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September 22, 2019  |  

Comparative transcriptomic and physiological analyses of Medicago sativa L. indicates that multiple regulatory networks are activated during continuous ABA treatment.

Alfalfa is the most extensively cultivated forage legume worldwide. However, the molecular mechanisms underlying alfalfa responses to exogenous abscisic acid (ABA) are still unknown. In this study, the first global transcriptome profiles of alfalfa roots under ABA treatments for 1, 3 and 12 h (three biological replicates for each time point, including the control group) were constructed using a BGISEQ-500 sequencing platform. A total of 50,742 isoforms with a mean length of 2541 bp were generated, and 4944 differentially expressed isoforms (DEIs) were identified after ABA deposition. Metabolic analyses revealed that these DEIs were involved in plant hormone signal transduction, transcriptional regulation, antioxidative defense and pathogen immunity. Notably, several well characterized hormone signaling pathways, for example, the core ABA signaling pathway, was activated, while salicylic acid, jasmonate and ethylene signaling pathways were mainly suppressed by exogenous ABA. Moreover, the physiological work showed that catalase and peroxidase activity and glutathione and proline content were increased after ABA deposition, which is in accordance with the dynamic transcript profiles of the relevant genes in antioxidative defense system. These results indicate that ABA has the potential to improve abiotic stress tolerance, but that it may negatively regulate pathogen resistance in alfalfa.

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September 22, 2019  |  

Transcriptome profiling using Illumina- and SMRT-based RNA-seq of hot pepper for in-depth understanding of genes involved in CMV infection.

Hot pepper (Capsicum annuum L.) is becoming an increasingly important vegetable crop in the world. Cucumber mosaic virus (CMV) is a destructive virus that can cause leaf distortion and fruit lesions, affecting pepper production. However, studies on the response to CMV infection in pepper at the transcriptional level are limited. In this study, the transcript profiles of pepper leaves after CMV infection were investigated using Illumina and single-molecule real-time (SMRT) RNA-sequencing (RNA-seq). A total of 2143 differentially expressed genes (DEGs) were identified at five different stages. Gene ontology (GO) and KEGG analysis revealed that these DEGs were involved in the response to stress, defense response and plant-pathogen interaction pathways. Among these DEGs, several key genes that consistently appeared in studies of plant-pathogen interactions had increased transcript abundance after inoculation, including chitinase, pathogenesis-related (PR) protein, TMV resistance protein, WRKY transcription factor and jasmonate ZIM-domain protein. Four of these DEGs were further validated by quantitative real-time RT-PCR (qRT-PCR). Furthermore, a total of 73, 597 alternative splicing (AS) events were identified in the pepper leaves after CMV infection, distributed in 12, 615 genes. The intron retention of WRKY33 (Capana09g001251) might be involved in the regulation of CMV infection. Taken together, our study provides a transcriptome-wide insight into the molecular basis of resistance to CMV infection in pepper leaves and potential candidate genes for improving resistance cultivars. Copyright © 2018 Elsevier B.V. All rights reserved.

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September 22, 2019  |  

De novo assembly of a Chinese soybean genome.

Soybean was domesticated in China and has become one of the most important oilseed crops. Due to bottlenecks in their introduction and dissemination, soybeans from different geographic areas exhibit extensive genetic diversity. Asia is the largest soybean market; therefore, a high-quality soybean reference genome from this area is critical for soybean research and breeding. Here, we report the de novo assembly and sequence analysis of a Chinese soybean genome for “Zhonghuang 13” by a combination of SMRT, Hi-C and optical mapping data. The assembled genome size is 1.025 Gb with a contig N50 of 3.46 Mb and a scaffold N50 of 51.87 Mb. Comparisons between this genome and the previously reported reference genome (cv. Williams 82) uncovered more than 250,000 structure variations. A total of 52,051 protein coding genes and 36,429 transposable elements were annotated for this genome, and a gene co-expression network including 39,967 genes was also established. This high quality Chinese soybean genome and its sequence analysis will provide valuable information for soybean improvement in the future.

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September 22, 2019  |  

Multiple regulatory networks are activated during cold stress in Medicago sativa L.

