A Novel Soybean Diacylglycerol Acyltransferase 1b Variant with Three Amino Acid Substitutions Increases Seed Oil Content.

Improving soybean (Glycine max) seed composition by increasing the protein and oil components will add significant value to the crop and enhance environmental sustainability. Diacylglycerol acyltransferase (DGAT) catalyzes the final rate-limiting step in triacylglycerol (TAG) biosynthesis and has a major impact on seed oil accumulation. We previously identified a soybean DGAT1b variant with 14 amino acid substitutions (GmDGAT1b-MOD) that increases total oil content by 3 percentage points when overexpressed in soybean seeds. In the present study, additional GmDGAT1b variants were generated to further increase oil with a reduced number of substitutions. Variants with one to four amino acid substitutions were screened in the model systems S. cerevisiae and transient N. benthamiana leaf. Promising GmDGAT1b variants resulting in high oil accumulation in the model systems were selected for over-expression in soybeans. One GmDGAT1b variant with three novel amino acid substitutions (GmDGAT1b-3aa) increased total soybean oil to levels near the previously discovered GmDGAT1b-MOD variant. In a multiple location field trial, GmDGAT1b-3aa transgenic events had significantly increased oil and protein by up to 2.3 and 0.6 percentage points, respectively. Modeling of the GmDGAT1b-3aa protein structure provided insights into the potential function of the three substitutions. These findings will guide efforts to improve soybean oil content and overall seed composition by CRISPR editing.

Seroepidemiological Analysis of Canine Leptospira Species Infections in Changchun, China.

Leptospirosis is a significant worldwide zoonotic infectious disease that infects a wide range of animals and humans. Leptospira will colonize the animal's urinary and reproductive systems and be excreted with urine, potentially causing a wide range of infections. Dogs are an essential host for Leptospira, and epidemiological investigation studies of leptospirosis must be conducted to clarify the prevalence of leptospirosis and to reduce the risk of transmission to humans. This study aimed to investigate the seroepidemiology of leptospiral infection in dogs from Changchun, China, using Microscopic Agglutination Test (MAT). A total of 1053 canine blood samples were collected and tested by MAT. The positive rate of MAT was approximately 19.1%. The main prevalent Leptospira serogroups were L. Icterohaemorrhagiae (8.1%), L. Canicola (7.6%), L. Australis (5.3%), L. Ballum (4.7%) and L. Pyrogenes (4.2%). No statistically significant difference among different varieties, sexes and sampling seasons (p > 0.05), except the age (p < 0.05). The seropositive rate was much higher in adult and aged dogs than in juvenile dogs. Our results showed the seroprevalence and the prevalent serogroup of Canine leptospirosis in Changchun, China.

RNAi and CRISPR-Cas silencing E3-RING ubiquitin ligase AIP2 enhances soybean seed protein content.

The majority of plant protein in the world's food supply is derived from soybean (Glycine max). Soybean is a key protein source for global animal feed and is incorporated into plant-based foods for people, including meat alternatives. Soybean protein content is genetically variable and is usually inversely related to seed oil content. ABI3-interacting protein 2 (AIP2) is an E3-RING ubiquitin ligase that targets the seed-specific transcription factor ABI3. Silencing both soybean AIP2 genes (AIP2a and AIP2b) by RNAi enhanced seed protein content by up to seven percentage points, with no significant decrease in seed oil content. The protein content enhancement did not alter the composition of the seed storage proteins. Inactivation of either AIP2a or AIP2b by a CRISPR-Cas9-mediated mutation increased seed protein content, and this effect was greater when both genes were inactivated. Transactivation assays in transfected soybean hypocotyl protoplasts indicated that ABI3 changes the expression of glycinin, conglycinin, 2S albumin, and oleosin genes, indicating that AIP2 depletion increased seed protein content by regulating activity of the ABI3 transcription factor protein. These results provide an example of a gene-editing prototype directed to improve global food security and protein availability in soybean that may also be applicable to other protein-source crops.

