Team:UZurich/Proof Of Concept

Proof of Concept

Introduction

Here we present that bacterial membrane vesicles from different bacteria can indeed trigger immune response in plants of different genotypes, and such immune response can be further enhanced by displaying proteinaceous elicitors (e.g., flg22 (BBa_K3989010) or elf18 (BBa_K3989011)) of plant immune response on OMV surface using ClyA protein (for more detail see our Design page).

Results

To prove our concept, the work entailed:

  1. proof of a successful engineering process.
  2. statistical evidence gained by analysing the data from our experiments and performing statistical test to draw the aforementioned statistical conclusions.


You can also read everything up in more detail on our Engineering Success and Experiments pages.

Regarding the second concern, we had to answer the following questions with our data analysis:

  1. Are OMVs and CMVs able to elicit an immune response?
    • a. If yes, do they differ in their impact?
    • b. If yes, does their concentration matter?
  2. Which bacterial strains membrane vesicles elicit the highest immune response?
    • a. Is the immune response, no matter which vesicles from which strains, dependent on the plants genotype?
    • b. Is the immune response from a certain treatment dependent on the plants genotype?
  3. Are vesicles with our constructs able to elicit a higher immune response?
    • a. If yes, do the constructs differ in said regard?

Figure 1: Plot of SGI experiment data comparing dosage of vesicles



From the first SGI in A. thaliana, we found answers to questions 1. , 1.a and 1.b:

Both OMVs and CMVs are able to induce immune responses in seedlings, in a dose-dependent manner (i.e. higher vesicle concentration leads to stronger immune response), but OMVs can trigger stronger immune responses than CMVs, supported by the two-way ANOVA results.

Figure 2: Plot of SGI experiment data with 4 phenotypes of A.thaliana


Another SGI experiment delivered answers to questions 2, 2.a and 2.b. Comparing six different treatments in four different A.thaliana genotypes, the results of the analysis (results illustrated in Fig.2) showed that the plant growth-promoting bacteria P. putida OMVs trigger the strongest immune response among all vesicles tested, which would make this strain a good choice to produce OMVs for real-world application. The plant’s genotype (p = 0.00671), as well as its interaction with the treatment (p = 0.00347) also explained variability in our data, indicating that plant immune response triggered by membrane vesicles is dependent on the presence of certain immune receptors.



Figure 3: Plot of ROS experiment data in A.thaliana Col-0 and efr-1






By conducting a ROS experiment, we found that the same applies for the treatment and the interaction, but not the plant’s genotype alone (see Figure 3).

Figure 4: Plot of ROS experiment data of our own constructs





To verify that our engineered OMVs with immune elicitors on the surface can trigger stronger plant immune response (questions 3. and 3.a), we first performed a ROS burst essay using OMV-overproducing strain E. coli omp8. It shows that engineered OMVs with elf18 displayed by ClyA do in fact elicit stronger immune responses than OMVs with only ClyA. In another OMV-overproducing strain, E. coli tolB, such engineering strategy also gave stronger plant immune response, which further proves our concept (Fig.4). is in accordance with the empirical observation in our lab that pure elf18 peptide can trigger stronger plant immune response than flg22 peptide.

Figure 5: Plot of SGI experiment data of our own constructs







In addition to ROS assay, seedling growth inhibition assays were also performed using engineered OMVs, which gave similar results that display of elf18 can enhance the immunogenicity of OMVs. Such results further prove that our strategy works (see Fig.5).

Limitations

We have proved that our construct works but not our proposed application method of spraying OMVs onto the plants. Before implementing our project, one would also have to do different field experiments, to assess if spraying on the OMVs works as well as.

We are aware that our elicitor elf18 is only perceived in Brassicacea. However, this was only used for proof of concept. As for real-world applications, it is better to use a more widely perceived elicitor, or customize the elicitor according to the target crops.

Want to see how we plan to implement our technology? Have a look at our Proposed Implemenation page!

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