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September 22, 2019  |  

Comparative genomic analyses reveal the features for adaptation to nematodes in fungi.

Nematophagous (NP) fungi are ecologically important components of the soil microbiome in natural ecosystems. Esteya vermicola (Ev) has been reported as a NP fungus with a poorly understood evolutionary history and mechanism of adaptation to parasitism. Furthermore, NP fungal genomic basis of lifestyle was still unclear. We sequenced and annotated the Ev genome (34.2 Mbp) and integrated genetic makeup and evolution of pathogenic genes to investigate NP fungi. The results revealed that NP fungi had some abundant pathogenic genes corresponding to their niche. A number of gene families involved in pathogenicity were expanded, and some pathogenic orthologous genes underwent positive selection. NP fungi with diverse morphological features exhibit similarities of evolutionary convergence in attacking nematodes, but their genetic makeup and microscopic mechanism are different. Endoparasitic NP fungi showed similarity in large number of transporters and secondary metabolite coding genes. Noteworthy, expanded families of transporters and endo-beta-glucanase implied great genetic potential of Ev in quickly perturbing nematode metabolism and parasitic behavior. These results facilitate our understanding of NP fungal genomic features for adaptation to nematodes and lay a solid theoretical foundation for further research and application.© The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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September 22, 2019  |  

An ancient integration in a plant NLR is maintained as a trans-species polymorphism

Plant immune receptors are under constant selective pressure to maintain resistance to plant pathogens. Nucleotide-binding leucine-rich repeat (NLR) proteins are one class of cytoplasmic immune receptors whose genes commonly show signatures of adaptive evolution. While it is known that balancing selection contributes to maintaining high intraspecific allelic diversity, the evolutionary mechanism that influences the transmission of alleles during speciation remains unclear. The barley Mla locus has over 30 described alleles conferring isolate-specific resistance to barley powdery mildew and contains three NLR families (RGH1, RGH2, and RGH3). We discovered (using sequence capture and RNAseq) the presence of a novel integrated Exo70 domain in RGH2 in the Mla3 haplotype. Allelic variation across barley accessions includes presence/absence of the integrated domain in RGH2. Expanding our search to several Poaceae species, we found shared interspecific conservation in the RGH2-Exo70 integration. We hypothesise that balancing selection has maintained allelic variation at Mla as a trans-species polymorphism over 24 My, thus contributing to and preserving interspecific allelic diversity during speciation.

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September 22, 2019  |  

Nuclear and mitochondrial genomes of the hybrid fungal plant pathogen Verticillium longisporum display a mosaic structure

Allopolyploidization, genome duplication through interspecific hybridization, is an important evolutionary mechanism that can enable organisms to adapt to environmental changes or stresses. This increased adaptive potential of allopolyploids can be particularly relevant for plant pathogens in their quest for host immune response evasion. Allodiploidization likely caused the shift in host range of the fungal pathogen plant Verticillium longisporum, as V. longisporum mainly infects Brassicaceae plants in contrast to haploid Verticillium spp. In this study, we investigated the allodiploid genome structure of V. longisporum and its evolution in the hybridization aftermath. The nuclear genome of V. longisporum displays a mosaic structure, as numerous contigs consists of sections of both parental origins. V. longisporum encountered extensive genome rearrangements, whereas the contribution of gene conversion is negligible. Thus, the mosaic genome structure mainly resulted from genomic rearrangements between parental chromosome sets. Furthermore, a mosaic structure was also found in the mitochondrial genome, demonstrating its bi-parental inheritance. In conclusion, the nuclear and mitochondrial genomes of V. longisporum parents interacted dynamically in the hybridization aftermath. Conceivably, novel combinations of DNA sequence of different parental origin facilitated genome stability after hybridization and consecutive niche adaptation of V. longisporum.

