Stress responses in an Arctic microalga (Pelagophyceae) following sudden salinity change revealed by gene expression analysis
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59 BASIC BIOLOGICAL SCIENCES↗
Engineering topics
Barry, Kerrie W.
Publications and source records attributed to Barry, Kerrie W..
Mechanically induced localisation of SECONDARY WALL INTERACTING bZIP is associated with thigmomorphogenic and secondary cell wall gene expression
Plant growth requires the integration of internal and external cues, perceived and transduced into a developmental programme of cell division, elongation and wall thickening. Mechanical forces contribute to this regulation, and thigmomorphogenesis typically includes reducing stem height, increasing stem diameter, and a canonical transcriptomic response. We present data on a bZIP transcription factor involved in this process in grasses. Brachypodium distachyon SECONDARY WALL INTERACTING bZIP (SWIZ) protein translocated into the nucleus following mechanostimulation. Classical touch-responsive genes were upregulated in B. distachyon roots following touch, including significant induction of the glycoside hydrolase 17 family, which may be unique to grass thigmomorphogenesis. SWIZ protein binding to an E-box variant in exons and introns was associated with immediate activation followed by repression of gene expression. SWIZ overexpression resulted in plants with reduced stem and root elongation. These data further define plant touch-responsive transcriptomics and physiology, offering insights into grass mechanotranduction dynamics.
Coomey, Joshua↗
Major impacts of widespread structural variation on sorghum
Genetic diversity is critical to crop breeding and improvement, and dissection of the genomic variation underlying agronomic traits can both assist breeding and give insight into basic biological mechanisms. Although recent genome analyses in plants reveal many structural variants (SVs), most current studies of crop genetic variation are dominated by single-nucleotide polymorphisms (SNPs). The extent of the impact of SVs on global trait variation, as well as their utility in genome-wide selection, is not yet understood. In this study, we built an SV data set based on whole-genome resequencing of diverse sorghum lines (n = 363), validated the correlation of photoperiod sensitivity and variety type, and identified SV hotspots underlying the divergent evolution of cellulosic and sweet sorghum. In addition, we showed the complementary contribution of SVs for heritability of traits related to sorghum adaptation. Importantly, inclusion of SV polymorphisms in association studies revealed genotype–phenotype associations not observed with SNPs alone. Three-way genome-wide association studies (GWAS) based on whole-genome SNP, SV, and integrated SNP + SV data sets showed substantial associations between SVs and sorghum traits. The addition of SVs to GWAS substantially increased heritability estimates for some traits, indicating their important contribution to functional allelic variation at the genome level. Our discovery of the widespread impacts of SVs on heritable gene expression variation could render a plausible mechanism for their disproportionate impact on phenotypic variation. This study expands our knowledge of SVs and emphasizes the extensive impacts of SVs on sorghum.
59 BASIC BIOLOGICAL SCIENCES↗
Haplotype‐resolved genome assembly of Populus tremula × P. alba reveals aspen‐specific megabase satellite DNA
SUMMARY Populus species play a foundational role in diverse ecosystems and are important renewable feedstocks for bioenergy and bioproducts. Hybrid aspen Populus tremula × P. alba INRA 717‐1B4 is a widely used transformation model in tree functional genomics and biotechnology research. As an outcrossing interspecific hybrid, its genome is riddled with sequence polymorphisms which present a challenge for sequence‐sensitive analyses. Here we report a telomere‐to‐telomere genome for this hybrid aspen with two chromosome‐scale, haplotype‐resolved assemblies. We performed a comprehensive analysis of the repetitive landscape and identified both tandem repeat array‐based and array‐less centromeres. Unexpectedly, the most abundant satellite repeats in both haplotypes lie outside of the centromeres, consist of a 147 bp monomer PtaM147, frequently span >1 megabases, and form heterochromatic knobs. PtaM147 repeats are detected exclusively in aspens (section Populus ) but PtaM147‐like sequences occur in LTR‐retrotransposons of closely related species, suggesting their origin from the retrotransposons. The genomic resource generated for this transformation model genotype has greatly improved the design and analysis of genome editing experiments that are highly sensitive to sequence polymorphisms. The work should motivate future hypothesis‐driven research to probe into the function of the abundant and aspen‐specific PtaM147 satellite DNA.
