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July 7, 2019

ALUMINUM RESISTANCE TRANSCRIPTION FACTOR 1 (ART1) contributes to natural variation in aluminum resistance in diverse genetic backgrounds of rice (O. sativa)

Abstract Transcription factors (TFs) regulate the expression of other genes to indirectly mediate stress resistance mechanisms. Therefore, when studying TF-mediated stress resistance, it is important to understand how TFs interact with genes in the genetic background. Here, we fine-mapped the aluminum (Al) resistance QTL Alt12.1 to a 44-kb region containing six genes. Among them is ART1, which encodes a C2H2-type zinc finger TF required for Al resistance in rice. The mapping parents, Al-resistant cv Azucena (tropical japonica) and Al-sensitive cv IR64 (indica), have extensive sequence polymorphism within the ART1 coding region, but similar ART1 expression levels. Using reciprocal near-isogenic lines (NILs) we examined how allele-swapping the Alt12.1 locus would affect plant responses to Al. Analysis of global transcriptional responses to Al stress in roots of the NILs alongside their recurrent parents demonstrated that the presence of the Alt12.1 from Al-resistant Azucena led to greater changes in gene expression in response to Al when compared to the Alt12.1 from IR64 in both genetic backgrounds. The presence of the ART1 allele from the opposite parent affected the expression of several genes not previously implicated in rice Al tolerance. We highlight examples where putatively functional variation in cis-regulatory regions of ART1-regulated genes interacts with ART1 to determine gene expression in response to Al. This ART1–promoter interaction may be associated with transgressive variation for Al resistance in the Azucena × IR64 population. These results illustrate how ART1 interacts with the genetic background to contribute to quantitative phenotypic variation in rice Al resistance.

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July 7, 2019

Generation of a collection of mutant tomato lines using pooled CRISPR libraries.

The high efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mutagenesis in plants enables the development of high-throughput mutagenesis strategies. By transforming pooled CRISPR libraries into tomato (Solanum lycopersicum), collections of mutant lines were generated with minimal transformation attempts and in a relatively short period of time. Identification of the targeted gene(s) was easily determined by sequencing the incorporated guide RNA(s) in the primary transgenic events. From a single transformation with a CRISPR library targeting the immunity-associated leucine-rich repeat subfamily XII genes, heritable mutations were recovered in 15 of the 54 genes targeted. To increase throughput, a second CRISPR library was made containing three guide RNAs per construct to target 18 putative transporter genes. This resulted in stable mutations in 15 of the 18 targeted genes, with some primary transgenic plants having as many as five mutated genes. Furthermore, the redundancy in this collection of plants allowed for the association of aberrant T0 phenotypes with the underlying targeted genes. Plants with mutations in a homolog of an Arabidopsis (Arabidopsis thaliana) boron efflux transporter displayed boron deficiency phenotypes. The strategy described here provides a technically simple yet high-throughput approach for generating a collection of lines with targeted mutations and should be applicable to any plant transformation system.© 2017 American Society of Plant Biologists. All Rights Reserved.

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July 7, 2019

Sequencing the genomic regions flanking S-linked PvGLO sequences confirms the presence of two GLO loci, one of which lies adjacent to the style-length determinant gene CYP734A50.

Primula vulgaris contains two GLOBOSA loci, one located adjacent to the style length determinant gene CYP734A50 which lies within the S -locus. Using a combination of BAC walking and PacBio sequencing, we have sequenced two substantial genomic contigs in and around the S-locus of Primula vulgaris. Using these data, we were able to demonstrate that two alleles of PvGlo (P) as well as PvGlo (T) can be present in the genome of a single plant, providing empirical evidence that these two forms of the MADS-box gene GLOBOSA are separate loci and not allelic as previously reported. We propose they should be renamed PvGLO1 and PvGLO2. BAC contigs extending from each GLOBOSA locus were identified and fully sequenced. No homologous genes were found between the contigs other than the GLOBOSA genes themselves, consistent with their identity as separate loci. Exons of the recently identified style-length determinant gene CYP734A50 were identified on one end of the contig containing PvGLO2 and these genes are adjacent in the genome, suggesting that PvGLO2 lies either within or at least very close to the S-locus. Current evidence suggests that both CYP734A50 and GLO2 are specific to the S-morph mating type and are hemizygous rather than heterozygous in the Primula genome. This finding contrasts classical models of the HSI locus, which propose that components of the S-locus are allelic, suggesting that these models may need to be reconsidered.

