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Walker, SS, Degen D, Nickbarg E, Carr D, Soriano A, Mandal M B, Painter RE, Sheth PR, Xiao L, Sher X et al..  2017.  Affinity selection-mass spectrometry identifies a novel antibacterial RNA polymerase inhibitor.. ACS Chemical Biology. 12:1346-1352. Abstract
The growing prevalence of drug-resistant Gram-negative bacteria is a significant global threat to human health. Rifampicin, an RNA polymerase-targeting agent, is an important part of the antibacterial armamentarium; however the emergence of resistance requires that it be used against only certain infections and usually in combination with another antibiotic. While rifampicin has significant clinical limitations, it does show that bacterial RNA polymerase can be an effective target for antibacterial intervention. To find new RNA polymerase inhibitors we initiated a screen of a collection of antibacterial bioactive molecules using affinity selection-mass spectrometry and purified Escherichia coli core RNA polymerase (subunits α, β, β', ω). Affinity selection screening identified a novel small molecule, MRL-436, that binds selectively to and inhibits RNA polymerase in vitro and inhibits RNA synthesis in the cell. Selection for resistance followed by whole genome sequencing identified a missense mutation in rpoC (β' subunit) and, separately, frameshift mutations in rpoZ (ω subunit) suggesting that MRL-436 targets RNA polymerase in the cell. In addition, cells lacking the rpoZ gene or purified RNA polymerase containing either a specific substitution in β' or lacking ω are selectively resistant to MRL-436. Molecular modeling indicates that the location of the substitution mutation in β' is closely juxtaposed with ω in a region of the complex thought to be important for transcription regulation during cellular response to amino acid starvation.
Wang, W, Kerstetter R, Michael TP..  2011.  Evolution of Genome Size in Duckweeds (Lemnaceae).. Journal of Botany.
Wang, W, Wu Y, Messing J.  2014.  RNA-Seq transcriptome analysis of Spirodela dormancy without reproduction. BMC Genomics. 15:60. AbstractWebsite
BACKGROUND: Higher plants exhibit a remarkable phenotypic plasticity to adapt to adverse environmental changes. The Greater Duckweed Spirodela, as an aquatic plant, presents exceptional tolerance to cold winters through its dormant structure of turions in place of seeds. Abundant starch in turions permits them to sink and escape the freezing surface of waters. Due to their clonal propagation, they are the fastest growing biomass on earth, providing yet an untapped source for industrial applications. RESULTS: We used next generation sequencing technology to examine the transcriptome of turion development triggered by exogenous ABA. A total of 208 genes showed more than a 4-fold increase compared with 154 down-regulated genes in developing turions. The analysis of up-regulated differential expressed genes in response to dormancy exposed an enriched interplay among various pathways: signal transduction, seed dehydration, carbohydrate and secondary metabolism, and senescence. On the other side, the genes responsible for rapid growth and biomass accumulation through DNA assembly, protein synthesis and carbon fixation are repressed. Noticeably, three members of late embryogenesis abundant protein family are exclusively expressed during turion formation. High expression level of key genes in starch synthesis are APS1, APL3 and GBSSI, which could artificially be reduced for re-directing carbon flow from photosynthesis to create a higher energy biomass. CONCLUSIONS: The identification and functional annotation of differentially expressed genes open a major step towards understanding the molecular network underlying vegetative frond dormancy. Moreover, genes have been identified that could be engineered in duckweeds for practical applications easing agricultural production of food crops.
Wang, W, Wu Y, Messing J.  2014.  RNA-Seq transcriptome analysis of Spirodela dormancy without reproduction. BMC Genomics. 15(60) Abstract
Higher plants exhibit a remarkable phenotypic plasticity to adapt to adverse environmental changes. The Greater Duckweed Spirodela, as an aquatic plant, presents exceptional tolerance to cold winters through its dormant structure of turions in place of seeds. Abundant starch in turions permits them to sink and escape the freezing surface of waters. Due to their clonal propagation, they are the fastest growing biomass on earth, providing yet an untapped source for industrial applications.
