Endosidin2-14 Targets the Exocyst Complex in Plants and Fungal Pathogens to Inhibit Exocytosis

Abstract

The evolutionarily conserved octameric exocyst complex tethers secretory vesicles to the site of membrane fusion during exocytosis. The plant exocyst complex functions in cell wall biosynthesis, polarized growth, stress responses, and hormone signaling. In fungal pathogens, the exocyst complex is required for growth, development, and pathogenesis. Endosidin2 (ES2) is known to inhibit exocytosis in plant and mammalian cells by targeting the EXO70 subunit of the exocyst complex. Here we show that an analog of ES2, ES2-14, targets plant and two fungal EXO70s. A lower dosage of ES2-14 than of ES2 is required to inhibit plant growth, plant exocytic trafficking, and fungal growth. ES2-14 treatments inhibit appressorium formation and reduce lesion sizes caused by Magnaporthe oryzae Inhibition of EXO70 by ES2-14 in Botrytis cinerea also reduces its virulence in Arabidopsis (Arabidopsis thaliana). Interestingly, ES2-14 did not affect EXO70 localization or transferrin recycling in mammalian cells. Overall, our results indicate that a minor change in ES2 affects its specificity in targeting EXO70s in different organisms and they demonstrate the potential of using ES2-14 to study the mechanisms of plant and fungal exocytosis and the roles of exocytosis in fungus-plant interactions.

Figure 1.

Inhibition of Arabidopsis root growth by ES2 and ES2-14. A, Molecular structures of ES2 and ES2-14. B, Representative images of 10-d-old Arabidopsis seedlings grown on one-half strength Murashige and Skoog (MS) media supplemented with 0.1% DMSO or different concentrations of ES2 and ES2-14. Bars = 1 cm. C, A time-course assay for root growth in Arabidopsis seedlings grown on one-half strength MS media with different concentrations of ES2 and ES2-14 at the indicated time points. Data represent the mean ± sd from three independent replicates (n = 15). Statistically significant differences were determined by one-way ANOVA test followed by Tukey’s multiple comparisons test. Lowercase letters indicate significant differences between groups (P < 0.05) with regard to root length of Arabidopsis seedlings at 10 d.

Figure 2.

Inhibition of PIN2 trafficking by treatment with ES2 and ES2-14. A, PIN2:GFP localization after treatment with DMSO or different concentrations of ES2 and ES2-14 for 2 h. Scale bars = 10 μm. B, Numbers of PVCs containing PIN2:GFP in Arabidopsis root epidermal cells treated with ES2 or ES2-14 for 2 h. ES2-14 is more effective in promoting PIN2 localization to the PVC. Data represent the means ± sd (n = 90 cells from eight seedlings). C, Boxplot showing the size distribution (Feret diameter) of PVCs containing PIN2:GFP, as shown in B. D, Quantification of the fluorescence intensity of PM-localized PIN2 on root epidermal cells treated with DMSO, ES2, or ES2-14. Data represent means ± sd (n = 120 cells from eight seedlings). E, Representative images of PIN2 localization after treatment with 40 μm of BFA for 1 h followed by 80 min recovery in one-half strength MS liquid media with 0.1% DMSO, 40 μm ES2, or 40 μm ES2-14. F, Numbers of BFA-induced compartments with PIN2:GFP in root epidermal cells after 80 min recovery in one-half strength liquid MS. ES2-14 is more effective than ES2 in reducing PIN2:GFP exocytic trafficking. Data represent the means ± sd (n = 110 cells from four seedlings). Asterisks indicate significant difference as determined by paired t test: *P < 0.05; **P < 0.01; ***P < 0.0001. Lowercase letters in C indicate significant differences between groups (P < 0.05) as determined by paired t test, and those in D indicate significant differences between groups (P < 0.05) as determined by one-way ANOVA test followed by Tukey’s multiple comparisons test.

Figure 3.

