ABC transporter genes ABC-C6 and ABC-G33 alter plant-microbe-parasite interactions in the rhizosphere

Abstract

Plants are master regulators of rhizosphere ecology, secreting a complex mixture of compounds into the soil, collectively termed plant root exudate. Root exudate composition is highly dynamic and functional, mediating economically important interactions between plants and a wide range of soil organisms. Currently we know very little about the molecular basis of root exudate composition, which is a key hurdle to functional exploitation of root exudates for crop improvement. Root expressed transporters modulate exudate composition and could be manipulated to develop beneficial plant root exudate traits. Using Virus Induced Gene silencing (VIGS), we demonstrate that knockdown of two root-expressed ABC transporter genes in tomato cv. Moneymaker, ABC-C6 and ABC-G33, alters the composition of semi-volatile compounds in collected root exudates. Root exudate chemotaxis assays demonstrate that knockdown of each transporter gene triggers the repulsion of economically relevant Meloidogyne and Globodera spp. plant parasitic nematodes, which are attracted to control treatment root exudates. Knockdown of ABC-C6 inhibits egg hatching of Meloidogyne and Globodera spp., relative to controls. Knockdown of ABC-G33 has no impact on egg hatching of Meloidogyne spp. but has a substantial inhibitory impact on egg hatching of G. pallida. ABC-C6 knockdown has no impact on the attraction of the plant pathogen Agrobacterium tumefaciens, or the plant growth promoting Bacillus subtilis, relative to controls. Silencing ABC-G33 induces a statistically significant reduction in attraction of B. subtilis, with no impact on attraction of A. tumefaciens. By inoculating selected differentially exuded compounds into control root exudates, we demonstrate that hexadecaonic acid and pentadecane are biologically relevant parasite repellents. ABC-C6 represents a promising target for breeding or biotechnology intervention strategies as gene knockdown leads to the repulsion of economically important plant parasites and retains attraction of the beneficial rhizobacterium B. subtilis. This study exposes the link between ABC transporters, root exudate composition, and ex planta interactions with agriculturally and economically relevant rhizosphere organisms, paving the way for new approaches to rhizosphere engineering and crop protection.

Figure 1

VIGS triggers target-specific knockdown of root-expressed ABC transporter genes in tomato. (A) The mean ratio of ABC-C6 abundance relative to the endogenous control gene, elongation factor 1 subunit alpha (EF-α), following blunt syringe inoculation of A. tumefaciens and pTRV plasmids. (B) The mean ratio of ABC-C6 abundance relative to the endogenous control gene following topical application of A. tumefaciens and pTRV plasmids. (C) The mean ratio of ABC-G33 abundance relative to the endogenous control gene following blunt syringe inoculation of A. tumefaciens and pTRV plasmids. (D) The mean ratio of ABC-G33 abundance relative to the endogenous control gene following topical application of A. tumefaciens and pTRV plasmids. Data represent three biological replicates sampled on day 21 post-inoculation, with each replicate consisting of three plants each; error bars represent SEM. One-way ANOVA and Tukey’s HSD tests were used to assess statistical significance between groups (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 2

ABC transporter gene knockdown modulates PPN hatching, attraction and invasion. Hatching of J2s in response to experimental treatment (root exudates or water) as a factor of time, for (A) M. incognita; (B) M. javanica; and (C) G. pallida. Error bars represent SEM. (D) Chemosensory challenge of Meloidogyne and Globodera spp. A positive chemosensory index (CI) equates to attraction towards tested exudates, and a negative CI infers repulsion from the exudates. Data represent five biological replicates for each nematode species in each treatment group. (E) The number of galls formed (Meloidogyne spp.) or developing females found on the root surface (G. pallida) six weeks post inoculation on VIGS plants (n = 10 plants per species). Asterisks indicate statistical significance relative to controls following one-way ANOVA and Tukey’s HSD tests: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3

Heatmap showing differences in the relative abundance of identified compounds across experimental exudates. The mean composition of 10 biological replicates (three plants per replicate) is plotted for each experimental group post-VIGS, and has been assessed by two-way ANOVA, and Tukey’s multiple comparison test. Statistical significance is indicated relative to the PDS knockdown control, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

PPN responses to selected exudate compounds. Chemotaxis responses to selected compounds inoculated into control plant root exudate (PDS knockdown treatment) for: (A) M. incognita; (B) M. javanica; (C) G. pallida. Data represent the mean of five biological replicates of root exudate, for which ten replicate assays are performed for each nematode species. Asterisks indicate statistical significance in chemosensory index relative to the PDS:DMSO control following one-way ANOVA and Tukey’s HSD tests. Hatch responses following inoculation of control exudate with selected compounds for: (D) M. incognita; (E) M. javanica; (F) G. pallida. The area under the curve of percentage hatch (AUCPH) was estimated by trapezoidal integration and compared by one-way ANOVA and Dunnett’s multiple comparisons test and asterisks indicate statistical significance in hatching relative to the PDS:DMSO control. **P < 0.01; ***P < 0.001; ****P < 0.0001, error bars represent SEM.

Figure 5

ABC transporter gene knockdown modulates the attraction of B. subtilis, but not A. tumefaciens. (A) Chemosensory response of B. subtilis (168) to root exudates collected following gene knockdown. (B) Chemosensory response of A. tumefaciens (AGL-1) to root exudates collected following gene knockdown. Data represent ten biological replicates for each species. **P < 0.01; ***P < 0.001; ****P < 0.0001, error bars represent SEM.