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author:("dwell, David")
1.  Differential gene expression and alternative splicing in insect immune specificity 
BMC Genomics  2014;15(1):1031.
Background
Ecological studies routinely show genotype-genotype interactions between insects and their parasites. The mechanisms behind these interactions are not clearly understood. Using the bumblebee Bombus terrestris/trypanosome Crithidia bombi model system (two bumblebee colonies by two Crithidia strains), we have carried out a transcriptome-wide analysis of gene expression and alternative splicing in bees during C. bombi infection. We have performed four analyses, 1) comparing gene expression in infected and non-infected bees 24 hours after infection by Crithidia bombi, 2) comparing expression at 24 and 48 hours after C. bombi infection, 3) determining the differential gene expression associated with the bumblebee-Crithidia genotype-genotype interaction at 24 hours after infection and 4) determining the alternative splicing associated with the bumblebee-Crithidia genotype-genotype interaction at 24 hours post infection.
Results
We found a large number of genes differentially regulated related to numerous canonical immune pathways. These genes include receptors, signaling pathways and effectors. We discovered a possible interaction between the peritrophic membrane and the insect immune system in defense against Crithidia. Most interestingly, we found differential expression and alternative splicing of immunoglobulin related genes (Dscam and Twitchin) are associated with the genotype-genotype interactions of the given bumblebee colony and Crithidia strain.
Conclusions
In this paper we have shown that the expression and alternative splicing of immune genes is associated with specific interactions between different host and parasite genotypes in this bumblebee/trypanosome model.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1031) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-1031
PMCID: PMC4302123  PMID: 25431190
Genotype-genotype; Peritrophic membrane; Social insects
2.  SIDECAR POLLEN suggests a plant-specific regulatory network underlying asymmetric microspore division in Arabidopsis 
Plant Signaling & Behavior  2011;6(3):416-419.
Asymmetric cell division is a universal strategy to generate diverse cell types necessary for patterning and proliferation of all eukaryotes. The development of haploid male gametophytes (pollen grains) in flowering plants is a remarkable example in which division asymmetry governs the functional specialization and germline differentiation essential for double fertilization. The male gametophyte is patterned via two mitotic divisions resulting in three highly differentiated daughter cells at maturity, a vegetative cell and two sperm cells. The first asymmetric division segregates a unique male germ cell from an undetermined haploid microspore and is executed in an elaborate sequence of cellular events. However the molecular mechanisms governing the division asymmetry in microspores are poorly understood. Recently we studied the phenotype of sidecar pollen (scp) mutants in detail, and demonstrated a requirement of SCP for both the correct timing and orientation of microspore division. SCP is a microspore-specific member of the LOB/AS2 domain family (LBD27/ASL29) showing that a plant-specific regulator plays a key role in oriented division of polarized microspores. Identification of SCP will serve as a new platform to further explore the largely unknown molecular networks regulating division asymmetry in microspores that establishes the male germline in flowering plants.
doi:10.4161/psb.6.3.14385
PMCID: PMC3142426  PMID: 21364317
sidecar pollen; microspore division; division asymmetry; male gametophyte development; male germline; LBD/ASL family protein
3.  A dynamic DUO of regulatory proteins coordinates gamete specification and germ cell mitosis in the angiosperm male germline 
Plant Signaling & Behavior  2009;4(12):1159-1162.
The production of two functional sperm cells within each male gametophyte is essential for double fertilization in flowering plants and involves a single mitotic division of the male germ cell and cell specification to produce functional gametes. Several proteins that are important regulators of male germ cell division have been identified as well as the R2R3 MYB protein DUO1 that has a dual role in cell division and cell specification. We recently identified a novel regulatory protein DUO3, that has overlapping roles with DUO1 in cell division and specification and shows similarity to GON4 related cell lineage regulators in animals. DUO3 also has important roles outside the germline and is required for embryo patterning and meristem function. We outline the regulatory roles of DUO3 in male germline development and its possible mechanisms of action as a lineage regulator in current models that link germ cell cycle control and gamete specification.
