VILLIN2 regulates cotton defense against Verticillium dahliae by modulating actin cytoskeleton remodeling

Abstract

The active structural change of actin cytoskeleton is a general host response upon pathogen attack. This study characterized the function of the cotton (Gossypium hirsutum) actin-binding protein VILLIN2 (GhVLN2) in host defense against the soilborne fungus Verticillium dahliae. Biochemical analysis demonstrated that GhVLN2 possessed actin-binding, -bundling, and -severing activities. A low concentration of GhVLN2 could shift its activity from actin bundling to actin severing in the presence of Ca2+. Knockdown of GhVLN2 expression by virus-induced gene silencing reduced the extent of actin filament bundling and interfered with the growth of cotton plants, resulting in the formation of twisted organs and brittle stems with a decreased cellulose content of the cell wall. Upon V. dahliae infection, the expression of GhVLN2 was downregulated in root cells, and silencing of GhVLN2 enhanced the disease tolerance of cotton plants. The actin bundles were less abundant in root cells of GhVLN2-silenced plants than in control plants. However, upon infection by V. dahliae, the number of actin filaments and bundles in the cells of GhVLN2-silenced plants was raised to a comparable level as those in control plants, with the dynamic remodeling of the actin cytoskeleton appearing several hours in advance. GhVLN2-silenced plants exhibited a higher incidence of actin filament cleavage in the presence of Ca2+, suggesting that pathogen-responsive downregulation of GhVLN2 could activate its actin-severing activity. These data indicate that the regulated expression and functional shift of GhVLN2 contribute to modulating the dynamic remodeling of the actin cytoskeleton in host immune responses against V. dahliae.

Figure 1.

The expression pattern of Gh_A02G1060/Gh_D03G0623. A) Transcriptional expression pattern of Gh_A02G1060/Gh_D03G0623 in various cotton tissues. R, root; S, stem; L, leaf; Pe, petal; Po, pollen. B) Relative Gh_A02G1060/Gh_D03G0623 transcript levels in cotton roots upon infection with V. dahliae. hpi, hours postinoculation. Values represent means ± Sd (n = 3). Asterisks indicate statistically significant differences between mock control (0) and V. dahliae inoculated samples (3 to 48), as determined by Student's t-test (*P < 0.05; **P < 0.01). Three biological repeats were performed.

Figure 2.

Actin-binding, -bundling, and -severing activities of GhVLN2. A) High-speed cosedimentation assay of actin and GhVLN2. Actin, 2 μM; GhVLN2, 2 μM; M, molecular weight marker; S, supernatant; P, pellet. B) Low-speed cosedimentation assay of actin and GhVLN2. Actin, 2 μM; GhVLN2, 2 μM; M, molecular weight marker; S, supernatant; P, pellet. C) Actin filaments visualized by fluorescence microscopy. Actin, 2 μM; GhVLN2, 2 μM; Scale bar = 10 μm. D) Quantitative analysis of the extent of actin filament bundling (skewness) in C). Values represent means ± Sd (n = 12). The asterisk indicates a statistically significant difference between actin alone and in the presence of GhVLN2 proteins, as determined by Student's t-test (**P < 0.01). Three biological repeats were performed.

Figure 3.

Actin-severing activity of GhVLN2. A) Actin-severing activities of GhVLN2 in the presence of different concentrations of CaCl₂. Actin, 2 μM. GhVLN2, 0.5 nM. Scale bar = 10 μm. B) The distribution of actin filament length in A). Values represent means ± Sd (n ≥ 9). Asterisks indicate statistically significant difference between actin alone and in the presence of GhVLN2 proteins with different concentrations of CaCl₂, as determined by Student's t-test (*P < 0.05; **P < 0.01). Three biological repeats were performed.

Figure 4.

Representative phenotypes of GhVLN2-silenced cotton plants. A) Efficiency of GhVLN2 knockdown by VIGS. Values represent means ± Sd (n = 3). Asterisks indicate statistically significant differences between control (TRV) and GhVLN2-silenced (TRV-GhVLN2) plants, as determined by Student's t-test (**P < 0.01). B–E) Phenotypes of control (TRV) and GhVLN2-silenced (TRV-GhVLN2) cotton plants. C) Leaf; Scale bar = 2 cm. D) Stem; Scale bars = 0.5 cm. E) Root; Scale bars = 1 cm. Actin filament arrays in control (TRV) and GhVLN2-silenced (TRV-GhVLN2) cotton leaf F), stem G), and root H) epidermal cells. Scale bars = 10 μm. Quantitative analyses of the actin filament percent occupancy (density) I) and the extent of bundling (skewness) J) in F–H). Values represent means ± Sd (n ≥ 20). Asterisks indicate statistically significant differences between control (TRV) and GhVLN2-silenced (TRV-GhVLN2) cells, as determined by Student's t-test (*P < 0.05). Three biological repeats were performed.