Wang, W, Wu Y, Messing J.  2012.  The mitochondrial genome of an aquatic plant, Spirodela polyrhiza. PloS one. 7:e46747. AbstractWebsite
BACKGROUND: Spirodela polyrhiza is a species of the order Alismatales, which represent the basal lineage of monocots with more ancestral features than the Poales. Its complete sequence of the mitochondrial (mt) genome could provide clues for the understanding of the evolution of mt genomes in plant. METHODS: Spirodela polyrhiza mt genome was sequenced from total genomic DNA without physical separation of chloroplast and nuclear DNA using the SOLiD platform. Using a genome copy number sensitive assembly algorithm, the mt genome was successfully assembled. Gap closure and accuracy was determined with PCR products sequenced with the dideoxy method. CONCLUSIONS: This is the most compact monocot mitochondrial genome with 228,493 bp. A total of 57 genes encode 35 known proteins, 3 ribosomal RNAs, and 19 tRNAs that recognize 15 amino acids. There are about 600 RNA editing sites predicted and three lineage specific protein-coding-gene losses. The mitochondrial genes, pseudogenes, and other hypothetical genes (ORFs) cover 71,783 bp (31.0%) of the genome. Imported plastid DNA accounts for an additional 9,295 bp (4.1%) of the mitochondrial DNA. Absence of transposable element sequences suggests that very few nuclear sequences have migrated into Spirodela mtDNA. Phylogenetic analysis of conserved protein-coding genes suggests that Spirodela shares the common ancestor with other monocots, but there is no obvious synteny between Spirodela and rice mtDNAs. After eliminating genes, introns, ORFs, and plastid-derived DNA, nearly four-fifths of the Spirodela mitochondrial genome is of unknown origin and function. Although it contains a similar chloroplast DNA content and range of RNA editing as other monocots, it is void of nuclear insertions, active gene loss, and comprises large regions of sequences of unknown origin in non-coding regions. Moreover, the lack of synteny with known mitochondrial genomic sequences shed new light on the early evolution of monocot mitochondrial genomes.
Wang, W, Zhang W, Wu Y, Maliga P, Messing J.  2015.  RNA Editing in Chloroplasts of Spirodela polyrhiza, an Aquatic Monocotelydonous Species.. PloS one. 10(10):e0140285. Abstract
RNA editing is the post-transcriptional conversion from C to U before translation, providing a unique feature in the regulation of gene expression. Here, we used a robust and efficient method based on RNA-seq from non-ribosomal total RNA to simultaneously measure chloroplast-gene expression and RNA editing efficiency in the Greater Duckweed, Spirodela polyrhiza, a species that provides a new reference for the phylogenetic studies of monocotyledonous plants. We identified 66 editing sites at the genome-wide level, with an average editing efficiency of 76%. We found that the expression levels of chloroplast genes were relatively constant, but 11 RNA editing sites show significant changes in editing efficiency, when fronds turn into turions. Thus, RNA editing efficiency contributes more to the yield of translatable transcripts than steady state mRNA levels. Comparison of RNA editing sites in coconut, Spirodela, maize, and rice suggests that RNA editing originated from a common ancestor.
Wang, W, Wu Y, Messing J.  2012.  The Mitochondrial Genome of an Aquatic Plant, Spirodela polyrhiza.. PloS one. 7(10):e46747. AbstractWebsite
Spirodela polyrhiza is a species of the order Alismatales, which represent the basal lineage of monocots with more ancestral features than the Poales. Its complete sequence of the mitochondrial (mt) genome could provide clues for the understanding of the evolution of mt genomes in plant.
