The Soybean Rhg1 locus for resistance to the soybean cyst nematode Heterodera glycines regulates the expression of a large number of stress- and defense-related genes in degenerating feeding cells.

To gain new insights into the mechanism of soybean (Glycine max) resistance to the soybean cyst nematode (Heterodera glycines), we compared gene expression profiles of developing syncytia in soybean near-isogenic lines differing at Rhg1 (for resistance to Heterodera glycines), a major quantitative trait locus for resistance, by coupling laser capture microdissection with microarray analysis. Gene expression profiling revealed that 1,447 genes were differentially expressed between the two lines. Of these, 241 (16.8%) were stress- and defense-related genes. Several stress-related genes were up-regulated in the resistant line, including those encoding homologs of enzymes that lead to increased levels of reactive oxygen species and proteins associated with the unfolded protein response. These results indicate that syncytia induced in the resistant line are undergoing severe oxidative stress and imbalanced endoplasmic reticulum homeostasis, both of which likely contribute to the resistance reaction. Defense-related genes up-regulated within syncytia of the resistant line included those predominantly involved in apoptotic cell death, the plant hypersensitive response, and salicylic acid-mediated defense signaling; many of these genes were either partially suppressed or not induced to the same level by a virulent soybean cyst nematode population for successful nematode reproduction and development on the resistant line. Our study demonstrates that a network of molecular events take place during Rhg1-mediated resistance, leading to a highly complex defense response against a root pathogen.

Sequence mining and transcript profiling to explore cyst nematode parasitism.

BACKGROUND: Cyst nematodes are devastating plant parasites that become sedentary within plant roots and induce the transformation of normal plant cells into elaborate feeding cells with the help of secreted effectors, the parasitism proteins. These proteins are the translation products of parasitism genes and are secreted molecular tools that allow cyst nematodes to infect plants. RESULTS: We present here the expression patterns of all previously described parasitism genes of the soybean cyst nematode, Heterodera glycines, in all major life stages except the adult male. These insights were gained by analyzing our gene expression dataset from experiments using the Affymetrix Soybean Genome Array GeneChip, which contains probeset sequences for 6,860 genes derived from preparasitic and parasitic H. glycines life stages. Targeting the identification of additional H. glycines parasitism-associated genes, we isolated 633 genes encoding secretory proteins using algorithms to predict secretory signal peptides. Furthermore, because some of the known H. glycines parasitism proteins have strongest similarity to proteins of plants and microbes, we searched for predicted protein sequences that showed their highest similarities to plant or microbial proteins and identified 156 H. glycines genes, some of which also contained a signal peptide. Analyses of the expression profiles of these genes allowed the formulation of hypotheses about potential roles in parasitism. This is the first study combining sequence analyses of a substantial EST dataset with microarray expression data of all major life stages (except adult males) for the identification and characterization of putative parasitism-associated proteins in any parasitic nematode. CONCLUSION: We have established an expression atlas for all known H. glycines parasitism genes. Furthermore, in an effort to identify additional H. glycines genes with putative functions in parasitism, we have reduced the currently known 6,860 H. glycines genes to a pool of 788 most promising candidate genes (including known parasitism genes) and documented their expression profiles. Using our approach to pre-select genes likely involved in parasitism now allows detailed functional analyses in a manner not feasible for larger numbers of genes. The generation of the candidate pool described here is an important enabling advance because it will significantly facilitate the unraveling of fascinating plant-animal interactions and deliver knowledge that can be transferred to other pathogen-host systems. Ultimately, the exploration of true parasitism genes verified from the gene pool delineated here will identify weaknesses in the nematode life cycle that can be exploited by novel anti-nematode efforts.

Soluble factors from neocortical astrocytes enhance neuronal differentiation of neural progenitor cells from adult rat hippocampus on micropatterned polymer substrates.

