J. Fosu-Nyarko

Gene silencing in root lesion nematodes (Pratylenchus spp.) significantly reduces reproduction in a plant host

Root lesion nematodes (RLNs, Pratylenchus species) are a group of economically important migratory endoparasitic plant pathogens that attack host roots of major crops such as wheat and sugarcane, and can reduce crop yields by 7-15%. Pratylenchus thornei and Pratylenchus zeae were treated with double stranded RNA (dsRNA) to study gene silencing, (RNA interference, RNAi), as a potential strategy for their control. Mixed stages of nematodes of both species ingested dsRNA when incubated in a basic soaking solution in the presence of the neurostimulant octopamine. Incubation for up to 16 h in soaking solutions containing 10-50 mM octopamine, 0.1-1.0 mg/mL FITC, and 0.5-6 mM spermidine did not affect vitality. Spermidine phosphate salt hexahydrate rather than spermidine or spermidine trihydrochloride increased uptake of FITC by nematodes, and this resulted in more effective gene silencing. Silencing pat-10 and unc-87 genes of P. thornei and P. zeae resulted in paralysis and uncoordinated movements in both species, although to a higher degree in P. thornei. There was also a greater reduction in transcript of both genes in P. thornei indicating that it may be more susceptible to RNAi. For P. thornei treated with dsRNA of pat-10 and unc-87 there was a significant reduction (77-81%) in nematode reproduction on carrot mini discs over a 5 week period. The results show that RLNs are clearly amenable to gene silencing, and that in planta delivery of dsRNA to target genes in these nematodes should confer host resistance. Moreover, for the two genes, dsRNA derived from either nematode species silenced the corresponding gene in both species. This implies cross-species control of nematodes via RNAi is possible.

Functional characterization of transcripts expressed in early‐stage Meloidogyne javanica‐induced giant cells isolated by laser microdissection

The root-knot nematode Meloidogyne javanica induces giant cells and feeds from them during its development and reproduction. To study the cellular processes underlying the formation of giant cells, laser microdissection was used to isolate the contents of early-stage giant cells 4 and 7 days post-infection (dpi) from tomato, and cDNA libraries from both stages were generated with 87 [250 expressed sequence tag (EST) clones] and 54 (309 EST clones) individual transcripts identified, respectively. These transcripts have roles in metabolism, stress response, protein synthesis, cell division and morphogenesis, transport, signal transduction, protein modification and fate, and regulation of cellular processes. The expression of 25 selected transcripts was studied further by real-time quantitative reverse transcriptase-polymerase chain reaction. Among them, 13 showed continuous up-regulation in giant cells from 4 to 7 dpi. The expression of two transcripts was higher than in controls at 4 dpi and remained at the same level at 7 dpi; a further five transcripts were highly expressed only at 7 dpi. The Phi-1 protein gene, a cell cycle-related homologue in tobacco, was expressed 8.5 times more strongly in giant cells than in control cells at 4 dpi, but was reduced to 6.7 times at 7 dpi. Using in situ hybridization, the expression of the Phi-1 gene was preferentially localized in the cytoplasm of giant cells at 4 dpi, together with a pectinesterase U1 precursor gene. The identification of highly expressed transcripts in developing giant cells adds to the knowledge of the plant genes responsive to nematode infection, and may provide candidate genes for nematode control strategies.

de novo analysis and functional classification of the transcriptome of the root lesion nematode, Pratylenchus thornei, after 454 GS FLX sequencing.

The migratory endoparasitic root lesion nematode Pratylenchus thornei is a major pest of the cereals wheat and barley. In what we believe to be the first global transcriptome analysis for P. thornei, using Roche GS FLX sequencing, 787,275 reads were assembled into 34,312 contigs using two assembly programs, to yield 6,989 contigs common to both. These contigs were annotated, resulting in functional assignments for 3,048. Specific transcripts studied in more detail included carbohydrate active enzymes potentially involved in cell wall degradation, neuropeptides, putative plant nematode parasitism genes, and transcripts that could be secreted by the nematode. Transcripts for cell wall degrading enzymes were similar to bacterial genes, suggesting that they were acquired by horizontal gene transfer. Contigs matching 14 parasitism genes found in sedentary endoparasitic nematodes were identified. These genes are thought to function in suppression of host defenses and in feeding site development, but their function in P. thornei may differ. Comparison of the common contigs from P. thornei with other nematodes showed that 2,039 were common to sequences of the Heteroderidae, 1,947 to the Meloidogynidae, 1,218 to Radopholus similis, 1,209 matched expressed sequence tags (ESTs) of Pratylenchus penetrans and Pratylenchus vulnus, and 2,940 to contigs of Pratylenchus coffeae. There were 2,014 contigs common to Caenarhabditis elegans, with 15.9% being common to all three groups. Twelve percent of contigs with matches to the Heteroderidae and the Meloidogynidae had no homology to any C. elegans protein. Fifty-seven percent of the contigs did not match known sequences and some could be unique to P. thornei. These data provide substantial new information on the transcriptome of P. thornei, those genes common to migratory and sedentary endoparasitic nematodes, and provide additional understanding of genes required for different forms of parasitism. The data can also be used to identify potential genes to study host interactions and for crop protection.