Wang, W, Haberer G, Gundlach H, Glasser C, Nussbaumer T, Luo MC, Lomsadze A, Borodovsky M, Kerstetter RA, Shanklin J et al..  2014.  The Spirodela polyrhiza genome reveals insights into its neotenous reduction fast growth and aquatic lifestyle. Nat Commun. 5:3311. AbstractWebsite
The subfamily of the Lemnoideae belongs to a different order than other monocotyledonous species that have been sequenced and comprises aquatic plants that grow rapidly on the water surface. Here we select Spirodela polyrhiza for whole-genome sequencing. We show that Spirodela has a genome with no signs of recent retrotranspositions but signatures of two ancient whole-genome duplications, possibly 95 million years ago (mya), older than those in Arabidopsis and rice. Its genome has only 19,623 predicted protein-coding genes, which is 28% less than the dicotyledonous Arabidopsis thaliana and 50% less than monocotyledonous rice. We propose that at least in part, the neotenous reduction of these aquatic plants is based on readjusted copy numbers of promoters and repressors of the juvenile-to-adult transition. The Spirodela genome, along with its unique biology and physiology, will stimulate new insights into environmental adaptation, ecology, evolution and plant development, and will be instrumental for future bioenergy applications.
Wang, Q., Dooner HK.  2012.  Dynamic evolution of bz orthologous regions in the Andropogoneae and other grasses.. The Plant journal : for cell and molecular biology. 72(2):212-21. Abstract
Genome structure exhibits remarkable plasticity within Zea mays. To examine how haplotype structure has evolved within the Andropogoneae tribe, we have analyzed the bz gene-rich region of maize (Zea mays), the Zea teosintes mays ssp. mexicana, luxurians and diploperennis, Tripsacum dactyloides, Coix lacryma-jobi and Sorghum propinquum. We sequenced and annotated BAC clones from these species and re-annotated the orthologous Sorghum bicolor region. Gene colinearity in the region is well conserved within the genus Zea. However, the orthologous regions of Coix and Sorghum exhibited several micro-rearrangements relative to Zea, including addition, truncation and deletion of genes. The stc1 gene, involved in the production of a terpenoid insect defense signal, is evolving particularly fast, and its progressive disappearance from some species is occurring by microhomology-mediated recombination. LTR retrotransposons are the main contributors to the dynamic evolution of the bz region. Common transposon insertion sites occur among haplotypes from different Zea mays sub-species, but not outside the species. As in Zea, different patterns of interspersion between genes and retrotransposons are observed in Sorghum. We estimate that the mean divergence times between maize and Tripsacum, Coix and Sorghum are 8.5, 12.1 and 12.4 million years ago, respectively, and that between Coix and Sorghum is 9.3 million years ago. A comparison of the bz orthologous regions of Zea, Sorghum and Coix with those of Brachypodium, Setaria and Oryza allows us to infer how the region has evolved by addition and deletion of genes in the approximately 50 million years since these genera diverged from a common progenitor.
Wang, W, Wu Y, Yan Y, Ermakova M, Kerstetter R, Messing J.  2010.  DNA barcoding of the Lemnaceae, a family of aquatic monocots. BMC Plant Biol. 10:205. AbstractWebsite
BACKGROUND: Members of the aquatic monocot family Lemnaceae (commonly called duckweeds) represent the smallest and fastest growing flowering plants. Their highly reduced morphology and infrequent flowering result in a dearth of characters for distinguishing between the nearly 38 species that exhibit these tiny, closely-related and often morphologically similar features within the same family of plants. RESULTS: We developed a simple and rapid DNA-based molecular identification system for the Lemnaceae based on sequence polymorphisms. We compared the barcoding potential of the seven plastid-markers proposed by the CBOL (Consortium for the Barcode of Life) plant-working group to discriminate species within the land plants in 97 accessions representing 31 species from the family of Lemnaceae. A Lemnaceae-specific set of PCR and sequencing primers were designed for four plastid coding genes (rpoB, rpoC1, rbcL and matK) and three noncoding spacers (atpF-atpH, psbK-psbI and trnH-psbA) based on the Lemna minor chloroplast genome sequence. We assessed the ease of amplification and sequencing for these markers, examined the extent of the barcoding gap between intra- and inter-specific variation by pairwise distances, evaluated successful identifications based on direct sequence comparison of the "best close match" and the construction of a phylogenetic tree. CONCLUSIONS: Based on its reliable amplification, straightforward sequence alignment, and rates of DNA variation between species and within species, we propose that the atpF-atpH noncoding spacer could serve as a universal DNA barcoding marker for species-level identification of duckweeds.
