Team:Wageningen UR/project/fungal inhibition

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Wageningen UR iGEM 2014

Fungal inhibition


Overview

In order to inhibit Fusarium oxysporum cubense growth, several fungal growth inhibitors 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 an antibiotic against plant pathogens[1]. Chitinase is a lytic enzyme that breaks down fungal cell walls. Dimethyldisulfide (DMDS) and dimethyltrisulfide (DMTS) are sulfur by-products produced by Pseudomonas putida and have shown to stimulate plant growth and inhibit F.oxyposrum respectively[2,3]. 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 fungal growth inhibitors our engineered P. putida will be able to better inhibit F. oxysporum.

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 is a broad spectrum antibiotic that has been shown to play a key role in the biological control of various plant pathogens including F. oxysporum[4]. In addition to that, 2,4-DAPG has also shown to induce systematic resistance in plants[5]. Since P. putida does not produce DAPG by itself, a gene cluster obtained from Pseudomonas fluorescens was introduced into P. putida. The phl gene cluster contains eight genes, from phlA to phlH. Gene cluster phlABCDE was used for this project as literature has shown that phlABCD are the DAPG synthesis genes(see figure 1) and phlE codes for an efflux pump[6,7]. phlABCD has been expressed before in P. putida and has shown to produce 2,4-DAPG[8].

Figure 1:2,4-DAPG synthesis pathway (Loper 2009)



Dimethyldisulfide (DMDS) and dimethyltrisulfide (DMTS)

When breaking down methionine for ammonium, methanethiol gets formed as a by-product. Then methanethiol gets oxidized into dimethyldisulphide (DMDS) and dimethyltrisulphide (DMTS) (see figure 2). DMTS was shown to have an inhibitory effect on F. oxysporum with an inhibition at the start of 78, but then slowly declined to 26%[3]. DMDS is used as plant growth promoter and at the same time also has shown a slight inhibition to F. oxysporum of 10%[2]. 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, which means less conversion of methanethiol, which then leads to low DMTS and DMDS production. So, we decided to overexpress an enzyme that has a higher affinity towards methionine and will therefore increase DMDS and DMTS production, this enyzme is a methionine-γ-lyase from Brevibacterium linens, which has shown to increase DMDS production in Lactocossus lactis[9]. This gene was codon-optimized and synthetsized by IDT for P. putida.

Figure 2:Dimethyldisulfate (DMDS) and dimethyltrisulfate pathway.

Pyoverdine

Pyoverdines are siderophores produced by P. putida[10,11]. Siderophores are green/yellow fluorescent compounds that have high affinity to iron(III), which by scavenging free iron that can in the end lead to iron limitation. Due to iron starvation, the growth of pathogenic fungi and bacteria in the rhizosphere will be restricted[12]. It was shown that there was a direct correlation of siderophore production and their inhibition to germination of chlamydospores of F. oxysporum[13]. In addition to that, siderophores have also been shown to induce resistance in radish plants[14]. However the effects of siderophore decreases when the disease incidence increases above 74%[15]. P. putida WCS358 is able to produce another sort of siderophore, pseudobactin 358 (PBS358), which has been shown to be involved in inhibition of Fusarium[16]. Pyoverdine production is iron dependent as it is regulated by a ferric uptake regulator protein (Fur)[17], see figure 4. The Fur protein inhibits expression of PfrI and PfrI is a transcription activator that is needed for activation of genes involved in pyoverdine synthesis[18]. So an overexpression was done of PfrI, which we expected to result in pyoverdine production, even when in an iron abundant environment.

Figure 3:Pyoverdine structure[19]

Figure 4:Fur regulation of siderophore genes[20]

Chitinase

Chitinase is a hydrolytic enzyme that breaks down hydrolytic bonds in chitin and is produced in both bacteria and plants. Chitinase has shown to be useful in biological control against fungi[21,22]. In bacteria their function is to attack shellfish animals or fungi, and degrade their chitin cell walls. In plants they are known as pathogen related 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 this gene was overexpressed to increase chitinase activity.


