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Reference gene selection and validation for the early responses to downy mildew infection in susceptible and resistant Vitis vinifera cultivars.

ABSTRACT: The pivotal role of cultivated grapevine (Vitis vinifera L.) in many countries economy is compromised by its high susceptibility to Plasmopara viticola, the causal agent of downy mildew disease. Recent research has identified a set of genes related to resistance which may be used to track downy mildew infection. Quantification of the expression of these resistance genes requires normalizing qPCR data using reference genes with stable expression in the system studied. In this study, a set of eleven genes (VATP16, 60 S, UQCC, SMD3, EF1?, UBQ, SAND, GAPDH, ACT, PsaB, PTB2) was evaluated to identify reference genes during the first hours of interaction (6, 12, 18 and 24 hpi) between two V. vinifera genotypes and P. viticola. Two analyses were used for the selection of reference genes: direct comparison of susceptible, Trincadeira, and resistant, Regent, V. vinifera cultivars at 0 h, 6, 12, 18 and 24 hours post inoculation with P. viticola (genotype effect); and comparison of each genotype with mock inoculated samples during inoculation time-course (biotic stress effect). Three statistical methods were used, GeNorm, NormFinder, and BestKeeper, allowing to identify UBQ, EF1? and GAPDH as the most stable genes for the genotype effect. For the biotic stress effect, EF1?, SAND and SMD3 were the most constant for the susceptible cultivar Trincadeira and EF1?, GAPDH, UBQ for the resistant cultivar Regent. In addition, the expression of three defense-related transcripts, encoding for subtilisin-like protein, CYP and PR10, was analysed, for both datasets, during inoculation time-course. Taken together, our results provide guidelines for reference gene(s) selection towards a more accurate and widespread use of qPCR to study the first hours of interaction between different grapevine cultivars and P. viticola.

SUBMITTER: Monteiro F

PROVIDER: S-EPMC3762845 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Reference gene selection and validation for the early responses to downy mildew infection in susceptible and resistant Vitis vinifera cultivars.

Monteiro Filipa F Sebastiana Mónica M Pais Maria Salomé MS Figueiredo Andreia A

PloS one 20130904 9

The pivotal role of cultivated grapevine (Vitis vinifera L.) in many countries economy is compromised by its high susceptibility to Plasmopara viticola, the causal agent of downy mildew disease. Recent research has identified a set of genes related to resistance which may be used to track downy mildew infection. Quantification of the expression of these resistance genes requires normalizing qPCR data using reference genes with stable expression in the system studied. In this study, a set of elev ...[more]

PMID: 24023800

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Project description:Downy mildew of spinach is caused by the obligate oomycete pathogen, Peronospora effusa. The disease causes significant economic losses, especially in the organic sector of the industry where the use of synthetic fungicides is not permitted for disease control. New pathotypes of this pathogen are increasingly reported which are capable of breaking resistance. In this study, we took advantage of new spinach genome resources to conduct RNA-seq analyses of transcriptomic changes in leaf tissue of resistant and susceptible spinach cultivars Solomon and Viroflay, respectively, at an early stage of pathogen establishment (48 hours post inoculation, hpi) to a late stage of symptom expression and pathogen sporulation (168 hpi). Fold change differences in gene expression were recorded between the two cultivars to identify candidate genes for resistance. In Solomon, the hypersensitive inducible genes such as pathogenesis-related gene PR-1, glutathione-S-transferase, phospholipid hydroperoxide glutathione peroxidase and peroxidase were significantly up-regulated uniquely at 48 hpi and genes involved in zinc finger CCCH protein, glycosyltransferase, 1-aminocyclopropane-1-carboxylate oxidase homologs, receptor-like protein kinases were expressed at 48 hpi through 168 hpi. The types of genes significantly up-regulated in Solomon in response to the pathogen suggests that salicylic acid and ethylene signaling pathways mediate resistance. Furthermore, many genes involved in the flavonoid and phenylpropanoid pathways were highly expressed in Viroflay compared to Solomon at 168 hpi. As anticipated, an abundance of significantly down-regulated genes was apparent at 168 hpi, reflecting symptom development and sporulation in cultivar Viroflay, but not at 48 hpi. In the pathogen, genes encoding RxLR-type effectors were expressed during early colonization of cultivar Viroflay while crinkler-type effector genes were expressed at the late stage of the colonization. Our results provide insights on gene expression in resistant and susceptible spinach-P. effusa interactions, which can guide future studies to assess candidate genes necessary for downy mildew resistance in spinach.

Project description:BACKGROUND:The glyoxalase system usually comprises two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII). This system converts cytotoxic methylglyoxal (MG) into non-toxic D-lactate in the presence of reduced glutathione (GSH) in two enzymatic steps. Recently, a novel type of glyoxalase III (GLYIII) activity has observed in Escherichia coli that can detoxify MG into D-lactate directly, in one step, without a cofactor. Investigation of the glyoxalase enzymes of a number of plant species shows the importance of their roles in response both to abiotic and to biotic stresses. Until now, glyoxalase gene families have been identified in the genomes of four plants, Arabidopsis, Oryza sativa, Glycine max and Medicago truncatula but no similar study has been done with the grapevine Vitis vinifera L. RESULTS:In this study, four GLYI-like, two GLYII-like and three GLYIII-like genes are identified from the genome database of grape. All these genes were analysed in detail, including their chromosomal locations, phylogenetic relationships, exon-intron distributions, protein domain organisations and the presence of conserved binding sites. Using quantitative real-time PCR analysis (qRT-PCR), the expression profiles of these genes were analysed in different tissues of grape, and also when under infection stress from downy mildew (Plasmopara viticola). The study reveals that most VvGLY-like genes had higher expressions in stem, leaf, tendril and ovule but lower expressions in the flower. In addition, most of the VvGLY-like gene members were P. viticola responsive with high expressions 6-12?h and 96-120?h after inoculation. However, VvGLYI-like1 was highly expressed 48?h after inoculation, similar to VvPR1 and VvNPR1 which are involved in the defence response. CONCLUSIONS:This study identified the GLYI-like, GLYII-like and GLYIII-like full gene families of the grapevine. Based on a phylogenetic analysis and the presence of conserved binding sites, we speculate that these glyoxalase-like genes in grape encode active glyoxalases. Moreover, our study provides a basis for discussing the roles of VvGLYI-like, VvGLYII-like and VvGLYIII-like genes in grape's response to downy mildew infection. Our results shed light on the selection of candidate genes for downy mildew tolerance in grape and lay the foundation for further functional investigations of these glyoxalase genes.

Dataset Information

Reference gene selection and validation for the early responses to downy mildew infection in susceptible and resistant Vitis vinifera cultivars.

Publications

Reference gene selection and validation for the early responses to downy mildew infection in susceptible and resistant Vitis vinifera cultivars.

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