Highly efficient heritable targeted deletions of gene clusters and non-coding regulatory regions in Arabidopsis using CRISPR/Cas9

Genome editing using CRISPR/Cas9 is considered the best instrument for genome engineering in plants. This methodology is based on the nuclease activity of Cas9 that is guided to specific genome sequences by single guide RNAs (sgRNAs) thus enabling researchers to engineer simple mutations or large chromosomal deletions. Current methodologies for targeted genome editing in plants using CRISPR/Cas9 are however largely inefficient, mostly due to low Cas9 activity, variable sgRNA efficiency and low heritability of genetic lesions. Here, we describe a newly developed strategy to enhance CRISPR/Cas9 efficiency in Arabidopsis thaliana focusing on the design of novel binary vectors (pUbiCAS9-Red and pEciCAS9-Red), the selection of highly efficient sgRNAs, and the use of direct plant regeneration from induced cell cultures. Our work demonstrates that by combining these three independent developments, heritable targeted chromosomal deletions of large gene clusters and intergenic regulatory sequences can be engineered at a high efficiency. Our results demonstrate that this improved CRISPR/Cas9 methodology can provide a fast, efficient and cost-effective tool to engineer targeted heritable chromosomal deletions, which will be instrumental for future high-throughput functional genomics studies in plants

Spatio-temporal expression patterns of Arabidopsis thaliana and Medicago truncatula defensin-like genes

Plant genomes contain several hundred defensin-like (DEFL) genes that encode short cysteine-rich proteins resembling defensins, which are well known antimicrobial polypeptides. Little is known about the expression patterns or functions of many DEFLs because most were discovered recently and hence are not well represented on standard microarrays. We designed a custom Affymetrix chip consisting of probe sets for 317 and 684 DEFLs from Arabidopsis thaliana and Medicago truncatula, respectively for cataloging DEFL expression in a variety of plant organs at different developmental stages and during symbiotic and pathogenic associations. The microarray analysis provided evidence for the transcription of 71% and 90% of the DEFLs identified in Arabidopsis and Medicago, respectively, including many of the recently annotated DEFL genes that previously lacked expression information. Both model plants contain a subset of DEFLs specifically expressed in seeds or fruits. A few DEFLs, including some plant defensins, were significantly up-regulated in Arabidopsis leaves inoculated with Alternaria brassicicola or Pseudomonas syringae pathogens. Among these, some were dependent on jasmonic acid signaling or were associated with specific types of immune responses. There were notable differences in DEFL gene expression patterns between Arabidopsis and Medicago, as the majority of Arabidopsis DEFLs were expressed in inflorescences, while only a few exhibited root-enhanced expression. By contrast, Medicago DEFLs were most prominently expressed in nitrogen-fixing root nodules. Thus, our data document salient differences in DEFL temporal and spatial expression between Arabidopsis and Medicago, suggesting distinct signaling routes and distinct roles for these proteins in the two plant species

Co2TiO4/reduced graphene oxide nanohybrids for electrochemical sensing applications

For the first time, the synthesis, characterization, and analytical application for hydrogen peroxide quantification of the hybrid materials of Co2TiO4 (CTO) and reduced graphene oxide (RGO) is reported, using in situ (CTO/RGO) and ex situ (CTO+RGO) preparations. This synthesis for obtaining nanostructured CTO is based on a one-step hydrothermal synthesis, with new precursors and low temperatures. The morphology, structure, and composition of the synthesized materials were examined using scanning electron microscopy, X-ray diffraction (XRD), neutron powder diffraction (NPD), and X-ray photoelectron spectroscopy (XPS). Rietveld refinements using neutron diffraction data were conducted to determine the cation distributions in CTO. Hybrid materials were also characterized by Brunauer-Emmett-Teller adsorption isotherms, Scanning Electron microscopy, and scanning electrochemical microscopy. From an analytical point of view, we evaluated the electrochemical reduction of hydrogen peroxide on glassy carbon electrodes modified with hybrid materials. The analytical detection of hydrogen peroxide using CTO/RGO showed 11 and 5 times greater sensitivity in the detection of hydrogen peroxide compared with that of pristine CTO and RGO, respectively, and a two-fold increase compared with that of the RGO+CTO modified electrode. These results demonstrate that there is a synergistic effect between CTO and RGO that is more significant when the hybrid is synthetized through in situ methodology.Fil: Venegas, Constanza J.. Universidad de Chile; Chile. Universidad de Santiago de Chile; ChileFil: Gutierrez, Fabiana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Eguílaz Rubio, Marcos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Marco, José F.. Consejo Superior de Investigaciones Científicas; EspañaFil: Reeves-McLaren, Nik. University Of Sheffield; Reino UnidoFil: Rivas, Gustavo Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Ruiz-León, Domingo. Universidad de Santiago de Chile; ChileFil: Bollo, Soledad. Universidad de Chile; Chil

