Seasonal dynamics and tissue distribution of two major viruses associated with grapevine Leafroll under cool climate condition

Shabanian, Mehdi; Xiao, Huogen; Meng, Bin

doi:10.1007/s10658-020-02137-z

Cited by 13 publications

(13 citation statements)

References 30 publications

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“…However, there lacks definitive evidence indicating that any of these reference genes identified in other plant species was also the most stable in grapevines. In addition, few studies were conducted to identify reference genes for differential gene expression analysis among the different grapevine tissues/organs that were infected with GLRaV-3 [ 36 ].…”

Section: Methodologies To Study Effects Of Glravs On Grapevinementioning

confidence: 99%

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

Song

Hanner

Meng

2021

Viruses

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Grapevine leafroll is one of the most widespread and highly destructive grapevine diseases that is responsible for great economic losses to the grape and wine industries throughout the world. Six distinct viruses have been implicated in this disease complex. They belong to three genera, all in the family Closteroviridae. For the sake of convenience, these viruses are named as grapevine leafroll-associated viruses (GLRaV-1, -2, -3, -4, -7, and -13). However, their etiological role in the disease has yet to be established. Furthermore, how infections with each GLRaV induce the characteristic disease symptoms remains unresolved. Here, we first provide a brief overview on each of these GLRaVs with a focus on genome structure, expression strategies and gene functions, where available. We then provide a review on the effects of GLRaV infection on the physiology, fruit quality, fruit chemical composition, and gene expression of grapevine based on the limited information so far reported in the literature. We outline key methodologies that have been used to study how GLRaV infections alter gene expression in the grapevine host at the transcriptomic level. Finally, we present a working model as an initial attempt to explain how infections with GLRaVs lead to the characteristic symptoms of grapevine leafroll disease: leaf discoloration and downward rolling. It is our hope that this review will serve as a starting point for grapevine virology and the related research community to tackle this vastly important and yet virtually uncharted territory in virus-host interactions involving woody and perennial fruit crops.

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Section: Methodologies To Study Effects Of Glravs On Grapevinementioning

confidence: 99%

“…The typical leaf symptoms of GLRaV-3 infection, downward rolling of leaf margins and interveinal discoloration (reddening in dark-berried grapevines and chlorosis in white-skinned cultivars) are readily seen. Photos in B are from Shabanian et al (2020) [ 36 ].…”

Section: Figurementioning

confidence: 99%

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

Song

Hanner

Meng

2021

Viruses

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“…We included both spring petioles and fall canes in all of our evaluations to account for possible false negative test results due to variable virus titer and uneven within-vine distribution. These issues have been documented with GLRaV-1, GLRaV-2, GLRaV-3 [46][47][48][49], GVA, GVB, GRSPaV [50], GFLV [51], and GRBV [52]. In the initial broad evaluation of our HTS-based assay, we observed six false negatives when using TNA extracted from spring petioles and fall canes (Table S4).…”

Section: Discussionmentioning

confidence: 71%

Quality Assessment and Validation of High-Throughput Sequencing for Grapevine Virus Diagnostics

Soltani

Stevens

Klaassen

et al. 2021

Viruses

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Development of High-Throughput Sequencing (HTS), also known as next generation sequencing, revolutionized diagnostic research of plant viruses. HTS outperforms bioassays and molecular diagnostic assays that are used to screen domestic and quarantine grapevine materials in data throughput, cost, scalability, and detection of novel and highly variant virus species. However, before HTS-based assays can be routinely used for plant virus diagnostics, performance specifications need to be developed and assessed. In this study, we selected 18 virus-infected grapevines as a test panel for measuring performance characteristics of an HTS-based diagnostic assay. Total nucleic acid (TNA) was extracted from petioles and dormant canes of individual samples and constructed libraries were run on Illumina NextSeq 500 instrument using a 75-bp single-end read platform. Sensitivity was 98% measured over 264 distinct virus and viroid infections with a false discovery rate (FDR) of approximately 1 in 5 positives. The results also showed that combining a spring petiole test with a fall cane test increased sensitivity to 100% for this TNA HTS assay. To evaluate extraction methodology, these results were compared to parallel dsRNA extractions. In addition, in a more detailed dilution study, the TNA HTS assay described here consistently performed well down to a dilution of 5%. In that range, sensitivity was 98% with a corresponding FDR of approximately 1 in 5. Repeatability and reproducibility were assessed at 99% and 93%, respectively. The protocol, criteria, and performance levels described here may help to standardize HTS for quality assurance and accreditation purposes in plant quarantine or certification programs.

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“…To examine the robustness of our optimized RNA isolation protocol in downstream analyses, total RNAs of Cabernet Franc (both leaf and berry samples) were used in RT-PCR with GLRaV-3-specific primers designed in our lab [2]. Total RNAs isolated from ripe berries of additional nine dark-skinned wine grape varieties (the infection status for GLRaV-3 was unknown) were used in RT-PCR for the amplification of phytoene desaturase (PDS) gene using primers PDS-853F and PDS-1252R, with an expected amplicon size of 500 bp.…”

Section: Rt-pcrmentioning

confidence: 99%

“…Over the past few decades, this woody fruit crop has been the focus of diverse molecular studies, including responses to abiotic or biotic stresses, developmental changes and impact on berry quality, and host-pathogen interactions. Studies often involved using nucleic-acid based assays such as RT-PCR and RT-qPCR [2][3][4][5][6][7][8][9][10][11], or highthroughput sequencing (HTS) such as microarray and RNA-Sequencing (RNA-Seq) [12][13][14]. While these are powerful technologies that deliver insights to our understanding of grapevine molecular biology, success of the analyses is heavily dependent on the integrity and yield of total RNA isolated from grapevine tissues.…”

Section: Introductionmentioning

confidence: 99%

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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Seasonal dynamics and tissue distribution of two major viruses associated with grapevine Leafroll under cool climate condition

Cited by 13 publications

References 30 publications

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

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

Quality Assessment and Validation of High-Throughput Sequencing for Grapevine Virus Diagnostics

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

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