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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

Genetic control of plasticity of oil yield for combined abiotic stresses using a joint approach of crop modelling and genome-wide association.

Understanding the genetic basis of phenotypic plasticity is crucial for predicting and managing climate change effects on wild plants and crops. Here, we combined crop modelling and quantitative genetics to study the genetic control of oil yield plasticity for multiple abiotic stresses in sunflower. First, we developed stress indicators to characterize 14 environments for three abiotic stresses (cold, drought and nitrogen) using the SUNFLO crop model and phenotypic variations of three commercial varieties. The computed plant stress indicators better explain yield variation than descriptors at the climatic or crop levels. In those environments, we observed oil yield of 317 sunflower hybrids and regressed it with three selected stress indicators. The slopes of cold stress norm reaction were used as plasticity phenotypes in the following genome-wide association study. Among the 65 534 tested Single Nucleotide Polymorphisms (SNPs), we identified nine quantitative trait loci controlling oil yield plasticity to cold stress. Associated single nucleotide polymorphisms are localized in genes previously shown to be involved in cold stress responses: oligopeptide transporters, lipid transfer protein, cystatin, alternative oxidase or root development. This novel approach opens new perspectives to identify genomic regions involved in genotype-by-environment interaction of a complex traits to multiple stresses in realistic natural or agronomical conditions.© 2017 John Wiley & Sons Ltd.

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July 7, 2019

Genomics-enabled analysis of the emergent disease cotton bacterial blight.

Cotton bacterial blight (CBB), an important disease of (Gossypium hirsutum) in the early 20th century, had been controlled by resistant germplasm for over half a century. Recently, CBB re-emerged as an agronomic problem in the United States. Here, we report analysis of cotton variety planting statistics that indicate a steady increase in the percentage of susceptible cotton varieties grown each year since 2009. Phylogenetic analysis revealed that strains from the current outbreak cluster with race 18 Xanthomonas citri pv. malvacearum (Xcm) strains. Illumina based draft genomes were generated for thirteen Xcm isolates and analyzed along with 4 previously published Xcm genomes. These genomes encode 24 conserved and nine variable type three effectors. Strains in the race 18 clade contain 3 to 5 more effectors than other Xcm strains. SMRT sequencing of two geographically and temporally diverse strains of Xcm yielded circular chromosomes and accompanying plasmids. These genomes encode eight and thirteen distinct transcription activator-like effector genes. RNA-sequencing revealed 52 genes induced within two cotton cultivars by both tested Xcm strains. This gene list includes a homeologous pair of genes, with homology to the known susceptibility gene, MLO. In contrast, the two strains of Xcm induce different clade III SWEET sugar transporters. Subsequent genome wide analysis revealed patterns in the overall expression of homeologous gene pairs in cotton after inoculation by Xcm. These data reveal important insights into the Xcm-G. hirsutum disease complex and strategies for future development of resistant cultivars.

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July 7, 2019

Antibody-independent mechanisms regulate the establishment of chronic Plasmodium infection.

Malaria is caused by parasites of the genus Plasmodium. All human-infecting Plasmodium species can establish long-lasting chronic infections(1-5), creating an infectious reservoir to sustain transmission(1,6). It is widely accepted that the maintenance of chronic infection involves evasion of adaptive immunity by antigenic variation(7). However, genes involved in this process have been identified in only two of five human-infecting species: Plasmodium falciparum and Plasmodium knowlesi. Furthermore, little is understood about the early events in the establishment of chronic infection in these species. Using a rodent model we demonstrate that from the infecting population, only a minority of parasites, expressing one of several clusters of virulence-associated pir genes, establishes a chronic infection. This process occurs in different species of parasites and in different hosts. Establishment of chronicity is independent of adaptive immunity and therefore different from the mechanism proposed for maintenance of chronic P. falciparum infections(7-9). Furthermore, we show that the proportions of parasites expressing different types of pir genes regulate the time taken to establish a chronic infection. Because pir genes are common to most, if not all, species of Plasmodium(10), this process may be a common way of regulating the establishment of chronic infections.

