No abstract

VirE2 is a ssDNA binding protein essential for virulence in Agrobacterium tumefaciens. A tetracysteine mutant (VirE2-TC) was prepared for in vitro and in vivo fluorescence imaging based on the ReAsH reagent. VirE2-TC was found to be biochemically active as it binds both ssDNA and the acidic secretion chaperone VirE1. It was also biologically functional in complementing virE2 null strains transforming Arabidopsis thaliana roots and Nicotiana tabacum leaves. In vitro experiments demonstrated a two-color fluorescent complex using VirE2-TC/ReAsH and Alexa Fluor 488 labeled ssDNA. In vivo, fluorescent VirE2-TC/ReAsH was detected in bacteria and in plant cells at time frames relevant to transformation. Importance Cell to cell transfer of proteins and nucleic acids lies at the heart of many biological processes. Detecting such transfer is often problematic or indirect. We show here that the biarsenical fluorescent reagent ReAsH can be used to reveal the presence of the secreted effector VirE2 from Agrobacterium tumefaciens in the cytoplasm of host plant cells. Our studies establish a new method for monitoring translocation of the VirE2 effector from A. tumefaciens to plant target cells during the infection process.

The transfer of single-stranded (ss) T-DNA from soil bacteria of the genus Agrobacterium with the help of the VirE2 protein, which possibly mediates the delivery of ss-T-DNA across the cell membrane, was demonstrated earlier, but how VirE2 participates in ssDNA transfer across artificial and natural membranes is not known. Using computational methods, we reconstructed model structures composed of two and four VirE2 proteins and showed by the MOLE program the formation of pores with channel diameters of 1.2-1.6 and 1.4-4.6 nm in a model structure formed from two and four VirE2 molecules, respectively. Using light scattering, we recorded the size distribution for recombinant VirE2-dependent complexes in aqueous solutions and found that VirE2 in a buffer solution is present as a complex made up of two or more proteins. We revealed single, long-lived jumps in voltage-dependent membrane conductance during coincubation of planar black membranes with the VirE2 protein. On the addition of VirE2 and FAM-labeled oligonucleotides to HeLa cells, the fluorescence intensity for the cells increased by 56% as compared to that for cells incubated only with oligonucleotides.

The literature and experimental analysis of maize embryo and endospermogenesis genes expression before and after pollination will be presented. Data on the CRISPR/Cas9 editing of maize embryo- and endospermogenesis genes are discussed.

Problems of agrobacterial transformation of maize by methods in planta and in vitro are described. Experimental data on optimization of regeneration from transformed embryogenic maize callus of the KM line are presented.