Rat adult hippocampal progenitor cells (AHPCs) are self-renewing, multipotent neural progenitors that have the ability to differentiate into neurons and glia. Previously, we demonstrated that coculture of AHPCs with postnatal day 2, type 1 cortical astrocytes on laminin-coated micropatterned polymer substrates facilitates selective neuronal differentiation of the AHPCs (Recknor et al., Biomaterials 2006;27:4098-4108). Under this condition, multidimensional cell-cell and/or cell-extracellular matrix interactions, as well as possible soluble factors released from astrocytes provided spatial and temporal control selectively enhancing neuronal differentiation and neurite alignment on topographically different regions of the same substrate. To investigate the potential role of astrocyte-derived soluble factors as cues involved in neuronal differentiation, a noncontact coculture system was used. Under control conditions, approximately 14% of the AHPCs were immunoreactive (IR) for the neuronal marker, class III beta-tubulin (TUJ1-IR). When cocultured in physical contact with astrocytes, neuronal differentiation increased significantly to about 25%, consistent with our previous results. Moreover, under noncontact coculture conditions using Transwell insert cultures, neuronal differentiation was dramatically increased to approximately 64%. Furthermore, neurite outgrowth from neuronal cell bodies was considerably greater on the patterned substrate when compared with the nonpatterned planar substrate under noncontact coculture conditions. Taken together, our results demonstrate that astrocyte-derived soluble factors provide cues for specific neuronal differentiation of AHPCs cultured on micropatterned substrates. In addition, a suppressive influence on neuronal differentiation appears to be mediated by contact with cocultured astrocytes. These results provide important insights into mechanisms for controlling neural progenitor/stem cell differentiation and facilitate development of strategies for CNS repair.

Overall type I error rate and power of multiple Dunnett's tests on rodent body weights in toxicology studies.

Body weight data are routinely collected in in vivo general toxicology studies, including 2-year carcinogenicity studies, to help assess the overall health of animals. The effect of the compound on body weight is statistically evaluated for each sex separately using a linear trend test or a many-to-one test by Dunnett. These tests are performed either in the framework of a one-factor analysis of variance (ANOVA) or a repeated measures ANOVA. The one-factor ANOVA with Dunnett's test at each time point is a common practice in industry. Although each individual test is conducted at the 0.05 significance level, one wonders about the overall type I error rate and power for performing many individual Dunnett's tests. A simulation study is conducted to answer this question for general toxicology studies of durations 1 month, 3 months, and 2 years. These results provide guidance to managing multiplicity of body weight analysis of general toxicology studies.

Fabp3 as a biomarker of skeletal muscle toxicity in the rat: comparison with conventional biomarkers.

Fatty acid binding protein 3 (Fabp3) has been used as a serological biomarker of cardiac injury, but its utility as a preclinical biomarker of injury to skeletal muscle is not well described. Fabp3 concentrations were determined for tissues from Sprague-Dawley rats and found to occur at highest concentrations in cardiac muscle and in skeletal muscles containing an abundance of type I fibers, such as the soleus muscle. Soleus is also a primary site of skeletal muscle (SKM) injury caused by lipid-lowering peroxisome proliferator-activated receptor alpha (PPAR-alpha) agonists. In rats administered repeat doses of a PPAR-alpha agonist, the kinetics and amplitude of plasma concentrations of Fabp3 were consistent with plasma compound concentrations and histopathology findings of swollen, hyalinized, and fragmented muscle fibers with macrophage infiltration. Immunohistochemical detection of Fabp3 revealed focal depletion of Fabp3 protein from injured SKM fibers which is consistent with increased serum Fabp3 concentrations in treated rats. We then assessed the predictivity of serological Fabp3 for SKM necrosis in short duration toxicology studies. Rats were treated with various doses of 27 different compounds, and the predictivity of serological biomarkers was assessed relative to histology in individual rats and in treatment groups. Under these study conditions, Fabp3 was the most useful individual biomarker based on concordance, sensitivity, positive and negative predictive values, and false negative rate. In addition, the combination of Fabp3 and aspartate aminotransferase (AST) had greater diagnostic value than the conventional combination of creatine kinase-MM isoenzyme (CK) and AST.

