All of the leafroll viruses selected for this study belong to the Closteroviridae family and all but one belong to the Ampelovirus genus; GLRaV-2 belongs to the genus Closterovirus. All of the GLRaVs used in this study contain a conserved replication gene block but are diverse outside of the RGB . In addition to host genotype and environment, the sequencespowdery and downy mildew in grapevine . The abundance of these genes varies among Vitis species and are particularly dense at resistance loci . The HR to viruses is mediated by resistance genes. HR is a means of prohibiting pathogen spread and can confer resistance when a corresponding dominant avirulence protein is produced by the pathogen . However, GLRaV infections are systemic and persist over time, and SA does not seem to play a preeminent role in the response to GLRaV infections. In a previous study of GLRaV-3 infections in Cabernet Sauvignon and Carmenère, the authors also remarked on the induction of expression of defence genes but their inability to impede systemic infection . Both SA and ABA can participate in the response to viruses, gallon pot though considerably less is understood about the role of ABA and its relationship to NBS-LRRs.
Notably, however, ABA deficiency is associated with an increase in R gene efficacy in incompatible interactions with Pseudomonas syringae and in a manner independent of SA . Hormones have been implicated in mediating defence- and development-related networks and are over-represented at network hubs . Hormones like ABA, SA, and JA act as important signalling molecules during ripening and defence. The pathways engaged under stress are often tailored to particular pathogens. This entails coordination between hormone pathways . Interestingly, the effects of GLRaVs on gene expression in several hormone signalling pathways differed between rootstocks. A subsequent effort could be made to measure the abundances of hormones not quantified here, like cytokinins, GAs, and ethylene. Of the hormones considered in this study, however, the abundance of ABA and ABA-GE tended to increase in GLRaV and this was influenced by rootstock. ABA can antagonize SA and JA signalling pathways and suppress ROS signalling . WRKY transcription factors regulate and/or are regulated by ABA, SA, and JA . SA and ABA both can interact with RNAi, which is a fundamental component of antiviral defence. AGO1 expression is positively correlated with ABA levels and the expression of miR168a, which regulates AGO1, and contains ABREs in its promoter . Levels of ABA and ABA-GE increase in tobacco mosaic virus-infected leaves. One way in which ABA might aid plant defence is by increasing callose deposition to impair virus movement ; a gene encoding callose synthase is upregulated in the leaves of grapevine virus B-infected plants .
Relatively more is known about ABA’s function as a ripening promoter , in response to drought stress , and in transmitting long-distance signals from roots to aerial organs and vice versa . In a study of the impact of GLRaV-3 infection, drought stress, and a combination of both on grapevine plantlets in vitro, individual stresses both induced increases in ABA levels . Drought stress increases ABA levels and induces the flavonoid pathway in both tea plants and grapes . Our findings, in which ABA abundance tends to increase in GLRaV , are different than that observed for red blotch virus-infected berries, in which ABA abundance and NCED expression decrease in infected fruits . The results of our analysis of metabolites associated with the phenylpropanoid and flavonoid pathways are mixed in their consistency with previous work. Though non-significant decreases in anthocyanin levels were observed, anthocyanin levels significantly increased in several GLRaV conditions, albeit usually in individual years. These findings differed from others; some observed significant decreases in anthocyanin levels in fruits from GLRaV-infected plants and others observed no significant changes in anthocyanin at harvest . In agreement with the results by Vega et al. , flavonol levels were elevated in GLRaV and the largest differences versus GLRaV occurred at the first two stages, FLS expression was downregulated in GLRaV at harvest, and CHS and MYBPA1 were upregulated at véraison and generally downregulated at harvest. In the present study, changes in the abundance of ABA and related metabolites distinguished the effects of GLRaVs between rootstocks. The parentage of the two rootstocks used in this study,Kober 5BB and MGT 101-14, includes Vitis riparia. The other parents of Kober 5BB and MGT 101-14 are Vitis berlandieri and Vitis rupestris, respectively.