Wang, W, Messing J.  2011.  High-throughput sequencing of three Lemnoideae (duckweeds) chloroplast genomes from total DNA. PLoS One. 6:e24670. AbstractWebsite
BACKGROUND: Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth. METHODS: We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs) using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform. CONCLUSIONS: This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power.
Wang, C, Shi X, Liu L, Li H, Ammiraju JS, Kudrna DA, Xiong W, Wang H, Dai Z, Zheng Y et al..  2013.  Genomic resources for gene discovery, functional genome annotation, and evolutionary studies of maize and its close relatives. Genetics. 195:723-37. AbstractWebsite
Maize is one of the most important food crops and a key model for genetics and developmental biology. A genetically anchored and high-quality draft genome sequence of maize inbred B73 has been obtained to serve as a reference sequence. To facilitate evolutionary studies in maize and its close relatives, much like the Oryza Map Alignment Project (OMAP) (www.OMAP.org) bacterial artificial chromosome (BAC) resource did for the rice community, we constructed BAC libraries for maize inbred lines Zheng58, Chang7-2, and Mo17 and maize wild relatives Zea mays ssp. parviglumis and Tripsacum dactyloides. Furthermore, to extend functional genomic studies to maize and sorghum, we also constructed binary BAC (BIBAC) libraries for the maize inbred B73 and the sorghum landrace Nengsi-1. The BAC/BIBAC vectors facilitate transfer of large intact DNA inserts from BAC clones to the BIBAC vector and functional complementation of large DNA fragments. These seven Zea Map Alignment Project (ZMAP) BAC/BIBAC libraries have average insert sizes ranging from 92 to 148 kb, organellar DNA from 0.17 to 2.3%, empty vector rates between 0.35 and 5.56%, and genome equivalents of 4.7- to 8.4-fold. The usefulness of the Parviglumis and Tripsacum BAC libraries was demonstrated by mapping clones to the reference genome. Novel genes and alleles present in these ZMAP libraries can now be used for functional complementation studies and positional or homology-based cloning of genes for translational genomics.
Wang, J, Barr MM.  2005.  RNA Interference in Caenorhabditis Elegans. Methods Enzymol. 392:36-55. Abstract
RNA interference (RNAi) was first discovered in the nematode Caenorhabditis elegans (Fire et al., 1998; Guo and Kemphues, 1995). The completion of the C. elegans genome in 1998 coupled with the advent of RNAi techniques to knock down gene function ushered in a new age in the field of functional genomics. There are four methods for double-stranded RNA (dsRNA) delivery in C. elegans: (1) injection of dsRNA into any region of the animal (Fire et al., 1998), (2) feeding with bacteria producing dsRNA (Timmons et al., 2001), (3) soaking in dsRNA (Tabara et al., 1998), and (4) in vivo production of dsRNA from transgenic promoters (Tavernarakis et al., 2000). In this chapter, we discuss the molecular genetic mechanisms, techniques, and applications of RNAi in C. elegans.
Wang, W, Haberer G, Gundlach H, Gläßer C, Nussbaumer T, Luo MC, Lomsadze A, Borodovsky M, Kerstetter RA, Shanklin J et al..  2014.  The Spirodela polyrhiza genome reveals insights into its neotenous reduction fast growth and aquatic lifestyle. 5 AbstractWebsite
The subfamily of the Lemnoideae belongs to a different order than other monocotyledonous species that have been sequenced and comprises aquatic plants that grow rapidly on the water surface. Here we select Spirodela polyrhiza for whole-genome sequencing. We show that Spirodela has a genome with no signs of recent retrotranspositions but signatures of two ancient whole-genome duplications, possibly 95 million years ago (mya), older than those in Arabidopsis and rice. Its genome has only 19,623 predicted protein-coding genes, which is 28% less than the dicotyledonous Arabidopsis thaliana and 50% less than monocotyledonous rice. We propose that at least in part, the neotenous reduction of these aquatic plants is based on readjusted copy numbers of promoters and repressors of the juvenile-to-adult transition. The Spirodela genome, along with its unique biology and physiology, will stimulate new insights into environmental adaptation, ecology, evolution and plant development, and will be instrumental for future bioenergy applications.