Biochemical assays for direct interaction of AtEXO70A1 with ES2 and ES2-14. A and B, DARTS assay for interaction between ES2 and recombinant AtEXO70A1 with different concentrations of pronase. A, Silver staining of proteins. B, Quantification of ratios of AtEXO70A1 and BSA intensities in samples treated with ES2 and DMSO, as shown in A. Data represent means ± sd (n = 3). C, MST assay for the interaction between AtEXO70A1 and ES2. The thermophoresis binding curve of the interaction of NT-647-labeled recombinant AtEXO70A1 with different concentrations of ES2 is shown. Data represent the means ± sd (n = 4). D to F, The same assays as in A to C, respectively, but with ES2-14 used instead of ES2. Data in E and F represent the means ± sd (n = 3). The raw fluorescence of NT-647-labeled recombinant AtEXO70A1 with different concentrations of ES2-14 is shown in F, because ES2-14 quenches NT-647 fluorescence at higher concentrations tested, which may be due to the interaction with the fluorophore at the binding region. For B and E, silver-staining gels from three independent DARTS assays were used for quantification. *P < 0.05 by paired t test. G to I, DSF assay showing the thermal stability of AtEXO70A1 in the presence of increasing concentrations of ES2 (G), ES2-14 (H), and Ampicillin (I; negative control). RFU, relative fluorescence units. Data shown are representative of three independent repeats.

Figure 4.

ES2-14 affects polar localization of AtEXO70A1 and has a genetic interaction with AtEXO70A1. A, Representative images of GFP-AtEXO70A1 in cells at the root elongation zone in plants treated with DMSO, ES2, or ES2-14 for 2 h. Polar localization of GFP-AtEXO70A1 at the outer lateral side of root epidermal cells was altered by ES2 and ES2-14 treatment. Scale bars = 10 μm. B, Quantification of the fluorescence intensity of GFP-AtEXO70A1 at the outer lateral sides of root epidermal cells as shown in A. Data represent means ± sd (n = 50 from nine seedlings). C, Representative 5-d-old heterozygous exo70A1-3 plants grown on one-half strength MS media supplemented with DMSO, ES2, or ES2-14. Bars = 1 cm. D, Quantification of the root length of seedlings grown on DMSO, ES2, or ES2-14 at the indicated concentration. Data represent means ± sd (n = 13). Asterisks indicate a significant difference as determined by paired t test: *P < 0.05; ***P < 0.0001.

Figure 5.

Inhibition of B. cinerea and M. oryzae growth by ES2 and ES2-14. A, Representative cultures of M. oryzae grown on complete minimal medium supplemented with 0.1% DMSO or different concentrations of ES2 or ES2-14 for 4 d. B, Diameter of 4-d-old complete minimal medium cultures of M. oryzae in the presence of ES2 or ES2-14. Data represent means ± sd (n = 6). C, Four-day-old V8 cultures of B. cinerea colonies with 0.1% DMSO or different concentrations of ES2 or ES2-14. D, Diameter of 4-d-old V8 B. cinerea colonies in the presence of different concentrations of ES2 or ES2-14. Data represent means ± sd (n = 6). The diameters of the petri dishes are 6 cm for M. oryzae and 10 cm for B. cinerea. **P < 0.01 (B and D) as compared with the DMSO control, determined by paired t test.

Figure 6.

Biochemical assays for the interaction of ES2 and ES2-14 with MoEXO70. A and B, DARTS assays for the interaction between ES2 and recombinant MoEXO70 with different concentrations of pronase. A, Silver staining of proteins in the DARTS assay of MoEXO70 with ES2. B, Quantification of ratios of MoEXO70 and BSA intensities in silver staining gels between samples treated with ES2 and DMSO. Data represent means ± sd (n = 3). C, MST assay for the interaction between MoEXO70 and ES2. Data represent means ± sd (n = 4). The thermophoresis binding curve of the interaction of GFP-MoEXO70 with different concentrations of ES2 is shown. The gray outlier data point at the highest ES2 concentration was not used for curve fitting and Kd estimation. D to F, The same assays as shown in A to C, respectively, except that ES2-14 was used instead of ES2. Data in E and F represent means ± sd (n = 3). Silver staining gels from three independent DARTS experiments were used to generate each chart in B and E. ES2 did not protect MoEXO70 from degradation by the dilutions of pronase that were tested. ES2-14 protected MoEXO70 from degradation by pronase. *P < 0.05, paired t test. G to I, DSF assay showing the thermal stability of MoEXO70 in the presence of increasing concentrations of ES2 (G), ES2-14 (H) and Ampicillin (I; negative control). RFU, relative fluorescence units. Data shown are representative of three independent repeats.

