Overexpression of snakin-1 gene enhances resistance to Rhizoctonia solani and Erwinia carotovora in transgenic potato plants

Snakin‐1 (SN1), a cysteine‐rich peptide with broad‐spectrum antimicrobial activity in vitro, was evaluated for its ability to confer resistance to pathogens in transgenic potatoes. Genetic variants of this gene were cloned from wild and cultivated Solanum species. Nucleotide sequences revealed highly evolutionary conservation with 91–98% identity values. Potato plants (S. tuberosum subsp. tuberosum cv. Kennebec) were transformed via Agrobacterium tumefaciens with a construct encoding the S. chacoense SN1 gene under the regulation of the ubiquitous CaMV 35S promoter. Transgenic lines were molecularly characterized and challenged with either Rhizoctonia solani or Erwinia carotovora to analyse whether constitutive in vivo overexpression of the SN1 gene may lead to disease resistance. Only transgenic lines that accumulated high levels of SN1 mRNA exhibited significant symptom reductions of R. solani infection such as stem cankers and damping‐off. Furthermore, these overexpressing lines showed significantly higher survival rates throughout the fungal resistance bioassays. In addition, the same lines showed significant protection against E. carotovora measured as: a reduction of lesion areas (from 46.5 to 88.1% with respect to the wild‐type), number of fallen leaves and thickened or necrotic stems. Enhanced resistance to these two important potato pathogens suggests in vivo antifungal and antibacterial activity of SN1 and thus its possible biotechnological application.Instituto de BiotecnologíaFil: Almasia, Natalia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina.Fil: Bazzini, Ariel Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina.Fil: Vazquez Rovere, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Almasia, Natalia Ines. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Área de Biotecnología; ArgentinaFil: Hopp, Horacio Esteban. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Área de Biotecnología; Argentin

The overexpression of antifungal genes enhances resistance to rhizoctonia solani in transgenic potato plants without affecting arbuscular mycorrhizal symbiosis

The biological control of fungal diseases through the use of genetically modified (GM) plants could decrease the input of chemical pesticides. To overcome possible losses in potato (Solanum tuberosum) yield because of susceptibility to soil fungal pathogens, researchers have developed potato transgenic lines expressing antifungal proteins. However, all GM crops must be monitored in their potentially detrimental effects on non-target soil microorganisms. Arbuscular mycorrhizal (AM) fungi are good candidates for this type of analysis, as good indicators of a normal rhizosphere structure and functionality. In this work, we have monitored potato lines with over-expression of genes encoding peptides with antifungal properties on their effects on the soil-borne fungal pathogen Rhizoctonia solani and AM fungi.The six GM potato lines (AG-1, AG-3, RC-1, RC-5, AGRC-8 and AGRC-12) evaluated showed higher reduction in infection indexes in comparison to untransformed plants when challenged with a highly virulent strain of R. solani. The growth of RC-1, RC-5 and AGRC-12 lines remained almost unaltered by the pathogen; which evidenced the maximum inhibition of R. solani infection. The level of root colonization by the AM fungus Rizophagus intraradices (pure in vitro isolated) did not significantly differ between transgenic and wild potato lines under in vitro and microcosm conditions. An increase in mycorrhization was evident with the addition of potato biomass residues of these GM lines in comparison to the addition of residues of the wild type potato line.In addition to the R. intraradices assays, we performed microcosm assays with soil samples from sites with at least100-year history of potato crop as inoculum source.The roots of AGRC-12 GM line showed significant higher levels of native mycorrhization and arbuscules development. In general, the potato lines apparently were less receptive to R. intraradices pure inoculum than to AM species from the natural inoculum. In this work, the selected GM potato lines did not have evident adverse effects on AM fungal colonization.Fil: Fernandez Bidondo, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; ArgentinaFil: Almasia, Natalia Ines. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bazzini, Ariel Alejandro. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Colombo, Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; ArgentinaFil: Hopp, E.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Vazquez Rovere, Cecilia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Godeas, Alicia Margarita. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

linc-mipep and linc-wrb encode micropeptides that regulate chromatin accessibility in vertebrate-specific neural cells

Thousands of long intergenic non-coding RNAs (lincRNAs) are transcribed throughout the vertebrate genome. A subset of lincRNAs enriched in developing brains have recently been found to contain cryptic open-reading frames and are speculated to encode micropeptides. However, systematic identification and functional assessment of these transcripts have been hindered by technical challenges caused by their small size. Here, we show that two putative lincRNAs (linc-mipep, also called lnc-rps25, and linc-wrb) encode micropeptides with homology to the vertebrate-specific chromatin architectural protein, Hmgn1, and demonstrate that they are required for development of vertebrate-specific brain cell types. Specifically, we show that NMDA receptor-mediated pathways are dysregulated in zebrafish lacking these micropeptides and that their loss preferentially alters the gene regulatory networks that establish cerebellar cells and oligodendrocytes - evolutionarily newer cell types that develop postnatally in humans. These findings reveal a key missing link in the evolution of vertebrate brain cell development and illustrate a genetic basis for how some neural cell types are more susceptible to chromatin disruptions, with implications for neurodevelopmental disorders and disease

Citrus psorosis virus 24K protein interacts with citrus miRNA precursors, affects their processing and subsequent miRNA accumulation and target expression