PMCID: PMC2819445  PMID: 20514235
DUO3; male germline development; cell cycle; cell specification; Arabidopsis; pollen; GON4-L
4.  MicroRNA and tasiRNA diversity in mature pollen of Arabidopsis thaliana 
BMC Genomics  2009;10:643.
Background
New generation sequencing technology has allowed investigation of the small RNA populations of flowering plants at great depth. However, little is known about small RNAs in their reproductive cells, especially in post-meiotic cells of the gametophyte generation. Pollen - the male gametophyte - is the specialised haploid structure that generates and delivers the sperm cells to the female gametes at fertilisation. Whether development and differentiation of the male gametophyte depends on the action of microRNAs and trans-acting siRNAs guiding changes in gene expression is largely unknown. Here we have used 454 sequencing to survey the various small RNA populations present in mature pollen of Arabidopsis thaliana.
Results
In this study we detected the presence of 33 different microRNA families in mature pollen and validated the expression levels of 17 selected miRNAs by Q-RT-PCR. The majority of the selected miRNAs showed pollen-enriched expression compared with leaves. Furthermore, we report for the first time the presence of trans-acting siRNAs in pollen. In addition to describing new patterns of expression for known small RNAs in each of these classes, we identified 7 putative novel microRNAs. One of these, ath-MIR2939, targets a pollen-specific F-box transcript and we demonstrate cleavage of its target mRNA in mature pollen.
Conclusions
Despite the apparent simplicity of the male gametophyte, comprising just two different cell types, pollen not only utilises many miRNAs and trans-acting siRNAs expressed in the somatic tissues but also expresses novel miRNAs.
doi:10.1186/1471-2164-10-643
PMCID: PMC2808329  PMID: 20042113
5.  The tobacco MAP215/Dis1-family protein TMBP200 is required for the functional organization of microtubule arrays during male germline establishment 
Journal of Experimental Botany  2009;61(4):969-981.
The haploid microspore division during pollen development in flowering plants is an intrinsically asymmetric division which establishes the male germline for sexual reproduction. Arabidopsis gem1 mutants lack the male germline as a result of disturbed microspore polarity, division asymmetry, and cytokinesis and represent loss-of-function mutants in MOR1/GEM1, a plant orthologue of the conserved MAP215/Dis1 microtubule associated protein (MAP) family. This provides genetic evidence for the role of MAP215/Dis1 in the organization of gametophytic microtubule arrays, but it has remained unknown how microtubule arrays are affected in gem1 mutant microspores. Here, novel male gametophytic microtubule-reporter Nicotiana tabacum plants were constructed, expressing a green fluorescent protein-α-TUBULIN fusion protein (GFP-TUA6) under the control of a microspore-specific promoter. These plants allow effective visualization of all major male gametophytic microtubule arrays and provide useful tools to study the regulation of microtubule arrays by MAPs and other effectors. Depletion of TMBP200, a tobacco homologue of MOR1/GEM1 in gametophytic microtubule-reporter plants using microspore-targeted RNA interference, induced defects in microspore polarity, division asymmetry and cytokinesis that were associated with striking defects in phragmoplast position, orientation, and structure. Our observations further reveal a requirement for TMBP200 in gametophytic spindle organization and a novel role in spindle position and orientation in polarized microspores. These results provide direct evidence for the function of MAP215/Dis1 family protein TMBP200 in the organization of microtubule arrays critical for male germline formation in plants.
doi:10.1093/jxb/erp367
PMCID: PMC2826647  PMID: 20022922
Asymmetric division; male germline; microspore; microtubule arrays; TMBP200 protein; tobacco pollen
6.  Imprinting of the Polycomb Group Gene MEDEA Serves as a Ploidy Sensor in Arabidopsis 
PLoS Genetics  2009;5(9):e1000663.
