Identification of Pepper CaSBP08 Gene in Defense Response Against Phytophthora capsici Infection

Abstract

Little information is available on the role of Squamosa promoter binding protein (SBP)-box genes in pepper plants. This family of genes is known to have transcription characteristics specific to plants and to regulate plant growth, development, stress responses, and signal transduction. To investigate their specific effects in pepper (Capsicum annuum), we screened pepper SBP-box family genes (CaSBP genes) for Phytophthora capsici (P. capsici) resistance genes using virus-induced gene silencing. CaSBP08, CaSBP11, CaSBP12, and CaSBP13, which are associated with plant defense responses against P. capsici, were obtained from among fifteen identified CaSBP genes. The function of CaSBP08 was identified in pepper defense response against P. capsici infection in particular. CaSBP08 protein was localized to the nucleus. Silencing of CaSBP08 enhanced resistance to P. capsici infection. Following P. capsici inoculation, the malondialdehyde content, peroxidase activity, and disease index percentage of the CaSBP08-silenced plants decreased compared to the control. Additionally, the expression levels of other defense-related genes, especially those of CaBPR1 and CaSAR8.2, were more strongly induced in CaSBP08-silenced plants than in the control. However, CaSBP08 overexpression in Nicotiana benthamiana enhanced susceptibility to P. capsici infection. This work provides a foundation for the further research on the role of CaSBP genes in plant defense responses against P. capsici infection.

Keywords: CaSBP08; Nicotiana benthamiana; Phytophthora capsici; SBP-box family genes; defense genes; pepper.

Figure 1

Phenotypes and silencing efficiency of the fifteen members of CaSBPs silenced plant. (A) Photographs were taken forty days after injection (the diameter of the pot is 7 cm). (B) Silencing efficiency of the fifteen members of CaSBPs in their corresponding silenced plants. (C) The expression level of the pepper SBP-box genes, which with the highest homology with their corresponding silenced gene. * and ** represent significant differences at P ≤ 0.05 and P ≤ 0.01 respectively. Mean values and SDs for three biological replicates are shown.

Figure 2

Phenotype and percentage of the lesion area of detached leaves of the fifteen members of CaSBPs ^, silenced and control plants after inoculation with P. capsici. (A) Phenotype of detached leaves of the fifteen members of CaSBPs ^, silenced and control plants after inoculation with P. capsici. Photographs were taken after inoculation with P. capsici three days. The diameter of the plug of P. capsici is 0.5cm. The red dotted line was used to label the lesion area in each leaf. (B) Percentage of the lesion area of the leaves three days after inoculation with P. capsici. Bars with different lower case letters indicate significant differences at P ≤ 0.05. Mean values and SDs for three biological replicates are shown.

Figure 3

Subcellular localization of the CaSBP08 protein. Agrobacterium tumefaciens strain GV3101 with pVBG2307:CaSBP08:GFP and pVBG : GFP (used as a control) vectors were transiently expressed in N. benthamiana leaves. The fluorescence was visualized using a laser scanning confocal microscope under bright and fluorescent fields. The photographs were taken in a dark field for green fluorescence and under bright light for the morphology of the cell. Bars in this picture are 75μm.

Figure 4

Loss function analysis of CaSBP08 in pepper plant defense response against P. capsici infection. (A) Phenotypes of detached leaves of CaSBP08 silenced and negative control plants after inoculation with P. capsici. Photographs were taken at two days after inoculation with P. capsici. The yellow dotted line was used to label the lesion area in each leaf. (B) The average diseased areas of the detached leaves of the CaSBP08 silenced and negative control plants. Data were collected, two days after inoculation with P. capsici. (C) Phenotypes of the CaSBP08 silenced and negative control plants after inoculation with P. capsici sixteen days. (D) The disease index percentage of the CaSBP08 silenced and negative control plants and data were collected sixteen days after inoculation with P. capsici. (E) Determination of MDA content (E-1), POD activity (E-2), and CAT activity (E-3) of CaSBP08 silenced and negative control plants after inoculating with P. capsici. Bars in Figure A are 0.5cm, and C are 4.5cm. Bars with different letters indicate significant differences at P ≤ 0.05. * Represent significant differences at P ≤ 0.05. Mean values and SDs for three biological replicates are shown.

Figure 5

The expression of CaSBP08 (A), and defense-related genes, CaDEF1 (B), CaSAR8.2 (C), CaPO1 (D), and CaBPR1 (E) after inoculation with P. capsici in CaSBP08 silenced and negative control plants. * and ** represent significant differences at P ≤ 0.05 and P ≤ 0.01 respectively. Mean values and SDs for three biological replicates are shown.

Figure 6

Function analysis of overexpression of CaSBP08 transgenic lines in defense response against P. capsici infection. (A) Phenotypes of the detached leaves of transgenic and wild-type plants after inoculation with P. capsici two days. The red dotted line was used to label the lesion area in each leaf. (B) The average diseased areas of the detached leaves of transgenic and wild-type plants. Data were collected, two days after inoculation with P. capsici. (C) Phenotypes of transgenic and wild-type plants after inoculation with P. capsici three days. The black arrow indicates the phenotype of the left transgenic and wild-type plants after removing the matrix. White arrows indicate the constricted area between root and stem. (D) Classification of disease index percentage of transgenic and wild-type plants after inoculation with P. capsici thirteen days. (E) Disease index percentage of transgenic and wild-type plants and data were collected thirteen days after inoculation with P. capsici. (F) The expression level of CaSBP08 in transgenic and wild-type plants. (G) The expression of defense-related genes in transgenic and wild-type plants after inoculation with P. capsici. Bars in Figure A are 0.4cm, C and D are 3.5cm. * and ** represent significant differences at P ≤ 0.05 and P ≤ 0.01 respectively. Mean values and SDs for three biological replicates are shown.