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.

QPCR analysis and RNAi define pharyngeal gland cell-expressed genes of Heterodera glycines required for initial interactions with the host.

Changes in transcript abundance of genes expressed in the three pharyngeal gland cells of Heterodera glycines after host invasion were monitored by quantitative polymerase chain reaction (qPCR) and the consequences of disrupting their expression studied by RNAi treatment prior to invasion. Two transcripts were known to be expressed in the two subventral gland cells (hg-pel and hg-eng-1), a further two in the single dorsal gland cell only (hg-gp and hg-syv46), and a fifth transcript (hg-cm) was expressed by both gland cell types. The qPCR study established that transcripts of hg-syv46 and hg-gp increased in abundance by 2 days postinfection (dpi), with the former remaining the most abundant. The hg-cm transcript level showed minor changes from 0 to 14 dpi but did fall by 21 dpi. In contrast, hg-eng-1 and hg-eng-2 messenger (m)RNA declined by 7 dpi and hg-pel by 14 dpi before it increased at 21 dpi. RNAi-targeting of hg-eng-1 reduced the number of females present on the plants at 10 days. Targeting of hg-gp, hg-cm, and hg-pel caused a change in sexual fate favoring male development on roots. Both effects were evident after targeting hg-syv46. Suppression of hg-eng-1 mRNA levels in second-stage juveniles (J2i) by RNAi was transient, with a recovery by 15 days of incubation in water after treatment. Presoaking H. glycines J2 with double-stranded RNA has value for studying gene function during the nematode's early interaction with a plant.