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July 7, 2019

A small secreted protein in Zymoseptoria tritici is responsible for avirulence on wheat cultivars carrying the Stb6 resistance gene.

Zymoseptoria tritici is the causal agent of Septoria tritici blotch, a major pathogen of wheat globally and the most damaging pathogen of wheat in Europe. A gene-for-gene (GFG) interaction between Z. tritici and wheat cultivars carrying the Stb6 resistance gene has been postulated for many years, but the genes have not been identified. We identified AvrStb6 by combining quantitative trait locus mapping in a cross between two Swiss strains with a genome-wide association study using a natural population of c. 100 strains from France. We functionally validated AvrStb6 using ectopic transformations. AvrStb6 encodes a small, cysteine-rich, secreted protein that produces an avirulence phenotype on wheat cultivars carrying the Stb6 resistance gene. We found 16 nonsynonymous single nucleotide polymorphisms among the tested strains, indicating that AvrStb6 is evolving very rapidly. AvrStb6 is located in a highly polymorphic subtelomeric region and is surrounded by transposable elements, which may facilitate its rapid evolution to overcome Stb6 resistance. AvrStb6 is the first avirulence gene to be functionally validated in Z. tritici, contributing to our understanding of avirulence in apoplastic pathogens and the mechanisms underlying GFG interactions between Z. tritici and wheat. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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July 7, 2019

Resolving multicopy duplications de novo using polyploid phasing

While the rise of single-molecule sequencing systems has enabled an unprecedented rise in the ability to assemble complex regions of the genome, long segmental duplications in the genome still remain a challenging frontier in assembly. Segmental duplications are at the same time both gene rich and prone to large structural rearrangements, making the resolution of their sequences important in medical and evolutionary studies. Duplicated sequences that are collapsed in mammalian de novo assemblies are rarely identical; after a sequence is duplicated, it begins to acquire paralog-specific variants. In this paper, we study the problem of resolving the variations in multicopy, long segmental duplications by developing and utilizing algorithms for polyploid phasing. We develop two algorithms: the first one is targeted at maximizing the likelihood of observing the reads given the underlying haplotypes using discrete matrix completion. The second algorithm is based on correlation clustering and exploits an assumption, which is often satisfied in these duplications, that each paralog has a sizable number of paralog-specific variants. We develop a detailed simulation methodology and demonstrate the superior performance of the proposed algorithms on an array of simulated datasets. We measure the likelihood score as well as reconstruction accuracy, i.e., what fraction of the reads are clustered correctly. In both the performance metrics, we find that our algorithms dominate existing algorithms on more than 93% of the datasets. While the discrete matrix completion performs better on likelihood score, the correlation-clustering algorithm performs better on reconstruction accuracy due to the stronger regularization inherent in the algorithm. We also show that our correlation-clustering algorithm can reconstruct on average 7.0 haplotypes in 10-copy duplication datasets whereas existing algorithms reconstruct less than one copy on average.

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July 7, 2019

Chromosome end repair and genome stability in Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum replicates within circulating red blood cells, where it is subjected to conditions that frequently cause DNA damage. The repair of DNA double-stranded breaks (DSBs) is thought to rely almost exclusively on homologous recombination (HR), due to a lack of efficient nonhomologous end joining. However, given that the parasite is haploid during this stage of its life cycle, the mechanisms involved in maintaining genome stability are poorly understood. Of particular interest are the subtelomeric regions of the chromosomes, which contain the majority of the multicopy variant antigen-encoding genes responsible for virulence and disease severity. Here, we show that parasites utilize a competitive balance between de novo telomere addition, also called “telomere healing,” and HR to stabilize chromosome ends. Products of both repair pathways were observed in response to DSBs that occurred spontaneously during routine in vitro culture or resulted from experimentally induced DSBs, demonstrating that both pathways are active in repairing DSBs within subtelomeric regions and that the pathway utilized was determined by the DNA sequences immediately surrounding the break. In combination, these two repair pathways enable parasites to efficiently maintain chromosome stability while also contributing to the generation of genetic diversity.IMPORTANCE Malaria is a major global health threat, causing approximately 430,000 deaths annually. This mosquito-transmitted disease is caused by Plasmodium parasites, with infection with the species Plasmodium falciparum being the most lethal. Mechanisms underlying DNA repair and maintenance of genome integrity in P. falciparum are not well understood and represent a gap in our understanding of how parasites survive the hostile environment of their vertebrate and insect hosts. Our work examines DNA repair in real time by using single-molecule real-time (SMRT) sequencing focused on the subtelomeric regions of the genome that harbor the multicopy gene families important for virulence and the maintenance of infection. We show that parasites utilize two competing molecular mechanisms to repair double-strand breaks, homologous recombination and de novo telomere addition, with the pathway used being determined by the surrounding DNA sequence. In combination, these two pathways balance the need to maintain genome stability with the selective advantage of generating antigenic diversity. Copyright © 2017 Calhoun et al.