Identification of differentially expressed gene categories in microarray studies using nonparametric multivariate analysis.

MOTIVATION: The field of microarray data analysis is shifting emphasis from methods for identifying differentially expressed genes to methods for identifying differentially expressed gene categories. The latter approaches utilize a priori information about genes to group genes into categories and enhance the interpretation of experiments aimed at identifying expression differences across treatments. While almost all of the existing approaches for identifying differentially expressed gene categories are practically useful, they suffer from a variety of drawbacks. Perhaps most notably, many popular tools are based exclusively on gene-specific statistics that cannot detect many types of multivariate expression change. RESULTS: We have developed a nonparametric multivariate method for identifying gene categories whose multivariate expression distribution differs across two or more conditions. We illustrate our approach and compare its performance to several existing procedures via the analysis of a real data set and a unique data-based simulation study designed to capture the challenges and complexities of practical data analysis. We show that our method has good power for differentiating between differentially expressed and non-differentially expressed gene categories, and we utilize a resampling based strategy for controlling the false discovery rate when testing multiple categories. AVAILABILITY: R code (www.r-project.org) for implementing our approach is available from the first author by request.

Divergent evolution of arrested development in the dauer stage of Caenorhabditis elegans and the infective stage of Heterodera glycines.

BACKGROUND: The soybean cyst nematode Heterodera glycines is the most important parasite in soybean production worldwide. A comprehensive analysis of large-scale gene expression changes throughout the development of plant-parasitic nematodes has been lacking to date. RESULTS: We report an extensive genomic analysis of H. glycines, beginning with the generation of 20,100 expressed sequence tags (ESTs). In-depth analysis of these ESTs plus approximately 1,900 previously published sequences predicted 6,860 unique H. glycines genes and allowed a classification by function using InterProScan. Expression profiling of all 6,860 genes throughout the H. glycines life cycle was undertaken using the Affymetrix Soybean Genome Array GeneChip. Our data sets and results represent a comprehensive resource for molecular studies of H. glycines. Demonstrating the power of this resource, we were able to address whether arrested development in the Caenorhabditis elegans dauer larva and the H. glycines infective second-stage juvenile (J2) exhibits shared gene expression profiles. We determined that the gene expression profiles associated with the C. elegans dauer pathway are not uniformly conserved in H. glycines and that the expression profiles of genes for metabolic enzymes of C. elegans dauer larvae and H. glycines infective J2 are dissimilar. CONCLUSION: Our results indicate that hallmark gene expression patterns and metabolism features are not shared in the developmentally arrested life stages of C. elegans and H. glycines, suggesting that developmental arrest in these two nematode species has undergone more divergent evolution than previously thought and pointing to the need for detailed genomic analyses of individual parasite species.

Distinct biphasic mRNA changes in response to Asian soybean rust infection.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is now established in all major soybean-producing countries. Currently, there is little information about the molecular basis of ASR-soybean interactions, which will be needed to assist future efforts to develop effective resistance. Toward this end, abundance changes of soybean mRNAs were measured over a 7-day ASR infection time course in mock-inoculated and infected leaves of a soybean accession (PI230970) carrying the Rpp2 resistance gene and a susceptible genotype (Embrapa-48). The expression profiles of differentially expressed genes (ASR-infected compared with the mock-inoculated control) revealed a biphasic response to ASR in each genotype. Within the first 12 h after inoculation (hai), which corresponds to fungal germination and penetration of the epidermal cells, differential gene expression changes were evident in both genotypes. mRNA expression of these genes mostly returned to levels found in mock-inoculated plants by 24 hai. In the susceptible genotype, gene expression remained unaffected by rust infection until 96 hai, a time period when rapid fungal growth began. In contrast, gene expression in the resistant genotype diverged from the mock-inoculated control earlier, at 72 h, demonstrating that Rpp2-mediated defenses were initiated prior to this time. These data suggest that ASR initially induces a nonspecific response that is transient or is suppressed when early steps in colonization are completed in both soybean genotypes. The race-specific resistance phenotype of Rpp2 is manifested in massive gene expression changes after the initial response prior to the onset of rapid fungal growth that occurs in the susceptible genotype.

