Identification of genomic loci conferring broad-spectrum resistance to multiple nematode species in exotic soybean accession PI 567305.

KEY MESSAGE: Genetic analysis identified a unique combination of major QTL for resistance to important soybean nematodes concurrently present in a single soybean accession, which has not been reported earlier. An exotic soybean [Glycine max (L.) Merr.] accession, PI 567305, was reported to be highly resistant to three important nematode species, soybean cyst (SCN), root-knot (RKN), and reniform (RN) nematodes. However, genetic basis controlling broad-spectrum resistance in this germplasm has not been investigated. We report results of genetic analysis to identify genomic loci conferring resistance to these nematode species. A bi-parental population consisting of 242 F8-derived recombinant inbred lines (RILs) was developed from a cross of a nematode susceptible cultivar, Magellan, and resistant accession, PI 567305. The RILs were phenotyped for nematode resistance to three SCN HG types. They were genotyped using the Infinium SoySNP6K BeadChips and genotype-by-sequencing (GBS) methods in an attempt to evaluate the cost-effectiveness and efficiency of these two genotyping platforms. Genetic analysis confirmed the major QTL on chromosomes (Chrs) 10 and 18 with broad-spectrum resistance to the three nematodes present in this germplasm. Haplotype and copy number variation analyses of SCN resistance QTL indicated that PI 567305 has a different haplotype, which is associated with likely a unique SCN resistance mechanism different from Peking- or PI 88788-type resistance. The evaluations of both Infinium Beadchip- and GBS-based genotyping technologies provided comprehensive insights for researchers to choose a cost-effective and efficient platform for QTL mapping and for other genomic studies in soybeans.

A comprehensive transcriptomic analysis of the effect of silicon on wheat plants under control and pathogen stress conditions.

The supply of soluble silicon (Si) to plants has been associated with many benefits that remain poorly explained and often contested. In this work, the effect of Si was studied on wheat plants under both control and pathogen stress (Blumeria graminis f. sp. tritici) conditions by conducting a large transcriptomic analysis (55,000 unigenes) aimed at comparing the differential response of plants under four treatments. The response to the supply of Si on control (uninfected) plants was limited to 47 genes of diverse functions providing little evidence of regulation of a specific metabolic process. Plants reacted to inoculation with B. graminis f. sp. tritici by an upregulation of many genes linked to stress and metabolic processes and a downregulation of genes linked to photosynthesis. Supplying Si to inoculated plants largely prevented disease development, a phenotypic response that translated into a nearly perfect reversal of genes regulated by the effect of B. graminis f. sp. tritici alone. These results suggest that Si plays a limited role on a plant's transcriptome in the absence of stress, even in the case of a high-Si-accumulating monocot such as wheat. On the other hand, the benefits of Si in the form of biotic stress alleviation were remarkably aligned with a counter-response to transcriptomic changes induced by the pathogen B. graminis f. sp. tritici.

Identification of genes potentially involved in the biocontrol activity of Pseudozyma flocculosa.

Flocculosin is an antifungal cellobiose lipid linked to the biocontrol activity of Pseudozyma flocculosa and whose structure is very similar to that of ustilagic acid produced by Ustilago maydis. In this work, homologs of the U. maydis cyp1 gene, involved in the biosynthesis of ustilagic acid, were isolated and sequenced from P. flocculosa and P. fusiformata, the latter species being also known to produce ustilagic acid. Interestingly, no homologs were found in four other closely related Pseudozyma spp. from which no evidence of ustilagic acid production has ever been obtained, thus supporting the specificity of cyp1 with ustilagic acid synthesis. In addition, a homolog of the U. maydis uat1 gene involved in the acetylation of the molecule and located next to the cyp1 gene was partially sequenced from P. flocculosa. All three newly sequenced genes showed strong sequence similarity to their counterparts in U. maydis. Cyp1 expression was monitored in conditions that were either conducive or repressive to flocculosin production. Expression increased markedly (>100x) when P. flocculosa was inoculated in a growth medium conducive to flocculosin production but was rapidly downregulated in a repressive medium (in vitro) or on powdery mildew-infected cucumber leaves (in vivo). This suggests that the molecule was preferentially synthesized early in the process of searching for a growth substrate. This study provides the first identification of genes involved in the production of flocculosin, a molecule potentially associated with the biocontrol properties of P. flocculosa.

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.

Alternative methods for genetic transformation of Pseudozyma antarctica, a basidiomycetous yeast-like fungus.

