Engineering Agrobacterium for improved plant transformation

Abstract

Outside of a few model systems and selected taxa, the insertion of transgenes and regeneration of modified plants are difficult or impossible. This is a major bottleneck both for biotechnology and scientific research with many important species. Agrobacterium-mediated transformation (AMT) remains the most common approach to insert DNA into plant cells, and is also an important means to stimulate regeneration of organized tissues. However, the strains and transformation methods available today have been largely unchanged since the 1990s. New sources of Agrobacterium germplasm and associated genomic information are available for hundreds of wild strains in public repositories, providing new opportunities for research. Many of these strains contain novel gene variants or arrangements of genes in their T-DNA, potentially providing new tools for strain enhancement. There are also several new techniques for Agrobacterium modification, including base editing, CRISPR-associated transposases, and tailored recombineering, that make the process of domesticating wild strains more precise and efficient. We review the novel germplasm, genomic resources, and new methods available, which together should lead to a renaissance in Agrobacterium research and the generation of many new domesticated strains capable of promoting plant transformation and/or regeneration in diverse plant species.

Keywords: CRISPR; gene editing; genetic transformation; recombineering; regeneration; transgenic; transposase.

Figure 1

Phylogeny of sequenced Agrobacterium strain Ti and Ri plasmids illustrates the lack of diversity present among common laboratory strains. (a) Phylogeny of Ti and Ri plasmids reconstructed from Weisberg et al. (2020). Arrows indicate sources of strains or vir plasmids in common laboratory strains. Note that C58 and closely related Ti plasmids sometimes cluster within type Ib Ti plasmids rather than type Ia (their classified group) depending on the source genes used to assemble the tree (pers. comm.). (b) Examples of laboratory strain origin including the chromosomal background and the disarmed or Ri plasmid introduced into a given strain. Text colors in (b) correspond to the source clade from the Ti/Ri phylogeny in (a).

Figure 2

Gene editing and recombineering approaches to rapidly domesticate wild strains and enhance their utility in a laboratory setting. (a) Base editing to introduce premature stop codons into useful gene targets on the Agrobacterium chromosome. These include the recA gene to increase introduced construct stability, and the thyA gene to induce auxotrophy to eliminate the need to wash transformed explants and prevent overgrowth. (b) INTEGRATE system to insert cargo into specific sites in the Agrobacterium genome. This relies on a CRISPR‐associated transposase to insert cargo where directed to by a gRNA. For disarmament, lox sites are inserted flanking a native T‐DNA, then a Cre expression plasmid is added to induce excision of the T‐DNA. For insertion of other tools, gRNAs can be targeted to the chromosome or regions of the vir/Ti/Ri plasmid. INTEGRATE could also be used to disrupt genes with a cargo by inserting them into a gene coding sequence. (c) Recombinases isolated from rhizobia genomes and λRed systems can be used to induce homologous recombination with a native sequence. Cargo vectors include homology arms to the target site, usually also inserting an antibiotic resistance gene to improve recombinant identification. These could be used for simultaneous disarmament and insertion of sequences like a GAANTRY landing pad. This approach is most similar to traditional double crossover via triparental mating. (d) Example of a heavily engineered strain with ideal laboratory qualities. These include disarmed native T‐DNAs, increased construct stability, auxotrophy, delivery of non‐T‐DNA transformation enhancing elements (NTEEs) to reduce plant defense, GAANTRY construct assembly, and novel hypervirulent vir gene combinations.

Figure 3

Hairy root transformation systems for recalcitrant plant species. (a) Wild strains with binary vectors containing a gene of interest for rapid trait assessment in liquid culture or composite plants. (b) Wild strains with binary vectors containing a gene of interest, using natural regenerability of mature root tissues to regenerate transgenic intact plants (cut–dip–budding method). (c) Wild strains with binary vectors containing a gene of interest and inducible morphogenic genes, allowing for highly efficient shoot regeneration in vivo. (d) Single vector systems in disarmed strains delivering hairy root‐inducing rol genes which are excised by a recombinase during regeneration, producing shoots with only the gene of interest integrated.