Wang, W, Zhang W, Wu Y, Maliga P, Messing J.  2015.  RNA Editing in Chloroplasts of Spirodela polyrhiza, an Aquatic Monocotelydonous Species. PLoS One. 10:e0140285. AbstractWebsite
RNA editing is the post-transcriptional conversion from C to U before translation, providing a unique feature in the regulation of gene expression. Here, we used a robust and efficient method based on RNA-seq from non-ribosomal total RNA to simultaneously measure chloroplast-gene expression and RNA editing efficiency in the Greater Duckweed, Spirodela polyrhiza, a species that provides a new reference for the phylogenetic studies of monocotyledonous plants. We identified 66 editing sites at the genome-wide level, with an average editing efficiency of 76%. We found that the expression levels of chloroplast genes were relatively constant, but 11 RNA editing sites show significant changes in editing efficiency, when fronds turn into turions. Thus, RNA editing efficiency contributes more to the yield of translatable transcripts than steady state mRNA levels. Comparison of RNA editing sites in coconut, Spirodela, maize, and rice suggests that RNA editing originated from a common ancestor.
Wang, W, Wu Y, Messing J.  2012.  The mitochondrial genome of an aquatic plant, Spirodela polyrhiza. PLoS ONE. 7(10) Abstract
Spirodela polyrhiza is a species of the order Alismatales, which represent the basal lineage of monocots with more ancestral features than the Poales. Its complete sequence of the mitochondrial (mt) genome could provide clues for the understanding of the evolution of mt genomes in plant.
Wang, W, Messing J.  2015.  Status of duckweed genomics and transcriptomics. Plant Biol (Stuttg). 17 Suppl 1:10-5. AbstractWebsite
Duckweeds belong to the smallest flowering plants that undergo fast vegetative growth in an aquatic environment. They are commonly used in wastewater treatment and animal feed. Whereas duckweeds have been studied at the biochemical level, their reduced morphology and wide environmental adaption had not been subjected to molecular analysis until recently. Here, we review the progress that has been made in using a DNA barcode system and the sequences of chloroplast and mitochondrial genomes to identify duckweed species at the species or population level. We also review analysis of the nuclear genome sequence of Spirodela that provides new insights into fundamental biological questions. Indeed, reduced gene families and missing genes are consistent with its compact morphogenesis, aquatic floating and suppression of juvenile-to-adult transition. Furthermore, deep RNA sequencing of Spirodela at the onset of dormancy and Landoltia in exposure of nutrient deficiency illustrate the molecular network for environmental adaption and stress response, constituting major progress towards a post-genome sequencing phase, where further functional genomic details can be explored. Rapid advances in sequencing technologies could continue to promote a proliferation of genome sequences for additional ecotypes as well as for other duckweed species.
Wang, W, Wu Y, Ermakova M, Kerstetter R, Messing J.  2010.  DNA barcoding of the Lemnaceae, a family of aquatic monocots. BMC Plant Biology. 10:205.