Electroporation and Agrobacterium tumefaciens-mediated transformation (ATMT) were adapted and optimized for genetic transformation of the basidiomycetous yeast-like fungus Pseudozyma antarctica as alternatives to the cumbersome PEG/CaCl(2)-mediated transformation of protoplasts. Electroporation yielded 100-200 transformants per mug of DNA per 10(8) cells after 3 days on selective medium. For its part, ATMT yielded 60-160 transformants per 10(6) input cfu after 5-10 days on a selective medium. Transformants obtained from both methods showed stable hygromycin resistance and strong expression of green fluorescent protein. Analysis of integration events revealed a limited number of predominantly tandem insertions in the genome of transformants, an improvement over PEG/CaCl(2)-mediated transformation. Both protocols relied on intact conidia of P. antarctica as starting material and thus eliminated the need for cell wall-degrading or weakening agents such as lytic enzymes or chemicals. Other advantages over protoplast transformation included higher yield of transformants and shorter recovery time of transformed colonies on selective medium.

The potential of Pseudozyma yeastlike epiphytes for the production of heterologous recombinant proteins.

Although Basidiomycetes represent the most evolved class of fungi, they have been neglected with regard to recombinant gene expression. In this work, basidiomycetous yeasts belonging to Pseudozyma spp. were studied with respect to their amenability to heterologous protein production. Single plasmid or cotransformation experiments routinely afforded 100 to 200 independent transformants for the two tested species of Pseudozyma. Green fluorescent protein (GFP) was expressed in the correctly folded conformation, as demonstrated by fluorescence microscopy, and hen egg white lysozyme (HEWL) was expressed in its active form, as revealed by its lytic activity on Micrococcus lysodeikticus cells. Protease analysis established that Pseudozyma spp. contained equivalent or less extracellular protease activity than yeasts and far less protease activity than ascomycetous filamentous fungi in similar culture conditions. This proteolytic activity was inhibited by over 97% with a combination of PMSF and Pepstatin A. N-glycosylation patterns of native Pseudozyma flocculosa secreted proteins were comprised of one or a few short glycan chains that possess a classic eukaryotic structure typical of higher fungi and animal cells. This is the first report of a Basidiomycete that possesses multiple intrinsic characteristics necessary for use as a heterologous gene expression system.

Giant spinal perimedullary fistula in hereditary haemorrhagic telangiectasia: diagnosis, endovascular treatment and review of the literature.

Hereditary hemorrhagic telangiectasia (HHT, Rendu-Osler-Weber disease) is a vascular disorder with dominant autosomal transmission characterised usually by multiple mucocutaneous and visceral abnormalities. Neurological manifestations due to the primary involvement of spinal cord by vascular malformations are rare. We present a young man with HHT associated with a central nervous system arteriovenous malformation and a giant perimedullary fistula, that was manifested as progressive myelopathy. The diagnosis was made coupling magnetic resonance imaging to selective spinal arteriography. The therapeutic option was endovascular treatment by mechanically detachable coils which resulted in full exclusion of the fistula with full improvement of symptoms. During follow-up a stable clinical and morphological outcome was achieved. Clinical manifestations in HHT, with emphasis on neurological symptoms, are reviewed as well as the therapeutic options to deal with giant perimedullary fistula.

Posterior cervical haemangiopericytoma with intracranial and skull base extension. Diagnostic and therapeutic challenge of a rare hypervascular neoplasm.

Haemangiopericytomas are rare hypervascular tumors arising from pericytes. They may occur anywhere in the body, but posterior cervical location is rather uncommon. A case of posterior cervical haemangiopericytoma with posterior fossa and temporal bone extension is reported. Although the patient had undergone preoperative endovascular embolization and surgical resection on three separate occasions, control of the skull base extension was not successful. Following endovascular embolization combined with radiotherapy, the patient has been asymptomatic for 48 months. Angiographic features may help in differentiating haemangiopericytomas from other hypervascular lesions. Preoperative endovascular embolization is recommended due to the pronounced tendency for haemorrhage throughout biopsy and surgical procedures.

An improved genetic linkage map for cowpea (Vigna unguiculata L.) combining AFLP, RFLP, RAPD, biochemical markers, and biological resistance traits.

