Essential role of a Plasmodium berghei heat shock protein (PBANKA_0938300) in gametocyte development

The continued existence of Plasmodium parasites in physiologically distinct environments during their transmission in mosquitoes and vertebrate hosts requires effector proteins encoded by parasite genes to provide adaptability. Parasites utilize their robust stress response system involving heat shock proteins for their survival. Molecular chaperones are involved in maintaining protein homeostasis within a cell during stress, protein biogenesis and the formation of protein complexes. Due to their critical role in parasite virulence, they are considered targets for therapeutic interventions. Our results identified a putative P. berghei heat shock protein (HSP) belonging to the HSP40 family (HspJ62), which is abundantly induced upon heat stress and expressed during all parasite stages. To determine the role HspJ62, a gene-disrupted P. berghei transgenic line was developed (ΔHspJ62), which resulted in disruption of gametocyte formation. Such parasites were unable to form subsequent sexual stages because of disrupted gametogenesis, indicating the essential role of HspJ62 in gametocyte formation. Transcriptomic analysis of the transgenic line showed downregulation of a number of genes, most of which were specific to male or female gametocytes. The transcription factor ApiAP2 was also downregulated in ΔHspJ62 parasites. Our findings suggest that the downregulation of ApiAP2 likely disrupts the transcriptional regulation of sexual stage genes, leading to impaired gametogenesis. This finding also highlights the critical role that HspJ62 indirectly plays in the development of P. berghei sexual stages and in facilitating the conversion from the asexual blood stage to the sexual stage. This study characterizes the HspJ62 protein as a fertility factor because parasites lacking it are unable to transmit to mosquitoes. This study adds an important contribution to ongoing research aimed at understanding gametocyte differentiation and formation in parasites. The molecule adds to the list of potential drug targets that can be targeted to inhibit parasite sexual development and consequently parasite transmission.

Generating and testing the efficacy of transgenic Cas9 in Tribolium castaneum

CRISPR/Cas9 genome editing has now expanded to many insect species, including Tribolium castaneum. However, compared to Drosophila melanogaster, the CRISPR toolkit of T. castaneum is limited. A particularly apparent gap is the lack of Cas9 transgenic animals, which generally offer higher editing efficiency. We address this by creating and testing transgenic beetles expressing Cas9. We generated two different constructs bearing basal heat shock promoter-driven Cas9, two distinct 3 UTRs, and one containing Cas9 fused to EGFP by a T2A peptide. For each construct, we were able to generate a line that is homozygous viable, though variable reductions in reproductive success with each construct were noted. Analyses of Cas9 activity in each transgenic line demonstrated that both designs are capable of inducing CRISPR- mediated changes in the genome in the absence of heat induction. Overall, these resources enhance the accessibility of CRISPR/Cas9 genome editing for the Tribolium research community and provide a benchmark against which to compare future transgenic Cas9 lines.

Maintenance and breeding of transgenic mouse strains at the scientific center of biomedical technologies of the FMBA of Russia

In this article, we analyze the specifics of health monitoring and maintenance of transgenic mouse strains. A method for keeping animals in laboratory conditions is discussed, the criteria for inbred crossing of homozygous transgenic mice lines are characterized. The obtained data can be useful for the staff of vivaria in maintaining the transgenic line of mice.

Over-Expression of Chorismate Mutase Enhances the Accumulation of Salicylic Acid, Lignin, and Antioxidants in Response to the White-Backed Planthopper in Rice Plants

The white-backed planthopper (WBPH) is a serious pest of rice crop and causes sever yield loss each year, especially in Asian countries. In this study, we used chorismate mutase (CM) transgenic line to examine the defense mechanism of rice plants against WBPH. The survival rate of WBPHs, infestation rate of plants, lignin biosynthesis, transcriptional regulation of related genes, salicylic acid (SA) accumulation and signaling and antioxidants regulation were investigated. The WBPH population decreased by 67% in OxCM-t, and the plant infestation rate was 3.5-fold higher in wild-type plants compared with transgenic plants. A substantial increase in lignin was found in the transgenic line (742%) and wild-type (417%) plants. Additionally, CM, phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), and chalcone isomerase (CHI) showed significant increases in their relative expression level in the transgenic line. Salicylic acid was significantly enhanced in the transgenic line compared with WBPH infestation. SA can activate pathogenesis related proteins-1 (PR1), PR2, antioxidants, and the expression of their related genes: superoxide dismutase (SOD) and catalase (CAT). WBPH infestation reduced the chlorophyll contents of both transgenic and wild-type plants, but the reduction was great in wild-type than transgenic plants. The sugar content was only significantly increased in the transgenic line, indicating that sugars are not heavily involved in WBPH stress. Phenylalanine, proline, aspartic acid, and total amino acids were increased in the transgenic line and reduced in the wild-type plants. Taken together, all the results suggest that overexpression of CM gene regulates the defense mechanisms and enhances the rice toward WBPH stress.

