Transient expression of anti-HrpE scFv antibody reduces the hypersensitive response in non-host plant against bacterial phytopathogen Xanthomonas citri subsp. citri

Abstract

Citrus canker is a bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that affects the citrus industry worldwide. Hrp pili subunits (HrpE), an essential component of Type III secretion system (T3SS) bacteria, play a crucial role in the pathogenesis of Xcc by transporting effector proteins into the host cell and causing canker symptoms. Therefore, development of antibodies that block HrpE can suppress disease progression. In this study, a specific scFv detecting HrpE was developed using phage display technique and characterized using sequencing, ELISA, Western blotting, and molecular docking. In addition, a plant expression vector of pCAMBIA-scFvH6 was constructed and agroinfiltrated into Nicotiana tabacum cv. Samson leaves. The hypersensitive response (HR) in the leaves of transformed and non-transformed plants was evaluated by inoculating leaves with Xcc. After three rounds of biopanning of the phage library, a specific human scFv antibody, named scFvH6, was identified that showed high binding activity against HrpE in ELISA and Western blotting. Molecular docking results showed that five intermolecular hydrogen bonds are involved in HrpE-scFvH6 interaction, confirming the specificity and high binding activity of scFvH6. Successful transient expression of pCAMBIA-scFvH6 in tobacco leaves was verified using immunoassay tests. The binding activity of plant-produced scFvH6 to detect HrpE in Western blotting and ELISA was similar to that of bacterial-produced scFvH6 antibody. Interestingly, tobacco plants expressing scFvH6 showed a remarkable reduction in HR induced by Xcc compared with control plants, so that incidence of necrotic lesions was significantly higher in non-transformed controls (≥ 1.5 lesions/cm²) than in the plants producing scFvH6 (≤ 0.5 lesions/cm²) after infiltration with Xcc inoculum. Our results revealed that the expression of scFvH6 in tobacco leaves can confer resistance to Xcc, indicating that this approach could be considered to provide resistance to citrus bacterial canker disease.

Keywords: Citrus canker; HrpE; Phage display; Single chain fragment variable; Transient expression.

Figure 1

A schematic diagram of the experimental setup used in this study. (a) Representation of expression and purification of rHrpE, (b) selection of monoclonal scFv antibody from phage display library and characterization of selected scFv (scFvH6) against HrpE, (c) transient expression of scFvH6 antibody in tobacco plant model and characterization of plant-produced scFvH6 against HrpE at in vitro and in vivo levels. A. radiobacter: Agrobacterium radiobacter; ELISA: enzyme-linked immunosorbent assay; E. coli: Escherichia coli; IMAC: immobilized-metal affinity chromatography; IPTG: isopropyl ß-D-1-thiogalactopyranoside; rHrpE: recombinant HrpE; scFv: single-chain variable fragment; Xcc: Xanthomonas citri subsp. citri.

Figure 2

(a) Enrichment of Tomlinson I scFv library against rHrpE through three rounds of biopanning. (b) Phage-ELISA for screening of 94 individual phage clones obtained from the third round of panning against rHrpE. (c) The amino acid sequences of selected scFv antibody (scFvH6) with binding specificity to rHrpE (including depiction of CDRs, His-tag and c-Myc tag). (d) Western blot analysis to assay purification of scFH6 using anti-His tag antibody (1:10,000). BSA was considered as negative control. Data are represented as mean ± SD from three independent experiments. ****P < 0.0001, significantly different from control. BSA: bovine serum albumin; CDRs: complementarity-determining regions; M: Marker PageRuler Prestained Protein Ladder (Thermo Scientific, USA); rHrpE: recombinant HrpE.

Figure 3

Characterization of in vitro binding activity of scFvH6 against rHrpE and native HrpE in Xcc-infected samples using (a) Western blot analysis and (b) indirect ELISA. (c) Titration ELISA to detect the working concentration of scFvH6. BSA was used as negative control. Data are represented as mean ± SD from three independent experiments. ****P < 0.0001, significantly different from control. BSA: bovine serum albumin; M: Marker PageRule Prestained Protein Ladder (Thermo Scientific, USA); rHrpE: recombinant HrpE; Xcc: Xanthomonas citri subsp. citri.

Figure 4

3D structure presentation of the binding poses of HrpE (blue) and scFvH6 (green). Hydrogen bonds involved in scFvH6-HrpE interaction are shown in the 3D structure as pink lines, and total intermolecular analyses containing hydrogen bonds and Van der Waals forces of the interaction are also presented in 2D structure (A: antibody and B: antigen). 2D: two-dimensional; 3D: three-dimensional.

Figure 5

Detection and characterization of expression of scFvH6 in leaves of Nicotiana tabacum cv. Samson. (a) Temporal analysis of the expression scFvH6 in transformed leaves during 1–3 days post inoculation (dpi) using Western blotting. (b) The binding activity of scFvH6 expressed in different transformed leaves to detect rHrpE using Western blotting. (c) The binding activity of plant-produced scFvH6 against rHrpE and native HrpE in Xcc-infected samples using Western blotting and (d) indirect ELISA. Data are represented as mean ± SD from three independent experiments. ****P < 0.0001, significantly different from control. B: bovine serum albumin; M: Marker PageRuler Prestained Protein Ladder (Thermo Scientific, USA); rH: recombinant HrpE; Xcc: Xanthomonas citri subsp. citri.

Figure 6

(a) Appearance of N. tabacum leaves infiltrated with agrobacterium bearing pCAMBIA-scFvH6w, agrobacterium bearing vector control (pCAMBIA1300), and PBS that after 3 days were inoculated with suspension of Xcc (10⁸ CFU/mL). (b) Comparison of the size of the necrotic lesions developed by Xcc in different treatments (2 dpi). Data are represented as mean ± SD from six independent experiments. ****P < 0.0001, significantly different from control. PBS: phosphate-buffered saline; Xcc: Xanthomonas citri subsp. citri.