Detection and phylogenetic analysis of tick-borne bacterial and protozoan pathogens in a forest province of eastern China.

Ticks, as obligate blood-sucking ectoparasites, feed on a broad range of vertebrates and transmit a great diversity of pathogenic microorganisms. Some tick-borne pathogens (TBPs) are endemic in China, whereas epidemiological studies are limited in Jiangxi, a forest province located in eastern China. Here, we have determined the positivity rates of TBPs in humans, rodents, dogs, goats and ticks, and performed the molecular characterization of TBPs in Jiangxi province. We found a high positivity rate of TBPs in the collected samples, demonstrating 23 (12.92%) samples positive for more than one TBPs. Of those, 11 (6.18%) samples were positive for Rickettsia spp., six (3.37%) Ehrlichia spp./Anaplasma spp., one (0.56%) Bartonella spp., two (1.12%) Borrelia spp., and five (2.81%) Babesia spp. The positivity rates of TBPs varied among ticks, animals, and humans as follow: goats (14/37, 37.84%), ticks (8/35, 22.86%), and dogs (1/11, 9.09%). Humans and rodents were negative for TBP presence. Phylogenetic analyses of these TBP sequences revealed the presence of Rickettsia japonica, Ehrlichia minasensis, and an unclassified Babesia spp. in goats, and Anaplasma phagocytophilum, Borrelia valaisiana, and an unclassified Bartonella spp. in ticks. Furthermore, R. japonica infection was exclusively found in goats with the positivity rate of 29.73%. Our study is the first report of R. japonica in goats around the world. These findings suggest high TBP positivity rates among goats, ticks, and dogs, and diverse TBPs in goats and ticks in the studied sites. Therefore, our results underscore the urgent need to assess TBP-tick-vertebrate-environment interactions and the risk of tick borne disease exposure in humans in the future.

Efficient gene targeting in soybean using Ochrobactrum haywardense-mediated delivery of a marker-free donor template.

Gene targeting (GT) for precise gene insertion or swap into pre-defined genomic location has been a bottleneck for expedited soybean precision breeding. We report a robust selectable marker-free GT system in soybean, one of the most economically important crops. An efficient Oh H1-8 (Ochrobactrum haywardense H1-8)-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants 6-8 weeks after transformation. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with ∼ 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without the need for a selectable marker within the template. High-resolution Southern-by-Sequencing analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment-mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require a selectable marker within the donor DNA, and generates nonchimeric plants with heritable GT.

Use of CRISPR/Cas9 for Crop Improvement in Maize and Soybean.

CRISPR/Cas enables precise improvement of commercially relevant crop species by transgenic and nontransgenic methodologies. We have used CRISPR/Cas with or without DNA repair template in both corn and soybean for a range of applications including enhancing drought tolerance, improving seed oil composition, and endowing herbicide tolerance. Importantly, by pairing CRISPR/Cas technology with recent advances in plant tissue culture, these changes can be introduced directly into commercially relevant genotypes. This powerful combination of technologies enables advanced breeding techniques for introducing natural genetic variations directly into product relevant lines with improved speed and quality compared with traditional breeding methods. Variation generated through such CRISPR/Cas enabled advanced breeding approaches can be indistinguishable from naturally occurring variation and therefore should be readily accessible for commercialization. The precision, reach, and flexibility afforded by CRISPR/Cas promise an important role for genome editing in future crop improvement efforts.

First molecular detection of tick-borne pathogens in dogs from Jiangxi, China.

In this study, blood samples obtained from 162 dogs in Jiangxi, China, were employed in molecular screening of canine tick-borne pathogens by PCR and sequencing. Babesia spp. gene fragment was detected in 12 (7.41%) dogs. All samples were negative for Hepatozoon spp., Ehrlichia canis, Coxiella spp., Borrelia spp., Rickettsia spp. and Anaplasma platys. Species-specific PCR analysis further confirmed that 8 (4.94%) and 4 (2.47%) dogs were infected by Babesia canis vogeli and Babesia gibsoni, respectively. Based on our analyses, Babesia spp. infection in Jiangxi appeared not related to age, gender, breed, usage, activity and health status or tick infestation history of the dogs. This is the first molecular report of Babesia canis vogeli and Babesia gibsoni in dogs from Jiangxi, China.

