The Soybean Cyst Nematode Effector Cysteine Protease 1 (CPR1) Targets a Mitochondrial Soybean Branched-Chain Amino Acid Aminotransferase (GmBCAT1).

The soybean cyst nematode (SCN; Heterodera glycines) facilitates infection by secreting a repertoire of effector proteins into host cells to establish a permanent feeding site composed of a syncytium of root cells. Among the diverse proteins secreted by the nematode, we were specifically interested in identifying proteases to pursue our goal of engineering decoy substrates that elicit an immune response when cleaved by an SCN protease. We identified a cysteine protease that we named Cysteine Protease 1 (CPR1), which was predicted to be a secreted effector based on transcriptomic data obtained from SCN esophageal gland cells, presence of a signal peptide, and lack of transmembrane domains. CPR1 is conserved in all isolates of SCN sequenced to date, suggesting it is critical for virulence. Transient expression of CPR1 in Nicotiana benthamiana leaves suppressed cell death induced by a constitutively active nucleotide binding leucine-rich repeat protein, RPS5, indicating that CPR1 inhibits effector-triggered immunity. CPR1 localizes in part to the mitochondria when expressed in planta. Proximity-based labeling in transgenic soybean roots, co-immunoprecipitation, and cleavage assays identified a branched-chain amino acid aminotransferase from soybean (GmBCAT1) as a substrate of CPR1. Consistent with this, GmBCAT1 also localizes to mitochondria. Silencing of the CPR1 transcript in the nematode reduced penetration frequency in soybean roots while the expression of CPR1 in soybean roots enhanced susceptibility. Our data demonstrates that CPR1 is a conserved effector protease with a direct target in soybean roots, highlighting it as a promising candidate for decoy engineering.

Comparative transcriptomic analysis of soybean cyst nematode inbred populations non-adapted or adapted on soybean rhg1-a/Rhg4-mediated resistance.

Soybean cyst nematode (SCN, Heterodera glycines) is most effectively managed through planting resistant soybean cultivars, but the repeated use of the same resistance sources has led to a widespread emergence of virulent SCN populations that can overcome soybean resistance. Resistance to SCN HG type 0 (Race 3) in soybean cultivar Forrest is mediated by an epistatic interaction between the soybean resistance genes rhg1-a and Rhg4. We previously developed two SCN inbred populations by mass-selecting SCN HG type 0 (Race 3) on susceptible and resistant recombinant inbred lines, derived from a cross between Forrest and the SCN-susceptible cultivar Essex, which differ for Rhg4. To identify SCN genes potentially involved in overcoming rhg1-a/Rhg4-mediated resistance, we conducted RNA-sequencing on early parasitic juveniles of these two SCN inbred populations infecting their respective hosts, only to discover a handful of differentially expressed genes (DEGs). However, in a comparison to early parasitic juveniles of an avirulent SCN inbred population infecting a resistant host, we discovered 59 and 171 DEGs uniquely up- or down-regulated in virulent parasitic juveniles adapted on the resistant host. Interestingly, the proteins coded by these 59 DEGs included vitamin B-associated proteins (reduced folate carrier, biotin synthase, and thiamine transporter) and nematode effectors known to play roles in plant defense suppression, suggesting that virulent SCN may exert a heightened transcriptional response to cope with enhanced plant defenses and an altered nutritional status of a resistant soybean host.

Esophageal Gland RNA-Seq Resource of a Virulent and Avirulent Population of the Soybean Cyst Nematode Heterodera glycines.

The soybean cyst nematode Heterodera glycines is the most economically devastating pathogen of soybean in the United States and threatens to become even more damaging through the selection of virulent nematode populations in the field that can overcome natural resistance mechanisms in soybean cultivars. This pathogen, therefore, demands intense transcriptomic/genomic research inquiries into the biology of its parasitic mechanisms. H. glycines delivers effector proteins that are produced in specialized gland cells into the soybean root to enable infection. The study of effector proteins, thus, is particularly promising when exploring novel management options against this pathogen. Here, we announce the availability of a gland cell-specific RNA-seq resource. These data represent an expression snapshot of gland cell activity during early soybean infection of a virulent and an avirulent H. glycines population, providing a unique and highly valuable resource for scientists examining effector biology and nematode virulence.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Shared data science infrastructure for genomics data.

