UC Berkeley

Xanthomonas spp. are the causative pathogens of bacterial spot and have caused major yield losses in commercial varieties of tomato and pepper. Bacterial spot disease is a threat to worldwide production of Solanaceous crops, especially in warm and humid environments. The Bacterial Spot 5 (Bs5) gene, which is conserved among Solanaceae, is composed of three exons, and encodes a small proline-rich putative tail-anchored protein with an unknown function. Bs5 is flanked by its paralog, Bs5-like, in a head-to-head orientation. The Bs5 protein is a putative member of the cysteine-rich transmembrane module (CYSTM) protein family, a diverse family found to be expressed in response to biotic and abiotic stress in many eukaryotes. A naturally occurring recessive allele, bs5, was found in pepper to confer disease resistance against a hypervirulent strain of Xanthomonas euvesicatoria in pepper (Capsicum annuum). The bs5 resistance phenotype is characterized by restricted pathogen growth and an absence of necrotic lesions characteristic of bacterial spot susceptibility. Pepper varieties containing the bs5 allele have shown durable resistance, impeding the emergence of bs5-resistant strains in commercial fields. Deployment of the bs5 allele in tomato has the potential to confer disease resistance against bacterial spot. The causative mutation of bs5 is an in-frame six nucleotide deletion in the third exon. It was unknown whether the precise six nucleotide deletion is the sole mutation that confers the resistance phenotype against bacterial spot. We set out to investigate whether introducing nucleotide insertions or deletions to the coding region of tomato Bs5 would be sufficient to introduce resistance. Using CRISPR/Cas9-mediated gene editing, we established two distinct mutant lines with mutations in exon 2 of Bs5 and Bs5-L. We also established a mutant line with mutations in exon 3, the location of the bs5 allele mutation in pepper. Our objective was to establish bs5 resistance in tomatoes using CRISPR/Cas9 based genome editing. We were able to generate several mutant tomato lines with alleles distinct from bs5 in the pepper allele. These tomato lines exhibited resistance against several species of xanthomonads.