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July 7, 2019

Human to yeast pathway transplantation: cross-species dissection of the adenine de novo pathway regulatory node

Pathway transplantation from one organism to another represents a means to a more complete understanding of a biochemical or regulatory process. The purine biosynthesis pathway, a core metabolic function, was transplanted from human to yeast. We replaced the entire Saccharomyces cerevisiae adenine de novo pathway with the cognate human pathway components. A yeast strain was humanized for the full pathway by deleting all relevant yeast genes completely and then providing the human pathway in trans using a neochromosome expressing the human protein coding regions under the transcriptional control of their cognate yeast promoters and terminators. The humanized yeast strain grows in the absence of adenine, indicating complementation of the yeast pathway by the full set of human proteins. While the strain with the neochromosome is indeed prototrophic, it grows slowly in the absence of adenine. Dissection of the phenotype revealed that the human ortholog of ADE4, PPAT, shows only partial complementation. We have used several strategies to understand this phenotype, that point to PPAT/ADE4 as the central regulatory node. Pathway metabolites are responsible for regulating PPATs protein abundance through transcription and proteolysis as well as its enzymatic activity by allosteric regulation in these yeast cells. Extensive phylogenetic analysis of PPATs from diverse organisms hints at adaptations of the enzyme-level regulation to the metabolite levels in the organism. Finally, we isolated specific mutations in PPAT as well as in other genes involved in the purine metabolic network that alleviate incomplete complementation by PPAT and provide further insight into the complex regulation of this critical metabolic pathway.

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July 7, 2019

Correspondence on Lovell et al.: response to Bornelöv et al.

While the analysis of Bornelöv et al. is informative, they provide evidence for the existence of only 3% of the reported avian missing genes set, and thus do not significantly challenge our main findings that specific groups of syntenic protein-coding genes are missing in birds.This is a response to the Correspondence article: https://www.dx.doi.org/10.1186/s13059-017-1231-1.

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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

Euglena gracilis genome and transcriptome: organelles, nuclear genome assembly strategies and initial features.

Euglena gracilis is a major component of the aquatic ecosystem and together with closely related species, is ubiquitous worldwide. Euglenoids are an important group of protists, possessing a secondarily acquired plastid and are relatives to the Kinetoplastidae, which themselves have global impact as disease agents. To understand the biology of E. gracilis, as well as to provide further insight into the evolution and origins of the Kinetoplastidae, we embarked on sequencing the nuclear genome; the plastid and mitochondrial genomes are already in the public domain. Earlier studies suggested an extensive nuclear DNA content, with likely a high degree of repetitive sequence, together with significant extrachromosomal elements. To produce a list of coding sequences we have combined transcriptome data from both published and new sources, as well as embarked on de novo sequencing using a combination of 454, Illumina paired end libraries and long PacBio reads. Preliminary analysis suggests a surprisingly large genome approaching 2 Gbp, with a highly fragmented architecture and extensive repeat composition. Over 80% of the RNAseq reads from E. gracilis maps to the assembled genome sequence, which is comparable with the well assembled genomes of T. brucei and T. cruzi. In order to achieve this level of assembly we employed multiple informatics pipelines, which are discussed here. Finally, as a preliminary view of the genome architecture, we discuss the tubulin and calmodulin genes, which highlight potential novel splicing mechanisms.

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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

Strategies for optimizing BioNano and Dovetail explored through a second reference quality assembly for the legume model, Medicago truncatula.

Third generation sequencing technologies, with sequencing reads in the tens- of kilo-bases, facilitate genome assembly by spanning ambiguous regions and improving continuity. This has been critical for plant genomes, which are difficult to assemble due to high repeat content, gene family expansions, segmental and tandem duplications, and polyploidy. Recently, high-throughput mapping and scaffolding strategies have further improved continuity. Together, these long-range technologies enable quality draft assemblies of complex genomes in a cost-effective and timely manner.Here, we present high quality genome assemblies of the model legume plant, Medicago truncatula (R108) using PacBio, Dovetail Chicago (hereafter, Dovetail) and BioNano technologies. To test these technologies for plant genome assembly, we generated five assemblies using all possible combinations and ordering of these three technologies in the R108 assembly. While the BioNano and Dovetail joins overlapped, they also showed complementary gains in continuity and join numbers. Both technologies spanned repetitive regions that PacBio alone was unable to bridge. Combining technologies, particularly Dovetail followed by BioNano, resulted in notable improvements compared to Dovetail or BioNano alone. A combination of PacBio, Dovetail, and BioNano was used to generate a high quality draft assembly of R108, a M. truncatula accession widely used in studies of functional genomics. As a test for the usefulness of the resulting genome sequence, the new R108 assembly was used to pinpoint breakpoints and characterize flanking sequence of a previously identified translocation between chromosomes 4 and 8, identifying more than 22.7 Mb of novel sequence not present in the earlier A17 reference assembly.Adding Dovetail followed by BioNano data yielded complementary improvements in continuity over the original PacBio assembly. This strategy proved efficient and cost-effective for developing a quality draft assembly compared to traditional reference assemblies.

