Selective control of parasitic nematodes using bioactivated nematicides.

Parasitic nematodes are a major threat to global food security, particularly as the world amasses 10 billion people amid limited arable land1-4. Most traditional nematicides have been banned owing to poor nematode selectivity, leaving farmers with inadequate means of pest control4-12. Here we use the model nematode Caenorhabditis elegans to identify a family of selective imidazothiazole nematicides, called selectivins, that undergo cytochrome-p450-mediated bioactivation in nematodes. At low parts-per-million concentrations, selectivins perform comparably well with commercial nematicides to control root infection by Meloidogyne incognita, a highly destructive plant-parasitic nematode. Tests against numerous phylogenetically diverse non-target systems demonstrate that selectivins are more nematode-selective than most marketed nematicides. Selectivins are first-in-class bioactivated nematode controls that provide efficacy and nematode selectivity.

Chia: Host Status for Meloidogyne incognita and Activity of Plant Extracts.

Chia (Salvia hispanica L.) seeds are used for food, drinks, oil, and animal feed, and all plant parts are employed in traditional medicine. The growing demand for the seed has created a need for improved disease management. Plant-parasitic nematodes have been found on other Salvia spp., but none have been reported from S. hispanica. Chia has also not been tested for production of compounds active against these nematodes. Therefore, aqueous extracts from shoots and roots of six chia lines, Brad's Organic, Cono, E2, G3, G5, and W13.1, were tested in laboratory assays. Some concentrations of all extracts were nematotoxic, killing about one-third of Meloidogyne incognita (Kofoid & White) Chitwood second-stage juveniles (J2s) in shoot extracts and up to nearly half of J2s in root extracts. Hatch was generally not affected by the extracts. In greenhouse trials, all six chia lines were hosts of M. incognita. Chia line G3 had approximately two times or more eggs per gram of root than Brad's Organic or Cono. When cucumber seedlings were transplanted into soil amended with chopped chia shoots (2.3 or 2.5% weight of fresh shoots/weight of dry soil), galling and egg production on cucumber roots were not suppressed. To our knowledge, this is the first report that chia is a host to M. incognita (or any phytoparasitic nematode) and that chia shoots and roots produce compounds active against a nematode.

Festulolium and fungal endophyte associations: host status for Meloidogyne incognita and nematotoxic plant extracts.

Festulolium hybrids are forage grasses used worldwide in temperate climates. They are associated with the fungal endophyte Epichloë uncinata, which aids in nutrient uptake, drought tolerance, and production of metabolites that protect against parasites and herbivores. Epichloë uncinata produces loline alkaloids, which can deter insect pests. Festulolium has not been widely studied for susceptibility to plant-parasitic nematodes, so Festulolium lines, with and without fungal endophytes, were tested in the greenhouse for host status to the root-knot nematode Meloidogyne incognita. All were poor hosts, regardless of line or endophyte status. Pepper seedlings planted into soil following removal of the Festulolium plants were infected by nematodes, likely because of surviving nematodes from the original inoculation combined with some reproduction on Festulolium. Lolines were found in shoots and roots of all endophyte-associated lines, and some types of lolines in roots increased after nematode infection. Methanolic extracts from roots and shoots of a tested Festulolium line did not inhibit egg hatch, but killed nearly a third of second-stage juveniles whether an endophyte was present or not. Further studies would indicate whether these Festulolium lines aid in suppressing field populations of M. incognita.Festulolium hybrids are forage grasses used worldwide in temperate climates. They are associated with the fungal endophyte Epichloë uncinata, which aids in nutrient uptake, drought tolerance, and production of metabolites that protect against parasites and herbivores. Epichloë uncinata produces loline alkaloids, which can deter insect pests. Festulolium has not been widely studied for susceptibility to plant-parasitic nematodes, so Festulolium lines, with and without fungal endophytes, were tested in the greenhouse for host status to the root-knot nematode Meloidogyne incognita. All were poor hosts, regardless of line or endophyte status. Pepper seedlings planted into soil following removal of the Festulolium plants were infected by nematodes, likely because of surviving nematodes from the original inoculation combined with some reproduction on Festulolium. Lolines were found in shoots and roots of all endophyte-associated lines, and some types of lolines in roots increased after nematode infection. Methanolic extracts from roots and shoots of a tested Festulolium line did not inhibit egg hatch, but killed nearly a third of second-stage juveniles whether an endophyte was present or not. Further studies would indicate whether these Festulolium lines aid in suppressing field populations of M. incognita.