Wang, W, Messing J.  2012.  Analysis of ADP-glucose pyrophosphorylase expression during turion formation induced by abscisic acid in Spirodela polyrhiza (greater duckweed). BMC Plant Biol. 12:5. AbstractWebsite
BACKGROUND: Aquatic plants differ in their development from terrestrial plants in their morphology and physiology, but little is known about the molecular basis of the major phases of their life cycle. Interestingly, in place of seeds of terrestrial plants their dormant phase is represented by turions, which circumvents sexual reproduction. However, like seeds turions provide energy storage for starting the next growing season. RESULTS: To begin a characterization of the transition from the growth to the dormant phase we used abscisic acid (ABA), a plant hormone, to induce controlled turion formation in Spirodela polyrhiza and investigated their differentiation from fronds, representing their growth phase, into turions with respect to morphological, ultra-structural characteristics, and starch content. Turions were rich in anthocyanin pigmentation and had a density that submerged them to the bottom of liquid medium. Transmission electron microscopy (TEM) of turions showed in comparison to fronds shrunken vacuoles, smaller intercellular space, and abundant starch granules surrounded by thylakoid membranes. Turions accumulated more than 60% starch in dry mass after two weeks of ABA treatment. To further understand the mechanism of the developmental switch from fronds to turions, we cloned and sequenced the genes of three large-subunit ADP-glucose pyrophosphorylases (APLs). All three putative protein and exon sequences were conserved, but the corresponding genomic sequences were extremely variable mainly due to the invasion of miniature inverted-repeat transposable elements (MITEs) into introns. A molecular three-dimensional model of the SpAPLs was consistent with their regulatory mechanism in the interaction with the substrate (ATP) and allosteric activator (3-PGA) to permit conformational changes of its structure. Gene expression analysis revealed that each gene was associated with distinct temporal expression during turion formation. APL2 and APL3 were highly expressed in earlier stages of turion development, while APL1 expression was reduced throughout turion development. CONCLUSIONS: These results suggest that the differential expression of APLs could be used to enhance energy flow from photosynthesis to storage of carbon in aquatic plants, making duckweeds a useful alternative biofuel feedstock.
Wang, Q, Dooner HK.  2006.  Remarkable variation in maize genome structure inferred from haplotype diversity at the bz locus. Proc. Natl. Acad. Sci. U.S.A.. 103:17644–17649. Abstract
Maize is probably the most diverse of all crop species. Unexpectedly large differences among haplotypes were first revealed in a comparison of the bz genomic regions of two different inbred lines, McC and B73. Retrotransposon clusters, which comprise most of the repetitive DNA in maize, varied markedly in makeup, and location relative to the genes in the region and genic sequences, later shown to be carried by two helitron transposons, also differed between the inbreds. Thus, the allelic bz regions of these Corn Belt inbreds shared only a minority of the total sequence. To investigate further the variation caused by retrotransposons, helitrons, and other insertions, we have analyzed the organization of the bz genomic region in five additional cultivars selected because of their geographic and genetic diversity: the inbreds A188, CML258, and I137TN, and the land races Coroico and NalTel. This vertical comparison has revealed the existence of several new helitrons, new retrotransposons, members of every superfamily of DNA transposons, numerous miniature elements, and novel insertions flanked at either end by TA repeats, which we call TAFTs (TA-flanked transposons). The extent of variation in the region is remarkable. In pairwise comparisons of eight bz haplotypes, the percentage of shared sequences ranges from 25% to 84%. Chimeric haplotypes were identified that combine retrotransposon clusters found in different haplotypes. We propose that recombination in the common gene space greatly amplifies the variability produced by the retrotransposition explosion in the maize ancestry, creating the heterogeneity in genome organization found in modern maize.
Wang, W, Messing J.  2012.  Analysis of ADP-glucose pyrophosphorylase expression during turion formation induced by abscisic acid in Spirodela polyrhiza (greater duckweed). BMC Plant Biology. 12(5) Abstract
Aquatic plants differ in their development from terrestrial plants in their morphology and physiology, but little is known about the molecular basis of the major phases of their life cycle. Interestingly, in place of seeds of terrestrial plants their dormant phase is represented by turions, which circumvents sexual reproduction. However, like seeds turions provide energy storage for starting the next growing season.