Cultivated alfalfa (Medicago sativa L.) is one of the most important perennial legume forages in the world, and it has considerable potential as a valuable forage crop for livestock. However, the molecular mechanisms underlying alfalfa responses to cold stress are largely unknown. In this study, the transcriptome changes in alfalfa under cold stress at 4 °C for 2, 6, 24, and 48 h (three replicates for each time point) were analyzed using the high-throughput sequencing platform, BGISEQ-500, resulting in the identification of 50,809 annotated unigenes and 5283 differentially expressed genes (DEGs). Metabolic pathway enrichment analysis demonstrated that the DEGs were involved in carbohydrate metabolism, photosynthesis, plant hormone signal transduction, and the biosynthesis of amino acids. Moreover, the physiological changes of glutathione and proline content, catalase, and peroxidase activity were in accordance with dynamic transcript profiles of the relevant genes. Additionally, some transcription factors might play important roles in the alfalfa response to cold stress, as determined by the expression pattern of the related genes during 48 h of cold stress treatment. These findings provide valuable information for identifying and characterizing important components in the cold signaling network in alfalfa and enhancing the understanding of the molecular mechanisms underlying alfalfa responses to cold stress.

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September 22, 2019  |  

Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis.

Astragalus membranaceus, also known as Huangqi in China, is one of the most widely used medicinal herbs in Traditional Chinese Medicine. Traditional Chinese Medicine formulations from Astragalus membranaceus have been used to treat a wide range of illnesses, such as cardiovascular disease, type 2 diabetes, nephritis and cancers. Pharmacological studies have shown that immunomodulating, anti-hyperglycemic, anti-inflammatory, antioxidant and antiviral activities exist in the extract of Astragalus membranaceus. Therefore, characterising the biosynthesis of bioactive compounds in Astragalus membranaceus, such as Astragalosides, Calycosin and Calycosin-7-O-ß-d-glucoside, is of particular importance for further genetic studies of Astragalus membranaceus. In this study, we reconstructed the Astragalus membranaceus full-length transcriptomes from leaf and root tissues using PacBio Iso-Seq long reads. We identified 27 975 and 22 343 full-length unique transcript models in each tissue respectively. Compared with previous studies that used short read sequencing, our reconstructed transcripts are longer, and are more likely to be full-length and include numerous transcript variants. Moreover, we also re-characterised and identified potential transcript variants of genes involved in Astragalosides, Calycosin and Calycosin-7-O-ß-d-glucoside biosynthesis. In conclusion, our study provides a practical pipeline to characterise the full-length transcriptome for species without a reference genome and a useful genomic resource for exploring the biosynthesis of active compounds in Astragalus membranaceus.

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September 22, 2019  |  

Analysis of the hybrid genomes of two field isolates of the soil-borne fungal species Verticillium longisporum.

Brassica plant species are attacked by a number of pathogens; among them, the ones with a soil-borne lifestyle have become increasingly important. Verticillium stem stripe caused by Verticillium longisporum is one example. This fungal species is thought to be of a hybrid origin, having a genome composed of combinations of lineages denominated A and D. In this study we report the draft genomes of 2 V. longisporum field isolates sequenced using the Illumina technology. Genomic characterization and lineage composition, followed by selected gene analysis to facilitate the comprehension of its genomic features and potential effector categories were performed.The draft genomes of 2 Verticillium longisporum single spore isolates (VL1 and VL2) have an estimated ungapped size of about 70 Mb. The total number of protein encoding genes identified in VL1 was 20,793, whereas 21,072 gene models were predicted in VL2. The predicted genome size, gene contents, including the gene families coding for carbohydrate active enzymes were almost double the numbers found in V. dahliae and V. albo-atrum. Single nucleotide polymorphisms (SNPs) were frequently distributed in the two genomes but the distribution of heterozygosity and depth was not independent. Further analysis of potential parental lineages suggests that the V. longisporum genome is composed of two parts, A1 and D1, where A1 is more ancient than the parental lineage genome D1, the latter being more closer related to V. dahliae. Presence of the mating-type genes MAT1-1-1 and MAT1-2-1 in the V. longisporum genomes were confirmed. However, the MAT genes in V. dahliae, V. albo-atrum and V. longisporum have experienced extensive nucleotide changes at least partly explaining the present asexual nature of these fungal species.The established draft genome of V. longisporum is comparatively large compared to other studied ascomycete fungi. Consequently, high numbers of genes were predicted in the two V. longisporum genomes, among them many secreted proteins and carbohydrate active enzyme (CAZy) encoding genes. The genome is composed of two parts, where one lineage is more ancient than the part being more closely related to V. dahliae. Dissimilar mating-type sequences were identified indicating possible ancient hybridization events.