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September 22, 2019  |  

Identification of candidate genes at the Dp-fl locus conferring resistance against the rosy apple aphid Dysaphis plantaginea

The cultivated apple is susceptible to several pests including the rosy apple aphid (RAA; Dysaphis plantaginea Passerini), control of which is mainly based on chemical treatments. A few cases of resistance to aphids have been described in apple germplasm resources, laying the basis for the development of new resistant cultivars by breeding. The cultivar ‘Florina’ is resistant to RAA, and recently, the Dp-fl locus responsible for its resistance was mapped on linkage group 8 of the apple genome. In this paper, a chromosome walking approach was performed by using a ‘Florina’ bacterial artificial chromosome (BAC) library. The walking started from the available tightly linked molecular markers flanking the resistance region. Various walking steps were performed in order to identify the minimum tiling path of BAC clones covering the Dp-fl region from both the “resistant” and “susceptible” chromosomes of ‘Florina’. A genomic region of about 279 Kb encompassing the Dp-fl resistance locus was fully sequenced by the PacBio technology. Through the development of new polymorphic markers, the mapping interval around the resistance locus was narrowed down to a physical region of 95 Kb. The annotation of this sequence resulted in the identification of four candidate genes putatively involved in the RAA resistance response.

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September 22, 2019  |  

By land, air, and sea: hemipteran diversity through the genomic lens

Thanks to a recent spate of sequencing projects, the Hemiptera are the first hemimetabolous insect order to achieve a critical mass of species with sequenced genomes, establishing the basis for comparative genomics of the bugs. However, as the most speciose hemimetabolous order, there is still a vast swathe of the hemipteran phylogeny that awaits genomic representation across subterranean, terrestrial, and aquatic habitats, and with lineage-specific and developmentally plastic cases of both wing polyphenisms and flightlessness. In this review, we highlight opportunities for taxonomic sampling beyond obvious pest species candidates, motivated by intriguing biological features of certain groups as well as the rich research tradition of ecological, physiological, developmental, and particularly cytogenetic investigation that spans the diversity of the Hemiptera.

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September 22, 2019  |  

Genome sequences of Chlorella sorokiniana UTEX 1602 and Micractinium conductrix SAG 241.80: implications to maltose excretion by a green alga.

Green algae represent a key segment of the global species capable of photoautotrophic-driven biological carbon fixation. Algae partition fixed-carbon into chemical compounds required for biomass, while diverting excess carbon into internal storage compounds such as starch and lipids or, in certain cases, into targeted extracellular compounds. Two green algae were selected to probe for critical components associated with sugar production and release in a model alga. Chlorella sorokiniana UTEX 1602 – which does not release significant quantities of sugars to the extracellular space – was selected as a control to compare with the maltose-releasing Micractinium conductrix SAG 241.80 – which was originally isolated from an endosymbiotic association with the ciliate Paramecium bursaria. Both strains were subjected to three sequencing approaches to assemble their genomes and annotate their genes. This analysis was further complemented with transcriptional studies during maltose release by M. conductrix SAG 241.80 versus conditions where sugar release is minimal. The annotation revealed that both strains contain homologs for the key components of a putative pathway leading to cytosolic maltose accumulation, while transcriptional studies found few changes in mRNA levels for the genes associated with these established intracellular sugar pathways. A further analysis of potential sugar transporters found multiple homologs for SWEETs and tonoplast sugar transporters. The analysis of transcriptional differences revealed a lesser and more measured global response for M. conductrix SAG 241.80 versus C. sorokiniana UTEX 1602 during conditions resulting in sugar release, providing a catalog of genes that might play a role in extracellular sugar transport.© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

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September 22, 2019  |  

Complete genome sequence of Bacillus velezensis 157 isolated from Eucommia ulmoides with pathogenic bacteria inhibiting and lignocellulolytic enzymes production by SSF.

Bacillus velezensis 157 was isolated from the bark of Eucommia ulmoides, and exhibited antagonistic activity against a broad spectrum of pathogenic bacteria and fungi. Moreover, B. velezensis 157 also showed various lignocellulolytic activities including cellulase, xylanase, a-amylase, and pectinase, which had the ability of using the agro-industrial waste (soybean meal, wheat bran, sugarcane bagasse, wheat straw, rice husk, maize flour and maize straw) under solid-state fermentation and obtained several industrially valuable enzymes. Soybean meal appeared to be the most efficient substrate for the single fermentation of B. velezensis 157. Highest yield of pectinase (19.15 ± 2.66 U g-1), cellulase (46.69 ± 1.19 U g-1) and amylase (2097.18 ± 15.28 U g-1) was achieved on untreated soybean meal. Highest yield of xylanase (22.35 ± 2.24 U g-1) was obtained on untreated wheat bran. Here, we report the complete genome sequence of the B. velezensis 157, composed of a circular 4,013,317 bp chromosome with 3789 coding genes and a G + C content of 46.41%, one circular 8439 bp plasmid and a G + C content of 40.32%. The genome contained a total of 8 candidate gene clusters (bacillaene, difficidin, macrolactin, butirosin, bacillibactin, bacilysin, fengycin and surfactin), and dedicates over 15.8% of the whole genome to synthesize secondary metabolite biosynthesis. In addition, the genes encoding enzymes involved in degradation of cellulose, xylan, lignin, starch, mannan, galactoside and arabinan were found in the B. velezensis 157 genome. Thus, the study of B. velezensis 157 broadened that B. velezensis can not only be used as biocontrol agents, but also has potentially a wide range of applications in lignocellulosic biomass conversion.

