Plant Resistance to Root-Knot Nematodes through RNA Interference Targeting a Parasitism Gene.

다국어 초록 (Multilingual Abstract)

Root-knot nematodes (RKN), members of the genus Meloidogyne, are sedentary plant-parasitic nematodes that infect multiple crop species and cause staggering economic losses worldwide. Proteins produced in specialized esophageal gland cells are secreted from the stylet of RKN to transform recipient host plant root cells into multinucleate giant-cells that are essential for nematode feeding. The peptide encoded by the Meloidogyne incognita 16D10 parasitism gene has previously been shown to be involved in giant-cell formation, and host-derived RNA interference (RNAi) against the 16D10 transcript in Arabidopsis thaliana resulted in resistance to the four major species of RKN (Huang et al., 2006b). The first objective of this dissertation was to transform two host crop species, tobacco and strawberry, with 16D10RNAi constructs and to test for resistance of the resulting transgenic plants to RKN species. Haploid plants of the Nicotiana tabacum cultivars TN90 (burley) and Hicks (flue-cured) were transformed with the 16D10RNAi constructs used by Huang et al. (2006b), and doubled haploid plants were produced. Expression of siRNA by the transgenic tobacco was confirmed by RT-PCR and high-throughput siRNA Ion Proton sequencing. Several lines of the 16D10RNAi transgenic tobacco were found to be significantly more resistant to root-knot nematodes than wild-type untransformed controls. A maximum of 62% reduction in Meloidogyne arenaria egg production and a maximum of 52% reduction in M. incognita egg production were observed in TN90 16D10RNAi tobacco roots, and a maximum of 73% reduction in egg production of M. arenaria was observed in roots of Hicks 16D10RNAi tobacco plants.
Transformation of strawberry with the 16D10-RNAi constructs was also attempted, however explant browning in tissue culture prevented the production of regenerated transgenic strawberry plants. The research described here addresses some of the challenges in strawberry plant tissue culture, and suggests methods for overcoming these issues.
The second objective of this research was to design and create new 16D10 siRNA expression vectors in an attempt to improve siRNA expression. Currently, all work with 16D10RNAi has been performed using the pHANNIBAL vector (Wesley et al., 2001), which utilizes the CaMV 35S promoter and the PDK intron as a spacer. The 16D10RNAi-expressing tobacco using the pHANNIBAL vector was found to be less resistant to root-knot nematodes than the 16D10RNAi Arabidopsis created by Huang et al. (2006b). The purpose of this research was to determine if altering the 16D10RNAi expression constructs could produce greater siRNA expression and potentially greater resistance to root-knot nematodes. The 35S promoter was exchanged for two new promoters, Gmubi and Ntcel7, and the GUS spacer was also substituted for the PDK intron to improve hairpin double-stranded RNA intron splicing. Gmubi is a constitutive promoter like 35S, but has been shown to result in greater expression than 35S in soybean (Chiera et al. 2007). The Ntcel7 promoter is a tissue-specific promoter that is only expressed in shoot and root meristematic tissue and weakly expressed in the vasculature, but has been shown to be up-regulated in giant-cells (Wang et al., 2007). All vectors were successfully assembled and electroporated into Agrobacterium tumefaciens for plant transformations. To test the efficacy of the new constructs, they were all transformed into Arabidopsis thaliana using the floral dip method. Self-pollination and selection of Arabidopsis to obtain homozygous lines is currently underway and the resulting lines will be tested for siRNA expression and root-knot nematode resistance.