CRISPRi in Xanthomonas demonstrates functional convergence of transcription activator-like effectors in two divergent pathogens.
Carlos Andrés Zárate-ChavesCorinne AudranCésar Augusto Medina CulmaAline EscalonStéphanie JavegnyLionel GagnevinEmilie ThomasLéa-Lou PimparéCamilo E LópezJonathan M JacobsLaurent D NoëlRalf KoebnikAdriana Jimena BernalBoris SzurekPublished in: The New phytologist (2023)
Functional analysis of large gene families in plant pathogens can be cumbersome using classical insertional mutagenesis. Additionally, Cas9 toxicity has limited the application of CRISPR-Cas9 for directed mutagenesis in bacteria. Here, we successfully applied a CRISPR interference strategy to investigate the cryptic role of the transcription activator-like effector (tale) multigene family in several plant-pathogenic Xanthomonas bacterial species, owing to their contribution to pathogen virulence. Single guide RNAs (sgRNAs) designed against Xanthomonas phaseoli pv manihotis tale conserved gene sequences efficiently silenced expression of all tales, with concomitant decrease in virulence and TALE-induced host gene expression. The system is readily translatable to other Xanthomonas species infecting rice, citrus, Brassica, and cassava, silencing up to 16 tales in a given strain using a single sgRNA. Complementation with plasmid-borne designer tales lacking the sgRNA-targeted sequence restored molecular and virulence phenotypes in all pathosystems. Our results evidenced that X. campestris pv campestris CN08 tales are relevant for symptom development in cauliflower. They also show that the MeSWEET10a sugar transporter is surprisingly targeted by the nonvascular cassava pathogen X. cassavae, highlighting a new example of TALE functional convergence between phylogenetically distant Xanthomonas. Overall, this novel technology provides a platform for discovery and rapid functional understanding of highly conserved gene families.
Keyphrases
- crispr cas
- genome editing
- antimicrobial resistance
- escherichia coli
- transcription factor
- genome wide identification
- pseudomonas aeruginosa
- gene expression
- genome wide
- staphylococcus aureus
- copy number
- biofilm formation
- high throughput
- dna methylation
- oxidative stress
- poor prognosis
- genome wide analysis
- gram negative
- nuclear factor
- squamous cell carcinoma
- small molecule
- dendritic cells
- lymph node metastasis
- inflammatory response
- quantum dots