Molecular analysis of Mi-mediated root-knot nematode resistance in tomato

The overall goals of this dissertation were to analyze the Mi-1.2 gene in the context of its gene family, study the function of the Mi-1.2 gene in nematode resistance at the molecular level, and examine the role of Mi-1.2 expression levels in nematode resistance. Comparing homologues and investigating the evolution of the genes in the Mi-1 locus in tomato showed that this gene family participates in sequences exchanges and is subject to diversifying selection. A novel class of DNA elements named Solanaceous Inverted Repeat elements were identified within the Mi-1 locus as well as other Solanaceous plant species that are frequently located near resistance genes. Domain swap and site-directed mutagenesis constructs of Mi-1.2 with homologue Mi-1.1 were assayed for nematode resistance phenotypes in transgenic hairy roots and transient expression phenotypes in tobacco. Results presented here, in contrast to prior reports, indicated that differences between the N-terminal regions of Mi-1.1 and Mi-1.2 are not critical for function. However, pairing of a specific region from Mi-1.1 with the leucinerich repeat domain of Mi-1.2 induced a hypersensitive response in tobacco and appeared to be lethal in tomato, implicating the corresponding region of the Mi-1.2 protein in regulation of activation. In addition, instability of certain Mi-1.2 constructs in regulation of activation. In addition, instability of certain Mi-1.2 constructs in Agrobacterium was documented, and a silent mutation that knocks out cell death in tobacco but not nematode resistance was identified. Finally, a variety of transgenic tomato fines containing different promoters with Mi-1.2 were screened for nematode resistance and expression of Mi-1.2 using quantitative RT-PCR. Constructs under the control of the CaMV 35S promoter displayed higher expression levels than wild-type and conferred strong nematode resistance that segregated with the transgene. A dosage effect was observed with Mi-1.2 under control of the Mi-1.1 promoter that may correlate Mi-1.2 transcript levels with resistance suggesting that a threshold expression level may be essential for proper function