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The uterine myometrium expands and maintains contractile quiescence before parturition. While the steroid hormone progesterone blocks labor, the role of progesterone signaling in myometrial expansion remains elusive. This study investigated the myometrial functions of the progesterone receptor, PGR. Pgr ablation in mouse smooth muscle leads to subfertility, oviductal embryo retention, and impaired myometrial adaptation to pregnancy. While gross morphology between mutant and control uteri are comparable, mutant uteri manifest a decrease of 76.6% oxytocin-stimulated contractility in a pseudopregnant context with a reduced expression of intracellular calcium homeostasis genes including Pde5a and Plcb4. At mid-pregnancy, the mutant myometrium exhibits discontinuous myofibers and disarrayed extracellular matrix at the conceptus site. Transcriptome of the mutant mid-pregnant uterine wall manifests altered muscle and extracellular matrix profiles and resembles that of late-pregnancy control tissues. A survey of PGR occupancy, H3K27ac histone marks, and chromatin looping annotates cis-acting elements that may direct gene expression of mid-pregnancy uteri for uterine remodeling. Further analyses suggest that major muscle and matrix regulators Myocd and Ccn2 and smooth muscle building block genes are PGR direct downstream targets. Cataloging enhancers that are topologically associated with progesterone downstream genes reveals distinctive patterns of transcription factor binding motifs in groups of enhancers and identifies potential regulatory partners of PGR outside its occupying sites. Finally, conserved correlations are found between estimated PGR activities and RNA abundance of downstream muscle and matrix genes in human myometrial tissues. In summary, PGR is pivotal to direct the molecular program for the uterus to remodel and support pregnancy.

Background & Aims Aberrant immune activation is associated with numerous inflammatory and autoimmune diseases and contributes to cancer development and progression. Within the stomach, inflammation drives a well-established sequence from gastritis to metaplasia, eventually resulting in adenocarcinoma. Unfortunately, the processes that regulate gastric inflammation and prevent carcinogenesis remain unknown. Tristetraprolin (TTP) is an RNA-binding protein that promotes the turnover of numerous proinflammatory and oncogenic messenger RNAs. Here, we assess the role of TTP in regulating gastric inflammation and spasmolytic polypeptide-expressing metaplasia (SPEM) development. Methods We used a TTP-overexpressing model, the TTPΔadenylate-uridylate rich element mouse, to examine whether TTP can protect the stomach from adrenalectomy (ADX)-induced gastric inflammation and SPEM. Results We found that TTPΔadenylate-uridylate rich element mice were completely protected from ADX-induced gastric inflammation and SPEM. RNA sequencing 5 days after ADX showed that TTP overexpression suppressed the expression of genes associated with the innate immune response. Importantly, TTP overexpression did not protect from high-dose-tamoxifen–induced SPEM development, suggesting that protection in the ADX model is achieved primarily by suppressing inflammation. Finally, we show that protection from gastric inflammation was only partially due to the suppression of Tnf, a well-known TTP target. Conclusions Our results show that TTP exerts broad anti-inflammatory effects in the stomach and suggest that therapies that increase TTP expression may be effective treatments of proneoplastic gastric inflammation. Transcript profiling: GSE164349.

Summary The loss of uterine epithelial Progesterone Receptor (PGR) is crucial for successful embryo implantation in both humans and mice. The two major isoforms PGRA and PGRB have divergent functions under both physiological and pathological conditions. The present study compares phenotypes and gene signatures of PGRA and PGRB in uterine epithelium using uterine epithelial-specific constitutively expressed PGRA or PGRB mouse models. The cistrome and transcriptome analysis reveals substantial overlap between epithelial PGRA and PGRB, and both disrupts embryo implantation through FOXO1 pathways. Constitutive epithelial PGRA and PGRB expression impairs ESR1 occupancy at the promoter of Lif leading to reduced Lif transcription, further exaggerates SGK1 expression leading to enhanced PI3K-SGK1 activities, and both contributes to the decline of nuclear FOXO1 expression. Our study demonstrates that PGRA and PGRB in the uterine epithelium act on a similar set of target genes and commonly regulate the LIF-SGK1-FOXO1 signaling pathway for embryo implantation.