These rootstocks were developed at different times. MGT 101-14 originated in France in 1882 and Kober 5BB originated in Austria in 1930 . Rootstocks are chosen for the advantages they confer to the scion given a particular set of circumstances, often having to do with resistance to Phylloxera, nematodes, scion vigour, soil type, and abiotic stress tolerance . V. riparia, V. rupestris, and V. berlandieri are asymptomatic hosts of GLRaVs . Yet, the particularly severe response to GLRaV-1,2 was not entirely unexpected in Kober 5BB-grafted vines . It would be interesting to determine whether differences in viral titre exist between Kober 5BB, MGT 101-14, and Cabernet Franc and between different infection conditions and whether such differences, if they exist, influence the severity of leafroll disease. Differences in wood abnormalities given different rootstocks have been observed for particular isolates causing grapevine rugose wood disease . Notably, nutritional deficiencies in phosphorus, magnesium, and potassium produce symptoms that resemble those typically observed in GLRaV-infected plants . Magnesium deficiency tolerance , the impact of phosphorus deficiency on canopy growth , and potassium uptake and channels are influenced by rootstock . Potassium uptake, channel activity, and related gene expression are also regulated by ABA , and the application of ABA to tomato roots by drip irrigation affects fruit mineral composition . Furthermore, elevated levels of potassium are observed in leafroll virus-infected Burger and Sultana fruits and in leaf petioles but potassium levels are lower in leaf blades . Perhaps potassium deficient and GLRaV phenotypes are similarly governed by ABA and fine-tuned by rootstocks. If some portion of scion ABA originates in roots and/or if rootstock can influence scion ABA levels and signalling genes, as observed here and by others , then perhaps this partially accounts for the variation in response observed between rootstocks. This experiment did not include a comprehensive survey of phytohormones, which would be beneficial, but ABA’s function in root–shoot communication, its role in ripening, and the results here make it a good candidate around which to study the basis of leafroll disease symptom variability going forward. In addition, the transport of RNAs across the graft junction may perform some function that affects scion disease severity, but this remains to be seen in the particular case of GLRaV . Together, these data support several conclusions. The majority of genes differentially expressed as a consequence of infection or between GLRaV plants with different rootstocks were yearspecific. A small subset of effects was consistently observed across experimental conditions and in both years. These shared changes in expression involved genes associated with pathogen detection, ABA signalling and transport, ROS-related signalling, gallon nursery pot cytoskeleton remodelling, vesicle trafficking, phenylpropanoid metabolism, sugar transport and conjugation, and leucine biosynthesis. The impacts of GLRaV-1,2 dual infection on Kober 5BB-grafted vines were the most distinctive and severe. Though there was variation between GLRaV infections observed, only the effects of GLRaV-1,2 were distinguishable overall from those of other infections. The particular effects of GLRaVs in plants grafted to different rootstocks were distinguishable overall at every developmental stage. ABA-related variables were among those that best distinguished the responses to GLRaVs in different rootstock conditions. This included the abundance of ABA, the abundance of ABA-GE, and the expression of genes associated with ABA and other hormone signalling pathways.
