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Agrobacterium tumefaciens VirD2 protein is one of the key elements of Agrobacterium-mediated plant transformation, a process of transfer of T-DNA sequence from the Agrobacterium tumour inducing plasmid into the nucleus of infected plant cells and its integration into the host genome. The VirD2 protein has been shown to be a substrate for a plant caspase-like protease activity (PCLP) in tobacco. We demonstrate here that mutagenesis of the VirD2 protein to prevent cleavage by PCLP increases the efficiency of reporter gene transfer and expression. These results indicate that PCLP cleavage of the Agrobacterium VirD2 protein acts to limit the effectiveness of T-DNA transfer and is a novel resistance mechanism that plants utilise to combat Agrobacterium infection.

The transfer of T-DNA from Agrobacterium to plant cells is mediated by a system which involves the virB operon of the Ti plasmid. We report that VirE2 and VirD2, two T-DNA-associated proteins, as well as VirF, a protein known to be secreted into plant cells, are present in the periplasm and supernatant fractions of growing cells of Agrobacterium as are VirJ and ChvE, two known periplasmic proteins. Two cytoplasmic proteins, Ros and chloramphenicol acetyl transferase, and a VirE2 :: green fluorescent protein construct were not detected in the above fraction. Export of VirE2 into the culture supernatant did not require any Ti plasmid genes, except for VirE1, a specific chaperone for VirE2. The levels of the VirE2 and VirD2 proteins in the supernatant increased significantly when cells were grown at 19 degrees C as compared with 28 degrees C. When Agrobacterium expressed the oncogenic suppressive activity protein (Osa), VirE2 and VirF proteins could not be detected in the supernatant or the periplasm and the level of VirD2 was greatly reduced. However, oncogenic suppressive activity protein did not block the accumulation of VirJ and ChvE in the periplasm. Our data suggest that VirD2, VirE2, and VirF are transported across the cytoplasmic membrane by a specific pathway, independent of virB. Thus, transfer of the T-complex of Agrobacterium may take place in two steps, the first mediated by an unidentified pathway and the second by the virB system.

In April 2019, hydroponically cultivated cucumber plants with characteristic symptoms of crazy root disease were found in two different commercial production cucumber greenhouses in Poland. Due to excessive and inappropriate root growth, this disease led to a reduction in yield and deterioration of the general conditions of infected plants. Bacteria isolated from the roots were subjected to a morphological evaluation, as well as molecular, biochemical and pathogenicity tests. To identify the bacteria causing the disease, Agrobacterium-like colonies were subjected to PCR with primers complementary to the pathogenicity-related genes located on the crazy root-inducing plasmid (Ri-plasmid): the virD2A + virD2E primers complementary to the virD2 gene and the rolBF + rolBR primers complementary to the rolB gene. The pathogenicity of the isolated strains was studied in sunflowers and cucumbers. Twelve strains positive for the Ri plasmid, as determined by PCR, and pathogenic to sunflowers were identified based on sequence analysis of the 16S rRNA and recA genes. One strain was classified as belonging to the genus Pararhizobium, three to Rhizobium, and eight to Agrobacterium biovar 1, with the highest similarity to genomospecies G3. The results of the analyses suggest that these strains may belong to a new, thus far, undescribed species. To confirm this hypothesis, further phylogenetic studies are required.

Agrobacterium-mediated transformation is a complex process that is widely utilized for generating transgenic plants. However, one of the major concerns of this process is the frequent presence of undesirable T-DNA vector backbone sequences in the transgenic plants. To mitigate this deficiency, a ternary strain of A. tumefaciens was modified to increase the precision of T-DNA border nicking such that the backbone transfer is minimized. This particular strain supplemented the native succinamopine VirD1/VirD2 of EHA105 with VirD1/VirD2 derived from an octopine source (pTi15955), the same source as the binary T-DNA borders tested here, residing on a ternary helper plasmid containing an extra copy of the succinamopine VirB/C/G operons and VirD1. Transformation of maize immature embryos was carried out with two different test constructs, pDAB101556 and pDAB111437, bearing the reporter YFP gene and insecticidal toxin Cry1Fa gene, respectively, contained in the VirD-supplemented and regular control ternary strains. Molecular analyses of ~ 700 transgenic events revealed a significant 2.6-fold decrease in events containing vector backbone sequences, from 35.7% with the control to 13.9% with the VirD-supplemented strain for pDAB101556 and from 24.9% with the control to 9.3% with the VirD-supplemented strain for pDAB111437, without compromising transformation efficiency. In addition, while the number of single copy events recovered was similar, there was a 24–26% increase in backbone-free events with the VirD-supplemented strain compared to the control strain. Thus, supplementing existing VirD1/VirD2 genes in Agrobacterium, to recognize diverse T-DNA borders, proved to be a useful tool to increase the number of high quality events in maize.

We have developed a simple genetic assay to detect active nuclear localization (NLS) and export signals (NES) on the basis of their function within yeast cells. The bacterial LexA protein was modified (mLexA) to abolish its intrinsic NLS and fused to the activation domain of the yeast Gal4p (Gal4AD) with or without the SV40 large T-antigen NLS. In the import assay, if a tested protein fused to mLexA-Gal4AD contains a functional NLS, it will enter the cell nucleus and activate the reporter gene expression. In the export assay, if a tested protein fused to mLexA-SV40 NLS-Gal4AD contains a functional NES, it will exit into the cytoplasm, decreasing the reporter gene expression. We tested this system with known NLS and NES and then used it to demonstrate a NES activity of the capsid protein of a plant geminivirus. This approach may help to identify, analyze, and select for proteins containing functional NLS and NES.

Agrobacterium tumefaciens‐ mediated plant transformation, a unique example of interkingdom gene transfer, has been widely adopted for the generation of transgenic plants. In vitro synthesized transferred DNA (T‐DNA) complexes comprising single‐stranded DNA and Agrobacterium virulence proteins VirD2 and VirE2, essential for plant transformation, were used to stably transfect HeLa cells. Both proteins positively influenced efficiency and precision of transgene integration by increasing overall transformation rates and by promoting full‐length single‐copy integration events. These findings demonstrate that the virulence proteins are sufficient for the integration of a T‐DNA into a eukaryotic genome in the absence of other bacterial or plant factors. Synthetic T‐DNA complexes are therefore unique protein:DNA delivery vectors with potential applications in the field of mammalian transgenesis.

We describe a plant protoplast transformation method that provides transformants with a simple pattern of integration of a foreign gene. The approach is to deliver into plant protoplasts by direct gene transfer the Agrobacterium virulence genes virD1 and virD2 with or without virE2, together with a target plasmid containing a gene of interest flanked by Agrobacterium T-DNA border repeat sequences of 25 bp. We present evidence of T-DNA formation in maize protoplasts and its integration into the maize genome. The frequency of VirD1-VirD2-mediated integration events was about 20-35% of the total number of transformants. The addition of virE2 doubled the transformation efficiency. The method described here is of sufficient efficiency and simplicity to be useful for the production of transgenic plants with single-copy well-defined transgenic inserts