Whitefly (Bemisia tabaci) genome project: analysis of sequenced clones from egg, instar, and adult (viruliferous and non-viruliferous) cDNA libraries

BACKGROUND: The past three decades have witnessed a dramatic increase in interest in the whitefly Bemisia tabaci, owing to its nature as a taxonomically cryptic species, the damage it causes to a large number of herbaceous plants because of its specialized feeding in the phloem, and to its ability to serve as a vector of plant viruses. Among the most important plant viruses to be transmitted by B. tabaci are those in the genus Begomovirus (family, Geminiviridae). Surprisingly, little is known about the genome of this whitefly. The haploid genome size for male B. tabaci has been estimated to be approximately one billion bp by flow cytometry analysis, about five times the size of the fruitfly Drosophila melanogaster. The genes involved in whitefly development, in host range plasticity, and in begomovirus vector specificity and competency, are unknown. RESULTS: To address this general shortage of genomic sequence information, we have constructed three cDNA libraries from non-viruliferous whiteflies (eggs, immature instars, and adults) and two from adult insects that fed on tomato plants infected by two geminiviruses: Tomato yellow leaf curl virus (TYLCV) and Tomato mottle virus (ToMoV). In total, the sequence of 18,976 clones was determined. After quality control, and removal of 5,542 clones of mitochondrial origin 9,110 sequences remained which included 3,843 singletons and 1,017 contigs. Comparisons with public databases indicated that the libraries contained genes involved in cellular and developmental processes. In addition, approximately 1,000 bases aligned with the genome of the B. tabaci endosymbiotic bacterium Candidatus Portiera aleyrodidarum, originating primarily from the egg and instar libraries. Apart from the mitochondrial sequences, the longest and most abundant sequence encodes vitellogenin, which originated from whitefly adult libraries, indicating that much of the gene expression in this insect is directed toward the production of eggs. CONCLUSION: This is the first functional genomics project involving a hemipteran (Homopteran) insect from the subtropics/tropics. The B. tabaci sequence database now provides an important tool to initiate identification of whitefly genes involved in development, behaviour, and B. tabaci-mediated begomovirus transmission

Genome sequence of the pattern forming Paenibacillus vortex bacterium reveals potential for thriving in complex environments

<p>Abstract</p> <p>Background</p> <p>The pattern-forming bacterium <it>Paenibacillus vortex </it>is notable for its advanced social behavior, which is reflected in development of colonies with highly intricate architectures. Prior to this study, only two other <it>Paenibacillus </it>species (<it>Paenibacillus </it>sp. JDR-2 and <it>Paenibacillus larvae</it>) have been sequenced. However, no genomic data is available on the <it>Paenibacillus </it>species with pattern-forming and complex social motility. Here we report the <it>de novo </it>genome sequence of this Gram-positive, soil-dwelling, sporulating bacterium.</p> <p>Results</p> <p>The complete <it>P. vortex </it>genome was sequenced by a hybrid approach using 454 Life Sciences and Illumina, achieving a total of 289× coverage, with 99.8% sequence identity between the two methods. The sequencing results were validated using a custom designed Agilent microarray expression chip which represented the coding and the non-coding regions. Analysis of the <it>P. vortex </it>genome revealed 6,437 open reading frames (ORFs) and 73 non-coding RNA genes. Comparative genomic analysis with 500 complete bacterial genomes revealed exceptionally high number of two-component system (TCS) genes, transcription factors (TFs), transport and defense related genes. Additionally, we have identified genes involved in the production of antimicrobial compounds and extracellular degrading enzymes.</p> <p>Conclusions</p> <p>These findings suggest that <it>P. vortex </it>has advanced faculties to perceive and react to a wide range of signaling molecules and environmental conditions, which could be associated with its ability to reconfigure and replicate complex colony architectures. Additionally, <it>P. vortex </it>is likely to serve as a rich source of genes important for agricultural, medical and industrial applications and it has the potential to advance the study of social microbiology within Gram-positive bacteria.</p

DC Respond to Cognate T Cell Interaction in the Antigen-Challenged Lymph Node

Dendritic cells (DC) are unrivaled in their potential to prime naive T cells by presenting antigen and providing costimulation. DC are furthermore believed to decode antigen context by virtue of pattern recognition receptors and to polarize T cells through cytokine secretion toward distinct effector functions. Diverse polarized T helper (TH) cells have been explored in great detail. In contrast, studies of instructing DC have to date largely been restricted to in vitro settings or adoptively transferred DC. Here we report efforts to unravel the DC response to cognate T cell encounter in antigen-challenged lymph nodes (LN). Mice engrafted with antigen-specific T cells were immunized with nanoparticles (NP) entrapping adjuvants and absorbed with antigen to study the immediate DC response to T cell encounter using bulk and single cell RNA-seq profiling. NP induced robust antigen-specific TH1 cell responses with minimal bystander activation. Fluorescent-labeled NP allowed identification of antigen-carrying DC and focus on transcriptional changes in DC that encounter T cells. Our results support the existence of a bi-directional crosstalk between DC and T cells that promotes TH1 responses, including involvement of the ubiquitin-like molecule Isg15 that merits further study

