Analysis of two host genes required for induction of root-knot nematode feeding sites [an abstract of entire text]

Root-knot nematodes are plant parasites that cause widespread loss in agriculture worldwide. Host infestation by root-knot nematodes is comprised of three stages. During the first invasion stage, nematode juveniles penetrate the root and migrate to target cells in the root tip. This is followed by the induction stage, when the nematode induces host cells to adopt a new enlarged “giant cell” morphology specialized for nutrient transfer. The nematode then feeds permanently from these giant cells for its remaining life-cycle. The induction stage is the key step that determines parasitic success and thus the development of effective control strategies requires an in-depth understanding of this process. However, the molecular mechanism(s) by which rootknot nematodes induce feeding sites remains unknown. In this study, two host genes were analysed to provide insight on the molecular mechanisms for feeding site induction. The first gene, FIE, was selected in a hypothesis-based approach for genes that may drive the global reprogramming process, whereas the second gene, ASTRAY, was selected based on infestation phenotype observed in mutant plants. 1. Functional study of the FIE Polycomb subunit during feeding site formation In plants, the Polycomb Repressive Complex 2 (PRC2) regulates global gene expression during developmental transitions. This study therefore tested the hypothesis that the plant PRC2 complex is used by root-knot nematodes to direct the reprogramming of giant cell fate during the induction stage. The role of PRC2 was tested by perturbing expression of the complex core subunit gene, FIE. An inducible RNAi vector system was used for temporal knockdown of FIE expression during root-knot nematode invasion and induction stage. Inducer treatments were established for use with nematode infestation assays and the inducer vector was confirmed to be effective at depleting FIE levels in a temporal manner. A transgenic hairy root system was successfully implemented for testing and optimisation, although poor nematode infestation was observed. Nevertheless, all knockdown lines obtained showed a phenotype with at least 50% reduction in number of enlarged nematodes, indicative of a defect in the induction stage following inducer treatment. This was consistent with a role for FIE in feeding site development and stable independent transgenic plant lines have now been successfully generated that will facilitate future in-depth studies. 2. A robust system for quantitative analysis of root-knot nematode infestation One limiting factor in understanding the molecular mechanisms of feeding site development has been the lack of a standardised quantitative assay, which should also enable downstream analyses and be free of any other biological sources of variability. As part of this study, a new robust and highly reproducible assay method has now been developed that solves these problems. This includes a new approach for preparation of high quality axenic nematode populations and optimised infestation conditions for the selected host plant Lotus japonicus, a model legume. In addition, an improved compatible hairy root transformation protocol was established facilitating mechanistic studies. The strategies developed here are also readily applied to different plant hosts.