BACKGROUND: Creating a scalable computational infrastructure to analyze the wealth of information contained in data repositories is difficult due to significant barriers in organizing, extracting and analyzing relevant data. Shared data science infrastructures like Boag is needed to efficiently process and parse data contained in large data repositories. The main features of Boag are inspired from existing languages for data intensive computing and can easily integrate data from biological data repositories. RESULTS: As a proof of concept, Boa for genomics, Boag, has been implemented to analyze RefSeq's 153,848 annotation (GFF) and assembly (FASTA) file metadata. Boag provides a massive improvement from existing solutions like Python and MongoDB, by utilizing a domain-specific language that uses Hadoop infrastructure for a smaller storage footprint that scales well and requires fewer lines of code. We execute scripts through Boag to answer questions about the genomes in RefSeq. We identify the largest and smallest genomes deposited, explore exon frequencies for assemblies after 2016, identify the most commonly used bacterial genome assembly program, and address how animal genome assemblies have improved since 2016. Boag databases provide a significant reduction in required storage of the raw data and a significant speed up in its ability to query large datasets due to automated parallelization and distribution of Hadoop infrastructure during computations. CONCLUSIONS: In order to keep pace with our ability to produce biological data, innovative methods are required. The Shared Data Science Infrastructure, Boag, provides researchers a greater access to researchers to efficiently explore data in new ways. We demonstrate the potential of a the domain specific language Boag using the RefSeq database to explore how deposited genome assemblies and annotations are changing over time. This is a small example of how Boag could be used with large biological datasets.

The genome of the soybean cyst nematode (Heterodera glycines) reveals complex patterns of duplications involved in the evolution of parasitism genes.

BACKGROUND: Heterodera glycines, commonly referred to as the soybean cyst nematode (SCN), is an obligatory and sedentary plant parasite that causes over a billion-dollar yield loss to soybean production annually. Although there are genetic determinants that render soybean plants resistant to certain nematode genotypes, resistant soybean cultivars are increasingly ineffective because their multi-year usage has selected for virulent H. glycines populations. The parasitic success of H. glycines relies on the comprehensive re-engineering of an infection site into a syncytium, as well as the long-term suppression of host defense to ensure syncytial viability. At the forefront of these complex molecular interactions are effectors, the proteins secreted by H. glycines into host root tissues. The mechanisms of effector acquisition, diversification, and selection need to be understood before effective control strategies can be developed, but the lack of an annotated genome has been a major roadblock. RESULTS: Here, we use PacBio long-read technology to assemble a H. glycines genome of 738 contigs into 123 Mb with annotations for 29,769 genes. The genome contains significant numbers of repeats (34%), tandem duplicates (18.7 Mb), and horizontal gene transfer events (151 genes). A large number of putative effectors (431 genes) were identified in the genome, many of which were found in transposons. CONCLUSIONS: This advance provides a glimpse into the host and parasite interplay by revealing a diversity of mechanisms that give rise to virulence genes in the soybean cyst nematode, including: tandem duplications containing over a fifth of the total gene count, virulence genes hitchhiking in transposons, and 107 horizontal gene transfers not reported in other plant parasitic nematodes thus far. Through extensive characterization of the H. glycines genome, we provide new insights into H. glycines biology and shed light onto the mystery underlying complex host-parasite interactions. This genome sequence is an important prerequisite to enable work towards generating new resistance or control measures against H. glycines.

The draft genome of Ditylenchus dipsaci.

Ditylenchus dipsaci is a devastating pest to many crops worldwide. We present the first genome sequence for this species, produced with PacBio sequencing and assembled with CANU.Ditylenchus dipsaci is a devastating pest to many crops worldwide. We present the first genome sequence for this species, produced with PacBio sequencing and assembled with CANU.