Somatodendritic Expression of JAM2 Inhibits Oligodendrocyte Myelination

Myelination occurs selectively around neuronal axons to increase the efficiency and velocity of action potentials. While oligodendrocytes are capable of myelinating permissive structures in the absence of molecular cues, structurally permissive neuronal somata and dendrites remain unmyelinated. Utilizing a purified spinal cord neuron-oligodendrocyte myelinating coculture system, we demonstrate that disruption of dynamic neuron-oligodendrocyte signaling by chemical crosslinking results in aberrant myelination of the somatodendritic compartment of neurons. We hypothesize that an inhibitory somatodendritic cue is necessary to prevent non-axonal myelination. Using next-generation sequencing and candidate profiling, we identify neuronal Junction Adhesion Molecule 2 (JAM2) as an inhibitory myelin-guidance molecule. Taken together, our results demonstrate that the somatodendritic compartment directly inhibits myelination, and suggest a model in which broadly indiscriminate myelination is tailored by inhibitory signaling to meet local myelination requirements

Transcriptional Reprogramming of CD11b+Esamhi Dendritic Cell Identity and Function by Loss of Runx3

Classical dendritic cells (cDC) are specialized antigen-presenting cells mediating immunity and tolerance. cDC cell-lineage decisions are largely controlled by transcriptional factor regulatory cascades. Using an in vivo cell-specific targeting of Runx3 at various stages of DC lineage development we show that Runx3 is required for cell-identity, homeostasis and function of splenic Esamhi DC. Ablation of Runx3 in DC progenitors led to a substantial decrease in splenic CD4+/CD11b+ DC. Combined chromatin immunoprecipitation sequencing and gene expression analysis of purified DC-subsets revealed that Runx3 is a key gene expression regulator that facilitates specification and homeostasis of CD11b+Esamhi DC. Mechanistically, loss of Runx3 alters Esamhi DC gene expression to a signature characteristic of WT Esamlow DC. This transcriptional reprogramming caused a cellular change that diminished phagocytosis and hampered Runx3-/- Esamhi DC capacity to prime CD4+ T cells, attesting to the significant role of Runx3 in specifying Esamhi DC identity and function

Dual role of starvation signaling in promoting growth and recovery

<div><p>Growing cells are subject to cycles of nutrient depletion and repletion. A shortage of nutrients activates a starvation program that promotes growth in limiting conditions. To examine whether nutrient-deprived cells prepare also for their subsequent recovery, we followed the transcription program activated in budding yeast transferred to low-phosphate media and defined its contribution to cell growth during phosphate limitation and upon recovery. An initial transcription wave was induced by moderate phosphate depletion that did not affect cell growth. A second transcription wave followed when phosphate became growth limiting. The starvation program contributed to growth only in the second, growth-limiting phase. Notably, the early response, activated at moderate depletion, promoted recovery from starvation by increasing phosphate influx upon transfer to rich medium. Our results suggest that cells subject to nutrient depletion prepare not only for growth in the limiting conditions but also for their predicted recovery once nutrients are replenished.</p></div

Two-wave induction of the phosphate starvation response.