MicroRNA (miRNA) abundance is tightly controlled by regulation of biogenesis and decay. Here we show that the mir-35 miRNA family undergoes selective decay at the transition from embryonic to larval development in C. elegans. The seed sequence of the miRNA is necessary and largely sufficient for this regulation. Sequences outside the seed (3’ end) regulate mir-35 abundance in the embryo but are not necessary for sharp decay at the transition to larval development. Enzymatic modifications of the miRNA 3’ end are neither prevalent nor correlated with changes in decay, suggesting that miRNA 3’ end display is not a core feature of this mechanism and further supporting a seed-driven decay model. Our findings demonstrate that seed sequence-specific decay can selectively and coherently regulate all redundant members of a miRNA seed family, a class of mechanism that has great biological and therapeutic potential for dynamic regulation of a miRNA family’s target repertoire.

Alternative polyadenylation (APA) is widespread among metazoans and has been shown to have important impacts on mRNA stability and protein expression. Beyond a handful of well-studied organisms, however, its existence and consequences have not been well investigated. We therefore turned to the deep-branching red alga

Phytophthora sojae is an oomycete that causes stem and root rot disease in soybean. P. sojae delivers many RxLR effector proteins, including Avr1b, into host cells to promote infection. We show here that Avr1b interacts with the soybean U-box protein, GmPUB1-1, in yeast two-hybrid, pull down, and bimolecular fluorescence complementation (BIFC) assays. GmPUB1-1, and a homeologous copy GmPUB1-2, are induced by infection and encode 403 amino acid proteins with U-Box domains at their N-termini. Non-synonymous mutations in the Avr1b C-terminus that abolish suppression of cell death also abolished the interaction of Avr1b with GmPUB1-1, while deletion of the GmPUB1-1 C-terminus, but not the U box, abolished the interaction. BIFC experiments suggested that the GmPUB1-1-Avr1b complex is targeted to the nucleus. In vitro ubiquitination assays demonstrated that GmPUB1-1 possesses E3 ligase activity. Silencing of the GmPUB1 genes in soybean cotyledons resulted in loss of recognition of Avr1b by gene products encoded by Rps1-b and Rps1-k. The recognition of Avr1k (which did not interact with GmPUB1-1) by Rps1-k plants was not, however, affected following GmPUB1-1 silencing. Furthermore, over-expression of GmPUB1-1 in particle bombardment experiments triggered cell death suggesting that GmPUB1 may be a positive regulator of effector-triggered immunity. In a yeast two-hybrid system, GmPUB1-1 also interacted with a number of other RxLR effectors including Avr1d, while Avr1b and Avr1d interacted with a number of other infection-induced GmPUB proteins, suggesting that the pathogen uses a multiplex of interactions of RxLR effectors with GmPUB proteins to modulate host immunity.

Uterine contractile dysfunction leads to pregnancy complications such as preterm birth and labor dystocia. In humans, it is hypothesized that progesterone receptor isoform PGR-B promotes a relaxed state of the myometrium, and PGR-A facilitates uterine contraction. This hypothesis was tested in vivo using transgenic mouse models that overexpress PGR-A or PGR-B in smooth muscle cells. Elevated PGR-B abundance results in a marked increase in gestational length compared to control mice (21.1 versus 19.1 d respectively, P < 0.05). In both ex vivo and in vivo experiments, PGR-B overexpression leads to prolonged labor, a significant decrease in uterine contractility, and a high incidence of labor dystocia. Conversely, PGR-A overexpression leads to an increase in uterine contractility without a change in gestational length. Uterine RNA sequencing at midpregnancy identified 1,174 isoform-specific downstream targets and 424 genes that are commonly regulated by both PGR isoforms. Gene signature analyses further reveal PGR-B for muscle relaxation and PGR-A being proinflammatory. Elevated PGR-B abundance reduces Oxtr and Trpc3 and increases Plcl2 expression, which manifests a genetic profile of compromised oxytocin signaling. Functionally, both endogenous PLCL2 and its paralog PLCL1 can attenuate uterine muscle cell contraction in a CRISPRa-based assay system. These findings provide in vivo support that PGR isoform levels determine distinct transcriptomic landscapes and pathways in myometrial function and labor, which may help further the understanding of abnormal uterine function in the clinical setting.

