French-American hybrids and North American grape species play a significant role in Canada’s grape and wine industry. Unfortunately, the occurrence of viruses and viral diseases among these locally ...important non-vinifera grapes remains understudied. We report here the results from a large-scale survey to assess the prevalence of 14 viruses among 533 composite samples representing 2665 vines from seven French-American hybrid wine grape cultivars, two North American juice grape cultivars (Concord and Niagara), and the table grape cultivar Sovereign coronation. Based on reverse transcription polymerase chain reaction (RT-PCR) assays, ten viruses were detected. Grapevine rupestris stem pitting-associated virus, grapevine leafroll-associated virus 3, grapevine Pinot gris virus and grapevine red blotch virus were detected with the highest frequency. As expected, mixed infections were common; 62% of the samples contained two or more viruses. Overall, hybrid wine grapes were infected with more viruses and a higher prevalence of individual viruses than juice and table grapes. To validate these findings and to refine the virome of these non-European grapes, high-throughput sequencing (HTS) analyses of five composite samples representing each category of grapevine cultivars was performed. Results from HTS agreed with those from RT-PCR. Importantly, Vidal, a widely grown white-wine grape with international recognition due to its use in the award-winning icewine, is host to 14 viruses, four of which comprise multiple and distinct genetic variants. This comprehensive survey represents the most extensive examination of viruses among French-American hybrids and North American grapes to date.
Grapevine leafroll-associated virus-
2 (GLRaV-2) and GLRaV-3 are both (+) ssRNA viruses of the family
Closteroviridae
and are involved in grapevine leafroll, the most destructive viral disease ...affecting the global grape/wine industry. Outbreaks of the disease were recently reported in Canada, causing serious concerns to the grape/wine industry. Reliable, sensitive and timely detection is key to the control of the disease. However, information on their seasonal dynamics and tissue distribution under cool climate conditions has been rather limited. We conducted a two-year comprehensive study to elucidate the temporal variation and spatial distribution of both viruses through symptom monitoring, Western blotting and RT-qPCR. Sampling was done monthly from commercial Cabernet Franc and Chardonnay vines from May to October in 2015 and 2016. Both viral RNA and capsid protein levels in leaves remained low during May and June, steadily increased from late July, and peaked in September or October. Interestingly, young berries collected in June contained high levels of viral RNA for both viruses. As expected, the viral RNA levels of GLRaV-2 detected by RT-qPCR using primers targeting the CP region were much higher than those by using primers targeting the genomic RNA. Surprisingly, virtually the same levels of viral RNA were detected for GLRaV-3 regardless of the targeted genomic regions. To ensure accurate detection of both viruses, we recommend using young berries early in the season and leaves and cambial scrapings from late July to harvest. To our knowledge, this is the first report on the seasonal dynamics and tissue distribution of two major grapevine viruses in Canada.
ABSTRACT
The C‐repeat (CRT)‐binding factor/dehydration‐responsive element (DRE) binding protein 1 (CBF/DREB1) transcription factors control an important pathway for increased freezing and drought ...tolerance in plants. Three CBF/DREB1‐like genes, CBF 1–3, were isolated from both freezing‐tolerant wild grape (Vitis riparia) and freezing‐sensitive cultivated grape (Vitis vinifera). The deduced proteins in V. riparia are 63–70% identical to each other and 96–98% identical to the corresponding proteins in V. vinifera. All Vitis CBF proteins are 42–51% identical to AtCBF1 and contain CBF‐specific amino acid motifs, supporting their identification as CBF proteins. Grape CBF sequences are unique in that they contain 20–29 additional amino acids and three serine stretches. Agro‐infiltration experiments revealed that VrCBF1b localizes to the nucleus. VrCBF1a, VrCBF1b and VvCBF1 activated a green fluorescent protein (GFP) or glucuronidase (GUS) reporter gene behind CRT‐containing promoters. Expression of the endogenous CBF genes was low at ambient temperature and enhanced upon low temperature (4 °C) treatment, first for CBF1, followed by CBF2, and about 2 d later by CBF3. No obvious significant difference was observed between V. riparia and V. vinifera genes. The expression levels of all three CBF genes were higher in young tissues than in older tissues. CBF1, 2 and 3 transcripts also accumulated in response to drought and exogenous abscisic acid (ABA) treatment, indicating that grape contains unique CBF genes.
In recent years, the Ontario grape and wine industry has experienced outbreaks of viral diseases across the province. Little is known about the prevalence of viruses and viral diseases in Ontario. ...Since 2015, we have conducted large-scale surveys for major viruses in commercial wine grapes in order to obtain a comprehensive understanding of the prevalence and severity of viral diseases in Ontario.