Results

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

PhlABCDE was successfully cloned and put into plasmid pSEVA254[23], which has a kanamycin resistance, a RFS1010 ori and a lacIq with a Ptrc promoter. A SEVA plasmid was used instead of the iGEM plasmid because it has shown to work in P. putida, it contains a LacI that controls an IPTG inducible Ptrc promoter and it has terminators behind the gene insert. Both E. coli and P. putida KT2440 were transformed with a pSEVA containing the phlABCDE gene cluster. This strain was further referred to as 2,4-DAPG P. putida. Succesfull transformants were verified via colony PCR (see journal). During colony PCR of P. putida, we were not able to get an expected 5.4kbp band. However, when we used a primer pair that forms a 1kbp product, we could see positive transformants. For detection of 2,4-DAPG we successfully set up a High-performance liquid chromatography (HPLC) method (see protocol). Standards were detected and a standard curve was made. However, 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 an extraction step.

Figure 5:2,4-DAPG HPLC standard curve.


In order to test the inhibition of pure 2,4-DAPG against F. oxysporum, an assay was done on Komada agar plates. Different concentrations of pure 2,4-DAPG were plated and then inoculated with a 5mm F. oxysporum plug. Plates were then incubated at 25°C for several days.



Figure 6:Inhibition assay with pure 2,4-DAPG concentrations (0, 25, 50, 100 and 200ug/ml) inoculated with 5mm F. oxysporum TR4 plug. t=6 days, upper row= face down plates, lower row= face up plates .

From figure 6 it can be seen that with an increase of 2,4-DAPG there is a decrease in F. oxysporum growth. However when it reached to 100ug/ml results were not reproducible with one plate having more growth than the other. Afterwards, another inhibition experiment was set up to test 2,4-DAPG concentration from 100-400ug/ml. However here spores (3.87E+07 spores/ml) were used as an inoculum, instead of a F. oxysporum plug, due to unavailability of a fresh F. oxysporum plug on agar plate. Plates were then incubated in 25°C for several days.



Figure 7:Inhibition assay with pure 2,4-DAPG with F. oxysporumTR4 spores (3.87E+07 spores/ml) with concentration range (100,200 and 400 ug/ml), t=5 days, upper row= face down plates, lower row= face up plates.

When comparing figure 6 and 7 it can be seen that when inoculated with spores even in 5 days there is absolutely no growth of F. oxysporum at a concentration of 100ug/ml 2,4-DAPG, whereas when for the F. oxysporum plug there is growth after 6 days with mixed results at the same concentration of 100ug/ml 2,4-DAPG. After testing inhibition with pure 2,4-DAPG, another inhibition experiments was done using our 2,4-DAPG P. putida, containing the phl gene cluster for 2,4-DAPG production. An overnight 2,4-DAPG P. putida liquid culture was spread on a LB agar plate containing 1mM IPTG for induction, grown overnight at 30°C and then inoculated with a 5mm F. oxysporum plug.

Figure 8:Inhibition profiles of F. oxysporumTR4 by 2,4-DAPG P. putida. A 5mm F. oxysporum TR4 plug was used, tests done in duplo (upper & lower row), t=7 days. Red circle indicates area occupated by F. oxysporum growth. Foc control=only F. oxysporum TR4, Wildtype= P. putida KT2440 and DAPG=2,4-DAPG P. putida containing phlABCDE gene cluster.

In figure 8, plate pictures were modified with a red circle to better visualize the fungal disc. It can be seen that the wild type P. putida KT2440 already inhibits F. oxysporum very well. Our 2,4-DAPG P. putida seems to show a slightly smaller F. oxysporum disc when comparing it with the wild type. It is very hard to see a big difference between the wild type and our 2,4-DAPG P. putida, as the wild type has shown to already inhibit F. oxysporum greatly when comparing wild type and foc control. Please note that phlABCDE was not yet made into the BioBrick standard, due to too many illegal sites and insufficient time to remove them.