Uterine contractile dysfunction leads to pregnancy complications such as preterm birth and labor dystocia. In humans, it is hypothesized that progesterone receptor isoform PGR-B promotes a relaxed state of the myometrium, and PGR-A facilitates uterine contraction. This hypothesis was tested in vivo using transgenic mouse models that overexpress PGR-A or PGR-B in smooth muscle cells. Elevated PGR-B abundance results in a marked increase in gestational length compared to control mice (21.1 versus 19.1 d respectively, P < 0.05). In both ex vivo and in vivo experiments, PGR-B overexpression leads to prolonged labor, a significant decrease in uterine contractility, and a high incidence of labor dystocia. Conversely, PGR-A overexpression leads to an increase in uterine contractility without a change in gestational length. Uterine RNA sequencing at midpregnancy identified 1,174 isoform-specific downstream targets and 424 genes that are commonly regulated by both PGR isoforms. Gene signature analyses further reveal PGR-B for muscle relaxation and PGR-A being proinflammatory. Elevated PGR-B abundance reduces Oxtr and Trpc3 and increases Plcl2 expression, which manifests a genetic profile of compromised oxytocin signaling. Functionally, both endogenous PLCL2 and its paralog PLCL1 can attenuate uterine muscle cell contraction in a CRISPRa-based assay system. These findings provide in vivo support that PGR isoform levels determine distinct transcriptomic landscapes and pathways in myometrial function and labor, which may help further the understanding of abnormal uterine function in the clinical setting.

Cancer is caused primarily by genomic alterations resulting in deregulation of gene regulatory circuits in key growth, apoptosis, or DNA repair pathways. Multiple genes associated with the initiation and development of tumors are also regulated at the level of mRNA decay, through the recruitment of RNA-binding proteins to AU-rich elements (AREs) located in their 3'-untranslated regions. One of these ARE-binding proteins, tristetraprolin (TTP; encoded by Zfp36), is consistently dysregulated in many human malignancies. Herein, using regulated overexpression or conditional ablation in the context of cutaneous chemical carcinogenesis, we show that TTP represents a critical regulator of skin tumorigenesis. We provide evidence that TTP controlled both tumor-associated inflammation and key oncogenic pathways in neoplastic epidermal cells. We identify Areg as a direct target of TTP in keratinocytes and show that EGFR signaling potentially contributed to exacerbated tumor formation. Finally, single-cell RNA-Seq analysis indicated that ZFP36 was downregulated in human malignant keratinocytes. We conclude that TTP expression by epidermal cells played a major role in the control of skin tumorigenesis., info:eu-repo/semantics/published

Aberrant immune activation is associated with numerous inflammatory and autoimmune diseases and contributes to cancer development and progression. Within the stomach, inflammation drives a well-established sequence from gastritis to metaplasia, eventually resulting in adenocarcinoma. Unfortunately, the processes that regulate gastric inflammation and prevent carcinogenesis remain unknown. Tristetraprolin (TTP) is an RNA-binding protein that promotes the turnover of numerous pro-inflammatory and oncogenic mRNAs. Here, we utilized a TTP-overexpressing model, the TTPΔARE mouse, to examine whether TTP can protect the stomach from adrenalectomy (ADX)-induced gastric inflammation and spasmolytic polypeptide-expressing metaplasia (SPEM). We found that TTPΔARE mice were completely protected from ADX-induced gastric inflammation and SPEM. RNA sequencing revealed that TTP overexpression suppressed the expression of genes associated with the innate immune response. Finally, we show that protection from gastric inflammation was only partially due to suppression ofTnf, a well-known TTP target. Our results demonstrate that TTP exerts broad anti-inflammatory effects in the stomach and suggest that therapies that increase TTP expression may be effective treatments of pro-neoplastic gastric inflammation.

Transcription factor FOXL2 exhibits an increase in mRNA levels in eutopic endometrial biopsy in endometriosis patients. While FOXL2 is known of regulating sex differentiation and reproductive function, the impact of elevated FOXL2 expression on uterine physiology remains unknown. To answer this question, we generated mice with over expression of FOXL2 (FOXL2OE) in the female reproductive tract by crossingFoxl2LsL/+with thePgrcremodel. FOXL2OEuterus showed severe morphological abnormality including abnormal epithelial stratification, blunted adenogenesis, increased endometrial fibrosis and disrupted myometrial morphology. In contrast, increasing FOXL2 levels specifically in uterine epithelium by crossing theFoxl2LsL/+with theLtficremice resulted in the eFOXL2OEmice with uterine epithelial stratification but without defects in endometrial fibrosis and adenogenesis, demonstrating a role of the endometrial stroma in the uterine abnormalities of the FOXL2OEmice. Transcriptomic analysis of 12 weeks oldPgrcreand FOXL2OEuterus at diestrus stage showed a positive correlation of FOXL2OEuterine transcriptome with human endometrium of endometriosis patients. Furthermore, we found FOXL2OEmice were sterile. The infertility was caused in part by a disruption of the hypophyseal ovarian axis resulting in an anovulatory phenotype. The FOXL2OEmice failed to show decidual responses during artificial decidualization in ovariectomized mice which demonstrates the uterine contribution to the infertility phenotype. These data supported that aberrantly increased FOXL2 expressions in the female reproductive tract can disrupt ovarian and uterine functions, particularly, may be involved in the progressions of endometriosis.

