Silencing of a Cotton Actin-Binding Protein GhWLIM1C Decreases Resistance against Verticillium dahliae Infection

Abstract

LIM proteins are widely spread in various types of plant cells and play diversely crucial cellular roles through actin cytoskeleton assembly and gene expression regulation. Till now, it has not been clear whether LIM proteins function in plant pathogen defense. In this study, we characterized a LIM protein, GhWLIM1C, in upland cotton (Gossypium hirsutum). We found that GhWLIM1C could bind and bundle the actin cytoskeleton, and it contains two LIM domains (LIM1 and LIM2). Both the two domains could bind directly to the actin filaments. Moreover, the LIM2 domain additionally bundles the actin cytoskeleton, indicating that it possesses a different biochemical activity than LIM1. The expression of GhWLIM1C responds to the infection of the cotton fungal pathogen Verticillium dahliae (V. dahliae). Silencing of GhWLIM1C decreased cotton resistance to V. dahliae. These may be associated with the down regulated plant defense response, including the PR genes expression and ROS accumulation in the infected cotton plants. In all, these results provide new evidence that a plant LIM protein functions in plant pathogen resistance and the assembly of the actin cytoskeleton are closely related to the triggering of the plant defense response.

Keywords: LIM protein; V. dahliae; actin cytoskeleton; cotton; defense response.

Figure 1

Expression analysis of cotton GhWLIM1C. (A) qRT-PCR analysis of GhWLIM1C expression in cotton roots in response to V. dahliae infection. Total RNA was extracted from cotton roots inoculated with V. dahliae spores (1 × 10⁶ conidia/mL) for 0, 6, 12, 24, 36, 48, 60, and 72 h, respectively, and 0 h indicates cotton plants without inoculation with the fungal spores. Error bars represent SD from three independent experiments (n = 3). Asterisks indicate statistically significant differences, as determined by Student’s t-test (** p < 0.01). The experiments were repeated three times with similar results. (B) qRT-PCR analysis of GhWLIM1C gene expression in root (R), stem (S), leaf (L), petal (P), anther (A), and fiber (F; 12 days post-anthesis) in cotton plants. The expression levels are indicated relative to cotton UBI gene. Error bars represent ± SE of three biological replicates.

Figure 2

GhWLIM1C silencing decreases resistance to V. dahliae infection in cotton. (A) Preliminary assay of the efficiency of VIGS under our experimental conditions. Ten-day-old cotton plants were infiltrated with Agrobacterium carrying TRV:PDS. The photographs were taken 2 weeks after infiltration. (B,C) Phenotypes of control (B) and GhWLIM1C silenced cotton plants (C). Cotton plants were infiltrated with Agrobacterium carrying VIGS-control vector (TRV:00) or (TRV:GhWLIM1C) and inoculated with a suspension of V. dahliae spores. Photographs were taken 2 weeks after V. dahliae infection. (D) qRT-PCR analysis of GhWLIM1C expression in cotton leaves and the root tissues infiltrated with VIGS-control vector (TRV:00) and TRV:GhWLIM1C. Error bars indicate SD from three technical replicates of one biological experiment. The experiments were repeated three times with similar results. (E,F) Rate of diseased plants and disease index of TRV:00 and TRV:GhWLIM1C cotton plants. Error bars represent the SD of three biological replicates (n = 36), and asterisks indicate statistically significant differences, as determined by Student’s t-test (** p < 0.01).

Figure 3

Subcellular localization of GhWLIM1C and its two LIM domains. Colocalization of GhWLIM1C (A), LIM1 (B), LIM2 (C). and ABD2-RFP in the N. benthamiana cells. Agrobacterium cells containing the indicated pair of GhWLIM1C-GFP, LIM1-GFP, LIM2-GFP, and ABD2-RFP plasmids were coinfiltrated into leaves of N. benthamiana. The signal was visualized by confocal microscopy. Bars = 20 µm.

Figure 4

GhWLIM1C bundles actin filaments in vitro. (A) Low-speed cosedimentation assay of the actin-bundling activity of GhWLIM1C. The presence of GhWLIM1C in the pellet indicates its cosedimentation with F-actin. Lane 1, actin alone (3 µM); lanes 2 to 8, actin (3 µM) incubated with 0.2, 0.5, 1, 2, 4, 8, and 10 µM GhWLIM1C, respectively. (B) Actin bundles visualized by fluorescence microscopy. F-actin alone (left); F-actin + 1 µM GhWLIM1C (right). Bars = 1 µm.

Figure 5

Fluorescence microscopy analysis of F-actin-binding/bundling activities of GhWLIM1C and the two LIM domains. Preassembled F-actin incubated with GhWLIM1C-RFP (A), LIM1-RFP (B), and LIM2-RFP (C). Then, the samples were stained with Alexa488-phalloidin and detected with fluorescence microscopy. Actin alone was used as a negative control (D). Bars = 1 µm.

Figure 6

Defense response analysis of GhWLIM1C-silencing plants. (A–D) H2DCFDA staining of the ROS in root cells of the TRV:00 (A) and three GhWLIM1C-silencing (B–D) cotton plants at 72 hpi. Fluorescence of H2DCFDA was detected with confocal microscopy. Bars = 20 µm. (E) Quantification of the fluorescence in (A–D). (F–H) qRT-PCR analysis of the expressions of defense-related genes, PR1 (F), PR5 (G) and PDF1.2 (H) in the root tissues of the TRV:00 and three GhWLIM1C silencing cotton plants at 72 hpi. Error bars represent the SD of three biological replicates (n = 36), and asterisks indicate statistically significant differences, as determined by Student’s t-test (** p < 0.01).