Thérèse Ouellet

Ectopic Expression of BABY BOOM Triggers a Conversion from Vegetative to Embryonic Growth

The molecular mechanisms underlying the initiation and maintenance of the embryonic pathway in plants are largely unknown. To obtain more insight into these processes, we used subtractive hybridization to identify genes that are upregulated during the in vitro induction of embryo development from immature pollen grains of Brassica napus (microspore embryogenesis). One of the genes identified, BABY BOOM (BBM), shows similarity to the AP2/ERF family of transcription factors and is expressed preferentially in developing embryos and seeds. Ectopic expression of BBM in Arabidopsis and Brassica led to the spontaneous formation of somatic embryos and cotyledon-like structures on seedlings. Ectopic BBM expression induced additional pleiotropic phenotypes, including neoplastic growth, hormone-free regeneration of explants, and alterations in leaf and flower morphology. The expression pattern of BBM in developing seeds combined with the BBM overexpression phenotype suggests a role for this gene in promoting cell proliferation and morphogenesis during embryogenesis.

An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance

Acetohydroxy acid synthase (AHAS) is an essential enzyme for many organisms as it catalyzes the first step in the biosynthesis of the branched-chain amino acids valine, isoleucine, and leucine. The enzyme is under allosteric control by these amino acids. It is also inhibited by several classes of herbicides, such as the sulfonylureas, imidazolinones and triazolopyrimidines, that are believed to bind to a relic quinone-binding site. In this study, a mutant allele of AHAS3 responsible for sulfonylurea resistance in a Brassica napus cell line was isolated. Sequence analyses predicted a single amino acid change (557 Trp→Leu) within a conserved region of AHAS. Expression in transgenic plants conferred strong resistance to the three classes of herbicides, revealing a single site essential for the binding of all the herbicide classes. The mutation did not appear to affect feedback inhibition by the branched-chain amino acids in plants.

Genome-wide identification and evaluation of novel internal control genes for Q-PCR based transcript normalization in wheat

To accurately quantify gene expression using quantitative PCR amplification, it is vital that one or more ideal internal control genes are used to normalize the samples to be compared. Ideally, the expression level of those internal control genes should vary as little as possible between tissues, developmental stages and environmental conditions. In this study, 32 candidate genes for internal control were obtained from the analysis of nine independent experiments which included 333 Affymetrix GeneChip Wheat Genome arrays. Expression levels of the selected genes were then evaluated by quantitative real-time PCR with cDNA samples from different tissues, stages of development and environmental conditions. Finally, fifteen novel internal control genes were selected and their respective expression profiles were compared using NormFinder, geNorm, Pearson correlation coefficients and the twofold-change method. The novel internal control genes from this study were compared with thirteen traditional ones for their expression stability. It was observed that seven of the novel internal control genes were better than the traditional ones in expression stability under all the tested cDNA samples. Among the traditional internal control genes, the elongation factor 1-alpha exhibited strong expression stability, whereas the 18S rRNA, Alpha-tubulin, Actin and GAPDH genes had very poor expression stability in the range of wheat samples tested. Therefore, the use of the novel internal control genes for normalization should improve the accuracy and validity of gene expression analysis.

Towards genomic and proteomic studies of protein phosphorylation in plant-pathogen interactions.

Phosphorylation is an effective method of post-translational protein modification but understanding its significance is hindered by its biological complexity. Many protein kinases and phosphatases have been identified that connect signal perception mechanisms to plant defence responses. Recent studies of mitogen-activated protein kinases, calcium-dependent protein kinases and other kinases and phosphatases have revealed some important mechanisms, but have also raised new questions. The regulation of any phosphorylation pathway is complex and dynamic. There are many protein kinases and phosphatases in the plant genome, which makes it hard to delineate the phosphorylation machinery fully. Genomics and proteomics have already identified new components and will continue to influence the study of phosphorylation profoundly in plant-pathogen interactions.