The myometrium undergoes structural and functional remodeling during pregnancy. We hypothesize that myometrial genomic elements alter correspondingly in preparation for parturition. Human myometrial tissues from nonpregnant (NP) and term pregnant (TP) human subjects were examined by RNAseq, ATACseq and PGR ChIPseq assays to profile transcriptome, assessible genome and PGR occupancy. NP and TP specimens exhibit 2890 differentially expressed genes, reflecting an increase of metabolic, inflammatory and PDGF signaling, among others, in adaptation to pregnancy. At the epigenome level, patterns of accessible genome change between NP and TP myometrium, leading to altered enrichment of binding motifs for hormone and muscle regulators such as the progesterone receptor (PGR), Krüppel-like factors and MEF2A transcription factors. PGR genome occupancy exhibits a significant difference between the two stages of the myometrium, concomitant with distinct transcriptomic profiles including genes such as ENO1, LHDA, and PLCL1 in the glycolytic and calcium signaling pathways. Over-representation of SRF, MYOD and STAT binding motifs in PGR occupying sites further suggests interactions between PGR and major muscle regulators for myometrial gene expression. In conclusion, changes in accessible genome and PGR occupancy are part of the myometrial remodeling process and may serve as mechanisms to formulate the state-specific transcriptome profiles.

Deleterious alleles are expected to be purged by purifying selection or maintained at low frequency. However, many additional evolutionary forces may shape the pattern of deleterious mutations across the genome and among populations, including selection, hitchhiking, recombination, and demographic history. We used exome capture data to estimate the genome-wide distribution of deleterious alleles across natural populations of the model tree black cottonwood (Populus trichocarpa). Although deleterious alleles were on average present at low frequency suggesting purifying selection, they were preferentially enriched both within genomic regions of low-recombination and in regions showing evidence of positive selection. The demographic history of this species also appeared to play a role in the distribution of deleterious alleles among populations, with peripheral populations having higher rates of deleterious homozygosity. This be due to less efficient selection arising from smaller effective population sizes at the edges of the range, and possibly also due to recent bottlenecks associated with postglacial recolonization. Finally, correlations between deleterious homozygosity and plant growth suggest a significant effect of deleterious load on fitness. Our results show that both genomic context and historical demography play a role in shaping the distribution of deleterious alleles across the genome and range of P. trichocarpa.

Few quantitative trait loci (QTL) have been mapped for the expression of partial resistance to in soybean and very little is known about the molecular mechanisms that contribute to this trait. Therefore, the objectives of this study were to identify additional QTL conferring resistance to and to identify candidate genes that may contribute to this form of defense. QTL on chromosomes 12, 13, 14, 17, and 19, each explaining 4 to 7% of the phenotypic variation, were identified using 186 RILs from a cross of the partially resistant cultivar ‘Conrad’ and susceptible cultivar ‘Sloan’ through composite interval mapping. Microarray analysis identified genes with significant differences in transcript abundances between Conrad and Sloan, both constitutively and following inoculation. Of these genes, 55 mapped to the five QTL regions. Ten genes encoded proteins with unknown functions, while the others encode proteins related to defense or physiological traits. Seventeen genes within the genomic region that encompass the QTL were selected and their transcript abundance was confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). These results suggest a complex QTL-mediated resistance network. This study will contribute to soybean resistance breeding by providing additional QTL for marker-assisted selection as well as a list of candidate genes which may be manipulated to confer resistance.