ROS-Scavengers, Osmoprotectants and Violaxanthin De-Epoxidation in Salt-Stressed Arabidopsis thaliana with Different Tocopherol Composition

To determine the role of α- and γ-tocopherol (TC), this study compared the response to salt stress (200 mM NaCl) in wild type (WT) Arabidopsis thaliana (L.) Heynh. And its two mutants: (1) totally TC-deficient vte1; (2) vte4 accumulating γ-TC instead of α-TC; and (3) tmt transgenic line overaccumulating α-TC. Raman spectra revealed that salt-exposed α-TC accumulating plants were more flexible in regulating chlorophyll, carotenoid and polysaccharide levels than TC deficient mutants, while the plants overaccumulating γ-TC had the lowest levels of these biocompounds. Tocopherol composition and NaCl concentration affected xanthophyll cycle by changing the rate of violaxanthin de-epoxidation and zeaxanthin formation. NaCl treated plants with altered TC composition accumulated less oligosaccharides than WT plants. α-TC deficient plants increased their oligosaccharide levels and reduced maltose amount, while excessive accumulation of α-TC corresponded with enhanced amounts of maltose. Salt-stressed TC-deficient mutants and tmt transgenic line exhibited greater proline levels than WT plants, lower chlorogenic acid levels, and lower activity of catalase and peroxidases. α-TC accumulating plants produced more methylated proline- and glycine- betaines, and showed greater activity of superoxide dismutase than γ-TC deficient plants. Under salt stress, α-TC demonstrated a stronger regulatory effect on carbon- and nitrogen-related metabolites reorganization and modulation of antioxidant patterns than γ-TC. This suggested different links of α- and γ-TCs with various metabolic pathways via various functions and metabolic loops.

Cardioluminescence in Transgenic Zebrafish Larvae: A Calcium Imaging Tool to Study Drug Effects and Pathological Modeling

Zebrafish embryos and larvae have emerged as an excellent model in cardiovascular research and are amenable to live imaging with genetically encoded biosensors to study cardiac cell behaviours, including calcium dynamics. To monitor calcium ion levels in three to five days post-fertilization larvae, we have used bioluminescence. We generated a transgenic line expressing GFP-aequorin in the heart, Tg(myl7:GA), and optimized a reconstitution protocol to boost aequorin luminescence. The analogue diacetyl h-coelenterazine enhanced light output and signal-to-noise ratio. With this cardioluminescence model, we imaged the time-averaged calcium levels and beat-to-beat calcium oscillations continuously for hours. As a proof-of-concept of the transgenic line, changes in ventricular calcium levels were observed by Bay K8644, an L-type calcium channel activator and with the blocker nifedipine. The β-adrenergic blocker propranolol decreased calcium levels, heart rate, stroke volume, and cardiac output, suggesting that larvae have a basal adrenergic tone. Zebrafish larvae treated with terfenadine for 24 h have been proposed as a model of heart failure. Tg(myl7:GA) larvae treated with terfenadine showed bradycardia, 2:1 atrioventricular block, decreased time-averaged ventricular calcium levels but increased calcium transient amplitude, and reduced cardiac output. As alterations of calcium signalling are involved in the pathogenesis of heart failure and arrhythmia, the GFP-aequorin transgenic line provides a powerful platform for understanding calcium dynamics.

Potential Roles for Pattern Molecule of PAMP-triggered Immunity in Improving Crop Cold Tolerance

Pathogen infection cross-activates cold response and increase cold tolerance of host plants. However it is not possible to use the infection to increase cold tolerance of field plants. Here flagellin 22 (flg22), the most widely-studied PAMP, was used to mimic the pathogen infection to cross-activate cold response. Flg22 treatment alleviated the injury caused by freezing in Arabidopsis, oilseed and tobacco. In Arabidopsis, flg22 activated the expression of immunity and cold-related genes. Moreover the flg22 induced alleviation of cold injury was lost in NahG transgenic line (SA-deficient), sid2-2 and npr1-1 mutant plants, and flg22-induced expression of cold tolerance-related genes, which indicating that salicylic acid signaling pathway is required for the alleviation of cold injury by flg22 treatment. In short flg22 application can be used to enhance cold tolerance in field via a salicylic acid-depended pathway.

