Overview

In order to inhibit Fusarium oxysporum cubense 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 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 anti-fungal substances 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’s a broad spectrum antibiotic has been shown to play a key role in the biological control of various plant pathogens including F. oxysporum[4]. In addition to that, it 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 flourescens was introduced into P. putida. The phl gene cluster contains eight genes, from phlA to phlH. Gene cluster phlABCDE was be 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].

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 against 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 shown a slight inhibition to foc 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.

Pyoverdine

Pyoverdines are siderophores produced by P. putida[10,11]. Siderophores are small green/yellow fluorescent compounds that have high affinity to iron(III), which by scavenging free iron can inhe end can 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 a 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. PfrI is a transcription activator that is needed for activation of genes involved in pyoverdine synthesis[18]. So an overexpression was be done of pfrI, which is expected to result in 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. 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]. This plasmid was used instead of the iGEM plasmid because it has shown to work before in P. putida, it contains LacI coupled with an IPTG inducible promoter and it has terminators behind the gene insert. Both E. coli and P. putida KT2440 were transformed with a SEVA plasmid containing the phlABCDE gene cluster. 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 using a primer pair that forms a 1kbp product, we could then see possible transformants. The method used in High-performance liquid chromatography (HPLC) was correct (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.

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

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 that the other. Afterwards, another in vitro 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.

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 in vitro assay, an in vivo assay was also done using our P. putida tranformants, containing the phl gene cluster for 2,4-DAPG production. An overnight P. putida liquid culture was spread on a LB agar plate, grown overnight at 30°C and then inoculated with a 5mm F. oxysporum plug.

In figure 8, plates were modified with a red circle to better visualize the fungal disc. It can be seen that the wild type P. putida KT24400 already inhibits F. oxysporum very well. Our transformant (DAPG) seems to show a slightly small F. oxysporum disc when comparing it with the wild type. It is very hard to see a big difference between the wildtype and our transformants, as the wildtype has shown to already inhibit F. oxysporum greatly when comparing wildtype 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. P. putida were grown, induced and harvested after 3 hours. Cell free extracts were obtained via sonification and were then used for an assay. DMDS and DMTS 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 in vivo assay was done co-inoculating our transformed P. putida with F. oxysporum.

In figure 9 it can be seen when comparing our transformant containing Methionine-γ-lyase with the wildtype P. putida there is a much small fungal disc, probably meaning a higher inhibition of our transformant.

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. Transformed in both E. coli and P. putida KT2440. Succesfull transformants were verified via colony PCR. Afterwards growth experiments were done in minimal M9 medium supplemented with iron and pyoverdine was measured using spectrophotomery. When grown overnight it was possible to see that Pfri transformants were slightly greener when compared to the control. Control used here was a P. putida containing an empty pSEVA254 plasmid.

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

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, an in vivo experiment was also done with pfri tranformants co-inoculated with a 5mm F. oxysporum plug.

In figure 12 it can be seen that pfri transformant has a smaller F. oxysporum disk when compared to the wildtype. Meaning it's inhibition is increased but only ever so slightly.

Chitinase

Chitinase was succesfully cloned into broad host range plasmid (pSEVA254) and transformed into P. putida. Succesfull tranformants were verified via colony PCR. Transformants were grown, induced and harvest 3 hours after induction. Cell free extracted were obtained via sonification of cells (see protocols). Transformants containing overexpression of chitinase were also co-inoculated with Fusarium in an in vivo assay, results in figure 13.

In figure 13, it can be seen that the tranformants with an overexpression of chitinase seems to have a smaller fungal disc as when compared to the wild type.

All transformants together

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

1. Chitinase, Methionine-γ-lyase and Pfri.
2. phlABCDE, Chitinase, Methionine-γ-lyase and Pfri

These two cocktail 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.

In figure 14, chit+MGL+Pfri seems to have a better inhibitory effect when compared to the wildtype as it can be seen that the fungal disc is smaller than the one of the wildtype. For the cocktail mix that contains all four, mix(all 4) the fungal disc is only slightly smaller when compared to the wildtype.

Conclusion

We engineered P. putida with four different genes, coding for anti-fungal 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 competition experiments with F. oxysporum to detect growth inhibition. Additionally for some antifungals we tested specific production or the effect of the pure anti-fungal only on F. oxysporum growth.

Out of the 4 anti-fungals, two 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 vitro assays that pure 2,4-DAPG is indeed a good anti-fungal against F. oxysporum TR4. With decrease 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 increase inhibition effect of the anti-fungal producing transformants 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 see a distinguish increased growth inhibition by our synthetic, anti-fungal 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 anti-fungal 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. Other works would be to try other anti-fungal genes to increase inhibition by using other anti-fungal compounds such as phenazine-1-carboxylic acid which was shown to also have anti-fungal effects

