Part:BBa_K4286205

Snakin-1, a cysteine-rich basic antimicrobial peptide (AMP)

The Snakin-1 peptide, a cysteine-rich antimicrobial peptide isolated from potato (Solanum tuberosum), has 63 amino acid residues, 12 of which are highly conserved cysteines. The Snakin-1 peptide is an important members of a group of evolutionarily conserved low molecular weight defense peptides, which are a vital component of the innate and the adaptive immune responses in plants and help deliver broad spectrum resistance against a wide variety of phytopathogens. Snakin-1 was indeed effective against the rice sheath blight fungus both in vitro and in planta without affecting the normal growth and development of the transgenic rice. The antifungal mode of action of Snakin-1 has not been deciphered yet. Due to the detection of a helix-turn-helix (HTH) motif in snakin and others studies, a hypothetical model of action of Snakin-1 was proposed that Snakin-1 affects the fungus by triggering apoptosis via multiple pathways. It also probably alters the cell membrane permeability and its cell surface hydrophobicity, therefore affecting its adhesion capabilities. The combination of all these factors accounts for its antifungal action against Rhizoctonia solani.

Figure 1. The expression of Snakin-1 by engineered Trichoderma atroviride

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 914
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 66
  

Assembly

Plasmid construction

Through homologous recombination, the coding sequence of Snakin-1 gene was integrated into plasmid pCAMBIA1302, and the strong constitutive promoter CaMV 35S promoter and NOS terminator of pCAMBIA plasmid vector were used to express Snakin-1 gene. In addition, we ligated 6× His tag at the end of sn1 CDS to facilitate protein purification in subsequent experiments. The following figure shows the recombinant plasmid, [sn1]-pCAMBIA1302.

Figure 2. [sn1]-pCAMBIA1302

Overproduction of recombinant plasmids

Since we needed to transfer the plasmids into Trichoderma, which would require a large number of plasmids, we transferred pCAMBIA1302 recombinant plasmids with Epl 1, Prb 1, and Snakin 1 into E. coli DH5a, to amplify them in large quantities, thus obtaining a constant and large number of plasmids.

After transforming the recombinant pCAMBIA1302 plasmid into DH5a competent cells, the recombinants were screened by the kana resistance gene on the plasmid. Subsequently, we first performed colony PCR on the isolated colonies and selected the successfully transformed isolated colonies for simple amplification with the extracted plasmids. Then we verified them by PCR and double digestion. We designed three pairs of primers with theoretical PCR fragment sizes of Epl 1-565bp, Prb 1-1388bp, and Snakin 1-425bp, respectively. The PCR results of three plasmids are shown in Figure 3, and all the selected plasmids were in expected positions, consistent with the positions of the positive control.

Figure 3: 1% agarose gel stained with Epl 1、Prb 1、Snakin 1 intergration of pCAMCIA1302 in E.coli was checked by PCR. Agarose gel was used to validate the pcr results. This figure shows PCR results of Epl 1, Prb 1, and Snakin 1, respectively. E+, P+, S+ are positive controls for Epl 1, Prb 1, and Snakin 1(positive clone is the synthetic plasmid). The capitalized word with a number represents the sample we choose.

In the double digestion verification, we used EcoRI and Bgl II enzymes to cut the plasmid into two segments, the longer segment was 9729bp, and the shorter segments of Prb 1 was 2065bp. As shown in the electrophoresis diagram of Prb 1 plasmid in Figure 4, the lower plasmid is in the superhelical state, followed by a band in the target position, which is probably the linear band of the plasmid.

Figure 4: 1% agarose gel stained with epl 1、prb 1、snakin 1 intergration of pCAMCIA1302 in E.coli was checked by enzyme digestion. (A) The results of double digestion of Epl 1 plasmid. E5-0 and E6-0 are controls that were not treated with enzymes. E5-2 and E6-2 represent the results of double digestion of E5 and E6.(B) The results of double digestion of Prb 1 plasmid. P5-0 and P6-0 are controls that were not treated with enzymes. P5-2 and P6-2 represent the results of double digestion of P5 and P6.(C) The results of double digestion of Snakin 1 plasmid. S0 is control ,S1 is the result of single enzyme digestion and S2 is double digestion of Snakin 1.

These results show that the selected separated colonies are positive and we then amplified and cultured these bacteria, and then extracted the plasmids in bulk for subsequent transformation of Trichoderma.

