Marco Adán Juárez Verdayes

Objective: To establish the optimal conditions for genetic transformation using Agrobacterium rhizogenes (A. rhizogenes) in three bean varieties. Design/methodology/approach: The experimental black bean (LEF-UA-03) and mustard (LEF-UA-01) lines, including the commercial cultivar Pinto Saltillo, were utilized to evaluate their susceptibility to transformation with A. rhizogenes K599. Subsequently, transformation efficiency was determined using both the A. rhizogenes K599 strain and the A. rhizogenes K599 (pH35GUSGFP) strain. The integration and expression of the transgene were confirmed through the visualization of green fluorescent protein (GFP) in transformed roots, as well as through the amplification of the GFP sequence from genomic DNA by PCR. Results: In the present study, the susceptibility of three bean varieties to hairy root formation was evaluated using A. rhizogenes K599 and A. rhizogenes K599 (pH35GUSGFP). The susceptibility of the varieties ranged from 69% to 76%. Limitations on study/implications: A limitation on this study is that transformation efficiency was evaluated only under a single set of experimental conditions. Therefore, genotype dependent variability and environmental factors such as bacterial concentration that may influence transformation efficiency were not extensively explored. The high susceptibility observed in all three varieties suggests that this protocol may serve as a reliable platform for functional genomics studies. Additionally, the successful generation of transgenic hairy roots indicates potential applications in the production of valuable metabolites using hairy root cultures in controlled bioreactor systems. Findings/conclusions: All bean varieties demonstrated high susceptibility to transformation with A. rhizogenes K599 > 69%. The successful expression and molecular confirmation of the GFP reporter gene validate the effectiveness of the transformation system. These results indicate that the evaluated protocol is suitable for generating transgenic hairy roots in common bean and represents a useful tool for functional gene analysis and biotechnological applications.

En este estudio, se propone una aproximación in silico de la hibridación sustractiva genómica (GSH por sus siglas en inglés) para diseñar oligonucleótidos especie‒específico que permitan identificar especies de lepidópteros por PCR. Se utilizaron dos especies de la familia Noctuidae como modelo: Spodoptera frugiperda (J. E. Smith, 1797) y Helicoverpa zea (Boddie, 1850), las cuales son importantes plagas agrícolas en México, causando daños significativos a cultivos como maíz, sorgo y algodón y otros cultivos, ya que son polífagos. La identificación de los lepidópteros es compleja debido a la variabilidad que presentan los diferentes estadios larvales incluso dentro de una misma especie (tamaño, color, forma y comportamiento). Los métodos actuales de monitoreo y control resultan ineficientes debido a la ambigüedad en la identificación taxonómica de las plagas. Si bien se han desarrollado herramientas moleculares para ciertos taxones, aún se carece de un protocolo molecular estandarizado que permita discriminar con precisión la totalidad de las especies de este orden. La técnica de PCR con oligonucleótidos especie‒específicos fue rápida y precisa para las especies modelo. Además de diferenciar el ADN genómico de ambas especies, esta herramienta ofrece una solución para la discriminación frente a otras especies congéneres morfológicamente similares. La identificación precisa de lepidópteros es importante en el manejo y control de plagas en la agricultura y evaluar la biodiversidad en diferentes ecosistemas. La técnica propuesta puede ser útil para monitorear cambios en las poblaciones de especies de importancia económica.

Summary: Developmental processes are regulated at multiple levels, including the epigenetic level. Among the epigenetic factors, histone H3 lysine 4 (H3K4) methyltransferases contribute to active transcription of target genes, and here, we explored how the H3K4 methyltransferase ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1) affects Arabidopsis thaliana lateral root (LR) primordium (LRP) morphogenesis. We examined LR development in a loss‐of‐function null mutant (atx1‐1) and a mutant affected in the ATX1 catalytic domain (atx1setm) through bright‐field and long‐term time‐lapse confocal microscopy, transcriptomics, and chromatin immunoprecipitation. LRP morphogenesis in both mutants was severely abnormal, resulting from altered principal growth directions, and was accompanied by extended cell cycle durations and slower transitions between LRP stages. Among the differentially expressed genes downregulated in atx1setm, the most enriched Gene Ontology categories were cell wall organization and H₂O₂ metabolism, the latter of which included PEROXIDASE35 (PRX35). The LRP morphogenesis abnormalities were similar in prx35 and atx1 mutants. Both the deposition of H3K4me3 at the PRX35 promoter and the PRX35 expression in the atx1setm mutant were significantly reduced. Our results reveal a link between LR development and a redox homeostasis controlled by ATX1 at epigenetic level by maintaining active transcription of PRX35 and thereby impacting root system formation.

DNA-based molecular techniques are crucial for the precise identification of microorganisms. Despite their speed and sensitivity, specificity hinges on primer or probe design. The search for unique sequences in organisms’ genomes has various applications such as microorganism identification. There are published bioinformatic tools that employ the concept of in silico genomic subtractive hybridization analysis to identify species-specific/unique regions within a genome of interest. However, they use deprecated tools and program[1]ming languages that are not currently used in bioinformatics, some of them are specialized or difficult to obtain because the repositories, where these tools were hosted, are not public or the web servers are currently down. To address these issues, we implemented the in silico genomic subtractive hybridization idea in a user-friendly, open-source, freely available, easy-to-install command pipeline application written in Python (called Not-alike3). We designed PCR primers over the sequence of unique regions identified in the genomes of two Mucorales species, Rhizopus oryzae and Cuninghamlla bertholletiae, employing Not-alike3 command pipeline; we made specificity tests to challenge these primers and we observed that they were species-specific.

Plant phospholipase C (PLC) proteins are phospholipid-degrading enzymes classified into two subfamilies: phosphoinositide-specific PLCs (PI-PLCs) and non-specific PLCs (NPCs). PI-PLCs have been widely studied in various biological contexts, including responses to abiotic and biotic stresses and plant development; NPCs have been less thoroughly studied. No PLC subfamily has been characterized in relation to the symbiotic interaction between Fabaceae (legume) species and the nitrogen-fixing bacteria called rhizobia. However, lipids are reported to be crucial to this interaction, and PLCs may therefore contribute to regulating legume–rhizobia symbiosis. In this work, we functionally characterized NPC4 from common bean ( Phaseolus vulgaris L.) during rhizobial symbiosis, finding evidence that NPC4 plays an important role in bean root development. The knockdown of PvNPC4 by RNA interference (RNAi) resulted in fewer and shorter primary roots and fewer lateral roots than were seen in control plants. Importantly, this phenotype seems to be related to altered auxin signaling. In the bean–rhizobia symbiosis, PvNPC4 transcript abundance increased 3 days after inoculation with Rhizobium tropici . Moreover, the number of infection threads and nodules, as well as the transcript abundance of PvEnod40 , a regulatory gene of early stages of symbiosis, decreased in PvNPC4 -RNAi roots. Additionally, transcript abundance of genes involved in autoregulation of nodulation (AON) was altered by PvNPC4 silencing. These results indicate that PvNPC4 is a key regulator of root and nodule development, underscoring the participation of PLC in rhizobial symbiosis.