Agrobacterium expressing a type III secretion system delivers Pseudomonas effectors into plant cells to enhance transformation

Agrobacterium tumefaciens transfers T-DNA and virulence proteins via a type IV secretion system (T4SS) and is widely used to generate transgenic plants. However, low transformation efficiency in many species is a major limitation, largely attributed to innate plant immune responses, including PAMP-triggered immunity (PTI) mediated by PRRs such as EFR recognizing EF-TU. Dampening basal immunity increases susceptibility to AMT, but field application is impractical when it requires generating transgenic plants. Alternatively, modifying host factors like histones can aid AMT but also necessitates transgenesis. Many Gram-negative phytopathogens deliver type III effectors (T3Es) via T3SS to suppress PTI. The hypothesis is that engineering A. tumefaciens with a functional T3SS to deliver defense-suppressing T3Es, or plant proteins that facilitate T-DNA integration, will suppress host defense and/or promote transformation machinery during infection, thereby increasing transient and stable AMT across diverse plant species, including recalcitrant crops.

Prior studies established that PTI limits AMT; Arabidopsis efr mutants are more susceptible to Agrobacterium. T3SSs from plant pathogens efficiently deliver T3Es that suppress PTI; heterologous expression of T3SSs can function in non-native bacteria. AvrPto interacts with PRR kinases (FLS2, EFR) to suppress PTI; inducible AvrPto expression in plants increases transient AMT, and NahG Arabidopsis (reduced salicylic acid) shows increased transient transformation. Altering plant host factors, including histones (e.g., H2A/HTA1), affects T-DNA integration and AMT, but transgenic approaches pose regulatory hurdles. GFP-sized fusions can hinder T3SS translocation; smaller reporters like PhiLOV and split-GFP have been used to track effector delivery. Optimization of AMT media often tracks PTI markers (FRK1, NHL10). Efficient, reproducible wheat AMT remains challenging with variable success (historically ~5–25% in cv. Fielder using optimized parameters). Previous attempts to express T3Es other than AvrPto in planta to boost transformation were unsuccessful when the effectors did not target early PTI at the receptor level, indicating the need to choose T3Es that act upstream on PRRs (e.g., AvrPtoB, HopAO1).

• Engineering Agrobacterium with T3SS: Introduced plasmid pLN18 carrying the Pseudomonas syringae pv. syringae 61 T3SS gene cluster into A. tumefaciens strains (GV2260, EHA105, A208, A348, AGL1) via triparental mating. Constructed pBBR1MCS5-based plasmids expressing T3Es (AvrPto, AvrPtoB, HopAO1) under their native promoters; effectors were tagged with PhiLOV or split-GFP fragments for visualization. Effectors included their N-terminal T3S export signals.
• Secretion and translocation assays: Cultured engineered Agrobacterium in hrp-derepressing medium (HDM). Performed immunoblotting on cell pellets and supernatants to detect AvrPto-PhiLOV secretion. Conducted in planta split-GFP assays in Nicotiana benthamiana by expressing GFP1-10 via T-DNA and delivering AvrPto-GFP11 via T3SS to reconstitute GFP fluorescence; also used FM4-64 staining for localization.
• Transient transformation assays: In Arabidopsis (Col-0) and N. benthamiana, infiltrated leaves with disarmed strains (EHA105 or GV2260) harboring a GUS-intron reporter T-DNA (pCAMBIA1301), with/without pLN18 and effector plasmids. Assessed GUS by histochemical staining and fluorometric 4-MUG assays.
• Stable transformation/tumorigenesis: Arabidopsis root tumor assays used tumorigenic A. tumefaciens A208 expressing T3Es ± pLN18; quantified percent root segments forming tumors after 4 weeks. N. benthamiana leaf disk tumor assays used tumorigenic A348; quantified fresh weight of tumors. Arabidopsis root callus selection used EHA105 with pCAS1 (bar/PPT selection).
• Crop transformations: Wheat (cv. Fielder) immature embryos infected with AGL1 carrying PANIC6B reporter and T3E constructs ± pLN18; regeneration under hygromycin selection; confirmation by GUS staining and PCR (hph, GUSPlus). Alfalfa (line R2336) leaflets infected with EHA105 (PANIC6B) ± AvrPto ± pLN18; selection and regeneration under hygromycin. Switchgrass (line NFCX01) calli infected with AGL1 (PANIC6B) ± AvrPto ± pLN18; selection/regeneration under hygromycin. Transformation efficiencies calculated as percentage of independent transgenic plants (wheat, switchgrass) or transgenic shoots per leaflet (alfalfa).
• Controls: Included strains lacking pLN18, lacking effector, or expressing non-effective effector HopAI1 (control construct referred as HopAI1/HopAll) to test specificity; tested T3SS-only and effector-only controls.
• Virulence and defense gene expression: RT-qPCR on A. tumefaciens vir genes (virA, virB2, virD2, virE3) in A208 ± T3SS/T3Es after acetosyringone induction. Measured Arabidopsis defense marker transcript levels (FRK1, NHL10) at 2 h and 16 h after root infection with A208 ± AvrPto ± pLN18, normalized to UBQ10.
• Delivery of plant proteins: Constructed codon-optimized Arabidopsis HTA1 and truncated HTA1 (tHTA1; first 39 aa) driven by promoters and N-terminal T3S signals of AvrRpm1 (AvrRpm1N) or AvrRps4 (AvrRps4N) for export via T3SS. Tested in Arabidopsis root tumors, N. benthamiana leaf disks, and in crop transformation (wheat, alfalfa, switchgrass).
• Imaging and analytics: Confocal microscopy for GFP/PhiLOV fluorescence; statistical analyses via ANOVA with Tukey’s post-hoc tests; variance assessed with Brown-Forsythe tests; replicates as specified per assay.