Balanced maternal and paternal genome contributions are a requirement for successful seed development. Unbalanced contributions often cause seed abortion, a phenomenon that has been termed “triploid block.” Misregulation of imprinted regulatory genes has been proposed to be the underlying cause for abnormalities in growth and structure of the endosperm in seeds with deviating parental contributions. We identified a mutant forming unreduced pollen that enabled us to investigate direct effects of unbalanced parental genome contributions on seed development and to reveal the underlying molecular mechanism of dosage sensitivity. We provide evidence that parent-of-origin–specific expression of the Polycomb group (PcG) gene MEDEA is causally responsible for seed developmental aberrations in Arabidopsis seeds with increased paternal genome contributions. We propose that imprinted expression of PcG genes is an evolutionary conserved mechanism to balance parental genome contributions in embryo nourishing tissues.
Author Summary
Crosses between plants of different ploidy often fail because seed development does not proceed normally and non-viable seeds are produced. It is assumed that abnormalities in growth and structure of the endosperm (the nutritional tissue of the seed) are the cause of triploid seed failure, consistent with the proposed role of the endosperm in reproductive isolation and angiosperm speciation. In many species, the ratio of maternal to paternal genomes in the endosperm is important for normal seed development, giving rise to the hypothesis that parent-of-origin–specific gene expression (imprinting) of regulatory genes in the endosperm is the underlying cause for developmental failure in seeds with deviating parental contributions. We tested this hypothesis using the jason mutant that forms unreduced male gametes and triploid seeds with increased paternal genome dosage. Based on the results of our study, we propose that imprinting of the FIS component MEDEA serves as a dosage sensor for increasing paternal genome contributions, establishing the molecular basis for dosage sensitivity. Our study provides strong evidence supporting the hypothesis that misbalanced expression of imprinted genes is the cause of seed development defects after interploidy crosses and demonstrates that MEDEA imprinting is a major origin of developmental defects caused by increased paternal genome contributions.
doi:10.1371/journal.pgen.1000663
PMCID: PMC2738949  PMID: 19779546
7.  A Plant Germline-Specific Integrator of Sperm Specification and Cell Cycle Progression 
PLoS Genetics  2009;5(3):e1000430.
The unique double fertilisation mechanism in flowering plants depends upon a pair of functional sperm cells. During male gametogenesis, each haploid microspore undergoes an asymmetric division to produce a large, non-germline vegetative cell and a single germ cell that divides once to produce the sperm cell pair. Despite the importance of sperm cells in plant reproduction, relatively little is known about the molecular mechanisms controlling germ cell proliferation and specification. Here, we investigate the role of the Arabidopsis male germline-specific Myb protein DUO POLLEN1, DUO1, as a positive regulator of male germline development. We show that DUO1 is required for correct male germ cell differentiation including the expression of key genes required for fertilisation. DUO1 is also necessary for male germ cell division, and we show that DUO1 is required for the germline expression of the G2/M regulator AtCycB1;1 and that AtCycB1:1 can partially rescue defective germ cell division in duo1. We further show that the male germline-restricted expression of DUO1 depends upon positive promoter elements and not upon a proposed repressor binding site. Thus, DUO1 is a key regulator in the production of functional sperm cells in flowering plants that has a novel integrative role linking gametic cell specification and cell cycle progression.