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September 22, 2019  |  

Reference quality genome assemblies of three Parastagonospora nodorum isolates differing in virulence on wheat.

Parastagonospora nodorum, the causal agent of Septoria nodorum blotch in wheat, has emerged as a model necrotrophic fungal organism for the study of host-microbe interactions. To date, three necrotrophic effectors have been identified and characterized from this pathogen, including SnToxA, SnTox1, and SnTox3. Necrotrophic effector identification was greatly aided by the development of a draft genome of Australian isolate SN15 via Sanger sequencing, yet it remained largely fragmented. This research presents the development of nearly finished genomes of P. nodorum isolates Sn4, Sn2000, and Sn79-1087 using long-read sequencing technology. RNAseq analysis of isolate Sn4, consisting of eight time points covering various developmental and infection stages, mediated the annotation of 13,379 genes. Analysis of these genomes revealed large-scale polymorphism between the three isolates, including the complete absence of contig 23 from isolate Sn79-1087, and a region of genome expansion on contig 10 in isolates Sn4 and Sn2000. Additionally, these genomes exhibit the hallmark characteristics of a “two-speed” genome, being partitioned into two distinct GC-equilibrated and AT-rich compartments. Interestingly, isolate Sn79-1087 contains a lower proportion of AT-rich segments, indicating a potential lack of evolutionary hotspots. These newly sequenced genomes, consisting of telomere-to-telomere assemblies of nearly all 23 P. nodorum chromosomes, provide a robust foundation for the further examination of effector biology and genome evolution. Copyright © 2018 Richards et al.

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September 22, 2019  |  

Induced salt tolerance of perennial ryegrass by a novel bacterium strain from the rhizosphere of a desert shrub Haloxylon ammodendron.

Drought and soil salinity reduce agricultural output worldwide. Plant-growth-promoting rhizobacteria (PGPR) can enhance plant growth and augment plant tolerance to biotic and abiotic stresses.Haloxylon ammodendron, a C4 perennial succulent xerohalophyte shrub with excellent drought and salt tolerance, is naturally distributed in the desert area of northwest China. In our previous work, a bacterium strain numbered as M30-35 was isolated from the rhizosphere ofH. ammodendronin Tengger desert, Gansu province, northwest China. In current work, the effects of M30-35 inoculation on salt tolerance of perennial ryegrass were evaluated and its genome was sequenced to identify genes associated with plant growth promotion. Results showed that M30-35 significantly enhanced growth and salt tolerance of perennial ryegrass by increasing shoot fresh and dry weights, chlorophyll content, root volume, root activity, leaf catalase activity, soluble sugar and proline contents that contributed to reduced osmotic potential, tissue K? content and K?/Na? ratio, while decreasing malondialdehyde (MDA) content and relative electric conductivity (REC), especially under higher salinity. The genome of M30-35 contains 4421 protein encoding genes, 12 rRNA, 63 tRNA-encoding genes and four rRNA operons. M30-35 was initially classified as a new species inPseudomonasand named asPseudomonassp. M30-35. Thirty-four genes showing homology to genes associated with PGPR traits and abiotic stress tolerance were identified inPseudomonassp. M30-35 genome, including 12 related to insoluble phosphorus solubilization, four to auxin biosynthesis, four to other process of growth promotion, seven to oxidative stress alleviation, four to salt and drought tolerance and three to cold and heat tolerance. Further study is needed to clarify the correlation between these genes from M30-35 and the salt stress alleviation of inoculated plants under salt stress. Overall, our research indicated that desert shrubs appear rich in PGPRs that can help important crops tolerate abiotic stress.

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September 22, 2019  |  

Vegetative compatibility groups partition variation in the virulence of Verticillium dahliae on strawberry.