Rapid proliferation and nucleolar organizer targeting centromeric retrotransposons in cotton.

Centromeric chromatin in most eukaryotes is composed of highly repetitive centromeric retrotransposons and satellite repeats that are highly variable even among closely related species. The evolutionary mechanisms that underlie the rapid evolution of centromeric repeats remain unknown. To obtain insight into the evolution of centromeric repeats following polyploidy, we studied a model diploid progenitor (Gossypium raimondii, D-genome) of the allopolyploid (AD-genome) cottons, G. hirsutum and G. barbadense. Sequence analysis of chromatin-immunoprecipitated DNA showed that the G. raimondii centromeric repeats originated from retrotransposon-related sequences. Comparative analysis showed that nine of the 10 analyzed centromeric repeats were absent from the centromeres in the A-genome and related diploid species (B-, F- and G-genomes), indicating that they colonized the centromeres of D-genome lineage after the divergence of the A- and D- ancestral species or that they were ancestrally retained prior to the origin of Gossypium. Notably, six of the nine repeats were present in both the A- and D-subgenomes in tetraploid G. hirsutum, and increased in abundance in both subgenomes. This finding suggests that centromeric repeats may spread and proliferate between genomes subsequent to polyploidization. Two repeats, Gr334 and Gr359 occurred in both the centromeres and nucleolar organizer regions (NORs) in D- and AD-genome species, yet localized to just the NORs in A-, B-, F-, and G-genome species. Contained within is a story of an established centromeric repeat that is eliminated and allopolyploidization provides an opportunity for reinvasion and reestablishment, which broadens our evolutionary understanding behind the cycles of centromeric repeat establishment and targeting.

In vivo modification of a maize engineered minichromosome.

Engineered minichromosomes provide efficient platforms for stacking transgenes in crop plants. Methods for modifying these chromosomes in vivo are essential for the development of customizable systems for the removal of selection genes or other sequences and for the addition of new genes. Previous studies have demonstrated that Cre, a site-specific recombinase, could be used to modify lox sites on transgenes on maize minichromosomes; however, these studies demonstrated somatic recombination only, and modified minichromosomes could not be recovered. We describe the recovery of an engineered chromosome composed of little more than a centromere plus transgene that was derived by telomere-mediated truncation. We used the fiber fluorescence in situ hybridization technique and detected a transgene on the minichromosome inserted among stretches of CentC centromere repeats, and this insertion was large enough to suggest a tandem insertion. By crossing the minichromosome to a plant expressing Cre-recombinase, the Bar selection gene was removed, leaving behind a single loxP site. This study demonstrates that engineered chromosomes can be modified in vivo using site-specific recombinases, a demonstration essential to the development of amendable chromosome platforms in plants.

CenH3 evolution in diploids and polyploids of three angiosperm genera.

BACKGROUND: Centromeric DNA sequences alone are neither necessary nor sufficient for centromere specification. The centromere specific histone, CenH3, evolves rapidly in many species, perhaps as a coevolutionary response to rapidly evolving centromeric DNA. To gain insight into CenH3 evolution, we characterized patterns of nucleotide and protein diversity among diploids and allopolyploids within three diverse angiosperm genera, Brassica, Oryza, and Gossypium (cotton), with a focus on evidence for diversifying selection in the various domains of the CenH3 gene. In addition, we compare expression profiles and alternative splicing patterns for CenH3 in representatives of each genus. RESULTS: All three genera retain both duplicated CenH3 copies, while Brassica and Gossypium exhibit pronounced homoeologous expression level bias. Comparisons among genera reveal shared and unique aspects of CenH3 evolution, variable levels of diversifying selection in different CenH3 domains, and that alternative splicing contributes significantly to CenH3 diversity. CONCLUSIONS: Since the N terminus is subject to diversifying selection but the DNA binding domains do not appear to be, rapidly evolving centromere sequences are unlikely to be the primary driver of CenH3 sequence diversification. At present, the functional explanation for the diversity generated by both conventional protein evolution in the N terminal domain, as well as alternative splicing, remains unexplained.

