Efficient agroinfection of Prunus domestica with infectious clones of Plum pox virus
Adam Achs 1 Zdeno Šubr 1
Agroinfection is a simple and effective method for transfection of plants with viral replicons via Agrobacterium-mediated gene delivery. Agrobacterium tumefaciens is a Gram-negative soil bacterium that naturally transfers a part of its tumor inducing (Ti) plasmid, called T-DNA, into plant cells. As the T-DNA can be engineered by replacing tumor inducing genes, the modified Ti plasmids have become useful vectors for gene delivery .
Agroinfiltration method was developed to deliver agrobacteria into the intracellular space of the leaf tissue, thus allowing access of agrobacteria to most leaf cells. In this method, a suspension of agrobacteria is introduced into a plant leaf by direct injection or by vaccum infiltration. The most amenable host plant for agroinfiltration is Nicotiana benthamiana and other related species of the genus Nicotiana including tobacco . However, technological improvements have allowed the application of agroinfiltration to many different plant species, including lettuce, tomato, Arabidopsis or some woody trees [2, 3].
Agroinfiltration of stone fruits of the genus Prunus has not yet been described. Here we report a successful agroinfection of Prunus domestica var. Toptaste with infectious clones of Plum pox virus (PPV), which is the causal agent of „sharka“ disease. Leaves of 3-week-old P. domestica seedlings were syringe-infiltrated with a suspension of A. tumefaciens EHA105 harboring a full-lenght copy of the PPV genome cloned in a binary vector. Ten of 12 seedlings (≈ 83 %) developed common symptoms of a systemic PPV infection 3 weeks post infiltration. Leaf symptoms included chlorotic spots, mosaics, vein clearing and leaf deformation. Subsequent Western blotting, as well as RT-PCR analyses confirmed the presence of PPV in symptomatic tissues.
Our results suggest an easier way to transfect Prunus species in laboratory conditions. Previous attempts to transfect PPV woody hosts with a modified PPV genome relied mainly on the in vitro shoot graft inoculation . Agroinfection represents a simpler and less time-consuming approach, so it is a promising tool for plant-pathogen interactions studies in Prunus species, as well as for production of foreign proteins in woody plants by virus-mediated expression.
 Wroblewski T., Tomczak A., Michelmore R. (2005) Plant Biotechnol. J., 3(2), p. 259-273
 Takata N., Eriksson M. E. (2012) Plant Methods, 8(1), p. 30
 Lansac M., Eyquard J. P., Salvador B., et al. (2005) J. Virol. Methods, 129, p. 125-133