Activation of Nicotiana benthamiana Domin against Pectobacterium atrosepticum phytopathogen by recombinant and synthetic peptides derived from the potato apyrase B-domain
摘要
A conserved B-domain within Pectobacterium atrosepticum parasite NTPDases, also identified in its potato (Solanum tuberosum) apyrase counterpart, is a target for the mammalian B-cell immune response. Here, the r-6His-DomB, a recombinant B-domain expressed in Escherichia coli, and synthesized peptides potB1LJ and potB2LJ, which span the B-domain of potato apyrase, were used to assess the Nicotiana benthamiana Domin protective capability against the phytopathogen P. atrosepticum. The evaluation of the plant susceptibility to the infection through disease progress marked an intense chlorosis, signs of senescence, wilting, and leaf rot, culminating in death after 20 d. Pre-treatment of plant leaves with those biomolecules, followed by a challenge with P. atrosepticum, prevented the spread of bacteria and cell death in the surrounding infiltrated areas. Therefore, as shown here for the first time, the isolated potato apyrase B-domain protected the plant organism against a pathogen infection. Furthermore, a chimeric gene containing B-domain, idealized and evaluated by in silico analysis for both transient and stable expression in Nicotiana benthamiana Domin, was considered to counteract the disease. Leaves agroinfiltrated exhibited resistance when challenged with P. atrosepticum. Vascular flow reduction, as well as both H2O2 production and callose deposition, was observed in the leaves. In addition, cell death was not detected by Trypan blue staining. Finally, the genetically transformed Nicotiana plants were challenged with P. atrosepticum. Integrated into the plant genome, the gene induced resistance to bacteria, preventing their proliferation and dispersion, as observed for at least 20 d, showing its ability to protect the plant against disease progression.