Effect of transgenes on global gene expression in soybean is within the natural range of variation of conventional cultivars.

Current safety assessment for novel crops, including transgenic crops, uses a targeted approach, which relies on compositional analysis. The possibility that transgene expression could lead to unintended effects remains a debated issue. This study used transcriptome profiling as a nontargeted approach to evaluate overall molecular changes in transgenic soybean cultivars. Global gene expression was measured in the first trifoliate leaves of two transgenic and three conventional soybean cultivars using the soybean Affymetrix GeneChip. It was found that gene expression differs more between the two conventional cultivars than between the transgenics and their closest conventional cultivar investigated and that the magnitudes of differences measured in gene expression and genotype (determined by SSR analysis) do not necessarily correlate. A MySQL database coupled with a CGI Web interface was developed to store and present the results ( http://soyxpress.agrenv.mcgill.ca/). By integrating the microarray data with gene annotations and other soybean data, a comprehensive view of differences in gene expression is explored between cultivars.

Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity.

The interaction between the viral protein linked to the genome (VPg) of turnip mosaic potyvirus (TuMV) and the translation eukaryotic initiation factor eIF(iso)4E of Arabidopsis thaliana has previously been reported. eIF(iso)4E binds the cap structure (m(7)GpppN, where N is any nucleotide) of mRNAs and has an important role in the regulation in the initiation of translation. In the present study, it was shown that not only did VPg bind eIF(iso)4E but it also interacted with the eIF4E isomer of A. thaliana as well as with eIF(iso)4E of Triticum aestivum (wheat). The interaction domain on VPg was mapped to a stretch of 35 amino acids, and substitution of an aspartic acid residue found within this region completely abolished the interaction. The cap analogue m(7)GTP, but not GTP, inhibited VPg-eIF(iso)4E complex formation, suggesting that VPg and cellular mRNAs compete for eIF(iso)4E binding. The biological significance of this interaction was investigated. Brassica perviridis plants were infected with a TuMV infectious cDNA (p35Tunos) and p35TuD77N, a mutant which contained the aspartic acid substitution in the VPg domain that abolished the interaction with eIF(iso)4E. After 20 days, plants bombarded with p35Tunos showed viral symptoms, while plants bombarded with p35TuD77N remained symptomless. These results suggest that VPg-eIF(iso)4E interaction is a critical element for virus production.

Identification of a 37 kDa plant protein that interacts with the turnip mosaic potyvirus capsid protein using anti-idiotypic-antibodies.

Experimental data are provided for the presence of a plant protein that interacts with the capsid protein (CP) of turnip mosaic potyvirus (TuMV). The receptor-like protein was identified by exploiting the molecular mimicry potential of anti-idiotypic antibodies. A single-chain Fv molecule derived from the monoclonal antibody 7A (Mab-7A), which recognizes the CP of TuMV, was produced in Escherichia coli and the recombinant protein was used to raise rabbit antibodies. The immune serum reacted with Mab-7A but not with a monoclonal antibody of the same isotype, indicating that anti-idiotypic antibodies were produced. These anti-idiotypic antibodies recognized a 37 kDa protein from Lactuca sativa. Complex formation between the anti-idiotypic antibodies and the plant protein was inhibited by the CP of TuMV which indicates that the plant protein interacts with the viral protein. The 37 kDa protein was localized in chloroplasts and was detected in other plant species.

Interaction of the viral protein genome linked of turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso) 4E of Arabidopsis thaliana using the yeast two-hybrid system.

The yeast LexA interaction trap was used to screen a cDNA library from Arabidopsis thaliana in order to identify proteins that interact with the viral protein genome linked (VPg)-proteinase of turnip mosaic potyvirus. The screen allowed the isolation of four candidate cDNA clones. Clones pHC4, pHC21, and pHC40 were partially sequenced but no homologies to known proteins were found. However, the amino acid sequence deduced from the complete nucleotide sequence of pSW56 revealed that it was the eukaryotic initiation factor (iso) 4E [eIF(iso)4E]. Deletion analysis indicated that the VPg domain was involved in the interaction with the plant protein. Interaction between the viral protein and the cellular protein was confirmed by ELISA-based binding experiments. eIF(iso)4E plays an essential role in the initiation of the translation of capped mRNAs and its association with VPg would point to a role of the viral protein in the translation of the virus.

