CaC3H14 encoding a tandem CCCH zinc finger protein is directly targeted by CaWRKY40 and positively regulates the response of pepper to inoculation by Ralstonia solanacearum

Abstract

Tandem CCCH zinc finger (TZnF) proteins have been implicated in plant defence, but their role in pepper (Capsicum annuum) is unclear. In the present study, the role of CaC3H14, a pepper TZnF protein, in the immune response of pepper plants to Ralstonia solanacearum infection was characterized. When fused to the green fluorescent protein, CaC3H14 was localized exclusively to the nuclei in leaf cells of Nicotiana benthamiana plants transiently overexpressing CaC3H14. Transcript abundance of CaC3H14 was up-regulated by inoculation with R. solanacearum. Virus-induced silencing of CaC3H14 increased the susceptibility of the plants to R. solanacearum and down-regulated the genes associated with the hypersensitive response (HR), specifically HIR1 and salicylic acid (SA)-dependent PR1a. By contrast, silencing resulted in the up-regulation of jasmonic acid (JA)-dependent DEF1 and ethylene (ET) biosynthesis-associated ACO1. Transient overexpression of CaC3H14 in pepper triggered an intensive HR, indicated by cell death and hydrogen peroxide (H₂ O₂ ) accumulation, up-regulated PR1a and down-regulated DEF1 and ACO1. Ectopic overexpression of CaC3H14 in tobacco plants significantly decreased the susceptibility of tobacco plants to R. solanacearum. It also up-regulated HR-associated HSR515, immunity-associated GST1 and the SA-dependent marker genes NPR1 and PR2, but down-regulated JA-dependent PR1b and ET-dependent EFE26. The CaC3H14 promoter and was bound and its transcription was up-regulated by CaWRKY40. Collectively, these results indicate that CaC3H14 is transcriptionally targeted by CaWRKY40, is a modulator of the antagonistic interaction between SA and JA/ET signalling, and enhances the defence response of pepper plants to infection by R. solanacearum.

Keywords: Capsicum annuum; Ralstonia solanacearum; CCCH-type zinc finger; immunity.

Figure 1

Relative expression levels of CaC3H14 and CaPR1a in pepper plants infected with Ralstonia solanacearum at various hours post‐inoculation (hpi) from quantitative real‐time polymerase chain reaction (PCR) analysis. The relative transcript levels of CaC3H14 were compared with those in control plants, which were assigned as unity. CaPR1a was used as a positive control. Data represent the mean ± standard deviation (SD) of two independent experiments, each with three replicates (n = 6). *P < 0.05; **P < 0.01 (Student–Newman–Keuls test).

Figure 2

Subcellular localization of CaC3H14. Nicotiana benthamiana leaves were infiltrated with cells of Agrobacterium tumefaciens strain GV3101 containing the 35S::CaC3H14‐GFP construct. Fluorescence of the green fluorescent protein (GFP) was detected 48 h after inoculation. Bar, 50 μm. DAPI, 4,6‐diamidino‐2‐phenylindole.

Figure 3

Virus‐induced gene silencing (VIGS) of CaC3H14 enhanced the susceptibility of pepper plants to Ralstonia solanacearum. (a) The silencing efficiency of CaC3H14 in TRV::CaC3H14 plants, with or without inoculation, detected by real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) at 24 and 48 h post‐inoculation (hpi). The relative transcription level of CaC3H14 in TRV::CaC3H14 plants was compared with that in mock‐treated TRV::00 plants, which was set as unity. (b) Ralstonia solanacearum growth in inoculated CaC3H14‐silenced or control plants at 48 hpi. CFU, colony‐forming unit. (c) Disease symptoms of TRV::00 and TRV::CaC3H14 plants at 6 days post‐inoculation (dpi) by root irrigation. (d) Dynamic disease index of TRV::00 and TRV::CaC3H14 pepper plants from 6 to 12 dpi; data represent the mean ± standard deviation (SD) of three biological replicates each comprising five plants. (e) The effect of CaC3H14 silencing on the relative expression of defence‐related genes in inoculated plants at 24 hpi. The relative transcription of immunity‐associated marker genes in R. solanacearum‐inoculated TRV::00 and TRV::CaC3H14 and mock‐treated TRV::CaC3H14 plants was compared with that in mock‐treated TRV::00 plants, which was set as unity. In (a), (b), (d) and (e), data represent the mean ± SD of two independent experiments, each with three replicates (n = 6). Different letters indicate significant difference as determined by Student–Newman–Keuls test (P < 0.01).

