An Effective Method of Ribes spp. Inoculation with Blackcurrant Reversion Virus under In Vitro Conditions

Blackcurrant reversion virus (BRV) is the most destructive currant-infecting and mite-transmitted pathogen from the genus Nepovirus. In this work, BRV transmission in the system Ribes ex vitro–Ribes in vitro was applied for the first time. Triple infection of BRV identified in blackcurrant cv. Gojai was used for phylogenetic analysis and inoculation assay. Transmission of BRV was successful due to its stability in the inoculum for up to 8 days at 4 °C; all BRV isolates were infectious. Our suggested inoculation method through roots was applied in six Ribes spp. genotypes with 100.0% reliability, and the expression levels of defence-related gene PR1 to biotic stress was observed. The prevalence of the virus in microshoots after 2–14 days post-inoculation (dpi) was established by PCR. In resistant genotypes, the BRV was identified up to 8 dpi; meanwhile, infection remained constant in susceptible genotypes. We established that BRV transmission under controlled conditions depends on the inoculum quality, post-inoculation cultivation temperature, and host-plant susceptibility to pathogen. This in vitro inoculation method opens possibilities to reveal the resistance mechanisms or response pathways to BRV and can be used for the selection of resistant Ribes spp. in breeding programs.

The BRV belongs to the subgroup c of the genus Nepovirus, family Secoviridae [10,11]. Virus isometric particles are approximately 27 nm in diameter, and the viral genome consists of two polyadenylated, single-stranded positive-sense RNA1 and RNA2 molecules [12,13]. It is known that the 3 nontranslated region (NTR) of RNAs are essential for virus lifecycles, promoting translation regulation and its efficiency [14], and consequently were used for BRV detection or phylogenetic analysis [6, [15][16][17].
The BRD according to symptoms is divided into two forms-European (E) and Russian (R) [4,18]. The main difference between the forms is the severity and rate of symptoms expressed in the infected plants. Both forms cause a transformation in leaf shape, a decrease in marginal serration and in the number of main veins and hair numbers, and an increase Table 1. Percent identity of BRV isolates at nucleotide level of RNA2 3 NTR regions found in Lithuanian cv. Gojai.
A phylogenetic dendrogram was constructed to determine the viral infection affinity using sequences of BRV identified in Lithuania and sequences submitted to the NCBI database ( Figure 1). Worldwide isolates of BRV formed two reliably distinct branches at 90.0% bootstrap. The tree provides information on the geographic origin of the isolates and the implicit migration of infection. According to the symptoms of the disease caused in host plants, virus isolates in the phylogenetic tree were classified as the R (Russian) form of BRD in most cases, except for the isolate from Canada or plants with virus symptoms. All isolates from Lithuania were grouped into the phylogenetic dendrogram's first branch. In this branch, BRV isolates were still reliably separated into several groups, and BRV_1_18_LT and BRV_7_18_LT formed a separate phylogenetic branch at 100.0% bootstrap. These sequences of RNA2 3 NTR were genetically close to each other but significantly genetically diverse from other BRV isolates worldwide. Isolate BRV_3_18_LT showed diversity from BRV_1_18_LT and BRV_7_18_LT but was genetically close to an isolate from Poland (AF321570).  Table S2 in Supplementary Materials.

Reliability of the Inoculation In Vitro
Symptomatic leaves of blackcurrant cv. Gojai containing heterogeneous infection o BRV, obtained from the plant genetic resources collection of LAMMC, were used in th preparation of inoculum. Stability of RNA2 of BRV in inoculum was investigated. Pre pared inoculum was maintained at 4 and 21 °C for up to 8 days. Specific primer pair ( Table 2) for RNA2 were used for BRV identification. Amplification results of BRV-specifi fragments in inoculum during different storage temperatures are presented in Figure 2 Virus RNA2 was detected in inoculum at 4 °C during the entire period of the experimen (Figure 2A), while BRV-specific fragments were amplified and clearly shown in the inoc ulum only after the first day in storage at 21 °C ( Figure 2B).   Table S2 in Supplementary Materials.