Nematotoxicity of Paeonia spp. Extracts and Camellia oleifera Tea Seed Cake and Extracts to Heterodera glycines and Meloidogyne incognita.

Tea-oil camellia (Camellia oleifera) is grown for tea seed oil production, with tea seed cake produced as a byproduct. Rather than disposing of the cake, agricultural uses increase the value of oil production. Constituents of C. oleifera are also utilized for traditional Chinese medicine, as are compounds produced by tree peony roots. Consequently, the unused C. oleifera cake, and stems from two tree peony species, Paeonia rockii and Paeonia suffruticosa, were studied for compounds antagonistic to soybean cyst nematode (Heterodera glycines) and root-knot nematode (Meloidogyne incognita). Extracts from C. oleifera cake and P. rockii stems suppressed hatch and were nematotoxic to second-stage juveniles (J2) of both nematode species. P. rockii extracts were more effective than P. suffruticosa extracts for decreasing M. incognita hatch and J2 viability. In greenhouse trials with soybean (Glycine max 'Essex'), powdered C. oleifera cake applied as a soil amendment suppressed H. glycines cysts/g root by up to 66% compared with nonamended controls. These results indicate that the extracts and cake contain compounds active against H. glycines and M. incognita, with activity varying between the two Paeonia species. C. oleifera tea seed cake, and constituents of the cake or of P. rockii, are candidates for further studies on management of these nematodes.

Activity of Vetiver Extracts and Essential Oil against Meloidogyne incognita.

Vetiver, a nonhost grass for certain nematodes, was studied for the production of compounds active against the southern root-knot nematode, Meloidogyne incognita . In laboratory assays studying the effects on second-stage juvenile (J2) activity and viability, crude vetiver root and shoot extracts were nematotoxic, resulting in 40% to 70% J2 mortality, and were also repellent to J2. Vetiver oil did not exhibit activity against J2 in these assays. Gas chromatography-mass spectrometry analyses of three crude vetiver root ethanol extracts and a commercial vetiver oil determined that two of the major components in each sample were the sesquiterpene acid 3,3,8,8-tetramethyltricyclo[5.1.0.0(2,4)]oct-5-ene-5-propanoic acid and the sesquiterpene alcohol 6-isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-2-ol. The acid was present in higher amounts in the extracts than in the oil. These studies demonstrating nematotoxicity and repellency of vetiver-derived compounds to M. incognita suggest that plant chemistry plays a role in the nonhost status of vetiver to root-knot nematodes, and that the chemical constituents of vetiver may be useful for suppressing nematode populations in the soil.

Arabidopsis genes, AtNPR1, AtTGA2 and AtPR-5, confer partial resistance to soybean cyst nematode (Heterodera glycines) when overexpressed in transgenic soybean roots.

BACKGROUND: Extensive studies using the model system Arabidopsis thaliana to elucidate plant defense signaling and pathway networks indicate that salicylic acid (SA) is the key hormone triggering the plant defense response against biotrophic and hemi-biotrophic pathogens, while jasmonic acid (JA) and derivatives are critical to the defense response against necrotrophic pathogens. Several reports demonstrate that SA limits nematode reproduction. RESULTS: Here we translate knowledge gained from studies using Arabidopsis to soybean. The ability of thirty-one Arabidopsis genes encoding important components of SA and JA synthesis and signaling in conferring resistance to soybean cyst nematode (SCN: Heterodera glycines) are investigated. We demonstrate that overexpression of three of thirty-one Arabidoposis genes in transgenic soybean roots of composite plants decreased the number of cysts formed by SCN to less than 50% of those found on control roots, namely AtNPR1(33%), AtTGA2 (38%), and AtPR-5 (38%). Three additional Arabidopsis genes decreased the number of SCN cysts by 40% or more: AtACBP3 (53% of the control value), AtACD2 (55%), and AtCM-3 (57%). Other genes having less or no effect included AtEDS5 (77%), AtNDR1 (82%), AtEDS1 (107%), and AtPR-1 (80%), as compared to control. Overexpression of AtDND1 greatly increased susceptibility as indicated by a large increase in the number of SCN cysts (175% of control). CONCLUSIONS: Knowledge of the pathogen defense system gained from studies of the model system, Arabidopsis, can be directly translated to soybean through direct overexpression of Arabidopsis genes. When the genes, AtNPR1, AtGA2, and AtPR-5, encoding specific components involved in SA regulation, synthesis, and signaling, are overexpressed in soybean roots, resistance to SCN is enhanced. This demonstrates functional compatibility of some Arabidopsis genes with soybean and identifies genes that may be used to engineer resistance to nematodes.