Dimethyldisulfide (DMDS) and dimethyltrisulfide (DMTS)

Methionine-γ-lyase was successfully made into a BioBrick standard format (Bba_K1493300). It was then put into plasmid pSEVA254 plasmid, which was transformed in both E. coli and P. putida KT2440. Succesfull transformants were verified via colony PCR for E. coli and P. putida. Transformants are further referred to as DMDS/DMTS P. putida. DMDS/DMTS P. putida was grown, induced and harvested after 3 hours. Cell free extracts were obtained via sonification and were then used for an assay. DMDS and DMTS compounds were supposed to be measured via Gas Chromatography (GC)(see protocol). But it was in the end not possible due to unexpected problems with the GC. However an inhibition assay was done co-inoculating our DMDS/DMTS P. putida with F. oxysporum.


Figure 9:Inhibition profiles of F. oxysporum TR4 by DMDS/DMTS P. putida. A 5mm F. oxysporum TR4 plug was used, tests done in duplo (upper & lower row), t=7 days. Red circle indicates area occupated by F. oxysporum growth. Foc control=only F. oxysporum TR4, Wildtype= P. putida KT2440 and DMDS/DMTS=DMDS/DMTS P. putida overexpressing Methionine-gamma-lyase.

In figure 9 it can be seen when comparing our DMDS/DMTS P. putida the wild type P. putida there is a much smaller fungal disc, probably cause by a higher inhibition of our DMDS/DMTS P. putida.


Pyoverdine

The pfri gene was successfully made into a BioBrick standard format (Bba_K1493200), validated and characterized. pfri was cloned and put into plasmid pSEVA254. Then transformed in both E. coli and P. putida KT2440. Successful transformants were verified via colony PCR. And are further referred to as pyoverdine P. putida. Afterwards growth experiments were done in minimal M9 medium supplemented with iron (31μM) and the pyoverdine compound was measured using spectrophotomery. When grown overnight it was possible to see that pyoverdine P. putida were slightly greener when compared to the control. Control used here was a P. putida containing an empty pSEVA254 plasmid.

Figure 10:Pyoverdine spectrum(350-400nm)OD corrected average from biological duplicate.


In figure 10 it can be seen that the peak (400nm) of pyoverdine P. putida is higher than the peak from a P. putida containing an empty plasmid.



Figure 11:Pyoverdine absorbance at 400nm with error bars, measured in duplicates, OD600 corrected.


When looking only at the aborbance at 400nm (figure 11), it can be seen that there is a 4-fold increase of pyoverdine production when pfri is overexpressed in P. putida. In addition to this experiment, inhibition assay was also done with pyoverdine P. putida co-inoculated with a 5mm F. oxysporum plug.


Figure 12:Inhibition profiles of F. oxysporum TR4 by pyoverdine P. putida. A 5mm F. oxysporum TR4 plug was used, tests done in duplo (upper & lower row), t=7 days. Red circle indicates area occupated by F. oxysporum growth. Foc control=only F. oxysporum TR4, Wild type= P. putida KT2440 and pyoverdine= pyoverdine P. putida with an overexpression of pfri.

In figure 12 it can be seen that pyoverdine P. putida has a smaller F. oxysporum disk when compared to the wild type. Meaning it's inhibition is increased but only ever so slightly.


Chitinase

PP3066 was succesfully cloned into broad host range plasmid (pSEVA254) and transformed into P. putida. Successful tranformants were verified via colony PCR. And are further referred to at chitinase P. putida. Chitinase P. putida was grown, induced and harvested 3 hours after induction. Cell free extracted were obtained via sonification of cells (see protocols), sadly there was not enough time to do a chitinase assay. Chitinase P. putida was co-inoculated with Fusarium in an inhibition assay, results in figure 13.


Figure 13:Inhibition profiles of F. oxysporum TR4 by chitinase P. putida. A 5mm F. oxysporum TR4 plug was used, tests done in duplo (upper & lower row), t=7 days. Red circle indicates area occupated by F. oxysporum growth. Foc control=Fusarium oxysporum cubense TR4, Wild type= P. putida KT2440 and chitinase=chitinase P. putida with an overexpression of PP3066 predicted to have chitinase activity.