Reliability of the Inoculation In Vitro
Symptomatic leaves of blackcurrant cv. Gojai containing heterogeneous infection of BRV, obtained from the plant genetic resources collection of LAMMC, were used in the preparation of inoculum. Stability of RNA2 of BRV in inoculum was investigated. Prepared inoculum was maintained at 4 and 21 • C for up to 8 days. Specific primer pairs (Table 2) for RNA2 were used for BRV identification. Amplification results of BRV-specific fragments in inoculum during different storage temperatures are presented in Figure 2. Virus RNA2 was detected in inoculum at 4 • C during the entire period of the experiment (Figure 2A), while BRV-specific fragments were amplified and clearly shown in the inoculum only after the first day in storage at 21 • C ( Figure 2B).  Table S2 in Supplementary Materials.

Reliability of the Inoculation In Vitro
Symptomatic leaves of blackcurrant cv. Gojai containing heterogeneous infection of BRV, obtained from the plant genetic resources collection of LAMMC, were used in the preparation of inoculum. Stability of RNA2 of BRV in inoculum was investigated. Prepared inoculum was maintained at 4 and 21 °C for up to 8 days. Specific primer pairs ( Table 2) for RNA2 were used for BRV identification. Amplification results of BRV-specific fragments in inoculum during different storage temperatures are presented in Figure 2 Virus RNA2 was detected in inoculum at 4 °C during the entire period of the experiment (Figure 2A), while BRV-specific fragments were amplified and clearly shown in the inoculum only after the first day in storage at 21 °C ( Figure 2B).   The standard curve for quantification of the virus concentration by RT-PCR with eight points was constructed ( Figure 3). Plasmid vector with RNA2 3 NTR of the BRV was used to determine the number of copies of the virus. Through the range from 1.98 × 10 3 to 1.98 × 10 10 copies per reaction, the threshold cycle (Ct) and copy numbers displayed a linear relationship with an R 2 of 0.9986 and the equation y = -3.6614x + 29.923 ( Figure 3). The value of Ct obtained in the inoculum was 21.73 (±0.15). The standard curve for quantification of the virus concentration by RT-PCR with eight points was constructed ( Figure 3). Plasmid vector with RNA2 3′ NTR of the BRV was used to determine the number of copies of the virus. Through the range from 1.98 × 10 3 to 1.98 × 10 10 copies per reaction, the threshold cycle (Ct) and copy numbers displayed a linear relationship with an R 2 of 0.9986 and the equation y = -3.6614x + 29.923 ( Figure 3). The value of Ct obtained in the inoculum was 21.73 (±0.15). To determine the efficiency of the inoculation method in vitro, microshoots with roots of R. aureum, R. dikuscha, and R. nigrum cvs. Aldoniai, Ben Gairn, Ben Tirran, and Vernisaz were inoculated by soaking in the same way as shown in Figure 4. The roots increase the contact area between the inoculum and the plant, and enhance the entry of the virus into the phloem. Based on our data in Figure 2, a post-inoculation period at 4 °C is necessary to ensure viability of the virus on the surface of the roots. In the post-inoculation period at 2, 4, 6, 8, 10, and 14 days, the virus infection in microshoots without roots was evaluated by PCR ( Figure 5). According to the data for 481 bplength BRV-specific fragment sequencing, all isolates of BRV were transmitted to infected plants by in vitro mechanical inoculation. The nucleotide sequences of these fragments completely coincided with the sequence provided to NCBI (MH891843, MH891844, and MH891845). This indicates that BRV strains obtained in the cultivar Gojai