Sensitivity of Fusarium graminearum to tebuconazole, metconazole, and prothioconazole fungicides

Non-Peer ReviewedTriazole fungicides, along with cultural disease control strategies, has been a useful tool in managing Fusarium head blight (FHB) in the absence of satisfactory levels of FHB resistance in the commercial wheat varieties. Although the triazoles have been used across the world since 1990's for FHB management, there are still very few reports on resistance built-up in the pathogen population. In this study we determined the sensitivity of 254 Fusarium graminearum isolates collected from Western Canada to prothioconazole, metconazole and tebuconazole. Phenotyping for fungicide sensitivity of F. graminearum isolates revealed phenotypic variation in the population for sensitivity to the three fungicides. There was no evidence of cross-resistance between prothioconazole, metconazole and tebuconazole

Establishing a mycotoxin quantification platform to support FHB research and breeding programs

Non-Peer ReviewedFusarium head blight (FHB), caused by Fusarium spp., is a destructive disease of small grain cereals, such as wheat, barley, oat and canaryseed. Apart from grain yield losses and reduced baking and seed quality, a major concern with FHB is crop contamination with Fusarium-produced trichothecene mycotoxins, specifically deoxynivalenol (DON), also known as vomitoxin. These mycotoxins accumulate in the grain making it unfit for consumption by humans and animals. Significant DON contamination may render a crop unmarketable, or reduces the market value by 40-65%. Breeding productive cultivars with high disease resistance and low mycotoxin contamination is a priority for wheat breeders. However, measurement of DON content is not always included in breeding programs due to the lack of efficient quantification methods. Some methods are easy-to-use, but they lack the needed accuracy and sensitive, such as enzyme-linked immunosorbent assay (ELISA); while some chromatographic-based methods have relatively higher accuracy and sensitivity compared to ELISA, but require complex extraction and cleanup steps and longer running time, which are not cost-efficient and environmentally friendly. In this study, we established a tandem mass spectrometry (MS/MS) method, which employed a one-step acetonitrile extraction protocol and flow injection analysis (FIA)-MS/MS method (i.e. no analytical column) to reduce the complexity, cost and time. This method is designed for FHB breeding programs or DON quantification for other purposes that require a fast, high throughput DON phenotyping, but provides relatively high selectivity, accuracy and sensitivity compared to existing assays. This method has been fully validated according to the US Food and Drug Administration (FDA) Guidance for Bioanalytical Method Validation, including selectivity, linearity, accuracy, precision, recovery, matrix effects, stability and dilution integrity. With ease of use, high sensitivity and accuracy, this high throughput DON quantification method will increase breeding efficiency and accelerate the screening progress for FHB resistant germplasm

Discovery of Functional Genes for Systemic Acquired Resistance in Arabidopsis Thaliana through Integrated Data Mining

Various data mining techniques combined with sequence motif information in the promoter region of genes were applied to discover functional genes that are involved in the defense mechanism of systemic acquired resistance (SAR) in Arabidopsis thaliana. A series of K-Means clustering with difference-in-shape as distance measure was initially applied. A stability measure was used to validate this clustering process. A decision tree algorithm with the discover-and-mask technique was used to identify a group of most informative genes. Appearance and abundance of various transcription factor binding sites in the promoter region of the genes were studied. Through the combination of these techniques, we were able to identify 24 candidate genes involved in the SAR defense mechanism. The candidate genes fell into 2 highly resolved categories, each category showing significantly unique profiles of regulatory elements in their promoter regions. This study demonstrates the strength of such integration methods and suggests a broader application of this approach.Diff\ue9rentes techniques d'exploration de donn\ue9es, combin\ue9es \ue0 de l'information sur le motif de s\ue9quence dans la r\ue9gion promotrice de g\ue8nes, ont \ue9t\ue9 appliqu\ue9es pour d\ue9couvrir les g\ue8nes fonctionnels qui interviennent dans le m\ue9canisme de d\ue9fense de la r\ue9sistance syst\ue9mique acquise (RSA ou SAR) chez Arabidopsis thaliana. On a initialement utilis\ue9 une s\ue9rie de classifications par les K moyennes et la diff\ue9rence de forme comme mesure de distance. On a utilis\ue9 une mesure de stabilit\ue9 pour valider ce processus de classification, et un algorithme d'arbre de d\ue9cision ainsi que la technique de d\ue9couverte et de masquage pour identifier un groupe de g\ue8nes sup\ue9rieurement informatifs. On a \ue9tudi\ue9 l'apparence et l'abondance de diff\ue9rents sites de liaison de facteurs de transcription dans la r\ue9gion promotrice des g\ue8nes. En combinant ces techniques, nous avons pu identifier 24 g\ue8nes candidats intervenant dans le m\ue9canisme de d\ue9fense de la RSA. Ces g\ue8nes candidats se classaient dans deux cat\ue9gories hautement r\ue9solues, chacune pr\ue9sentant des profils v\ue9ritablement uniques d'\ue9l\ue9ments r\ue9gulateurs dans leurs r\ue9gions promotrices. Cette \ue9tude d\ue9montre le potentiel de pareilles m\ue9thodes d'int\ue9gration et laisse entrevoir une plus vaste application de cette approche.Peer reviewed: YesNRC publication: Ye

