A mRNA Vaccine Encoding for a RBD 60-mer Nanoparticle Elicits Neutralizing Antibodies and Protective Immunity Against the SARS-CoV-2 Delta Variant in Transgenic K18-hACE2 Mice.

Two years into the COVID-19 pandemic there is still a need for vaccines to effectively control the spread of novel SARS-CoV-2 variants and associated cases of severe disease. Here we report a messenger RNA vaccine directly encoding for a nanoparticle displaying 60 receptor binding domains (RBDs) of SARS-CoV-2 that acts as a highly effective antigen. A construct encoding the RBD of the Delta variant elicits robust neutralizing antibody response, and also provides protective immunity against the Delta variant in a widely used transgenic mouse model. We ultimately find that the proposed mRNA RBD nanoparticle-based vaccine provides a flexible platform for rapid development and will likely be of great value in combatting current and future SARS-CoV-2 variants of concern.

Heterodera glycines utilizes promiscuous spliced leaders and demonstrates a unique preference for a species-specific spliced leader over C. elegans SL1.

Spliced leader trans-splicing (SLTS) plays a part in the maturation of pre-mRNAs in select species across multiple phyla but is particularly prevalent in Nematoda. The role of spliced leaders (SL) within the cell is unclear and an accurate assessment of SL occurrence within an organism is possible only after extensive sequencing data are available, which is not currently the case for many nematode species. SL discovery is further complicated by an absence of SL sequences from high-throughput sequencing results due to incomplete sequencing of the 5'-ends of transcripts during RNA-seq library preparation, known as 5'-bias. Existing datasets and novel methodology were used to identify both conserved SLs and unique hypervariable SLs within Heterodera glycines, the soybean cyst nematode. In H. glycines, twenty-one distinct SL sequences were found on 2,532 unique H. glycines transcripts. The SL sequences identified on the H. glycines transcripts demonstrated a high level of promiscuity, meaning that some transcripts produced as many as nine different individual SL-transcript combinations. Most uniquely, transcriptome analysis revealed that H. glycines is the first nematode to demonstrate a higher SL trans-splicing rate using a species-specific SL over well-conserved Caenorhabditis elegans SL-like sequences.

Propagation of cell death in dropdead1, a sorghum ortholog of the maize lls1 mutant.

We describe dropdead1-1 (ded1), an EMS-induced recessive lesion mimic mutant of sorghum. It is characterized by the formation of spreading necrotic lesions that share many attributes with those associated with the maize lethal leaf spot1 (lls1) and Arabidopsis accelerated cell death1 (acd1) mutation. We show that as in lls1, ded1 lesions are initiated by wounding and require light for continued propagation, and that loss of chloroplast integrity is responsible for ded1 cell death. Consistent with these parallels, we demonstrate that ded1 is an ortholog of lls1 and encodes pheophorbide a oxidase (PaO) with 93% identity at the protein level. The mutant ded1 allele resulted from a stop codon-inducing single base pair change in exon 6 of the sorghum ortholog of lls1. The ded1 transcript was rapidly and transiently induced after wounding and substantially elevated in leaves containing ded1 lesions. Given that PaO is a key enzyme of the chlorophyll degradation pathway, its dysfunction would result in the accumulation of pheophorbide, a potent photosensitizer that results in the production of singlet oxygen. Consistent with this, cell death associated with ded1 lesions is most likely caused by singlet oxygen as our results exclude superoxide and H2O2 from this role. We explore the signal responsible for the propagation of lesions affecting both ded1 and lls1 lesions and find that both developmental age and ethylene increase the rate of lesion expansion in both mutants.

Construction of high-density linkage maps for mapping quantitative trait loci for multiple traits in field pea (Pisum sativum L.).