have the potential to contaminate and spread in an in vitro system. To determine the efficiency of the inoculation method in vitro, microshoots with roots of R. aureum, R. dikuscha, and R. nigrum cvs. Aldoniai, Ben Gairn, Ben Tirran, and Vernisaz were inoculated by soaking in the same way as shown in Figure 4. The roots increase the contact area between the inoculum and the plant, and enhance the entry of the virus into the phloem. Based on our data in Figure 2, a post-inoculation period at 4 • C is necessary to ensure viability of the virus on the surface of the roots.
The standard curve for quantification of the virus concentration by RT-PCR with eight points was constructed ( Figure 3). Plasmid vector with RNA2 3′ NTR of the BRV was used to determine the number of copies of the virus. Through the range from 1.98 × 10 3 to 1.98 × 10 10 copies per reaction, the threshold cycle (Ct) and copy numbers displayed a linear relationship with an R 2 of 0.9986 and the equation y = -3.6614x + 29.923 ( Figure 3). The value of Ct obtained in the inoculum was 21.73 (±0.15). To determine the efficiency of the inoculation method in vitro, microshoots with roots of R. aureum, R. dikuscha, and R. nigrum cvs. Aldoniai, Ben Gairn, Ben Tirran, and Vernisaz were inoculated by soaking in the same way as shown in Figure 4. The roots increase the contact area between the inoculum and the plant, and enhance the entry of the virus into the phloem. Based on our data in Figure 2, a post-inoculation period at 4 °C is necessary to ensure viability of the virus on the surface of the roots. In the post-inoculation period at 2, 4, 6, 8, 10, and 14 days, the virus infection in microshoots without roots was evaluated by PCR ( Figure 5). According to the data for 481 bplength BRV-specific fragment sequencing, all isolates of BRV were transmitted to infected plants by in vitro mechanical inoculation. The nucleotide sequences of these fragments completely coincided with the sequence provided to NCBI (MH891843, MH891844, and MH891845). This indicates that BRV strains obtained in the cultivar Gojai have the potential to contaminate and spread in an in vitro system. In the post-inoculation period at 2, 4, 6, 8, 10, and 14 days, the virus infection in microshoots without roots was evaluated by PCR ( Figure 5). According to the data for 481 bp-length BRV-specific fragment sequencing, all isolates of BRV were transmitted to infected plants by in vitro mechanical inoculation. The nucleotide sequences of these fragments completely coincided with the sequence provided to NCBI (MH891843, MH891844, and MH891845). This indicates that BRV strains obtained in the cultivar Gojai have the potential to contaminate and spread in an in vitro system. In vitro plants of R. dikuscha and R. nigrum cvs. Aldoniai or Ben Gairn remained symptom-free in the post-inoculation period. Clearly, symptoms of response to biotic stress were visible on R. aureum microplants after 6 dpi ( Figure 6). The leaves changed their colour, and the clear synthesis of the stress-characteristic phenolic compounds became visible. The incidence rate of BRV in all Ribes cultivars and species was 100% for 20 microshoots in each treatment. However, the persistence of the infection in the plant was different. Until the end of the experiment, 2 weeks after inoculation, the infection remained constant in the BRV-susceptible genotypes: R. aureum and R. nigrum cvs. Ben Tirran and Vernisaz. The viral infection was detected in the resistant plants, R. dikuscha and R. nigrum cv. Ben Gairn, until 6 dpi, and in R. nigrum cv. Aldoniai until 8 days after infection ( Figure 5).

