Overview
Our project combines two major components to take control measures against gray mold through RNAi biopesticides and plant immunity. During the infection of B. cinerea, virulence factors will be secreted by B. cinerea to help tomato infection, and tomato will secrete immune substances to defend against the attack of B. cinerea under stress. Therefore, RNAi pesticides and plant immune agents could play a role in the interaction between B. cinerea and tomato.
- - Blocking B. cinerea infection: If the tomato has been infected by B. cinerea, the RNAi molecules can silence the virulence factor genes helping to infect and the key genes relating to fungus's own growth.
- - Enhancing tomato immunity: If the tomato has not been infected, applying a signal to the tomato to stimulate its immunity can improve its anti-fungal ability.
By applying RNAi biopesticides and plant immune preparations, the balance of the interaction between tomato and B. cinerea began to shift in tomato's favor.
RNAi
For RNAi therapy, we have selected two classes of targets for our shRNA.
- 1. A type of shRNAs targeted genes that played a key role in the infection of tomato by B. cinerea. These include Bcpme1 (BBa_K4653007, BBa_K4653008), a pectin methylesterase that penetrates plant cell walls, and BcOAH (BBa_K4653005, BBa_K4653006), which helps synthesize oxalate to poison cells. Bcdcl1 (BBa_K4653009, BBa_K4653010) and Bcdcl2(BBa_K4653011, BBa_K4653012) were synthesized to silence plant immune-related genes.
- 2. Another type of shRNAs targeted genes essential for the survival of B. cinerea. Including BcchsIIIa (BBa_K4653001, BBa_K4653002), a gene that synthetes chitin, a major component of the cell wall of pathogen. And the ergosterol synthesis gene Bccyp51 (BBa_K4653003, BBa_K4653004) maintains the cell membrane bioactivity of B. cinerea.
Two shRNAs were designed for each of the above genes, and their effects were verified at the phenotypic and molecular levels in tomato infected by B. cinerea. After the effective shRNA was screened step by step, to improve the delivery efficiency of shRNA, we also combined the cell transmembrane peptide BP100-(KH)9(BBa_K3224003) with shRNA and verified the enhancement effect of CPP on shRNA. Then, in order to make shRNA molecules more stable and reduce the cost for large-scale production, two of the effective shRNAs were spliced together to form bi-shRNA(BBa_K4653015), which proved to be more effective when combined with CPP at both phenotypic and molecular levels.
| Part Numbers | Name | Type | Part Description |
|---|---|---|---|
| BBa_K4653907 |
T7 promoter - shRNA(Pme1)-2 - T7 Terminator |
Composite part | Producing the shRNA silencing chitin (CHS) of B. cinerea in E. coli DH5α |
| BBa_K4653001 | shRNA(CHSIIIa)-1 | RNA (new basic part) |
The shRNA silencing chitin (CHS) of B. cinerea. |
| BBa_K4653002 | shRNA(CHSIIIa)-2 | RNA (new basic part) |
The shRNA silencing chitin (CHS) of B. cinerea. |
| BBa_K4653003 | shRNA(cyp51)-1 | RNA (new basic part) |
The shRNA silencing ergosterol (ERG) of B. cinerea. |
| BBa_K4653004 | shRNA(cyp51)-2 | RNA (new basic part) |
The shRNA silencing ergosterol (ERG) of B. cinerea. |
| BBa_K4653005 | shRNA(OAH)-1 | RNA (new basic part) |
The shRNA silencing oxaloacetate hydrolase (OAH) of B. cinerea. |
| BBa_K4653006 | shRNA(OAH)-2 | RNA (new basic part) |
The shRNA silencing oxaloacetate hydrolase (OAH) of B. cinerea. |
| BBa_K4653007 | shRNA(Pme1)-1 | RNA (new basic part) |
The shRNA silencing pectin methylesterase (PME) of B. cinerea. |
| BBa_K4653008 | shRNA(Pme1)-2 | RNA (new basic part) |
The shRNA silencing pectin methylesterase (PME) of B. cinerea. |
| BBa_K4653009 | shRNA(DCL1)-1 | RNA (new basic part) |
The shRNA silencing Dicer-like proteins (DCL1) of B. cinerea. |
| BBa_K4653010 | shRNA(DCL1)-2 | RNA (new basic part) |
The shRNA silencing Dicer-like proteins (DCL1) of B. cinerea. |
| BBa_K4653011 | shRNA(DCL2)-1 | RNA (new basic part) |
The shRNA silencing Dicer-like proteins (DCL2) of B. cinerea. |
| BBa_K4653012 | shRNA(DCL2)-2 | RNA (new basic part) |
The shRNA silencing Dicer-like proteins (DCL2) of B. cinerea. |
| BBa_K4653014 | shRNA(Box-infect) | RNA (new basic part) |
The tetra-shRNA silencing the genes that play a key role in the infection of B. cinerea. |
| BBa_K4653015 | shRNA(Box-survival) | RNA (new basic part) |
The bi-shRNA silencing the genes that maintain the survival of B. cinerea. |
| BBa_K3224003 | BP100-(KH)9 | Protein coding sequences | The short peptides that can penetrate the cell membrane and deliver a wide range of cargoes into the cell. |
| BBa_R0187 | T7 promoter (lacI repressible) | Regulatory | A lac repressible T7 promoter. |
| BBa_M50060 | T7 Terminator | Terminator | Terminator for bacterial expression. |
Thus, we have completed the iteration of shRNA - CPP+shRNA - CPP+bi-shRNA. More information can be found in Engineering.