BACKGROUND: The objective of this research was to map quantitative trait loci (QTLs) of multiple traits of breeding importance in pea (Pisum sativum L.). Three recombinant inbred line (RIL) populations, PR-02 (Orb x CDC Striker), PR-07 (Carerra x CDC Striker) and PR-15 (1-2347-144 x CDC Meadow) were phenotyped for agronomic and seed quality traits under field conditions over multiple environments in Saskatchewan, Canada. The mapping populations were genotyped using genotyping-by-sequencing (GBS) method for simultaneous single nucleotide polymorphism (SNP) discovery and construction of high-density linkage maps. RESULTS: After filtering for read depth, segregation distortion, and missing values, 2234, 3389 and 3541 single nucleotide polymorphism (SNP) markers identified by GBS in PR-02, PR-07 and PR-15, respectively, were used for construction of genetic linkage maps. Genetic linkage groups were assigned by anchoring to SNP markers previously positioned on these linkage maps. PR-02, PR-07 and PR-15 genetic maps represented 527, 675 and 609 non-redundant loci, and cover map distances of 951.9, 1008.8 and 914.2 cM, respectively. Based on phenotyping of the three mapping populations in multiple environments, 375 QTLs were identified for important traits including days to flowering, days to maturity, lodging resistance, Mycosphaerella blight resistance, seed weight, grain yield, acid and neutral detergent fiber concentration, seed starch concentration, seed shape, seed dimpling, and concentration of seed iron, selenium and zinc. Of all the QTLs identified, the most significant in terms of explained percentage of maximum phenotypic variance (PVmax) and occurrence in multiple environments were the QTLs for days to flowering (PVmax = 47.9%), plant height (PVmax = 65.1%), lodging resistance (PVmax = 35.3%), grain yield (PVmax = 54.2%), seed iron concentration (PVmax = 27.4%), and seed zinc concentration (PVmax = 43.2%). CONCLUSION: We have identified highly significant and reproducible QTLs for several agronomic and seed quality traits of breeding importance in pea. The QTLs identified will be the basis for fine mapping candidate genes, while some of the markers linked to the highly significant QTLs are useful for immediate breeding applications.

Genetic diversity and association mapping of iron and zinc concentrations in chickpea (Cicer arietinum L.).

Chickpea (Cicer arietinum L.) is the world's second most important pulse crop after common bean. Chickpea has historically been an important daily staple in the diet of millions of people, especially in the developing countries. Current chickpea breeding programs have mainly been directed toward high yield, biotic and abiotic stress resilience that has increased global production, but less attention has been directed toward improving micronutrient concentrations in seeds. In an effort to develop micronutrient-dense chickpea lines, a study to examine the variability and to identify SNP alleles associated with seed iron and zinc concentrations was conducted using 94 diverse accessions of chickpea. The results indicated that there is substantial variability present in chickpea germplasm for seed iron and zinc concentrations. In the current set of germplasm, zinc is negatively correlated with grain yield across all locations and years; whereas the negative correlation between iron and grain yield was only significant at the Elrose locality. Eight SNP loci associated with iron and (or) zinc concentrations in chickpea seeds were identified. One SNP located on chromosome 1 (chr1) is associated with both iron and zinc concentrations. On chr4, three SNPs associated with zinc concentration and two SNPs for iron concentration were identified. Two additional SNP loci, one on chr6 and the other on chr7, were also found to be associated with iron and zinc concentrations, respectively. The results show potential opportunity for molecular breeding for improvement of seed iron and zinc concentrations in chickpea.

Gene-based SNP discovery and genetic mapping in pea.

KEY MESSAGE: Gene-based SNPs were identified and mapped in pea using five recombinant inbred line populations segregating for traits of agronomic importance. Pea (Pisum sativum L.) is one of the world's oldest domesticated crops and has been a model system in plant biology and genetics since the work of Gregor Mendel. Pea is the second most widely grown pulse crop in the world following common bean. The importance of pea as a food crop is growing due to its combination of moderate protein concentration, slowly digestible starch, high dietary fiber concentration, and its richness in micronutrients; however, pea has lagged behind other major crops in harnessing recent advances in molecular biology, genomics and bioinformatics, partly due to its large genome size with a large proportion of repetitive sequence, and to the relatively limited investment in research in this crop globally. The objective of this research was the development of a genome-wide transcriptome-based pea single-nucleotide polymorphism (SNP) marker platform using next-generation sequencing technology. A total of 1,536 polymorphic SNP loci selected from over 20,000 non-redundant SNPs identified using deep transcriptome sequencing of eight diverse Pisum accessions were used for genotyping in five RIL populations using an Illumina GoldenGate assay. The first high-density pea SNP map defining all seven linkage groups was generated by integrating with previously published anchor markers. Syntenic relationships of this map with the model legume Medicago truncatula and lentil (Lens culinaris Medik.) maps were established. The genic SNP map establishes a foundation for future molecular breeding efforts by enabling both the identification and tracking of introgression of genomic regions harbouring QTLs related to agronomic and seed quality traits.

