Editing of SlWRKY29 by CRISPR-activation promotes somatic embryogenesis in Solanum lycopersicum cv. Micro-Tom.

At present, the development of plants with improved traits like superior quality, high yield, or stress resistance, are highly desirable in agriculture. Accelerated crop improvement, however, must capitalize on revolutionary new plant breeding technologies, like genetically modified and gene-edited crops, to heighten food crop traits. Genome editing still faces ineffective methods for the transformation and regeneration of different plant species and must surpass the genotype dependency of the transformation process. Tomato is considered an alternative plant model system to rice and Arabidopsis, and a model organism for fleshy-fruited plants. Furthermore, tomato cultivars like Micro-Tom are excellent models for tomato research due to its short life cycle, small size, and capacity to grow at high density. Therefore, we developed an indirect somatic embryo protocol from cotyledonary tomato explants and used this to generate epigenetically edited tomato plants for the SlWRKY29 gene via CRISPR-activation (CRISPRa). We found that epigenetic reprogramming for SlWRKY29 establishes a transcriptionally permissive chromatin state, as determined by an enrichment of the H3K4me3 mark. A whole transcriptome analysis of CRISPRa-edited pro-embryogenic masses and mature somatic embryos allowed us to characterize the mechanism driving somatic embryo induction in the edited tomato cv. Micro-Tom. Furthermore, we show that enhanced embryo induction and maturation are influenced by the transcriptional effector employed during CRISPRa, as well as by the medium composition and in vitro environmental conditions such as osmotic components, plant growth regulators, and light intensity.

CRISPRa-mediated transcriptional activation of the SlPR-1 gene in edited tomato plants.

With the continuous deterioration of arable land due to an ever-growing population, improvement of crops and crop protection have a fundamental role in maintaining and increasing crop productivity. Alternatives to the use of pesticides encompass the use of biological control agents, generation of new resistant crop cultivars, the application of plant activator agrochemicals to enhance plant defenses, and the use of gene editing techniques, like the CRISPR-Cas system. Here, we test the hypothesis that epigenome editing, via CRISPR activation (CRISPRa), activate tomato plant defense genes to confer resistance against pathogen attack. We provide evidence that edited tomato plants for the PATHOGENESIS-RELATED GENE 1 gene (SlPR-1) show enhanced disease resistance to Clavibacter michiganensis subsp. michiganensis infection. Resistance was assessed by evaluating disease progression and symptom appearance, pathogen accumulation, and changes in SlPR-1 gene expression at different time points. We determined that CRISPRa-edited plants develop enhanced disease-resistant to the pathogen without altering their agronomic characteristics and, above all, preventing the advancement of disease symptoms, stem canker, and plant death.

PRIMER REPORTE DE BEGOMOVIRUS INFECTANDO CULTIVOS DE AJÍ (Capsicum spp.) EN COLOMBIA / First report of begomoviruses infecting pepper (Capsicum spp.) crops in Colombia

RESUMEN Virus del género Begomovirus infectan cultivos de importancia económica en todo el mundo, incluyendo ají. A la fecha, en Colombia no hay reportes de la presencia de begomovirus infectando este cultivo, por lo que el objetivo de esta investigación fue identificar la presencia de virus de este género en ají empleando estrategias moleculares. Se colectaron 197 muestras de ají en diez municipios del Valle del Cauca. Se extrajo el DNA genómico total vegetal y mediante PCR se detectó la presencia de begomovirus. Para establecer la identidad molecular del virus se amplificaron fragmentos de 1,4 kb de muestras colectadas en Palmira y Vijes. Los fragmentos fueron clonados, secuenciados y analizados. Se encontró que el 85,7 % de las muestras de ají evaluadas fueron positivas para begomovirus. Los análisis de secuencia de los fragmentos virales de 1,4 kb arrojaron una identidad de 91,8 % entre ellos y los de secuencia de nucleótidos de los virus aislados en Vijes y Palmira mostró que éstos presentan los valores de identidad más altos (87,2 % y 86,6 %) con el virus de la distorsión de la hoja de maracuyá, un begomovirus aislado de maracuyá en Colombia. Estos análisis estarían indicando que este begomovirus aislado de ají podría ser una nueva especie. De acuerdo con la literatura, este es el primer reporte de un begomovirus infectando cultivos de ají en Colombia.

ABSTRACT Viruses of the genus Begomovirus infect crops of economic importance around the world, including pepper. To date, in Colombia there are no reports of the presence of begomoviruses infecting this crop; therefore, this research work aimed to identify the presence of viruses of this genus in pepper using molecular strategies. Around 197 pepper samples were collected in ten municipalities in Valle del Cauca. Total plant genomic DNA was extracted, and the presence of begomoviruses was detected by using PCR. In order to establish the molecular identity of the virus, fragments of 1.4 kb were amplified from samples collected in Palmira and Vijes municipalities. The fragments obtained were cloned, sequenced, and analyzed. The results show that about 85.7 % of the pepper samples evaluated were positive for begomoviruses. Sequence analysis of the viral fragments of 1.4 kb showed an identity of 91.8 % among them. The nucleotide sequence analysis of the begomoviruses isolated in Vijes and Palmira showed its highest identity values (87.2 % and 86.6 %) with the passion fruit leaf distortion virus, a begomovirus that is affecting passion fruit crops in Colombia. These sequences analyze would indicate that this begomovirus isolated from pepper could be a new species that has not been reported worldwide. To our knowledge, this is the first report of a begomovirus infecting pepper crops in Colombia.