Verticillium dahliae infection of strawberry (Fragaria x ananassa) is a major cause of disease-induced wilting in soil-grown strawberries across the world. To understand what components of the pathogen are affecting disease expression, the presence of the known effector VdAve1 was screened in a sample of Verticillium dahliae isolates. Isolates from strawberry were found to contain VdAve1 and were divided into two major clades, based upon their vegetative compatibility groups (VCG); no UK strawberry isolates contained VdAve1. VC clade was strongly related to their virulence levels. VdAve1-containing isolates pathogenic on strawberry were found in both clades, in contrast to some recently published findings. On strawberry, VdAve1-containing isolates had significantly higher virulence during early infection, which diminished in significance as the infection progressed. Transformation of a virulent non-VdAve1 containing isolate, with VdAve1 was found neither to increase nor decrease virulence when inoculated on a susceptible strawberry cultivar. There are therefore virulence factors that are epistatic to VdAve1 and potentially multiple independent routes to high virulence on strawberry in V. dahliae lineages. Genome sequencing a subset of isolates across the two VCGs revealed that isolates were differentiated at the whole genome level and contained multiple changes in putative effector content, indicating that different clonal VCGs may have evolved different strategies for infecting strawberry, leading to different virulence levels in pathogenicity tests. It is therefore important to consider both clonal lineage and effector complement as the adaptive potential of each lineage will differ, even if they contain the same race determining effector.

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September 22, 2019  |  

Rhizospheric microbial communities are driven by Panax ginseng at different growth stages and biocontrol bacteria alleviates replanting mortality

The cultivation of Panax plants is hindered by replanting problems, which may be caused by plant-driven changes in the soil microbial community. Inoculation with microbial antagonists may efficiently alleviate replanting issues. Through high-throughput sequencing, this study revealed that bacterial diversity decreased, whereas fungal diversity increased, in the rhizosphere soils of adult ginseng plants at the root growth stage under different ages. Few microbial community, such as Luteolibacter, Cytophagaceae, Luteibacter, Sphingomonas, Sphingomonadaceae, and Zygomycota, were observed; the relative abundance of microorganisms, namely, Brevundimonas, Enterobacteriaceae, Pandoraea, Cantharellales, Dendryphion, Fusarium, and Chytridiomycota, increased in the soils of adult ginseng plants compared with those in the soils of 2-year-old seedlings. Bacillus subtilis 50-1, a microbial antagonist against the pathogenic Fusarium oxysporum, was isolated through a dual culture technique. These bacteria acted with a biocontrol efficacy of 67.8%. The ginseng death rate and Fusarium abundance decreased by 63.3% and 46.1%, respectively, after inoculation with B. subtilis 50-1. Data revealed that microecological degradation could result from ginseng-driven changes in rhizospheric microbial communities; these changes are associated with the different ages and developmental stages of ginseng plants. Biocontrol using microbial antagonists alleviated the replanting problem.

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September 22, 2019  |  

Deciphering lignocellulose deconstruction by the white rot fungus Irpex lacteus based on genomic and transcriptomic analyses.

Irpex lacteus is one of the most potent white rot fungi for biological pretreatment of lignocellulose for second biofuel production. To elucidate the underlying molecular mechanism involved in lignocellulose deconstruction, genomic and transcriptomic analyses were carried out for I. lacteus CD2 grown in submerged fermentation using ball-milled corn stover as the carbon source.Irpex lacteus CD2 efficiently decomposed 74.9% lignin, 86.3% cellulose, and 83.5% hemicellulose in corn stover within 9 days. Manganese peroxidases were rapidly induced, followed by accumulation of cellulase and hemicellulase. Genomic analysis revealed that I. lacteus CD2 possessed a complete set of lignocellulose-degrading enzyme system composed mainly of class II peroxidases, dye-decolorizing peroxidases, auxiliary enzymes, and 182 glycoside hydrolases. Comparative transcriptomic analysis substantiated the notion of a selection mode of degradation. These analyses also suggested that free radicals, derived either from MnP-organic acid interplay or from Fenton reaction involving Fe2+ and H2O2, could play an important role in lignocellulose degradation.The selective strategy employed by I. lacteus CD2, in combination with low extracellular glycosidases cleaving plant cell wall polysaccharides into fermentable sugars, may account for high pretreatment efficiency of I. lacteus. Our study also hints the importance of free radicals for future designing of novel, robust lignocellulose-degrading enzyme cocktails.