Ectopic expression of AtPAD4 broadens resistance of soybean to soybean cyst and root-knot nematodes.

BACKGROUND: The gene encoding PAD4 (PHYTOALEXIN-DEFICIENT4) is required in Arabidopsis for expression of several genes involved in the defense response to Pseudomonas syringae pv. maculicola. AtPAD4 (Arabidopsis thaliana PAD4) encodes a lipase-like protein that plays a regulatory role mediating salicylic acid signaling. RESULTS: We expressed the gene encoding AtPAD4 in soybean roots of composite plants to test the ability of AtPAD4 to deter plant parasitic nematode development. The transformed roots were challenged with two different plant parasitic nematode genera represented by soybean cyst nematode (SCN; Heterodera glycines) and root-knot nematode (RKN; Meloidogyne incognita). Expression of AtPAD4 in soybean roots decreased the number of mature SCN females 35 days after inoculation by 68 percent. Similarly, soybean roots expressing AtPAD4 exhibited 77 percent fewer galls when challenged with RKN. CONCLUSIONS: Our experiments show that AtPAD4 can be used in an economically important crop, soybean, to provide a measure of resistance to two different genera of nematodes.

Engineered resistance and hypersusceptibility through functional metabolic studies of 100 genes in soybean to its major pathogen, the soybean cyst nematode.

During pathogen attack, the host plant induces genes to ward off the pathogen while the pathogen often produces effector proteins to increase susceptibility of the host. Gene expression studies of syncytia formed in soybean root by soybean cyst nematode (Heterodera glycines) identified many genes altered in expression in resistant and susceptible roots. However, it is difficult to assess the role and impact of these genes on resistance using gene expression patterns alone. We selected 100 soybean genes from published microarray studies and individually overexpressed them in soybean roots to determine their impact on cyst nematode development. Nine genes reduced the number of mature females by more than 50 % when overexpressed, including genes encoding ascorbate peroxidase, ß-1,4-endoglucanase, short chain dehydrogenase, lipase, DREPP membrane protein, calmodulin, and three proteins of unknown function. One gene encoding a serine hydroxymethyltransferase decreased the number of mature cyst nematode females by 45 % and is located at the Rhg4 locus. Four genes increased the number of mature cyst nematode females by more than 200 %, while thirteen others increased the number of mature cyst nematode females by more than 150 %. Our data support a role for auxin and ethylene in susceptibility of soybean to cyst nematodes. These studies highlight the contrasting gene sets induced by host and nematode during infection and provide new insights into the interactions between host and pathogen at the molecular level. Overexpression of some of these genes result in a greater decrease in the number of cysts formed than recognized soybean cyst nematode resistance loci.

The pJan25 vector series: an enhancement of the Gateway-compatible vector pGWB533 for broader promoter testing applications.