Genome wide SNP identification in chickpea for use in development of a high density genetic map and improvement of chickpea reference genome assembly.

BACKGROUND: In the whole genome sequencing, genetic map provides an essential framework for accurate and efficient genome assembly and validation. The main objectives of this study were to develop a high-density genetic map using RAD-Seq (Restriction-site Associated DNA Sequencing) genotyping-by-sequencing (RAD-Seq GBS) and Illumina GoldenGate assays, and to examine the alignment of the current map with the kabuli chickpea genome assembly. RESULTS: Genic single nucleotide polymorphisms (SNPs) totaling 51,632 SNPs were identified by 454 transcriptome sequencing of Cicer arietinum and Cicer reticulatum genotypes. Subsequently, an Illumina GoldenGate assay for 1,536 SNPs was developed. A total of 1,519 SNPs were successfully assayed across 92 recombinant inbred lines (RILs), of which 761 SNPs were polymorphic between the two parents. In addition, the next generation sequencing (NGS)-based GBS was applied to the same population generating 29,464 high quality SNPs. These SNPs were clustered into 626 recombination bins based on common segregation patterns. Data from the two approaches were used for the construction of a genetic map using a population derived from an intraspecific cross. The map consisted of 1,336 SNPs including 604 RAD recombination bins and 732 SNPs from Illumina GoldenGate assay. The map covered 653 cM of the chickpea genome with an average distance between adjacent markers of 0.5 cM. To date, this is the most extensive genetic map of chickpea using an intraspecific population. The alignment of the map with the CDC Frontier genome assembly revealed an overall conserved marker order; however, a few local inconsistencies within the Cicer arietinum pseudochromosome 1 (Ca1), Ca5 and Ca8 were detected. The map enabled the alignment of 215 unplaced scaffolds from the CDC Frontier draft genome assembly. The alignment also revealed varying degrees of recombination rates and hotspots across the chickpea genome. CONCLUSIONS: A high-density genetic map using RAD-Seq GBS and Illumina GoldenGate assay was developed and aligned with the existing kabuli chickpea draft genome sequence. The analysis revealed an overall conserved marker order, although some localized inversions between draft genome assembly and the genetic map were detected. The current analysis provides an insight of the recombination rates and hotspots across the chickpea genome.

Effective and specific in planta RNAi in cyst nematodes: expression interference of four parasitism genes reduces parasitic success.

Cyst nematodes are highly evolved sedentary plant endoparasites that use parasitism proteins injected through the stylet into host tissues to successfully parasitize plants. These secretory proteins likely are essential for parasitism as they are involved in a variety of parasitic events leading to the establishment of specialized feeding cells required by the nematode to obtain nourishment. With the advent of RNA interference (RNAi) technology and the demonstration of host-induced gene silencing in parasites, a new strategy to control pests and pathogens has become available, particularly in root-knot nematodes. Plant host-induced silencing of cyst nematode genes so far has had only limited success but similarly should disrupt the parasitic cycle and render the host plant resistant. Additional in planta RNAi data for cyst nematodes are being provided by targeting four parasitism genes through host-induced RNAi gene silencing in transgenic Arabidopsis thaliana, which is a host for the sugar beet cyst nematode Heterodera schachtii. Here it is reported that mRNA abundances of targeted nematode genes were specifically reduced in nematodes feeding on plants expressing corresponding RNAi constructs. Furthermore, this host-induced RNAi of all four nematode parasitism genes led to a reduction in the number of mature nematode females. Although no complete resistance was observed, the reduction of developing females ranged from 23% to 64% in different RNAi lines. These observations demonstrate the relevance of the targeted parasitism genes during the nematode life cycle and, potentially more importantly, suggest that a viable level of resistance in crop plants may be accomplished in the future using this technology against cyst nematodes.

