Engineering Resistance to Orobanche aegyptiaca: Evidence of Sarcotoxin IA as an Anti-Parasite Protein and Macromolecule Movement From Host to Parasite

Orobanche species are parasitic weeds that subsist on the roots of many dicotyledonous plants. These parasites form symplastic and apoplastic connections with their hosts and act as strong sinks for the uptake of water, minerals, and photosynthates, often causing severe damage to the hosts. Although the uptake of small molecules such as sugars and herbicides by Orobanche has been documented, movement of macromolecules between host and parasite has not been characterized. The objectives of this research were to 1) determine whether, and by what route, host macromolecules can be translocated to the parasite, and 2) engineer host resistance based on inducible expression of sarcotoxin IA, an anti-microbial peptide from the flesh fly (Sarcophaga peregrina). To address the first objective, transgenic plants expressing GFP localized to either the host cell cytosol (symplast) or secreted to the extra-cellular space (apoplast) were parasitized by O. aegyptiaca. Observations of green fluorescence in O. aegyptiaca tubercles growing on these plants indicate that the 27 kDa GFP molecule was translocated to the parasite via both symplastic and apoplastic routes. This work was supported by studies with xylem- and phloem-specific dyes, which showed that fluorescent dextrans as large as 70 kDa moved into the parasite through xylem connections. The second objective was addressed using tobacco (Nicotiana tabacum L. cv. Xanthi) plants expressing the sarcotoxin IA transgene under control of the parasite-inducible HMG2 promoter. In soil experiments, transgenic tobacco plants had greater height and biomass, and showed up to 90% reduction in O. ramosa parasitism as measured by the fresh weight of parasite tubercles. In a semi-hydroponic growth system, where Orobanche tubercles can be visualized at early stages of growth, O. aegyptiaca parasites growing on plants expressing sarcotoxin IA were smaller and had an increased number of senescent tubercles compared to those growing on non-transformed plants. Considering the relatively small size of sarcotoxin IA (4 kDa), it is likely that this peptide moves from host to the parasite, where it accumulates to phytotoxic concentrations. In addition to increasing our knowledge of host-Orobanche interactions, this research used an antibiotic peptide to engineer partial Orobanche resistance into a highly susceptible crop. This strategy has broad implications for the control of other parasitic weeds.