Engineered Agrobacterium improves transformation by mitigating plant immunity detection

Abstract

Summary Agrobacterium tumefaciens microbe‐associated molecular pattern elongation factor Tu (EF‐Tu) is perceived by orthologs of the Arabidopsis immune receptor EFR activating pattern‐triggered immunity (PTI) that causes reduced T‐DNA‐mediated transient expression. We altered EF‐Tu in A. tumefaciens to reduce PTI and improved transformation efficiency. A robust computational pipeline was established to detect EF‐Tu protein variation in a large set of plant bacterial species and identified EF‐Tu variants from bacterial pathogen Pseudomonas syringae pv. tomato DC3000 that allow the pathogen to escape EFR perception. Agrobacterium tumefaciens strains were engineered to substitute EF‐Tu with DC3000 variants and examined their transformation efficiency in plants. Elongation factor Tu variants with rarely occurred amino acid residues were identified within DC3000 EF‐Tu that mitigates recognition by EFR. Agrobacterium tumefaciens strains were engineered by expressing DC3000 EF‐Tu instead of native agrobacterial EF‐Tu and resulted in decreased plant immunity detection. These engineered A. tumefaciens strains displayed an increased efficiency in transient expression in both Arabidopsis thaliana and Camelina sativa. The results support the potential application of these strains as improved vehicles to introduce transgenic alleles into members of the Brassicaceae family.

Natural variation of immune epitopes reveals intrabacterial antagonism

“…Similarly, weakly ROS immunogenic EF-80, was previously demonstrated to be unable to induce robust callose deposition, a late-stage immune output. 5, 37…”

“…While immune outcomes of weakly-immunogenic variants from Streptomyces, Xanthomonas, and Pseudomonas are comparable, their polymorphisms are unique and their respective EF-Tu proteins do not cluster phylogenetically, indicating convergent evolution (Figure 3A, 3F). 37 Across all elf18 variants, polymorphisms were position-dependent, showcasing peptide variation may be constrained by EF-Tu function (Figure 3A).…”

“…Similarly, weakly ROS immunogenic EF-80, was previously demonstrated to be unable to induce robust callose deposition, a late-stage immune output. 5,37 To understand the relationship between immunogenic outcomes and protein evolution, we developed a maximum-likelihood tree of EF-Tu and mapped on genera information, the percent amino acid similarity to the consensus, and the immunogenicity output by ROS (Figure 3F). The EF-Tu tree structure was indicative of taxonomic origin (Figure 3F).…”

Genetically refactoredAgrobacterium-mediated transformation

“…By mobilizing the transformation phenotype via pDimples into R. rhizogenes , we open the door to another promising avenue of AMT engineering: transferring the complex vir machinery to other bacteria. As A. fabrum can elicit plant immunity that impede transformation, multiple efforts have been made recently to circumvent this either through mutation of known immunogenic loci 53 or the addition of immune suppressing systems 54 . This work lays the foundation to developing synthetic pTi that function in bacteria that elicit minimal immune responses across plant species, potentially enabling the transformation of those that have traditionally been recalcitrant to genetic modification.…”

A rapid and highly efficient sorghum transformation strategy using GRF4‐GIF1/ternary vector system

“…Enhancing the infection efficiency of Agrobacterium is another approach to improve plant transformation efficiencies (van der Fits et al, 2000). Decreased plant immunity detection by engineering Agrobacterium could increase T-DNA mediated transient expression and then improve transformation efficiency in Arabidopsis and Camelina sativa (Yang et al, 2023). Ternary vector system that expresses diverse virulent genes could enhance T-DNA transfer to plant cells, leading to increased transformation efficiency (Che et al, 2018;van der Fits et al, 2000;Zhang et al, 2020).…”