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September 22, 2019  |  

Genomic diversity in the endosymbiotic bacterium Rhizobium leguminosarum.

Rhizobium leguminosarum bv. viciae is a soil a-proteobacterium that establishes a diazotrophic symbiosis with different legumes of the Fabeae tribe. The number of genome sequences from rhizobial strains available in public databases is constantly increasing, although complete, fully annotated genome structures from rhizobial genomes are scarce. In this work, we report and analyse the complete genome of R. leguminosarum bv. viciae UPM791. Whole genome sequencing can provide new insights into the genetic features contributing to symbiotically relevant processes such as bacterial adaptation to the rhizosphere, mechanisms for efficient competition with other bacteria, and the ability to establish a complex signalling dialogue with legumes, to enter the root without triggering plant defenses, and, ultimately, to fix nitrogen within the host. Comparison of the complete genome sequences of two strains of R. leguminosarum bv. viciae, 3841 and UPM791, highlights the existence of different symbiotic plasmids and a common core chromosome. Specific genomic traits, such as plasmid content or a distinctive regulation, define differential physiological capabilities of these endosymbionts. Among them, strain UPM791 presents unique adaptations for recycling the hydrogen generated in the nitrogen fixation process.

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September 22, 2019  |  

Aberration or analogy? The atypical plastomes of Geraniaceae

A number of plant groups have been proposed as ideal systems to explore plastid inheritance, plastome evolution and plastome-nuclear genome coevolution. Quick generation times and a compact nuclear genome in Arabidopsis thaliana, the relative ease of plastid isolation from Spinacia oleracea and the tractability of plastid transformation in Nicotiana tabacum are all desirable attributes in a model system; however, these and most other groups all lack novelty in terms of plastome structure and nucleotide sequence evolution. Contemporary sequencing and assembly technologies have facilitated analyses of atypical plastomes and, as predicted by early investigations, Geraniaceae plastomes have experienced unprecedented rearrangements relative to the canonical structure and exhibit remarkably high rates of synonymous and nonsynonymous nucleotide substitutions. While not the only lineage with unusual plastome features, likely no other group represents the array of aberrant phenomena recorded for the family. In this chapter, Geraniaceae plastomes will be discussed and, where possible, compared with other taxa.

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September 22, 2019  |  

Molecular characterization of NBS-LRR genes in the soybean Rsv3 locus reveals several divergent alleles that likely confer resistance to the soybean mosaic virus.

The divergence patterns of NBS – LRR genes in soybean Rsv3 locus were deciphered and several divergent alleles ( NBS_C, NBS_D and Columbia NBS_E ) were identified as the likely functional candidates of Rsv3. The soybean Rsv3 locus, which confers resistance to the soybean mosaic virus (SMV), has been previously mapped to a region containing five nucleotide binding site-leucine-rich repeats (NBS-LRR) genes (referred to as nbs_A-E) in Williams 82. In resistant cultivars, however, the number of NBS-LRR genes in this region and their divergence from susceptible alleles remain unclear. In the present study, we constructed and screened a bacterial artificial chromosome (BAC) library for an Rsv3-possessing cultivar, Zaoshu 18. Sequencing two positive BAC inserts on the Rsv3 locus revealed that Zaoshu 18 possesses the same gene content and order as Williams 82, but two of the NBS-LRR genes, NBS_C and NBS_D, exhibit distinct features that were not observed in the Williams 82 alleles. Obtaining these NBS-LRR genes from eight additional cultivars demonstrated that the NBS_A-D genes diverged into two different alleles: the nbs_A-D alleles were associated with the rsv3-type cultivars, whereas the NBS_A-D alleles were associated with the Rsv3-possessing cultivars. For the NBS_E gene, the cultivar Columbia possesses an allele (NBS_E) that differed from that in Zaoshu 18 and rsv3-type cultivars (nbs_E). Exchanged fragments were further detected on alleles of the NBS_C-E genes, suggesting that recombination is a major force responsible for allele divergence. Also, the LRR domains of the NBS_C-E genes exhibited extremely strong signals of positive selection. Overall, the divergence patterns of the NBS-LRR genes in Rsv3 locus elucidated by this study indicate that not only NBS_C but also NBS_D and Columbia NBS_E are likely functional alleles that confer resistance to SMV.

