160 - Microbiología Molecular y Fisiología

CRISPR-CAS MEDIATED BASE EDITING IN PHYTOPATHOGENIC BACTERIA Xanthomonas vesicatoria

Ponso, M. Agustina¹ - Martino, Roman² - Smania, Andrea² - Bianco, M. Isabel³ - Yaryura, Pablo M¹.

1) Instituto Multidisciplinario de Investigación y Transferencia Agroalimentario y Biotecnológica (IMITAB, UNVM-CONICET). Instituto Académico Pedagógico de Ciencias Básicas y Aplicadas - Villa María - Córdoba - Argentina.
2) Departamento de Química Biológica Ranwel-Caputto (CIQUIBIC-CONICET), Facultad de Ciencias Químicas, Universidad Nacional de Córdoba - Córdoba - Argentina.
3) Instituto de Ciencia y Tecnología “Dr. César Milstein”, Consejo Nacional de Investigaciones Científicas y Técnicas (ICT Milstein - CONICET); Fundación Pablo Cassará - Ciudad Autónoma de Buenos Aires - Buenos Aires - Argentina.
Contacto: aponso@unvm.edu.ar

Xanthomonas spp. are gram-negative phytopathogenic bacteria that affect a wide variety of different crops worldwide. Among them, Xanthomonas vesicatoria (Xv) infects tomato (Solanum lycopersicum) and pepper (Capsicum spp.), causing bacterial spot. This disease is the reason for substantial yield loss leading to great economic loss. Current management of this disease relies on copper-based bactericides, which are only partially effective and pose environmental concerns. Xanthan, the main exopolysaccharide (EPS) produced by Xanthomonas spp., plays an important role in bacterial virulence, influencing factors such as motility, biofilm formation, resistance to stress agents, colonization and survival in the plant. The production of xanthan is regulated by the gum operon in which gumB is the first gene that codes for an outer membrane protein. In other Xanthomonas species, gumB mutants were unable to polymerize and export xanthan. We therefore believe that the same effect will be observed when editing gumB in Xv. The genomic modifications in phytopathogenic bacteria as Xv by traditional methods are laborious and time-consuming. Instead, the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated proteins) based genome editing technology has become increasingly important in prokaryotic research over the years, but is rarely used in phytopathogenic strains. In this study we used a cytosine base editor (CBE) mediated by CRISPR-Cas to edit the gumB gene in Xv strain BNM 208, to obtain a xanthan-deficient Xv strain. The editing strategy lies on the use of CBE to generate a C>T base change causing a newly premature stop codon through the design of an specific RNA guide (gRNA) to edit gumB. Here, we construct the editing vector and transform BNM 208 in order to obtain the edited strain, ΔgumB. We further characterized virulence associated phenotypic traits as EPS production (precipitation of xanthan), biofilm formation (by violet cristal technique) and swarming motility (in a semisolid medium). The ΔgumB strain lacks the capacity for xanthan production, swarming motility and biofilm formation. We find interesting the study of mutant strains deficient in the function of different virulence factors being crucial for understanding bacterial virulence factors, pathogenesis and colonization mechanisms. This will help us identify action targets that will lead to development of a disease management alternative in the future. Moreover, the use of CRISPR technology in Xanthomonas spp., and even in phytopathogenic bacteria is still scarce. This is the first strain of Xv to be edited using CRISPR-Cas mediated technologies.

Palabras clave: Xanthomonas - cytosine base editor - CRISPR