Agrobacterium-mediated transformation of plants has enhanced our ability to progress more rapidly in plant genetic engineering. Development of binary vectors for Agrobacterium has played a major role in advancing plant biology. Here, we report new features added to the Gateway-compatible vector pGWB533 for promoter testing with the reporter gene encoding ß-glucuronidase (GUS). The original vector contains the spectinomycin/streptomycin adenylyltransferase (aadA) gene for bacterial selection and the hygromycin phosphotransferase gene (hpt) for transformed plant selection. However, some bacterial strains used to transform plants, such as Agrobacterium rhizogenes strain K599, have elevated tolerance to spectinomycin and streptomycin, thus making bacterial selection of pGWB533 inefficient. Although pGWB533 confers chemical selection for transgenic plants using hygromycin resistance, the plasmid has no visual marker that enables visual selection of transformed plants or transgenic tissue. In this regard, adding a gene to constitutively express green fluorescent protein (eGFP) makes it easier to visually select the transformed tissue and trim out the non-transformed. In this report we describe a series of vectors, pJan25S (NCBI: KC416200), pJan25T (NCBI: KC416201) and pJan25X (NCBI: KC416202), that are enhancements of pGWB533 for promoter testing. All three vectors contain the gene encoding eGFP as a visual marker for transformed tissue. However, in pJan25S and pJan25T, eGFP is controlled by the rolD promoter for root-specific expression, while in pJan25X it is controlled by the CaMV35S promoter for constitutive expression in all plant tissues. Spectinomycin and streptomycin resistance remains in pJan25S for bacterial selection; however, pJan25T and pJan25X contain the gene encoding tetracycline resistance (tet) for bacterial selection. These changes resulted in enhanced vectors with better visual and chemical selection that should have broad application in promoter studies.

Two-dimensional proteome reference maps for the soybean cyst nematode Heterodera glycines.

2-DE reference maps of Heterodera glycines were constructed. After in-gel digestion with trypsin, 803 spots representing 426 proteins were subsequently identified by LC-MS/MS. Proteins with annotated function were further categorized by Gene Ontology. The results showed that proteins involved in metabolic, developmental and biological regulation processes were the most abundant.

Microarray Detection Call Methodology as a Means to Identify and Compare Transcripts Expressed within Syncytial Cells from Soybean (Glycine max) Roots Undergoing Resistant and Susceptible Reactions to the Soybean Cyst Nematode (Heterodera glycines).

Background. A comparative microarray investigation was done using detection call methodology (DCM) and differential expression analyses. The goal was to identify genes found in specific cell populations that were eliminated by differential expression analysis due to the nature of differential expression methods. Laser capture microdissection (LCM) was used to isolate nearly homogeneous populations of plant root cells. Results. The analyses identified the presence of 13,291 transcripts between the 4 different sample types. The transcripts filtered down into a total of 6,267 that were detected as being present in one or more sample types. A comparative analysis of DCM and differential expression methods showed a group of genes that were not differentially expressed, but were expressed at detectable amounts within specific cell types. Conclusion. The DCM has identified patterns of gene expression not shown by differential expression analyses. DCM has identified genes that are possibly cell-type specific and/or involved in important aspects of plant nematode interactions during the resistance response, revealing the uniqueness of a particular cell population at a particular point during its differentiation process.

Population-specific gene expression in the plant pathogenic nematode Heterodera glycines exists prior to infection and during the onset of a resistant or susceptible reaction in the roots of the Glycine max genotype Peking.

