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pT7 - BvEP - 6�His

BvEP is an elicitor protein phosphopentomutase from Bacillus velezensis LJ02 which improves the tomato resistance to B. cinerea. High purity BvEP proteins induces the hypersensitivity response (HR) in Nicotiana tabacum. BvEP reduced the rotting rate and lesion diameter of tomato fruits caused by B. cinerea, and increased the expression of Pattern-triggered Immunity (PTI), Effector-triggered Immunity (ETI), systemic acquired resistance (SAR) related genes, ROS content, SOD and POD activities in tomato fruits, while there was no significant effect on weight loss and VC contents of tomato fruits. To help tomatoes resist gray mold, we first used E. coli BL21 (DE3) as a chassis to overexpress BvEP. The gene circuit of BVEP is constructed on the pET-28a plasmid vector, including T7 promoter, Lac operator, RBS, BvEP gene, 6×His affinity tag, and T7 terminator.

Sequence and Features

Design

The fragment was amplified from the total DNA of B. Velezensis LJ02 by specific primers. We retrieved the total genome of Bacillus Velez and entered the primer sequence after opening its FASTA file on Snapgene.

The fragments simulated by primers were used as BvEP sequences, which were blasted and showed high homology among the bacillus. However, we found that the fragment amplified by the primer was incomplete and lacked the start codon, so we performed blastp on the incomplete sequence in NCBI to obtain the complete amino acid sequence. Finally, the complete sequence of the protein was obtained on the whole genome.

Plasmid construction

The obtained target gene sequence was assembled into the polyclonal site of the pET28a (+) plasmid, and the 6*His protein purification tag sequence was linked to facilitate subsequent protein extraction and purification experiments. The recombinant plasmid was transferred into the proteinase-deficient E. coli BL21 (DE3), and the fermentation of BvEP was induced by IPTG.

Functional verification

In our experiment, the target protein was extracted and purified according to the protein label and applied to the leaves of tomato. BvEP can stimulate the outbreak of reactive oxygen species in tomato leaves as an immune response. Hydrogen peroxide, a kind of ROS, can be used to calculate or predict the degree of immune response by observing or counting the relative gray value of leaves after the brown red precipitates generated by DAB staining are decolorized by ethanol.

Recombinant plasmid electrophoresis

In our experiment, the target protein was extracted and purified according to the protein label and treated to the leaves of tomato. BvEP can stimulate the outbreak of reactive oxygen species in tomato leaves as an immune response. Hydrogen peroxide, a kind of ROS, can be used to calculate or predict the degree of immune response by observing or counting the relative gray value of leaves after the brown red precipitates generated by DAB staining are decolorized by ethanol.

Figure 4. Results of amplification of different recombinant plasmids by specific primers. (a) PCR of pGS21a-PehA. M: DL2000 DNA marker; 1: PehA.(b) PCR of 1x Flg22, 3x Flg22, Flagellin full length and BvEP in vector pET-28a. M: DL2000 DNA marker; 1-4: 1x Flg22, 3x Flg22, Flagellin full length and BvEP.

Expression of Proteins

The expression of immune protein factors is regulated by lactose operon, so IPTG is applied to induce our protein expression, and BvEP protein is extracted and purified by His labeled protein purification kit. The protein concentration was determined by BCA method. The expression level of BvEP was very low under conventional induction. We then optimized the experiment and found that protein expression levels were higher when induced at 100 rpm in a shaker at 20℃. The protein concentration in the 4.5mL induction culture was measured at 4h, 8h and 16h. The expression concentration of BvEP protein was the highest after 16h induction, reaching 0.530 mg/ml.

Figure 5. BCA standard curve

In addition, we verified the extracted target proteins by SDS-PAGE after 16 h of induction. As shown in the figure, Figure 6a shows the BvEP protein electrophoresis after 16h of IPTG induction. By comparing with the total protein of E. coli BL21(DE3) without induction, it can be seen that there is a clear band between 14.4kD and 4kD. The predicted size of BvEP protein was 12.1kD, indicating that this band was our target protein. Similarly, in Figure 6b, an obvious band between 35kD and 25kD appeared after 16 h of IPTG induction compared with the non-induced sample, while the size of our Flagellin full length protein was 30.6kD, showing that it was successfully induced.

Figure 6. The induced expression of target proteins detected by SDS-PAGE (a) Induced expression of BvEP; (b) Induced expression of Flagellin full length

In addition, our target proteins were also verified in Western Blot experiments. As can be seen from Figure 7, there is a specific band between 15kD and 10kD, and the protein size of BvEP is 12.1kD, indicating that the protein extracted from the induced E. coli is indeed our target protein.

Figure 7. Western Blot validation of BvEP induced expression

Proof Of Concept: DAB staining experiment

The burst of ROS is a typical response to the activation of plant immunity, so we performed DAB staining experiments to examine the degree of ROS burst in tomato leaves after treatment with different concentrations of BvEP. The darker the DAB staining results, the more ROS burst in the leaves. In that experiment, our control group 1 was treated with clean water and control group 2 was treated with total protein extracted in E. coli BL21(DE3) without induction (Figure. 8).

Figure 8. DAB staining to detect the intensity of immunity triggered by different concentrations of BvEP (a) Soaking method to apply BvEP. Low: 10ug/ul, Mid: 25ug/ml, High: 50ug/ml. (b) Dropping method to apply BvEP. Low: 40ug, Mid: 100ug, High: 200ug.

After staining, ImageJ software was used to perform gray scale analysis of the leaves treated with the two different BvEP methods to quantify the immune intensity, followed by t-test to determine the significant difference between the different BvEP concentrations. Lower gray levels, that is, more brown precipitates in leaves, indicate a stronger burst of ROS. Each treatment contains a sample number of 9, and the resulting data are shown in Figure 9.

Figure 9. Gray levels in different leaves measured by ImageJ software. (a)The gray value of leaves treated protein after soaking. (b) The gray value of leaves after dropping. (ns P > 0.05; * P  < 0.05; ** P  < 0.01; *** P  < 0.001; **** P < 0.0001 )

As can be seen from the figure above, the degree of ROS burst in leaves increases with the concentration of applied BvEP, whether using the soaking or dropping method. Through the comparison of the two methods, we can obviously observe that the ROS burst in the leaves treated by soaking method is more than that by dropping method.

References

[1] Li Z, Hu J, Sun Q, Zhang X, Chang R, Wang Y. A novel elicitor protein phosphopentomutase from Bacillus velezensis LJ02 enhances tomato resistance to Botrytis cinerea. Front Plant Sci. 2022 Nov 29;13:1064589. doi: 10.3389/fpls.2022.1064589.
[2] Daudi, A. and O’Brien, J. A. (2012). Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves. Bio-protocol 2(18): e263. DOI: 10.21769/BioProtoc.263.
[3] Pruitt RN, Gust AA, Nürnberger T. Plant immunity unified. Nat Plants. 2021 Apr;7(4):382-383. doi: 10.1038/s41477-021-00903-3.
[4] Yuan M, Ngou BPM, Ding P, Xin XF. PTI-ETI crosstalk: an integrative view of plant immunity. Curr Opin Plant Biol. 2021 Aug;62:102030. doi: 10.1016/j.pbi.2021.102030. Epub 2021 Mar 5.
[5] Hu J, Chang R, Yuan Y, Li Z, Wang Y. Identification of Key Residues Essential for the Activation of Plant Immunity by Subtilisin From Bacillus velezensis LJ02. Front Microbiol. 2022 Aug 15;13:869596. doi: 10.3389/fmicb.2022.869596.