Bioengineering Tomato (Solanum Lycopersicum Mill) With Peptide Aptamer For Meloidogyne Incognita Chorismate Mutase As A Strategy For Nematode Resistance

ABSTRACT

Root knot nematodes (RKNs) of (Meloidogyne spp.) are among the most devastating soil-borne plant parasitic pests infecting many food crops. They have been shown to result in over 50% loss of crop yield in tomato (Lycopersicon esculentum Mill). Various control measures including agronomic practices and nematicides have been employed in the past to manage the parasite with only modest gains. These are also costly to small-scale farmers especially in developing countries. There is need therefore for a feasible and effective biotech-based solution for controlling the nematodes in tomato. Infective, second stage juveniles (J2) of these species induce elaborate modifications in selected root cells to form enlarged, multinucleate feeding cells that serve as the sole food source for growth and reproduction of the subsequent sedentary parasitic stages. The single dorsal and two subventral esophageal gland cells of RKNs have evolved into enlarged secretory cells that produce secretions released from the stylet and these play a central role in plant parasitism. These secretions contains protein products of parasitism genes that plays a direct role in host’s basal defense suppression, cell wall degradation and feeding site induction and maintenance. M. incognita chorismate mutase 1, coded by the gene Mi-cm-1 is a key effector in successful parasitism playing integral roles in suppression of the host basal defense response, establishment and maintenance of nematode feeding sites. Reverse genetics via peptide interference to inhibit functionality of this protein therefore offers a feasible control strategy. The target chorismate mutase protein MI-CM-1 was aligned along 5 CM proteins from 3 phytonematode genus and MI-CM-2 for class identity and function comparison. Due to the genotype-dependent nature of tomato genetic transformation and regeneration, specific in vitro conditions were determined. First, cotyledons were chosen as explants of choice over hypocotyledons and primary leaves with a shoot induction frequency of 43.46%. Secondly, a cytokinin/auxin ratio of zeatin 2mg/l and IAA 0.1 mg/l combination was chosen for initiation of regeneration with a shoot induction frequency of 51.60%. Finally, kanamycin concentration of 150 mg/l was chosen as ideal selection with explant survival rate of 32.78%. A peptide aptamer against M. incognita chorismate mutase (PA-Mi-CM-1) gene was transformed into tomato cv. vf36 using Agrobacterium tumefaciens strain EHA105 carrying the gene construct pART27::PA-Mi-CM-1. The regeneration of transformed tomato cells followed the prior optimized parameters. The developed putative T0 transgenic plants were analyzed through PCR. Further molecular analysis and nematode stress assays on the transgenic tomato developed are necessary for validation of conferred resistance. Development of tomato varieties with in-build resistance to RKNs would help mitigate the effect of these parasites and overcome the challenges faced by the conventional control methods in a concerted effort to boost global tomato production.