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Plant-Bacillus Interaction: Gain of Function Project

Periodic Reporting for period 1 - PBIGP (Plant-Bacillus Interaction: Gain of Function Project)

Reporting period: 2016-03-17 to 2018-03-16

The application to the H2020 -MSCA individual fellowship program was done to explore and facilitate the re-integration of the applicant in European scientific community as he was out of the European network for more than 10 years. The stay of the applicant in the department of the Microbiology, Adaptation and Pathology (UMR5240; Lyon, France) that is part of the CNRS (French National Center for Scientific Research) was primarily used built a European network, apply for professional positions (in public and private sectors) in Europe, extend his experience in teaching while developing a new research program that would support his integration within the French or European research system. The main mission of the fellowship was achieved successfully as the applicant had to abandon his fellowship to take a new role a group leader in a private company in France. The applicant had this opportunity from the new network built and new skills acquired during his stay in the host institution.

Even if none of the original scientific milestones could be achieved, this fellowship allowed the completions and release to the public domain via publications of synthetic biology tools developed to support plant engineering. The most important outcome was the development of a robust DNA assembly tools that allow support combinatorial and rapid gene assemblies and gene stacking to support the engineering of Agrobaterium transformation compatible hosts (e.g. plants). This tool was completed with the development of a two-component-switch for plants allowing precise and robust control of transgene. This system is a negative switch composed of a RNase that recognizes a specific RNA module. Binding of the RNase prevents the translation of any mRNA sequence that contains the RNA module by cleaving it. It adds further sophistication to the fine-tuning of expression of metabolic pathways, for example, by allowing multiple genes to be switched off in response to an external cue and/or in specific tissues This system was also validated to simultaneously repress the expression of two proteins in a specific plant tissue
There is not really any reportable data generated on the main proposed project however results from lateral projects resulted in 2 publications and all data are summarized above and detailed in these open access publications.
Regarding Training activities: I was trained to work with pathogenic microorganisms (fungi and bacteria), it includes all necessary precaution for safe manipulations and stewardships. Additional trainings were focused on acquisition of teaching skills, as I had several opportunities to give lectures in class setting to students from undergraduate to graduate levels. It gave me more credibility to apply for professorship positions. Moreover, with my host-team; I had the opportunity to practice my applications for faculty and governmental positions.
Even if none of the original scientific milestones could be achieved, this fellowship allowed the completions and release to the public domain via publications of synthetic biology tools developed to support plant engineering. One of the tools was based on the generation of standardized gene parts (Promoters, Coding sequences, terminators) following a synthax defined by an international plant community to support combinatorial and rapid gene assembly compatible with Golden Gate cloning based method. This tool was completed by the development of a trinary plant transformation vector allowing its replication in three hosts: Yeast, E.coli and A. tumefaciens. This vector allows an organized assembly of multiple DNA fragments (e.g. genes) in one step using the capacity of yeast to perform robustly homologous recombination. The use of E. coli allows the isolation and amplification of the vector assembled in yeast and A. tumefaciens the transfer of the assemble DNA clusters (e.g. gene stack) into plants hosts. Even if this system was developed for plants, it is compatible for any hosts that are compatible for agrobacterium transformation. This system allows rapid design of genes and sharing gene parts across the community and will facilitate rapid crop engineering. It was also tested and validated to isolate plant chromosomal regions from one plant species and transfer it to another species, meaning that should support fine mapping, validation and transfer of agronomical QTLs.
Gene stacking summary