Developmental transcript profiling of cyst nematode feeding cells in soybean roots.

Cyst nematodes of the genus Heterodera are obligate, sedentary endoparasites that have developed highly evolved relationships with specific host plant species. Successful parasitism involves significant physiological and morphological changes to plant root cells for the formation of specialized feeding cells called syncytia. To better understand the molecular mechanisms that lead to the development of nematode feeding cells, transcript profiling was conducted on developing syncytia induced by the soybean cyst nematode Heterodera glycines in soybean roots by coupling laser capture microdissection with high-density oligonucleotide microarray analysis. This approach has identified pathways that may play intrinsic roles in syncytium induction, formation, and function. Our data suggest interplay among phytohormones that likely regulates synchronized changes in the expression of genes encoding cell-wall-modifying proteins. This process appears to be tightly controlled and coordinately regulated with cell wall rigidification processes that may involve lignification of feeding cell walls. Our data also show local downregulation of jasmonic acid biosynthesis and responses in developing syncytia, which suggest a local suppression of plant defense mechanisms. Moreover, we identified genes encoding putative transcription factors and components of signal transduction pathways that may be important in the regulatory processes governing syncytium formation and function. Our analysis provides a broad mechanistic picture that forms the basis for future hypothesis-driven research to understand cyst nematode parasitism and to develop effective management tools against these pathogens.

Parallel genome-wide expression profiling of host and pathogen during soybean cyst nematode infection of soybean.

Global analysis of gene expression changes in soybean (Glycine max) and Heterodera glycines (soybean cyst nematode [SCN]) during the course of infection in a compatible interaction was performed using the Affymetrix GeneChip soybean genome array. Among 35,611 soybean transcripts monitored, we identified 429 genes that showed statistically significant differential expression between uninfected and nematode-infected root tissues. These included genes encoding enzymes involved in primary metabolism; biosynthesis of phenolic compounds, lignin, and flavonoids; genes related to stress and defense responses; cell wall modification; cellular signaling; and transcriptional regulation. Among 7,431 SCN transcripts monitored, 1,850 genes showed statistically significant differential expression across different stages of nematode parasitism and development. Differentially expressed SCN genes were grouped into nine different clusters based on their expression profiles during parasitism of soybean roots. The patterns of gene expression we observed in SCN suggest coordinated regulation of genes involved in parasitism. Quantitative real-time reverse-transcription polymerase chain reaction confirmed the results of our microarray analysis. The simultaneous genome-wide analysis of gene expression changes in the host and pathogen during a compatible interaction provides new insights into soybean responses to nematode infection and the first profile of transcript abundance changes occurring in the nematode as it infects and establishes a permanent feeding site within a host plant root.

Gene expression profiling in Salmonella Choleraesuis-infected porcine lung using a long oligonucleotide microarray.

Understanding the transcriptional response to pathogenic bacterial infection within food animals is of fundamental and applied interest. To determine the transcriptional response to Salmonella enterica serovar Choleraesuis (SC) infection, a 13,297-oligonucleotide swine array was used to analyze RNA from control, 24-h postinoculation (hpi), and 48-hpi porcine lung tissue from pigs infected with SC. In total, 57 genes showed differential expression (p < 0.001; false discovery rate = 12%). Quantitative real-time PCR (qRT-PCR) of 61 genes was used to confirm the microarray results and to identify pathways responding to infection. Of the 33 genes identified by microarray analysis as differentially expressed, 23 were confirmed by qRT-PCR results. A novel finding was that two transglutaminase family genes (TGM1 and TGM3) showed dramatic increases in expression postinoculation; combined with several other apoptotic genes, they indicated the induction of apoptotic pathways during SC infection. A predominant T helper 1-type immune response occurred during infection, with interferon gamma (IFNG) significantly increased at 48 hpi. Genes induced by IFNs (GBP1, GBP2, C1S, C1R, MHC2TA, PSMB8, TAP1, TAP2) showed increased expression during porcine lung infection. These data represent the first thorough investigation of gene regulation pathways that control an important porcine respiratory and foodborne bacterial infection.

