Characterisation by RNAi of pioneer genes expressed in the dorsal pharyngeal gland cell of Heterodera glycines and the effects of combinatorial RNAi.

Changes in transcript abundance of 24 genes expressed in the dorsal pharyngeal gland cell of Heterodera glycines encoding for putative secretions of unknown function were monitored by quantitative PCR (qPCR) at 0, 2, 7, 14 and 21 days post-invasion (pi) of soybean plantlets. Five groups of temporal patterns (A, B1, B2, C and D) were defined for the 24 genes plus data for two previously studied genes expressed in the same cell. Group D (two genes) showed no significant increase between 0 and 2 days pi and were the least abundantly expressed at 7-21 days pi. Transcripts of group C (nine genes including one studied previously) increased in abundance from 0 to 2 days pi but were the second least expressed for 7-21 days pi. Groups A (three genes), B1 (seven genes) and B2 (five genes including one studied previously) were all abundant at 7-21 days pi. B1 and B2 were discriminated by their relative abundance at 0 and 2 days pi. RNA interference (RNAi) targeting two genes of group A and one each of B1 and B2 in nematodes prior to invasion resulted in phenotypic effects on total parasites per plant and sexual fate at 10 days pi. Phenotype penetrance was reduced for three genes showing such effects and one with a strong effect in earlier work when two genes rather than one were concurrently targeted for RNAi. One gene (dg13) was more abundantly expressed after combinatorial RNAi than for either control nematodes or when targeting singly by RNAi. This work reports the unexpected elevation in mRNA expression after combinatorial RNAi that requires understanding before combinatorial RNAi can be advanced for highly effective cyst nematode control via plant biotechnology.