A chromosome-level genome assembly of the disco clam, Ctenoides ales.

The bivalve subclass Pteriomorphia, which includes the economically important scallops, oysters, mussels, and ark clams, exhibits extreme ecological, morphological, and behavioral diversity. Among this diversity are five morphologically distinct eye types, making Pteriomorphia an excellent setting to explore the molecular basis for the evolution of novel traits. Of pteriomorphian bivalves, Limida is the only order lacking genomic resources, greatly limiting the potential phylogenomic analyses related to eyes and phototransduction. Here, we present a limid genome assembly, the disco clam, Ctenoides ales, which is characterized by invaginated eyes, exceptionally long tentacles, and a flashing light display. This genome assembly was constructed with PacBio long reads and Dovetail Omni-CTM proximity-ligation sequencing. The final assembly is ∼2.3Gb and over 99% of the total length is contained in 18 pseudomolecule scaffolds. We annotated 41,064 protein coding genes and report a BUSCO completeness of 91.9% for metazoa_obd10. Additionally, we report a complete and annotated mitochondrial genome, which also had been lacking from Limida. The ∼20Kb mitogenome has 12 protein coding genes, 22 tRNAs, 2 rRNA genes, and a 1,589 bp duplicated sequence containing the origin of replication. The C. ales nuclear genome size is substantially larger than other pteriomorphian genomes, mainly accounted for by transposable element sequences. We inventoried the genome for opsins, the signaling proteins that initiate phototransduction, and found that, unlike its closest eyed-relatives, the scallops, C. ales lacks duplication of the rhabdomeric Gq-protein coupled opsin that is typically used for invertebrate vision. In fact, C. ales has uncharacteristically few opsins relative to the other pteriomorphian families, all of which have unique expansions of xenopsins, a recently discovered opsin subfamily. This chromosome-level assembly, along with the mitogenome, will be valuable resources for comparative genomics and phylogenetics in bivalves and particularly for the understudied but charismatic limids.

Chromosome-Level Assembly and Annotation of the Genome of the Endangered Giant Patagonian Bumble Bee Bombus dahlbomii.

This article describes a genome assembly and annotation for Bombus dahlbomii, the giant Patagonian bumble bee. DNA from a single, haploid male collected in Argentina was used for PacBio (HiFi) sequencing, and Hi-C technology was then used to map chromatin contacts. Using Juicer and manual curation, the genome was scaffolded into 18 main pseudomolecules, representing a high-quality, near chromosome-level assembly. The sequenced genome size is estimated at 265 Mb. The genome was annotated based on RNA sequencing data of another male from Argentina, and BRAKER3 produced 15,767 annotated genes. The genome and annotation show high completeness, with >95% BUSCO scores for both the genome and annotated genes (based on conserved genes from Hymenoptera). This genome provides a valuable resource for studying the biology of this iconic and endangered species, as well as for understanding the impacts of its decline and designing strategies for its preservation.

Multiple maize minichromosomes in meiosis.

In this study, four distinct minichromosomes derived from the maize B chromosome, were increased in copy number using the B chromosome's accumulation mechanism, namely nondisjunction at the second pollen mitosis and preferential fertilization of the egg. These minichromosomes provide the unique opportunity to examine the behavior of many copies of a single chromosome in an otherwise diploid background. While multiple copies were associated in multivalent configurations, they often dissociated into univalents or bivalents prior to metaphase I. The largest mini's behavior closely resembled the progenitor B chromosome, but all smaller chromosomes showed failure of sister chromatid cohesion. In addition to the meiotic behavior, we observed many anomalies of univalent behavior and possible heterochromatic fusions of B repeat associated heterochromatin.

The Complete Nucleotide Sequence of Barley Yellow Dwarf Virus-PAV from Wheat in Turkey.

