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.

Keywords: Xanthomonas; type 3 secretion system; Chlorophyll Fluorescence Imaging; Emergence; non-pathogenic strains.

Figure 1

Phylogenetic positioning of the non‐pathogenic strains CFBP 7698 and CFBP 7700. Strains CFBP 7698 and CFBP 7700 were positioned amongst the 82 genomes described by Merda et al. (2017). The phylogeny was constructed based on the 968 protein sequences shared by the 84 strains, using CVTree software, with a K‐mer size of 6. Bar, 0.02 substitutions per site. The non‐pathogenic strain CFBP 7698 clusters with strains belonging to the species Xanthomonas cannabis, and CFBP 7700 clusters with strains belonging to the species Xanthomonas campestris. The major independent acquisition events of a T3SS (as proposed by Merda et al., 2017) are indicated with a red arrow. Clades A, B, C and D are defined as proposed by Merda et al. (2017).

Figure 2

Map of the Tn5 insertions in the hrp cluster of Xcc 8004 cloned in pIJ3225. The hrp cluster is represented on the basis of the genome sequence of Xcc 8004 (He et al., 2007). Approximate positions of Tn5 insertions were determined on the basis of the physical map provided by Arlat et al. (1991). The exact positions of Tn5 insertions were determined by sequencing amplicons obtained using primers designed on Tn5 and the putative target genes. Vertical bars show the position of Tn5 insertions in pIJ3225. Genes involved in the suppression of the hypersensitive response (HR)‐like necrosis are represented in red. The intensity of the colour is representative of the impact of the Tn5 insertions on the ability of pIJ3225 to suppress the HR‐like necrosis induced by the non‐pathogenic strains CFBP 7698 and CFBP 7700. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

The type 3 secretion system (T3SS) encoded on pIJ3225 is functional in non‐pathogenic strains of Xanthomonas naturally devoid of T3SS. Capsicum annuum ECW (A) and ECW20R carrying the Bs2 resistance gene (B, C) were infiltrated with Xanthomonas strains to test for a hypersensitive response (HR). 1: Strain Xcc 8004 serves as an HR positive control on ECW and ECW20R to ensure that inoculated leaves are fully responsive. 2: Strain 7634R serves as a negative control: it does not induce HR on ECW. Strain 7634R possesses AvrBs2, but no T3SS encoding genes, and therefore does not induce HR on ECW20R either. 3: Strain 7634R pIJ3225: HR is observed specifically on ECW20R plants but not on ECW plants, showing that HR is caused by the specific recognition of AvrBs2 by Bs2. Therefore, pIJ3225 encodes a T3SS that enables the translocation of AvrBs2 into plant cells in a non‐pathogenic Xanthomonas background. 4a: Strain 7634R G9: the disruption of hrcJ inactivates the secretion through the T3SS. The absence of HR on ECW20R plants shows that the transfer of AvrBs2 is T3SS dependent. 4b: Strain 7634R H7: the disruption of hpa2 leads to an HR that appears milder than that observed in 3 (strain 7634R pIJ3225). Thus the translocation of AvrBs2 in 4b may be less efficient than that in 3. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

The acquisition of pIJ3225 enables the suppression of the hypersensitive response (HR)‐like necrosis on Nicotiana benthamiana. Leaves were inoculated with mock, wild‐type strains 7698R and 7700R, as well as their transconjugants carrying pIJ3225 or the pIJ3225::G9 (hrcJ ^‐) construct. The development of HR‐like necrosis was followed over 6 days post‐inoculation (dpi). The acquisition of a functional T3SS on pIJ3225 enables the suppression of the HR‐like necrosis on N. benthamiana. The acquisition of a non‐functional T3SS on plasmid pIJ3225::G9 does not enable the suppression of the HR‐like necrosis on N. benthamiana. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

(A–G) Clustering based on multiparametric information. Thresholds for the segmentation of chlorophyll fluorescence images were determined within each leaf based on the phenotypes obtained in areas infiltrated with control strains. (A) Each leaf features four infiltrated areas: (a) infiltrated with either strain 7698R or 7700R, and used as a positive control for the onset of hypersensitive response (HR)‐like necrosis on each leaf; (b) infiltrated with strain 7698R pIJ3225 or 7700R pIJ3225, and used as a control for the ability of pIJ3225 to suppress HR‐like necrosis on each leaf; (c) infiltrated with strain 7698R or 7700R carrying Tn5 derivatives of pIJ3225 (Table 2, Arlat et al., 1991), and used to test whether Tn5 insertions alter the ability of pIJ3225 to suppress HR‐like necrosis; (d) water‐inoculated control. (B, C) Chlorophyll fluorescence imaging of the inoculated leaves: (B) minimum fluorescence (F _o); (C) maximum fluorescence (F _m). (D, E) Distribution of chlorophyll fluorescence values amongst pixels in the areas inoculated with control strains. Histograms associated with F _o and F _m images (D, E) show that distributions of fluorescence values differ amongst control areas. (F, G) Clustering the pixels of the area inoculated with the test strains. Thresholds for clustering are determined on the basis of the distributions of chlorophyll fluorescence values obtained with control strains. For each control area (D, E), the upper and lower thresholds were fixed to correspond to the 20th quantile, i.e. splitting 1/20 of the F _o and F _m pixel distribution values. They were determined for each leaf to take into consideration the variability between leaves. These thresholds were then applied to histograms associated with F _o and F _m images of inoculated area (c) to cluster pixels that display fluorescence values similar to each control area (F, G). (H) Four clusters were determined. Clusters 1 and 2 correspond to tissues found on the HR‐like necrosis control (cluster 1, most impacted tissues; cluster 2, less impacted tissues). Cluster 4 corresponds to tissues found in the areas inoculated with pIJ3225 control strains (suppression control). Cluster 3 corresponds to fluorescence values intermediate between HR‐like necrosis and the suppression controls. We conclude the presence of HR‐like necrosis in the area inoculated with the test strain when the number of cumulated pixels belonging to clusters 1–3 is superior to the 20th quantile. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

The acquisition of a functional type 3 secretion system (T3SS) impairs the multiplication of non‐pathogenic strains of Xanthomonas after inoculation on Nicotiana benthamiana. For each strain, inocula calibrated at 10⁸ colony‐forming units (cfu)/mL were infiltrated into the leaves of N. benthamiana. Bacterial population sizes were determined by dilution plating. The charts show the combined results representative of two independent experiments, each experiment involving six independent leaves per strain for each time point. The top panel shows the results obtained with strain 7698R and its derivatives, and the bottom panel shows the results obtained with strain 7700R and its derivatives. Means marked with the same letter were not significantly different using a Kruskall–Wallis non‐parametric test. dpi, days post‐inoculation; wt, wild‐type. [Color figure can be viewed at wileyonlinelibrary.com]