Relative Gene Expression in Response to BRV Infection
The quantitative expression profiles of the pathogenesis-related 1 (PR1) gene after BRV infection were compared in both resistant and susceptible genotypes for treatment periods 2, 4, 6, and 8 dpi (Figure 7). We established that PR1 was rapidly induced after BRV infection in Ribes spp. The expression levels were significantly higher in all genotypes in comparison with the control plants during the entire period of inoculation. The highest expression values of PR1 were presented in virus-susceptible genotypes R. nigrum cvs. Ben Tirran and Vernisaz at 4-and 8-days post-inoculation. Relative PR1 expression confirms successful virus infection in all microshoots in vitro; gene response to biotic stress was found in all treatments after using our proposed in vitro inoculation method through roots. In vitro plants of R. dikuscha and R. nigrum cvs. Aldoniai or Ben Gairn remained symptom-free in the post-inoculation period. Clearly, symptoms of response to biotic stress were visible on R. aureum microplants after 6 dpi ( Figure 6). The leaves changed their colour, and the clear synthesis of the stress-characteristic phenolic compounds became visible. In vitro plants of R. dikuscha and R. nigrum cvs. Aldoniai or Ben Gairn remained symptom-free in the post-inoculation period. Clearly, symptoms of response to biotic stress were visible on R. aureum microplants after 6 dpi ( Figure 6). The leaves changed their colour, and the clear synthesis of the stress-characteristic phenolic compounds became visible. The incidence rate of BRV in all Ribes cultivars and species was 100% for 20 microshoots in each treatment. However, the persistence of the infection in the plant was different. Until the end of the experiment, 2 weeks after inoculation, the infection remained constant in the BRV-susceptible genotypes: R. aureum and R. nigrum cvs. Ben Tirran and Vernisaz. The viral infection was detected in the resistant plants, R. dikuscha and R. nigrum cv. Ben Gairn, until 6 dpi, and in R. nigrum cv. Aldoniai until 8 days after infection ( Figure 5).