We report the sequence of an assembled genome of Barley yellow dwarf virus-PAV (BYDV-PAV) from Turkey. This 5,672 nucleotide RNA encodes seven known open reading frames and a possible eighth. This genome from wheat is closely related to BYDV-PAVs in Pakistan, Brazil, and Australia, including one sequenced 34 years ago.

The genome and lifestage-specific transcriptomes of a plant-parasitic nematode and its host reveal susceptibility genes involved in trans-kingdom synthesis of vitamin B5.

Plant-parasitic nematodes are a major threat to crop production in all agricultural systems. The scarcity of classical resistance genes highlights a pressing need to find new ways to develop nematode-resistant germplasm. Here, we sequence and assemble a high-quality phased genome of the model cyst nematode Heterodera schachtii to provide a platform for the first system-wide dual analysis of host and parasite gene expression over time, covering all major parasitism stages. Analysis of the hologenome of the plant-nematode infection site identified metabolic pathways that were incomplete in the parasite but complemented by the host. Using a combination of bioinformatic, genetic, and biochemical approaches, we show that a highly atypical completion of vitamin B5 biosynthesis by the parasitic animal, putatively enabled by a horizontal gene transfer from a bacterium, is required for full pathogenicity. Knockout of either plant-encoded or now nematode-encoded steps in the pathway significantly reduces parasitic success. Our experiments establish a reference for cyst nematodes, further our understanding of the evolution of plant-parasitism by nematodes, and show that congruent differential expression of metabolic pathways in the infection hologenome represents a new way to find nematode susceptibility genes. The approach identifies genome-editing-amenable targets for future development of nematode-resistant crops.

Recovery of a telomere-truncated chromosome via a compensating translocation in maize.

Maize-engineered minichromosomes are easily recovered from telomere-truncated B chromosomes but are rarely recovered from A chromosomes. B chromosomes lack known genes, and their truncation products are tolerated and transmitted during meiosis. In contrast, deficiency gametes resulting from truncated A chromosomes prevent their transmission. We report here a de novo compensating translocation that permitted recovery of a large truncation of chromosome 1 in maize. The truncation (trunc-1) and translocation with chromosome 6 (super-6) occurred during telomere-mediated truncation experiments and were characterized using single-gene fluorescent in situ hybridization (FISH) probes. The truncation contained a transgene signal near the end of the broken chromosome and transmitted together with the compensating translocation as a heterozygote to approximately 41%-55% of progeny. Transmission as an addition chromosome occurred in ~15% of progeny. Neither chromosome transmitted through pollen. Transgene expression (Bar) cosegregated with trunc-1 transcriptionally and phenotypically. Meiosis in T1 plants revealed eight bivalents and one tetravalent chain composed of chromosome 1, trunc-1, chromosome 6, and super-6 in diplotene and diakinesis. Our data suggest that de novo compensating translocations allow recovery of truncated A chromosomes by compensating deficiency in female gametes and by affecting chromosome pairing and segregation. The truncated chromosome can be maintained as an extra chromosome or together with the super-6 as a heterozygote.

A chromosomal assembly of the soybean cyst nematode genome.

The soybean cyst nematode (Heterodera glycines) is a sedentary plant parasite that exceeds billion USD annually in yield losses. This problem is exacerbated by H. glycines populations overcoming the limited sources of natural resistance in soybean and by the lack of effective and safe alternative treatments. Although there are genetic determinants that render soybeans resistant to nematode genotypes, resistant soybeans are increasingly ineffective because their multiyear usage has selected for virulent H. glycines populations. Successful H. glycines infection relies on the comprehensive re-engineering of soybean root cells into a syncytium, as well as the long-term suppression of host defences to ensure syncytial viability. At the forefront of these complex molecular interactions are effectors, the proteins secreted by H. glycines into host root tissues. The mechanisms that control genomic effector acquisition, diversification, and selection are important insights needed for the development of essential novel control strategies. As a foundation to obtain this understanding, we created a nine-scaffold, 158 Mb pseudomolecule assembly of the H. glycines genome using PacBio, Chicago, and Hi-C sequencing. A Mikado consensus gene prediction produced an annotation of 22,465 genes using short- and long-read expression data. To evaluate assembly and annotation quality, we cross-examined synteny among H. glycines assemblies, and compared BUSCO across related species. To describe the predicted proteins involved in H. glycines' secretory pathway, we contrasted expression between preparasitic and parasitic stages with functional gene information. Here, we present the results from our assembly and annotation of the H. glycines genome and contribute this resource to the scientific community.

