Probing into the Effects of Grapevine Leafroll-Associated Viruses on the Physiology, Fruit Quality and Gene Expression of Grapes

Song, Yashu; Hanner, Robert; Meng, Bin

doi:10.3390/v13040593

Cited by 37 publications

(45 citation statements)

References 151 publications

(259 reference statements)

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“…GLRaV-3 was reported to form viral replication complexes in association with mitochondrial membrane of infected host cells [102][103][104]. It is hypothesized that GLRaV-3 infections cause damages to mitochondria; and that genes associated with mitochondrial processes would be differentially regulated in GLRaV-3-infected grapevines [70]. Interestingly, results from our study and those reported by others [44,57,86,97] have identified some of the genes involved in respiration as being stably expressed.…”

Section: Bioinformatics Analyses Of Rna-seq Dataset Enabled Identification Of a Set Of Stably Expressed Genes Involved In Several Biologisupporting

confidence: 63%

“…The most characteristic symptoms of GLRaV-3-infection in dark-skinned grapevine cultivars are the downward curling of leaf margins and red to purple pigmentation of interveinal regions in leaves [70]. The discoloration of GLRaV-3-infected leaves is associated with increased biosynthesis of anthocyanins, likely resulting from the up-regulation of UFGT gene [4].…”

Section: Novel Reference Genes Outperformed Traditional Reference Genes In Transcripts Quantificationmentioning

confidence: 99%

“…Grapevine leafroll-associated virus 3 (GLRaV-3) is a positive-sense, single-stranded RNA (ssRNA) virus of the Ampelovirus genus within the family Closteroviridae [69]. It is one of the major grapevine viruses with a worldwide prevalence and is the main agent associated with grapevine leafroll disease (GLRD); the most prevalent and destructive viral disease of grapevine that afflicts global grape and wine production [70][71][72]. Infection with GLRaV-3 can result in significant yield reduction and altered fruit chemistry that negatively impact the quality of fresh fruits, juice and wine, as well as the profitability and lifespan of vineyards [71,73].…”

Section: Introductionmentioning

confidence: 99%

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Genome-wide screening of novel RT-qPCR reference genes for study of GLRaV-3 infection in wine grapes and refinement of an RNA isolation protocol for grape berries

2021

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Background Grapevine, as an essential fruit crop with high economic values, has been the focus of molecular studies in diverse areas. Two challenges exist in the grapevine research field: (i) the lack of a rapid, user-friendly and effective RNA isolation protocol for mature dark-skinned berries and, (ii) the lack of validated reference genes that are stable for quantification of gene expression across desired experimental conditions. Successful isolation of RNA with sufficient yield and quality is essential for downstream analyses involving nucleic acids. However, ripe berries of dark-skinned grape cultivars are notoriously challenging in RNA isolation due to high contents of polyphenolics, polysaccharides, RNase and water. Results We have optimized an RNA isolation protocol through modulating two factors at the lysis step that could impact results of RNA isolation - 2-ME concentration and berry mass. By finding the optimal combination among the two factors, our refined protocol was highly effective in isolating total RNA with high yield and quality from whole mature berries of an array of dark-skinned wine grape cultivars. Our protocol takes a much shorter time to complete, is highly effective, and eliminates the requirement for hazardous organic solvents. We have also shown that the resulting RNA preps were suitable for multiple downstream analyses, including the detection of viruses and amplification of grapevine genes using reverse transcription-polymerase chain reaction (RT-PCR), gene expression analysis via quantitative reverse transcription PCR (RT-qPCR), and RNA Sequencing (RNA-Seq). By using RNA-Seq data derived from Cabernet Franc, we have identified seven novel reference gene candidates (CYSP, NDUFS8, YLS8, EIF5A2, Gluc, GDT1, and EF-Hand) with stable expression across two tissue types, three developmental stages and status of infection with grapevine leafroll-associated virus 3 (GLRaV-3). We evaluated the stability of these candidate genes together with two conventional reference genes (actin and NAD5) using geNorm, NormFinder and BestKeeper. We found that the novel reference gene candidates outperformed both actin and NAD5. The three most stable reference genes were CYSP, NDUFS8 and YSL8, whereas actin and NAD5 were among the least stable. We further tested if there would be a difference in RT-qPCR quantification results when the most stable (CYSP) and the least stable (actin and NAD5) genes were used for normalization. We concluded that both actin and NAD5 led to erroneous RT-qPCR results in determining the statistical significance and fold-change values of gene expressional change. Conclusions We have formulated a rapid, safe and highly effective protocol for isolating RNA from recalcitrant berry tissue of wine grapes. The resulting RNA is of high quality and suitable for RT-qPCR and RNA-Seq. We have identified and validated a set of novel reference genes based on RNA-Seq dataset. We have shown that these new reference genes are superior over actin and NAD5, two of the conventional reference genes commonly used in early studies.