Sweet orange (Citrus sinensis), one of the most important fruit crops worldwide, may suffer from disease symptoms induced by virus infections, thus resulting in dramatic economic losses. Here, we show that the infection of sweet orange plants with two isolates of Citrus psorosis virus (CPsV) expressing different symptomatology alters the accumulation of a set of endogenous microRNAs (miRNAs). Within these miRNAs, miR156, miR167 and miR171 were the most down-regulated, with almost a three-fold reduction in infected samples. This down-regulation led to a concomitant up-regulation of some of their targets, such as Squamosa promoter-binding protein-like 9 and 13, as well as Scarecrow-like 6. The processing of miRNA precursors, pre-miR156 and premiR171, in sweet orange seems to be affected by the virus. For instance, virus infection increases the level of unprocessed precursors, which is accompanied by a concomitant decrease in mature species accumulation. miR156a primary transcript accumulation remained unaltered, thus strongly suggesting a processing deregulation for this transcript. The co-immunoprecipitation of viral 24K protein with pre-miR156a or pre-miR171a suggests that the alteration in the processing of these precursors might be caused by a direct or indirect interaction with this particular viral protein. This result is also consistent with the nuclear localization of both miRNA precursors and the CPsV 24K protein. This study contributes to the understanding of the manner in which a virus can alter host regulatory mechanisms, particularly miRNA biogenesis and target expression.Fil: Reyes, Carina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Ocolotobiche, Eliana Evelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Marmisollé, Facundo E. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Robles Luna, Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Borniego, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Bazzini, Ariel Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Asurmendi, Sebastian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: García, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Optimized CRISPR-RfxCas13d system for RNA targeting in zebrafish embryos

CRISPR-Cas systems have been used to induce DNA mutagenesis for gene function discovery. However, the development of tools to eliminate RNAs provides complementary and unique approaches to disrupt gene expression. Here, we present a workflow to perform specific, efficient, and cost-effective mRNA knockdown in zebrafish embryos using our in vivo optimized CRISPR-RfxCas13d (CasRx) system. Although the described protocol focuses on mRNA knockdown in zebrafish embryos, it can also be applied to other vertebrates.This work was supported by Ramon y Cajal (RyC-2017-23041), PGC2018-097260-B-I00 grant and MDM-2016-0687 program (Spanish Ministerio de Ciencia, Innovación y Universidades, and European Union), Universidad Pablo de Olavide (UPO) Research and the Springboard programs from UPO and CABD, respectively (M.A.M.-M.). This study was supported by the Stowers Institute for Medical Research. A.A.B. was awarded a Pew Innovation Fund and the US National Institutes of Health (R01 GM136849). This work was performed as part of the research for the obtainment of a G.dS.P degree., Graduate School of the Stowers Institute for Medical Research. The CABD is an institution funded by Pablo de Olavide University, Consejo Superior de Investigaciones Científicas (CSIC), and Junta de Andalucía. L.H.-H. is a recipient of a predoctoral fellowship from Ministerio de Ciencia

Optimizing new RNA-targeting CRISPR-Cas systems in vivo

Trabajo presentado en EMBO Workshop CRISPR-Cas: From biology to therapeutic applications, celebrado en Sevilla (España) del 07 al 10 de noviembre de 2023.CRISPR-RfxCas13d system induces effective mRNA knockdown in vertebrate embryos, including zebrafish and mouse embryos. Until recently it was considered a precise tool to induce RNA depletion in an efficient and specific manner, however, this has been up to debate due to the recent discovery of collateral activity in eukaryotic cells. This phenomenon previously described in bacteria and in vitro consists in the degradation of non-target RNAs by the Cas13 protein in a non-controlled manner upon the previous specific targeting mediated by the gRNA. The need to further study the activity of this tool in different contexts has brought us to characterize the collateral activity of RfxCas13d in zebrafish embryos and to optimize other RNA-targeting CRISPR-Cas tools. Here, we demonstrate that CRISPR-RfxCas13d can be used to reliably deplete the vast majority of naturally present RNAs in zebrafish embryos without inducing collateral activity. We also discuss the potential of two other CRISPR-Cas systems, Cas7-11 and DjCas13d, as well as a high-fidelity version of RfxCas13d, as alternatives for the depletion of extremely abundant RNAs in zebrafish embryos, analysing their on-target and collateral activity. Overall, this work will contribute to enhance CRISPR-Cas approaches to target RNA in vivo, that ultimately will help to potentially use this technology as a precise therapeutic tool

CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos

Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to degrade RNA in yeast, plants, and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-RfxCas13d (CasRx) is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that zygotically expressed and maternally provided transcripts are efficiently targeted, resulting in a 76% average decrease in transcript levels and recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish, and mouse embryos. Altogether, our results demonstrate that CRISPR-RfxCas13d is an efficient knockdown platform to interrogate gene function in animal embryos.This work was supported by Ramon y Cajal program (RyC-2017-23041) and grants PGC2018-097260-B-I00 and MDM-2016-0687 from Spanish Ministerio de Ciencia, Innovación y Universidades and the Springboard program from CABD (M.A.M.-M.) and the Stowers Institute for Medical Research (A.A.B.). M.A.M.-M. was the recipient of the Genome Engineer Innovation 2019 Grant from Synthego. A.A.B. was awarded with Pew Innovation Fund. J.R.M.-M. is supported by BFU2017-86339-P and MDM-2016-0687 grants (Spanish Ministerio de Ciencia, Innovación y Universidades). E.M.-T. and J.A.-N.d.P. are supported by INNOVATE PERÚ grant 168-PNICP-PIAP-2015 and FONDECYT travel grant 043-2019