North American Grape ‘Norton’ is Resistant to Grapevine Vein Clearing Virus

Grapevine (Vitis spp.) is host to nearly 70 viruses from 17 families (Martelli 2017). An individual grapevine can be infected with a single virus species, or simultaneously with multiple viruses across families. Virus-associated diseases reduce berry quality and shorten the grapevine’s lifespan in a vineyard. Perennial growth of grapevines in both native habitats and vineyards exposes them continually to potential virus infection. In native habitats, insect vectors are the main transmission routes of viruses, and incidence of viral infection increases with dynamic epidemics of insect populations. In commercial production, vegetative propagation is the major means of disseminating viruses (Rowhani et al. 2005). Incidence of virus-associated diseases in vineyards increases with demand for propagating more grapevines in the expansion of viticulture.

In recent years, new viruses have emerged in vineyards and in wild grapevines (Maliogka et al. 2015; Perry et al. 2016; Qiu and Schoelz 2017; Sudarshana et al. 2015). They are bringing new and constant challenges to grape production, and grapevine vein clearing virus (GVCV) is one of these emerging challenges (Cieniewicz et al. 2020). GVCV belongs to the genus Badnavirus in the family Caulimoviridae (Zhang et al. 2011). Infection by GVCV stunts grapevine growth over a period of 3 to 7 years, deforms berry clusters, and even kills susceptible vines. The virus spreads among grape cultivars (Guo et al. 2014) as well as in wild Vitis species (Beach et al. 2017) and Ampelopsis cordata plants of the Vitaceae family (Petersen et al. 2019). Incidence of GVCV in wild Vitis species and A. cordata vines averages 10% and 34%, respectively. The grape aphid (Aphis illinosensis) is a reported insect vector (Petersen et al. 2019). Natural reservoirs of GVCV and grape aphids as transmission vectors provide multiple opportunities for virus infection in vineyards. Discovering GVCV-resistant grape cultivars would help alleviate losses of vineyards and provide germplasm for breeding virus-resistant grapevines.

Genetic sources of natural resistance to grapevine viruses are scarce. One study reported that a wild Middle Eastern V. vinifera, a V. rotundifolia cultivar, and a hybrid grape of V. vinifera and V. rotundifolia were resistant to grapevine fanleaf virus (GFLV) (Walker et al. 1985). After screening 223 Vitis species and interspecific hybrids for natural resistance to GFLV, arabis mosaic virus (ArMV), grapevine leafroll-associated virus 1 (GLRaV-1) and GLRaV-3, Lahogue and Boulard did not find a single virus-resistant accession (Lahogue and Boulard 1996). No sources of natural resistance in Vitis germplasm or rootstocks of cultivated grapes have been found to grapevine viruses that are associated with leafroll disease, rugose wood disease complex, or fleck disease complex (Oliver and Fuchs 2011). A collection of wild and cultivated Vitis and Muscadinia species were screened for virus resistance, but no germplasm was proven to be resistant to GFLV or GLRaVs (Oliver and Fuchs 2011). Therefore, continuous screening of Vitis germplasm for resistance against viruses is much needed (Oliver and Fuchs 2011). In our previous study (Guo et al. 2014), we reported that the hybrid grape ‘Chambourcin’ is resistant to GVCV. In this study, we assessed the resistance of the North American grape ‘Norton’ to GVCV.

‘Norton’ has been a major hybrid grape for red wine production in the midwestern to southeastern regions of the U.S. for over 180 years (Ambers 2013). ‘Norton’ berries have a high anthocyanin content and unique profiles of anthocyanins and proanthocyanidins (Ali et al. 2011). ‘Norton’ is resistant to major fungal pathogens including powdery mildew (Erysiphe necator) (Fung et al. 2008) and downy mildew (Plasmopara viticola) (Sapkota et al. 2018). High contents of salicylic acids and secondary metabolites are constitutively associated with high levels of ‘Norton’ disease resistance (Fung et al. 2008). However, whether ‘Norton’ is resistant to viruses has not yet been addressed.