Plant immunity
I.Inducing factor
To trigger the immune system in tomato, we selected the following elements as immune inducers:
- 1. BBa_K4653101 encodes a pectin enzyme PehA, which can digest pectin and produce oligo-galacturonic acid that can activate downstream immune pathways in plants.
- 2. BBa_K4653102 encodes a 22-amino acid N-terminal peptide of flagellin from Pseudomonas aeruginosa (Flg22), which can activate downstream immune pathways after binding to plant cell surface receptors. BBa_K4653103 encodes three Flg22 repeat peptides in tandem to verify its better immune triggering effect. In addition, we also used the full-length flagellin sequence (BBa_K4653104) in our experiments. The effective immune-inducing factors were screened out among the three parts.
- 3. BBa_K4653105 encodes a protein phosphatase BvEP from Bacillus velezensis, which has been shown to induce PTI and ETI immune responses in tomato to activate a more durable and effective defense in tomato.
| Part Numbers | Name | Type | Part Description |
|---|---|---|---|
| BBa_K4653113 | pET28a - BvEP - 6x His tag | Composite part | Overexpressing BvEP, in E. coli BL21 (DE3). The gene circuit of BVEP is constructed on the pET-28a plasmid vector, including T7 promoter, BvEP gene, 6x His affinity tag, and T7 terminator. |
| BBa_K4653114 | pMA5 - Veg promoter - Epr - BvEP - 2x fd Terminator | Composite part | Expressing BvEP in B. subtilisWB800. Using pMA5 as a plasmid carrier, strong promoter Veg promoter, Epr signal peptide, BvEP expression gene, and T7TE terminator. |
| BBa_K4653101 | PehA | Protein coding sequences (new part) |
A polygalacturonase from R. solanacearum, can hydrolyze pectin and produce Oligogalacturonides as DAMP to induce plant immunity. |
| BBa_K4653102 | 1x Flg22 | Protein coding sequences (new part) |
A conserved 22-amino acid sequence from the N-terminal of P. aeruginosa flagellin, is a PAMP to induce plant immunity. |
| BBa_K4653103 | 3x Flg22 | Protein coding sequences (new part) |
Repeating the sequence of Flg22 three times to increase the probability of the PAMP reaching the plant pattern recognition receptor to enhance the plant immune response. |
| BBa_K4653104 | Flagellin [Pseudomonas aeruginosa] | Protein coding sequences (new part) |
Flagellin from P. aeruginosa has 161 amino acids. The N-terminal sequence can be recognized by the pattern recognition receptors in plant cells. |
| BBa_K4653105 | BvEP | Protein coding sequences (new part) |
BvEP is an elicitor protein phosphopentomutase from Bacillus velezensis LJ02 which can improve the tomato resistance to B. cinerea. |
| BBa_R0187 | T7 promoter (lacI repressile) | Regulatory | A lac repressible T7 promoter. |
| BBa_K157011 | 6x His Tag | Tag | C-term His tag for purification and identification of protein of interest. |
| BBa_K316001 | Veg Promoter | promoter | pVeg Constitutive promoter for Veg locus from B. Subtilis. |
| BBa_B0034 | RBS | RBS | Efficient ribosome binding site from bacteriophage T7 gene 10. |
| BBa_K143021 | SpoVG RBS | RBS | SpoVG ribosome binding site (RBS) for B. subtilis for MoClo Level 0 with 6bp scar/spacer. |
| BBa_K4317100 | Epr signal peptide | Protein coding sequences | Epr signal peptide of B. subtilis minor extracellular serine protease. |
| BBa_M50060 | T7 Terminator | Terminator | Terminator for bacterial expression. |
| BBa_K4653107 | Fd Terminator | Terminator | Central terminator from bacteriophage fd. |
| BBa_K4653108 | 2xFd Terminator | Terminator | Double terminator consisting of central terminator from bacteriophage fd. |
II. Sucrose-induced suicide switch
Considering safety, we designed a sucrose-induced suicide switch, which prevents engineered B. subtilis from escaping and bringing negative effects to the field ecological environment.
- 1.The circuit of our condition-triggered suicide switch ( BBa_K4653121 ) is based on sacB biobricks and MazEF system.
- 2.SacB promoter (BBa_K4043004) is one of the strongest inducible promoters in the fungal kingdom.
- 3.MazEF (BBa_K2292002) is a common toxin-antitoxin system.
| Part Numbers | Name | Type | Part Description |
|---|---|---|---|
| BBa_K4653121 | SacB promoter-SpoVG RBS-MazE-MazF-Fd Terminator | Composite part | A suicide switch applied based on sucrose-dependent mazEF toxin-based antitoxin system in B subtilis. |
| BBa_K2292002 | MazEF kill switch | Protein coding sequences | MazF causes cell death by cleaving mRNA at a specific site. On the other hand, MazE plays a role of antitoxin which can antagonize the toxin MazF. |
| BBa_K4202001 | MazF | Protein coding sequences | MazF causes cell death by cleaving mRNA at a specific site. |
| BBa_K4202046 | MazE | Protein coding sequences | MazE plays a role of antitoxin which can antagonize the toxin MazF. |
| BBa_K4043004 | SacB promoter | promoter | The transcription of the PsacB promoter is not strictly regulated by sucrose, and can be transcribed without an inducer, but the intensity is 100 times lower than that of sucrose induction. |
| BBa_K143021 | SpoVG RBS | RBS | SpoVG ribosome binding site (RBS) for B. subtilis for MoClo Level 0 with 6bp scar/spacer |
| BBa_K4653107 | Fd Terminator | Terminator | Central terminator from bacteriophage fd. |
| BBa_K4653108 | 2xFd Terminator |
Terminator new part |
Double terminator consisting of central terminator from bacteriophage fd. |