Tomato R-gene-mediated resistance against Fusarium wilt originates in roots and extends to shoots via xylem to limit pathogen colonization

Abstract

Vascular wilt disease, caused by the soil-borne fungus Fusarium oxysporum (Fo), poses a threat to many crop species. Four different tomato resistance (R) genes (I-1, I-2, I-3, and I-7) have been identified to confer protection against Fo f.sp. lycopersici (Fol). These I genes are root-expressed and mount an immune response upon perception of the invading fungus. Despite immune activation, Fol is still able to colonize the xylem vessels of resistant tomato lines. Yet, the fungus remains localized in the vessels and does not colonize adjacent tissues or cause disease. The molecular mechanism constraining Fol in the vascular system of the stem remains unclear. We here demonstrate that an I-2-resistant rootstock protects a susceptible scion from Fusarium wilt, notwithstanding fungal colonization of the susceptible scion. Proteomic analyses revealed the presence of fungal effectors in the xylem sap of infected plants, showing that the lack of fungal pathogenicity is not due to its inability to express its virulence genes. To identify mobile root-derived proteins, potentially involved in controlling fungal proliferation, comparative xylem sap proteomics was performed. We identified distinct pathogenesis-related (PR) protein profiles in xylem sap from Fol-inoculated I-1, I-2, I-3, and I-7 resistant lines. Despite structural diversity, all four immune receptors trigger the accumulation of a common set of four PR proteins: PR-5x, PR-P2, and two glucan endo-1,3-β-D-glucosidases. This research provides insights into Fusarium resistance mechanisms and identifies a core set of proteins whose abundance correlates with defense against Fusarium wilt.

Keywords: R-genes; effectors; pathogenesis-related proteins; plant-fungal interaction; proteomics; tomato grafting; wilt disease.

Figure 1

Resistant (I-2) tomato rootstocks protect susceptible scions from Fusarium wilt disease. Fol007 (race 2) resistant (Res) and susceptible (Sus) plants were grafted in four different combinations (rootstock | scion). Approximately five-week-old chimeric plants were either mock- or Fol007-inoculated. Disease scoring of the grafted genotypes occurred three wpi. (A) Tukey boxplot shows plant fresh weight above the cotyledon level of three independent repetitions. Each data point is represented by a filled black circle (Mock) or an open black circle (Fol). Significant differences between mock- and Fol-inoculated plants were determined using Student’s unpaired t-test (*p ≤ 0.05; **p ≤ 0.01) ns = non-significant. (B) The dot plot shows the disease index of grafted genotypes. The scores from three repetitions were merged and tested using a Mann-Whitney U statistical test (*p ≤ 0.05; ****p ≤<0.0001).

Figure 2

I-2 resistant (Res) tomato rootstocks reduce colonization of Fol in susceptible (Sus) scions. (A) Stem sections from below the graft (BG), above the graft (AG), cotyledon (CL), and 1^st true leaf node (1TLN) were monitored for the presence of Fol007 (race 2). The absence of Fol from each stem level was described as “no colonization” (NC). (B) The dot plot shows fungal stem colonization scores of different grafting combinations (rootstockscion). The scores from three repetitions were merged and tested using a Mann-Whitney U statistical test (*p ≤ 0.05). (C) Representative scan of plates from the 2^nd repetition showing Fol outgrowth from BG and AG stem slices. Each plate includes five stem slices from individual plants. (D) The column chart shows the surface area (cm^2) of Fol outgrowth from BG and AG stem slices from two repetitions. Open black circles (2^nd repetition) and filled black circles (3^rd repetition) represent the average of four to eight stem slices per grafting combination. The scores from two repetitions were merged and tested using an ordinary one-way ANOVA (**p ≤ 0.01). ns = non-significant.

Figure 3

Fungal SIX proteins are present in xylem sap of resistant I-1, I-2, I-3, and I-7 lines inoculated with Fol. Log2 fold change values indicate relative abundance differences of fungal secretory proteins SIX1 (Avr3), SIX3 (Avr2), and SIX6 between Fol- and mock-inoculated resistant lines (four or five repetitions). Grey bars represent differentially accumulating SIX proteins based on a Student’s T-test.

Figure 4

I-1, I-2, I-3, and I-7 R-gene mediated resistance effects on tomato xylem sap proteome. Volcano plots represent -log10 p-values and log2 protein abundance ratios by comparing mock- and Fol- inoculated resistant lines (four or five repetitions). (A) Volcano plots compare mock- and Fol004- inoculated I-1 resistant line, (B) mock- and Fol007- inoculated I-2 resistant lines, (C) mock- and Fol029- inoculated I-3 resistant lines, and (D) mock- and Fol029- inoculated I-7 resistant lines. Grey dots represent non-differentially accumulating proteins (DAP), blue dots are downregulated DAPs (-log10 p-value > 1.3, log2 protein abundance ratio < -1), and orange dots are upregulated DAPs (-log10 p-value > 1.3, log2 protein abundance ratio >1). Four upregulated DAPs have been identified in all resistant lines and are highlighted in each volcano plot: PR-5x, PR-P2, and two glucan endo-1,3-β-D-glucosidase (endo.3BDG-a/b).

Figure 5

I-1, I-2, I-3, and I-7 induce the accumulation of unique and shared DAPs. (A) UpSet plot compares proteins differentially upregulated during R-gene-mediated resistances. The horizontal bar graph indicates the total number of DAPs per resistant line. The intersection bar graph displays the number of DAPs that are unique (black dot) or shared (colored dot) between resistant lines. (B) The tile plot shows the presence-absence of DAPs that are present in at least two R-gene lines. The tile plot color corresponds to the Upset plot dots.