The obtained complete sequences of RNA1 and RNA2 of ToTV-Wal'17 have been deposited in the NCBI database (MW729382 and MW729383). The lengths of RNA1 and RNA2 of the ToTV-Wal'17 were calculated and showed to have 7419 and 5390 nts (excluding polyA tail), respectively, indicating that RNA1 of Wal'17 was 394 nts shorter than the corresponding RNA strand from ToTV-Wal'03. The performed comparative sequence analyses of RNA1 of ToTV-Wal'17 showed 99% (in a range of 1-6826 nts), and 77% (6899–7419 nts) sequence identity within the corresponding RNA of ToTV-Wal'03 (EU563948), respectively. On the other hand, the entire RNA2 had 99% sequence identity with the corresponding RNA of Wal'03 (EU563947). All indicated nucleotide substitutions in both RNAs of Wal'17 were listed in Table 1.
Prot - protease; Hel - helicase, RdRp – RNA dependent polymerase, UTR- untranslated region, ORF- open reading frame, 3A- protein involved in virus movement, 11K- domain
Analysis of 3'untranslated regions (3'UTRs)
The RNA1 and RNA2 3'UTRs of ToTV-Wal'17 had 836 and 1092 nts, respectively. In general, in all ToTV isolates sequenced so far, the 3'UTR in RNA1 is longer than in RNA2. Surprisingly, contrary to ToTV-Wal'03, the 3'UTR in RNA1 of ToTV-Wal'17 was 256 nts shorter than that in RNA2. Moreover, by comparing sequence length, it was shown that 3'UTR in RNA1 of ToTV-Wal'17 was 394 nts shorter (32%) than the corresponding sequence from ToTV-Wal'03. On the other hand, the RNA2 3'UTR of ToTV-Wal'17 was the same in length as the analogous sequence from ToTV-Wal'03.
Previously it was shown that 3'UTR in ToTV RNAs were predicted to form structured regions . Additionally, RNA1 and RNA2 3'UTRs of European isolates of ToTV are generally long (over 1000 nucleotides), and they are organised according to the following scheme: stop codon, followed by variable region (D-VR, 241 and 103 nts in RNA1 and RNA2, respectively), then conserved region (D-CR, 989 nts in RNA1 and RNA2) with terminal polyA tail. Moreover, in the CR in the 3'UTRs direct repeats were identified and arranged in the following repetitive motifs: D1b – D2a – D1a – D2b .
Concerning the ToTV-Wal'17, sequence comparison of its RNA1 3'UTR indicated a short variable region D-VR was preserved in the viral RNA. However, the D-CR region in RNA1 shared only 77% identity with the corresponding D-CR region from ToTV-Wal'03. Due to the high sequence variability, the repeated sequenced D1b – D2a – D1a – D2b were not identified. Notably, within the D-CR of the 3'UTR of RNA1 in ToTV-Wal'17, three other repetitive sequences of 70–71 nts in length (designed as CRRNA1-R1 (position 6807–6877), CRRNA1-R2 (6899–6969), CRRNA1-R3 (7171–7240)) were identified (Fig. 1). The three mentioned repetitive sequences share 90–96% identity with the D2b region ToTV-Wal'03  .
The RNA2 3'UTR of ToTV-Wal'17 shares 99% identity with the corresponding region of ToTV-Wal'03 and in the CR region, the repetitive motifs D1b – D2a – D1a – D2b were still present.
Additionally, performed phylogenetic studies based on the 3'UTR of RNA1 and RNA2 confirmed the results from comparative sequences studies mentioned above. In the dendrogram generated based on 3'UTR RNA1, we showed that the ToTV-Wal'17 isolate clustered with the recently reported South African isolate, and they both are phylogenetically distant from the other Polish and Spanish isolates of ToTV. The 3'UTR of RNA2 sequence analysis showed that the ToTV-Wal'17 clustered together with the ToTV-Wal'03 and had a short evolutionary distance with other Polish and Spanish isolates (Fig. 2).
Here we characterised new mutations (silent and missense) accumulated in ToTV-Wal'17 genome after mechanical passages from host-to-host over time. We observed that the performed mechanical transmission of the virus did not affect its pathogenicity: neither attenuated nor boosted virus severity on S. lycopersicum or N. benthamiana.
The high mutation rate of plant RNA viruses is directed by virus RNA polymerase without proofreading activity . However, environmental pressure favours such viral RNA molecules (genomes) that can efficiently replicate, infect and spread in the host . Therefore, it is not surprising that a few new mutations changing amino acid context in virus polyproteins were described in the coding region of the ToTV-Wal'17 genome after thirteen years of mechanical passages through the same hosts (tomato and N. benthamiana). However, what might be surprising is a new genetic variant of virus RNA1 with substantially shortened 3'UTR. So significantly shortened sequence and its high variability have not been described so far for other European isolates of ToTV. The comparative sequence studies of 3'UTRs of RNA1 of ToTV isolates deposited in GenBank between 2007–2014 showed similar lengths; only in Polish isolates Kra and Ros ToTV the additional deletion variants were observed.
On the other hand, the new ToTV isolate reported in 2020 by Moodley in comparison to the present Wal'17 ToTV indicates a further reduction in this region of the viral genome. However, in the Wal'17 isolate, the deletions occurred in the D-CR region, which is almost identical in both RNA strands, suggesting that this region might be, to some extent, redundant. Thus, the question arises, what was the reason of significant RNA1 3'UTR shortening in these two geographically distant isolates, including the fact that Wal'17 evolved from a much longer prototype variant of Wal'03.
Our previous studies described the genetic variability of 3'UTR in RNA1 of ToTV-Kra and ToTV-Ros, associated with diverse deletions concerning only the D-VR region [20, 21]. However, in the mentioned ToTV isolates, the D-CR regions remained relatively unchanged, with preserved repetitive motifs possibly involved in virus adaptation to hosts. It was discussed then that this sequence conservation in D-CR of RNA1 and RNA2 3'UTR fixated the presence of predicted secondary (and possibly tertiary) intramolecular interactions  that were anticipated to be created within the RNAs. Budziszewska et al. discussed the phenomenon of genetic heterogeneity in RNA1 of Polish ToTV isolates, suggesting that the variable in lengths variants of 3'UTRs may have a potential role in the virus replication process and/or transmission by insect vector . The sequencing data and greenhouse observation obtained in a present study suggest that the regulatory elements (involved in virus replication) might be located outwardly in the D-CR region. The mechanism leading to the shortening of RNA1 3'UTR remains unknown. It might have been spontaneous or could have been forced by virus adaptability to plant host due to long-term passaging. Further experiments explaining the effect of identified variability in RNA1 and the function of characteristic direct repeats in ToTV 3'UTRs RNAs are necessary to be performed to understand the ToTV evolution concerning plant-virus-vector interaction.