To investigate if ‘Norton’ is resistant to GVCV, we inoculated ‘Norton’ grapevines with GVCV via graft-transmission. We performed wedge-grafting experiments using green cuttings of GVCV-infected ‘Chardonel’ grapevines and GVCV-free ‘Norton’ grapevines. In one set of three graftings, GVCV-infected ‘Chardonel’ grapevine was used as scion and ‘Norton’ grapevine as rootstock, and only one grafted vine survived for further assessment (Table 1). In the reciprocal grafting, ‘Norton’ grapevines served as scion and GVCV-infected ‘Chardonel’ grapevines as rootstock and six grafted vines survived (Table 1). The grafting of ‘Norton’ as rootstock or scion ensures systemic movement of GVCV from shoots to roots or vice versa. DNA was extracted from young leaves on the upper part of each branch. Polymerase chain reaction (PCR) was used to detect GVCV as previously described (Beach et al. 2017; Petersen et al. 2019). Detection of GVCV was carried out on grafted vines that were grown in the greenhouse periodically over 3 years after grafting (Table 1). GVCV was detected in both ‘Chardonel’ (not represented in Fig. 1) and ‘Cabernet Sauvignon’ scions that were grafted onto GVCV-infected ‘Chardonel’ rootstock (Fig. 1, lane 1 and 2). In contrast, GVCV was not detected in ‘Norton,’ either as scion (Fig. 1, lane 3 to 8) or rootstock (Fig. 1, lane 9 and 10). In the first case, it shows that GVCV did not move downward into the ‘Norton’ rootstock from the infected scion. In the second case, it indicates that GVCV did not move upward into the ‘Norton’ scion from the infected rootstock. These results suggest that either GVCV cannot move through the vascular tissues via the graft union or cannot replicate in the grafted ‘Norton’ grapevine. Although inoculation of a virus by graft-transmission cannot control the amount of virus inoculum (Oliver and Fuchs 2011) and the viral titer is too high for accurately assessing natural resistance (Laimer et al. 2009), graft-transmission ensures the presence of abundant viral inoculum. The PCR assay for GVCV was repeated periodically for 3 years after grafting. Absence of GVCV in ‘Norton’ as scion or rootstock clearly indicates that ‘Norton’ is resistant to GVCV up to 3 years after inoculation.

Table 1. Assessment of ‘Norton’ resistance via graft-transmission to grapevine vein clearing virus (GVCV) by polymerase chain reaction (PCR)

Scion	Rootstock	Vines grafted	Vines analyzed	Age of grafted vines in years	Detection of GVCV
					Scion	Rootstock
Norton	GVCV-Chardonela	6	6	1–3	–	+
GVCV-Chardonel	Norton	3	1	3	+	–
Cabernet Sauvignon	GVCV-Chardonel	3	1	1	+	+
Chardonel	GVCV-Chardonel	3	1	1	+	+
GVCV-Chardonel	Cabernet Sauvignon	3	1	3	+	+

^a GVCV-infected ‘Chardonel’ grapevine was used as virus inoculum.

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The above experiments were done under greenhouse conditions. We further evaluated ‘Norton’ resistance to GVCV in vineyards where both viral inoculum (reservoir) and an insect vector were present. The results from evaluation of ‘Norton’ resistance to GVCV in vineyards would reflect a real-world status of viruses and ‘Norton’ performance in commercial vineyards. Fortunately, a vineyard was available for us to perform this evaluation. In 2007, we planted virus-tested grape hybrids in a foundation vineyard at the Missouri State Fruit Experiment Station, Mountain Grove, Missouri, U.S.A. After we discovered that GVCV was associated with grapevine vein-clearing and vine decline disease (Zhang et al. 2011), we conducted a survey of GVCV in 86 ‘Chardonel’ vines in 2014, 2016, and 2018. GVCV was detected in 13%, 4%, and 3% of ‘Chardonel,’ respectively. Decreased incidence is a result of removing GVCV-infected vines as soon as they were discovered. It was clear that GVCV had spread in the foundation vineyard since 2007. ‘Vidal blanc’ was infected with GVCV at a 30% infection rate in 2016, and thus removed from the foundation vineyard. In 2018, we conducted a survey of GVCV in all 74 ‘Norton’ vines and found no incidence of this virus. ‘Norton’ and ‘Chardonel’ vines were planted in two rows each, 32 feet (9.8 m) apart, in the same foundation vineyard in 2007. It is clear that ‘Norton’ had not acquired GVCV in 12 years, although both inoculum source and insect vector had been present in the vineyard.

Small RNAs have been shown to transport through the graft union and systemically move through the entire plant (Melnyk et al. 2011; Molnar et al. 2010). Viral small RNAs (vsRNAs) of GVCV have been detected in susceptible grapevines (Howard and Qiu 2017). To verify the above results and also investigate possible mechanisms for viral resistance in ‘Norton,’ we began by determining if we could detect GVCV vsRNAs in the graft-inoculated ‘Norton.’ Total RNA was sent to Admera Health (126 Corporate Blvd, South Plainfield, NJ 07080) for acquiring sequences of small RNAs. The cDNA library construction and RNA size selection were carried out using NEBNext Multiplex Small RNA Library Prep Set for Illumina sequencing (New England Biolabs Inc, Ipswich, MA, U.S.A.) following the manufacturer’s protocol. The library was sequenced on the Illumina HiSeq for a length of 150 nt. After primer sequence removal with Cutadapt 1.9.1, the reads were checked for quality using a cutoff of Phred score 20 and a cutoff length of 17 nt (NGS QC V2.3.3). The subsequent procedures of aligning reads to the GVCV reference genome, calculating reads per million, and assembling the GVCV genome from vsRNAs have been described previously (Howard and Qiu 2017). We were able to assemble the GVCV genome with 100% and 99.6% coverage, respectively, from vsRNAs in the infected ‘Chardonel’ grapevines as rootstock or scion (Table 2). The whole GVCV genome with 100% coverage was also assembled in the GVCV-grafted ‘Cabernet Sauvignon.’ The 21-nt small RNAs are predominant among the vsRNAs in all samples, revealing a characteristic feature of the RNA silencing pathway in grapevines. Only 4.7 and 9.4 reads per million (RPM) were mapped to the GVCV genome in the grafted ‘Norton’ scion and rootstock in comparison with the 33,029 and 3,398 RPM in GVCV-infected rootstock and scion (Table 2). These results show that only a very small number of GVCV-specific vsRNAs may have been transferred to ‘Norton’ rootstock or scion through the graft union. We will need to investigate if this small number of vsRNAs would play a role in ‘Norton’s resistance to GVCV, or they are mere background of the RNA-seq technology.