<p>(A) Schematic illustration of the phosphate starvation response: Reduction in internal phosphate leads to the nuclear localization of the transcription factor Pho4 and the induction of its target genes. Genes induced by Pho4 include high-affinity transporters (e.g., <i>PHO84</i>) and an inhibitor of low-affinity transporters (<i>SPL2</i>), genes involved in phosphate mobilization in and out of vacuole storage as PolyP (e.g., <i>PHM3</i>), and phosphatases, which scavenge phosphate from molecules inside or outside the cell. (B) Cells grow for a limited number of generations following transfer to media containing different low phosphate levels: Shown is the temporal increase in cellular density (measured by optical density [OD]) following transfer to low-phosphate media, as indicated (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#sec010" target="_blank">Materials and methods</a>). The data are the mean and standard error of 2 replicates. (C–D) Sequential induction of the phosphate transcription response: Wild-type (WT) cells were transferred from rich medium into media containing the indicated low Pi level and followed for 24.7 hours (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#sec010" target="_blank">Materials and methods</a>). Samples for RNA sequencing were taken at the indicated time points. Shown in (C) is the log₂(expression) change in Pho4-target genes, stress response genes (Stress), and genes coding for ribosomal proteins (Protein Synthesis), as defined in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.ref020" target="_blank">20</a>] (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#sec010" target="_blank">Materials and methods</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s009" target="_blank">S2 Data</a>). Data were normalized as described in the Materials and methods, and values under detectable levels (not a number [NaN]) are depicted in grey. Vertical lines indicate the second transcription wave, as defined by clustering (using k-means) of the Pearson correlation matrix, shown in (D). Note that when transferred to 0.06 mM Pi, cells transiently induced the second wave approximately 3–4.5 hours following the transfer, followed by a stable induction at approximately 7 hours after the transfer. This transient induction is likely due to the double-feedback design of the system, as we showed [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.ref021" target="_blank">21</a>]. The reduction in growth rate (see E–F below) is observed already with first induction of the second transcription wave. The data in (C–D) are from 1 replicate. Additional biological (and experimental) replicates for growth in 0.06 mM and 0.2 mM Pi are found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.g003" target="_blank">Fig 3C</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s001" target="_blank">S1C Fig</a>, respectively, and for 0 mM Pi, see [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.ref021" target="_blank">21</a>]. For further supporting data for (C), see (E). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s001" target="_blank">S1A and S1B Fig</a> for definition of Pho4-target genes. (E–F) Phosphate becomes growth limiting concomitant with the induction of the second transcription wave: The PHO84p-Venus reporter is up-regulated following transfer of cells to low-phosphate media, as indicated (E). The cell growth rate was calculated by the logarithmic slope of the OD curve, shown in (B), and is plotted as a function of reporter expression (F). To control for density-dependent effects, the growth rate was normalized by that of cells transferred to rich phosphate medium (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#sec010" target="_blank">Materials and methods</a>). The data in (E–F) are the mean and standard error of 2 replicates. Note that growth rate begins to decrease when the PHO84p-Venus reporter crosses a given threshold, independently of the incubating conditions. This crossing of the activation threshold coincides with the time at which the second transcription wave is induced, as defined by clustering (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s001" target="_blank">S1E Fig</a>). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s001" target="_blank">S1D Fig</a> for the normalized growth rate as a function of time. The raw data for (B, E–F) are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s008" target="_blank">S1 Data</a>, and those for (C–D) are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s009" target="_blank">S2 Data</a>. Pi, inorganic phosphate; WT, wild type.</p

Lab evolution selects for <i>PHO84</i> mutations that rescue the recovery phenotype of PHO84^C.

<p>(A) Experimental scheme: 16 independent lines expressing constitutively high levels of <i>PHO84</i> were subjected to 10 cycles of alternated growth in high and low phosphate for a total of 300 generations. (B) <i>PHO84</i> mutations identified in selected lines: Single-cell isolates (evolved for approximately 150 and approximately 300 generations) were selected from the 10 cultures showing improved recovery and were sent for whole-genome sequencing. All isolates showing an adaptive phenotype contained mutations in the Pho84 open reading frame (ORF), with 56 out of the 60 identified mutations mapped to the same amino-acid substitution (L74F). (C–E) Selected <i>PHO84</i> mutation rescues the recovery phenotype: The <i>PHO84</i> mutations identified in the selected stains were introduced into the wild-type allele and their effect on the growth in low Pi and on the recovery phenotype was examined when <i>PHO84</i> was expressed at wild-type levels or constitutively (both the constitutive overexpression and the evolved mutations were introduced into <i>PHO84</i> endogenous site). Shown is the recovery of the constitutive strain with and without the amino-acid substitution (Pho84-L74F(ttG/ttC)) following 24.7 hours incubation in 0.2 mM Pi (C, D) and the corresponding pho4-target gene expression during recovery (E). Note the rescue of the recovery phenotype and the immediate down-regulation of pho4-target genes. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s006" target="_blank">S6B–SBJ Fig</a> for the growth in low-phosphate media and recovery phenotypes of the selected mutations: Pho84-L74F(ttG/ttC), Pho84-L259P, and Pho84-V383L. The data in (C) are from 1 replicate; see (D) for additional supporting data. The data in (D) are the mean and standard error of 3 replicates. The data in (E) are from 1 replicate. Additional data as in (E) for recovery (from 0.06 mM Pi) are found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s006" target="_blank">S6E Fig</a>. The conclusions of (E) are further supported by experiments with the Venus reporter (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s011" target="_blank">S4 Data</a>). The raw data for (C–D) are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s011" target="_blank">S4 Data</a>. The raw data for (E) are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s009" target="_blank">S2 Data</a>. Gen, generations; NaN, not a number; Pi, inorganic phosphate; WT, wild type.</p

The starvation program promotes recovery once phosphate is replenished.