Zhou, Ran↗
DNA hypomethylation of the host tree impairs interaction with mutualistic ectomycorrhizal fungus
Summary Ectomycorrhizas are an intrinsic component of tree nutrition and responses to environmental variations. How epigenetic mechanisms might regulate these mutualistic interactions is unknown. By manipulating the level of expression of the chromatin remodeler DECREASE IN DNA METHYLATION 1 ( DDM1 ) and two demethylases DEMETER‐LIKE ( DML ) in Populus tremula × Populus alba lines, we examined how host DNA methylation modulates multiple parameters of the responses to root colonization with the mutualistic fungus Laccaria bicolor . We compared the ectomycorrhizas formed between transgenic and wild‐type (WT) trees and analyzed their methylomes and transcriptomes. The poplar lines displaying lower mycorrhiza formation rate corresponded to hypomethylated overexpressing DML or RNAi‐ddm1 lines. We found 86 genes and 288 transposable elements (TEs) differentially methylated between WT and hypomethylated lines (common to both OX‐ dml and RNAi‐ ddm1 ) and 120 genes/1441 TEs in the fungal genome suggesting a host‐induced remodeling of the fungal methylome. Hypomethylated poplar lines displayed 205 differentially expressed genes ( cis and trans effects) in common with 17 being differentially methylated ( cis ). Our findings suggest a central role of host and fungal DNA methylation in the ability to form ectomycorrhizas including not only poplar genes involved in root initiation, ethylene and jasmonate‐mediated pathways, and immune response but also terpenoid metabolism.
Vigneaud, Julien↗
Salinity tolerance mechanisms of an Arctic Pelagophyte using comparative transcriptomic and gene expression analysis
Abstract Little is known at the transcriptional level about microbial eukaryotic adaptations to short-term salinity change. Arctic microalgae are exposed to low salinity due to sea-ice melt and higher salinity with brine channel formation during freeze-up. Here, we investigate the transcriptional response of an ice-associated microalgae over salinities from 45 to 8. Our results show a bracketed response of differential gene expression when the cultures were exposed to progressively decreasing salinity. Key genes associated with salinity changes were involved in specific metabolic pathways, transcription factors and regulators, protein kinases, carbohydrate active enzymes, and inorganic ion transporters. The pelagophyte seemed to use a strategy involving overexpression of Na + -H + antiporters and Na + -Pi symporters as salinity decreases, but the K + channel complex at higher salinities. Specific adaptation to cold saline arctic conditions was seen with differential expression of several antifreeze proteins, an ice-binding protein and an acyl-esterase involved in cold adaptation.
59 BASIC BIOLOGICAL SCIENCES↗
Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA
As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar.
59 BASIC BIOLOGICAL SCIENCES↗
Genomic Signatures of a Major Adaptive Event in the Pathogenic Fungus Melampsora larici-populina
The recent availability of genome-wide sequencing techniques has allowed systematic screening for molecular signatures of adaptation, including in nonmodel organisms. Host–pathogen interactions constitute good models due to the strong selective pressures that they entail. We focused on an adaptive event which affected the poplar rust fungus Melampsora larici-populina when it overcame a resistance gene borne by its host, cultivated poplar. Based on 76 virulent and avirulent isolates framing narrowly the estimated date of the adaptive event, we examined the molecular signatures of selection. Using an array of genome scan methods based on different features of nucleotide diversity, we detected a single locus exhibiting a consistent pattern suggestive of a selective sweep in virulent individuals (excess of differentiation between virulent and avirulent samples, linkage disequilibrium, genotype–phenotype statistical association, and long-range haplotypes). Our study pinpoints a single gene and further a single amino acid replacement which may have allowed the adaptive event. Although our samples are nearly contemporary to the selective sweep, it does not seem to have affected genome diversity further than the immediate vicinity of the causal locus, which can be explained by a soft selective sweep (where selection acts on standing variation) and by the impact of recombination in mitigating the impact of selection. Therefore, it seems that properties of the life cycle of M. larici-populina, which entails both high genetic diversity and outbreeding, has facilitated its adaptation.