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September 22, 2019  |  

Complete genome sequence of N2-fixing model strain Klebsiella sp. nov. M5al, which produces plant cell wall-degrading enzymes and siderophores.

The bacterial strain M5al is a model strain for studying the molecular genetics of N2-fixation and molecular engineering of microbial production of platform chemicals 1,3-propanediol and 2,3-butanediol. Here, we present the complete genome sequence of the strain M5al, which belongs to a novel species closely related toKlebsiella michiganensis. M5al secretes plant cell wall-degrading enzymes and colonizes rice roots but does not cause soft rot disease. M5al also produces siderophores and contains the gene clusters for synthesis and transport of yersiniabactin which is a critical virulence factor forKlebsiellapathogens in causing human disease. We propose that the model strain M5al can be genetically modified to study bacterial N2-fixation in association with non-legume plants and production of 1,3-propanediol and 2,3-butanediol through degradation of plant cell wall biomass.

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September 22, 2019  |  

The complete replicons of 16 Ensifer meliloti strains offer insights into intra- and inter-replicon gene transfer, transposon-associated loci, and repeat elements.

Ensifer meliloti (formerly Rhizobium meliloti and Sinorhizobium meliloti) is a model bacterium for understanding legume-rhizobial symbioses. The tripartite genome of E. meliloti consists of a chromosome, pSymA and pSymB, and in some instances strain-specific accessory plasmids. The majority of previous sequencing studies have relied on the use of assemblies generated from short read sequencing, which leads to gaps and assembly errors. Here we used PacBio-based, long-read assemblies and were able to assemble, de novo, complete circular replicons. In this study, we sequenced, de novo-assembled and analysed 10 E. meliloti strains. Sequence comparisons were also done with data from six previously published genomes. We identified genome differences between the replicons, including mol% G+C and gene content, nucleotide repeats, and transposon-associated loci. Additionally, genomic rearrangements both within and between replicons were identified, providing insight into evolutionary processes at the structural level. There were few cases of inter-replicon gene transfer of core genes between the main replicons. Accessory plasmids were more similar to pSymA than to either pSymB or the chromosome, with respect to gene content, transposon content and G+C content. In our population, the accessory plasmids appeared to share an open genome with pSymA, which contains many nodulation- and nitrogen fixation-related genes. This may explain previous observations that horizontal gene transfer has a greater effect on the content of pSymA than pSymB, or the chromosome, and why some rhizobia show unstable nodulation phenotypes on legume hosts.

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September 22, 2019  |  

Genomic changes associated with the evolutionary transitions of Nostoc to a plant symbiont.

Cyanobacteria belonging to the genus Nostoc comprise free-living strains and also facultative plant symbionts. Symbiotic strains can enter into symbiosis with taxonomically diverse range of host plants. Little is known about genomic changes associated with evolutionary transition of Nostoc from free-living to plant symbiont. Here, we compared the genomes derived from 11 symbiotic Nostoc strains isolated from different host plants and infer phylogenetic relationships between strains. Phylogenetic reconstructions of 89 Nostocales showed that symbiotic Nostoc strains with a broad host range, entering epiphytic and intracellular or extracellular endophytic interactions, form a monophyletic clade indicating a common evolutionary history. A polyphyletic origin was found for Nostoc strains which enter only extracellular symbioses, and inference of transfer events implied that this trait was likely acquired several times in the evolution of the Nostocales. Symbiotic Nostoc strains showed enriched functions in transport and metabolism of organic sulfur, chemotaxis and motility, as well as the uptake of phosphate, branched-chain amino acids, and ammonium. The genomes of the intracellular clade differ from that of other Nostoc strains, with a gain/enrichment of genes encoding proteins to generate l-methionine from sulfite and pathways for the degradation of the plant metabolites vanillin and vanillate, and of the macromolecule xylan present in plant cell walls. These compounds could function as C-sources for members of the intracellular clade. Molecular clock analysis indicated that the intracellular clade emerged ca. 600 Ma, suggesting that intracellular Nostoc symbioses predate the origin of land plants and the emergence of their extant hosts.

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September 22, 2019  |  

Coordinated regulation of core and accessory genes in the multipartite genome of Sinorhizobium fredii.