Relative Gene Expression in Response to BRV Infection
The quantitative expression profiles of the pathogenesis-related 1 (PR1) gene after BRV infection were compared in both resistant and susceptible genotypes for treatment periods 2, 4, 6, and 8 dpi (Figure 7). We established that PR1 was rapidly induced after BRV infection in Ribes spp. The expression levels were significantly higher in all genotypes in comparison with the control plants during the entire period of inoculation. The highest expression values of PR1 were presented in virus-susceptible genotypes R. nigrum cvs. Ben Tirran and Vernisaz at 4-and 8-days post-inoculation. Relative PR1 expression confirms successful virus infection in all microshoots in vitro; gene response to biotic stress was found in all treatments after using our proposed in vitro inoculation method through roots. The incidence rate of BRV in all Ribes cultivars and species was 100% for 20 microshoots in each treatment. However, the persistence of the infection in the plant was different. Until the end of the experiment, 2 weeks after inoculation, the infection remained constant in the BRV-susceptible genotypes: R. aureum and R. nigrum cvs. Ben Tirran and Vernisaz. The viral infection was detected in the resistant plants, R. dikuscha and R. nigrum cv. Ben Gairn, until 6 dpi, and in R. nigrum cv. Aldoniai until 8 days after infection ( Figure 5).

Relative Gene Expression in Response to BRV Infection
The quantitative expression profiles of the pathogenesis-related 1 (PR1) gene after BRV infection were compared in both resistant and susceptible genotypes for treatment periods 2, 4, 6, and 8 dpi (Figure 7). We established that PR1 was rapidly induced after BRV infection in Ribes spp. The expression levels were significantly higher in all genotypes in comparison with the control plants during the entire period of inoculation. The highest expression values of PR1 were presented in virus-susceptible genotypes R. nigrum cvs. Ben Tirran and Vernisaz at 4-and 8-days post-inoculation. Relative PR1 expression confirms successful virus infection in all microshoots in vitro; gene response to biotic stress was found in all treatments after using our proposed in vitro inoculation method through roots.

Discussion
A sensitive plant-testing system is an important tool for targeted and accelerated selection of resistant genotypes in Ribes breeding programs. The inoculation method under in vitro conditions is appropriate to screen genotypes for their responses to pathogens and could be a useful tool for better understanding the virus-host interaction and resistance mechanisms. Virus inoculation systems suitable for in vitro conditions have been developed in herbaceous plants such as potatoes, lettuce, and tomatoes [24][25][26]. However, inoculation systems for woody perennial plants under in vitro conditions are unexplored. In general, an in vitro system for orchard and berry cultures is usually used for virus elimination and micropropagation [27].
BRV is one of the most serious pathogens in blackcurrant plantations; its control is complicated and the consequences are economically damaging [28]. Heterogenic infection of BRV in the same host plant of field-grown cv. Gojai was shown by the diversity of the sequences (Table 1) and the data of phylogenetic analysis (Figure 1). In this study, we found that multiple infections of BRV with different origins could be detected in one host plant. The natural vector transmitting BRV among bushes is the gall mite. Under controlled conditions, transmission assays showed that 3 h of mite feeding on blackcurrant seedlings was sufficient for virus transmission [21]. However, previous studies have shown that using the gall mite infection method in vitro had difficulties in controlling both the infection pressure and the influence of environmental conditions on the spread of mites and BRV [28,29].
The BRV genome consists of two molecules of positive-sense RNAs: RNA1 and RNA2, which are packed in viral particles. BRV particles are composed of two capsid proteins (54 and 55 kDa) and can replicate in a low range of hosts [22,30]. The 3′ NTRs of BRV are highly similar and the most stable parts of the genome, and are therefore used for detection by PCR [15,31]. Genetic identity among isolates, from 94.6 to 99.6% in the 3′ nontranslated region of RNA2, was also found in our study (Table 1). Only virus particles formed from the 54 kDa fragment can be used for indicator plant infection by mechanical inoculation, but the resulting progeny viruses contain both forms [32]. For the first time, blackcurrant reversion virus was successfully mechanically transmitted from symptomatic blackcurrant leaves to Chenopodium quinoa [22] and to other herbaceous plants, Nicotiana occidentalis and Nicotiana tabacum [17]. These cases of mechanical inoculation are the only two reported in the literature where the virus was transmitted from blackcurrants to herbaceous plants, despite the attempts of scientists to repeat it. Despite these difficul-