Sources and genetic structure of a cluster of genes for resistance to three pathogens in lettuce.

The second largest cluster of resistance genes in lettuce contains at least two downy mildew resistance specificities, Dm5/8 and Dm10, as well as Tu, providing resistance against turnip mosaic virus, and plr, a recessive gene conferring resistance against Plasmopara lactucae-radicis, a root infecting downy mildew. In the present paper four additional genetic markers have been added to this cluster, three RAPD markers and one RFLP marker, CL1795. CL1795 is a member of a multigene family related to triose phosphate isomerase; other members of this family map to the other two major clusters of resistance genes in lettuce. Seven RAPD markers in the region were converted into sequence characterized amplified regions (SCARs) and used in the further analysis of the region and the mapping of Dm10. Three different segregating populations were used to map the four resistance genes relative to molecular markers. There were no significant differences in gene order or rate of recombination between the three crosses. This cluster of resistance genes spans 6.4 cM, with Dm10 1.2 cM from Dm8. Marker analysis of 20 cultivars confirmed multiple origins for Dm5/8 specificity. Two different Lactuca serriola origins for the Du5/8 specificity had previously been described and originally designated as either Dm5 or Dm8. Some ancient cultivars also had the same specificity. Previously, due to lack of recombination in genetic analyses and the same resistance specificities, it was assumed that Dm5 and Dm8 were determined by the same gene. However, molecular marker analysis clearly identified genotypes characteristic of each source. Therefore, Dm5/8 specificity is either ancient and widespread in L. serriola and some L. sativa, or else has arisen on multiple occasions as alleles at the same locus or at linked loci.

Pooled DNA for linkage analysis: practical and statistical considerations.

In an F2 population, the alleles at two loci with a recombination fraction r < 0.5 are in linkage disequilibrium. If r is small, then a pool of DNA from k diploid individuals that are fixed at one locus has a relatively high probability (P = (1 − r)2k) of containing only the coupled allele at the second locus. Based on this principle, several methods have been described to detect linkage (using one or two pools) or to estimate r (using a group of n pools). This report compares maximum likelihood and approximate estimators of r for use in pooled-DNA analysis and illustrates the use of this analysis for dominant markers. Expected values and expected mean squares for estimators of r were computed for varying levels of r, k, and n. Both estimators were biased, but the bias and variability were slightly smaller for the maximum-likelihood estimator. Bias was not severe except when k was large relative to r and (or) n. Methods for optimizing k are discussed relative to several criteria: minimizing variance, minimizing bias, minimizing the probability that linkage cannot be detected, and minimizing the number of samples that must be screened.

A method for detecting DNA polymorphism in large populations.

Molecular markers linked to loci of interest can be used for fine mapping a particular area of a genome or for marker-assisted selection. We present an approach for screening individual plants with polymorphic markers that facilitates phenotyping in large populations. Polymorphic DNA fragments, amplified by PCR, are labelled with digoxigenin and used as probes on slot blots of amplified DNA from the individual plants to be tested. DNA is obtained by a simple two-tube purification method. The colorimetric detection of alleles on the blots is more reliable, and more amenable to automation, than conventional staining of electrophoresis gels.

Genetic mapping of turnip mosaic virus resistance in Lactuca sativa.

