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      Wheat differential gene expression induced by different races of Puccinia triticina

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          Abstract

          Puccinia triticina, the causal agent of wheat leaf rust, causes significant losses in wheat yield and quality each year worldwide. During leaf rust infection, the host plant recognizes numerous molecules, some of which trigger host defenses. Although P. triticina reproduces clonally, there is still variation within the population due to a high mutation frequency, host specificity, and environmental adaptation. This study explores how wheat responds on a gene expression level to different P. triticina races. Six P. triticina races were inoculated onto a susceptible wheat variety and samples were taken at six days post inoculation, just prior to pustule eruption. RNA sequence data identified 63 wheat genes differentially expressed between the six races. A time course, conducted over the first seven days post inoculation, was used to examine the expression pattern of 63 genes during infection. Forty-seven wheat genes were verified to have differential expression. Three common expression patterns were identified. In addition, two genes were associated with race specific gene expression. Differential expression of an ER molecular chaperone gene was associated with races from two different P. triticina lineages. Also, differential expression in an alanine glyoxylate aminotransferase gene was associated with races with virulence shifts for leaf rust resistance genes.

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          Identification and validation of reference genes for quantitative RT-PCR normalization in wheat

          Background Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR. Results The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and α-tubulin. Conclusion The present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability under all the tested conditions. The new reference genes will enable more accurate normalization and quantification of gene expression in wheat and will be helpful for designing primer pairs targeting orthologous genes in other plant species.
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            The role and regulation of programmed cell death in plant-pathogen interactions.

            It is commonly known that animal pathogens often target and suppress programmed cell death (pcd) pathway components to manipulate their hosts. In contrast, plant pathogens often trigger pcd. In cases in which plant pcd accompanies disease resistance, an event called the hypersensitive response, the plant surveillance system has learned to detect pathogen-secreted molecules in order to mount a defence response. In plants without genetic disease resistance, these secreted molecules serve as virulence factors that act through largely unknown mechanisms. Recent studies suggest that plant bacterial pathogens also secrete antiapoptotic proteins to promote their virulence. In contrast, a number of fungal pathogens secrete pcd-promoting molecules that are critical virulence factors. Here, we review recent progress in determining the role and regulation of plant pcd responses that accompany both resistance and susceptible interactions. We also review progress in discerning the mechanisms by which plant pcd occurs during these different interactions.
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              Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway.

              Virus-induced gene silencing (VIGS) is an important tool for the analysis of gene function in plants. In VIGS, viruses engineered to carry sequences derived from plant gene transcripts activate the host's sequence-specific RNA degradation system. This mechanism targets the RNAs of the viral genome for degradation, and as the virus contains transcribed plant sequence, homologous host mRNAs are also targeted for destruction. While routinely used in some dicots, no VIGS system was known for monocot plants until the recent report of silencing in barley (Hordeum vulgare) by barley stripe mosaic virus (BSMV). Here, we report development of protocols for use of BSMV to efficiently silence genes in hexaploid wheat (Triticum aestivum). The VIGS system was first optimized in studies silencing phytoene desaturase expression. Next, we used it to assay genes functioning in leaf rust resistance mediated by Lr21, which encodes a nucleotide binding site-leucine-rich repeat class resistance gene product. We demonstrated that infection with BSMV constructs carrying a 150-bp fragment of Lr21 caused conversion of incompatible interactions to compatible, whereas infection with a control construct or one that silences phytoene desaturase had no effect on resistance or susceptibility. Additionally, silencing the RAR1, SGT1, and HSP90 genes, known to be required in many but not all nucleotide binding site-leucine-rich repeat resistance pathways in diverse plant species, resulted in conversion to compatibility, indicating that these genes are essential in Lr21-mediated resistance. These studies indicate that BSMV-VIGS is a powerful tool for dissecting the genetic pathways of disease resistance in hexaploid wheat.
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                Author and article information

                Contributors
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: SoftwareRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                7 June 2018
                2018
                : 13
                : 6
                : e0198350
                Affiliations
                [1 ] Department of Plant Pathology, Kansas State University, Manhattan, KS, United States of America
                [2 ] USDA- ARS, Hard Winter Wheat Genetics Research Unit, Manhattan, KS, United States of America
                University of Nebraska-Lincoln, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                [¤]

                Current address: USDA-ARS, Small Grains and Potato Germplasm Research Unit, Aberdeen, ID, United States of America

                Author information
                http://orcid.org/0000-0003-3872-2909
                Article
                PONE-D-17-35294
                10.1371/journal.pone.0198350
                5991701
                29879135
                a1d73cf0-1fae-4697-bfb9-0abaa9188aaa

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 29 September 2017
                : 17 May 2018
                Page count
                Figures: 4, Tables: 2, Pages: 15
                Funding
                This work was supported by the US Congress and the US Department of Agriculture. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Eukaryota
                Plants
                Grasses
                Wheat
                Biology and Life Sciences
                Genetics
                Gene Expression
                Biology and Life Sciences
                Plant Science
                Plant Anatomy
                Leaves
                Biology and life sciences
                Biochemistry
                Proteins
                RNA-binding proteins
                Biology and Life Sciences
                Genetics
                Fungal Genetics
                Biology and Life Sciences
                Mycology
                Fungal Genetics
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Endoplasmic Reticulum
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Secretory Pathway
                Endoplasmic Reticulum
                Biology and Life Sciences
                Organisms
                Eukaryota
                Fungi
                Biology and Life Sciences
                Mycology
                Fungal Structure
                Custom metadata
                Data are available in GenBank (Accession numbers JZ976938-JZ976982).

                Uncategorized
                Uncategorized

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