Figure 7.

Biochemical assays for the interaction of ES2 and ES2-14 with BcEXO70. A and B, DARTS assays for the interaction between ES2 and recombinant BcEXO70 with different concentrations of pronase. A, Silver staining of proteins in the DARTS assay of BcEXO70 with ES2. B, Quantification of ratios of BcEXO70 and BSA intensities in the silver staining gels shown in A, between samples treated with ES2 and DMSO. Data represent means ± sd (n = 3). C, MST assay for the interaction between BcEXO70 and ES2. Data represent means ± sd (n = 6). The thermophoresis binding curve for the interaction of GFP-BcEXO70 with different concentrations of ES2 is shown. D to F, The same assays as A to C, respectively, except that ES2-14 was used instead of ES2. Data in E represent means ± sd (n = 3). Data in F represent means ± sd (n = 3). Silver staining gels from three independent DARTS experiments were used to generate each chart in B and E. ES2 did not protect BcEXO70 from degradation by the dilutions of pronase that were tested. ES2-14 protected BcEXO70 from degradation by pronase. *P < 0.05, paired t test. In C and F, the gray outlier data points at the highest ES2 (C) and ES2-14 (F) concentrations was not used for curve fitting and Kd estimation. G to I, DSF assay showing the thermal stability of BcEXO70 in the presence of increasing concentrations of ES2 (G), ES2-14 (H), and Ampicillin (I; negative control). RFU, relative fluorescence units. Data shown are representative of three independent repeats.

Figure 8.

ES2 and ES2-14 affect cellular localization of MoEXO70. A, Representative images of MoEXO70-GFP in conidia treated with DMSO, ES2, or ES2-14 for 3 h. ES2 and ES2-14 reduce the localization of MoEXO70-GFP at the tips of germinated conidia. The arrow points to the tip of a germinated conidium. Scale bars = 5 μm outside the insets and 2.5 μm in the insets. B, Quantification of the intensity of MoEXO70-GFP at the tips of germinated conidia, as shown in A. Data represent means ± sd (n = 10). *P < 0.05, paired t test.

Figure 9.

Inhibitory effects of ES2 and ES2-14 on the pathogenicity of M. oryzae and B. cinerea. A, Inhibition of appressorium formation in M. oryzae by ES2 and ES2-14. The percentage of germ tubes that formed appressoria was examined after incubating spore suspensions with the indicated concentrations of ES2 or ES2-14 on plastic coverslips for 24 h. Whereas ES2 only slightly inhibited appressorium formation at 80 μm, ES2-14 completely blocked the formation of appressoria at 40 μm. Data represent means ± sd (n = 300). B, Rice leaves inoculated with M. oryzae spores mixed with DMSO, ES2, or ES2-14 were photographed at 6 dpi. C, Average size of lesions on rice leaves inoculated with M. oryzae spores mixed with the indicated concentrations of DMSO, ES2, or ES2-14 at 6 dpi. Data represent means ± sd (n = 20 from 10 plants). D, Arabidopsis leaves inoculated with B. cinerea spores mixed with DMSO, ES2, or ES2-14 were examined at 3 dpi. E, Average size of lesions caused by B. cinerea in treatments with DMSO, ES2, or ES2-14. Data represent means ± sd (n = 10 from 10 plants). Scale bars = 1 cm (B and D). *P < 0.05; **P < 0.01, ***P < 0.001, as determined by paired t test.