Part:BBa_K4286202

Prb1, a serine protease plays an important role in the destruction of plant pathogens

The PRB1 gene encodes a serine protease which plays an important role in the destruction of the plant pathogens. It is widely found in various trichoderma species which attack a large variety of phytopathogenic fungi responsible for major crop diseases. During the interaction, Trichoderma penetrates into the host mycelium, by partial degradation of its cell wall. It appears that the main mechanism involved in the antagonism to pathogenic fungi by Trichoderma is the release of lytic enzymes including PRB1. PRB1 production in Trichoderma is controlled by two mechanisms: it is induced by the presence of a phytopathogenic fungus, or its cell walls, and repressed by glucose. Evidence suggests that over-expression of the proteinase-encoding gene prb1 improved biocontrol activity of Trichoderma. To prevent Rice Sheath Blight, we choose Trichoderma atroviride(T.atroviride) as our chassis to overexpress Prb 1.

Assembly

Plasmid construction

Through homologous recombination, the coding sequence of PRB1 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 PRB1 gene. In addition, we ligated 6× His tag at the end of prb1 CDS to facilitate protein purification in subsequent experiments. The following figure shows the recombinant plasmid, [prb1]-pCAMBIA1302.

[parts-K4286202-figure1] Figure 1. [prb1]-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 Prb 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 a pair of primers with theoretical PCR fragment sizes of prb 1-1388bp. The PCR result of the recombinant plasmid [prb1]-pCAMBIA1302 is shown in Figure 2, and all the selected plasmids were in expected positions, consistent with the positions of the positive control.

[parts-figure2] Figure 2: 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 3, 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.

[parts-figure3] Figure 3: 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 medthod 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.

[parts-figure4] Figure 4: 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.

[parts-figure5] Figure 5: 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

Prb 1 protein is a kind of serine protease, so we first test the enzyme activity of wild-type T.a(WT) and engineered T.a. Our Trichoderma were grown in Mini medium and we use the supernate to test the enzyme activity.

[parts-figure6] Figure 6: Enzyme activity of Prb 1. Prb 1, a protein associated with mycoparacitism, enables Trichoderma to better parasitize and suppress the fungus. We compared the inhibition effect of wild type Trichoderma and Prb 1 engineered Trichoderma. [parts-figure7] Figure 7: The inhibition test of wild type T.a and engineered T.a.

In summary, our engineered Trichoderma has become more effective at inhibiting R.solani.

Sequencing

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 1105
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 205
  Illegal NgoMIV site found at 250
  Illegal NgoMIV site found at 544
  Illegal NgoMIV site found at 658
  Illegal AgeI site found at 1174
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 391
  Illegal BsaI.rc site found at 268
  

References

Flores A, Chet I, Herrera-Estrella A. Improved biocontrol activity of Trichoderma harzianum by over-expression of the proteinase-encoding gene prb1. Curr Genet. 1997 Jan;31(1):30-7. doi: 10.1007/s002940050173.