An improved genetic linkage map has been constructed for cowpea (Vigna unguiculata L. Walp.) based on the segregation of various molecular markers and biological resistance traits in a population of 94 recombinant inbred lines (RILs) derived from the cross between 'IT84S-2049' and '524B'. A set of 242 molecular markers, mostly amplified fragment length polymorphism (AFLP), linked to 17 biological resistance traits, resistance genes, and resistance gene analogs (RGAs) were scored for segregation within the parental and recombinant inbred lines. These data were used in conjunction with the 181 random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), AFLP, and biochemical markers previously mapped to construct an integrated linkage map for cowpea. The new genetic map of cowpea consists of 11 linkage groups (LGs) spanning a total of 2670 cM, with an average distance of 6.43 cM between markers. Astonishingly, a large, contiguous portion of LG1 that had been undetected in previous mapping work was discovered. This region, spanning about 580 cM, is composed entirely of AFLP markers (54 in total). In addition to the construction of a new map, molecular markers associated with various biological resistance and (or) tolerance traits, resistance genes, and RGAs were also placed on the map, including markers for resistance to Striga gesnerioides races 1 and 3, CPMV, CPSMV, B1CMV, SBMV, Fusarium wilt, and root-knot nematodes. These markers will be useful for the development of tools for marker-assisted selection in cowpea breeding, as well as for subsequent map-based cloning of the various resistance genes.

Establishment of a gene transfer system for Pseudozyma flocculosa, an antagonistic fungus of powdery mildew fungi.

A reliable DNA-mediated transformation system has been developed for Pseudozyma flocculosa, a fungus that is antagonistic to powdery-mildew fungi. Plasmids harboring various selectable markers under the control of different promoters were tested. Molecular analyses demonstrated that successful transformation could be achieved using a plasmid that confers resistance to hygromycin B under the control of the Ustilago maydis hsp70 promoter and terminator sequences. On average, 1-40 (mean = 20) transformants were obtained per 10 microg of linearized DNA per 10(8) protoplasts. Southern analysis of the transformants revealed that, in each case, the vector had integrated in multiple tandem copies into the genome of P. flocculosa, and that integration events were random. Pulsed-field gel electrophoresis was employed to separate the genome of P. flocculosa into at least 11 chromosomes with sizes ranging from 0.55 Mb to 2.9 Mb. Hybridization with the plasmid indicated that integration of vector DNA had occurred in one to several chromosomes depending on the transformant examined.

Bacillus thuringiensis cry3Ca1 protein is toxic to the Colorado potato beetle, Leptinotarsa decemlineata (Say).

We expressed the wild-type cry3Aa3 and cry3Ca1 Bacillus thuringiensis genes, which code for insecticidal proteins, in an Escherichia coli expression system. Highly purified preparations of the soluble delta-endotoxins were used to perform comparative bioassays with third-instar larvae of the Colorado potato beetle (CPB). Acute mortality data showed that Cry3Ca1 (LD(50) = 320.1 ng) was 2-fold more toxic than Cry3Aa3 (LD(50) = 672.9 ng). We also compared the chronic effects of sublethal doses of these toxins by measuring the consumption of untreated foliage and monitoring survival and development for 6 days after intoxication. No significant additional mortality was recorded, but we found that surviving larvae fed Cry3Ca1 consumed foliage at a slower rate than the larvae fed Cry3Aa3, suggesting more damage to their digestive epithelium. This study, the first assessment of the toxicity of cry3Ca1 in third-instar CPB, suggests cry3Ca1 will prove useful for the control of this important insect pest.

Premature polyadenylation contributes to the poor expression of the Bacillus thuringiensis cry3Ca1 gene in transgenic potato plants.

The cry genes that code for the insecticidal crystal proteins of Bacillus thuringiensis (B.t.) have been widely used to develop insect-resistant transgenic plants. The cry3Ca1 gene has been reported to code for a crystal protein which is particularly potent against the Colorado potato beetle (CPB). To explore the biotechnological potential of cry3Ca1, we introduced this gene into transgenic potato plants under the control of the CaMV 35S promoter. In the resulting transformants, the cry3-Ca1 gene was very poorly expressed. In fact, no full-length transcript (2300 nt) could be detected. Instead, only short transcripts of approximately 1100 nt were observed. Analysis of these short transcripts by Northern hybridization, RT-PCR as well as by cloning and sequencing showed that they resulted from premature polyadenylation. These processing events occurred at four sites within the cry3Ca1 coding region (at positions 652, 669, 914 and 981 relative to the translation start site). The sites at which premature polyadenylation took place were not those that showed the highest degree of identity to the canonical AAUAAA motif. Together with other recent data, our findings suggest that premature polyadenylation is an important mechanism which can contribute to the poor expression of transgenes in a foreign host.

Hairpin elements, the first family of foldback transposons (FTs) in Arabidopsis thaliana.

