Aptamer-based peptide interference with type III effectors for broad-spectrum and durable resistance of crop plants – PAPTiCROPs

In this project, we focused on two widely conserved effectors as targets for functional inactivation: Transcription-Activator Like Effectors (TALEs) from rice-pathogenic Xanthomonas bacteria and RipH effectors from tomato-infecting Ralstonia bacteria. Escape from effector inactivation should be unlikely due to the presence of several binding sites for the interfering, anti-bacterial protein (TALEs) or due to binding of several interfering, anti-bacterial proteins targeting distinct surface epitopes of the target protein (RipH). Our strategy was therefore expected to lead to broad-spectrum and durable crop protection as bacteria should not be able to overcome this type of resistance by effector inactivation.
Bacteriophage display and yeast two-hybrid screens were considered to fish effector interfering, anti-bacterial proteins. Candidates with satisfying binding properties were planned to be produced from within the plant cell in a transgenic approach, which were then expected to bind – and perhaps inactivate – the targeted virulence protein.

We succeeded to isolate proteins belonging to two different classes and binding to one or the other effector, as indicated by genetic and/or biochemical means. This work involved a new collaboration with the team of Philippe Minard at I2BC Paris-Saclay. However, it turned out that none of them was able to functionally interfere with the virulence activity of the targeted effectors under our experimental conditions, which involved transient expression of the candidate anti-bacterial proteins in the model plant Nicotiana benthamiana. Thus, using these non-interfering interactors, there was no way to generate resistant crop plants.

We consider that, in the future, the identified effector-binding proteins could serve as decoys in synthetic resistance proteins and generate resistant plants.

Since we could not provide a proof of concept, we were not able to publish our data in a scientific journal, nor could we apply for patents or use our data to create a start-up company.

Bacterial plant pathogens are among the major constraints for stable food production. Many of them, including devastating pathogens as Ralstonia solanacearum and Xanthomonas spp, depend on the activity of a Type III Secretion System, which injects bacterial Type III Effector proteins (T3Es) into the host’s plant cells to the benefit of the pathogen. In this project, we propose to disarm bacterial phytopathogens by inhibiting the activity of conserved, key T3Es in such a way that would prevent the emergence of mutant bacteria that overcome our intervention. Specifically, we will isolate, validate and apply peptide aptamers belonging to three different molecule classes that are able to bind T3Es and target them to the degradation pathway of the plant cell. This peptide aptamer interference will be applied to tomato and rice crops, which are highly susceptible to R. solanacearum and Xanthomonas oryzae attack.