Characterising putative parasitism genes for root lesion nematodes and their use in RNA interference studies

Tan, Jo-Anne Chiew Hwa (2015) Characterising putative parasitism genes for root lesion nematodes and their use in RNA interference studies. PhD thesis, Murdoch University.

Abstract

Root lesion nematodes (RLNs, Pratylenchus spp.) are economically important migratory endoparasitic pests of crop plants. The overall aim of the work in this thesis was to undertake molecular studies on Pratylenchus thornei to characterise genes potentially involved in plant parasitism, and to determine if they are amenable to gene silencing (RNA interference). The recent availability of the transcriptomes of three RLN species, Pratylenchus coffeae, P. thornei and P. zeae significantly expanded the resources available for this study. Amongst the transcriptome data, putative parasitism genes (PPGs) were identified. A common assembly platform was also used to analyse transcriptome data to determine whether differences between PPGs identified here and those reported previously were consistent when the same assembly was used. Bioinformatic analysis was also undertaken to compare PPGs between different Pratylenchus spp. The results showed that there were some differences in the PPGs identified in P. coffeae, P. thornei and P. zeae. From the transcriptome of P. thornei, genes encoding potential protein or peptide effectors were identified and three aspects of plant parasitism were studied – cell wall modifying proteins (CWMPs) needed to overcome the physical barrier of cell walls in host plant roots, proteins required for detoxification of reactive oxygen species produced as a host defence response, and those thought to have other roles in parasitism. There was no information available on whether RLNs would take up dsRNA by ‘soaking’, and so conditions for optimisation of dsRNA uptake were established using P. thornei and P. zeae. Using these conditions, down-regulation of target genes in P. thornei by RNAi was studied. Mixed stage populations of nematodes of P. thornei and P. zeae were induced to ingest dsRNA when incubated in a basic soaking solution (M9 buffer, 0.05 % gelatine, 3 mM spermidine) in the presence of the 10-50 mM neurostimulant octopamine, 0.1-1.0 mg/mL FITC, and 0.5-6 mM spermidine for 16h. Under these conditions, nematodes vitality was not affected. Knockdown of pat-10 and unc-87 genes of P. thornei and P. zeae resulted in aberrant movement in both species, more so in P. thornei. A greater reduction of transcript expression of both genes in P. thornei also indicates that it may be more susceptible to RNAi than P. zeae. P. thornei treated with dsRNA of pat-10 and unc-87 have reduced replication in mini carrot discs, showing an 81 % and 77 % decrease in numbers of nematodes harvested after 5 weeks culture following RNAi. Hairy roots generated with constructs containing Ptpat-10 also resulted in significant decrease in P. thornei reproduction. Additionally, dsRNA constructs of either nematode species elicited RNAi effects in both species indicating a possible cross-species control of nematodes via RNAi. After soaking mixed stages of P. thornei in dsRNA for 16 hr and transfer to carrot mini-discs for 5 weeks, down-regulation of genes encoding CWMPs significantly decreased nematode reproduction by between 86-95%, respectively, showing that secretion of cellulase, pectate lyase and polygalacturonase are required for P. thornei to infect host plants effectively. Similarly, reproduction of P. thornei was also reduced when RNAi was initiated against cathepsins, fatty acid and retinol binding protein, peroxiredoxin, transthyretin-like protein and venom allergen-like protein (46-88%, respectively). However, RNAi did not reduce replication of P. thornei when RNAi was directed against galectin and thioredoxin. These results show that RLNs are amenable to RNAi, and indicate that the efficiency of RNAi-based gene silencing can differ depending on the target gene chosen, whether it is a member of a multi-gene family, the specific sequence chosen and also details of experimental treatments. This research described here also contributes to describing genes needed for entry into and migration in host roots and evading host defences. However, genes encoding effectors required by sedentary endoparasitic nematodes for feeding site formation were not found in RLNs.

Item Type:	Thesis (PhD)
Murdoch Affiliation(s):	School of Veterinary and Life Sciences
Supervisor(s):	Jones, Michael and Fosu-Nyarko, John
URI:	http://researchrepository.murdoch.edu.au/id/eprint/29163

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