The genome structure of the 1-FEH genes in wheat (Triticum aestivum L.): new markers to track stem carbohydrates and grain filling QTLs in breeding

Terminal drought tolerance of wheat is a major target in many areas in the world and is a particular focus in Western Australia. It is widely considered to relate to water soluble carbohydrate (WSC) levels such as fructan in the stem, as the head is maturing. Fructan exohydrolases are key enzymes during both fructan biosynthesis and mobilization. The wheat genome sequences of three fructan 1-exohydrolase (1-FEH) genes with seven exons and six introns were isolated by using the available 1-FEH w2 cDNA sequence. The major size differences among the three genes were located in intron 1 and intron 4. The three 1-FEH genes were mapped to Chinese Spring chromosome 6A, 6B and 6D based on polymerase chain reaction (PCR) polymorphisms and Southern hybridization. 1-FEH-6A, -6B and -6D corresponded to published cDNA sequences 1-FEH w1, w3 and w2, respectively. The overall correlation of the mRNA accumulation profile for the 1-FEH genes in stem and sheath leaf tissue in relation to the profile of soluble carbohydrate accumulation was consistent with their postulated role in stem soluble carbohydrate accumulation. The accumulation of the 1-FEH-6B (1-FEH w3) mRNA was 300 fold greater than that of 1-FEH-6A and -6D. The mRNA accumulation continued after the stem water soluble carbohydrate concentrations reached a peak, consistent with a role of 1-FEH-6B in the breakdown of soluble carbohydrate. The relationship between the 1-FEH genes and soluble carbohydrate accumulation is discussed and the 1-FEH-6B gene in particular is suggested to provide a new class of molecular marker for this trait.

The complete nucleotide sequence of Subterranean clover mottle virus

Summary The complete nucleotide sequence of Subterranean clover mottle virus (SCMoV) genomic RNA has been determined. The SCMoV genome is 4,258 nucleotides in length. It shares most nucleotide and amino acid sequence identity with the genome of Lucerne transient streak virus (LTSV). SCMoV RNA encodes four overlapping open reading frames and has a genome organisation similar to that of Cocksfoot mottle virus (CfMV). ORF1 and ORF4 are predicted to encode single proteins. ORF2 is predicted to encode two proteins that are derived from a −1 translational frameshift between two overlapping reading frames (ORF2a and ORF2b). A search of amino acid databases did not find a significant match for ORF1 and the function of this protein remains unclear. ORF2a contains a motif typical of chymotrypsin-like serine proteases and ORF2b has motifs characteristically present in positive-stranded RNA-dependent RNA polymerases. ORF4 is likely to be expressed from a subgenomic RNA and encodes the viral coat protein. The ORF2a/ORF2b overlapping gene expression strategy used by SCMoV and CfMV is similar to that of the poleroviruses and differ from that of other published sobemoviruses. These results suggest that the sobemoviruses could now be divided into two distinct subgroups based on those that express the RNA-dependent RNA polymerase from a single, in-frame polyprotein, and those that express it via a −1 translational frameshifting mechanism.

Genome-level identification of cell wall invertase genes in wheat for the study of drought tolerance

In wheat (Triticum aestivum L.) drought-induced pollen sterility is a major contributor to grain yield loss and is caused by the downregulation of the cell wall invertase gene IVR1. The IVR1 gene catalyses the irreversible hydrolysis of sucrose to glucose and fructose, the essential energy substrates which support pollen development. Downregulation of IVR1 in response to drought is isoform specific and shows variation in temporal and tissue-specific expression. IVR1 is now prompting interest as a candidate gene for molecular marker development to screen wheat germplasm for improved drought tolerance. The aim of this study was to define the family of IVR1 genes to enable: (1) individual isoforms to be assayed in gene expression studies; and (2) greater accuracy in IVR1 mapping to the wheat genetic map and drought tolerance QTL analysis. Using a cell wall invertase-specific motif as a probe, wheat genomics platforms were screened for the presence of unidentified IVR1 isoforms. Wheat genomics platforms screened included the IWGSC wheat survey sequence, the wheat D genome donor sequence from Aegilops tauschii Coss, and the CCG wheat chromosome 3B assembly: contig506. Chromosome-specific sequences homologous to the query motif were isolated and characterised. Sequence annotation results showed five previously unidentified IVR1 isoforms exist on multiple chromosome arms and on all three genomes (A, B and D): IVR1-3A, IVR1-4A, IVR1-5B, IVR1.2-3B and IVR1-5D. Including three previously characterised IVR1 isoforms (IVR1.1-1A, IVR1.2-1A and IVR1.1-3B), the total number of isoform gene family members is eight. The IVR1 isoforms contain two motifs common to cell wall invertase (NDPN and WECPDF) and a high degree of conservation in exon 4, suggesting conservation of functionality. Sequence divergence at a primary structure level in other regions of the gene was evident amongst the isoforms, which likely contributes to variation in gene regulation and expression in response to water deficit within this subfamily of IVR1 isoforms in wheat.

