, an increased permeability of the spores to exogenous substances, and production of inviable spores, whereas the single gas2 and gas4 null diploids were similar to the parental strain. An analysis of spore ultrastructure indicated that the loss of Gas2 and Gas4 proteins affected the proper attachment of the glucan to the chitosan layer, probably as a consequence of the lack of coherence of the glucan layer. The ectopic expression of GAS2 and GAS4 genes in a gas1 null mutant revealed that these proteins are redundant versions of Gas1p specialized to function in a compartment at a pH value close to neutral.

Phospholipase D and the SNARE Sso1p are necessary for vesicle fusion during sporulation in yeast.

Spore formation in Saccharomyces cerevisiae requires the de novo formation of prospore membranes. The coalescence of secretory vesicles into a membrane sheet occurs on the cytoplasmic surface of the spindle pole body. Spo14p, the major yeast phospholipase D, is necessary for prospore membrane formation; however, the specific function of Spo14p in this process has not been elucidated. We report that loss of Spo14p blocks vesicle fusion, leading to the accumulation of prospore membrane precursor vesicles docked on the spindle pole body. A similar phenotype was seen when the t-SNARE Sso1p, or the partially redundant t-SNAREs Sec9p and Spo20p were mutated. Although phosphatidic acid, the product of phospholipase D action, was necessary to recruit Spo20p to the precursor vesicles, independent targeting of Spo20p to the membrane was not sufficient to promote fusion in the absence of SPO14. These results demonstrate a role for phospholipase D in vesicle fusion and suggest that phospholipase D-generated phosphatidic acid plays multiple roles in the fusion process.

Morphogenetic pathway of spore wall assembly in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae spore is protected from environmental damage by a multilaminar extracellular matrix, the spore wall, which is assembled de novo during spore formation. A set of mutants defective in spore wall assembly were identified in a screen for mutations causing sensitivity of spores to ether vapor. The spore wall defects in 10 of these mutants have been characterized in a variety of cytological and biochemical assays. Many of the individual mutants are defective in the assembly of specific layers within the spore wall, leading to arrests at discrete stages of assembly. The localization of several of these gene products has been determined and distinguishes between proteins that likely are involved directly in spore wall assembly and probable regulatory proteins. The results demonstrate that spore wall construction involves a series of dependent steps and provide the outline of a morphogenetic pathway for assembly of a complex extracellular structure.

Interspore bridges: a new feature of the Saccharomyces cerevisiae spore wall.

The Saccharomyces cerevisiae spore wall is a multilaminar coat that surrounds individual spores and protects them from environmental insult. Scanning electron microscopy reveals that the four spores of an ascus are connected by interspore bridges. Transmission electron microscopy of spores indicates that these bridges are continuous with the outer layers of the spore wall. In chs3 mutants, which lack the chitosan and dityrosine layers of the spore wall, bridges are absent. By contrast, in dit1 mutants, which lack only the dityrosine layer, bridges are present, suggesting that the bridges may be composed of chitosan. Interspore bridges are shown to be necessary to hold spores together after release from the ascus. A function for these bridges in the maintenance of heterozygous markers in a homothallic yeast population is proposed.

Genetic evidence of a role for membrane lipid composition in the regulation of soluble NEM-sensitive factor receptor function in Saccharomyces cerevisiae.

SEC9 and SPO20 encode SNARE proteins related to the mammalian SNAP-25 family. Sec9p associates with the SNAREs Sso1/2p and Snc1/2p to promote the fusion of vesicles with the plasma membrane. Spo20p functions with the same two partner SNAREs to mediate the fusion of vesicles with the prospore membrane during sporogenesis. A chimeric molecule, in which the helices of Sec9p that bind to Sso1/2p and Snc1/2p are replaced with the homologous regions of Spo20p, will not support vesicle fusion in vegetative cells. The phosphatidylinositol-4-phosphate-5-kinase MSS4 was isolated as a high-copy suppressor that permits this chimera to rescue the temperature-sensitive growth of a sec9-4 mutant. Suppression by MSS4 is specific to molecules that contain the Spo20p helical domains. This suppression requires an intact copy of SPO14, encoding phospholipase D. Overexpression of MSS4 leads to a recruitment of the Spo14 protein to the plasma membrane and this may be the basis for MSS4 action. Consistent with this, deletion of KES1, a gene that behaves as a negative regulator of SPO14, also promotes the function of SPO20 in vegetative cells. These results indicate that elevated levels of phosphatidic acid in the membrane may be required specifically for the function of SNARE complexes containing Spo20p.