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July 7, 2019

Large scale and significant expression from pseudogenes in Sodalis glossinidius – a facultative bacterial endosymbiont

The majority of bacterial genomes have high coding efficiencies, but there are some genomes of intracellular bacteria that have low gene density. The genome of the endosymbiont Sodalis glossinidius contains almost 50% pseudogenes containing mutations that putatively silence them at the genomic level. We have applied multiple omic strategies, combining: Illumina and Pacific Biosciences Single-Molecule Real Time DNA-sequencing and annotation; stranded RNA-sequencing; and proteome analysis to better understand the transcriptional and translational landscape of Sodalis pseudogenes, and potential mechanisms for their control. Between 53% and 74% of the Sodalis transcriptome remains active in cell-free culture. Mean sense transcription from Coding Domain Sequences (CDS) is four-times greater than that from pseudogenes. Comparative genomic analysis of six Illumina-sequenced Sodalis isolates from different host Glossina species shows pseudogenes make up ~40% of the 2,729 genes in the core genome, suggesting are stable and/or Sodalis is a recent introduction across the Glossina genus as a facultative symbiont. These data further shed light on the importance of transcriptional and translational control in deciphering host-microbe interactions, and demonstrate that pseudogenes are more complex than a simple degrading DNA sequence. The combination of genomics, transcriptomics and proteomics give a multidimensional perspective for studying prokaryotic genomes with a view to elucidating evolutionary adaptation to novel environmental niches.

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July 7, 2019

The genome of the cotton bacterial blight pathogen Xanthomonas citri pv. malvacearum strain MSCT1.

Xanthomonas citri pv. malvacearum is a major pathogen of cotton, Gossypium hirsutum L.. In this study we report the complete genome of the X. citri pv. malvacearum strain MSCT1 assembled from long read DNA sequencing technology. The MSCT1 genome is the first X. citri pv. malvacearum genome with complete coding regions for X. citri pv. malvacearum transcriptional activator-like effectors. In addition functional and structural annotations are presented in this study that will provide a foundation for future pathogenesis studies with MSCT1.

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July 7, 2019

Improved PKS gene expression with strong endogenous promoter resulted in geldanamycin yield increase.

The type I polyketide geldanamycin is a potent anti-tumor reagent. Its biosynthesis includes three steps: the biosynthesis of precursors, such as 3-amino-5-hydroxybenzoic acid (AHBA), the polyketide synthase (PKS) chain extension, and the post-PKS modifications. According to the genomic and transcriptomic analysis, the PKS chain extension was deduced to be the rate-limiting step for geldanamycin production in Streptomyces hygroscopicus XM201. In order to improve the expression of PKS genes, a strong endogenous promoter 5063p was obtained based on the transcriptomic analysis and XylE enzymatic assay. By replacing the native PKS promoter gdmA1p with 5063p, the expression of the PKS genes during geldanamycin fermentation was increased by 4-141-folds, and the geldanamycin yield was increased by 39%. Interestingly, AHBA feeding experiment showed that the supply of AHBA in turn become a new rate-limiting factor for geldanamycin production. Further combined overexpression of the 6-gene AHBA biosynthetic cassette and PKS genes increased the yield of geldanamycin by 88%, from 773?mg?L(-1) of the wild-type to 1450?mg?L(-1) in the derived strain. Our results suggested that improved expression of all PKS genes in a particular biosynthetic gene cluster is important for the yield increase of the corresponding polyketide natural product.© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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July 7, 2019

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

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July 7, 2019

Genome diversity and evolution in the budding yeasts (Saccharomycotina).

Considerable progress in our understanding of yeast genomes and their evolution has been made over the last decade with the sequencing, analysis, and comparisons of numerous species, strains, or isolates of diverse origins. The role played by yeasts in natural environments as well as in artificial manufactures, combined with the importance of some species as model experimental systems sustained this effort. At the same time, their enormous evolutionary diversity (there are yeast species in every subphylum of Dikarya) sparked curiosity but necessitated further efforts to obtain appropriate reference genomes. Today, yeast genomes have been very informative about basic mechanisms of evolution, speciation, hybridization, domestication, as well as about the molecular machineries underlying them. They are also irreplaceable to investigate in detail the complex relationship between genotypes and phenotypes with both theoretical and practical implications. This review examines these questions at two distinct levels offered by the broad evolutionary range of yeasts: inside the best-studied Saccharomyces species complex, and across the entire and diversified subphylum of Saccharomycotina. While obviously revealing evolutionary histories at different scales, data converge to a remarkably coherent picture in which one can estimate the relative importance of intrinsic genome dynamics, including gene birth and loss, vs. horizontal genetic accidents in the making of populations. The facility with which novel yeast genomes can now be studied, combined with the already numerous available reference genomes, offer privileged perspectives to further examine these fundamental biological questions using yeasts both as eukaryotic models and as fungi of practical importance. Copyright © 2017 by the Genetics Society of America.

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