Finally, this work alone is insufficient to recommend the use of one rootstock or another, but the disparity in sensitivity and symptom severity observed in berries from Cabernet Franc vines grafted to different rootstocks suggests that rootstock selection should be further explored as a strategy to mitigate some of the negative consequences of leafroll virus infections, should vectors of the virus encroach upon a vineyard.The experimental vineyard used in this study was established in 2010 and consists of Cabernet Franc clone 04 grapevines grafted on different rootstocks and infected with zero, individual, or pairs of GLRaVs. The rootstock portion of these plants was inoculated with chip buds carrying each virus in 2009 . All rootstocks and Cabernet Franc scions were tested for grapevine pathogens. Total nucleic acid extracts were prepared from all rootstocks and Cabernet Franc scions as described by Al Rwahnih et al. . Extracted TNA samples were analysed by reverse transcription quantitative PCR using TaqMan probes on the QuantStudio 6 Flex Real-Time PCR System as described previously . The samples were screened for the following pathogens: GLRaV-1, GLRaV-3, GLRaV-4 ; GLRaV-2 ; GLRaV-7 ; grapevine fleck virus ; grapevine rupestris vein feathering virus ; grapevine fanleaf virus, tobacco ringspot virus, and tomato ringspot virus ; grapevine virus A, grapevine virus B, grapevine virus D, grapevine virus E, and grapevine virus F ; grapevine red blotch virus ; and grapevine rupestris stem pitting-associated virus , phytoplasmas, and Xylella fastidiosa. In autumn 2008, Cabernet Franc grapevines were bench-grafted onto rootstocks, including MGT 101-14 and Kober 5BB. These plants were subsequently grown in a greenhouse. Between 2009 and 2011, the rootstock portions of these plants were inoculated with two chip buds from single leaf roll-infected plants. Infected plants used for chip buds were reconfirmed by RT-qPCR. Plants infected with a single species of GLRaV received two identical chip buds. Plants infected with two species of GLRaVs also received two chip buds, each carrying a single virus. Plants infected with GLRaV-1, GLRaV-2, and/or GLRaV-3 were inoculated with two or more isolates of each species of GLRaV. The inoculated plants were kept in a greenhouse for approximately 1 month, acclimatized, and then planted in the field. Healthy controls included nonchip budded plants and plants chip budded from a healthy source. The vines were planted in a randomized complete block design, with 7 feet between vines and 9 feet between rows. One group of five vines was planted per rootstock × infection condition in each of three blocks. Healthy vines were distributed throughout each block to monitor the spread of viruses, and experimental vines were sampled yearly to test and reaffirm the vines’ infection status. A buffer zone of healthy vines was planted as a barrier between the leafroll vineyard and other vineyards in the area. Vines were trained with a bilateral cordon and spur pruned.High-quality genomic DNA was isolated from grape leaves using the method described in Chin et al. . SMRTbell libraries were prepared for Cabernet Franc clone 04 as described by Massonnet et al. . Final libraries were evaluated for quantity and quality using a Bioanalyzer 2100 and sequenced on a PacBio RS II . De novo assembly of Cabernet Franc clone 04 was performed using FALCON-Unzip as described in Minio et al. . Repetitive sequences were masked before and after read error correction using the TANmask and REPmask modules in Damasker . Contigs were polished with Quiver . The primary assembly was scaffolded to reduce sequence fragmentation. First, primary contigs were scaffolded with SSPACE-LongRead v. 1.1; . Junctions supported by at least 20 reads were allowed. Hi-C data and the proprietary HiRise software were used for hybrid scaffolding. A Dovetail Hi-C library was prepared by Dovetail Genomics as described in Lieberman-Aiden et al. and sequenced on an Illumina platform, generating 2 × 150-bp pairedend reads. The repeat and gene annotation were performed as reported in Vondras et al. . Briefly, RepeatMasker and a custom V. vinifera repeat library were applied to identify repetitive elements in the genome. Publicly available data sets were used as evidence for gene prediction. Transcriptional evidence included Vitis expressed sequence tags, Cabernet Sauvignon corrected Iso-Seq reads , Tannat , Corvina , and Cabernet Sauvignon transcriptomes , and previously published RNA-Seq data . Swissprot Viridiplantae data and Vitis data were used as experimental evidence. Each RNA-Seq sample was trimmed with Trimmomatic v. 0.36 , assembled with Stringtie v. 1.3.3 , and mapped onto the genome using Exonerate v. 2.2.0 and PASA v. 2.1.0 . Alignments and ab initio predictions generated with SNAP v. 2006-07-28 , Augustus v. 3.0.3 , and GeneMark-ES v. 4.32 were used as input for EVidenceModeler v. 1.1.1 . EVidenceModeler was used to identify consensus gene structures.