In figure 13, it can be seen that chitinase P. putida seem to have a smaller fungal disc as when compared to the wild type.


All fungal growth inhibitors together

In addition to testing just one tranformant with F. oxysporum TR4, two culture mixes were made.

  1. Chitinase, DMDS/DMTS and pyoverdine
  2. 2,4-DAPG, Chitinase, DMDS/DMTS and pyoverdine

These two culture mix were then spread on agar plates using overnight liquid culture, grown overnight in 30°C and co-inoculated with F. oxysporum the following day.

Figure 14:Inhibition profiles of F. oxysporum TR4 by pyoverdine P. putida. A 5mm F. oxysporum TR4 plug was used, tests done in duplo (upper & lower row), t=7 days. Red circle indicates area occupated by F. oxysporum growth. Foc control=Fusarium oxysporum cubense TR4, WT=wild type P. putida KT2440, chitinase+DMDS/DMDS+pyoverdine= culture mix 1, mix(all 4)= culture mix 2.

In figure 14, chit+DMDS/DMDS+pyoverdine seems to have a better inhibitory effect when compared to the wild type as it can be seen that the fungal disc is smaller than the one of the wild type. For the culture mix that contains all four, mix(all 4) the fungal disc is only slightly smaller when compared to the wild type. The latter we cannot explain, and shows more and different assay are needed to thoroughly investigate the combined inhibition by our engineered P. putida strains.


Conclusion

We engineered P. putida with four different genes on four different pSEVa plasmids, coding for fungal growth inhibitor resistance, in order to increase its inhibitory effects against F. oxysporum. All four different transformants were obtained and have been further tested. We tested the P. putida transformants in inhibition experiments with F. oxysporum to detect growth inhibition. Additionally for some fungal growth inhibitors, we tested specific production or the effect of the pure fungal growth inhibitor only on F. oxysporum growth.

Out of the 4 fungal growth inhibitors, methionine-γ-lyase and pfri were already made into BioBricks format, Bba_K1493300 and Bba_K1493200 respectively. With both BioBricks validated, and for PfrI characterized. PfrI has shown to give a four-fold increase of pyoverdine production in the pressence of iron in the medium.

Even though production of 2,4-DAPG could not clearly be verified via HPLC because of extraction problems in the assay. But it was also shown in inhibition assays that pure 2,4-DAPG is indeed a good fungal growth inhibitor against F. oxysporum TR4. With decreased growth of F. oxysporum when there is an increase of 2,4-DAPG in the plates (figure 6 and 7).

In general, it was hard to distinguish the increased inhibition effect of the fungal growth inhibitor producing P. putida against F. oxysporum. This is because the P. putida chassis we have chosen is already very good at inhibiting F. oxysporum naturally, which probably makes it hard to distinguish increased growth inhibition by our synthetic, fungal growth inhibitor producing P. putida strains. However, with the DMDS/DMTS production strain, we have a clear indication that there is an increase of growth inhibition of F. oxysporum (figure 9) and with others, producing 2,4-DAPG, chitinase or pyoverdine, we can say that there is an indication of a slight increase of growth inhibition (figure 8, 12 and 13) on top of the natural inhibition.



Future work

For future work, it would be nice if all the fungal growth inhibitor genes are coupled together behind the fusaric acid promoter in order to test production rate that is induced by fusaric acid. Also to test the strains in the green house with actual banana plants, by a co-inoculating the plant roots it with F. oxysporum and our engineered strains. This could demonstrate if the production of the fungal growth inhibitors can actually affect to F. oxysporum in the soil. Other works would be to try other fungal growth inhibitor genes to increase inhibition by using other fungal growth inhibitor compounds such as phenazine-1-carboxylic acid which was shown to also have fungal growth inhibitor effects[24].



Continue to Green House >>

References

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