Production and analysis of composite tomato plants Solanum lycopersicum L. carrying pea genes encoding the receptors to rhizobial signal molecules

The development of legume-rhizobial symbiosis is based on signal exchange between partners, which ensures their mutual recognition and activation of the infection process and the program of nodule organogenesis. In this regard, it is of great interest to study the possibility of acquisition by non-legume plants of the ability to perceive lipochito-oligosaccharide signal molecules of rhizobia, the Nod factors, and subsequent activation of signal transduction pathway. To study this possibility in our work, we carried out the transfer of the genes encoding receptors to Nod factors of legume plant pea Pisum sativum L. into tomato Solanum lycopersicum L. (Carmello cultivar) using the transformation with Agrobacterium rhizogenes . In pea, two receptor kinases, SYM10 and K1, were previously identified, which are necessary for the recognition of Nod factors during the initiation of symbiosis with rhizobia. Upon reception of Nod factors, a complex is formed between these two receptor kinases, which leads to signal transduction. In the present work, we carried out the transfer of two genes encoding LysM-RLK SYM10 and K1 in pea P. sativum into tomato plants S. lycopersicum using agrobacterial transformation. In composite plants transformed with PsSym10 or PsK1 genes, the possibility of expression activation of introduced receptor genes in response to inoculation with a typical rhizobial strain Rhizobium leguminosarum bv. viciae CIAM1026 was shown. It was also shown that, under the influence of receptors in genetically transformed roots of composite plants, the expression of genes is increased, which can be regulated by components of the “common” signal pathway. The aim of this work was to study the possibility of acquiring the ability of S. lycopersicum plants to recognize signal molecules of rhizobia after transfer of the genes encoding receptors for Nod factors in the legume plant P. sativum . Two types of constructs in the pKm43GW vector were obtained and used, in which the PsSym10 or PsK1 genes encoding receptors were cloned under the pSlEXT1 promotor of tomato extensin gene -pSlEXT1:: PsSym10 - 3xFLAG ::T35S and pSlEXT1:: PsK1-RFP ::T35S. Young tomato seedlings of S. lycopersicum cv. Carmello were transformed with the Agrobacterium rhizogenes Arqua 1 strain. The transformed seedlings were placed on Murashige-Skoog (MS) agar medium without sucrose in Petri dishes and cultured in an upright position in a phytotron until callus is appeared. After that, the plants were transferred to MS medium with 3 % sucrose containing 0.3 mg/ml of the antibiotic cefotaxime and incubated until transgenic roots are appeared. Composite plants were transferred into vermiculite poured with 0.5½ Fahreus medium and incubated under high humidity conditions for 2-3 days. The plants were then inoculated with R. leguminosarum bv. viciae CIAM1026 containing the uidA glucuronidase gene ( GUS ). For the analysis we used transformed roots of composite tomato plants without rhizobial inoculation (control, 7 days), as well as transformed roots at 7 and 21 days after inoculation. The analysis of gene expression was performed by quantitative PCR combined with reverse transcription (RT-PCR). In genetically transformed roots of tomato plants the expression of both PsSym10 and PsK1 genes was observed under the pSlEXT1 promoter, moreover the expression was enhanced under the influence of rhizobial inoculation. A significant (approximately 2.0-2.5-fold) increase in the expression of the PsSym10 gene was shown in response to inoculation with rhizobia both at 7 and 21 days. The level of PsK1 expression was found to be the highest 7 days after inoculation in the transformed roots of composite tomato plants as compared to the control. To determine whether the components of the “common” signal pathway will be activated under the influence of transferred receptors in composite tomato plants, the changes in the expression of S. lycopersicum SlD27 , SlNSP2 , SlRAM1 , and SlMAPK6 ge