Adaptation to climate across latitude and altitude reflects shared climatic constraints, which may lead to parallel adaptation. However, theory predicts that higher gene flow should favor more concentrated genomic architectures, which would lead to fewer locally maladapted recombinants. We used exome capture to resequence the gene space along a latitudinal and two altitudinal transects in the model tree Populus trichocapra. Adaptive trait phenotyping was coupled with FST outlier tests and sliding window analysis to assess the degree of parallel adaptation as well as the genomic distribution of outlier loci. Up to 51% of outlier loci overlapped between transect pairs and up to 15% of these loci overlapped among all three transects. Genomic clustering of adaptive loci was more pronounced for altitudinal than latitudinal transects. In both altitudinal transects, there was a larger number of these 'islands of divergence', which were on average longer and included several of exceptional physical length. Our results suggest that recapitulation of genetic clines over latitude and altitude involves extensive parallelism, but that steep altitudinal clines generate islands of divergence. This suggests that physical proximity of genes in coadapted complexes may buffer against the movement of maladapted alleles from geographically proximal but climatically distinct populations.

Local adaptation to climate in temperate forest trees involves the integration of multiple physiological, morphological, and phenological traits. Latitudinal clines are frequently observed for these traits, but environmental constraints also track longitude and altitude. We combined extensive phenotyping of 12 candidate adaptive traits, multivariate regression trees, quantitative genetics, and a genome-wide panel of SNP markers to better understand the interplay among geography, climate, and adaptation to abiotic factors in Populus trichocarpa. Heritabilities were low to moderate (0.13 to 0.32) and population differentiation for many traits exceeded the 99th percentile of the genome-wide distribution of FST, suggesting local adaptation. When climate variables were taken as predictors and the 12 traits as response variables in a multivariate regression tree analysis, evapotranspiration (Eref) explained the most variation, with subsequent splits related to mean temperature of the warmest month, frost-free period (FFP), and mean annual precipitation (MAP). These grouping matched relatively well the splits using geographic variables as predictors: the northernmost groups (short FFP and low Eref) had the lowest growth, and lowest cold injury index; the southern British Columbia group (low Eref and intermediate temperatures) had average growth and cold injury index; the group from the coast of California and Oregon (high Eref and FFP) had the highest growth performance and the highest cold injury index; and the southernmost, high-altitude group (with high Eref and low FFP) performed poorly, had high cold injury index, and lower water use efficiency. Taken together, these results suggest variation in both temperature and water availability across the range shape multivariate adaptive traits in poplar.

Verticillium wilt of oilseed rape (Brassica napus) is caused primarily by Verticillium longisporum and has become a serious problem in northern Europe. In order to evaluate whether V. longisporum and V. dahliae differ in their interaction with oilseed rape, phenotypical and molecular assessments were made. Oilseed rape plants for fungal assessments were inoculated with V. longisporum and V. dahliae via root-dipping and samples were taken from roots, stems, leaves, flowers, pods and seeds during plant development. The infection by V. longisporum was found to start mainly in lateral roots and root-hairs, followed by colonization of the xylem vessels and extensive spread in stems and leaves, whereas V. dahliae infected the main roots and remained in the region below the cotyledon node of the plants. Re-isolation studies, together with PCR analysis of samples taken from early growth stages through to fully ripe plants, showed that the onset of flowering was a critical phase for V. longisporum to colonize plants. No seeds infected with V. longisporum were found. Mycelial growth from V. dahliae but not V. longisporum was significantly reduced on media containing tissue from a low glucosinolate B. napus genotype compared with growth on media containing tissue from a high glucosinolate cultivar. The results of this study suggest that V. longisporum favours B. napus as host and that the transition from the vegetative to the generative phase is of importance for the spread of the fungus in oilseed rape plants.

Molecular and genetic approaches were used to evaluate the genetic relatedness among isolates of the fungus Phoma macrostoma Montagne originating from Canada and Europe and to other species in the genus Phoma. Distinct differences were observed in genetic variation among nine species of the genus Phoma. Randomly amplified polymorphic DNA (RAPD) revealed the presence of intraspecific genetic variation among the isolates of P. macrostoma, with the isolates being used for biological weed control being distributed in a distinct phylogenetic cluster. Additional variation within the biocontrol isolate cluster in P. macrostoma was revealed by pulsed field gel electrophoresis (PFGE), which showed that biocontrol isolates generated two different chromosomal profiles, however the profiles did not relate to their Canadian ecozone origin. Mating studies showed that biocontrol isolates of P. macrostoma from Canada did not produce sexual reproductive structures and were incapable of crossing. These studies also confirmed that no obvious differentiation exists among the biocontrol isolates of P. macrostoma from Canadian Ecozones 3 and 4.