BACKGROUND: A single Glycine max (soybean) genotype (Peking) reacts differently to two different populations of Heterodera glycines (soybean cyst nematode) within the first twelve hours of infection during resistant (R) and susceptible (S) reactions. This suggested that H. glycines has population-specific gene expression signatures. A microarray analysis of 7539 probe sets representing 7431 transcripts on the Affymetrix soybean GeneChip were used to identify population-specific gene expression signatures in pre-infective second stage larva (pi-L2) prior to their infection of Peking. Other analyses focused on the infective L2 at 12 hours post infection (i-L2(12h)), and the infective sedentary stages at 3 days post infection (i-L2(3d)) and 8 days post infection (i-L2/L3(8d)). RESULTS: Differential expression and false discovery rate (FDR) analyses comparing populations of pi-L2 (i.e., incompatible population, NL1-RHg to compatible population, TN8) identified 71 genes that were induced in NL1-RHg as compared to TN8. These genes included putative gland protein G23G12, putative esophageal gland protein Hgg-20 and arginine kinase. The comparative analysis of pi-L2 identified 44 genes that were suppressed in NL1-RHg as compared to TN8. These genes included a different Hgg-20 gene, an EXPB1 protein and a cuticular collagen. By 12 h, there were 7 induced genes and 0 suppressed genes in NL1-RHg. By 3d, there were 9 induced and 10 suppressed genes in NL1-RHg. Substantial changes in gene expression became evident subsequently. At 8d there were 13 induced genes in NL1-RHg. This included putative gland protein G20E03, ubiquitin extension protein, putative gland protein G30C02 and beta-1,4 endoglucanase. However, 1668 genes were found to be suppressed in NL1-RHg. These genes included steroid alpha reductase, serine proteinase and a collagen protein. CONCLUSION: These analyses identify a genetic expression signature for these two populations both prior to and subsequently as they undergo an R or S reaction. The identification of genes like steroid alpha reductase and serine proteinase that are involved in feeding and nutritional uptake as being highly suppressed during the R response at 8d may indicate genes that the plant is targeting. The analyses also identified numerous putative parasitism genes that are differentially expressed. The 1668 genes that are suppressed in NL1-RHg, and hence induced in TN8 may represent genes that are important during the parasitic stages of H. glycines development. The potential for different arrays of putative parasitism genes to be expressed in different nematode populations may indicate how H. glycines evolve mechanisms to overcome resistance.

A correlation between host-mediated expression of parasite genes as tandem inverted repeats and abrogation of development of female Heterodera glycines cyst formation during infection of Glycine max.

Host-mediated (hm) expression of parasite genes as tandem inverted repeats was investigated as a means to abrogate the formation of mature Heterodera glycines (soybean cyst nematode) female cysts during its infection of Glycine max (soybean). A Gateway-compatible hm plant transformation system was developed specifically for these experiments in G. max. Three steps then were taken to identify H. glycines candidate genes. First, a pool of 150 highly conserved H. glycines homologs of genes having lethal mutant phenotypes or phenocopies from the free living nematode Caenorhabditis elegans were identified. Second, annotation of those 150 genes on the Affymetrix soybean GeneChip allowed for the identification of a subset of 131 genes whose expression could be monitored during the parasitic phase of the H. glycines life cycle. Third, a microarray analyses identified a core set of 32 genes with induced expression (>2.0-fold, log base 2) during the parasitic stages of infection. H. glycines homologs of small ribosomal protein 3a and 4 (Hg-rps-3a [accession number CB379877] and Hg-rps-4 [accession number CB278739]), synaptobrevin (Hg-snb-1 [accession number BF014436]) and a spliceosomal SR protein (Hg-spk-1 [accession number BI451523.1]) were tested for functionality in hm expression studies. Effects on H. glycines development were observed 8 days after infection. Experiments demonstrated that 81-93% fewer females developed on transgenic roots containing the genes engineered as tandem inverted repeats. The effect resembles RNA interference. The methodology has been used here as an alternative approach to engineer resistance to H. glycines.

Protease inhibitor expression in soybean roots exhibiting susceptible and resistant interactions with soybean cyst nematode.

Protease inhibitors play a role in regulating proteases during cellular development and in plant defense. We cloned and sequenced cDNA encoding six protease inhibitors expressed in soybean roots infected with soybean cyst nematode (SCN) and determined their expression patterns. Four of these protease inhibitors are novel and have not been reported previously. Using RT-PCR, we measured the relative transcript levels of each protease inhibitor in roots of the soybean cv. Peking inoculated with either SCN TN8 to examine the expression of protease inhibitors during the susceptible interaction or with SCN NL1-RHg representing the resistant interaction. Within 12 to 24 hours, mRNA transcripts encoding five of the six protease inhibitors were more highly elevated in soybean roots exhibiting the susceptible interaction than the resistant interaction. Transcripts encoding two protease inhibitors possessing Kunitz trypsin inhibitor domains were induced 37- and 27-fold in the susceptible interaction within 1 dpi, but were induced only 5- to 7-fold in roots displaying the resistant interaction. Our results indicate that soybean roots recognize differences between these two SCN populations before the nematodes initiate a feeding site, and accordingly the roots express transcripts encoding soybean protease inhibitors differentially. These transcripts were generally less abundant in roots exhibiting the resistant interaction.