A guardian of grasses: specific origin and conservation of a unique disease-resistance gene in the grass lineage.

The maize Hm1 gene provides protection against a lethal leaf blight and ear mold disease caused by Cochliobolus carbonum race 1 (CCR1). Although it was the first disease-resistance (DR) gene to be cloned, it remains a novelty because, instead of participating in the plant recognition and response system as most DR genes do, Hm1 disarms the pathogen directly. It does so by encoding an NADPH-dependent reductase, whose function is to inactivate Helminthosporium carbonum (HC) toxin, an epoxide-containing cyclic tetrapeptide, which the pathogen produces as a key virulence factor to colonize maize. Although CCR1 is strictly a pathogen of maize, orthologs of Hm1 and the HC-toxin reductase activity are present in the grass family, suggesting an ancient and evolutionarily conserved role of this DR trait in plants. Here, we provide proof for such a role by demonstrating its involvement in nonhost resistance of barley to CCR1. Barley leaves in which expression of the Hm1 homologue was silenced became susceptible to infection by CCR1, but only if the pathogen was able to produce HC toxin. Phylogenetic analysis indicated that Hm1 evolved exclusively and early in the grass lineage. Given the devastating ability of CCR1 to kill maize, these findings imply that the evolution and/or geographical distribution of grasses may have been constrained if Hm1 did not emerge.

Maize Brittle stalk2 encodes a COBRA-like protein expressed in early organ development but required for tissue flexibility at maturity.

The maize (Zea mays) brittle stalk2 (bk2) is a recessive mutant, the aerial parts of which are easily broken. The bk2 phenotype is developmentally regulated and appears 4 weeks after planting, at about the fifth-leaf stage. Before this time, mutants are indistinguishable from wild-type siblings. Afterward, all organs of the bk2 mutants turn brittle, even the preexisting ones, and they remain brittle throughout the life of the plant. Leaf tension assays and bend tests of the internodes show that the brittle phenotype does not result from loss of tensile strength but from loss in flexibility that causes the tissues to snap instead of bend. The Bk2 gene was cloned by a combination of transposon tagging and a candidate gene approach and found to encode a COBRA-like protein similar to rice (Oryza sativa) BC1 and Arabidopsis (Arabidopsis thaliana) COBRA-LIKE4. The outer periphery of the stalk has fewer vascular bundles, and the sclerids underlying the epidermis possess thinner secondary walls. Relative cellulose content is not strictly correlated with the brittle phenotype. Cellulose content in mature zones of bk2 mature stems is lowered by 40% but is about the same as wild type in developing stems. Although relative cellulose content is lowered in leaves after the onset of the brittle phenotype, total wall mass as a proportion of dry mass is either unchanged or slightly increased, indicating a compensatory increase in noncellulosic carbohydrate mass. Fourier transform infrared spectra indicated an increase in phenolic ester content in the walls of bk2 leaves and stems. Total content of lignin is unaffected in bk2 juvenile leaves before or after appearance of the brittle phenotype, but bk2 mature and developing stems are markedly enriched in lignin compared to wild-type stems. Despite increased lignin in bk2 stems, loss of staining with phloroglucinol and ultraviolet autofluorescence is observed in vascular bundles and sclerid layers. Consistent with the infrared analyses, levels of saponifiable hydroxycinnamates are elevated in bk2 leaves and stems. As Bk2 is highly expressed during early development, well before the onset of the brittle phenotype, we propose that Bk2 functions in a patterning of lignin-cellulosic interactions that maintain organ flexibility rather than having a direct role in cellulose biosynthesis.