Several isolates of the fungus Phoma macrostoma demonstrated bioherbicidal activity against dandelion seedlings when applied to soil. Weed control ranged from 36 to 100% depending on the isolates and the doses applied. Using microbiological and molecular genetic techniques, the ability of these isolates to colonize target, and nontarget plants and to disperse and persist in soil were determined. PCR primers highly specific to the biocontrol isolates of P. macrostoma, were used to detect the isolates at rates of application between 4 and 1000 g/m2. Based on the results from representative isolates tested, it was concluded that P. macrostoma colonized root tissues of both resistant and susceptible crop species and a susceptible weed species grown in treated soil, and the frequency of fungal isolation declined with time. It was occasionally detected on untreated plant tissues, which may have resulted from either natural occurrences on seed, or contamination of soil. The biocontrol fungus appeared to have limited mobility in the soil since it was not often detected away from the area where it was placed. It persisted in the soil at detectable levels for up to 4 months, but then its presence declined with time. One year post application, P. macrostoma was either not present or significantly reduced in both soil and plant samples depending on the year of sampling. The results suggested that the isolates of P. macrostoma used for biological weed control would have minimal environmental impact due to its ubiquitous nature, limited mobility, and weak persistence over seasons.

Extant variation in temperate and boreal plant species has been influenced by both demographic histories associated with Pleistocene glacial cycles and adaptation to local climate. We used sequence capture to investigate the role of these neutral and adaptive processes in shaping diversity in black cottonwood (Populus trichocarpa). Nucleotide diversity and Tajima's D were lowest at replacement sites and highest at intergenic sites, while LD showed the opposite pattern. With samples grouped into three populations arrayed latitudinally, effective population size was highest in the north, followed by south and centre, and LD was highest in the south followed by the north and centre, suggesting a possible northern glacial refuge. FST outlier analysis revealed that promoter, 5′-UTR and intronic sites were enriched for outliers compared with coding regions, while no outliers were found among intergenic sites. Codon usage bias was evident, and genes with synonymous outliers had 30% higher average expression compared with genes containing replacement outliers. These results suggest divergent selection related to regulation of gene expression is important to local adaptation in P. trichocarpa. Finally, within-population selective sweeps were much more pronounced in the central population than in putative northern and southern refugia, which may reflect the different demographic histories of the populations and concomitant effects on signatures of genetic hitchhiking from standing variation.

Background High-throughput re-sequencing is rapidly becoming the method of choice for studies of neutral and adaptive processes in natural populations across taxa. As re-sequencing the genome of large numbers of samples is still cost-prohibitive in many cases, methods for genome complexity reduction have been developed in attempts to capture most ecologically-relevant genetic variation. One of these approaches is sequence capture, in which oligonucleotide baits specific to genomic regions of interest are synthesized and used to retrieve and sequence those regions. Results We used sequence capture to re-sequence most predicted exons, their upstream regulatory regions, as well as numerous random genomic intervals in a panel of 48 genotypes of the angiosperm tree Populus trichocarpa (black cottonwood, or ‘poplar’). A total of 20.76Mb (5%) of the poplar genome was targeted, corresponding to 173,040 baits. With 12 indexed samples run in each of four lanes on an Illumina HiSeq instrument (2x100 paired-end), 86.8% of the bait regions were on average sequenced at a depth ≥10X. Few off-target regions (>250bp away from any bait) were present in the data, but on average ~80bp on either side of the baits were captured and sequenced to an acceptable depth (≥10X) to call heterozygous SNPs. Nucleotide diversity estimates within and adjacent to protein-coding genes were similar to those previously reported in Populus spp., while intergenic regions had higher values consistent with a relaxation of selection. Conclusions Our results illustrate the efficiency and utility of sequence capture for re-sequencing highly heterozygous tree genomes, and suggest design considerations to optimize the use of baits in future studies.

