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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.

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.

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.

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.

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.

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.

Expression of three proteins

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.

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

Sequencing

Sequence and Features

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.