Prokaryotes benefit from having accessory genes, but it is unclear how accessory genes can be linked with the core regulatory network when developing adaptations to new niches. Here we determined hierarchical core/accessory subsets in the multipartite pangenome (composed of genes from the chromosome, chromid and plasmids) of the soybean microsymbiont Sinorhizobium fredii by comparing twelve Sinorhizobium genomes. Transcriptomes of two S. fredii strains at mid-log and stationary growth phases and in symbiotic conditions were obtained. The average level of gene expression, variation of expression between different conditions, and gene connectivity within the co-expression network were positively correlated with the gene conservation level from strain-specific accessory genes to genus core. Condition-dependent transcriptomes exhibited adaptive transcriptional changes in pangenome subsets shared by the two strains, while strain-dependent transcriptomes were enriched with accessory genes on the chromid. Proportionally more chromid genes than plasmid genes were co-expressed with chromosomal genes, while plasmid genes had a higher within-replicon connectivity in expression than chromid ones. However, key nitrogen fixation genes on the symbiosis plasmid were characterized by high connectivity in both within- and between-replicon analyses. Among those genes with host-specific upregulation patterns, chromosomal znu and mdt operons, encoding a conserved high-affinity zinc transporter and an accessory multi-drug efflux system, respectively, were experimentally demonstrated to be involved in host-specific symbiotic adaptation. These findings highlight the importance of integrative regulation of hierarchical core/accessory components in the multipartite genome of bacteria during niche adaptation and in shaping the prokaryotic pangenome in the long run.

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September 22, 2019  |  

Nucleotide-binding resistance gene signatures in sugar beet, insights from a new reference genome.

Nucleotide-binding (NB-ARC), leucine-rich-repeat genes (NLRs) account for 60.8% of resistance (R) genes molecularly characterized from plants. NLRs exist as large gene families prone to tandem duplication and transposition, with high sequence diversity among crops and their wild relatives. This diversity can be a source of new disease resistance, but difficulty in distinguishing specific sequences from homologous gene family members hinders characterization of resistance for improving crop varieties. Current genome sequencing and assembly technologies, especially those using long-read sequencing, are improving resolution of repeat-rich genomic regions and clarifying locations of duplicated genes, such as NLRs. Using the conserved NB-ARC domain as a model, 231 tentative NB-ARC loci were identified in a highly contiguous genome assembly of sugar beet, revealing diverged and truncated NB-ARC signatures as well as full-length sequences. The NB-ARC-associated proteins contained NLR resistance gene domains, including TIR, CC, and LRR, as well as other integrated domains. Phylogenetic relationships of partial and complete domains were determined, and patterns of physical clustering in the genome were evaluated. Comparison of sugar beet NB-ARC domains to validated R genes from monocots and eudicots suggested extensive B. vulgaris-specific subfamily expansions. The NLR landscape in the rhizomania resistance conferring Rz region of Chromosome 3 was characterized, identifying 26 NLR-like sequences spanning 20 MB. This work presents the first detailed view of NLR family composition in a member of the Caryophyllales, builds a foundation for additional disease resistance work in B. vulgaris, and demonstrates an additional nucleic-acid-based method for NLR prediction in non-model plant species. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

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September 22, 2019  |  

Evolutionary history of bacteriophages in the genus Paraburkholderia.

The genus Paraburkholderia encompasses mostly environmental isolates with diverse predicted lifestyles. Genome analyses have shown that bacteriophages form a considerable portion of some Paraburkholderia genomes. Here, we analyzed the evolutionary history of prophages across all Paraburkholderia spp. Specifically, we investigated to what extent the presence of prophages and their distribution affect the diversity/diversification of Paraburkholderia spp., as well as to what extent phages coevolved with their respective hosts. Particular attention was given to the presence of CRISPR-Cas arrays as a reflection of past interactions with phages. We thus analyzed 36 genomes of Paraburkholderia spp., including those of 11 new strains, next to those of three Burkholderia species. Most genomes were found to contain at least one full prophage sequence. The highest number was found in Paraburkholderia sp. strain MF2-27; the nine prophages found amount to up to 4% of its genome. Among all prophages, potential moron genes (e.g., DNA adenine methylase) were found that might be advantageous for host cell fitness. Co-phylogenetic analyses indicated the existence of complex evolutionary scenarios between the different Paraburkholderia hosts and their prophages, including short-term co-speciation, duplication, host-switching and phage loss events. Analysis of the CRISPR-Cas systems showed a record of diverse, potentially recent, phage infections. We conclude that, overall, different phages have interacted in diverse ways with their Paraburkholderia hosts over evolutionary time.

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