Author Summary
Flowering plants, unlike animals, require not one, but two sperm cells for successful fertilisation—one sperm cell to join with the egg cell to produce the embryo and the other to join with the central cell to produce the nutrient-rich endosperm tissue inside the seed. A mystery in this “double fertilization” process was how each single pollen grain could produce the pair of sperm cells needed for fertility and seed production. Here, we report the discovery of a dual role for DUO1, a regulatory gene required for plant sperm cell production. We show that the DUO1 gene is required to promote the division of sperm precursor cells, while at the same time promoting their differentiation into functional sperm cells. DUO1 is required for the expression of a key cell cycle regulator and for the expression of genes that are critical for gamete differentiation and fertilisation. This work provides the first molecular insight into the mechanisms through which cell cycle progression and gamete differentiation are coordinated in flowering plants. This knowledge will be helpful in understanding the mechanisms and evolution of gamete development in flowering plants and may be useful in the control of gene flow and crossing behaviour.
doi:10.1371/journal.pgen.1000430
PMCID: PMC2653642  PMID: 19300502
8.  MADS-complexes regulate transcriptome dynamics during pollen maturation 
Genome Biology  2007;8(11):R249.
Pollen transcript profiling of mutants defective in MADS-domain MIKC* protein complexes suggests they control a transcriptional network directing cellular differentiation during pollen maturation.
Background
Differentiation processes are responsible for the diversity and functional specialization of the cell types that compose an organism. The outcome of these processes can be studied at molecular, physiologic, and biochemical levels by comparing different cell types, but the complexity and dynamics of the regulatory processes that specify the differentiation are largely unexplored.
Results
Here we identified the pollen-specific MIKC* class of MADS-domain transcription factors as major regulators of transcriptome dynamics during male reproductive cell development in Arabidopsis thaliana. Pollen transcript profiling of mutants deficient in different MIKC* protein complexes revealed that they control a transcriptional switch that directs pollen maturation and that is essential for pollen competitive ability. We resolved the functional redundancy among the MIKC* proteins and uncovered part of the underlying network by identifying the non-MIKC* MADS-box genes AGL18 and AGL29 as downstream regulators of a subset of the MIKC* MADS-controlled genes.
Conclusion
Our results provide a first, unique, and compelling insight into the complexity of a transcription factor network that directs cellular differentiation during pollen maturation, a process that is essential for male reproductive fitness in flowering plants.
doi:10.1186/gb-2007-8-11-r249
PMCID: PMC2258202  PMID: 18034896
9.  Arabidopsis Gene Family Profiler (aGFP) – user-oriented transcriptomic database with easy-to-use graphic interface 
BMC Plant Biology  2007;7:39.
Background
Microarray technologies now belong to the standard functional genomics toolbox and have undergone massive development leading to increased genome coverage, accuracy and reliability. The number of experiments exploiting microarray technology has markedly increased in recent years. In parallel with the rapid accumulation of transcriptomic data, on-line analysis tools are being introduced to simplify their use. Global statistical data analysis methods contribute to the development of overall concepts about gene expression patterns and to query and compose working hypotheses. More recently, these applications are being supplemented with more specialized products offering visualization and specific data mining tools. We present a curated gene family-oriented gene expression database, Arabidopsis Gene Family Profiler (aGFP; ), which gives the user access to a large collection of normalised Affymetrix ATH1 microarray datasets. The database currently contains NASC Array and AtGenExpress transcriptomic datasets for various tissues at different developmental stages of wild type plants gathered from nearly 350 gene chips.
Results
The Arabidopsis GFP database has been designed as an easy-to-use tool for users needing an easily accessible resource for expression data of single genes, pre-defined gene families or custom gene sets, with the further possibility of keyword search. Arabidopsis Gene Family Profiler presents a user-friendly web interface using both graphic and text output. Data are stored at the MySQL server and individual queries are created in PHP script. The most distinguishable features of Arabidopsis Gene Family Profiler database are: 1) the presentation of normalized datasets (Affymetrix MAS algorithm and calculation of model-based gene-expression values based on the Perfect Match-only model); 2) the choice between two different normalization algorithms (Affymetrix MAS4 or MAS5 algorithms); 3) an intuitive interface; 4) an interactive "virtual plant" visualizing the spatial and developmental expression profiles of both gene families and individual genes.