Massive amounts of transcriptomic data documenting plant responses to changes in environment continue to accumulate in online databases. Unfortunately, many of these data sets have not been analyzed in full detail, especially those that involve time course experiments. To gain more knowledge of the successive gene expression events that occur when stress is initiated in one organ and then relayed to another, we have chosen stress response data for Arabidopsis shoots and roots from the detailed time course study of Killian et al. as a promising source to mine. Using refined statistical analysis, modified vector analysis, and a GO enrichment algorithm, more information was revealed concerning the effects of salt and UVB on gene expression events in shoots and roots over a 24-h time period. GeneMania, with in-house modifications, was used to further analyze abscisic acid (ABA) and jasmonic acid-related (JA) gene expression events in salt-stressed roots and shoots. JA effects appeared to be quite distinct in roots when compared to shoots, especially with respect to the expression of members of the negative regulatory JAZ gene family. In contrast, ABA-related gene expression events were more similar in the two organs. Instances of crosstalk between hormones were observed, as were early responses of regulatory genes involved in both auxin and cytokinin signaling. In the case of each hormone class examined, hormone biosynthesis genes were coexpressed with the genes encoding negative regulators of the corresponding signaling pathway. Hypotheses to explain this finding and future experiments to further explore these nonlinear phenomena are proposed.

Oomycete plant pathogens such as Phytophthora species and downy mildews cause destructive diseases in an enormous variety of crop plant species as well as forests and native ecosystems. These pathogens are most closely related to algae in the kingdom Stramenopiles, and hence have evolved plant pathogenicity independently of other plant pathogens such as fungi. We have used bioinformatic analysis of genome sequences and EST collections, together with functional genomics to identify plant and pathogen genes that may be key players in the interaction between the soybean pathogen Phytophthora sojae and its host. In P. sojae, we have identified many rapidly diversifying gene families that encode potential pathogenicity factors including protein toxins, and a class of proteins (avirulence or effector proteins) that appear to have the ability to penetrate plant cells. Transcriptomic analysis of quantitative or multigenic resistance against P. sojae in soybean has revealed that there are widespread adjustments in host gene expression in response to infection, and that some responses are unique to particular resistant cultivars. These observations lay the foundation for dissecting the interplay between pathogen and host genes during infection at a whole-genome level.

Six unique expressed sequence tag (EST) libraries were generated from four developmental stages of Phytophthora sojae P6497. RNA was extracted from mycelia, swimming zoospores, germinating cysts, and soybean (Glycine max (L.) Merr.) cv. Harosoy tissues heavily infected with P. sojae. Three libraries were created from mycelia growing on defined medium, complex medium, and nutrient-limited medium. The 26,943 high-quality sequences obtained clustered into 7,863 unigenes composed of 2,845 contigs and 5,018 singletons. The total number of P. sojae unigenes matching sequences in the genome assembly was 7,412 (94%). Of these unigenes, 7,088 (90%) matched gene models predicted from the P. sojae sequence assembly, but only 2,047 (26%) matched P. ramorum gene models. Analysis of EST frequency from different growth conditions and morphological stages revealed genes that were specific to or highly represented in particular growth conditions and life stages. Additionally, our results indicate that, during infection, the pathogen derives most of its carbon and energy via glycolysis of sugars in the plant. Sequences identified with putative roles in pathogenesis included avirulence homologs possessing the RxLR motif, elicitins, and hydrolytic enzymes. This large collection of P. sojae ESTs will serve as a valuable public genomic resource.

Background High throughput methods, such as high density oligonucleotide microarray measurements of mRNA levels, are popular and critical to genome scale analysis and systems biology. However understanding the results of these analyses and in particular understanding the very wide range of levels of transcriptional changes observed is still a significant challenge. Many researchers still use an arbitrary cut off such as two-fold in order to identify changes that may be biologically significant. We have used a very large-scale microarray experiment involving 72 biological replicates to analyze the response of soybean plants to infection by the pathogen Phytophthora sojae and to analyze transcriptional modulation as a result of genotypic variation. Results With the unprecedented level of statistical sensitivity provided by the high degree of replication, we show unambiguously that almost the entire plant genome (97 to 99% of all detectable genes) undergoes transcriptional modulation in response to infection and genetic variation. The majority of the transcriptional differences are less than two-fold in magnitude. We show that low amplitude modulation of gene expression (less than two-fold changes) is highly statistically significant and consistent across biological replicates, even for modulations of less than 20%. Our results are consistent through two different normalization methods and two different statistical analysis procedures. Conclusion Our findings demonstrate that the entire plant genome undergoes transcriptional modulation in response to infection and genetic variation. The pervasive low-magnitude remodeling of the transcriptome may be an integral component of physiological adaptation in soybean, and in all eukaryotes.