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Section: Bioinformatics Analyses Of Rna-seq Dataset Enabled Identification Of a Set Of Stably Expressed Genes Involved In Several Biologisupporting

confidence: 63%

Section: Novel Reference Genes Outperformed Traditional Reference Genes In Transcripts Quantificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Genome-wide screening of novel RT-qPCR reference genes for study of GLRaV-3 infection in wine grapes and refinement of an RNA isolation protocol for grape berries

2021

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“…The use of molecular sequence data is an essential component of many analyses across various fields of scientific study, including virology ( Radford et al 2012 ), gene expression studies ( Song et al 2021 ), evolutionary biology ( Hudson 2008 ), taxonomy and species identifications ( Hubert and Hanner 2015 ) and biodiversity surveys ( Young et al 2021 ). One of the strengths of molecular approaches is in the comparability of the data, even when obtained by different labs and researchers.…”

Section: Introductionmentioning

confidence: 99%

Molecular Acquisition, Cleaning and Evaluation in R (MACER) - A tool to assemble molecular marker datasets from BOLD and GenBank

Young

Gill

Gillis

et al. 2021

BDJ

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Molecular sequence data is an essential component for many biological fields of study. The strength of these data is in their ability to be centralised and compared across research studies. There are many online repositories for molecular sequence data, some of which are very large accumulations of varying data types like NCBI’s GenBank. Due to the size and the complexity of the data in these repositories, challenges arise in searching for data of interest. While data repositories exist for molecular markers, taxa and other specific research interests, repositories may not contain, or be suitable for, more specific applications. Manually accessing, searching, downloading, accumulating, dereplicating and cleaning data to construct project-specific datasets is time-consuming. In addition, the manual assembly of datasets presents challenges with reproducibility. Here, we present the MACER package to assist researchers in assembling molecular datasets and provide reproducibility in the process.

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“…Virus diseases in the grapevine have detrimental effects on the agronomic, ecological performance, and morphological changes of vines. In addition to reduced yield and quality, viruses also damage photosynthesis, chloroplast level, functions and ultrastructure, accumulation of photoassimilates (6) . Viruses also may determine leaf and cluster of morphological modifications which aid phenotypic expression of disease symptoms and exertion of plenty of other pathological changes in the plant.…”

Section: Introductionmentioning

confidence: 99%

Lipid Peroxidation Level and Peroxidase Activity in (Vitis vinifera L.) Leaves Infected with Grapevine Leafroll-Associated Virus 3

Bayramova¹,

Sultanova²,

Aliyeva³

et al. 2021

IJST

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Probing into the Effects of Grapevine Leafroll-Associated Viruses on the Physiology, Fruit Quality and Gene Expression of Grapes

Cited by 37 publications

References 151 publications

Genome-wide screening of novel RT-qPCR reference genes for study of GLRaV-3 infection in wine grapes and refinement of an RNA isolation protocol for grape berries

Genome-wide screening of novel RT-qPCR reference genes for study of GLRaV-3 infection in wine grapes and refinement of an RNA isolation protocol for grape berries

Molecular Acquisition, Cleaning and Evaluation in R (MACER) - A tool to assemble molecular marker datasets from BOLD and GenBank

Lipid Peroxidation Level and Peroxidase Activity in (Vitis vinifera L.) Leaves Infected with Grapevine Leafroll-Associated Virus 3

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