RNA interference is of value in determining gene function in many organisms. Plant parasitic nematodes are refractory to microinjection as a means of introducing RNA and do not show any oral uptake until they are within plants. We have used octopamine to stimulate uptake by preparasitic second stage juveniles of two cyst nematodes, Heterodera glycines and Globodera pallida. This new technique was used to facilitate uptake of double stranded RNA (dsRNA) together with fluoroscein isothiocyanate as a visual marker. Targeting cysteine proteinases did not reduce the number of parasites but caused a shift from the normal female/male ratio of 3:1 to 1:1 by 14 days postinfection (dpi). Exposure of H. glycines to dsRNA corresponding to a newly characterized protein with homology to C-type lectins did not affect sexual fate, but 41% fewer parasites were recovered from the plants. As expected, treatment with dsRNA corresponding to the major sperm protein (MSP) had no effect on either parasite development or sexual fate over 14 days. Northern analysis showed lower transcript abundance for the two targeted mRNAs that occur in J2, plus a later inhibition for MSP transcripts when males developed sperm at 15 dpi. These findings establish a procedure for RNAi of plant parasitic nematodes.

The cysteine proteinase inhibitor, oryzacystatin-I (Oc-I), and several engineered Oc-I variants have been tested for efficacy in inhibiting growth and development of both the free-living nematode, Caenorhabditis elegans, and the plant parasitic nematode Globodera pallida. To assist in the design of protein engineering experiments to improve the efficacy of Oc-I, an alignment of 28 cystatins and a molecular model of Oc-I were generated. Inhibitory activities (Ki) of wild-type and variant forms of Oc-I against both papain and the C. elegans cysteine proteinase, gcp-1, were measured. For one variant, in which residue Asp86 was deleted (Oc-I deltaD86), the Ki was reduced by 13- to 14-fold. LD50 studies to test the effect of Oc-I and Oc-I delta D86 against C. elegans showed the relative median potency of Oc-I delta D86 to be 0.76 that of wild-type Oc-I. When expressed in tomato hairy roots both Oc-I and Oc-I delta D86 had a detrimental effect on growth and development of G. pallida. This effect was significantly greater on Oc-I deltaD86-expressing roots leading to a reduction in size of G. pallida females to a level at which fecundity is profoundly affected.

Plant nematodes are major pests of agriculture. Transgenic plant technology has been developed based on the use of proteinase inhibitors as nematode anti-feedants. The approach offers prospects for novel plant resistance and reduced use of environmentally damaging nematicides. A modified rice cystatin, Oc-I delta D86, expressed as a transgene in Arabidopsis thaliana, has a profound effect on the size and fecundity of females for both Heterodera schachtii (beet-cyst nematode) and Meloidogyne incognita (root-knot nematode). No females of either species achieved the minimum size they require for egg production. Ingestion of Oc-I delta D86 from the plant was correlated with loss of cysteine proteinase activity in the intestine thereby suppressing normal growth, as required of an effective antifeedant plant defence.

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.

Plant defence strategies usually involve the action of several gene products. Transgenic resistance strategies are likely to have enhanced efficacy when they involve more than one transgene. Here we explore possible mechanisms for the co-delivery of multiple effectors via a single transgene. As an example we report the co-delivery of two distinct proteinase inhibitors in Arabidopsis thaliana (L.) Heynh. to examine resistance against plant parasitic nematodes. A cysteine and serine proteinase inhibitor have been joined as translational fusions by one of two peptide linkers. One linker, part of the spacer region of a plant metallothionein-like protein (PsMTa), was selected to be cleaved in planta. A second linker, derived from the fungal enzyme galactose oxidase (GO) was chosen to be refractory to cleavage in planta. Western blot analysis of cell extracts confirmed the expected pattern of predominantly single inhibitors derived from the PsMTa construct and a primarily dual inhibitor from the GO construct. Analysis of cyst and root-knot nematodes recovered from transgenic Arabidopsis expressing inhibitors as single or dual molecules revealed the uptake of inhibitors with the exception of those linked by the PsMTa linker. This unexpected result may be due to residues of the PsMTa linker interacting with cell membranes. Despite lack of ingestion, PsMTa-linked cowpea trypsin inhibitor (CpTI) affected the sexual development of the cyst nematodes, indicating an external site of action. The engineered cystatin (Oc-I delta D86) component from the PsMTa constuct had no effect, indicating that ingestion is necessary for the cystatin to be effective. The delivery of dual inhibitors linked by the GO linker showed a clear additive effect over either inhibitor delivered singly. The application of this technology to other plant pathogens is discussed.