Mutant Agrobacterium VirD2 Strains for Transient Plant Genome Editing
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Solution Overview
Problem
Current methods for genetic engineering of plants using Agrobacterium species often result in transgenic plants due to integration of T-DNA into the genome, which is regulated and not feasible for plants with long generation times or those propagated vegetatively, such as trees, bananas, and sweet potatoes.
Innovation Solution
Development of mutant Agrobacterium strains with modified VirD2 genes that facilitate transient transformation of T-DNA without integration into the plant genome, allowing for genome editing without leaving a transgenic trait, using specific mutations and insertions in the VirD2 protein to reduce stable transformation efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Agrobacterium species are used to transfer T-DNA into plants, then genetic transformation is achieved, but T-DNA integrates into the plant genome creating transgenic plants which are regulated and not feasible for certain applications
Solution Approach 1:
The invention extracts and removes the integration function from the T-DNA delivery system by using mutant Agrobacterium strains with modified VirD2 proteins that can transfer T-DNA into plant cells but cannot integrate it into the genome. This separates the useful transformation function from the harmful integration function, enabling transient expression without creating transgenic plants.
Solution Approach 2:
The invention changes the molecular parameters of the VirD2 protein through specific mutations (such as in the ω domain) that alter its functionality. These parameter changes in the protein structure enable T-DNA transfer while disabling the integration capability, thus resolving the contradiction between efficient transformation and unwanted integration.
2Stability of the object's composition
If T-DNA is integrated into the plant genome to achieve stable transformation, then transgenic plants are created, but this is subject to regulation and not feasible for plants with long generation times or those propagated vegetatively
Solution Approach 1:
Instead of achieving stability through integration (the conventional approach), the invention inverts the approach by using transient expression without integration. This allows the T-DNA to temporarily express genes in plant cells and tissues without becoming part of the genome, thus providing application flexibility for regulated or asexual propagation scenarios while still achieving the desired genetic modification effect.
Data Source
AI summary
Agrobacterium strains with mutations in the VirD2 gene and protein and expression constructs and genome-editing systems for leveraging the same. Methods are also provided for Agrobacterium-mediated incorporation of exogenous expressible nucleic acids into a host plant material using the Agrobacterium strains, expression constructs, and/or genome-editing systems.