Development of TALE-adenine base editors in plants

Base editors enable precise nucleotide changes at targeted genomic loci without requiring double-stranded DNA breaks or repair templates. TALE-adenine base editors (TALE-ABEs) are genome editing tools, composed of a DNA-binding domain from transcription activator-like effectors (TALEs), an engineered adenosine deaminase (TadA8e), and a cytosine deaminase domain (DddA), that allow A•T-to-G•C editing in human mitochondrial DNA. However, the editing ability of TALE-ABEs in plants apart from chloroplast DNA has not been described, so far, and the functional role how DddA enhances TadA8e is still unclear. We tested a series of TALE-ABEs with different deaminase fusion architectures in Nicotiana benthamiana and rice. The results indicate that the double-stranded DNA-specific cytosine deaminase DddA can boost the activities of single-stranded DNA-specific deaminases (TadA8e or APOBEC3A) on double-stranded DNA. We analysed A•T-to-G•C editing efficiencies in a β-glucuronidase reporter system and showed precise adenine editing in genomic regions with high product purity in rice protoplasts. Furthermore, we have successfully regenerated rice plants with A•T-to-G•C mutations in the chloroplast genome using TALE-ABE. Consequently, TALE-adenine base editors provide alternatives for crop improvement and gene therapy by editing nuclear or organellar genomes

Laser-based molecular delivery and its applications in plant science

Lasers enable modification of living and non-living matter with submicron precision in a contact-free manner which has raised the interest of researchers for decades. Accordingly, laser technologies have drawn interest across disciplines. They have been established as a valuable tool to permeabilize cellular membranes for molecular delivery in a process termed photoinjection. Laser-based molecular delivery was first reported in 1984, when normal kidney cells were successfully transfected with a frequency-multiplied Nd:YAG laser. Due to the rapid development of optical technologies, far more sophisticated laser platforms have become available. In particular, near infrared femtosecond (NIR fs) laser sources enable an increasing progress of laser-based molecular delivery procedures and opened up multiple variations and applications of this technique. This review is intended to provide a plant science audience with the physical principles as well as the application potentials of laser-based molecular delivery. The historical origins and technical development of laser-based molecular delivery are summarized and the principle physical processes involved in these approaches and their implications for practical use are introduced. Successful cases of laser-based molecular delivery in plant science will be reviewed in detail, and the specific hurdles that plant materials pose will be discussed. Finally, we will give an outlook on current limitations and possible future applications of laser-based molecular delivery in the field of plant science

Epigenetic features improve TALE target prediction

Background: The yield of many crop plants can be substantially reduced by plant-pathogenic Xanthomonas bacteria. The infection strategy of many Xanthomonas strains is based on transcription activator-like effectors (TALEs), which are secreted into the host cells and act as transcriptional activators of plant genes that are beneficial for the bacteria.The modular DNA binding domain of TALEs contains tandem repeats, each comprising two hyper-variable amino acids. These repeat-variable diresidues (RVDs) bind to their target box and determine the specificity of a TALE.All available tools for the prediction of TALE targets within the host plant suffer from many false positives. In this paper we propose a strategy to improve prediction accuracy by considering the epigenetic state of the host plant genome in the region of the target box. Results: To this end, we extend our previously published tool PrediTALE by considering two epigenetic features: (i) chromatin accessibility of potentially bound regions and (ii) DNA methylation of cytosines within target boxes. Here, we determine the epigenetic features from publicly available DNase-seq, ATAC-seq, and WGBS data in rice.We benchmark the utility of both epigenetic features separately and in combination, deriving ground-truth from RNA-seq data of infections studies in rice. We find an improvement for each individual epigenetic feature, but especially the combination of both.Having established an advantage in TALE target predicting considering epigenetic features, we use these data for promoterome and genome-wide scans by our new tool EpiTALE, leading to several novel putative virulence targets. Conclusions: Our results suggest that it would be worthwhile to collect condition-specific chromatin accessibility data and methylation information when studying putative virulence targets of Xanthomonas TALEs

Adaptation of a Danish online version of the Oxford Physical Activity Questionnaire (OPAQ) for secondary school students-a pilot study