Detection of AtRKD4 protein during induction of somatic embryogenesis in Dendrobium lineale Rolfe transgenic orchids carrying 35S::GR::AtRKD4

Dendrobium lineale is an Indonesian native orchid from the Spatulata section in Orchidaceae Family. This orchid is important because it is usually used as a parental plant in orchid breeding and is predicted to have a potential phytochemistry compound. In addition, in their natural habitat, this orchid is threatened due to forest exploitation and natural disaster. Therefore the precision mass propagation techniques for this orchid need to be conducted. Biotechnological approaches through inserting embryo gene such as AtRKD4 from Arabidopsis thaliana has already been successfully conducted. This study aims to check the integration stability of T-DNA harboring 35S::GR::AtRKD4 from ten selection transformants and to detect the existence of AtRKD4 protein after induction by Dexamethasone and/ Thidiazuron. The result showed that T-DNA were stably integrated into the genome of D. lineale transformants and the AtRKD4 protein with a molecular weight of 28.53 kDa was detected in D. lineale transformant plants after being induced by 15 µM DEX and 3 mgL-1 TDZ for 5 days

A new role for histone demethylases in the maintenance of plant genome integrity

Histone modifications deposited by the Polycomb repressive complex 2 (PRC2) play a critical role in the control of growth, development, and adaptation to environmental fluctuations of most multicellular eukaryotes. The catalytic activity of PRC2 is counteracted by Jumonji-type (JMJ) histone demethylases, which shapes the genomic distribution of H3K27me3. Here, we show that two JMJ histone demethylases in Arabidopsis, EARLY FLOWERING 6 (ELF6) and RELATIVE OF EARLY FLOWERING 6 (REF6), play distinct roles in H3K27me3 and H3K27me1 homeostasis. We show that failure to reset these chromatin marks during sexual reproduction results in the transgenerational inheritance of histone marks, which cause a loss of DNA methylation at heterochromatic loci and transposon activation. Thus, Jumonji-type histone demethylases play a dual role in plants by helping to maintain transcriptional states through development and safeguard genome integrity during sexual reproduction

A novel signaling pathway required for Arabidopsis endodermal root organization shapes the Rhizosphere microbiome

The Casparian strip (CS) constitutes a physical diffusion barrier to water and nutrients in plant roots, which is formed by the polar deposition of lignin polymer in the endodermis tissue. The precise pattern of lignin deposition is determined by the scaffolding activity of membrane-bound Casparian Strip domain proteins (CASPs), but little is known of the mechanism(s) directing this process. Here, we demonstrate that Endodermis-specific Receptor-like Kinase 1 (ERK1) and, to a lesser extent, ROP Binding Kinase1 (RBK1) are also involved in regulating CS formation, with the former playing an essential role in lignin deposition as well as in the localization of CASP1. We show that ERK1 is localized to the cytoplasm and nucleus of the endodermis and that together with the circadian clock regulator, Time for Coffee (TIC), forms part of a novel signaling pathway necessary for correct CS organization and suberization of the endodermis, with their single or combined loss of function resulting in altered root microbiome composition. In addition, we found that other mutants displaying defects in suberin deposition at the CS also display altered root exudates and microbiome composition. Thus, our work reveals a complex network of signaling factors operating within the root endodermis that establish both the CS diffusion barrier and influence the microbial composition of the rhizosphere

Progress and challenges for the machine learning-based design of fit-for-purpose monoclonal antibodies

