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SpyCatcher-VDAL-CPPs (R9-Tag)

To enhance plant immunity, a novel fusion protein, SpyCatcher-VDAL-CPPs, was designed. This construct incorporates a SpyCatcher tag^[1] for conjugation, VDAL for immune activation^[2], and the R9 cell-penetrating peptide for intracellular delivery^[3]. Functional studies demonstrated the capacity of this fusion protein to penetrate plant cell membranes and induce an ETI response. Molecular characterization and DAB staining confirmed the successful construction and immunogenic activity of the fusion protein.

The design was verified as shown in Figure 1.

Usage and Biology

VDAL, an Aspf2-like protein derived from Verticillium dahliae, has been shown to activate PTI responses when expressed extracellularly ^[4] and can also induce endogenous ETI immune responses when expressed intracellularly. To verify the function of the constructed circuit, a pET28a-based expression system was employed, with the T₇ promoter under the control of the lac operon. The circuit includes the engineered SpyCatcher-VDAL-CPPs protein, SpyTag-6*His protein, and SpyCatcher-CPPs protein. The SpyCatcher-SpyTag (SpyC/SpyT) system is derived from the CnaB2 domain of the Streptococcus pyogenes fibronectin-binding protein. SpyC is an immunoglobulin-like protein with a molecular weight of approximately 12 kDa, while SpyT is a short peptide consisting of 13 residues. These two components can specifically recognize each other and spontaneously form an isopeptide bond, enabling high-affinity binding. The designers aimed to utilize this covalent linkage during co-expression to verify the functionality of the SpyCatcher-SpyTag system and facilitate the purification of the target protein.

The constructed plasmid vector is illustrated in Figure 2.

Experimental Verification

Transformation

After chemical transformation using calcium chloride, the constructed plasmid was introduced into E. coli BL21 (DE3). The transformed cells were plated on LB agar plates supplemented with kanamycin and incubated at 37℃ for 16 hours. Colony PCR was performed on individual colonies to verify the presence of the plasmid.

The results of the colony PCR are presented in Figure 3.

SDS-PAGE

SDS-PAGE detection of bacterial total protein

    1) The single colony confirmed by sequencing was inoculated into a shake flask and cultured at 37°C for 6 hours until the OD₆₀₀ reached 0.6.

    2) IPTG was then added to a final concentration of 1 mM, and the culture was further incubated at 16°C for 18 hours.

    3) Cells were harvested by centrifugation at 8000 rpm, 4°C for 10 minutes, washed with PBS, resuspended, and centrifuged again.

    4) Finally, the cell pellet was resuspended in 1 ml of PBS to obtain a cell suspension.

    5) The cell suspension was mixed with 5X Protein Loading Buffer and heated at 95°C for 10 minutes.E. coli BL21 (DE3) harboring the empty pET28a plasmid was processed in the same manner as a control.

    6) Samples were analyzed by 12% SDS-PAGE, and a protein band corresponding to the expected size of SpyCatcher-VDAL-CPPs + SpyTag-6*His protein (53.7 kDa) was observed.

The results are presented in Figure 4.

    1) The single colony confirmed by sequencing was inoculated into a shake flask and cultured at 37°C for 6 hours until the OD₆₀₀ reached 0.6.

    2) IPTG was then added to a final concentration of 1 mM, and the culture was further incubated at 16°C for 18 hours.

    3) Cells were harvested by centrifugation at 8000 rpm, 4°C for 10 minutes, washed with PBS, resuspended, and centrifuged again.

    4) Finally, the cell pellet was resuspended in 2 ml of Binding Washing Buffer (Sangon Biotech, Shanghai, China) containing 10 mM imidazole and 80 μl of PMSF (Sangon Biotech, Shanghai, China).

    5) The cell suspension was then sonicated for 10 minutes with a 1-second pulse followed by a 2-second pause.

    6) The cell lysate was centrifuged at 12000 rpm at 4°C for 15 minutes.

    7) The supernatant was transferred to a new Eppendorf tube, and the pellet was resuspended in 1 ml of PBS for storage.

    8) The supernatant was mixed with an equal volume of Binding Buffer to prepare the sample.

    9) The storage solution was slowly drained, and the Ni column was equilibrated with 5 ml of Washing Buffer.

    10) The sample was loaded onto the column in two bed volumes, and the first flow-through was reloaded.

    11) The column was washed with two bed volumes of Washing Buffer, and the flow-through was collected until the absorbance at 280 nm reached the baseline.

    12) The protein was eluted with two bed volumes of Elution Buffer, collecting 2 ml fractions each time, until the absorbance at 280 nm reached the baseline.

    13) The purified protein was obtained. (Specific experimental procedures were followed from HyPur T Ni-NTA 6FF (His-Tag) PrePacked Gravity Column Kit, Sangon Biotech, Shanghai, China)

    14) The harvested bacterial cell pellet, the supernatant after cell lysis, the purified protein sample, and the E. coli BL21 (DE3) cell suspension containing the empty pET28a plasmid were mixed with 5X Protein Loading Buffer and heated at 95°C for 10 minutes.

    15) Samples were separated by 12% SDS-PAGE and stained with Coomassie Brilliant Blue G250 for 12 hours, followed by destaining.