A decline in transcript abundance for Heterodera glycines homologs of Caenorhabditis elegans uncoordinated genes accompanies its sedentary parasitic phase.

BACKGROUND: Heterodera glycines (soybean cyst nematode [SCN]), the major pathogen of Glycine max (soybean), undergoes muscle degradation (sarcopenia) as it becomes sedentary inside the root. Many genes encoding muscular and neuromuscular components belong to the uncoordinated (unc) family of genes originally identified in Caenorhabditis elegans. Previously, we reported a substantial decrease in transcript abundance for Hg-unc-87, the H. glycines homolog of unc-87 (calponin) during the adult sedentary phase of SCN. These observations implied that changes in the expression of specific muscle genes occurred during sarcopenia. RESULTS: We developed a bioinformatics database that compares expressed sequence tag (est) and genomic data of C. elegans and H. glycines (CeHg database). We identify H. glycines homologs of C. elegans unc genes whose protein products are involved in muscle composition and regulation. RT-PCR reveals the transcript abundance of H. glycines unc homologs at mobile and sedentary stages of its lifecycle. A prominent reduction in transcript abundance occurs in samples from sedentary nematodes for homologs of actin, unc-60B (cofilin), unc-89, unc-15 (paromyosin), unc-27 (troponin I), unc-54 (myosin), and the potassium channel unc-110 (twk-18). Less reduction is observed for the focal adhesion complex gene Hg-unc-97. CONCLUSION: The CeHg bioinformatics database is shown to be useful in identifying homologs of genes whose protein products perform roles in specific aspects of H. glycines muscle biology. Our bioinformatics comparison of C. elegans and H. glycines genomic data and our Hg-unc-87 expression experiments demonstrate that the transcript abundance of specific H. glycines homologs of muscle gene decreases as the nematode becomes sedentary inside the root during its parasitic feeding stages.

Manipulation of two α-endo-ß-1,4-glucanase genes, AtCel6 and GmCel7, reduces susceptibility to Heterodera glycines in soybean roots.

Plant endo-ß-1,4-glucanases (EGases) include cell wall-modifying enzymes that are involved in nematode-induced growth of syncytia (feeding structures) in nematode-infected roots. EGases in the α- and ß-subfamilies contain signal peptides and are secreted, whereas those in the γ-subfamily have a membrane-anchoring domain and are not secreted. The Arabidopsis α-EGase At1g48930, designated as AtCel6, is known to be down-regulated by beet cyst nematode (Heterodera schachtii) in Arabidopsis roots, whereas another α-EGase, AtCel2, is up-regulated. Here, we report that the ectopic expression of AtCel6 in soybean roots reduces susceptibility to both soybean cyst nematode (SCN; Heterodera glycines) and root knot nematode (Meloidogyne incognita). Suppression of GmCel7, the soybean homologue of AtCel2, in soybean roots also reduces the susceptibility to SCN. In contrast, in studies on two γ-EGases, both ectopic expression of AtKOR2 in soybean roots and suppression of the soybean homologue of AtKOR3 had no significant effect on SCN parasitism. Our results suggest that secreted α-EGases are likely to be more useful than membrane-bound γ-EGases in the development of an SCN-resistant soybean through gene manipulation. Furthermore, this study provides evidence that Arabidopsis shares molecular events of cyst nematode parasitism with soybean, and confirms the suitability of the Arabidopsis-H. schachtii interaction as a model for the soybean-H. glycines pathosystem.

Laser capture microdissection (LCM) and comparative microarray expression analysis of syncytial cells isolated from incompatible and compatible soybean (Glycine max) roots infected by the soybean cyst nematode (Heterodera glycines).

