Toxoplasma gondii metacaspase 2 is an important factor that influences bradyzoite formation in the Pru strain.

Toxoplasma gondii is an important zoonotic protozoan of the phylum Apicomplexa that can infect nearly all warm-blooded animals. The parasite can exist as the interconvertible tachyzoite or bradyzoite forms, leading to acute or latent infection, respectively. No drug has been reported to penetrate the cyst wall and reduce bradyzoite survival and proliferation till now. The transcriptional level of metacaspases 2 (TgMCA2) in T. gondii is significantly upregulated during the formation of bradyzoites in the Pru strain, indicating that it may play an important role in the formation of bradyzoites. To further explore the function of TgMCA2, we constructed a TgMCA2 gene-knockout variant of the Pru strain (Δmca2). Comparative analysis revealed that the proliferative capacity of Pru Δmca2 increased, while the invasion and egressing properties were not affected by the knockout. Further data shows that the tachyzoites of Δmca2 failed to induce differentiation and form bradyzoites in vitro, and the transcriptional levels of some of the bradyzoite-specific genes (such as BAG1, LDH2, and SAG4A) in Δmca2 were significantly lower compared with that in the Pru strain at the bradyzoite stage. In vivo, no cysts were detected in Δmca2-infected mice. Further determination of parasite burden in Δmca2- and Pru-infected mice brain tissue at the genetic level showed that the gene load was significantly lower than that in Pru. In summary, we confirmed that TgMCA2 contributes to the formation of bradyzoites, and could provide an important foundation for the development of attenuated vaccines for the prevention of T. gondii infection.

A simple and rapid approach to manipulate pseudorabies virus genome by CRISPR/Cas9 system.

OBJECTIVES: The broad host range of pseudorabies virus (PRV) and large capacity for foreign DNA make it a promising vector for the development of vaccines and agents of gene therapy. RESULTS: We show that up to 100 % viral gene disrupting efficiency was achieved by simple co-transfection of the purified PRV genomes with the clustered regularly-interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) into cells. Furthermore, CRISPR/Cas9-mediated knock-in of >4-kb-long DNA cassettes into the PRV genome at a positive rate of 50 % by a homology-independent DNA repair mechanism without constructing homology arms. This approach requires only a simple plasmid construction and is applicable to knock-in of other foreign genes. CONCLUSION: Our studies offered simple and efficient methods to manipulate PRV.

Multi-Species Prediction of Physiological Traits with Hyperspectral Modeling.

Lack of high-throughput phenotyping is a bottleneck to breeding for abiotic stress tolerance in crop plants. Efficient and non-destructive hyperspectral imaging can quantify plant physiological traits under abiotic stresses; however, prediction models generally are developed for few genotypes of one species, limiting the broader applications of this technology. Therefore, the objective of this research was to explore the possibility of developing cross-species models to predict physiological traits (relative water content and nitrogen content) based on hyperspectral reflectance through partial least square regression for three genotypes of sorghum (Sorghum bicolor (L.) Moench) and six genotypes of corn (Zea mays L.) under varying water and nitrogen treatments. Multi-species models were predictive for the relative water content of sorghum and corn (R2 = 0.809), as well as for the nitrogen content of sorghum and corn (R2 = 0.637). Reflectances at 506, 535, 583, 627, 652, 694, 722, and 964 nm were responsive to changes in the relative water content, while the reflectances at 486, 521, 625, 680, 699, and 754 nm were responsive to changes in the nitrogen content. High-throughput hyperspectral imaging can be used to predict physiological status of plants across genotypes and some similar species with acceptable accuracy.