A 14-3-3 Protein Ca16R Acts Positively in Pepper Immunity against Ralstonia solanacearum by Interacting with CaASR1

Abstract

Although 14-3-3 proteins have been implicated in plant growth, development, and stress response, their roles in pepper immunity against R. solanacearum remain poorly understood. In this study, a 14-3-3-encoding gene in pepper, Ca16R, was found to be upregulated by R. solanacearum inoculation (RSI), its silencing significantly reduced the resistance of pepper plants to RSI, and its overexpression significantly enhanced the resistance of Nicotiana benthamiana to RSI. Consistently, its transient overexpression in pepper leaves triggered HR cell death, indicating that it acts positively in pepper immunity against RSI, and it was further found to act positively in pepper immunity against RSI by promoting SA but repressing JA signaling. Ca16R was also found to interact with CaASR1, originally using pull-down combined with a spectrum assay, and then confirmed using bimolecular fluorescence complementation (BiFC) and a pull-down assay. Furthermore, we found that CaASR1 transient overexpression induced HR cell death and SA-dependent immunity while repressing JA signaling, although this induction and repression was blocked by Ca16R silencing. All these data indicate that Ca16R acts positively in pepper immunity against RSI by interacting with CaASR1, thereby promoting SA-mediated immunity while repressing JA signaling. These results provide new insight into mechanisms underlying pepper immunity against RSI.

Keywords: 14-3-3; ASR; R. solanacearum; pepper.

Figure 1

Ca16R was upregulated by R. solanacearum inoculation. (A) Schematic diagram of the distribution of cis-elements on the promoter of Ca16R. (B) Ca16R was upregulated by R. solanacearum inoculation from 1 to 48 hpi, reaching the highest expression level at 12 hpi; FPKM: fragments per kilobase of exon model per million mapped fragments. (C) Ca16R was upregulated by the exogenous application of SA but downregulated by exogenously applied MeJA. Data in (B,C) represent the mean ± SD from four independent experiments. Error bars indicate SD. Uppercase letters above the bars indicate significant differences (p < 0.01) calculated with Fisher’s protected t-test.

Figure 2

Silencing of Ca16R significantly reduced pepper resistance to R. solanacearum inoculation. (A) The transcript level of Ca16R in R. solanacearum TRV::Ca16R plants inoculated after 12 h is only 10% of that in the wild-type plants. (B) TRV::Ca16R pepper plants exhibited a significantly lower level of resistance to RSI compared to the wild-type plants. (C) TRV::Ca16R pepper plants exhibited a significantly higher level of dynamic disease index from 3 to 12 dpi compared to the wild-type plants. (D) TRV::Ca16R pepper plants provided a higher level of bacterial growth than the wild-type plants. Data are shown as the mean ± standard error of eight replicates. Different uppercase letters above the bars indicate significant differences (p < 0.01) calculated using Fisher’s protected t-test; CFU/cm²: the number of R. solanacearum colonies in 1 cm² of leaf. (E) The R. solanacearum TRV::Ca16R pepper plants inoculated after 12 h exhibited a lower expression level of CaPR1 and CaNPR1 but a higher expression level of CaDEF1 and CaCOI1. Data in (A,E) represent the mean ± SD from four independent experiments. Error bars indicate SD. Uppercase letters above the bars indicate significant differences (p < 0.01) calculated using Fisher’s protected t-test.