Presence of the dominant Tu gene in Lactuca sativa is sufficient to confer resistance to infection by turnip mosaic virus (TuMV). In order to obtain an immunological assay for the presence of TuMV in inoculated plants, the TuMV coat protein (CP) gene was cloned by amplification of a cDNA corresponding to the viral genome using degenerate primers designed from conserved potyvirus CP sequences. The TuMV CP was overexpressed in Escherichia coli, and polyclonal antibodies were produced. To locate Tu on the L. sativa genetic map, F3 families from a cross between cvs "Cobbham Green" (resistant to TuMV) and "Calmar" (susceptible) were genotyped for Tu. Families known to be recombinant in the region containing Tu were infected with TuMV and tested by the indirect enzyme-linked immunosorbent assay (ELISA) using the anti-CP serum. This assay placed Tu between two random amplified polymorphic DNA (RAPD) markers and 3.2 cM from Dm5/8 (which confers resistance to Bremia lactucae). Also, bulked segregant analysis was used to screen for additional RAPD markers tightly linked to the Tu locus. Five new markers linked to Tu were identified in this region, and their location on the genetic map was determined using informative recombinants in the region. Six markers were identified as being linked within 2.5 cM of Tu.

Random amplified polymorphic DNA and pedigree relationships in spring barley.

We investigated random amplified polymorphic DNA (RAPD) in 27 inbred barley lines with varying amounts of common ancestry and in 20 doubled-haploid (DH) lines from a biparental cross. Of 33 arbitrary 10 base primers that were tested, 19 distinguished a total of 31 polymorphisms. All polymorphisms were scored as dominant genetic markers except for 1, where Southern analysis indicated the presence of two codominant amplification products. The inheritance of 19 RAPD polymorphisms and one morphological trait was studied in the DH lines. There was no evidence for segregation distortion, but a group of four tightly linked loci was detected. The frequencies of RAPD polymorphism in pairs of inbred lines were used to compute values of genetic distance (d), which were compared to kinship coefficients (r) between the same pairs of lines. A linear relationship between r and d was evident, but low values of r gave poor predictions of d. Cluster analysis showed that groups of inbred lines based on r were similar to those based on d with some notable exceptions. RAPD markers can be used to gain information about genetic similarities or differences that are not evident from pedigree information.

Reduced levels of hsp90 compromise steroid receptor action in vivo.

Signalling by steroid hormones is mediated by receptor proteins that bind hormonal ligands and regulate the transcription of specific genes. The heat-shock protein hsp90 seems to associate selectively with unliganded receptors (aporeceptors), but it has not been determined whether this interaction affects receptor function in vivo. To address the role of hsp90, we have taken advantage of the capacity of mammalian steroid receptors to function in yeast. We constructed a strain of Saccharomyces cerevisiae in which hsp90 expression was regulatable and could be reduced more than 20-fold relative to wild type. At low levels of hsp90, aporeceptors seem to be mostly hsp90-free, yet fail to enhance transcription; on hormone addition, the receptors are activated but with markedly reduced efficiency. Thus hsp90 does not inhibit receptor function solely by steric interference; rather, hsp90 seems to facilitate the subsequent response of the aporeceptor to the hormonal signal. This is the first biological evidence that hsp90 acts in the signal transduction pathway for steroid receptors.

Several nodulins of soybean share structural domains but differ in their subcellular locations.

Four soybean cDNA nodule-specific clones encoding nodulin-23, -26b, -27 and -44 were observed to cross-hybridize under low stringency conditions. Nucleotide sequence analysis revealed that the cDNAs contain three distinct domains: two domains with 70 to 95% homology separated by a third domain unique to each cDNA. Despite a number of nucleotide insertions and deletions, the protein sequences are conserved in the two domains which correlate with the homologous nucleotide domains. The amino terminal domain of each nodulin contains putative signal sequences for membrane translocation, although only two (nodulin-23 and -44) meet all the criteria for a functional signal. Immuno-precipitation of hybrid-release translation products of the four cDNAs revealed that nodulin-23 is associated with the peribacteroid membrane while nodulin-27 is in the cytoplasmic fraction of the nodule. These four nodulins are members of a diverse family with conserved structural features and the genes encoding them appear to have recently evolved from a common ancestor.

Nodulin-26, a peribacteroid membrane nodulin is expressed independently of the development of the peribacteroid compartment.