A pseudomolecule assembly of the Rocky Mountain elk genome.

Rocky Mountain elk (Cervus canadensis) populations have significant economic implications to the cattle industry, as they are a major reservoir for Brucella abortus in the Greater Yellowstone area. Vaccination attempts against intracellular bacterial diseases in elk populations have not been successful due to a negligible adaptive cellular immune response. A lack of genomic resources has impeded attempts to better understand why vaccination does not induce protective immunity. To overcome this limitation, PacBio, Illumina, and Hi-C sequencing with a total of 686-fold coverage was used to assemble the elk genome into 35 pseudomolecules. A robust gene annotation was generated resulting in 18,013 gene models and 33,422 mRNAs. The accuracy of the assembly was assessed using synteny to the red deer and cattle genomes identifying several chromosomal rearrangements, fusions and fissions. Because this genome assembly and annotation provide a foundation for genome-enabled exploration of Cervus species, we demonstrate its utility by exploring the conservation of immune system-related genes. We conclude by comparing cattle immune system-related genes to the elk genome, revealing eight putative gene losses in elk.

Transcriptomic Analysis of Shiga Toxin-Producing Escherichia coli during Initial Contact with Cattle Colonic Explants.

Foodborne pathogens are a public health threat globally. Shiga toxin-producing Escherichia coli (STEC), particularly O26, O111, and O157 STEC, are often associated with foodborne illness in humans. To create effective preharvest interventions, it is critical to understand which factors STEC strains use to colonize the gastrointestinal tract of cattle, which serves as the reservoir for these pathogens. Several colonization factors are known, but little is understood about initial STEC colonization factors. Our objective was to identify these factors via contrasting gene expression between nonpathogenic E. coli and STEC. Colonic explants were inoculated with nonpathogenic E. coli strain MG1655 or STEC strains (O26, O111, or O157), bacterial colonization levels were determined, and RNA was isolated and sequenced. STEC strains adhered to colonic explants at numerically but not significantly higher levels compared to MG1655. After incubation with colonic explants, flagellin (fliC) was upregulated (log2 fold-change = 4.0, p < 0.0001) in O157 STEC, and collectively, Lon protease (lon) was upregulated (log2 fold-change = 3.6, p = 0.0009) in STEC strains compared to MG1655. These results demonstrate that H7 flagellum and Lon protease may play roles in early colonization and could be potential targets to reduce colonization in cattle.

Modulating Pathway Performance by Perturbing Local Genetic Context.

Combinatorial engineering is a preferred strategy for attaining optimal pathway performance. Previous endeavors have been concentrated on regulatory elements (e.g., promoters, terminators, and ribosomal binding sites) and/or open reading frames. Accumulating evidence indicates that noncoding DNA sequences flanking a transcriptional unit on the genome strongly impact gene expression. Here, we sought to mimic the effect imposed on expression cassettes by the genome. We created variants of the model yeast Saccharomyces cerevisiae with significantly improved fluorescence or cellobiose consumption rate by randomizing the sequences adjacent to the GFP expression cassette or the cellobiose-utilization pathway, respectively. Interestingly, nucleotide specificity was observed at certain positions and showed to be essential for achieving optimal cellobiose assimilation. Further characterization suggested that the modulation effects of the short sequences flanking the expression cassettes could be potentially mediated by remodeling DNA packaging and/or recruiting transcription factors. Collectively, these results indicate that the often-overlooked contiguous DNA sequences can be exploited to rapidly achieve balanced pathway expression, and the corresponding approach could be easily stacked with other combinatorial engineering strategies.