Table 2. Assessment of ‘Norton’ resistance to grapevine vein clearing virus (GVCV) by RNAseq of small RNAs in GVCV-grafted ‘Norton’ versus GVCV-grafted ‘Cabernet Sauvignon’ 3 years after graft-transmission

Scion/Rootstock	vsRNA profiles by RNAseq
	Reads mapped to GVCV in RPMa	Percentage of genome assembled	21 nt	22 nt	24 nt	>24 nt
Norton/	4.7	0	51	8	2	0
GVCV-Chardonel	33,029.0	100	403,627	93,338	15,303	5,595
GVCV-Chardonel/	3,398.8	99.59	37,310	13,414	1,674	1,108
Norton	9.4	0	100	24	5	1
GVCV-Chardonel/	8,237	100	79,226	8,259	1,313	2,715
Cabernet Sauvignon	40,713	100	319,114	83,238	13,476	12,332

^aRPM: reads per million.

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A plant virus replicates in a single cell, moves locally to the neighboring cells, and then systemically through vascular tissues to establish infection in the entire plant. In response, the plant resists a virus via gene silencing, R-gene mediated innate immunity, or the blocking of local and systemic movement. Some plants may not possess host factors that are essential for viral replication and movement (Kang et al. 2005). In a previous study, we compiled GVCV-derived small RNAs (Howard and Qiu 2017). We searched these vsRNAs against the sequenced draft genome of ‘Norton’ and did not find homologous regions on the ‘Norton’ genome that align with GVCV-derived vsRNAs. This suggests that ‘Norton’ may not encode miRNAs that participate in the active defense pathways of degrading GVCV-derived RNAs. Inability of GVCV to move through the graft union suggests that ‘Norton’ as scion or rootstock cannot accept GVCV into vascular tissues, either blocking GVCV movement or lacking host factors assisting GVCV movement or replication.

Genetic background plays a significant role in plants’ resistance to viruses (Gallois et al. 2018). V. aestivalis is native to the Midwest region of the U.S. where GVCV is present at 34% incidence in wild native A. cordata plants (Petersen et al. 2019) and in wild Vitis plants (Beach et al. 2017). Coevolution of V. aestivalis and GVCV selects for resistant wild V. aestivalis accessions. ‘Norton’ genetic background derives mainly from wild V. aestivalis (Ambers 2013; Sawler et al. 2013). Multiple genes from wild V. aestivalis may contribute to form innate immunity to GVCV. Host factors also contribute to each phase of a virus’s life cycle: translation, replication, transcription, movement, and virion assembly (Garcia-Ruiz 2018), and thus are referred to as susceptibility genes. The lack of these host factors forms another line of resistance in that the virus is unable to complete the infection cycle in a host that does not possess these cellular factors (Garcia-Ruiz 2018). One possible scenario is that ‘Norton’ may not encode the genes that are required for GVCV replication and movement. Therefore, genetic and molecular mechanisms underlying ‘Norton’ resistance to GVCV merit further investigation.

Conclusions

‘Norton’ is found to be resistant to GVCV by graft transmission. The conclusion is further supported by absence of GVCV in ‘Norton’ grapevines in vineyards. This unveils a new trait of ‘Norton’ grapevine. Therefore, ‘Norton’ can be used as a parent in breeding GVCV-resistant varieties. Given that virus-resistant Vitis germplasm is scarce, it is worthwhile to assess ‘Norton’ resistance to grapevine viruses other than GVCV. Further study on the genetic and molecular mechanisms underlying ‘Norton’ resistance to GVCV will provide novel insights on understanding a woody perennial plant’s resistance to a virus and interconnectedness of ‘Norton’ resistance to multiple pathogens.

Acknowledgments

We are thankful to those undergraduate and graduate students over the last five years who have contributed to this project.