Expression characteristics of CsPG23 in citrus and analysis of its interacting protein

Abstract

Exploring the resistance genes of citrus to Huanglongbing (HLB) is the foundation and key to citrus disease-resistant breeding. Through the analysis of comparative transcriptome data, we identified six cell wall degradation genes that respond to citrus infection with CaLas. We selected one of the genes with high differential expression levels and cloned it, naming it CsPG23. The subcellular localization results of tobacco indicated that the CsPG23 protein is localized in the nucleus, cytoplasm, and cell membrane. Real-time fluorescence quantitative PCR (RT-qPCR) analysis showed that the expression of CsPG23 is related to variety tolerance, tissue location, and symptom development. In addition, we constructed overexpression and silencing vectors for CsPG23 and obtained CsPG23 silencing plants, overexpression and silencing hairy roots, and analyzed the expression characteristics of CsPG23 in response to SA, JA, MeSA and H₂O₂ induction through RT-qPCR. Using Protein-Protein Interaction (PPI) to predict and screen for a citrus protein CsAGD8 that may interact with CsPG23, and preliminarily verifying its interaction with CsPG23 protein through Yeast Two-hybrid (Y2H). We constructed overexpression and silencing vectors for CsAGD8 and obtained CsAGD8 overexpression and silencing hairy roots. In summary, it is indicated that CsPG23 may interact with CsAGD8 in response to CaLas infection.

Keywords: Citrus; CsAGD8; CsPG23; hairy roots.

Figure 1.

Subcellular location of CsPG23 protein in tobacco epidermis cell. (a) Schematic diagram of the constructs used for agroinfiltration. A 35S, CaMV 35S promoter; NOS, the nopaline synthase terminator. (b) Subcellular localization of CsPG23:GFP fusion protein as observed by confocal microscopy. A histone 2B (H2B) fusion with the red fluorescent protein (RFP) was used as a marker for the nucleus.

Figure 2.

Analysis of the expression characteristics of CsPG23 gene in citrus. (a) Relative expression of CsPG23 before and after showing symptoms in sweet oranges. (b) The relative expression level of CsPG23 in healthy and CaLas-infected sweet oranges. (c) Relative expression levels of CsPG23 in roots and leaf veins in CaLas-infected sweet oranges. (d) Relative expression of CsPG23 in CaLas-infected sour pomelo and sweet oranges. Values are expressed as means ± standard deviation of three independent tests. *on top of the bars indicates a significant difference (p < 0.05, Student’s t-test).

Figure 3.

Transgenic plants silencing CsPG23. (a) A 35S, 35S promoter; NOSt, the nopaline synthase terminator; LB, left border; RB, right border. (b) Identification of silencing plants by PCR. M, DNA marker; T, TRV plasmid; WT, wild-type control; TRV-#, silencing plants. (c) Phenotypic observation of silencing plants. (d) Relative expression levels of CsPG23 in silencing plants. Relative expression of CsPG23 in silencing plants was normalized against its expression in wild-type control using the citrus GAPDH gene as internal reference. Values are expressed as means ± standard deviation of three independent tests. *on top of the bars indicates significant differences compared to WT control (p < 0.05, Student’s t-test).

Figure 4.

Transgenic hairy roots overexpressing CsPG23. (a) A 35S, 35S promoter; NOSt, the nopaline synthase terminator; LB, left border; RB, right border. (b) Identification of transgenic hairy roots by PCR. M, DNA marker; P, p35S: CsPG23 plasmid; WT, wild-type control; OE-#, transgenic hairy roots. (c) Statistics of transgenic hairy roots. (d) Observation on symptoms of transgenic hairy roots. (e) Number statistics of wild-type and transgenic hairy roots. (f) Length statistics of wild-type and transgenic hairy roots. Values are means ± SEMs (n = 3). Lowercase letters indicate significant differences among different treatments (p < 0.05; Duncan’s multiple comparisons test). (g) Relative expression levels of CsPG23 in transgenic hairy roots. Relative expression of CsPG23 in transgenic hairy roots was normalized against its expression in wild-type control using the citrus GAPDH gene as internal reference. Standard errors were calculated from three hairy roots per line. Values are expressed as means ± standard deviation of three independent tests. *on top of the bars indicates significant differences compared to WT control (p < 0.05, Student’s t-test).