Cas9-Guide RNA Directed Genome Editing in Soybean.

Recently discovered bacteria and archaea adaptive immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) endonuclease has been explored in targeted genome editing in different species. Streptococcus pyogenes Cas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis, gene integration, and gene editing in soybean (Glycine max). Two genomic sites, DD20 and DD43 on chromosome 4, were mutagenized with frequencies of 59% and 76%, respectively. Sequencing randomly selected transgenic events confirmed that the genome modifications were specific to the Cas9-gRNA cleavage sites and consisted of small deletions or insertions. Targeted gene integrations through homology-directed recombination were detected by border-specific polymerase chain reaction analysis for both sites at callus stage, and one DD43 homology-directed recombination event was transmitted to T1 generation. T1 progenies of the integration event segregated according to Mendelian laws and clean homozygous T1 plants with the donor gene precisely inserted at the DD43 target site were obtained. The Cas9-gRNA system was also successfully applied to make a directed P178S mutation of acetolactate synthase1 gene through in planta gene editing.

Stacking multiple transgenes at a selected genomic site via repeated recombinase-mediated DNA cassette exchanges.

Recombinase-mediated DNA cassette exchange (RMCE) has been successfully used to insert transgenes at previously characterized genomic sites in plants. Following the same strategy, groups of transgenes can be stacked to the same site through multiple rounds of RMCE. A gene-silencing cassette, designed to simultaneously silence soybean (Glycine max) genes fatty acid ω-6 desaturase 2 (FAD2) and acyl-acyl carrier protein thioesterase 2 (FATB) to improve oleic acid content, was first inserted by RMCE at a precharacterized genomic site in soybean. Selected transgenic events were subsequently retransformed with the second DNA construct containing a Yarrowia lipolytica diacylglycerol acyltransferase gene (DGAT1) to increase oil content by the enhancement of triacylglycerol biosynthesis and three other genes, a Corynebacterium glutamicum dihydrodipicolinate synthetase gene (DHPS), a barley (Hordeum vulgare) high-lysine protein gene (BHL8), and a truncated soybean cysteine synthase gene (CGS), to improve the contents of the essential amino acids lysine and methionine. Molecular characterization confirmed that the second RMCE successfully stacked the four overexpression cassettes to the previously integrated FAD2-FATB gene-silencing cassette. Phenotypic analyses indicated that all the transgenes expressed expected phenotypes.

High yield recombinant silk-like protein production in transgenic plants through protein targeting.

DP1B is a synthetic analogue of spider dragline silk protein. It can be spun to form silk fiber. Previously, it had been expressed in transgenic plants, showing the general feasibility of the plant-based DP1B production. However, success of such a plant-based platform requires a great increase of DP1B productivity in plant cells to reduce production cost. This report describes a protein targeting approach to accumulate DP1B in apoplast, ER lumen, and vacuole in Arabidopsis cells, by utilizing appropriate combinations of sporamin-targeting determinant peptides and ER retention peptide. The approach has dramatically enhanced DP1B accumulation, resulting in high production yield. The accumulation can be as high as 8.5 and 6.7% total soluble protein in leaf tissue by targeting to apoplast and ER lumen, respectively, or as high as 18 and 8.2% total soluble protein in seeds by targeting to ER lumen and vacuole, respectively. However, the vacuole targeting in leaves and the apoplast targeting in seeds have failed to accumulate full length DP1B molecules or any DP1B at all, respectively, suggesting that they may not be suitable for applications in leaf tissues and seeds. Data in this study recommend a combination of seed-specific expression and ER-targeting as one of the best strategies for yield enhancement of plant-based DP1B production.