Wang, W, Wu Y, Messing J.  2016.  Genome-wide analysis of pentatricopeptide-repeat proteins of an aquatic plant.. Planta. Abstract
A large proportion of genes in plant genomes are organized as gene families. Whereas most gene families in the aquative plant Spirodela are reduced in their copy number, the PPR gene family is expanded, which match the RNA editing sites in organelles, providing us with new insights in the evolution of flowering plants. Pentatricopeptide-repeat proteins (PPRs) are nuclear-encoded proteins that are targeted to mitochondria and plastids to stabilize and edit mRNA transcribed from organellar genomes. They have been described for many terrestrial plant species from a diverse spectrum of sequenced genomes. To further increase our understanding of the evolution of this gene family across angiosperms, we analyzed the PPR genes in the aquatic species Spirodela polyrhiza in the order of the Alismatales (monocotyledonous plants). Because we had generated next generation sequencing data from transcripts and had sequenced the genome of Spirodela polyrhiza, we were able to identify its PPR genes and determine the level of their expression. In total, we could identify 556 PPR proteins, of which 238 members belong to the P (P motif) subfamily that is mainly involved in RNA stabilization and 318 ones to the PLS (P, Longer P, shorter P motif) subfamily responsible for RNA editing. Compared to other angiosperms, this is a large increase in the copy number of the PLS-PPRs subfamily and the expansion correlates with the increase of the number of RNA editing sites of organellar transcripts. Expression of PPR was generally stable even during growing and dormant stages, indicating that their function was critical throughout development. However, PPRs, especially those of the PLS subfamily, were expressed at relatively low levels, suggesting a delicate fine-tuning of its trans-acting function in the post-transcriptional regulation of gene expression. Thus, understanding PPR evolution and expression will help decipher the PPR code for their binding sites, which could genetically engineer RNA-binding proteins toward desired sequence.
Wei, F, Coe E, Nelson W, Bharti AK, Engler F, Butler E, Kim H, Goicoechea JL, Chen M, Lee S et al..  2007.  Physical and Genetic Structure of the Maize Genome Reflects Its Complex Evolutionary History. PLoS Genet. 3:e123. AbstractWebsite
Maize (Zea mays L.) is one of the most important cereal crops and a model for the study of genetics, evolution, and domestication. To better understand maize genome organization and to build a framework for genome sequencing, we constructed a sequence-ready fingerprinted contig-based physical map that covers 93.5% of the genome, of which 86.1% is aligned to the genetic map. The fingerprinted contig map contains 25,908 genic markers that enabled us to align nearly 73% of the anchored maize genome to the rice genome. The distribution pattern of expressed sequence tags correlates to that of recombination. In collinear regions, 1 kb in rice corresponds to an average of 3.2 kb in maize, yet maize has a 6-fold genome size expansion. This can be explained by the fact that most rice regions correspond to two regions in maize as a result of its recent polyploid origin. Inversions account for the majority of chromosome structural variations during subsequent maize diploidization. We also find clear evidence of ancient genome duplication predating the divergence of the progenitors of maize and rice. Reconstructing the paleoethnobotany of the maize genome indicates that the progenitors of modern maize contained ten chromosomes.
Weiss, LA, Harrison PG, Nickels BE, Glickman MS, Campbell EA, Darst SA, Stallings CL.  2012.  Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis. J Bacteriol. 194:5621-5631. AbstractWebsite
Mycobacterium tuberculosis infection continues to cause substantial human suffering. New chemotherapeutic strategies, which require insight into the pathways essential for M. tuberculosis pathogenesis, are imperative. We previously reported that depletion of the CarD protein in mycobacteria compromises viability, resistance to oxidative stress and fluoroquinolones, and pathogenesis. CarD associates with the RNA polymerase (RNAP), but it has been unknown which of the diverse functions of CarD are mediated through the RNAP; this question must be answered to understand the CarD mechanism of action. Herein, we describe the interaction between the M. tuberculosis CarD and the RNAP beta subunit and identify point mutations that weaken this interaction. The characterization of mycobacterial strains with attenuated CarD/RNAP beta interactions demonstrates that the CarD/RNAP beta association is required for viability and resistance to oxidative stress but not for fluoroquinolone resistance. Weakening the CarD/RNAP beta interaction also increases the sensitivity of mycobacteria to rifampin and streptomycin. Surprisingly, depletion of the CarD protein did not affect sensitivity to rifampin. These findings define the CarD/RNAP interaction as a new target for chemotherapeutic intervention that could also improve the efficacy of rifampin treatment of tuberculosis. In addition, our data demonstrate that weakening the CarD/RNAP beta interaction does not completely phenocopy the depletion of CarD and support the existence of functions for CarD independent of direct RNAP binding.