Conclusion
Arabidopsis GFP gives users the possibility to analyze current Arabidopsis developmental transcriptomic data starting with simple global queries that can be expanded and further refined to visualize comparative and highly selective gene expression profiles.
doi:10.1186/1471-2229-7-39
PMCID: PMC1963329  PMID: 17645793
10.  gemini pollen 2, a male and female gametophytic cytokinesis defective mutation 
Sexual plant reproduction  2004;17(2):63-70.
Gametophytic cytokinesis is essential for the development and function of the male and female gametophytes. We have previously described the isolation and characterisation of the gemini pollen 1 (gem1) that acts gametophytically to disturb asymmetric division and cytokinesis at pollen mitosis I in Arabidopsis. Here we describe the genetic and cytological analysis of an independent gametophytic mutant, gem2, with similar characteristics to gem1, but which maps to a different genetic locus. gem2 shows reduced genetic transmission through both male and female gametes and leads to the production of divided or twin-celled pollen. Developmental analysis revealed that gem2 does not affect karyokinesis at pollen mitosis I, but leads to repositioning of the cell plate and partial or complete failure of cytokinesis, resulting in symmetrical divisions or binucleate pollen grains respectively. Symmetrical divisions lead to altered pollen cell fate with both sister cells displaying vegetative cell fate. Moreover, we demonstrate that the predominant female defect in gem2 is a lack of cellularization of the embryo sac during megagametogenesis. GEM2 therefore defines an independent genetic locus that is involved in the correct specification of both male and female gametophytic cytokinesis.
doi:10.1007/s00497-004-0216-x
PMCID: PMC1855439  PMID: 17464359
gemini pollen; Arabidopsis thaliana; pollen mitosis I; gametophytic cytokinesis; cellularization
11.  MOR1/GEM1 plays an essential role in the plant-specific cytokinetic phragmoplast 
Nature cell biology  2002;4(9):711-714.
MOR1 is a member of the MAP215 family of microtubule-associated proteins and is required to establish interphase cortical microtubule arrays in plant cells.1 Here we show that MOR1 binds microtubules in vivo, localising to both cortical microtubules and to areas of overlapping microtubules in the phragmoplast. We demonstrate an essential role for MOR1 in cytokinesis through genetic complementation of the cytokinesis defective gem1-1 mutation with MOR1. Phenotypic analysis of gem1-1 and a T-DNA insertion allele gem1-2 confirm that MOR1/GEM1 is essential for regular patterns of cytokinesis. Both mutations, gem1-1 and gem1-2, cause the truncation of MOR1/GEM1. Moreover, the C-terminal domain absent in both mutants binds microtubules in vitro. These data demonstrate that MOR1/GEM1 plays an essential role in the cytokinetic phragmoplast.
doi:10.1038/ncb844
PMCID: PMC1802096  PMID: 12198497
12.  Identification of microspore-active promoters that allow targeted manipulation of gene expression at early stages of microgametogenesis in Arabidopsis 
BMC Plant Biology  2006;6:31.
Background
The effective functional analysis of male gametophyte development requires new tools enabling the spatially and temporally controlled expression of both marker genes and modified genes of interest. In particular, promoters driving expression at earlier developmental stages including microspores are required.
Results
Transcriptomic datasets covering four progressive stages of male gametophyte development in Arabidopsis were used to select candidate genes showing early expression profiles that were male gametophyte-specific. Promoter-GUS reporter analysis of candidate genes identified three promoters (MSP1, MSP2, and MSP3) that are active in microspores and are otherwise specific to the male gametophyte and tapetum. The MSP1 and MSP2 promoters were used to successfully complement and restore the male transmission of the gametophytic two-in-one (tio) mutant that is cytokinesis-defective at first microspore division.
Conclusion
We demonstrate the effective application of MSP promoters as tools that can be used to elucidate gametophytic gene functions in microspores in a male-specific manner.
doi:10.1186/1471-2229-6-31
PMCID: PMC1769379  PMID: 17184530
13.  Generalization of DNA microarray dispersion properties: microarray equivalent of t-distribution 
Biology Direct  2006;1:27.