The peribacteroid membrane (pbm) of root nodules is derived from the plant cell plasma membrane but contains in addition several nodule-specific host proteins (nodulins). Antibodies raised against purified pbm of soybean were used to immunoprecipitate polysomes to isolate an RNA fraction that served as a template for the synthesis of a cDNA probe for screening a nodule-specific cDNA library. Clone p1B1 was found to encode a 26.5 kDa polypeptide (nodulin-26) which is immunoprecipitable specifically with the anti-pbm serum. Nodulin-26 has features of a transmembrane protein and its structure differs from that of nodulin-24 which appears to be a surface protein of pbm. The expression of these two pbm nodulins was examined in nodules induced by Bradyrhizobium japonicum Tn5 mutants that arrest nodule development at different stages of pbm biosynthesis. Nodules that do not show release of bacteria from the infection thread express nodulin-24 at a very low level. In contrast, the expression of nodulin-26 occurs fully in nodules that form infection threads only and is not affected by the release of bacteria from the threads.

Nodulins and nodulin genes of Glycine max.

Nodulins are organ-specific plant proteins induced during symbiotic nitrogen fixation. Nodulins play both metabolic and structural roles within infected and uninfected nodule cells. In soybean, several nodulin genes, coding for abundant nodulins, have been identified and isolated. Structural analysis of some of these genes has revealed their possible mode of regulation and the subcellar location of the protein product. Studies of ineffective symbiosis based on cultivar-strain genotype differences suggested that both partners influence the expression of nodulin genes. Concomitant with nodule organogenesis, the Rhizobium undergoes substantial differentiation leading to the accumulation of nodule-specific bacterial proteins, bacteroidins. The major structural alteration occuring in the infected cell is the formation of a membrane enclosing the bacteroid (peribacteroid membrane). A number of nodulins are specifically targetted to this membrane during endosymbiosis. The induction of nodulins and bacteroidins leads to the formation of an effective nodule. Nodulin genes can be induced in vitro by factors derived from nodules suggesting that trans-activators may be involved in derepression of the host genes necessary for Rhizobium-legume symbiosis.

Specific targeting of membrane nodulins to the bacteroid-enclosing compartment in soybean nodules.

Rhizobium bacteroids in nodule cells are surrounded by the peribacteroid membrane (pbm), which is derived from the host plasma membrane during infection. The pbm was purified from R. japonicum 61A76-induced soybean nodules and analyzed by comparing it with the host cell plasma membrane for the presence of nodulins, nodule-specific plant proteins. Nodulins were found in pbm by reacting Western blots with a nodule-specific antiserum raised against the pbm. Peribacteroid fluid (the fluid enclosed in the pbm) was also found to contain several nodulins. The pbm nodulins were confirmed to be of plant origin by in vitro translation of poly(A) nodule mRNA followed by immunoprecipitation by the nodule-specific antiserum. Antibodies raised against a synthetic peptide corresponding to a repeated domain in nodulin-24, a pbm nodulin, and the nodule-specific pbm antiserum reacted exclusively with the pbm. The absence of pbm-nodulins in the plasma membrane suggests that the infected cells direct the intracellular transport of the pbm nodulins exclusively to this de novo synthesized subcellular compartment essential for symbiotic nitrogen fixation.

Presence of Host-Plasma Membrane Type H-ATPase in the Membrane Envelope Enclosing the Bacteroids in Soybean Root Nodules.

An improved method is described for the isolation of membrane envelope enclosing the bacteroids (peribacteroid membrane) from soybean (Glycine max L.) root nodules. The ATPase activity of the peribacteroid membrane from infected roots is compared with that of the plasma membrane from uninfected roots. The two ATPases are similar in terms of their vanadate sensitivities, pH optima, and mineral cation requirements, and show antigenic cross-reactivity. However, the ATPase of peribacteroid membrane is more sensitive to stimulation by NH(4) (+). ATP-dependent proton translocation across the peribacteroid membrane was demonstrated in broken protoplasts of infected cells, by the use of fluorescence microscopy with acridine orange. It is suggested that acidification of the peribacteroid space by the peribacteroid membrane ATPase results in the conversion of NH(3) to NH(4) (+) in this space and thereby facilitates the removal of fixed-nitrogen from the bacteroid.