Overview

In order to inhibit F.oxysproum growth, several anti-fungal substances will be produced when fusaric acid is sensed. Those being 2,4-DAPG, chitinase, DMDS,DMTS and pyoverdine. 2,4-DAPG or 2,4-Diacetylphloroglucinol is a widely used antiobiotic against plant pathogens. Chitinase is a lytic enzyme that breaks down fungal cell walls. Dimethyldisulfide (DMDS) and dimethyltrisulfide (DMTS) are sulfur by-products produced by P.putida and have shown to produce plant growth and inhibit F.oxyposrum respectively. And lastly, pyoverdines are iron chelating compounds that are produced when iron is limited in order to scavenge for iron to induce iron competition. With these anti-fungal substances our engineered P.putidawill be able to sense F.oxypsroum and in turn produce substances that will eliminate or inhibit F.oxysporum from in the soil.

2,4-Diacetylphloroglucinol(2,4-DAPG)

2,4-DAPG, full name 2,4-diacetylphloroglucinol is an antibiotic that is widely used in the agricultural industry against pathogens. It’s a broad spectrum antibiotic has been shown to play a key role in the biological control of various plant pathogens including F. oxysporum(Belgrove 2007). In addition to that, it has also shown to induce systematic resistance (ISR) in plants (Rezzonico, Zala et al. 2007). Since P. putida does not produce DAPG by itself, a gene cluster obtained from P.flourescens will be introduced into P. putida. The phl gene cluster contains eight genes, from phlA to phlH. Gene cluster phlABCDE will be used for this project as literature has shown that phlABCD are synthesis genes and phlE codes for an efflux pump (Bangera and Thomashow 1999) (Abbas, McGuire et al. 2004). phlABCD has been expressed before in P.putida and has shown to produce 2,4-DAPG (Bakker, Glandorf et al. 2002).

Dimethyldisulfide (DMDS) and dimethyltrisulfide (DMTS)

When breaking down methionine for ammonium, methanethiol gets formed as a side product. Then methanethiol gets oxidized into dimethyldisulphide (DMDS) and dimethyltrisulphide (DMTS) (see figure 2, pathway 1). DMTS was shown to have an inhibitory effect against foc with starting inhibition of 78% but then slowly declined to 26% (Zhang, Mallik et al. 2013). DMDS is used as plant growth promoter and at the same time also shown a slight inhibition to foc of 10% (Meldau, Meldau et al. 2013). Both DMTS and DMDS are naturally produced in P. putida but a higher production is desired. One thing that causes low yield of DMDS is the low affinity of methionine-γ-lyase to methionine meaning less formation of methanethiol, which then leads to low DMTS and DMDS production. So it was thought to overexpress an enzyme that has a higher affinity towards methionine and will therefor increase DMDS and DMTS production, this enyzme is a Methyl-γ-lyase from Brevibacterium linens that has shown to increase DMDS in Lactocossus lactis (Hanniffy, Philo et al. 2009). This gene was codon optimized and synthetically made for P.putida.

Pyoverdine

Pyoverdines are siderophores produced by P.putida (Ravel and Cornelis 2003, Matthijs, Laus et al. 2009). Siderophores are small green/yellow fluorescent compounds that have high affinity to Fe(III) that in the end can lead to iron competition. Due to iron starvation, the growth of pathogenic fungi and bacteria in the rhizosphere will be restricted (Duijff, Meijer et al. 1993). It was shown that there was a direct correlation of siderophore production and their inhibition to germination of chlamydospores of F.oxysporum (Elad and Baker 1985). In addition to that siderophores have also been shown to induce resistance in radish plants (Nagarajkumar, Bhaskaran et al. 2004). However the effects of siderophore decreases when the disease incidence increases above 74% (Duijff, Bakker et al. 1994). P. putida WCS358 is able to produce a siderophore, pseudobactin 358 (PBS358), which has been shown to be involved in inhibition of Fusarium (Lemanceau, Bakker et al. 1992). Pyoverdine production is iron dependent as it is regulated by a Fur (ferric update regulator protein) protein (Dos Santos, Heim et al. 2004). PfrI is a transcription activator that is needed for activation of genes involved in pyoverdine synthesis (Venturi, Ottevanger et al. 1995). So an overexpression will be done of pfrI this will be expected to increase pyoverdine production, even when in an iron abundant environment.

Chitinase

Chitinase is a hydrolytic enzyme that breaks down hydrolytic bonds in chitin and is produced in both bacteria and plants and has shown to be useful in biological control against fungi (Mauch, Mauch-Mani et al. 1988, Herrera-Estrella and Chet 1999). In bacteria their function is to attack shellfish animals or fungi, and degrade their cell wall. In plants they are known as pathogen related (PR) proteins that are involved in the induced systematic resistance of plants in order to defend themselves against pathogens. P. putida KT2440 has a lytic enzyme PP3066 that is predicted to have chitinase activity, so a possible overexpression of this gene was done for this project.

Results

2,4-DAPG

PhlABCDE was successfully cloned and put into SEVA 254 plasmid. Transformed in both E.coli and P.putida KT2440. Transformants were verified via colony PCR. For P.putida there was difficulty when doing colony PCR with Taq polymerase. It was never successful to get a full 5.4kbp band doing colony PCR with P.putida but when using a primer pair that forms a 1kbp product, it was then possible to see positive transformants.Verifying production of 2,4-DAPG via HPLC turned out to be difficult due to a lot of background noise of the samples, even after doing an extraction step. In order to test 2,4-DAPG against Fusarium, a in vitro assay was done on agar plates. Where different concentration of pure 2,4-DAPG were plated and <

Dimethyldisulfide (DMDS) and dimethyltrisulfide (DMTS)

Methionine-γ-lyase was successfully cloned and put into SEVA 254 plasmid. Transformed in both E.coli and P.putida KT2440. Transformants could be verified via colony PCR for E.coli but not for P.putida.

Pyoverdine

PfrI was successfully cloned and put into SEVA 254 plasmid. Transformed in both E.coli and P.putida KT2440 and checked via colony PCR. Growth experiments were done in minimal M9 medium supplemented with Iron was done with pyoverdine measured using spectrophotometer. Results can be seen in the (graph). As it seems that pfrI does not seem to affect the production of pyoverdine when in an iron rich environment.

Chitinase

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Future work

For future work, it would be nice it all the antifungal genes are coupled together behind the fusaric acid promoter in order to test production rate that is induced by fusaric acid. Also testing in the green house with a co-inoculation of F.oxysporum in order to see if production of the anti-fungals can reach high enough levels that it causes inhibition affect to F.oxysporum. This really depends on the amount of fusaric acid present in the soil when F.oxysporum is present.

References