OBJECTIVE: To adapt and partly validate a Danish online version of the patient-reported outcome measure (PROM) Oxford Physical Activity Questionnaire (“OPAQ”) and evaluate mobile phones and tablets as data capturing tool to identify potential problems and deficiencies in the PROM prior to implementation in the full study. METHODS: The OPAQ was translated into Danish by a formalised forward-backward translation procedure. Face validity was examined by interviewing 12 school students aged 10–15, recruited from two Danish public schools. After modifications, the online version of the Danish OPAQ was pilot tested in a convenience sample of seven school students for 1 week. Simultaneous objective accelerometer data were captured during the registration period. RESULTS: No major challenges were identified when translating OPAQ. Based on the interviews, the Danish version of OPAQ was perceived to be easy to understand in general, and the questions were relevant for tracking activities during the week. Five of the 12 participants had difficulties with understanding the introductory question: “what is your cultural background” in the original OPAQ. The interviews revealed that the participants recalling 7 days forgot to record some of the physical activity they had done during the week, indicating issues with the weekly recall method. After transforming to the online version, this was reported to be easy and quick to fill in (taking 1–3 min per day), and participants reported the daily design was helpful to remember activities. There was good correspondence between the online version and objective actigraphs with a tendency to underreport. Six participants reported 10–60 min less moderate to vigorous physical activity compared to the actigraphs, while one participant reported 3 min more. CONCLUSION: Participants found the online OPAQ quick and easy to complete during a 1-week period. Completing daily rather than weekly may help limit issues with recall. Overall, there was good agreement between the objective actigraphs and the OPAQ, though the OPAQ tended to slightly underreport moderate to vigorous physical activity. The Danish online version of OPAQ may be useful for capturing school students’ physical activity when objective measures are not feasible. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40814-022-01108-x

TAL effectors from Xanthomonas: design of a programmable DNA-binding specificity

Abstract Xanthomonas spp. are Gram-negative bacteria with powerful molecular weapons to attack their plant hosts. Key for pathogenicity of Xanthomonas is a type III secretion system that injects a cocktail of effector proteins into plant cells to function as potent virulence factors. TAL (transcription activator-like) effectors from Xanthomonas function as transcriptional activators of plant genes in the plant nucleus. They contain a central domain of tandem, near-identical 34 amino-acid repeats. Each repeat recognizes a single base pair in a contiguous DNA sequence and two adjacent hypervariable amino acids per repeat specify the base that is bound. This modular DNA-binding code allows a simple reprogramming of DNA-binding specificity, a feature with high potential for biotechnology. We developed a method called "Golden TAL Technology" that allows a flexible assembly of TAL proteins with a designed order of repeats

Cytoplasmic Injection of Zygotes to Genome Edit Naturally Occurring Sequence Variants Into Bovine Embryos

Genome editing provides opportunities to improve current cattle breeding strategies through targeted introduction of natural sequence variants, accelerating genetic gain. This can be achieved by harnessing homology-directed repair mechanisms following editor-induced cleavage of the genome in the presence of a repair template. Introducing the genome editors into zygotes and editing in embryos has the advantage of uncompromised development into live animals and alignment with contemporary embryo-based improvement practices. In our study, we investigated the potential to introduce sequence variants, known from the pre-melanosomal protein 17 (PMEL) and prolactin receptor (PRLR) genes, and produce non-mosaic, edited embryos, completely converted into the precision genotype. Injection of gRNA/Cas9 editors into bovine zygotes to introduce a 3 bp deletion variant into the PMEL gene produced up to 11% fully converted embryos. The conversion rate was increased to up to 48% with the use of TALEN but only when delivered by plasmid. Testing three gRNA/Cas9 editors in the context of several known PRLR sequence variants, different repair template designs and delivery as DNA, RNA or ribonucleoprotein achieved full conversion rates up to 8%. Furthermore, we developed a biopsy-based screening strategy for non-mosaic embryos which has the potential for exclusively producing non-mosaic animals with intended precision edits. Copyright © 2022 Wei, Brophy, Cole, Moormann, Boch and Laible

Analysis of resistance gene-mediated defense responses in Arabidopsis thaliana plants carrying a mutation in CPR5

In resistant plants, pathogen attack often leads to rapid activation of defense responses that limit multiplication and spread of the pathogen. To investigate the signaling mechanisms underlying this process, we carried out a screen for mutants in the signaling pathway governing resistance in Arabidopsis thaliana to the bacterial pathogen Pseudomonas syringae. This involved screening for suppressor mutations that restored resistance to a susceptible line carrying a mutation in the RPS2 resistance gene. A mutant that conferred resistance by activating defense responses in the absence of pathogens was isolated. This mutant, which carries a mutation at the CPR5 locus and was thus designated cpr5-2, exhibited resistance to P. syringae, spontaneous development of necrotic lesions, elevated PR gene expression in the absence of pathogens, and abnormal trichomes. Resistance gene-mediated defenses, including the hypersensitive response, restriction of pathogen growth, and induction of defense-related gene expression, were functional in cpr5-2 mutant plants. Additionally, in cpr5-2 plants RPS2-mediated induction of PR-1 expression was enhanced, whereas RPM1-mediated induction of ELI3 was not. These findings suggest that CPR5 encodes a negative regulator of the RPS2 signal transduction pathway