Exogenous H2O2 and catalase treatments interfere with Tri genes expression in liquid cultures of Fusarium graminearum

Effect of exogenous H2O2 and catalase was tested in liquid cultures of the deoxynivalenol and 15‐acetyldeoxynivalenol‐producing fungus Fusarium graminearum. Accordingly to previous results, H2O2 supplementation of the culture medium leads to increased toxin production. This study indicates that this event seems to be linked to a general up regulation of genes involved in the deoxynivalenol and 15‐acetyldeoxynivalenol biosynthesis pathway, commonly named Tri genes. In catalase‐treated cultures, toxin accumulation is reduced, and Tri genes expression is significantly down regulated. Furthermore, kinetics of expression of several Tri genes is proposed in relation to toxin accumulation. Biological meanings of these findings are discussed.

The γ-gliadin multigene family in common wheat (Triticum aestivum) and its closely related species

BackgroundThe unique properties of wheat flour primarily depend on gluten, which is the most important source of protein for human being. γ-Gliadins have been considered to be the most ancient of the wheat gluten family. The complex family structure of γ-gliadins complicates the determination of their function. Moreover, γ-gliadins contain several sets of celiac disease epitopes. However, no systematic research has been conducted yet.ResultsA total of 170 γ-gliadin genes were isolated from common wheat and its closely related species, among which 138 sequences are putatively functional. The ORF lengths of these sequences range from 678 to 1089 bp, and the repetitive region is mainly responsible for the size heterogeneity of γ-gliadins. The repeat motif P(Q/L/S/T/I/V/R/A)F(S/Y/V/Q/I/C/L)P(R/L/S/T/H/C/Y)Q1–2(P(S/L/T/A/F/H)QQ)1–2is repeated from 7 to 22 times. Sequence polymorphism and linkage disequilibrium analyses show that γ-gliadins are highly diverse. Phylogenic analyses indicate that there is no obvious discrimination between Sitopsis and Ae. tauschii at the Gli-1 loci, compared with diploid wheat. According to the number and placement of cysteine residues, we defined nine cysteine patterns and 17 subgroups. Alternatively, we classified γ-gliadins into two types based on the length of repetitive domain. Amino acid composition analyses indicate that there is a wide range of essential amino acids in γ-gliadins, and those γ-gliadins from subgroup SG-10 and SG-12 and γ-gliadins with a short repetitive domain are more nutritional. A screening of toxic epitopes shows that γ-gliadins with a pattern of C9 and γ-gliadins with a short repetitive domain almost lack any epitopes.Conclusionγ-Gliadin sequences in wheat and closely related Aegilops species are diverse. Each group/subgroup contributes differently to nutritional quality and epitope content. It is suggested that the genes with a short repetitive domain are more nutritional and valuable. Therefore, it is possible to breed wheat varieties, the γ-gliadins of which are less, even non-toxic and more nutritional.

Effect of salicylic acid on Fusarium graminearum, the major causal agent of fusarium head blight in wheat

Salicylic acid (SA) is one of the key signal molecules in regulating plant resistance to diverse pathogens. In Arabidopsis thaliana, it is predominantly associated with resistance against biotrophic and hemibiotrophic pathogens, and triggering systemic acquired resistance. In contrast, the effect of SA on the defence efficiency of wheat against fusarium head blight (FHB) and its causal agent, Fusarium graminearum, is still poorly understood. Here we show that the F. graminearum mycelial growth and conidia germination were significantly inhibited, and eventually halted in the presence of increasing concentration of SA in both liquid and solid media. Addition of SA also significantly reduced the production of the mycotoxin deoxynivalenol (DON). However the inhibitory effect of SA required acidic growth conditions to be observed while basic conditions allowed F. graminearum to use SA as a carbon source. High performance liquid chromatography (HPLC) analysis confirmed the capacity of F. graminearum to metabolize SA. To better understand the effect of SA on F. graminearum mycelial growth, we have compared the expression profiles of SA-treated and untreated F. graminearum liquid cultures after 8 and 24 h of treatment, using an F. graminearum custom-commercial microarray. The microarray analysis suggested that F. graminearum can metabolize SA through either the catechol or gentisate pathways that are present in some fungal species. Inoculation of F. graminearum conidia in a SA-containing solution has led to reduced FHB symptoms in the very susceptible Triticum aestivum cv. Roblin. In contrast, no inhibition was observed when SA and conidia were inoculated sequentially. The expression patterns for the wheat PR1, NPR1, Pdf1.2, and PR4 genes, a group of indicator genes for the defence response, suggested that SA-induced resistance contributed little to the reduction of symptoms in our assay conditions. Our results demonstrate that, although F. graminearum has the capacity to metabolize SA, SA has a significant and direct impact on F. graminearum through a reduction in efficiency of germination and growth at higher concentrations.