A total of 657 composite leaf samples representing 3285 vines collected from 137 vine blocks of 33 vineyards from three appellations: Niagara Peninsula, Lake Erie North Shore and Prince Edward County. These samples covered six major red cultivars and five major white grape cultivars. Using a multiplex RT-PCR format, we tested these samples for 17 viruses including those involved in all major viral diseases of the grapevine, such as five grapevine leafroll-associated viruses (GLRaV-1, 2, 3, 4, 7), grapevine red blotch virus (GRBV), grapevine Pinot gris virus (GPGV), grapevine rupestris stem sitting-associated virus (GRSPaV), grapevine virus A (GVA), grapevine virus B (GVB), grapevine fleck virus (GFkV), arabis mosaic virus (ArMV), tomato ringspot virus (ToRSV), trapevine fanleaf virus (GFLV), among others.
Fourteen of the 17 viruses were detected from these samples and the predominant viruses are GRSPaV, GLRaV-3, GFkV, GPGV and GRBaV with an incidence of 84.0, 47.9, 21.8, 21.6 and 18.3%, respectively. As expected, mixed infections with multiple viruses are common. 95.6% of the samples included in the survey were infected with at least one virus; 67% of the samples with 2-4 viruses and 4.7% of the samples with 5-6 viruses. The major grape cultivars all tested positive for these major viruses. The results also suggested that the use of infected planting material may have been one of the chief factors responsible for the recent outbreaks of viral diseases across the province.
This is the first such comprehensive survey for grapevine viruses in Ontario and one of the most extensive surveys ever conducted in Canada. The recent outbreaks of viral diseases in Ontario vineyards were likely caused by GLRaV-3, GRBV and GPGV. Findings from this survey provides a baseline for the grape and wine industry in developing strategies for managing grapevine viral diseases in Ontario vineyards.
Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widely prevalent and economically important of the complex of RNA viruses associated with grapevine leafroll disease (GLD). Phylogenetic ...studies have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus species. In addition, new divergent variants have been discovered recently around the world. Accurate identification of the virus is an essential component in the management and control of GLRaV-3; however, the diversity of GLRaV-3, coupled with the limited sequence information, have complicated the development of a reliable detection assay. In this study, GLRaV-3 sequence data available in GenBank and those generated at Foundation Plant Services, University of California-Davis, was used to develop a new RT-qPCR assay with the capacity to detect all known GLRaV-3 variants. The new assay, referred to as FPST, was challenged against samples that included plants infected with different GLRaV-3 variants and originating from 46 countries. The FPST assay detected all known GLRaV-3 variants, including the highly divergent variants, by amplifying a small highly conserved region in the 3' untranslated terminal region (UTR) of the virus genome. The reliability of the new RT-qPCR assay was confirmed by an enzyme linked immunosorbent assay (ELISA) that can detect all known GLRaV-3 variants characterized to date. Additionally, three new GLRaV-3 divergent variants, represented by four isolates, were identified using a hierarchical testing process involving the FPST assay, GLRaV-3 variant-specific assays and high-throughput sequencing analysis. These variants were distantly related to groups I, II, III, V, VI, VII and IX, but much similar to GLRaV-3 variants with no assigned group; thus, they may represent new clades. Finally, based on the phylogenetic analysis, a new GLRaV-3 subclade is proposed and named as group X.
Syrah decline, first identified in Southern France in the 1990s, has become a major concern in the global grape and wine industry. This disease mainly affects Syrah (Shiraz) grapevines. ...Characteristic symptoms include the bright and uniform reddening of leaves throughout the canopy in late summer or early fall; the appearance of abnormalities on the trunk, mainly at the graft union (swelling, pits, grooves, and necrosis); and a reduction in vine vigor, yield and berry quality. Diseased vines may die a few years after disease onset. Damages to the vine are even more pronounced in cool climate regions such as Ontario (Canada), where the affected vines are subjected to very cold and prolonged winters, leading to large numbers of vine deaths. Despite the extensive efforts of the global grape research community over the past few decades, the etiology of this disease remains unclear. In this study, we conducted extensive analyses of viruses in declining Syrah vines identified in commercial vineyards in the Niagara region (Ontario, Canada) through high-throughput sequencing, PCR, RT-PCR and the profiling of genetic variants of select viruses. Multiple viruses and viral strains, as well as three viroids, were identified. However, an unequivocal causal relationship cannot be established between Syrah decline and any of these viruses, although the possibility that certain virus or genetic variants, or both in combination, may contribute to the disease cannot be excluded. Gleaning all information that is available to date, we feel that the traditional approach and an insistence on finding a single cause for such a complex disorder in a woody perennial fruit crop involving grafting will prove to be futile. We hope that this study offers new conceptual perspectives on the etiology of this economically important but enigmatic disease complex that affects the global grape and wine industry.
