Role of the acquisition of a type 3 secretion system in the emergence of novel pathogenic strains of Xanthomonas

Abstract

Cases of emergence of novel plant-pathogenic strains are regularly reported that reduce the yields of crops and trees. However, the molecular mechanisms underlying such emergence are still poorly understood. The acquisition by environmental non-pathogenic strains of novel virulence genes by horizontal gene transfer has been suggested as a driver for the emergence of novel pathogenic strains. In this study, we tested such an hypothesis by transferring a plasmid encoding the type 3 secretion system (T3SS) and four associated type 3 secreted proteins (T3SPs) to the non-pathogenic strains of Xanthomonas CFBP 7698 and CFBP 7700, which lack genes encoding T3SS and any previously known T3SPs. The resulting strains were phenotyped on Nicotiana benthamiana using chlorophyll fluorescence imaging and image analysis. Wild-type, non-pathogenic strains induced a hypersensitive response (HR)-like necrosis, whereas strains complemented with T3SS and T3SPs suppressed this response. Such suppression depends on a functional T3SS. Amongst the T3SPs encoded on the plasmid, Hpa2, Hpa1 and, to a lesser extent, XopF1 collectively participate in suppression. Monitoring of the population sizes in planta showed that the sole acquisition of a functional T3SS by non-pathogenic strains impairs growth inside leaf tissues. These results provide functional evidence that the acquisition via horizontal gene transfer of a T3SS and four T3SPs by environmental non-pathogenic strains is not sufficient to make strains pathogenic. In the absence of a canonical effector, the sole acquisition of a T3SS seems to be counter-selective, and further acquisition of type 3 effectors is probably needed to allow the emergence of novel pathogenic strains.

A Computation Method Based on the Combination of Chlorophyll Fluorescence Parameters to Improve the Discrimination of Visually Similar Phenotypes Induced by Bacterial Virulence Factors

“…Strain 7698R is an environmental strain belonging to the species X. cannabis isolated from bean seeds, but not pathogenic on bean. As largely documented in Meline et al (2019), when inoculated on N. benthamiana, this strain induces a necrosis of leaf tissues at 6 dpi. Strain 7698R was complemented with the plasmid pIJ3225 that carries a 20 kb hrp cluster encoding a major bacterial virulence determinant: the Type 3 Secretion System and four Type 3 Secreted Proteins (Arlat et al, 1991).…”

“…When inoculated on the non-host plant Nicotiana benthamiana, strain 7698R induces a rapid necrosis similar to the hypersensitive reaction (HR) observed in non-host resistance. The complementation of this non-pathogenic strain with a plasmid harboring genes encoding a T3SS, suppressed the onset of this HR-like necrosis, and only a mild chlorosis could be observed on inoculated tissues at 6 days post-inoculation (6 dpi) (Meline et al, 2019). Such a suppression of a defense layer could constitute a first step toward the emergence of novel pathogenic strains.…”

“…Mutant strains of Xanthomonas were already described in Meline et al (2019). Strain 7698R is an environmental strain belonging to the species X. cannabis isolated from bean seeds, but not pathogenic on bean.…”

“…On N. benthamiana at 6 dpi, the complemented strain 7698R pIJ3225 does not induce any necrosis, the inoculated tissues only display a chlorotic phenotype. Derivatives of pIJ3225 harboring a Tn5 insertion that inactivates one gene in the hrp cluster were characterized in Meline et al (2019). These derivatives were transformed into strain 7698R to obtain strains 7698R pIJ3225::Tn5 (G9, G2, F2, F15, C3, or G1).…”

“…It featured 85 leaves of N. benthamiana at 3 dpi. Each leaf was inoculated with three controls and one tested-strain as described in Meline et al (2019). The three controls were, (i) strain 7698R used as necrosis control, (ii) strain 7698R pIJ3225 used as chlorosis control, and (iii) mockinoculated control used as no symptom control.…”