Catherine J. Lilley

Ingestion of Double-Stranded RNA by Preparasitic Juvenile Cyst Nematodes Leads to RNA Interference

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.

Identification of Genes Involved in the Response of Arabidopsis to Simultaneous Biotic and Abiotic Stresses

Arabidopsis responds to simultaneous water stress and nematode infection by activating a unique program of gene expression that is distinct from the response to individual stresses. In field conditions, plants may experience numerous environmental stresses at any one time. Research suggests that the plant response to multiple stresses is different from that for individual stresses, producing nonadditive effects. In particular, the molecular signaling pathways controlling biotic and abiotic stress responses may interact and antagonize one another. The transcriptome response of Arabidopsis (Arabidopsis thaliana) to concurrent water deficit (abiotic stress) and infection with the plant-parasitic nematode Heterodera schachtii (biotic stress) was analyzed by microarray. A unique program of gene expression was activated in response to a combination of water deficit and nematode stress, with 50 specifically multiple-stress-regulated genes. Candidate genes with potential roles in controlling the response to multiple stresses were selected and functionally characterized. RAPID ALKALINIZATION FACTOR-LIKE8 (AtRALFL8) was induced in roots by joint stresses but conferred susceptibility to drought stress and nematode infection when overexpressed. Constitutively expressing plants had stunted root systems and extended root hairs. Plants may produce signal peptides such as AtRALFL8 to induce cell wall remodeling in response to multiple stresses. The methionine homeostasis gene METHIONINE GAMMA LYASE (AtMGL) was up-regulated by dual stress in leaves, conferring resistance to nematodes when overexpressed. It may regulate methionine metabolism under conditions of multiple stresses. AZELAIC ACID INDUCED1 (AZI1), involved in defense priming in systemic plant immunity, was down-regulated in leaves by joint stress and conferred drought susceptibility when overexpressed, potentially as part of abscisic acid-induced repression of pathogen response genes. The results highlight the complex nature of multiple stress responses and confirm the importance of studying plant stress factors in combination.

The genome and life-stage specific transcriptomes of Globodera pallida elucidate key aspects of plant parasitism by a cyst nematode

BackgroundGlobodera pallida is a devastating pathogen of potato crops, making it one of the most economically important plant parasitic nematodes. It is also an important model for the biology of cyst nematodes. Cyst nematodes and root-knot nematodes are the two most important plant parasitic nematode groups and together represent a global threat to food security.ResultsWe present the complete genome sequence of G. pallida, together with transcriptomic data from most of the nematode life cycle, particularly focusing on the life cycle stages involved in root invasion and establishment of the biotrophic feeding site. Despite the relatively close phylogenetic relationship with root-knot nematodes, we describe a very different gene family content between the two groups and in particular extensive differences in the repertoire of effectors, including an enormous expansion of the SPRY domain protein family in G. pallida, which includes the SPRYSEC family of effectors. This highlights the distinct biology of cyst nematodes compared to the root-knot nematodes that were, until now, the only sedentary plant parasitic nematodes for which genome information was available. We also present in-depth descriptions of the repertoires of other genes likely to be important in understanding the unique biology of cyst nematodes and of potential drug targets and other targets for their control.ConclusionsThe data and analyses we present will be central in exploiting post-genomic approaches in the development of much-needed novel strategies for the control of G. pallida and related pathogens.

Continual Green-Fluorescent Protein Monitoring of Cauliflower Mosaic Virus 35S Promoter Activity in Nematode-Induced Feeding Cells in Arabidopsis thaliana

The responsiveness of the cauliflower mosaic virus 35S promoter in feeding sites developed by both sexes of Heterodera schachtii and female Meloidogyne incognita has been studied. The objective was to establish the value of green-fluorescent protein (GFP) as a nondestructive reporter gene system for characterizing promoter activity at nematode feeding sites in vivo. Growth units were devised that allowed individual feeding sites in roots of Arabidopsis thaliana to be observed by both bright-field and epifluorescent illumination. Changes in GFP expression were visually observed under experimental conditions that resulted in chloroplast formation in syncytia but not other root cells. Changes in GFP levels altered the extent of quenching, by this protein, of red light emitted by chlorophyll within the chloroplasts under violet excitation. Image analysis provided a semiquantitative basis for simultaneous measurement of changes in GFP fluorescence and the unquenched emission by chlorophyll. GFP levels were constant in cells surrounding the syncytium induced by H. schachtii, but they fell progressive from 10 to 35 days postinfection within this structure. Significant reduction in GFP levels was not limited to the early part of the time course but also occurred between 27 and 35 days postinfection. GFP was detected by immunoblotting in females of M. incognita but not in H. schachtii parasitizing similar GFP-expressing roots.

RNA interference in plant parasitic nematodes: a summary of the current status.

SUMMARYRNA interference (RNAi) has emerged as an invaluable gene-silencing tool for functional analysis in a wide variety of organisms, particularly the free-living model nematode Caenorhabditis elegans. An increasing number of studies have now described its application to plant parasitic nematodes. Genes expressed in a range of cell types are silenced when nematodes take up double stranded RNA (dsRNA) or short interfering RNAs (siRNAs) that elicit a systemic RNAi response. Despite many successful reports, there is still poor understanding of the range of factors that influence optimal gene silencing. Recent in vitro studies have highlighted significant variations in the RNAi phenotype that can occur with different dsRNA concentrations, construct size and duration of soaking. Discrepancies in methodology thwart efforts to reliably compare the efficacy of RNAi between different nematodes or target tissues. Nevertheless, RNAi has become an established experimental tool for plant parasitic nematodes and also offers the prospect of being developed into a novel control strategy when delivered from transgenic plants.