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September 22, 2019  |  

Targeted sequencing by gene synteny, a new strategy for polyploid species: sequencing and physical structure of a complex sugarcane region.

Sugarcane exhibits a complex genome mainly due to its aneuploid nature and high ploidy level, and sequencing of its genome poses a great challenge. Closely related species with well-assembled and annotated genomes can be used to help assemble complex genomes. Here, a stable quantitative trait locus (QTL) related to sugar accumulation in sorghum was successfully transferred to the sugarcane genome. Gene sequences related to this QTL were identified in silico from sugarcane transcriptome data, and molecular markers based on these sequences were developed to select bacterial artificial chromosome (BAC) clones from the sugarcane variety SP80-3280. Sixty-eight BAC clones containing at least two gene sequences associated with the sorghum QTL were sequenced using Pacific Biosciences (PacBio) technology. Twenty BAC sequences were found to be related to the syntenic region, of which nine were sufficient to represent this region. The strategy we propose is called “targeted sequencing by gene synteny,” which is a simpler approach to understanding the genome structure of complex genomic regions associated with traits of interest.

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September 22, 2019  |  

Secretome analysis identifies potential pathogenicity/virulence factors of Tilletia indica, a quarantined fungal pathogen inciting Karnal bunt disease in wheat.

Tilletia indica is a smut fungus that incites Karnal bunt in wheat. It has been considered as quarantine pest in more than 70 countries. Despite its quarantine significance, there is meager knowledge regarding the molecular mechanisms of disease pathogenesis. Moreover, various disease management strategies have proven futile. Development of effective disease management strategy requires identification of pathogenicity/virulence factors. With this aim, the present study was conducted to compare the secretomes of T. indica isolates, that is, highly (TiK) and low (TiP) virulent isolates. About 120 and 95 protein spots were detected reproducibly in TiK and TiP secretome gel images. Nineteen protein spots, which were consistently observed as upregulated/differential in the secretome of TiK isolate, were selected for their identification by MALDI-TOF/TOF. Identified proteins exhibited homology with fungal proteins playing important role in fungal adhesion, penetration, invasion, protection against host-derived reactive oxygen species, production of virulence factors, cellular signaling, and degradation of host cell wall proteins and antifungal proteins. These results were complemented with T. indica genome sequence leading to identification of candidate pathogenicity/virulence factors homologs that were further subjected to sequence- and structure-based functional annotation. Thus, present study reports the first comparative secretome analysis of T. indica for identification of pathogenicity/virulence factors. This would provide insights into pathogenic mechanisms of T. indica and aid in devising effective disease management strategies.© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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September 22, 2019  |  

Comparative genomics of smut pathogens: Insights from orphans and positively selected genes into host specialization.

Host specialization is a key evolutionary process for the diversification and emergence of new pathogens. However, the molecular determinants of host range are poorly understood. Smut fungi are biotrophic pathogens that have distinct and narrow host ranges based on largely unknown genetic determinants. Hence, we aimed to expand comparative genomics analyses of smut fungi by including more species infecting different hosts and to define orphans and positively selected genes to gain further insights into the genetics basis of host specialization. We analyzed nine lineages of smut fungi isolated from eight crop and non-crop hosts: maize, barley, sugarcane, wheat, oats, Zizania latifolia (Manchurian rice), Echinochloa colona (a wild grass), and Persicaria sp. (a wild dicot plant). We assembled two new genomes: Ustilago hordei (strain Uhor01) isolated from oats and U. tritici (strain CBS 119.19) isolated from wheat. The smut genomes were of small sizes, ranging from 18.38 to 24.63 Mb. U. hordei species experienced genome expansions due to the proliferation of transposable elements and the amount of these elements varied among the two strains. Phylogenetic analysis confirmed that Ustilago is not a monophyletic genus and, furthermore, detected misclassification of the U. tritici specimen. The comparison between smut pathogens of crop and non-crop hosts did not reveal distinct signatures, suggesting that host domestication did not play a dominant role in shaping the evolution of smuts. We found that host specialization in smut fungi likely has a complex genetic basis: different functional categories were enriched in orphans and lineage-specific selected genes. The diversification and gain/loss of effector genes are probably the most important determinants of host specificity.

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