Discussion
A sensitive plant-testing system is an important tool for targeted and accelerated selection of resistant genotypes in Ribes breeding programs. The inoculation method under in vitro conditions is appropriate to screen genotypes for their responses to pathogens and could be a useful tool for better understanding the virus-host interaction and resistance mechanisms. Virus inoculation systems suitable for in vitro conditions have been developed in herbaceous plants such as potatoes, lettuce, and tomatoes [24][25][26]. However, inoculation systems for woody perennial plants under in vitro conditions are unexplored. In general, an in vitro system for orchard and berry cultures is usually used for virus elimination and micropropagation [27].
BRV is one of the most serious pathogens in blackcurrant plantations; its control is complicated and the consequences are economically damaging [28]. Heterogenic infection of BRV in the same host plant of field-grown cv. Gojai was shown by the diversity of the sequences (Table 1) and the data of phylogenetic analysis (Figure 1). In this study, we found that multiple infections of BRV with different origins could be detected in one host plant. The natural vector transmitting BRV among bushes is the gall mite. Under controlled conditions, transmission assays showed that 3 h of mite feeding on blackcurrant seedlings was sufficient for virus transmission [21]. However, previous studies have shown that using the gall mite infection method in vitro had difficulties in controlling both the infection pressure and the influence of environmental conditions on the spread of mites and BRV [28,29].
The BRV genome consists of two molecules of positive-sense RNAs: RNA1 and RNA2, which are packed in viral particles. BRV particles are composed of two capsid proteins (54 and 55 kDa) and can replicate in a low range of hosts [22,30]. The 3 NTRs of BRV are highly similar and the most stable parts of the genome, and are therefore used for detection by PCR [15,31]. Genetic identity among isolates, from 94.6 to 99.6% in the 3 nontranslated region of RNA2, was also found in our study (Table 1). Only virus particles formed from the 54 kDa fragment can be used for indicator plant infection by mechanical inoculation, but the resulting progeny viruses contain both forms [32]. For the first time, blackcurrant reversion virus was successfully mechanically transmitted from symptomatic blackcurrant leaves to Chenopodium quinoa [22] and to other herbaceous plants, Nicotiana occidentalis and Nicotiana tabacum [17]. These cases of mechanical inoculation are the only two reported in the literature where the virus was transmitted from blackcurrants to herbaceous plants, despite the attempts of scientists to repeat it. Despite these difficulties, sap from C. quinoa symptom-bearing leaves was transmitted from 4 to 11 days using carborundum to other herbaceous plants: C. amaranticolor, C. murale, N. benthamiana, N. clevelandii, N. debnyi, and N. occidentalis [16,22]. The successful slash inoculation of young tissue-culture-propagated blackcurrants with the isolated virus from C. quinoa was performed by A. Lemmetty and K. Lehto [23]. This plant material is more sensitive due to its soft tissues and low content of phenolic compounds. The first symptoms on the leaves were observed after 5-7 months. Furthermore, the visual symptoms of the disease appeared only after 4-5 years when inoculating blackcurrant woody plants. Additional fulfilling of Koch's postulates was achieved when mechanically inoculated plants developed typical BRD symptoms on leaves and flower buds [15,18].
It was observed that some plant viruses are difficult to inoculate mechanically because they are transmitted by arthropods during feeding and their localisation to vascular tissue (usually phloem) is limited [33]. The BRV can reach low titres at the beginning of the spread of the infection in the Ribes plants, and does not give a titre in genetically resistant plants under natural conditions. Specific antibodies for BRV proteins are not commercially available [10,20]; therefore, polymerase chain reaction is the most sensitive and reliable detection method at present. Using PCR in this study, BRV infection was approved until 8 dpi in BRV-resistant plants R. dikuscha, cv. Aldoniai (R. dikuscha in progeny) [34] and cv. Ben Gairn (R. uva-crispa in progeny) [5].
The amount of virus in the inoculum is an important factor for mechanical infection transmission. Too high or too low virus concentration in the inoculum leads to unsuccessful plant infection [35]. For example, wheat streak mosaic virus (WSMV) inoculum with 2.21 × 10 6 concentration was most suitable for mechanical inoculation in wheat [36]. In this study, for inoculum preparation we mixed the inoculation buffer with plant sap and triple BRV infection (approximately 2.24 × 10 4 copies in µL) (Figure 3). All three isolates of BRV ( Figure 1) appeared infectious according to the homology of 481 bp fragment ( Figure 5) sequencing in this study.
Pathogenesis-related 1 gene is the most abundant gene family of PRs, and the expression of PR1 is used as a molecular marker to indicate plant defence response to different types of insects and pathogens [37]. In our previous study, the biotic stress defence response PR1 homolog emphasizing the R. nigrum genome was found, and the primer pair identifying this gene was designed [38]. Therefore, in this work, RT-PCR was used to establish the host-plant defence response to viral infection, as biotic stress, for showing the success of the suggested inoculation method (Figure 7). We determined the expression profiles of PR1 in resistant and susceptible genotypes during the entire period of the experiment after mechanical BRV inoculation in vitro. The defence response pathway with the determined PR1 expression was characteristic for all genotypes, regardless of their resistance to BRV. The expression levels of PR1 in susceptible genotypes R. nigrum cvs. Ben Tirran and Vernisaz were higher compared to susceptible R. aureum and resistant genotypes. However, all susceptible genotypes had a similar tendency showing significant increase in PR1 at 4 and 8 dpi. It was found that in the Ribes spp., as in other plants, PRs are necessary for the establishment of systemic acquired resistance (SAR) in tissues distant from the primary infection site [39].
The in vitro inoculation technique described here is the first reported in the literature where the BRV was successfully transmitted from blackcurrants to blackcurrants with maximum reliability. Several virus transmission methods (leaf damage with carborundum powder; stem soaking in the inoculum; inoculum dripping with syringe into the stem) have been tested on microshoots during our research work, but all of them have failed. Possibilities to successful viral infection through inoculation of Ribes plants grown in vitro by using a mechanical approach are possible when the appropriate conditions are used (Figure 4). Rooted microshoots were the most suitable plant material for easy virus entry and movement in the plant because of its soft tissues and low content of phenolic compounds. Based on studies by P. Susi [10] and our data (Figure 2), cold storage (4 • C) in the first period after inoculation is necessary to ensure virus viability and for causing additional abiotic stress in the plant.
It is suggested that the procedure described in this study may be widely applicable for blackcurrant reversion virus mechanical inoculation using sap from R. nigrum. The principle for the inoculation method depends on soaking the roots of microshoots in the PBS buffer containing an infectious BRV isolate. The method was used successfully to inoculate susceptible Ribes spp. plants with BRV and to study the genetic response for BRV in resistance plants.