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July 7, 2019

Complete genome sequencing and targeted mutagenesis reveal virulence contributions of Tal2 and Tal4b of Xanthomonas translucens pv. undulosa ICMP11055 in bacterial leaf streak of wheat

Bacterial leaf streak caused by Xanthomonas translucens pv. undulosa (Xtu) is an important disease of wheat (Triticum aestivum) and barley (Hordeum vulgare) worldwide. Transcription activator-like effectors (TALEs) play determinative roles in many of the plant diseases caused by the different species and pathovars of Xanthomonas, but their role in this disease has not been characterized. ICMP11055 is a highly virulent Xtu strain from Iran. The aim of this study was to better understand genetic diversity of Xtu and to assess the role of TALEs in bacterial leaf streak of wheat by comparing the genome of this strain to the recently completely sequenced genome of a U.S. Xtu strain, and to several other draft X. translucens genomes, and by carrying out mutational analyses of the TALE (tal) genes the Iranian strain might harbor. The ICMP11055 genome, including its repeat-rich tal genes, was completely sequenced using single molecule, real-time technology (Pacific Biosciences). It consists of a single circular chromosome of 4,561,583 bp, containing 3,953 genes. Whole genome alignment with the genome of the United States Xtu strain XT4699 showed two major re-arrangements, nine genomic regions unique to ICMP11055, and one region unique to XT4699. ICMP110055 harbors 26 non-TALE type III effector genes and seven tal genes, compared to 25 and eight for XT4699. The tal genes occur singly or in pairs across five scattered loci. Four are identical to tal genes in XT4699. In addition to common repeat-variable diresidues (RVDs), the tal genes of ICMP11055, like those of XT4699, encode several RVDs rarely observed in Xanthomonas, including KG, NF, Y*, YD, and YK. Insertion and deletion mutagenesis of ICMP11055 tal genes followed by genetic complementation analysis in wheat cv. Chinese Spring revealed that Tal2 and Tal4b of ICMP11055 each contribute individually to the extent of disease caused by this strain. A largely conserved ortholog of tal2 is present in XT4699, but for tal4b, only a gene with partial, fragmented RVD sequence similarity can be found. Our results lay the foundation for identification of important host genes activated by Xtu TALEs as targets for the development of disease resistant varieties.

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July 7, 2019

Comparative sequence analysis of multidrug-resistant IncA/C plasmids from Salmonella enterica

Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in plasmids, advances in plasmid sequencing, and phylogenetic analyses, and important insights about how MDR evolution occurs across diverse serotypes from different animal sources, particularly in agricultural settings where antimicrobial drug use practices vary.

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July 7, 2019

Long-read sequencing offers path to more accurate drug metabolism profiles

In the complex drug discovery process, one of the looming questions for any new compound is how it will be metabolised in a human bodyWhi|e there are several methods for evaluating this, one of the most common involves CYP2D6,the enzyme encoded by the cytochrome P450—2D6 gene.This enzyme is involved in metabolising a quarter of all commonly used medications, making it an important target for ADME and pharmacogenomics studies. It is known to activate some drugs and to play a role in the deactivation or excretion of others.

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July 7, 2019

The evolution of the natural killer complex; a comparison between mammals using new high-quality genome assemblies and targeted annotation.