Low temperature induced defence gene expression in winter wheat in relation to resistance to snow moulds and other wheat diseases.

Cold hardening of winter wheat at 2 °C for 1-6 wks increased resistance to the snow mould pathogens LTB, Typhula incarnata, and Microdochium nivale as well as to powdery mildew (Blumaria graminis f. sp. graminis) and stripe rust (Puccinia striiformis). Using microarrays and hardening of winter wheat for 0.25, 0.5, 1, 7, 21 and 49 d, an upregulation of a wide range of stress-response genes that include defence-related and abiotic stress-related genes, transcription factors including several lipoxygenases and ethylene responsive factors, and WRKY genes was observed. For the majority of these genes, the upregulation occurred later in the 21-49 d hardening treatments and coincided with the highest expression levels of snow mould resistance. Defence-related sequences were upregulated to a greater extent and were more numerous in the snow mould resistant line CI14106 compared to cold hardy DH+268. Transcript profiling of candidate defence and other stress-related genes under prolonged conditions at -3 °C with or without snow mould infection showed that there was a decline in transcripts of the defence-related genes PR1.1b and NPR3 during the 12wks incubation. Additionally, 14 d hardening was insufficient to permit full expression of the jasmonic acid synthesis gene, allene oxide synthase (AOS) and the fructan degrading enzyme β-fructofuranosidase compared the 42 d hardening treatment. The snow mould resistant line CI14106 was able to maintain higher transcript levels of AOS for longer conditions compared to the susceptible line Norstar under artificial snow mould conditions. These results explain the nature of cold-induced resistance to snow moulds and provide direction on establishing selection criteria for improving resistance and cold tolerance in winter wheat.

RNA profiling of fusarium head blight-resistant wheat addition lines containing the Thinopyrum elongatum chromosome 7E

Abstract Fusarium graminearum is the major causal agent of fusarium head blight (FHB) of wheat, a fungal disease causing significant yield losses and reduction in grain quality due to the production and deposition of mycotoxins in the seed. Relatively few sources of resistance to FHB have been identified in wheat and other cereal crops. Thinopyrum elongatum (2n = 14, EE genome), a wild relative of wheat, was identified as a new source with a high level of resistance to FHB. Greenhouse inoculation experiments have determined that the resistance is located on the long arm of its chromosome 7E. To improve our understanding of the molecular mechanisms involved in that resistance, we have used gene expression profiling by microarray to compare the susceptible wheat ‘Chinese Spring’ (CS), with two resistant addition lines containing either the 7E chromosome or a long arm telocentric of that chromosome in the CS background. The results have shown that many genes were affected similarly by F. graminearum in the resistant and susceptible lines. Seventy genes were selected for further characterization. Those genes were grouped into five major expression patterns: genes expressed at higher or lower level in both F. graminearum- and water-treated samples in resistant lines, genes that were upregulated faster or to a higher level in resistant lines and genes that were downregulated more significantly in the susceptible line. Pathogenesis-related genes, genes from the phenylpropanoid pathway or from the jasmonic acid biosynthesis and signalling pathway, a group of kinase proteins and many other genes with unknown function are considered in this report.

Host-preferential Fusarium graminearum gene expression during infection of wheat, barley, and maize

Fusarium graminearum is a broad host pathogen threatening cereal crops in temperate regions around the world. To better understand how F. graminearum adapts to different hosts, we have performed a comparison of the transcriptome of a single strain of F. graminearum during early infection (up to 4 d post-inoculation) of barley, maize, and wheat using custom oligomer microarrays. Our results showed high similarity between F. graminearum transcriptomes in infected wheat and barley spike tissues. Quantitative RT-PCR was used to validate the gene expression profiles of 24 genes. Host-specific expression of genes was observed in each of the three hosts. This included expression of distinct sets of genes associated with transport and secondary metabolism in each of the three crops, as well as host-specific patterns for particular gene categories such as sugar transporters, integral membrane protein PTH11-like proteins, and chitinases. This study identified 69 F. graminearum genes as preferentially expressed in developing maize kernels relative to wheat and barley spikes. These host-specific differences showcase the genomic flexibility of F. graminearum to adapt to a range of hosts.