Plant-like bacterial expansins play contrasting roles in two tomato vascular pathogens

Abstract

Expansin proteins, which loosen plant cell walls, play critical roles in normal plant growth and development. The horizontal acquisition of functional plant-like expansin genes in numerous xylem-colonizing phytopathogenic bacteria suggests that bacterial expansins may also contribute to virulence. To investigate the role of bacterial expansins in plant diseases, we mutated the non-chimeric expansin genes (CmEXLX2 and RsEXLX) of two xylem-inhabiting bacterial pathogens, the Actinobacterium Clavibacter michiganensis ssp. michiganensis (Cmm) and the β-proteobacterium Ralstonia solanacearum (Rs), respectively. The Cmm ΔCmEXLX2 mutant caused increased symptom development on tomato, which was characterized by more rapid wilting, greater vascular necrosis and abundant atypical lesions on distant petioles. This increased disease severity correlated with larger in planta populations of the ΔCmEXLX2 mutant, even though the strains grew as well as the wild-type in vitro. Similarly, when inoculated onto tomato fruit, ΔCmEXLX2 caused significantly larger lesions with larger necrotic centres. In contrast, the Rs ΔRsEXLX mutant showed reduced virulence on tomato following root inoculation, but not following direct petiole inoculation, suggesting that the RsEXLX expansin contributes to early virulence at the root infection stage. Consistent with this finding, ΔRsEXLX attached to tomato seedling roots better than the wild-type Rs, which may prevent mutants from invading the plant's vasculature. These contrasting results demonstrate the diverse roles of non-chimeric bacterial expansins and highlight their importance in plant-bacterial interactions.

Keywords: Clavibacter michiganensis; Ralstonia solanacearum; bacterial plant pathogenesis; expansin; horizontal gene transfer.

Figure 1

Atypical tomato petiole lesions associated with the Clavibacter michiganensis ssp. michiganensis expansin mutant ΔCmEXLX2. (A) An individual petiole lesion rupturing a vascular bundle. Necrosis appeared to be confined to the vasculature with healthy plant tissue surrounding the petiole lesions. (B) Multiple lesions localized to the vasculature of an individual petiole. Black arrows highlight the large canker lesions present along multiple vascular tracks.

Figure 2

Deletion of Clavibacter michiganensis ssp. michiganensis (Cmm) expansin gene CmEXLX2 accelerates tomato fruit lesion development. (A) Lesion and halo sizes on tomato fruit (n = 100) inoculated with the three Cmm strains. Fruit lesion distances followed by the same letter are not significantly different. Differences amongst treatments for mean lesion size were determined with PROC GLIMMIX (P < 0.05) followed by Tukey–Kramer post‐test (P < 0.01). (B) Representative images of tomato fruit lesions: Cmm0317 (wild‐type), ΔCmEXLX2 (expansin mutant) and CΔCmEXLX2⁺ (complement). Scale bar, 1 mm. Error bars correspond to the standard error.

Figure 3

Virulence and root attachment of Ralstonia solanacearum (Rs) expansin mutant ΔRsEXLX. (A) Bacteria were directly inoculated into tomato stem vasculature by placing 500 colony‐forming units (CFUs) onto the surface of a freshly cut leaf petiole [P = 0.1056, repeated‐measures analysis of variance (ANOVA)]. (B) Plants were naturalistically inoculated by drenching the soil around the plants with approximately 5 × 10⁸ CFU bacteria/g soil (P = 0.0465, repeated‐measures ANOVA). Experiments were repeated three times with total n = 45 plants per strain. (C) 10⁴ CFU Rs were incubated for 2 h with sterile tomato seedling roots. The percentage of bacteria attached to roots after washing was measured by serial dilution plating of ground roots. *P < 0.0001 by ANOVA. Error bars correspond to the standard error.

Figure 4

Maximum‐likelihood phylogenetic tree for bacterial expansins represented by Clavibacter michiganensis, Ralstonia solanacearum and Bacillus subtilis. Alignment gaps were excluded, and the total number of sites used was 188 with 1000 repetitions. Bootstrap values are shown at the nodes if greater than 50%. The tree is drawn to scale, with branch lengths measured as the number of substitutions per site (Tamura et al., 2011).