<p>(A) Recovery time depends on the conditions leading to starvation: Cells were incubated in media containing different low phosphate levels and maintained in the starvation conditions for 21 hours before being transferred back into a rich medium. The number of generations that cells underwent 3 or 5.7 hours after the transfer to rich medium is shown as a function of the phosphate level in the incubating media. The data in (A) are the mean and standard error of 2 replicates. (B) Pho4-target genes are rapidly down-regulated when phosphate is replenished: Recovery expression profiles of cells grown for 24.7 hours in low-Pi media (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.g001" target="_blank">Fig 1C</a>) and recovered in rich medium. The data in (B) are from 1 replicate. Additional replicates for recovery from 0.2 mM and 0.06 mM Pi are found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.g006" target="_blank">Fig 6E</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s006" target="_blank">S6E Fig</a>, respectively. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s011" target="_blank">S4 Data</a> for related experiments using a reporter gene. (C) The starvation program promotes recovery: Competition experiments, as shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.g002" target="_blank">Fig 2</a>, were performed to compare the growth of Δ<i>pho4</i> and wild-type cells following recovery in rich medium. Wild-type and Δ<i>pho4</i> cells were coincubated in media containing different levels of low phosphate, grown to saturation, and maintained in the starvation conditions for 24.7 hours before being transferred back into rich medium. The (log₂) relative abundance of Δ<i>pho4</i> cells at different times following return to rich medium is shown. Note that Δ<i>pho4</i> cells are outcompeted by wild-type cells during recovery from starvation that was induced by incubation in media containing intermediate phosphate (e.g., 0.2 mM Pi). These conditions introduce the longest time gap between the first and second wave of Pho4-target gene induction (“preparation” time). The data in (C) are the mean and standard error of 2 replicates. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s004" target="_blank">S4A Fig</a> for similar experiments using Δ<i>pho81</i> deleted cells. (D, E) Constitutive expression of PHO84 partially rescues the recovery phenotype of Δpho4 cells: Experiments as shown in (C) were repeated for cells that constitutively express the PHO84 transporter (PHO84^C) in a wild-type or Δ<i>pho4</i> background. The (log₂) relative abundance of mutant cells during recovery from starvation induced by incubating in media containing 0.2 mM Pi is shown in (D). In (D), the mean of 2 replicates is shown for Δ<i>pho4</i>, and 1 replicate is shown for PHO84^C with and without Δ<i>pho4</i>. These data are supported by the experiments shown in (E) and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.g005" target="_blank">Fig 5B</a>. (E) The experiments in (D) were repeated using a variety of starvation media, and the fitness difference of mutant and wild-type cells was measured following 24.7 hours of incubation in rich medium. This fitness difference is shown as a function of the initial phosphate level in the starvation media. Note that deletion of <i>PHO4</i> impaired recovery when cells were first incubated at intermediate phosphate levels (around 0.2 mM Pi), allowing sufficient preparation time, and that constitutive expression of <i>PHO84</i> partially rescued this phenotype. The data in (E) are the mean and standard error of 3 replicates. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s004" target="_blank">S4B Fig</a> for similar experiments using Δ<i>pho81</i> cells. (F–I) Phosphate influx limits recovery from starvation: Competition experiments as in (C) were repeated for cells of the indicated genotype (F–I). Shown are the (log₂) relative abundance of the indicated mutant strains (F–G) and the level of the PHO84p-Venus reporter in the mutants and wild-type cells (H–I) during competitive growth. In (F, H) are shown several time points within the first 10 hours of recovery (into 20 mM Pi), and those after 9 hours of recovery into 20 mM Pi are shown in (G, I). The data in (F, H) are from 1 replicate and are supported by data in (G, I), which show the mean and standard error of 2 replicates. See (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s004" target="_blank">S4C and S4D Fig</a>) for recovery into a high Pi of 7.3 mM Pi. The raw data for (C, D, and F–I) are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s011" target="_blank">S4 Data</a>. The raw data for (A, B, and E) are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s008" target="_blank">S1</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s009" target="_blank">S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s012" target="_blank">S5</a> Data, respectively. NaN, not a number; Pi, inorganic phosphate; WT, wild type.</p