• Functional T3SS in Agrobacterium: A. tumefaciens carrying pLN18 secreted AvrPto-PhiLOV into culture supernatants, whereas strains lacking pLN18 did not. Split-GFP assays showed successful translocation of AvrPto-GFP11 (and AvrPtoB/AvrB) into plant cells, reconstituting GFP at the plasma membrane.
• Increased transformation in Arabidopsis and N. benthamiana:
  • Arabidopsis transient AMT: EHA105 with pLN18 + AvrPto significantly increased GUS activity compared to controls (qualitative and quantitative assays).
  • Arabidopsis stable AMT: A208 with pLN18 + AvrPto increased percent root segments forming tumors; EHA105 pCAS1 root callus assay also significantly increased PPT-resistant calli; floral dip transformation increased ~2-fold at low inoculum.
  • N. benthamiana: GV2260 transient GUS expression and A348 leaf disk tumor fresh weight significantly increased when T3SS + AvrPto were co-delivered.
• Other effectors: AvrPtoB and HopAO1 delivered via T3SS significantly increased Arabidopsis root tumor formation and N. benthamiana leaf disk tumor weights; negative control effector (HopAI1/HopAll) did not increase transformation.
• Crop species improvements:
  • Wheat (cv. Fielder): AGL1 delivering AvrPto, AvrPtoB, or HopAO1 via T3SS markedly increased the percentage of independent transgenic plants; AvrPto achieved ~400% of control (up to ~63% efficiency vs ~15% conventional in this study). HopAI1 control had no effect.
  • Alfalfa (R2336): Engineered A. tumefaciens delivering AvrPto increased transformation efficiency by ~260%.
  • Switchgrass (NFCX01): AvrPto delivery increased transformation efficiency by ~400%.
• Mechanism: No major increase in Agrobacterium vir gene expression with T3SS + T3E constructs, indicating effects are not due to elevated virulence gene induction. In Arabidopsis roots, PTI marker genes FRK1 and NHL10 were induced at 2 h post-infection across treatments, but were significantly reduced at 16 h when AvrPto was delivered via T3SS, supporting defense suppression.
• Delivery of plant proteins: T3SS-mediated delivery of HTA1 or tHTA1 increased stable transformation in Arabidopsis (root tumors), N. benthamiana (leaf disk tumors), and enhanced transformation efficiency in wheat, alfalfa, and switchgrass.
• Statistical significance: Across assays, improvements were significant by ANOVA with Tukey’s post-hoc tests (p-values reported per figure; e.g., wheat p=0.0003 for T3Es; Arabidopsis defense gene variance tests non-significant).

Engineering A. tumefaciens to express a heterologous Pseudomonas T3SS enables direct delivery of PTI-suppressing effectors or plant proteins during infection, addressing a key bottleneck in AMT—host defense. The observed increases in both transient and stable transformation across model and crop species, especially large gains in wheat, show that suppressing early immune signaling at the PRR level facilitates T-DNA delivery/integration without altering Agrobacterium vir gene expression. The approach generalizes to multiple effectors (AvrPto, AvrPtoB, HopAO1) and to delivery of beneficial plant proteins (HTA1/tHTA1), demonstrating a platform to modulate the host cellular environment non-transgenically during transformation. This has important implications for plant biotechnology, including enhancing transformation of recalcitrant genotypes and potentially enabling DNA-free or protein-only delivery strategies. The T3SS platform could complement or offer an alternative to T4SS-mediated protein translocation for delivering genome editing proteins (e.g., Cas9) or morphogenic factors, potentially reducing off-target effects and bypassing stable transgene integration. Protein size/structure constraints inherent to T3SS must be considered; nonetheless, smaller reporters (PhiLOV) and split-protein approaches mitigate such limitations. Overall, this work provides mechanistic evidence (reduced FRK1/NHL10 expression) linking effector delivery to defense suppression and improved AMT outcomes.

The study demonstrates that equipping Agrobacterium with a functional Pseudomonas T3SS enables delivery of PTI-suppressing T3Es and plant proteins into host cells, significantly enhancing both transient and stable transformation in Arabidopsis, Nicotiana benthamiana, wheat, alfalfa, and switchgrass. In wheat cv. Fielder, transformation efficiency increased up to ~63% with AvrPto, representing ~400% of control. Effector delivery reduced expression of PTI marker genes, supporting a defense suppression mechanism independent of Agrobacterium vir gene upregulation. Furthermore, T3SS-mediated delivery of histone HTA1/tHTA1 improved transformation, indicating the platform’s versatility for delivering plant factors that aid T-DNA integration. Future research should: expand the effector repertoire targeting various immunity and regeneration pathways; optimize T3SS expression and cargo design to overcome size/structure constraints; integrate T3SS-mediated delivery of genome-editing proteins (e.g., Cas9, base/prime editors) for DNA-free editing; and combine with morphogenic factors to improve regeneration in recalcitrant genotypes.

• Regeneration dependency: The approach improves transformation steps but does not overcome limitations in plant regeneration; thus, it may not benefit varieties recalcitrant to regeneration.
• Cargo constraints: T3SS has size/structural limits; large, tightly folded proteins (e.g., full-length GFP fusions) hinder secretion/translocation, necessitating small tags or split-protein systems.
• Species/effector specificity: While multiple species benefited, increases in already highly transformable species were incremental. Not all effectors are effective; a control effector (HopAI1/HopAll) targeting downstream MAPKs did not enhance transformation.
• Scope of testing: Effects were demonstrated in selected genotypes (e.g., wheat cv. Fielder, alfalfa R2336, switchgrass NFCX01); generalizability to broader germplasm and field conditions remains to be validated.