59 BASIC BIOLOGICAL SCIENCES↗
Genetic determinants of endophytism in the Arabidopsis root mycobiome
The roots of Arabidopsis thaliana host diverse fungal communities that affect plant health and disease states. Here, we sequence the genomes of 41 fungal isolates representative of the A. thaliana root mycobiota for comparative analysis with other 79 plant-associated fungi. Our analyses indicate that root mycobiota members evolved from ancestors with diverse lifestyles and retain large repertoires of plant cell wall-degrading enzymes (PCWDEs) and effector-like small secreted proteins. We identify a set of 84 gene families associated with endophytism, including genes encoding PCWDEs acting on xylan (family GH10) and cellulose (family AA9). Transcripts encoding these enzymes are also part of a conserved transcriptional program activated by phylogenetically-distant mycobiota members upon host contact. Recolonization experiments with individual fungi indicate that strains with detrimental effects in mono-association with the host colonize roots more aggressively than those with beneficial activities, and dominate in natural root samples. Furthermore, we show that the pectin-degrading enzyme family PL1_7 links aggressiveness of endophytic colonization to plant health.
59 BASIC BIOLOGICAL SCIENCES↗
Evolutionary transition to the ectomycorrhizal habit in the genomes of a hyperdiverse lineage of mushroom-forming fungi
The ectomycorrhizal (ECM) symbiosis has independently evolved from diverse types of saprotrophic ancestors. In this study, we seek to identify genomic signatures of the transition to the ECM habit within the hyperdiverse Russulaceae. We present comparative analyses of the genomic architecture and the total and secreted gene repertoires of 18 species across the order Russulales, of which 13 are newly sequenced, including a representative of a saprotrophic member of Russulaceae, Gloeopeniophorella convolvens. The genomes of ECM Russulaceae are characterized by a loss of genes for plant cell wall-degrading enzymes (PCWDEs), an expansion of genome size through increased transposable element (TE) content, a reduction in secondary metabolism clusters, and an association of small secreted proteins (SSPs) with TE ‘nests’, or dense aggregations of TEs. Here, some PCWDEs have been retained or even expanded, mostly in a species-specific manner. The genome of G. convolvens possesses some characteristics of ECM genomes (e.g. loss of some PCWDEs, TE expansion, reduction in secondary metabolism clusters). Functional specialization in ECM decomposition may drive diversification. Accelerated gene evolution predates the evolution of the ECM habit, indicating that changes in genome architecture and gene content may be necessary to prime the evolutionary switch.
59 BASIC BIOLOGICAL SCIENCES↗
Pests, diseases, and aridity have shaped the genome of Corymbia citriodora
Corymbia citriodora is a member of the predominantly Southern Hemisphere Myrtaceae family, which includes the eucalypts (Eucalyptus, Corymbia and Angophora; ~800 species). Corymbia is grown for timber, pulp and paper, and essential oils in Australia, South Africa, Asia, and Brazil, maintaining a high-growth rate under marginal conditions due to drought, poor-quality soil, and biotic stresses. To dissect the genetic basis of these desirable traits, we sequenced and assembled the 408 Mb genome of Corymbia citriodora, anchored into eleven chromosomes. Comparative analysis with Eucalyptus grandis reveals high synteny, although the two diverged approximately 60 million years ago and have different genome sizes (408 vs 641 Mb), with few large intra-chromosomal rearrangements. C. citriodora shares an ancient whole-genome duplication event with E. grandis but has undergone tandem gene family expansions related to terpene biosynthesis, innate pathogen resistance, and leaf wax formation, enabling their successful adaptation to biotic/abiotic stresses and arid conditions of the Australian continent.
59 BASIC BIOLOGICAL SCIENCES↗
Experimentally Validated Reconstruction and Analysis of a Genome-Scale Metabolic Model of an Anaerobic Neocallimastigomycota Fungus
Recent genomic analyses have revealed that anaerobic gut fungi possess both the largest number and highest diversity of lignocellulolytic enzymes of all sequenced fungi, explaining their ability to decompose lignocellulosic substrates, e.g., agricultural waste, into fermentable sugars. Despite their potential, the development of engineering methods for these organisms has been slow due to their complex life cycle, understudied metabolism, and challenging anaerobic culture requirements.