Figure 5.

P1300GMN-CsPG23RNAi transgenic hairy roots. (a) A 35S, 35S promoter; NOSt, the nopaline synthase terminator; LB, left border; RB, right border. (b) Identification of transgenic hairy roots by PCR. M, DNA marker; T, P1300GMN-RNAi plasmid; WT, wild-type control; P1300GMN-RNAi #, transgenic hairy roots. (c) Statistics of transgenic hairy roots. (d) Observation on symptoms of transgenic hairy roots. (e) Number statistics of wild-type and transgenic hairy roots. (f) Length statistics of wild-type and transgenic hairy roots. Values are means ± SEMs (n = 3). Lowercase letters indicate significant differences among different treatments (p < 0.05; Duncan’s multiple comparisons test). (g) Relative expression levels of CsPg23rnai in transgenic hairy roots. Relative expression of CsPg23rnai in transgenic hairy roots was normalized against its expression in wild-type control using the citrus GAPDH gene as internal reference. Standard errors were calculated from three hairy roots per line. Values are expressed as means ± standard deviation of three independent tests. *on top of the bars indicates significant differences compared to WT control (p < 0.05, student’s t-test).

Figure 6.

Determination of CsPG23 transgenic hairy roots hormone content. (a-d) characteristics of SA, JA, MeSA and ROS contents in transgenic hairy roots compared to WT control. Values are expressed as means ± standard deviation of three independent tests. *on top of the bars indicates a significant difference (p < 0.05, Student’s t-test). WT, wild type; OE-#, transgenic plants.

Figure 7.

CsPG23 up-regulated the expression of defense-related genes in transgenic hairy roots. The GAPDH gene was used as an endogenous control. Values are expressed as means ± standard deviation of three independent tests. *on top of the bars indicates significant differences compared to WT control (p < 0.05, student’s t-test).

Figure 8.

CsPG23 interacts with CsAGD8. Y2H assays show that CsPG23 interacts with AGD8. BD indicates pGBKT7 vector. AD indicates pGADT7 vector. BD-53 + AD-T is a positive control, BD-Lam + AD-T is a negative control.

Figure 9.

Transgenic plants overexpressing CsAGD8. (a) T-DNA structure of plant expression vector for the genetic transformation of citrus. (b) Identification of transgenic plants by PCR. M, DNA marker, P, p35S: CsAGD8 plasmid; WT, wildtype control; OE-#, transgenic plants. (c) Statistics of transgenic hairy roots. (d) Observation on symptoms of transgenic hairy roots. (e) Number statistics of wild-type and transgenic hairy roots. (f) Length statistics of wild-type and transgenic hairy roots. Values are means ± SEMs (n = 3). Lowercase letters indicate significant differences among different treatments (p < 0.05; Duncan’s multiple comparisons test). (g) Relative expression levels of CsAGD8 in transgenic plants. Relative expression of CsAGD8 in transgenic plants was normalized against its expression in wild-type control using the citrus GAPDH gene as internal reference. Values are expressed as means ± standard deviation of three independent tests. *on top of the bars indicates significant differences compared to WT control (p < 0.05, student’s t-test).

Figure 10.

P1300GMN-CsAGD8RNAi transgenic hairy roots. (a) A 35S, 35S promoter; NOSt, the nopaline synthase terminator; LB, left border; RB, right border. (b) Identification of transgenic hairy roots by PCR. M, DNA marker; T, P1300GMN-RNAi plasmid; WT, wild-type control; P1300GMN-RNAi #, transgenic hairy roots. (c) Statistics of transgenic hairy roots. (D) Observation on symptoms of transgenic hairy roots. (e) Number statistics of wild-type and transgenic hairy roots. (f) Length statistics of wild-type and transgenic hairy roots. Values are means ± SEMs (n = 3). Lowercase letters indicate significant differences among different treatments (p < 0.05; Duncan’s multiple comparisons test). (g) Relative expression levels of CsAgd8rnai in transgenic hairy roots. Relative expression of CsAgd8rnai in transgenic hairy roots was normalized against its expression in wild-type control using the citrus GAPDH gene as internal reference. Standard errors were calculated from three hairy roots per line. Values are expressed as means ± standard deviation of three independent tests. *on top of the bars indicates significant differences compared to WT control (p < 0.05, student’s t-test).