Rapid apple decline disease (RAD) has been affecting orchards in the USA and Canada. Although the primary cause for RAD remains unknown, viruses may contribute to the incidence or severity of the ...disease. We examined the diversity and prevalence of viruses in orchards affected by RAD in the Okanagan and Similkameen Valleys (British Columbia, Canada). Next-generation sequencing identified 20 previously described plant viruses and one viroid, as well as a new ilarvirus, which we named apple ilarvirus 2 (AIV2). AIV2 was related to subgroup 2 ilarviruses (42–71% nucleotide sequence identity). RT-PCR assays of 148 individual leaf samples revealed frequent mixed infections, with up to eight viruses or viroid detected in a single tree. AIV2 was the most prevalent, detected in 64% of the samples. Other prevalent viruses included three ubiquitous viruses from the family Betaflexiviridae and citrus concave gum-associated virus. Apple rubbery wood virus 1 and 2 and apple luteovirus 1 were also readily detected. The thirteen most prevalent viruses/viroid were detected not only in trees displaying typical RAD symptoms, but also in asymptomatic trees. When compared with reports from orchards affected by RAD in Pennsylvania, New York State, and Washington State, regional differences in relative virus prevalence were noted.
Dehydrins are proteins that accumulate in vegetative tissues subjected to various dehydrating stress conditions such as cold, drought, and salinity and in seeds at later stages of embryogenesis. ...Here, we report on two highly identical dehydrin genes, DHN1a and DHN1b, in wild and cultivated grapes, Vitis riparia and Vitis vinifera, and their expression in different tissues and under different environmental conditions. The two genes and their transcripts can easily be distinguished by RT-PCR because DHN1b has an 18 bp deletion compared to DHN1a. V. riparia expressed only DHN1a; V. vinifera expressed both DHN1a and DHN1b. Spliced transcripts, DHN1-S, encoding a putative YSK2-type dehydrin were present in low amounts in control leaves, but in high amounts in buds and seeds. Unspliced transcripts, DHN1-U, accumulated to high levels in buds and seeds. Cold, drought, and ABA treatment increased accumulation of both DHN1-S and DHN1-U in leaves, whereas short-day treatment increased only DHN1-S. The possible relation of these results with the difference in freezing stress tolerance between V. riparia and V. vinifera is discussed.
The CBF/DREB1 transcription factors control an important pathway for increased freezing and drought tolerance in plants. We report here the isolation of one CBF/DREB1-like gene, CBF4, from both ...freezing-tolerant wild grape (Vitis riparia) and freezing-sensitive cultivated grape (Vitis vinifera). The deduced protein in V. riparia is 99% identical to the corresponding protein in V. vinifera; 45-48% to three other Vitis CBF proteins reported earlier and 57% to AtCBF1, and contains CBF-specific amino acid motifs. Agroinfiltration experiments in tobacco leaves revealed that VrCBF4 activates expression from reporter genes driven by a CRT-containing promoter. Expression of the endogenous Vitis CBF4 genes was low at ambient temperature, but enhanced upon exposure to low temperature (4 °C). Uncommon for CBF genes, this expression was maintained for several days. No significant difference in expression pattern was observed between V. riparia and V. vinifera. Vitis CBF4 was expressed in both young and mature tissue, in contrast to the previously described Vitis CBF1, 2 and 3. Together, these results suggest that CBF4 represents a second type of CBF in grape that might be more important for the over-wintering of grape plants.
Plasmepsin I (PMI) is one of the four vacuolar pepsin-like proteases responsible for hemoglobin degradation by the malarial parasite
Plasmodium falciparum, and the only one with no crystal structure ...reported to date. Due to substantial functional redundancy of these enzymes, lack of inhibition of even a single plasmepsin can defeat efforts in creating effective antiparasitic agents. We have now solved crystal structures of the recombinant PMI as apoenzyme and in complex with the potent peptidic inhibitor, KNI-10006, at the resolution of 2.4 and 3.1
Å, respectively. The apoenzyme crystallized in the orthorhombic space group
P2
12
12
1 with two molecules in the asymmetric unit and the structure has been refined to the final
R-factor of 20.7%. The KNI-10006 bound enzyme crystallized in the tetragonal space group
P4
3 with four molecules in the asymmetric unit and the structure has been refined to the final
R-factor of 21.1%. In the PMI–KNI-10006 complex, the inhibitors were bound identically to all four enzyme molecules, with the opposite directionality of the main chain of KNI-10006 relative to the direction of the enzyme substrates. Such a mode of binding of inhibitors containing an allophenylnorstatine-dimethylthioproline insert in the P1–P1′ positions, previously reported in a complex with PMIV, demonstrates the importance of satisfying the requirements for the proper positioning of the functional groups in the mechanism-based inhibitors towards the catalytic machinery of aspartic proteases, as opposed to binding driven solely by the specificity of the individual enzymes. A comparison of the structure of the PMI–KNI-10006 complex with the structures of other vacuolar plasmepsins identified the important differences between them and may help in the design of specific inhibitors targeting the individual enzymes.
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