Syncytial cells in soybean (Glycine max cultivar [cv.] Peking) roots infected by incompatible and compatible populations of soybean cyst nematode (SCN [Heterodera glycines]) were collected using laser capture microdissection (LCM). Gene transcript abundance was assayed using Affymetrix soybean GeneChips, each containing 37,744 probe sets. Our analyses identified differentially expressed genes in syncytial cells that are not differentially expressed in the whole root analyses. Therefore, our results show that the mass of transcriptional activity occurring in the whole root is obscuring identification of transcriptional events occurring within syncytial cells. In syncytial cells from incompatible roots at three dpi, genes encoding lipoxygenase (LOX), heat shock protein (HSP) 70, superoxidase dismutase (SOD) were elevated almost tenfold or more, while genes encoding several transcription factors and DNA binding proteins were also elevated, albeit at lower levels. In syncytial cells formed during the compatible interaction at three dpi, genes encoding prohibitin, the epsilon chain of ATP synthase, allene oxide cyclase and annexin were more abundant. By 8 days, several genes of unknown function and genes encoding a germin-like protein, peroxidase, LOX, GAPDH, 3-deoxy-D-arabino-heptolosonate 7-phosphate synthase, ATP synthase and a thioesterase were abundantly expressed. These observations suggest that gene expression is different in syncytial cells as compared to whole roots infected with nematodes. Our observations also show that gene expression is different between syncytial cells that were isolated from incompatible and compatible roots and that gene expression is changing over the course of syncytial cell development as it matures into a functional feeding site.

A time-course comparative microarray analysis of an incompatible and compatible response by Glycine max (soybean) to Heterodera glycines (soybean cyst nematode) infection.

The development of an infection in soybean [Glycine max L. cultivar (cv.) Peking] roots by incompatible (I) and compatible (C) populations of soybean cyst nematode (SCN) (Heterodera glycines) was assayed using an AffymetriX soybean GeneChip. This time-course microarray analysis, using 37,744 probe sets, measured transcript abundance during I and C. These analyses reveal that infection by individual I and C H. glycines populations influence the transcription of G. max genes differently. A substantial difference in gene expression is present between I and C at 12 h post infection. Thus, G. max can differentiate between I and C nematode populations even before they have begun to select their feeding sites. The microarray analysis identified genes induced earlier in infection during I than C. MA also identified amplitude differences in transcript abundance between I and C reactions. Some of the probe sets measuring increased transcript levels during I represented no apical meristem (NAM) and WRKY transcription factors as well as NBS-LRR kinases. Later during I, heat shock protein (HSPs) probe sets (i.e. HSP90, HSP70, ClpB/HSP101) measured increased transcript abundance. These results demonstrate that G. max roots respond very differently to the different H. glycines races even before their feeding site selection has occurred. The ability of G. max to engage an I reaction, thus, appears to be dependent on the ability of root cells to recognize the different races of H. glycines because these experiments were conducted in the identical G. max genetic background.

Timecourse microarray analyses reveal global changes in gene expression of susceptible Glycine max (soybean) roots during infection by Heterodera glycines (soybean cyst nematode).

Changes in gene expression within roots of Glycine max (soybean), cv. Kent, susceptible to infection by Heterodera glycines (the soybean cyst nematode [SCN]), at 6, 12, and 24 h, and 2, 4, 6, and 8 days post-inoculation were monitored using microarrays containing more than 6,000 cDNA inserts. Replicate, independent biological samples were examined at each time point. Gene expression was analyzed statistically using T-tests, ANOVA, clustering algorithms, and online analytical processing (OLAP). These analyses allow the user to query the data in several ways without importing the data into third-party software. RT-PCR confirmed that WRKY6 transcription factor, trehalose phosphate synthase, EIF4a, Skp1, and CLB1 were differentially induced across most time-points. Other genes induced across most timepoints included lipoxygenase, calmodulin, phospholipase C, metallothionein-like protein, and chalcone reductase. RT-PCR demonstrated enhanced expression during the first 12 h of infection for Kunitz trypsin inhibitor and sucrose synthase. The stress-related gene, SAM-22, phospholipase D and 12-oxophytodienoate reductase were also induced at the early time-points. At 6 and 8 dpi there was an abundance of transcripts expressed that encoded genes involved in transcription and protein synthesis. Some of those genes included ribosomal proteins, and initiation and elongation factors. Several genes involved in carbon metabolism and transport were also more abundant. Those genes included glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase and sucrose synthase. These results identified specific changes in gene transcript levels triggered by infection of susceptible soybean roots by SCN.