Transcriptional profiling of myostatin-knockout mice implicates Wnt signaling in postnatal skeletal muscle growth and hypertrophy.

Myostatin is an inhibitor of skeletal muscle growth. Disruption of the Mstn gene in mice results in muscles that weigh two to three times those of controls, but precisely how myostatin signals to exert its effects on muscle is unclear. We used the Affymetrix GeneChip system to identify differences in gene expression between myostatin null and wild-type mice. The results indicated a switch in muscle fiber type, from slow to fast, in the absence of myostatin. They also indicated that myostatin may act upstream of Wnt pathway components. Notably, it repressed expression of Wnt4. Wnt4 was capable of stimulating satellite cell proliferation, while inhibition of Wnt signaling down-regulated satellite cell proliferation. This evidence points to a role for Wnt/calcium signaling in the growth and maintenance of postnatal skeletal muscle. This study offers new insight into potential downstream targets of myostatin, which will be beneficial for elucidation of the mechanism through which myostatin acts to inhibit muscle growth.

Validation of a first-generation long-oligonucleotide microarray for transcriptional profiling in the pig.

A first-generation porcine oligonucleotide set, representing 13,297 cDNAs and ESTs, has been designed by Qiagen-Operon for transcriptional profiling. To validate this set, microarrays containing each 70-mer oligonucleotide, referred to as the Qiagen-NRSP8 array, were hybridized with targets from porcine adult liver, lung, muscle, or small intestine. Transcriptome analyses showed that 11,328 of the oligonucleotides demonstrated expression in at least one tissue. Statistical analyses revealed that 1810 genes showed differential expression among tissues (Bonferroni adjusted p < 0.05). Biological pathways identified by DAVID/EASE analysis using a list of 423 tissue-selective genes matched archetypal pathways in the corresponding human or mouse tissue. Real-time quantitative PCR confirmed expression patterns for 9 of 11 genes tested. Our results demonstrate that this first-generation porcine oligonucleotide array is informative and the specificity is high. This is essential validation for investigators using the Qiagen-NRSP8 array for porcine functional genomics and for using the pig in modeling important physiological problems.

Transcriptional profiling using a novel cDNA array identifies differential gene expression during porcine embryo elongation.

A novel porcine cDNA array, containing 1,015 PCR products selected for embryonic expression, was used for transcriptional profiling of conceptuses at four stages of peri-implantation development. Total conceptus RNA from small spherical, large spherical, tubular, and filamentous stages was amplified, converted to cDNA, and hybridized to membranes. Initially, normalized signal intensities obtained using cDNA from total RNA or from amplified RNA were compared. Uniform distribution of P-values associated with t-tests conducted for each gene indicated no evidence that amplification introduced bias. Analysis of data obtained by using amplified targets and the novel array identified genes differentially expressed across stages. Such genes were identified by testing for significant stage effects in gene-specific mixed models. A total of nine genes were declared differentially expressed. Six of the nine genes had P-values less than 0.001, and a false discovery rate of approximately 17% was associated with this significance threshold. Two out of six genes were significant when using the Bonferroni method to control the probability of one or more false positives. The other three genes had P-values between 0.001 and 0.01 and exhibited differences greater than twofold between stages. All four genes selected for confirmation (steroidogenic acute regulatory protein, interleukin 1 beta, transforming growth factor beta 3, and thymosin beta 10) were shown to be differentially expressed by using quantitative real time RT-PCR. Our study shows that RNA amplification is useful for transcriptional profiling with limiting porcine embryonic RNA, and that this novel targeted array can detect differential gene expression during trophoblastic elongation. Finally, our results contribute to an increased understanding of the temporal patterns of expression of known genes controlling conceptus development, as well as identify novel genes also differentially regulated during implantation.