Identification and functional characterization of effectors in expressed sequence tags from various life cycle stages of the potato cyst nematode Globodera pallida

In this article, we describe the analysis of over 9000 expressed sequence tags (ESTs) from cDNA libraries obtained from various life cycle stages of Globodera pallida. We have identified over 50 G. pallida effectors from this dataset using bioinformatics analysis, by screening clones in order to identify secreted proteins up-regulated after the onset of parasitism and using in situ hybridization to confirm the expression in pharyngeal gland cells. A substantial gene family encoding G. pallida SPRYSEC proteins has been identified. The expression of these genes is restricted to the dorsal pharyngeal gland cell. Different members of the SPRYSEC family of proteins from G. pallida show different subcellular localization patterns in plants, with some localized to the cytoplasm and others to the nucleus and nucleolus. Differences in subcellular localization may reflect diverse functional roles for each individual protein or, more likely, variety in the compartmentalization of plant proteins targeted by the nematode. Our data are therefore consistent with the suggestion that the SPRYSEC proteins suppress host defences, as suggested previously, and that they achieve this through interaction with a range of host targets.

Recent progress in the development of RNA interference for plant parasitic nematodes

SUMMARY RNA interference (RNAi), first described for Caenorhabditis elegans, has emerged as a powerful gene silencing tool for investigating gene function in a range of organisms. Recent studies have described its application to plant parasitic nematodes. Genes expressed in a range of cell types are silenced when preparasitic juvenile nematodes take up double-stranded (ds)RNA that elicits a systemic RNAi response. Important developments over the last year have shown that in planta expression of a dsRNA targeting a nematode gene can successfully induce silencing in parasitizing nematodes. When the targeted gene has an essential function, a resistance effect is observed paving the way for the potential use of RNAi technology to control plant parasitic nematodes.

Characterization of two cDNAs encoding cysteine proteinases from the soybean cyst nematode Heterodera glycines

Two cDNAs encoding cysteine proteinases were isolated from a cDNA library constructed from feeding females of Heterodera glycines. The library was screened with a cysteine proteinase gene fragment originally amplified from cDNA of H. glycines. Database searches predict that 1 cDNA (hgcp-I) encodes a cathepsin L-like proteinase, while the second (hgcp-II) encodes a cathepsin S-like enzyme. Both predicted proteins contain a short secretion signal sequence, a long propeptide and a mature protein of 219 amino acids. Southern blot analysis suggests that the cathepsin S-like enzyme, HGCP-II, is encoded by a single-copy gene in contrast to the cathepsin L-like proteinase, HGCP-I which may have 2 homologues. The regions encoding the mature proteinases were cloned into an expression vector and recombinant protein produced in E. coli. HGCP-I was shown, after refolding, to cleave the synthetic peptide Z-Phe-Arg-AMC, and this activity could be inhibited by the engineered rice cystatin Oc-I delta D86. HGCP-II showed no activity against the synthetic substrates tested. The knowledge gained from these studies will improve our understanding of plant nematode proteinases and aid the development of a rational proteinase inhibitor-based approach to plant nematode resistance.

Molecular aspects of cyst nematodes

UNLABELLED SUMMARY Taxonomy: Superkingdom Eukaryota; kingdom Metazoa; phylum Nematoda; class Chromadorea; order Tylenchida; suborder Tylenchina; superfamily Tylenchoidea; family Heteroderidae; subfamily Heteroderinae; main genera Heterodera and Globodera. HOST RANGE Cyst nematodes comprise approximately 100 known species in six genera. They are pathogens of temperate, subtropical and tropical plant species and the host range of many species is narrow. The most economically important species are within the Globodera and Heterodera genera. Globodera pallida and G. rostochiensis are important pathogens of potato crops. There are many economic species in the Heterodera genus, including Heterodera glycines (soybean cyst nematode), H. avenae (cereal cyst nematode) and H. schachtii (sugar beet cyst nematode), the last of which attacks a range of Chenopodiaceae and Cruciferae, including Arabidopsis thaliana. Disease symptoms: Field symptoms of severe cyst nematode infection are often stunting, wilting and chlorosis, but considerable yield loss can occur without obvious symptoms. The only unique indicator of cyst nematode infection is the presence of adult female nematodes attached to host roots after several weeks of parasitism. Disease control: This is usually achieved by using integrated pest management involving cultural practices such as crop rotation, resistant cultivars if available and chemical control when economically justified.

Characterization of intestinally active proteinases of cyst nematodes

Cryostat sections of juvenile and adult female stages of the soybean cyst-nematode, Heterodera glycines, were incubated with 4 different naphthylamide-linked peptide substrates to localize and characterize proteinase activity within the animal. Detected activity was restricted to the intestine and 2 distinct classes of proteinase were identified on the basis of substrate specificity and sensitivity to plant proteinase inhibitors. A cathepsin L-like cysteine proteinase activity capable of hydrolysing the synthetic substrates Z-Ala-Arg-Arg-MNA and Z-Phe-Arg-MNA but not Z-Arg-Arg-MNA or L-Arg-NA was inhibited by an engineered variant of a cysteine proteinase inhibitor from rice (Oc-I delta D86). The cleavage of Z-Phe-Arg-MNA was sensitive to inhibition by a combination of Oc-I delta D86 and cowpea trypsin inhibitor (CpTI). Degenerate oligonucleotide primers were used to amplify fragments of cysteine proteinase genes from 2 cyst-nematodes, H. glycines and Globodera pallida. Comparison of the H. glycines fragment with known genes established highest homology to cathepsin L-like genes. In contrast, the amplified G. pallida fragment displayed greatest homology to cathepsin B-like genes from Caenorhabditis elegans.