The relatively large size of the target protein, which was tagged with His using the SpyCatcher-SpyTag (SpyC/SpyT) system, resulted in weaker binding affinity to the Ni-NTA column. To ensure protein purification, a Binding Wash Buffer with a reduced imidazole concentration was used. However, this led to increased contamination by non-specific proteins, although three distinct bands could still be observed near the expected molecular weight.

Western Blot

80μl of each purified sample was mixed with 20 μl of 5x protein loading buffer and incubated at 98°C for 15 minutes. Samples were then loaded onto an SDS-PAGE gel and electrophoresed at 80V for 2 hours. Proteins were transferred onto a nitrocellulose membrane overnight at 4°C. The membrane was blocked and then incubated with a primary antibody (mouse anti-His-tag monoclonal antibody). After washing, the membrane was incubated with a secondary antibody (goat anti-mouse IgG). Following washing, the membrane was developed using a chemiluminescent substrate, and the results were visualized by exposure to film.

The obtained results are shown in Figure 6.

The relatively high level of contaminating proteins in the purified sample, likely due to the His-tag, suggests a potential limitation of the system. However, the presence of three distinct bands near the expected molecular weight confirms the functionality of the SpyCatcher-SpyTag (SpyC/SpyT) system. The lower intensity of the target protein band may be attributed to the relatively large size of the SpyCatcher-VDAL-CPPs fusion protein and its lower expression level.

DAB-based ROS assay

Protocol

    1) Protein Quantification and Preparation: The purified protein sample containing SpyCatcher-VDAL-CPPs was quantified using the Bradford assay to a concentration of 100 μg/mL.

    2) Plant Material Preparation: Three healthy Arabidopsis thaliana Columbia wild-type plants of similar size and growth stage were selected. Leaves of comparable size and shape were excised and washed three times with ddH₂O, then blotted dry. Three leaves were grouped together, and a total of four groups were prepared.

    3)Treatment: Each group of leaves was incubated for 36 hours at 28°C in one of the following solutions: ddH₂O, 100 μM salicylic acid (SA) solution, 50 μg/mL SpyCatcher-VDAL-CPPs protein solution, or a mixture of 100 μM SA solution and 50 μg/mL SpyCatcher-VDAL-CPPs protein solution.

    4)DAB Staining: After incubation, the leaves were washed three times with ddH₂O and blotted dry. Samples were immersed in DAB staining solution and subjected to negative pressure (-0.1 MPa) for 30 minutes, followed by room temperature incubation in the dark for 12 hours until positive sites appeared dark brown. Samples were then rinsed three times with ddH₂O and blotted dry.

    5)Decolorization: Samples were immersed in tissue decolorization solution and incubated at 75°C for 30 minutes until the background color was completely removed.

    6) The samples were then rinsed three times with distilled water and blotted dry. Subsequently, they were immersed in a tissue preservation solution for 30 minutes before being imaged. (DAB staining kit and procedure were obtained from Beijing Solarbio Science & Technology Co., Ltd.)

    7) All leaves were arranged on a white background and flattened to ensure complete expansion. Images were captured under consistent lighting conditions, as shown in Figure 7.

(Note that the ddH₂O and SA groups in this experiment served as negative and positive controls, respectively, and were identical to those used in the BBa_K5335026 element verification.)</p>

    8) The acquired images were imported into ImageJ software and converted to 8-bit grayscale images. Subsequently, the images were inverted. Given that DAB staining results in the deposition of brown-colored precipitates in regions with reactive oxygen species (ROS), with the intensity of the color directly correlating with ROS levels, the inverted images displayed lighter regions corresponding to higher ROS content.

    8)Using the grayscale measurement tool in ImageJ, the contours of each leaf were outlined, and the average grayscale value for each leaf was calculated. After data normalization and analysis.

Analysis of the images revealed that VDAL-CPPs significantly induced the production of reactive oxygen species (ROS). Furthermore, the co-incubation of SA and VDAL-CPPs resulted in a notable interference in their respective abilities to induce ROS.

Reference

     1.Gilbert C, Howarth M, Harwood CR, Ellis T. Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis. ACS Synth Biol. 2017 Jun 16;6(6):957-967. doi: 10.1021/acssynbio.6b00292. Epub 2017 Mar 7.

     2.Ma A, Zhang D, Wang G, Wang K, Li Z, Gao Y, Li H, Bian C, Cheng J, Han Y, Yang S, Gong Z, Qi J. Verticillium dahliae effector VDAL protects MYB6 from degradation by interacting with PUB25 and PUB26 E3 ligases to enhance Verticillium wilt resistance. Plant Cell. 2021 Dec 3;33(12):3675-3699. doi: 10.1093/plcell/koab221.

     3.Soliman A, Laurie J, Bilichak A, Ziemienowicz A. Applications of CPPs in Genome Editing of Plants. Methods Mol Biol. 2022;2383:595-616. doi: 10.1007/978-1-0716-1752-6_39.

     4.Jiang S, Zheng W, Li Z, Tan J, Wu M, Li X, Hong SB, Deng J, Zhu Z, Zang Y. Enhanced Resistance to Sclerotinia sclerotiorum in Brassica rapa by Activating Host Immunity through Exogenous Verticillium dahliae Aspf2-like Protein (VDAL) Treatment. Int J Mol Sci. 2022 Nov 12;23(22):13958. doi: 10.3390/ijms232213958.

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Sequence and Features

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