Figure 3

Transient overexpression of Ca16R triggered clear HR cell death and upregulated the expression of SA-dependent genes while repressing JA-dependent immunity-related genes. (A) Confirmation of the transient overexpression of Ca16R-GFP with RT-qPCR and with Western blotting using anti-GFP. (B) Transient overexpression of Ca16R after 96 hpi triggered HR cell death displayed by darker trypan blue staining. (C) Transient overexpression of Ca16R triggered higher ion leakage displayed by conductivity at 48 hpi. Data are shown as the mean ± standard error of six replicates. Different uppercase letters above the bars indicate significant differences (p < 0.01) based on Fisher’s protected t-test; µS/cm: microsiemens per centimeter. (D) SA-dependent CaPR1 and CaNPR1 were upregulated but JA-dependent CaDEF1 and CaCOI1 were downregulated by the transient overexpression of Ca16R. Data in (A,D) represent the mean ± SD from four independent experiments. Error bars indicate SD. Uppercase letters above the bars indicate significant differences (p < 0.01) calculated using Fisher’s protected LSD test. The RSI pepper leaves were harvested 12 h after R. solanacearum inoculation.

Figure 4

Overexpression of Ca16R-GFP enhanced the resistance of N. benthamiana to RSI. (A) The success of Ca16R with RT-qPCR and with Western blotting using anti-GFP. (B) The plants of Ca16R-overexpressing N. benthamiana lines #1 and #2 exhibited enhanced resistance to RSI compared to the wild-type plants. (C) The plants of Ca16R-overexpressing N. benthamiana lines #1 and #2 exhibited a lower disease index from 2 to 12 dpi compared to the wild-type plants. Data are shown as the mean ± standard error of twelve replicates. (D) The R. solanacearum-inoculated plants of Ca16R-overexpressing N. benthamiana lines #1 and #2 supported a lower level of bacterial growth at 48 and 96 hpi compared to the wild-type plants. Uppercase letters above the bars indicate significant differences between mean values (p < 0.01), as calculated with an LSD test. The center line represents the median value and the boundaries indicate the 25th percentile (upper) and the 75th percentile (lower). Whiskers extend to the largest and smallest values. (E) The R. solanacearum-inoculated plants of Ca16R-overexpressing N. benthamiana lines #1 and #2 exhibited a higher level of NbPR1 expression and a lower level of NbCOI1 expression compared to the wild-type plants. Data in (A,E) represent the mean ± SD from four independent experiments. Error bars indicate SD. Uppercase letters above the bars indicate significant differences (p < 0.01) calculated using Fisher’s protected LSD test. The RSI N. benthamiana leaves were harvested 12 h after R. solanacearum inoculation.

Figure 5

Ca16R interacted with CaASR1 using BiFC and a pull-down assay. (A) The data from BiFC showed that CaASR1 interacted with Ca16R in plasma membrane and cytoplasm in epidermal cells of N. benthamiana leaves; bar is 25 um. (B) The data from the pull-down assay showed that CaASR1 interacted with Ca16R. Ni Smart beads and CaASR1-6×His were incubated with Ca16R-GST for three hours at 4 °C with gentle rotation. Eluting the bound proteins from the beads, they were found using either an anti-GST or an anti-6×His antibody.

Figure 6

Transient overexpression of CaASR1 induced immunity, including HR cell death, but this induction was repressed by Ca16R silencing. (A) Using the RT-qPCR assay, the success of CaASR1 transient overexpression and Ca16R silencing through virus-induced gene silencing in pepper plants. (B,C) Hypersensitive response-like cell death was observed by means of UV, trypan blue staining, and intensive ion leakage displayed by conductivity. CaASR1 was transiently overexpressed in WT and Ca16R-silenced pepper leaves, and silenced Ca16R weakened the hypersensitive response. Data are shown as the mean ± standard error of six replicates. Different uppercase letters above the bars indicate significant differences (p < 0.01) according to Fisher’s protected LSD test. (D) The activation of SA-dependent CaPR1 and CaNPR1 by CaASR1 was weakened by Ca16R silencing upon RSI at 48 hpi with RT-qPCR. In (A,D), the mean ± SD of four duplicate results are shown. Based on Fisher’s least significant difference (LSD) test, discrete capital letters on the bar graphs denote statistically significant differences (p < 0.01) between mean values. The RSI pepper leaves were harvested 12 h after R. solanacearum inoculation.