Transgenic increases in seed oil content are associated with the differential expression of novel Brassica-specific transcripts

<p>Abstract</p> <p>Background</p> <p>Seed oil accumulates primarily as triacylglycerol (TAG). While the biochemical pathway for TAG biosynthesis is known, its regulation remains unclear. Previous research identified microsomal diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) as controlling a rate-limiting step in the TAG biosynthesis pathway. Of note, overexpression of <it>DGAT1 </it>results in substantial increases in oil content and seed size. To further analyze the global consequences of manipulating <it>DGAT1 </it>levels during seed development, a concerted transcriptome and metabolome analysis of transgenic <it>B. napus </it>prototypes was performed.</p> <p>Results</p> <p>Using a targeted <it>Brassica </it>cDNA microarray, about 200 genes were differentially expressed in two independent transgenic lines analyzed. Interestingly, 24–33% of the targets showing significant changes have no matching gene in <it>Arabidopsis </it>although these represent only 5% of the targets on the microarray. Further analysis of some of these novel transcripts indicated that several are inducible by ABA in microspore-derived embryos. Of the 200 <it>Arabidopsis </it>genes implicated in lipid biology present on the microarray, 36 were found to be differentially regulated in DGAT transgenic lines. Furthermore, kinetic reverse transcriptase Polymerase Chain Reaction (k-PCR) analysis revealed up-regulation of genes encoding enzymes of the Kennedy pathway involved in assembly of TAGs. Hormone profiling indicated that levels of auxins and cytokinins varied between transgenic lines and untransformed controls, while differences in the pool sizes of ABA and catabolites were only observed at later stages of development.</p> <p>Conclusion</p> <p>Our results indicate that the increased TAG accumulation observed in transgenic <it>DGAT1 </it>plants is associated with modest transcriptional and hormonal changes during seed development that are not limited to the TAG biosynthesis pathway. These might be associated with feedback or feed-forward effects due to altered levels of DGAT1 activity. The fact that a large fraction of significant amplicons have no matching genes in <it>Arabidopsis </it>compromised our ability to draw concrete inferences from the data at this stage, but has led to the identification of novel genes of potential interest.</p

Localization of DIR1 at the tissue, cellular and subcellular levels during Systemic Acquired Resistance in Arabidopsis using DIR1:GUS and DIR1:EGFP reporters

<p>Abstract</p> <p>Background</p> <p>Systemic Acquired Resistance (SAR) is an induced resistance response to pathogens, characterized by the translocation of a long-distance signal from induced leaves to distant tissues to prime them for increased resistance to future infection. DEFECTIVE in INDUCED RESISTANCE 1 (DIR1) has been hypothesized to chaperone a small signaling molecule to distant tissues during SAR in <it>Arabidopsis</it>.</p> <p>Results</p> <p>DIR1 promoter:DIR1-GUS/<it>dir1-1 </it>lines were constructed to examine DIR1 expression. DIR1 is expressed in seedlings, flowers and ubiquitously in untreated or mock-inoculated mature leaf cells, including phloem sieve elements and companion cells. Inoculation of leaves with SAR-inducing avirulent or virulent <it>Pseudomonas syringae </it>pv <it>tomato </it>(<it>Pst</it>) resulted in Type III Secretion System-dependent suppression of DIR1 expression in leaf cells. Transient expression of fluorescent fusion proteins in tobacco and intercellular washing fluid experiments indicated that DIR1's ER signal sequence targets it for secretion to the cell wall. However, DIR1 expressed without a signal sequence rescued the <it>dir1-1 </it>SAR defect, suggesting that a cytosolic pool of DIR1 is important for the SAR response.</p> <p>Conclusions</p> <p>Although expression of DIR1 decreases during SAR induction, the protein localizes to all living cell types of the vasculature, including companion cells and sieve elements, and therefore DIR1 is well situated to participate in long-distance signaling during SAR.</p