Genetic transformation of Trichoderma

To transfer recombinant plasmids into Trichoderma, we first tried nanomaterials-mediated transformation as well as using cell penetrating peptides to transfer the plasmids, but neither of them succeeded. After that, we tried a more traditional way protoplasted-mediated transformation. However, this CaCl2-PEG induction method didn't work. All of these methods and tries can be viewed in Protocol and Notebook. Finally, we decided to use Agrobacterium-mediated transformation (AMT).

We first transferred the three recombinant plasmids into agrobacterium GV3101 and these were screened by Kanamycin and colony PCR.

Figure 5: Agarose gel stained with epl 1、prb 1、snakin 1 integration of pCAMCIA1302 in GV3101 was checked by colony PCR. (A)E1 and E2 represent the isolated Epl 1 transformed GV3101 colony. Their were in expected positions. (B)P1 and P2 represent the isolated Prb 1 transformed GV3101 colony. Their were in expected positions. (C)S1 and s2 represent the isolated Snakin 1 transformed GV3101 colony. Their were in expected positions.

These gel results showed that the recombinant plasmids had already been transformed into agrobacterium GV3101 correctly.

Then we used positive agrobacterium GV3101 to transform T.atroviride. After several attempts and having got advice from our PI, we finally obtained the transformed T.atroviride. We selected the recombinant T.atroviride by 50ug/ml Hygromycin-B and PCR after extracting its genome. Each potential transformant was selected by 50ug/ml Hygromycin-B 4 times in case of unstable genetic inheritance caused by gene fragment inserting in cytoplasmic genome.

Figure 6: Agarose gel stained with epl 1、prb 1、snakin 1 integration of T.atroviride genome was checked by PCR. (A) 1% agarose gel stained with Prb 1 integration PCR results. P+ is positive control which is Prb 1 plasmid checked by PCR and P1 and P2 represent recombinants we chose. It is shown on the picture that P2 is consist with P+, which means P2 is positive.(B) 2% agarose gel stained with Snakin 1 integration PCR results. S+ is positive control which is Snakin 1 plasmid checked by PCR and S1 represents recombinant we chose.

According to our PCR results, we can initially confirm that we have transformed Prb 1 and Snakin 1 into T.atroviride successfully. Epl 1 transformant failed to grow up in the second time of selecting.

Characterization

Inhibition test

Snakin 1 is a potato derived cysteine-rich antimicrobial peptide which has a significant inhibition effect on R.solani. We want our T.atroviride express Snankin 1 to increase its ability to kill R.solani,so we compared the inhibition effect of wild type Trichoderma and Snakin 1 engineered Trichoderma.

We also conducted a three-day standoff experiment. Both R.solani and T.atrovoride were grown in PDA for 3 days for activation and were placed in a new PDA through a 5mm hole puncher for a three-day standoff experiment. After using the algorithm to calculate the area of the R.soalni and calculating the area according to the following formula:

We got Figure 7, the comparison of inhibition rate between wild-type T.a and Snakin 1 integrated T.a. The Snakin 1 transformant has an 10% increase in inhibiting R.solani compared with wild-type T.a.

Figure 7: The inhibition test of wild-type T.a and Snakin 1 integreatd T.a. Wild -type represents the inhibition rate of wild-type T.a and Snakin 1 + refers to the inhibition rate of Snakin 1 integrated T.a. After T test, the significance between wild-type T.a and Snakin 1+ is confirmed.

References

[1]Molla KA, Karmakar S, Molla J, Bajaj P, Varshney RK, Datta SK, Datta K. Understanding sheath blight resistance in rice: the road behind and the road ahead. Plant Biotechnol J. 2020 Apr;18(4):895-915. doi: 10.1111/pbi.13312. Epub 2020 Jan 29.

[2]Almasia NI, Bazzini AA, Hopp HE, Vazquez-Rovere C. Overexpression of snakin-1 gene enhances resistance to Rhizoctonia solani and Erwinia carotovora in transgenic potato plants. Mol Plant Pathol. 2008 May;9(3):329-38. doi: 10.1111/j.1364-3703.2008.00469.x.

[3]Kuddus MR, Yamano M, Rumi F, Kikukawa T, Demura M, Aizawa T. Enhanced expression of cysteine-rich antimicrobial peptide snakin-1 in Escherichia coli using an aggregation-prone protein coexpression system. Biotechnol Prog. 2017 Nov;33(6):1520-1528. doi: 10.1002/btpr.2508. Epub 2017 Jun 12.