Analysis of the Transcriptome of the Infective Stage of the Beet Cyst Nematode, H. schachtii

The beet cyst nematode, Heterodera schachtii, is a major root pest that significantly impacts the yield of sugar beet, brassicas and related species. There has been limited molecular characterisation of this important plant pathogen: to identify target genes for its control the transcriptome of the pre-parasitic J2 stage of H. schachtii was sequenced using Roche GS FLX. Ninety seven percent of reads (i.e., 387,668) with an average PHRED score > 22 were assembled with CAP3 and CLC Genomics Workbench into 37,345 and 47,263 contigs, respectively. The transcripts were annotated by comparing with gene and genomic sequences of other nematodes and annotated proteins on public databases. The annotated transcripts were much more similar to sequences of Heterodera glycines than to those of Globodera pallida and root knot nematodes (Meloidogyne spp.). Analysis of these transcripts showed that a subset of 2,918 transcripts was common to free-living and plant parasitic nematodes suggesting that this subset is involved in general nematode metabolism and development. A set of 148 contigs and 183 singletons encoding putative homologues of effectors previously characterised for plant parasitic nematodes were also identified: these are known to be important for parasitism of host plants during migration through tissues or feeding from cells or are thought to be involved in evasion or modulation of host defences. In addition, the presence of sequences from a nematode virus is suggested. The sequencing and annotation of this transcriptome significantly adds to the genetic data available for H. schachtii, and identifies genes primed to undertake required roles in the critical pre-parasitic and early post-parasitic J2 stages. These data provide new information for identifying potential gene targets for future protection of susceptible crops against H. schachtii.

De novo analysis of the transcriptome of Pratylenchus zeae to identify transcripts for proteins required for structural integrity, sensation, locomotion and parasitism

The root lesion nematode Pratylenchus zeae, a migratory endoparasite, is an economically important pest of major crop plants (e.g. cereals, sugarcane). It enters host roots, migrates through root tissues and feeds from cortical cells, and defends itself against biotic and abiotic stresses in the soil and in host tissues. We report de novo sequencing of the P. zeae transcriptome using 454 FLX, and the identification of putative transcripts encoding proteins required for movement, response to stimuli, feeding and parasitism. Sequencing generated 347,443 good quality reads which were assembled into 10,163 contigs and 139,104 singletons: 65% of contigs and 28% of singletons matched sequences of free-living and parasitic nematodes. Three-quarters of the annotated transcripts were common to reference nematodes, mainly representing genes encoding proteins for structural integrity and fundamental biochemical processes. Over 15,000 transcripts were similar to Caenorhabditis elegans genes encoding proteins with roles in mechanical and neural control of movement, responses to chemicals, mechanical and thermal stresses. Notably, 766 transcripts matched parasitism genes employed by both migratory and sedentary endoparasites in host interactions, three of which hybridized to the gland cell region, suggesting that they might be secreted. Conversely, transcripts for effectors reported to be involved in feeding site formation by sedentary endoparasites were conspicuously absent. Transcripts similar to those encoding some secretory-excretory products at the host interface of Brugia malayi, the secretome of Meloidogyne incognita and products of gland cells of Heterodera glycines were also identified. This P. zeae transcriptome provides new information for genome annotation and functional analysis of possible targets for control of pratylenchid nematodes.

Advances in Understanding the Molecular Mechanisms of Root Lesion Nematode Host Interactions

Root lesion nematodes (RLNs) are one of the most economically important groups of plant nematodes. As migratory endoparasites, their presence in roots is less obvious than infestations of sedentary endoparasites; nevertheless, in many instances, they are the major crop pests. With increasing molecular information on nematode parasitism, available data now reflect the differences and, in particular, similarities in lifestyle between migratory and sedentary endoparasites. Far from being unsophisticated compared with sedentary endoparasites, migratory endoparasites are exquisitely suited to their parasitic lifestyle. What they lack in effectors required for induction of permanent feeding sites, they make up for with their versatile host range and their ability to move and feed from new host roots and survive adverse conditions. In this review, we summarize the current molecular data available for RLNs and highlight differences and similarities in effectors and molecular mechanisms between migratory and sedentary endoparasitic nematodes.

Application of Laser Microdissection to Study Plant–Fungal Pathogen Interactions

Laser microdissection (LM) has become an important tool for isolating individual cells or cell types from suitably prepared tissue samples. The technique can be used to isolate both fungal and host plant cells after pathogen infection for molecular studies. Sample preparation is a crucial step in LM and involves fixing samples with appropriate fixatives to preserve the integrity of cell morphology and target metabolites (e.g., RNA), and embedding the fixed tissue in paraffin wax for sectioning onto microscope slides. The sample sections are then deparaffinised, rehydrated, and cells are dissected by a laser focused through a microscope. LM samples are collected into protective (e.g., RNAse-free) medium for isolation of RNA. The RNA can then be subjected to gene expression studies such as quantitative RT-PCR and microarray analysis after a linear RNA amplification process.