A systems genomics and genetics approach to identify the genetic regulatory network for lignin content in Brassica napus seeds

Seed quality traits of oilseed rape, Brassica napus (B. napus), exhibit quantitative inheritance determined by its genetic makeup and the environment via the mediation of a complex genetic architecture of hundreds to thousands of genes. Thus, instead of single gene analysis, network-based systems genomics and genetics approaches that combine genotype, phenotype, and molecular phenotypes offer a promising alternative to uncover this complex genetic architecture. In the current study, systems genetics approaches were used to explore the genetic regulation of lignin traits in B. napus seeds. Four QTL (qLignin_A09_1, qLignin_A09_2, qLignin_A09_3, and qLignin_C08) distributed on two chromosomes were identified for lignin content. The qLignin_A09_2 and qLignin_C08 loci were homologous QTL from the A and C subgenomes, respectively. Genome-wide gene regulatory network analysis identified eighty-three subnetworks (or modules); and three modules with 910 genes in total, were associated with lignin content, which was confirmed by network QTL analysis. eQTL (expression quantitative trait loci) analysis revealed four cis-eQTL genes including lignin and flavonoid pathway genes, cinnamoyl-CoA-reductase (CCR1), and TRANSPARENT TESTA genes TT4, TT6, TT8, as causal genes. The findings validated the power of systems genetics to identify causal regulatory networks and genes underlying complex traits. Moreover, this information may enable the research community to explore new breeding strategies, such as network selection or gene engineering, to rewire networks to develop climate resilience crops with better seed quality

Mining biological information from 3D short time-series gene expression data: the OPTricluster algorithm

<p>Abstract</p> <p>Background</p> <p>Nowadays, it is possible to collect expression levels of a set of genes from a set of biological samples during a series of time points. Such data have three dimensions: gene-sample-time (GST). Thus they are called 3D microarray gene expression data. To take advantage of the 3D data collected, and to fully understand the biological knowledge hidden in the GST data, novel subspace clustering algorithms have to be developed to effectively address the biological problem in the corresponding space.</p> <p>Results</p> <p>We developed a subspace clustering algorithm called Order Preserving Triclustering (OPTricluster), for 3D short time-series data mining. OPTricluster is able to identify 3D clusters with coherent evolution from a given 3D dataset using a combinatorial approach on the sample dimension, and the order preserving (OP) concept on the time dimension. The fusion of the two methodologies allows one to study similarities and differences between samples in terms of their temporal expression profile. OPTricluster has been successfully applied to four case studies: immune response in mice infected by malaria (<it>Plasmodium chabaudi</it>), systemic acquired resistance in <it>Arabidopsis thaliana</it>, similarities and differences between inner and outer cotyledon in <it>Brassica napus </it>during seed development, and to <it>Brassica napus </it>whole seed development. These studies showed that OPTricluster is robust to noise and is able to detect the similarities and differences between biological samples.</p> <p>Conclusions</p> <p>Our analysis showed that OPTricluster generally outperforms other well known clustering algorithms such as the TRICLUSTER, gTRICLUSTER and K-means; it is robust to noise and can effectively mine the biological knowledge hidden in the 3D short time-series gene expression data.</p

Role of transcription factors in storage lipid accumulation in plants

With few exceptions, seeds oil accumulates as triacylglycerols; three fatty acid molecules esterified to a glycerol backbone. The synthesis of normal fatty acids and unusual fatty acids, their assembly into triacylglycerols and subsequent packaging to form oil bodies have been reviewed elsewhere, and appear as separate contributions to the AOCS Lipid Library by Harwood, Hildebrand, Weselake et al., and Huang, respectively. These processes are highly regulated, involving spatial compartmentalization between different organelles and the exquisite control of several biosynthetic steps by one or more of a variety of biochemical mechanisms. The important role of transcriptional regulation towards seed oil accumulation has recently emerged. Not only are mRNA levels of many genes involved in seed oil biosynthesis coordinated during seed development, but altering the levels of certain transcription factors can affect the expression of multiple lipid biosynthetic genes, resulting in altered seed oil content. This article reviews salient features of plant transcription factors that have been implicated in the control of seed oil deposition. To set the stage, fundamental concepts in transcription factor biology are first summarized. Subsequently, the role of key transcription factors is highlighted by describing seed oil phenotypes associated with altering their expression levels by mutagenesis or transgenic means, and providing information on the putative target genes and pathways affected. The location of the transcription factors in hierarchical cascades and the modes of transcriptional regulation are also discussed, when this information is known. A summary of the information presented is depicted in Figure 1.NRC publication: Ye