Xanthomonas transcriptome inside cauliflower hydathodes reveals bacterial virulence strategies and physiological adaptations at early infection stages

Abstract

Xanthomonas campestris pv. campestris (Xcc) is a seed‐transmitted vascular pathogen causing black rot disease on cultivated and wild Brassicaceae. Xcc enters the plant tissues preferentially via hydathodes, which are organs localized at leaf margins. To decipher both physiological and virulence strategies deployed by Xcc during early stages of infection, the transcriptomic profile of Xcc was analysed 3 days after entry into cauliflower hydathodes. Despite the absence of visible plant tissue alterations and despite a biotrophic lifestyle, 18% of Xcc genes were differentially expressed, including a striking repression of chemotaxis and motility functions. The Xcc full repertoire of virulence factors had not yet been activated but the expression of the HrpG regulon composed of 95 genes, including genes coding for the type III secretion machinery important for suppression of plant immunity, was induced. The expression of genes involved in metabolic adaptations such as catabolism of plant compounds, transport functions, sulphur and phosphate metabolism was upregulated while limited stress responses were observed 3 days postinfection. We confirmed experimentally that high‐affinity phosphate transport is needed for bacterial fitness inside hydathodes. This analysis provides information about the nutritional and stress status of bacteria during the early biotrophic infection stages and helps to decipher the adaptive strategy of Xcc to the hydathode environment.

Genome-wide identification of fitness determinants in theXanthomonas campestrisbacterial pathogen during early stages of plant infection

“…The mutant library encompassed 51,853 out of the 77,290 possible mariner insertion sites (TA dinucleotides) in the Xcc 8004 genome. With a four-fold coverage and one insertion every 100 bp on average, the mutagenesis was saturated and allowed the study of 3,674 out of 4,617 annotated CDS (Luneau et al, 2022a)…”

“…Deletion mutants were constructed with the SacB double recombination method (Schäfer et al, 1994) using pK18 derivatives (Table S4). Complemented strains were obtained by genomic integration in trans of selected genes under the constitutive p tac promoter using pK18_CompR3 plasmid derivatives (Luneau et al, 2022a; Table S4). Primers designed for all constructions are listed in Table S4.…”

“…We evaluated bacterial aggressiveness of the mutant strains by piercing-inoculation in the midrib of cauliflower leaves using a needle dipped in a Xcc inoculum adjusted at OD 600 = 0.1. We assessed disease progression at 10 days post-inoculation according to the annotation scale presented in (Luneau et al, 2022a) in three independent biological replicates of five individual plants each.…”

“…To initiate infection, Xcc enters host tissues via the hydathode pores. After a six-day long biotrophic phase, Xcc switches to necrotrophic behavior and destroys the inner epithemal tissue of hydathodes (Cerutti et al, 2017; Luneau et al, 2022a). Simultaneously, Xcc accesses xylem vessels and progressively spreads systemically in the plant.…”

“…Altogether, these traits contribute to the fitness of the bacterium during the infection. However, while previous studies have investigated in depth the virulence factors deployed by Xcc to implement its pathogenic lifestyle at late stages of infection, very few have looked at the genetic determinants of fitness at early stages of plant infection (An et al, 2020; Timilsina et al, 2020; Luneau et al, 2022a). Xcc colonization of hydathodes is associated with sedentary behavior, activation of pathogenicity determinants (T3SS) and expression of high-affinity transporters for nutrient uptake ( e.g .…”