We report here on the identification in Arabidopsis thaliana of a new family of transposable elements named Hairpin. These elements are related to foldback transposons (FTs), a large and heterogeneous group of transposable elements first described in Drosophila and recently in Solanaceae. Hairpin elements are the first family of FTs reported in Arabidopsis thaliana and the first family of FTs of type 3 to be described in the plant kingdom. In contrast to previous FTs described, Hairpin appears to be a homogeneous family in size (238 +/- 7 bp) as well as in structure. Hairpin elements are dispersed in the Arabidopsis genome and Southern hybridization revealed that they are present in relatively low copy numbers. Finally, we discuss the potential usefulness of these elements in studying the phylogenetic relationship between Arabidopsis ecotypes.

Four mismatch repair paralogues coexist in Arabidopsis thaliana: AtMSH2, AtMSH3, AtMSH6-1 and AtMSH6-2.

By using degenerate oligonucleotides based on the sequence homology between known MutS homologues, three MSH cDNAs belonging to the MSH2, MSH3 and MSH6 families, as defined in eukaryotes, have been isolated from Arabhidopsis thaliana (ecotype Columbia). Genomic sequences for two of these genes (AtMSH2 and AtMSH6-2) were also isolated and determined, whereas the genomic sequence of AtMSH3 was obtained through the Arabidopsis sequencing project, as was the sequence of a second, distinct AtMSH6 homologue (AtMSH6-1). Comparative analysis of the AtMSH2 Landsberg erecta genomic sequence (reported here) and the previously described AtMSH2 Columbia allele revealed several polymorphisms, including the presence of a small, transposon-like element in the 3' untranscribed region of the former allele. Arabidopsis is the first organism to show such divergence of two AtMSH6 genes; the divergence is strongly supported by sequence data and phylogenetic analysis. Southern analysis revealed that the three genes we have isolated exist as single copies, and genetic mapping indicated that AtMSH2 and AtMSH6-2 both reside on chromosome III. Finally, expression of these three genes could only be observed in suspensions of A. thaliana cells. Such a cell suspension divides actively after subculture, and the AtMSH genes are most strongly expressed at this stage.

Random chromosome segregation without meiotic arrest in both male and female meiocytes of a dmc1 mutant of Arabidopsis.

In yeast, the DMC1 gene is required for interhomolog recombination, which is an essential step for bivalent formation and the correct partition of chromosomes during meiosis I. By using a reverse genetics approach, we were able to identify a T-DNA insertion in AtDMC1, the Arabidopsis homolog of DMC1. Homozygotes for the AtDMC1 insertion failed to express AtDMC1, and their residual fertility was 1.5% that of the wild type. Complete fertility was restored in mutant plants when a wild-type copy of the AtDMC1 gene was reintroduced. Cytogenetical analysis points to a correlation of the sterility phenotype with severely disturbed chromosome behavior during both male and female meiosis. In this study, our data demonstrate that AtDMC1 function is crucial for meiosis in Arabidopsis. However, meiosis can be completed in the Arabidopsis dmc1 mutant, which is not the case for mouse or some yeast mutants.

Isolation and characterization of AtMLH1, a MutL homologue from Arabidopsis thaliana.

DNA mismatch repair systems play an essential role in the maintenance of genetic information in living organisms and are also implicated in genetic recombination and genome stability. Using degenerate primers, we have cloned the first plant homologue of the E. coli MutL gene, which we have called AtMLH1 for Arabidopsis thaliana MutL-homologue 1. AtMLH1 is present as a single-copy gene in the Arabidopsis genome and is located on the top arm of chromosome 4. Sequence analysis revealed that the product of this gene shows extensive sequence homology with other eukaryotic MLH1 proteins. As mlh1-deficient lines would be useful for studying the biological function of this gene, several populations that had been mutagenized using T-DNA and transposon insertions were screened to identify such mutants. One line that carries a T-DNA insertion in the promoter region of the AtMLH1 gene was isolated. Surprisingly, although the insertion occurred only approximately 80 bp upstream of the putative transcription start site, Northern analyses revealed very low but similar amounts of AtMLH1 transcript in both the wild type and the T-DNA insertion lines. RT-PCR analyses suggest, however, that transcription is initiated further upstream in the insertion line and that the T-DNA may supply this novel initiation site. Finally, no increase in microsatellite instability - a phenotype often associated with mutations in mismatch repair genes - was observed in plants homozygous for this insertion.

Regional insertional mutagenesis on chromosome III of Arabidopsis thaliana using the maize Ac element.