Figure 4

Transient overexpression of CaC3H14 induced cell death and defence‐related gene expression in pepper plants. (a) Photographs of pepper leaves which had been infiltrated with Agrobacterium tumefaciens strain GV3101 carrying the 35S::00 (empty vector) or 35S::CaC3H14 construct. Left panel: infiltrated pepper leaves at 4 days post‐inoculation (dpi); middle panel: after staining with 3,3′‐diaminobenzidine (DAB) to detect hydrogen peroxide; right panel: after staining with trypan blue to assess cell death. (b) Electrolyte leakage of pepper leaves measured by conductivity after agro‐infiltration with A. tumefaciens strain GV3101 cells containing the 35S::00 or 35S::CaC3H14 construct. (c) Relative expression levels of defence‐related genes, assessed by quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) analysis, in leaves transiently overexpressing 35S::CaC3H14 or 35S::00 at 24 h post‐inoculation (hpi). The transcript levels of defence‐related genes in CaC3H14 transient overexpressing leaves were compared with those in control plants, which were assigned as unity. In (b) and (c), data represent the mean ± standard deviation (SD) of two independent experiments, each with three replicates (n = 6). **P < 0.01 (Student–Newman–Keuls test).

Figure 5

Ectopic overexpression of CaC3H14 decreased susceptibility of the transgenic tobacco plants to Ralstonia solanacearum strain FJC100301 compared with wild‐type (WT) plants. (a) Disease symptoms of R. solanacearum‐inoculated tobacco plants at 7 days post‐inoculation (dpi) by root irrigation. (b) Growth of R. solanacearum in the third leaves of R. solanacearum‐inoculated plants of transgenic lines (CaC3H14‐OE‐2 and CaC3H14‐OE‐8) and WT control at 36 h post‐inoculation (hpi). (c) Dynamic disease index of plants inoculated with R. solanacearum using root irrigation, from 6 to 12 dpi. Data represent the mean ± standard deviation (SD) of three biological replicates, each comprising five plants. **P < 0.01 (Student–Newman–Keuls test).

Figure 6

Relative expression of defence‐related genes in transgenic tobacco lines overexpressing CaC3H14 or wild‐type (WT) tobacco plants, assessed using quantitative reverse transcription‐polymerase chain reaction (RT‐PCR). Plants were inoculated with Ralstonia solanacearum using root irrigation and assessed 48 h later. Controls were mock‐treated WT plants and transgenic plants. Expression in the former was set to unity. Data represent the mean ± standard deviation (SD) of two independent experiments, each with three replicates (n = 6). Different letters indicate significant difference as determined by Student–Newman–Keuls test (P < 0.01).

Figure 7

CaC3H14 is directly targeted and transcriptionally regulated by CaWRKY40. (a) The distribution of W‐boxes in the CaC3H14 promoter and the primer pairs used in chromatin immunoprecipitation (ChIP) analyses. PW1–2, PW3, primer pairs of the fragment containing W‐box 1 and 2, which are close to each other, and W‐box 3, respectively. CP, control primer pair (negative control), which was designed based on a region without a W‐box. (b) Assay of binding of CaWRKY40 to the CaC3H14 promoter determined by ChIP‐polymerase chain reaction (PCR) with different specific primer pairs. Pepper leaves were inoculated with Agrobacterium tumefaciens strain GV3101 cells containing the construct of 35S::CaWRKY40‐HA and harvested 48 h after inoculation. The immunoprecipitated DNA was used as a template for PCR. (c) Interaction of CaWRKY40 with the promoter fragment of CaC3H14 by microscale thermophoresis in solution. Data represent the mean ± standard deviation (SD) of three replicates. (d) The binding of CaWRKY40 to the CaC3H14 promoter was enhanced by Ralstonia solanacearum inoculation. Pepper leaves were inoculated with Agrobacterium tumefaciens strain GV3101 cells containing a construct of 35S::CaWRKY40‐HA; at 24 h post‐inoculation, they were inoculated with R. solanacearum, and 24 h later they were harvested. The PW3 primer pair was used for real‐time PCR; the data represent the mean ± SD of two independent experiments, each with three replicates (n = 6). **P < 0.01 (Student–Newman–Keuls test). (e) Assay of binding of CaWRKY27 and CaWRKY58 to the CaC3H14 promoter determined by ChIP‐PCR with different specific primer pairs using CaWRKY40 as positive control; the Anti‐HA+ derived from CaWRKY27 or CaWRKY58 transient overexpressing leaves was used as template with PW3 as the primer pair for PCR. Input, total DNA–protein complex; HA, human influenza haemagglutinin; Anti‐HA+, DNA–protein complex immunoprecipitated with anti‐HA antibody (α‐HA).

Figure 8

The transcription of CaC3H14 in pepper was up‐regulated by transient overexpression of CaWRKY40 and down‐regulated by CaWRKY40‐silencing using virus‐induced gene silencing (VIGS). (a) Relative expression levels of CaC3H14 in plants in which CaWRKY40 was transiently overexpressed. Expression in mock‐treated controls (35S::00) was set to 1. (b) Relative expression levels of CaC3H14 in CaWRKY40‐silenced plants. Expression in mock‐treated control plants (35S::00 or TRV::00) was set to 1. dpi; days post inoculation with R. solanacearum; data represent mean ± SD of two independent experiments, each with three replicates (n = 6); *, p < 0.05; **, p < 0.01 (Student–Newman–Keuls test).