Natural killer (NK) cells are a diverse population of lymphocytes with a range of biological roles including essential immune functions. NK cell diversity is in part created by the differential expression of cell surface receptors which modulate activation and function, including multiple subfamilies of C-type lectin receptors encoded within the NK complex (NKC). Little is known about the gene content of the NKC beyond rodent and primate lineages, other than it appears to be extremely variable between mammalian groups. We compared the NKC structure between mammalian species using new high-quality draft genome assemblies for cattle and goat; re-annotated sheep, pig, and horse genome assemblies; and the published human, rat, and mouse lemur NKC. The major NKC genes are largely in the equivalent positions in all eight species, with significant independent expansions and deletions between species, allowing us to propose a model for NKC evolution during mammalian radiation. The ruminant species, cattle and goats, have independently evolved a second KLRC locus flanked by KLRA and KLRJ, and a novel KLRH-like gene has acquired an activating tail. This novel gene has duplicated several times within cattle, while other activating receptor genes have been selectively disrupted. Targeted genome enrichment in cattle identified varying levels of allelic polymorphism between the NKC genes concentrated in the predicted extracellular ligand-binding domains. This novel recombination and allelic polymorphism is consistent with NKC evolution under balancing selection, suggesting that this diversity influences individual immune responses and may impact on differential outcomes of pathogen infection and vaccination.

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July 7, 2019

Comparative genomic analysis of Mycobacterium tuberculosis Beijing-like strains revealed specific genetic variations associated with virulence and drug resistance.

Isolates of the Mycobacterium tuberculosis lineage 2/East-Asian are considered one of the most successful strains due to their increased pathogenicity, hyper-virulence associated with drug resistance, and high transmission. Recent studies in Colombia have shown that the Beijing-like genotype is associated with multidrug-resistance and high prevalence in the southwest of the country, but the genetic basis of its success in dissemination is unknown. In contribution to this matter, we obtained the whole sequences of six genomes of clinical isolates assigned to the Beijing-like genotype. The genomes were compared with the reference genome of M. tuberculosis H37Rv and 53 previously published M. tuberculosis genomes. We found that the six Beijing-like isolates belong to a modern Beijing sub-lineage and share specific genomic variants: i.e. deletion in the PPE8 gene, in Rv3806c (ubiA) responsible of high ethambutol resistance and in Rv3862c (whiB6) which is involved in granuloma formation and virulence, are some of them. Moreover, each isolated has exclusively single nucleotide polymorphisms (SNPs) in genes related with cell wall processes and cell metabolism. We identified polymorphisms in genes related to drug resistance that could explain the drug-resistant phenotypes found in the six isolates from Colombia. We hypothesize that changes due to these genetic variations contribute to the success of these strains. Finally, we analyzed the IS6110 insertion sequences finding very low variance between them, suggesting that SNPs is the major cause of variability found in Beijing-like strains circulating in Colombia. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

The blaOXA-23-associated transposons in the genome of Acinetobacter spp. represent an epidemiological situation of the species encountering carbapenems.

High rates of carbapenem resistance in the human pathogen Acinetobacter baumannii threaten public health and need to be scrutinized.A total of 356 A. baumannii and 50 non-baumannii Acinetobacter spp. (NBA) strains collected in 2013 throughout South Korea were studied. The type of blaOXA-23 transposon was determined by PCR mapping and molecular epidemiology was assessed by MLST. Twelve representative strains and two comparative A. baumannii were entirely sequenced by single-molecule real-time sequencing.The carbapenem resistance rate was 88% in A. baumannii, mainly due to blaOXA-23, with five exceptional cases associated with ISAba1-blaOXA-51-like. The blaOXA-23 gene in A. baumannii was carried either by Tn2006 (44%) or Tn2009 (54%), with a few exceptions carried by Tn2008 (1.6%). Of the NBA strains, 14% were resistant to carbapenems, two with blaOXA-58 and five with blaOXA-23 associated with Tn2006. The Tn2006-possessing strains belonged to various STs, whereas Tn2008- and Tn2009-possessing strains were limited to ST208 and ST191, respectively. The three transposons were often multiplied in the chromosome, and the gene copy number and the carbapenem MICs presented linear relationships either very strongly for Tn2008 or moderately for Tn2006 and Tn2009.The dissemination of Tn2006 was facilitated by its capability for intercellular transfer and that of Tn2009 was attributable to successful dissemination of the ST191 bacterial host carrying the transposon. Tn2008 was infrequent because of its insufficient ability to undergo intercellular transfer and the scarce bacterial host A. baumannii ST208. Gene amplification is an adaptive mechanism for bacteria that encounter antimicrobial drugs.© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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