Description

Ralstonia solanacearum is the causal agent of bacterial wilt disease of more than 250 plant species, and is considered as one of the most destructive plant pathogens (Mansfield et al., 2012). R. solanacearum is a soil-borne bacterium that enters plants through the roots, eventually reaching the vascular system; bacterial proliferation in xylem vessels causes vascular blockage, which leads to plant wilting and death (Xue et al., 2020). We have recently shown that R. solanacearum promotes the production of gamma-aminobutyric acid (GABA) in plant cells, which in turn can be used as a nutrient to support bacterial replication and pathogenesis inside the plant (Xian et al., 2020). In other gram-negative bacteria, such as R. eutropha (also known as Cupriavidus necator) or Pseudomonas syringae, GABA catabolism involves GABA transaminases (GabT), which convert GABA into succinate semialdehyde (SSA); SSA is used by succinate semialdehyde dehydrogenases (SSA-DH / GabD) to generate succinate, which can be incorporated into the TCA cycle (Lutke-Eversloh & Steinbuchel, 1999; Park et al., 2010; Figure 1A). The genome of R. solanacearum contains a single copy of the gabT gene (RSc0029), which is located upstream of the gabD gene (RSc0028) in the same operon (Figure 1B). Interestingly, a ΔgabT knockout mutant was unable to use GABA as a nutrient, and severely impaired in its ability to cause disease symptoms in the model plant Arabidopsis thaliana and to grow in stems of tomato plants, their natural host (Xian et al., 2020). These results suggest that R. solanacearum requires the GabT transaminase to cause disease. However, given that the gabT and gabD genes belong to the same operon, it is formally possible that the mutation in gabT affects the expression of gabD, which could also contribute to the reduced virulence observed in the ΔgabT mutant. In order to address this possibility, we complemented the ΔgabT mutant using an integrative plasmid that leads to the expression of the gabT gene driven by its own promoter (see methods; Figure 1C). First, we inoculated the ΔgabT mutant in tomato plants by soil-drenching, an inoculation method that mimics the natural infection route of R. solanacearum, and found that, as previously shown in Arabidopsis, the ΔgabT mutant is significantly impaired in its ability to cause disease symptoms in tomato, in comparison to the GMI1000 wild-type reference strain (Figure 1D and 1E). Importantly, the symptoms induced by the complementation strain expressing gabT in the ΔgabT mutant background (ΔgabT/gabT) were similar to those induced by the GMI1000 wild-type strain (Figure 1D and 1E). Moreover, upon injection in tomato stems, expression of the gabT gene was able to rescue the attenuation in growth observed in the ΔgabT mutant (Figure 1F). Altogether, these results indicate that the virulence attenuation observed in the ΔgabT mutant (Xian et al., 2020; Figure 1D and 1E) is indeed caused by mutation of the gabT gene. These results also suggest that either the gabD gene is still expressed in the ΔgabT mutant, or the gabD gene is not required for virulence. However, if the gabD gene is not required for virulence, how does R. solanacearum catabolize SSA to complete the GABA catabolic pathway? In R. eutropha it has been shown that a gabD mutant still displays SSA-DH activity (Lutke-Eversloh & Steinbuchel, 1999), suggesting the presence of additional SSA-DH enzymes, and such non-GabD SSA-DH enzymes have been identified in other bacteria, such as Escherichia coli or Klebsiella pneumoniae (Marek & Henson, 1988; Sanchez et al., 1989). Similarly, even if the gabD gene were affected in the ΔgabT mutant, it is possible that R. solanacearum uses other SSA-DH enzymes to complete the GABA catabolic pathway in the ΔgabT/gabT complementation strain.