Figure 5.

VIGS-mediated silencing of GhVLN2 increases the resistance of cotton plants to V. dahliae infection. A) Disease symptoms of control (TRV) and GhVLN2-silenced (TRV-GhVLN2) plants 2 wk after inoculation with V. dahliae. B, Disease index for control (TRV) and GhVLN2-silenced (TRV-GhVLN2) plants. Values represent means ± Sd. The asterisk indicates statistically significant differences between control (TRV) and GhVLN2-silenced (TRV-GhVLN2) plants, as determined by Student's t-test (**P < 0.01). Three biological repeats were performed. C) Fungal recovery assay. The stem sections from control (TRV) and GhVLN2-silenced (TRV-GhVLN2) cotton plants were collected 2 wk after V. dahliae infection and plated on potato dextrose agar medium for 6 d. D) Semi-quantitative analysis of V. dahliae biomass by PCR in A). The ITS region of the ribosomal DNA of V. dahliae was amplified using specific primers ITS1-F and ST-VE1-R. Cotton HISTONE H3 gene was used as an internal control.

Figure 6.

Downregulation of GhVLN2 affects cellulose deposition in cotton plants. A) Brittle stem of GhVLN2-silenced (TRV-GhVLN2) plant in comparison with control (TRV). B) Calcofluor white staining of freehand cross-sections of control (TRV) and GhVLN2-silenced (TRV-GhVLN2) cotton stem. ep, Epidermal cells; co, cortex; ph, phloem; xy, xylem. Scale bars = 100 μm. C) The statistical analysis of fluorescent intensity in the vasculature. At least 10 stem sections of control and GhVLN2-silenced plants were used for calculation. Values represent means ± Sd. D) Representative profile of the cellulose deposition in cotton stems in B). High peaks indicate intense cellulose staining, whereas lower peaks represent weak cellulose staining. The locations of the measurement were marked with the yellow lines in B) (from left to right). E) The content of cellulose in cotton stems. AIR, alcohol-insoluble residue. Values represent means ± Se (n = 3). F) Relative expression of cellulose biosynthesis genes in cotton root. Values represent means ± Sd (n = 3). G) Relative transcript levels of pathogenesis-related genes in control and GhVLN2-silenced cotton plants. Values represent means ± Sd (n = 3). Asterisks indicate statistically significant differences between control (TRV) and GhVLN2-silenced (TRV-GhVLN2) plants, as determined by Student's t-test (*P < 0.05; **P < 0.01). Three biological repeats were performed.

Figure 7.

Actin filament array in control and GhVLN2-silenced cotton root epidermal cells in response to V. dahliae infection. A) Representative confocal images of the actin filament structure in the control (TRV) and GhVLN2-silenced (TRV-GhVLN2) cotton root epidermal cells before (0) and 3, 6, 12 h post inoculation (hpi) with V. dahliae. Scale bars = 10 μm. Quantitative analyses of actin filament percent occupancy (density) B) and the extent of bundling (skewness) C) in A). Values represent means ± Sd (n = 18). Asterisks indicate statistically significant differences, as determined by Student's t-test (*P < 0.05). Three biological repeats were performed.

Figure 8.

The actin-severing activity in control and GhVLN2-silenced cotton leaf epidermal cells. A) Actin-severing activity analysis in control (TRV) and GhVLN2-silenced (TRV-GhVLN2) cotton leaf epidermal cells. Scale bars = 2 μm. B) Actin-severing activity analysis in control (TRV) and GhVLN2-silenced (TRV-GhVLN2) cotton leaf epidermal cells in the presence of 10 μM CaCl₂. Scale bars = 2 μm. Arrowheads indicate the severing events in actin filaments. C) Quantitative analysis of actin filament severing frequency in A) and B). Values represent means ± Sd (n ≥ 12). Asterisks indicate statistically significant differences, as determined by Student's t-test (*P < 0.05). Three biological repeats were performed.