The barley HvSTP13GR mutant triggers resistance against biotrophic fungi

High-yielding and stress-resistant crops are essential to ensure future food supply. Barley is an important crop to feed livestock and to produce malt, but the annual yield is threatened by pathogen infections. Pathogens can trigger an altered sugar partitioning in the host plant, which possibly leads to an advantage for the pathogen. Hampering these processes represents a promising strategy to potentially increase resistance. We analysed the response of the barley monosaccharide transporter HvSTP13 towards biotic stress and its potential use for plant protection. The expression of HvSTP13 increased on bacterial and fungal pathogen-associated molecular pattern (PAMP) application, suggesting a PAMP-triggered signalling that converged on the transcriptional induction of the gene. Promoter studies indicate a region that is probably targeted by transcription factors downstream of PAMP-triggered immunity pathways. We confirmed that the nonfunctional HvSTP13GR variant confers resistance against an economically relevant biotrophic rust fungus in barley. Our experimental setup provides basal prerequisites to further decode the role of HvSTP13 in response to biological stress. Moreover, in line with other studies, our experiments indicate that the alteration of sugar partitioning pathways, in a host–pathogen interaction, is a promising approach to achieve broad and durable resistance in plants

Does children's healthcare seeking change after participation in a musculoskeletal study?:A register-based study

BACKGROUND: Participating in research studies often involves interactions with healthcare professionals, potentially influencing the participant's future help-seeking behaviour. We investigated whether participating in the Childhood Health Activity and Motor Performance School Study - Denmark (CHAMPS) (2008-2014), which involved telephone consultations and clinical assessments by healthcare professionals with participants experiencing musculoskeletal complaints, changed frequency of contacts with primary public healthcare services among participants over the subsequent five-years-period, compared to non-participating children.METHODS: Using Danish health register data from 1998 to 2020, we compared CHAMPS participant's and two control group's contacts with private physiotherapists, chiropractors (outside hospitals), and general practitioners: a random 10% sample of children from Denmark (National Controls), and a secondary local control group (Local Controls) during three periods: Before (1998-31.10.2008), during (01.11.2008-20.06.2014), and after (21.06.2014-31.12.2019) the CHAMPS-study. Separate multivariable Poisson regression models were used to assess the differences between groups for the outcome variables: contacts with physiotherapists, chiropractors, and general practitioners, and overall contacts.RESULTS: Compared to National Controls, the CHAMPS-Group had fewer physiotherapy contacts before the study with an estimated mean of 0.01 vs 0.02 per person-year, and after (0.13 vs 0.18 per person-year), corresponding to a crude incidence rate ratio (IRR) of 0.69 (95% confidence intervals (CI): 0.58-0.83) after the study period. However, they had more chiropractor contacts before (0.05 vs 0.03), and after (0.21 vs 0.09) the study, with a crude IRR of 2.29 (95% CI: 1.93-2.71) after the study period. General practice contacts were equal for the CHAMPS-group compared to national controls (5.84 vs 5.84) before the study but reduced during and after (3.21 vs 3.71), with a crude IRR of 0.86 (95% CI: 0.83-0.90) after the study. Comparable patterns of contacts changes from before to after the study were observed between the CHAMPS-group and the Local Controls except for physiotherapy which was equal between the two groups after the study.CONCLUSION: Our findings suggest that research studies involving systematic engagement with participants experiencing musculoskeletal complaints can influence subsequent healthcare-seeking behaviour. Future research should address the influence of health literacy, health education, and healthcare provider recommendations on healthcare decisions during such research studies.</p

Rice breeding in the new era: Comparison of useful agronomic traits

Understanding agronomic traits at a genetic level enables the leveraging of this knowledge to produce crops that are more productive and resilient, have better quality and are adjusted for consumer preferences. In the last decade, rice has become a model to validate the function of specific genes, resulting in valuable but scattered information. Here, we aimed to identify particular genes in rice related to traits that can be targeted by different mutation techniques in the breeding of crops. We selected gain of function, misfunction, and specific mutations associated with phenotypes of agronomic interest. The review includes specific trait-related genes involved in domestication, stress, herbicide tolerance, pathogen resistance, grain number/quality/weight, plant structure, nitrogen use, and others. The information presented can be used for rice, other cereals, and orphan crops to achieve a superior and sustainable production in challenging farming conditions