Background
DNA microarrays are a powerful technology that can provide a wealth of gene expression data for disease studies, drug development, and a wide scope of other investigations. Because of the large volume and inherent variability of DNA microarray data, many new statistical methods have been developed for evaluating the significance of the observed differences in gene expression. However, until now little attention has been given to the characterization of dispersion of DNA microarray data.
Results
Here we examine the expression data obtained from 682 Affymetrix GeneChips® with 22 different types and we demonstrate that the Gaussian (normal) frequency distribution is characteristic for the variability of gene expression values. However, typically 5 to 15% of the samples deviate from normality. Furthermore, it is shown that the frequency distributions of the difference of expression in subsets of ordered, consecutive pairs of genes (consecutive samples) in pair-wise comparisons of replicate experiments are also normal. We describe a consecutive sampling method, which is employed to calculate the characteristic function approximating standard deviation and show that the standard deviation derived from the consecutive samples is equivalent to the standard deviation obtained from individual genes. Finally, we determine the boundaries of probability intervals and demonstrate that the coefficients defining the intervals are independent of sample characteristics, variability of data, laboratory conditions and type of chips. These coefficients are very closely correlated with Student's t-distribution.
Conclusion
In this study we ascertained that the non-systematic variations possess Gaussian distribution, determined the probability intervals and demonstrated that the Kα coefficients defining these intervals are invariant; these coefficients offer a convenient universal measure of dispersion of data. The fact that the Kα distributions are so close to t-distribution and independent of conditions and type of arrays suggests that the quantitative data provided by Affymetrix technology give "true" representation of physical processes, involved in measurement of RNA abundance.
Reviewers
This article was reviewed by Yoav Gilad (nominated by Doron Lancet), Sach Mukherjee (nominated by Sandrine Dudoit) and Amir Niknejad and Shmuel Friedland (nominated by Neil Smalheiser).
doi:10.1186/1745-6150-1-27
PMCID: PMC1586001  PMID: 16959036
14.  Transcriptome analysis of haploid male gametophyte development in Arabidopsis 
Genome Biology  2004;5(11):R85.
A transcriptome analysis of male gametophyte development in Arabidopsis uncovers distinct temporal classes of gene expression and opens the door to detailed studies of the regulatory pathways involved.
Background
The haploid male gametophyte generation of flowering plants consists of two- or three-celled pollen grains. This functional specialization is thought to be a key factor in the evolutionary success of flowering plants. Moreover, pollen ontogeny is also an attractive model in which to dissect cellular networks that control cell growth, asymmetric cell division and cellular differentiation. Our objective, and an essential step towards the detailed understanding of these processes, was to comprehensively define the male haploid transcriptome throughout development.
Results
We have developed staged spore isolation procedures for Arabidopsis and used Affymetrix ATH1 genome arrays to identify a total of 13,977 male gametophyte-expressed mRNAs, 9.7% of which were male-gametophyte-specific. The transition from bicellular to tricellular pollen was accompanied by a decline in the number of diverse mRNA species and an increase in the proportion of male gametophyte-specific transcripts. Expression profiles of regulatory proteins and distinct clusters of coexpressed genes were identified that could correspond to components of gametophytic regulatory networks. Moreover, integration of transcriptome and experimental data revealed the early synthesis of translation factors and their requirement to support pollen tube growth.
Conclusions
The progression from proliferating microspores to terminally differentiated pollen is characterized by large-scale repression of early program genes and the activation of a unique late gene-expression program in maturing pollen. These data provide a quantum increase in knowledge concerning gametophytic transcription and lay the foundations for new genomic-led studies of the regulatory networks and cellular functions that operate to specify male gametophyte development.
doi:10.1186/gb-2004-5-11-r85
PMCID: PMC545776  PMID: 15535861

Results 1-14 (14)