A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish

Maternal products are those mRNAs and proteins deposited during oogenesis, which play critical roles in controlling oocyte formation, fertilization, and early embryonic development. However, loss-of-function studies for these maternal factors are still lacking, mainly because of the prolonged period of transgenerational screening and technical barriers that prevent the generation of maternal (M) and maternal and zygotic (MZ) mutant embryos. By the transgenic expression of multiple sgRNAs targeting a single gene of interest in the background of a transgenic line Tg(zpc:zcas9) with oocyte-specific cas9 expression, we have successfully obtained maternal or maternal–zygotic mutant for single genes in F1 embryos. In this work, we tandemly connected a maternal GFP marker and eight sgRNA expression units to target dvl2 and dvl3a simultaneously and introduced this construct to the genome of Tg(zpc:zcas9) by meganuclease I-Sce I. As expected, we confirmed the existence of Mdvl2;Mdvl3a embryos with strong defective convergence and extension movement during gastrulation among outcrossed GFP positive F1 offspring. The MZdvl2;MZdvl3a embryos were also obtained by crossing the mutant carrying mosaic F0 female with dvl2+/−;dvl3a−/− male fish. This proof-of-principle thus highlights the potential of this conditional knockout strategy to circumvent the current difficulty in the study of genes with multiple functionally redundant paralogs.

Characterization of genomic DNA sequence of the candidate gene for FB_Mfu10 associated with fire blight resistance in Malus species

Objective The proposed candidate gene underlying the Malus fusca fire blight resistance locus on chromosome 10 was previously predicted to possess 880 amino acids and 8 exons. Eight base pair (8 bp) insertion/deletion in the first exon potentially distinguished resistant genotypes from susceptible ones. This study aimed at analyzing the candidate gene sequence in another set of original resistant and susceptible progeny, characterizing the sequence in a transgenic line transformed with the candidate gene under its own native promoter, as well as deciphering the potential genomic differences between this candidate gene and its homolog in the ‘Golden Delicious’ doubled haploid genome (GDDH13). Results Sequences of amplicons of part of the candidate gene amplified in two progenies that showed resistant and susceptible fire blight phenotypes, confirmed the 8 bp insertion that distinguishes susceptible and resistant progenies. The transgenic line was positive for the candidate gene sequence, confirming a successful transfer into the background of apple cultivar ‘Pinova’, and possessed the same genomic sequence as the progeny with a resistant phenotype. Sequence analysis showed that the homolog gene on GDDH13 possesses a significant 18 bp deletion in exon 1 leading to a difference of 15 amino acid from the protein sequence of the candidate gene.

The roles of NADPH oxidases during adult zebrafish fin regeneration

Sustained elevated levels of reactive oxygen species (ROS) have been shown to be essential for whole body, appendage and organ regeneration in various organisms, including planarians, Hydra, zebrafish, axolotl, Xenopus, geckos and mice. In the majority of cases these roles have been shown via the use of NADPH oxidase pharmacological inhibitors, which generally target all NAPDH oxidases (NOXes). To identify the specific NOX or NOXes essential for ROS production during adult fin regeneration in zebrafish, we generated nox mutants for duox, nox5 and cyba (a key subunit of NOXes 1-4). We also crossed these mutant lines to a transgenic line ubiquitously expressing HyPer, which permits the measurement of ROS levels in adult zebrafish fins. Using this approach, we found that homozygous duox mutants have significantly attenuated ROS levels following fin amputation, and this correlated with a significantly diminished rate of fin regeneration. While the other nox homozygous mutants (nox5 and cyba) showed less of an effect on ROS levels or adult fin regeneration, duox/cyba double mutants showed a more diminished rate of fin regeneration than duox mutants alone, suggesting that Nox1-4 do play a role during regeneration, but one that is secondary to that of Duox. This work also serendipitously found that ROS levels in amputated adult zebrafish fins oscillate during the day with a circadian rhythm.