59 BASIC BIOLOGICAL SCIENCES↗
Desert truffle genomes reveal their reproductive modes and new insights into plant–fungal interaction and ectendomycorrhizal lifestyle
Desert truffles are edible hypogeous fungi forming ectendomycorrhizal symbiosis with plants of Cistaceae family. Knowledge about the reproductive modes of these fungi and the molecular mechanisms driving the ectendomycorrhizal interaction is lacking. Genomes of the highly appreciated edible desert truffles Terfezia claveryi Chatin and Tirmania nivea Trappe have been sequenced and compared with other Pezizomycetes. Transcriptomes of T. claveryi × Helianthemum almeriense mycorrhiza from well-watered and drought-stressed plants, when intracellular colonizations is promoted, were investigated. We have identified the fungal genes related to sexual reproduction in desert truffles and desert-truffles-specific genomic and secretomic features with respect to other Pezizomycetes, such as the expansion of a large set of gene families with unknown Pfam domains and a number of species or desert-truffle-specific small secreted proteins differentially regulated in symbiosis. A core set of plant genes, including carbohydrate, lipid-metabolism, and defence-related genes, differentially expressed in mycorrhiza under both conditions was found. Our results highlight the singularities of desert truffles with respect to other mycorrhizal fungi while providing a first glimpse on plant and fungal determinants involved in ecto to endo symbiotic switch that occurs in desert truffle under dry conditions.
59 BASIC BIOLOGICAL SCIENCES↗
Large-scale genome sequencing of mycorrhizal fungi provides insights into the early evolution of symbiotic traits
Mycorrhizal fungi are mutualists that play crucial roles in nutrient acquisition in terrestrial ecosystems. Mycorrhizal symbioses arose repeatedly across multiple lineages of Mucoromycotina, Ascomycota, and Basidiomycota. Considerable variation exists in the capacity of mycorrhizal fungi to acquire carbon from soil organic matter. Here, we present a combined analysis of 135 fungal genomes from 73 saprotrophic, endophytic and pathogenic species, and 62 mycorrhizal species, including 29 new mycorrhizal genomes. This study samples ecologically dominant fungal guilds for which there were previously no symbiotic genomes available, including ectomycorrhizal Russulales, Thelephorales and Cantharellales. Our analyses show that transitions from saprotrophy to symbiosis involve (1) widespread losses of degrading enzymes acting on lignin and cellulose, (2) co-option of genes present in saprotrophic ancestors to fulfill new symbiotic functions, (3) diversification of novel, lineage-specific symbiosis-induced genes, (4) proliferation of transposable elements and (5) divergent genetic innovations underlying the convergent origins of the ectomycorrhizal guild.
59 BASIC BIOLOGICAL SCIENCES↗
Modulation of Plant and Fungal Gene Expression Upon Cd Exposure and Symbiosis in Ericoid Mycorrhizal Vaccinium myrtillus
The success of Ericaceae in stressful habitats enriched in heavy metals has been ascribed to the distinctive abilities of their mycorrhizal fungal partners to withstand heavy metal stress and to enhance metal tolerance in the host plant. Whereas heavy metal tolerance has been extensively investigated in some ericoid mycorrhizal (ERM) fungi, the molecular and cellular mechanisms that extend tolerance to the host plant are currently unknown. Here, we show a reduced Cd content in Cd-exposed mycorrhizal roots of Vaccinium myrtillus colonized by a metal tolerant isolate of the fungus Oidiodendron maius as compared to non-mycorrhizal roots. To better understand this phenotype, we applied Next Generation Sequencing technologies to analyze gene expression in V. myrtillus and O. maius Zn grown under normal and Cd-stressed conditions, in the free living and in the mycorrhizal status. The results clearly showed that Cd had a stronger impact on plant gene expression than symbiosis, whereas fungal gene expression was mainly regulated by symbiosis. The higher abundance of transcripts coding for stress related proteins in non-mycorrhizal roots may be related to the higher Cd content. Regulated plant metal transporters have been identified that may play a role in reducing Cd content in mycorrhizal roots exposed to this metal.
Cd tolerance, transcriptomics↗
Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9
Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops.
59 BASIC BIOLOGICAL SCIENCES↗