Search

The Xanthomonas genus includes several plant pathogens responsible for significant crop losses worldwide. Most of the functional studies of virulence in this genus have been carried out by directed mutagenesis through homologous recombination. However, this strategy is cumbersome for the study of gene families. CRISPR interference (CRISPRi) allows precise silencing of target genes by using a catalytically dead Cas9 (dCas9) which interferes with gene expression. Because of its RNA-directed nature, this technology can be used for silencing several genes in a single experiment in bacteria and other organisms. We implemented a CRISPRi strategy to silence several members of the Transcriptional activatorlike effectors (TALE) gene family at once in four different species of Xanthomonas. Our results underscore the importance of the activation of the SWEET gene family in cassava upon infection by Xanthomonas phaseoli pv. manihotis. Remarkably, we report the importance of this gene family in the infection of this host by the non-vascular pathogen X. cassavae. In addition, we successfully silenced several TALE genes in a total of five species, including X. oryzae pv. oryzae, X. citri pv. citri and X. campestris pv. campestris using CRISPRi, confirming the importance of this gene family in these pathosystems. The CRISPRi tool can be further modulated to silence sets of genes within a gene family for functional studies in Xanthomonas and other plant pathogenic bacteria.

Childhood maltreatment is theorized as impeding the development of reflective functioning (RF; ability to perceive and interpret oneself and others in terms of mental states). However, previous research typically failed to support this association or yielded small sized and mixed associations. This study aims to provide a deeper look at the association between childhood maltreatment and RF by characterizing two non-mentalizing categories. One-hundred-and-sixteen pregnant women (mean age = 27.62, SD = 4.52) from the community (48.3% with a university degree, 96.5% in a relationship with the other parent) retrospectively reported on childhood abuse and neglect using the Childhood Trauma Questionnaire. They also participated in the Adult Attachment Interview subsequently coded using the Reflective Functioning Scale. Participants with poor to low RF were allocated to one of two groups (disavowal-distancing or distorted-inconsistent) using indicators provided in the RF Scale. No association was found between childhood maltreatment and overall RF when controlling for education level. A multinomial logistic regression revealed that childhood maltreatment was strongly predictive of a disrupted, over-analytical and inconsistent reflection about mental states but not of a tendency to discourse little about mental states. This tendency was rather only predicted by education level. Findings suggest that childhood maltreatment would lead to specific impairments in RF and that not considering how individuals fail to mentalize about attachment relationships may mask strong associations between RF and its determinants and correlates, including childhood maltreatment.

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.