Molecular and bioinformatic analyses of nematode-derived proteins involved in plant-parasitism

Plant parasitic nematodes comprise several groups; the most economically damaging of these are the sedentary endoparasites. Sedentary endoparasitic nematodes modify host root tissues, using a suite of effector proteins to create and maintain a feeding site that is their sole source of nutrition. Ultrastructural studies of plant-nematode interactions, and in particular those of the feeding sites, have identified two key feeding structures; feeding tubes and feeding plugs. Sedentary plant-parasitic nematodes feed by withdrawing host cell assimilate from the feeding site though a feeding tube. The function, composition and molecular characteristics of both feeding tubes and feeding plugs are poorly characterised. It is hypothesised that the apparent selective uptake of certain proteins from the feeding site is attributed to feeding tube size exclusion. A novel method is proposed to predict protein size based on protein database coordinates in silico. The validity of these predictions was tested using travelling wave ion mobility spectrometry – mass spectrometry, where predictions and measured values were within approximately 6% of each other. In silico predictions coupled with experimental techniques, such as mass spectrometry, analytical ultracentrifugation and protein electrophoresis, aimed to resolve seemingly-conflicting results of previous size exclusion experiments. Together these provided a pragmatic measurement of the upper limit for cyst nematode feeding tube size exclusion. Putative feeding-structure genes were identified from the genome sequence of the potato cyst nematode Globodera pallida using a series of reasoned assumptions about their characteristics. As a result, several large gene families were identified, one of which displayed highly complex genomic variation within a population. Subsequent characterisation of these candidate genes informed their function. The expression of several candidates was demonstrated in tissues with the capacity to secrete proteins into the host, implicating their role in host-pathogen interactions. In addition, for the 444 gene family, the protein was detected in the apoplasm, between the anterior end of the nematode and the feeding site. In planta host induced gene silencing targeting 444s reduced nematode infection by > 50%; further supporting their important role in successful parasitism. Feeding structure candidate genes were identified in de novo transcriptome assemblies of two related species (Globodera rostochiensis and Rotylenchulus reniformis). Differential expression analysis identified those candidates with congruent expression between species. 444 and 448 genes appear to be “core” effectors present in three genera of plant-parasitic nematodes that infect mono- and di-cotyledonous crop species.