References

1. Loper, H. G. a. J. E. (2009). "Genomics of secondary metabolite production by Pseudomonas spp." from http://pubs.rsc.org/en/content/articlehtml/2009/np/b817075b#cit62.
2. Meldau, D. G., S. Meldau, L. H. Hoang, S. Underberg, H. Wunsche and I. T. Baldwin (2013). "Dimethyl disulfide produced by the naturally associated bacterium bacillus sp B55 promotes Nicotiana attenuata growth by enhancing sulfur nutrition." Plant Cell 25(7): 2731-2747.
3. Zhang, H., A. Mallik and R. S. Zeng (2013). "Control of Panama disease of banana by rotating and intercropping with Chinese chive (Allium tuberosum Rottler): role of plant volatiles." J Chem Ecol 39(2): 243-252.
4. Belgrove, A. (2007). Biological Control of Fusarium Oxysporum F.sp. Cubense Using Non-pathogenic F. Oxysporum Endophytes, University of Pretoria.
5. Rezzonico, F., M. Zala, C. Keel, B. Duffy, Y. Moënne-Loccoz and G. Défago (2007). "Is the ability of biocontrol fluorescent pseudomonads to produce the antifungal metabolite 2,4-diacetylphloroglucinol really synonymous with higher plant protection?" New Phytologist 173(4): 861-872.
6. Bangera, M. G. and L. S. Thomashow (1999). "Identification and characterization of a gene cluster for synthesis of the polyketide antibiotic 2,4-diacetylphloroglucinol from Pseudomonas fluorescens Q2-87." J Bacteriol 181(10): 3155-3163.
7. Abbas, A., J. E. McGuire, D. Crowley, C. Baysse, M. Dow and F. O'Gara (2004). "The putative permease PhlE of Pseudomonas fluorescens F113 has a role in 2,4-diacetylphloroglucinol resistance and in general stress tolerance." Microbiology 150(Pt 7): 2443-2450.
8. Bakker, P. H. M., D. M. Glandorf, M. Viebahn, T. M. Ouwens, E. Smit, P. Leeflang, K. Wernars, L. Thomashow, J. Thomas-Oates and L. van Loon (2002). "Effects of Pseudomonas putida modified to produce phenazine-1-carboxylic acid and 2,4-diacetylphloroglucinol on the microflora of field grown wheat." 81(1-4): 617-624.
9. Hanniffy, S. B., M. Philo, C. Pelaez, M. J. Gasson, T. Requena and M. C. Martinez-Cuesta (2009). "Heterologous production of methionine-gamma-lyase from Brevibacterium linens in Lactococcus lactis and formation of volatile sulfur compounds." Appl Environ Microbiol 75(8): 2326-2332.
10. 23. Ravel, J. and P. Cornelis (2003). "Genomics of pyoverdine-mediated iron uptake in pseudomonads." Trends in Microbiology 11(5): 195-200.
11. Matthijs, S., G. Laus, J. M. Meyer, K. Abbaspour-Tehrani, M. Schafer, H. Budzikiewicz and P. Cornelis (2009). "Siderophore-mediated iron acquisition in the entomopathogenic bacterium Pseudomonas entomophila L48 and its close relative Pseudomonas putida KT2440." Biometals 22(6): 951-964.
12. Duijff, B., J. Meijer, P. H. M. Bakker and B. Schippers (1993). "Siderophore-mediated competition for iron and induced resistance in the suppression of fusarium wilt of carnation by fluorescent Pseudomonas spp." 99(5-6): 277-289.
13. Elad, Y. and R. Baker (1985). "The role of competition for iron and carbon in suppression of chlamydospore germination of Fusarium spp. by Pseudomonas spp." Phytopathology 75(9): 1053-1059.
14. Nagarajkumar, M., R. Bhaskaran and R. Velazhahan (2004). "Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight pathogen." Microbiological Research 159(1): 73-81.
15. Duijff, B. J., P. A. H. M. Bakker and B. Schippers (1994). "Suppression of fusarium wilt of carnation by Pseudomonas putida WCS358 at different levels of disease incidence and iron availability." Biocontrol Science and Technology 4(3): 279-288.
16. Lemanceau, P., P. A. Bakker, W. J. De Kogel, C. Alabouvette and B. Schippers (1992). "Effect of pseudobactin 358 production by Pseudomonas putida WCS358 on suppression of fusarium wilt of carnations by nonpathogenic Fusarium oxysporum Fo47." Applied and Environmental Microbiology 58(9): 2978-2982.
17. Dos Santos, V. A. P. M., S. Heim, E. R. B. Moore, M. Strätz and K. N. Timmis (2004). "Insights into the genomic basis of niche specificity of Pseudomonas putida KT2440." Environmental Microbiology 6(12): 1264-1286.
18. Venturi, V., C. Ottevanger, M. Bracke and P. Weisbeek (1995). "Iron regulation of siderophore biosynthesis and transport in Pseudomonas putida WCS358: involvement of a transcriptional activator and of the Fur protein." Molecular Microbiology 15(6): 1081-1093.
19. Atkinson, R. A., A. L. M. Salah El Din, B. Kieffer, J.-F. Lefèvre and M. A. Abdallah (1998). "Bacterial Iron Transport: 1H NMR Determination of the Three-Dimensional Structure of the Gallium Complex of Pyoverdin G4R, the Peptidic Siderophore of Pseudomonas putida G4R†,‡." Biochemistry 37(45): 15965-15973.
20. Venturi, V., P. Weisbeek and M. Koster (1995). "Gene regulation of siderophore-mediated iron acquisition in Pseudomonas: not only the Fur repressor." Mol Microbiol 17(4): 603-610.
21. Mauch, F., B. Mauch-Mani and T. Boller (1988). "Antifungal Hydrolases in Pea Tissue: II. Inhibition of Fungal Growth by Combinations of Chitinase and β-1,3-Glucanase." Plant Physiology 88(3): 936-942.
22. Herrera-Estrella, A. and I. Chet (1999). "Chitinases in biological control." Exs 87: 171-184.
23. Silva-Rocha, R., E. Martínez-García, B. Calles, M. Chavarría, A. Arce-Rodríguez, A. de las Heras, A. D. Páez-Espino, G. Durante-Rodríguez, J. Kim and P. I. Nikel (2013). "The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex