Stable recombinase-mediated cassette exchange in Arabidopsis using Agrobacterium tumefaciens.

Site-specific integration is an attractive method for the improvement of current transformation technologies aimed at the production of stable transgenic plants. Here, we present a Cre-based targeting strategy in Arabidopsis (Arabidopsis thaliana) using recombinase-mediated cassette exchange (RMCE) of transferred DNA (T-DNA) delivered by Agrobacterium tumefaciens. The rationale for effective RMCE is the precise exchange of a genomic and a replacement cassette both flanked by two heterospecific lox sites that are incompatible with each other to prevent unwanted cassette deletion. We designed a strategy in which the coding region of a loxP/lox5171-flanked bialaphos resistance (bar) gene is exchanged for a loxP/lox5171-flanked T-DNA replacement cassette containing the neomycin phosphotransferase (nptII) coding region via loxP/loxP and lox5171/lox5171 directed recombination. The bar gene is driven by the strong 35S promoter, which is located outside the target cassette. This placement ensures preferential selection of RMCE events and not random integration events by expression of nptII from this same promoter. Using root transformation, during which Cre was provided on a cotransformed T-DNA, 50 kanamycin-resistant calli were selected. Forty-four percent contained a correctly exchanged cassette based on PCR analysis, indicating the stringency of the selection system. This was confirmed for the offspring of five analyzed events by Southern-blot analysis. In four of the five analyzed RMCE events, there were no additional T-DNA insertions or they easily segregated, resulting in high-efficiency single-copy RMCE events. Our approach enables simple and efficient selection of targeting events using the advantages of Agrobacterium-mediated transformation.

Positive charge is an important feature of the C-terminal transport signal of the VirB/D4-translocated proteins of Agrobacterium.

Several human pathogens and the plant pathogen Agrobacterium tumefaciens use a type IV secretion system for translocation of effector proteins into host cells. How effector proteins are selected for transport is unknown, but a C-terminal transport signal is present in the proteins translocated by the A. tumefaciens VirB/D4 type IV secretion system. We characterized this signal in the virulence protein VirF by alanine scanning and further site-directed mutagenesis. The Cre recombinase was used as a reporter to measure the translocation efficiency of Cre-Vir fusions from A. tumefaciens to Arabidopsis. The data unambiguously showed that positive charge is an essential characteristic of the C-terminal transport signal. We increased the sensitivity of this translocation assay by modifying the Cre-induced readout in host cells from kanamycin resistance to GFP expression. This improvement allowed us to detect translocation of the VirD2 relaxase protein in the absence of transferred DNA, showing that attachment to the transferred DNA is not essential for transport by the VirB/D4 system. We also found another translocated effector protein, namely the VirD5 protein encoded by the tumor-inducing plasmid. According to secondary structure predictions, the C termini of all VirB/D4-translocated proteins identified so far are unstructured; however, they contain a characteristic hydropathic profile. Based on sequence alignments and mutational analysis of VirF, we conclude that the C-terminal transport signal for recruitment and translocation of effector proteins by the A. tumefaciens VirB/D4 system is hydrophilic and has a net positive charge with a consensus motif of R-X(7)-R-X-R-X-R-X-X(n)>.

Recognition of the Agrobacterium tumefaciens VirE2 translocation signal by the VirB/D4 transport system does not require VirE1.

Agrobacterium tumefaciens uses a type IV secretion system to deliver a nucleoprotein complex and effector proteins directly into plant cells. The single-stranded DNA-binding protein VirE2, the F-box protein VirF and VirE3 are delivered into host cells via this VirB/D4 encoded translocation system. VirE1 functions as a chaperone of VirE2 by regulating its efficient translation and preventing VirE2-VirE2 aggregation in the bacterial cell. We analyzed whether the VirE1 chaperone is also essential for transport recognition of VirE2 by the VirB/D4 encoded type IV secretion system. In addition, we assayed whether translocation of VirF and VirE3, which also forms part of the virE operon, is affected by the absence of VirE1. We employed the earlier developed CRAFT (Cre recombinase Reporter Assay For Translocation) assay to detect transfer of Cre::Vir fusion proteins from A. tumefaciens into plants, monitored by stable reconstitution of a kanamycin resistance marker, and into yeast, screened by loss of the URA3 gene. We show that the C-terminal 50 amino acids of VirE2 and VirE3 are sufficient to mediate Cre translocation into host cells, confirming earlier indications of a C-terminal transport signal. This transfer was independent of the presence or absence of VirE1. Besides, the translocation efficiency of VirF is not altered in a virE1 mutant. The results unambiguously show that the VirE1 chaperone is not essential for the recognition of the VirE2 transport signal by the transport system and the subsequent translocation across the bacterial envelope into host cells.