Plant Material
Leaves of R. nigrum cv. Gojai with symptoms of BRD presented in field conditions were used for preparation of inoculum. Inoculum containing BRV was tested for virus stability, infectivity, and genetic analysis. Rooted microshoots of the species R. aureum, R. dikuscha and R. nigrum cvs. Aldoniai, Ben Gairn, Ben Tirran, Vernisaz were used for inoculation treatments under in vitro conditions.

In Vitro Culture of Ribes spp.
All manipulations with plants in the in vitro system were performed using modified MS medium (Table S1 in Supplementary Materials) [40]. Microshoots of Ribes species and cultivars were propagated on medium I (3 weeks), grown on medium II up to 3-4 cm (2 weeks), and rooted on nutrition medium III (4 weeks). Plants grown in this way were used in the inoculation assay in vitro. Micropropagation and rooting of Ribes were carried out under controlled conditions in a growth chamber at 21 ± 2 • C, 50-150 µmol m -2 s -1 light intensity, and 16/8 h photoperiod.

Evaluation of BRV Stability in the Inoculum
Aliquots of the prepared inoculum were stored at 4 and 21 • C for 8 days to assess virus stability. RNA was extracted from each treatment of inoculums (fresh and after 1, 2, 4, 6, and 8 days). Virus RNA was evaluated by the PCR method using 3 primer pairs from different sites of the RNA2 3 NTR regions in the BRV genome (Table 2).  20 primer was used in the reaction.

BRV Detection by PCR
The detection of BRV was carried out by polymerase chain reaction (PCR) in the inoculum and in the Ribes spp. microshoots after mechanical inoculation in vitro. BRV-specific oligonucleotide primers and their characteristics are presented in Table 2 (Table 2), 72 • C for 40 s, and the final elongation step at 72 • C for 10 min. The amplification products were analysed in 1.5% (w/v) agarose gel using electrophoresis and visualized by ethidium bromide staining and UV illumination. The size of the PCR products was determined with O'GeneRuler 1kb DNA Ladder (Thermo Scientific, Vilnius, Lithuania).

Purification, Cloning, Sequencing, and Digestion of RNA2 3 NTR of BRV
Amplified fragments for sequencing with primer pair BCP11/P2 (Table 2) were purified using a GeneJET Gel Extraction Kit (Thermo Scientific, Vilnius, Lithuania). Fragments were ligated into pJET 1.2 blunt vector using a CloneJET™ PCR Cloning Kit (Thermo Scientific, Vilnius, Lithuania). The strain E. coli JM107 was transformed using Transfor-mAid Bacterial Transformation Kit (Thermo Scientific, Vilnius, Lithuania). Ten samples of plasmids with BRV insertions were sequenced using a Big Dye Terminator v 3.1 Cycle Sequencing Kit and performed on a 3130 Genetic Analyzer (Applied Biosystem, Waltham, MA, USA).

Plant Inoculation In Vitro
Before inoculation, virus-free rooted microshoots were placed in the dark for 48 h at 21 ± 2 • C. Under sterile conditions, roots of 4-week-old microshoots were shortened with a scalpel to a length of 4 mm and soaked for 2 min in sterile tubes with fresh inoculum containing BRV (Figure 4). Inoculated plants were replanted on nutrition medium II (Table S1 in Supplementary Materials) and cultivated for 2 weeks at 4 • C, 16/8 h photoperiod. Mock-inoculated microshoots in PBS buffer were used as the negative control. For RNA isolation, 20 microshoots of each genotype were frozen in liquid nitrogen after cutting their roots and immediately used for RNA isolation.

Statistical Analysis
Sequences of the three Lithuanian BRV isolates were uploaded into the NCBI database (accession numbers MH891843, MH891844, and MH891845). A percent identity matrix among the 3 NTR of RNA2 sequences of BRV was created by the Clustal 2.1 program.
The phylogenetic tree was constructed comparing 3 Lithuanian BRV sequences with 21 sequences of homologous region of BRV with reference to the NCBI database. Phylogenetic analysis was performed using the maximum likelihood method implemented in the PhyML program; a bootstrap analysis with 100 replications was performed [41]. Relative expression was assessed by the 2(-Delta Delta C (T)) method [42]. An actin gene, having constant expression levels (data not shown), was used to normalize raw data and calculate relative transcript levels. Means and SEM (standard error of the mean) from independent experiments were subjected to STAT-ENG.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants11131635/s1, Table S1: Composition of modified MS [35] nutrition medium for propagation, growth, and rooting of blackcurrant microshoots, Table S2: Accession numbers and details of blackcurrant BRV sequences obtained from the NCBI database and included in the phylogenetic tree of this study.