The authors describe the production and characterization of a collection of Arabidopsis lines each carrying a transposed Ac (trAc) element. A total of 507 lines were obtained following germinal transpositions of a single Ac element located on the upper portion of chromosome III. Southern analysis revealed that up to 90% of the lines in this collection harbour distinct insertions of Ac in the Arabidopsis genome. As previous studies on the behaviour of Ac in Arabidopsis have indicated that approximately two out of three transposition events occur to linked loci, the authors hypothesized that this collection could be of great use in isolating insertional mutants for genes located in the vicinity of the donor locus. PCR and phenotypic screens were performed to identify mutations in five loci located within a 40 cm region of chromosome III centered on the donor locus. Molecular analyses confirmed the presence of germinal insertions of Ac in three of the loci (NPTII, ABI3 and EST #210A22). At a fourth locus (AtDMC1), despite the absence of a germinal insertion, one line in which somatic insertions occurred regularly was identified and may be of use in isolating a germinal insertion. This collection of trAc lines constitutes a useful complement to the existing collection of T-DNA insertion lines and will soon be made available through the Arabidopsis Biological Resource Center.

The use of confocal microscopy in the investigation of cell structure and function in the heart, vascular endothelium and smooth muscle cells.

In recent years, fluorescence microscopy imaging has become an important tool for studying cell structure and function. This non invasive technique permits characterization, localisation and qualitative quantification of free ions, messengers, pH, voltage and a pleiad of other molecules constituting living cells. In this paper, we present results using various commercially available fluorescent probes as well as some developed in our laboratory and discuss the advantages and limitations of these probes in confocal microscopy studies of the cardiovascular system.

Increased Ac excision (iae): Arabidopsis thaliana mutations affecting Ac transposition.

The maize transposable element Ac is highly active in the heterologous hosts tobacco and tomato, but shows very much reduced levels of activity in Arabidopsis. A mutagenesis experiment was undertaken with the aim of identifying Arabidopsis host factors responsible for the observed low levels of Ac activity. Seed from a line carrying a single copy of the Ac element inserted into the streptomycin phosphotransferase (SPT) reporter fusion, and which displayed typically low levels of Ac activity, were mutagenized using gamma rays. Nineteen mutants displaying high levels of somatic Ac activity, as judged by their highly variegated phenotypes, were isolated after screening the M2 generation on streptomycin-containing medium. The mutations fall into two complementation groups, iae1 and iae2, are unlinked to the SPT::Ac locus and segregate in a Mendelian fashion. The iae1 mutation is recessive and the iae2 mutation is semi-dominant. The iae1 and iae2 mutants show 550- and 70-fold increases, respectively, in the average number of Ac excision sectors per cotyledon. The IAE1 locus maps to chromosome 2, whereas the SPT::Ac reporter maps to chromosome 3. A molecular study of Ac activity in the iae1 mutant confirmed the very high levels of Ac excision predicted using the phenotypic assay, but revealed only low levels of Ac re-insertion. Analyses of germinal transposition in the iae1 mutant demonstrated an average germinal excision frequency of 3% and a frequency of independent Ac re-insertions following germinal excision of 22%. The iae mutants represents a possible means of improving the efficiency of Ac/Ds transposon tagging systems in Arabidopsis, and will enable the dissection of host involvement in Ac transposition and the mechanisms employed for controlling transposable element activity.

Inefficient and incorrect processing of the Ac transposase transcript in iae1 and wild-type Arabidopsis thaliana.

As part of the analysis of the Arabidopsis mutant iae1-1 (increased Ac excision), quantitative studies of the Ac transposase transcript were conducted. The primary transcript of Ac contains three small introns (introns 1-3; mean size 89 bp) and one larger intron (intron 4; 387 bp). We analysed the splicing of intron 3 and intron 4 in wild-type Arabidopsis and the iae1-1 mutant. Our results demonstrate that the splicing of Ac introns 3 and 4 is inefficient (splicing efficiencies 57 and 30% respectively) compared with that of an intron of an endogenous Arabidopsis gene (PHYB intron 1; splicing efficiency 90%). The poor splicing efficiency of Ac intron 4 was found to correlate with aberrant processing. Steady state levels of total Ac transcript were higher in the iae1-1 mutant than wild-type, but the same aberrant processing occurred. The inefficient processing of Ac in Arabidopsis prompted us to construct an Ac element lacking introns (Ac::cDNA) in an attempt to increase transposition frequencies. Autonomous activity of the Ac::cDNA element was undetectable in Arabidopsis, despite its ability to transpose at high frequency in response to a strong transposase source (35S::transposase) in trans, and the demonstrable autonomy of the same element in tobacco. A number of smaller transcripts were detected in Arabidopsis lines containing Ac::cDNA or Ac. Analysis of these smaller transcripts revealed a high frequency of premature polyadenylation in exon 2 and splicing of cryptic introns.