Although the therapeutic efficacy and commercial success of monoclonal antibodies (mAbs) are tremendous, the design and discovery of new candidates remain a time and cost-intensive endeavor. In this regard, progress in the generation of data describing antigen binding and developability, computational methodology, and artificial intelligence may pave the way for a new era of in silico on-demand immunotherapeutics design and discovery. Here, we argue that the main necessary machine learning (ML) components for an in silico mAb sequence generator are: understanding of the rules of mAb-antigen binding, capacity to modularly combine mAb design parameters, and algorithms for unconstrained parameter-driven in silico mAb sequence synthesis. We review the current progress toward the realization of these necessary components and discuss the challenges that must be overcome to allow the on-demand ML-based discovery and design of fit-for-purpose mAb therapeutic candidates

Evaluating the extent and impact of the extreme Storm Gloria on Posidonia oceanica seagrass meadows

Extreme storms can trigger abrupt and often lasting changes in ecosystems by affecting foundational (habitat-forming) species. While the frequency and intensity of extreme events are projected to increase under climate change, its impacts on seagrass ecosystems remain poorly documented. In January 2020, the Spanish Mediterranean coast was hit by Storm Gloria, one of the most devastating recent climate events in terms of intensity and duration. We conducted rapid surveys of 42 Posidonia oceanica meadows across the region to evaluate the extent and type of impact (burial, unburial and uprooting). We investigated the significance of oceanographic (wave impact model), geomorphological (latitude, depth, exposure), and structural (patchiness) factors in predicting impact extent and intensity. The predominant impact of Storm Gloria was shoot unburial. More than half of the surveyed sites revealed recent unburial, with up to 40 cm of sediment removed, affecting over 50 % of the meadow. Burial, although less extensive, was still significant, with 10–80 % of meadow cover being buried under 7 cm of sediment, which is considered a survival threshold for P. oceanica. In addition, we observed evident signs of recently dead matte in some meadows and large amounts of detached drifting shoots on the sea bottom or accumulated as debris on the beaches. Crucially, exposed and patchy meadows were much more vulnerable to the overall impact than sheltered or continuous meadows. Given how slow P. oceanica is able to recover after disturbances, we state that it could take from decades to centuries for it to recoup its losses. Seagrass ecosystems play a vital role as coastal ecological infrastructure. Protecting vulnerable meadows from anthropogenic fragmentation is crucial for ensuring the resilience of these ecosystems in the face of the climate crisis.This study was funded by the CSIC project “Effects of storm Gloria on the western Mediterranean meadows (202030E052) and “Storms of change: as phenomena extreme weather alters Mediterranean coastal ecosystems, their services and their perception by society" (PID2020-113745RB-I00), state program of I+D+I Oriented to the Challenges of the Society and within the framework of the activities of the Spanish Government through the "Maria de Maeztu Centre of Excellence” accreditation to IMEDEA (CSIC-UIB) (CEX2021-001198). We want to thank the SPAS (Society of Fishing and Underwater Activities of Mataró) and the Mataró City Council, which has financed 25 years of the Alguer de Mataró project

HSFA1a modulates plant heat stress responses and alters the 3D chromatin organization of enhancer-promoter interactions

The complex and dynamic three-dimensional organization of chromatin within the nucleus makes understanding the control of gene expression challenging, but also opens up possible ways to epigenetically modulate gene expression. Because plants are sessile, they evolved sophisticated ways to rapidly modulate gene expression in response to environmental stress, that are thought to be coordinated by changes in chromatin conformation to mediate specific cellular and physiological responses. However, to what extent and how stress induces dynamic changes in chromatin reorganization remains poorly understood. Here, we comprehensively investigated genome-wide chromatin changes associated with transcriptional reprogramming response to heat stress in tomato. Our data show that heat stress induces rapid changes in chromatin architecture, leading to the transient formation of promoter-enhancer contacts, likely driving the expression of heat-stress responsive genes. Furthermore, we demonstrate that chromatin spatial reorganization requires HSFA1a, a transcription factor (TF) essential for heat stress tolerance in tomato. In light of our findings, we propose that TFs play a key role in controlling dynamic transcriptional responses through 3D reconfiguration of promoter-enhancer contacts