Viruses of the genus Begomovirus (family Geminiviridae) have emerged as important plant pathogens in tropical and subtropical regions [1]. Begomoviruses have small, circular, single-stranded DNA genomes and are vectored by whiteflies of the Bemisia tabaci cryptic species complex. The genome is arranged either in two components, referred to as DNA-A and DNA-B, or in a single component [2]. Begomoviruses have a wide diversity due to high mutation [3] and recombination frequencies [4]. Notably, tomato plants are hosts to many begomoviruses in Brazil, and the emergence of new species has been continuously observed [5]. Here, we describe a new bipartite tomato begomovirus sequence discovered in Brazil.
During a survey in a processing tomato field in Luziânia, Goiás state, Brazil (16°18’50.8” S, 47°42’35.2” W) in April 2020, a tomato plant (cv. AP533) with strong symptoms of mottling, leaf distortion, chlorotic spots, vein clearing, and mosaic (Fig. 1a) was observed. Total DNA was extracted, and the viral circular DNA was amplified by rolling-circle amplification (RCA, New England Biolabs – NEB, Ipswich, USA). Then, the RCA product was partially digested with PstI, BglII, NcoI, and EcoRI (NEB), pooled and sequenced using the Ligation Sequencing Kit (SQK-LSK109) (Oxford Nanopore Technologies - ONT, Oxford, UK). Briefly, the digested DNA was treated with NEBNext Ultra™ II End Repair/dA-Tailing Module (NEB), ligated to AMX sequencing adapter with NEB Blunt/TA ligation Master Mix (NEB), and loaded onto the Flongle flow cell (FLO-FLG106) on a MinION Mk1B (ONT). Raw FAST5 files were base-called under high accuracy mode (Guppy 4.4.2). Draft genomes were generated with TideHunter [6] and polished with Medaka v1.2.3 (https://github.com/nanoporetech/medaka). The contigs were transferred to Geneious 8.1.9 software (Biomatters Ltd. Auckland, New Zealand) and analyzed using the BLAST algorithm. Contigs presenting high identity with DNA-A and -B of begomoviruses were detected, and the full genome sequence of a potential new bipartite begomovirus was identified. To confirm it, the DNA-A was Sanger sequenced from the RCA product, and a dimeric genome was cloned into pCAMBIA0380 (Cambia Laboratory, Canberra, Australia) after partial digestion by NcoI (Thermo Fisher Scientific, Waltham, USA).
The assembled DNA-A genomes from the RCA product, 2 clones, and the Nanopore sequence contig were highly similar with 0-2 nucleotides mismatch. Clone 16 (GenBank MW561191) was 2596 nt-long, while the DNA-B consensus sequence from Nanopore was 2578 nt-long (GenBank MW650837). The genome organization was typical of bipartite New World begomoviruses (Fig. 1b; DNA-A: CP, Rep, TrAP, REn, AC4; DNA-B: NSP, MP). The conserved geminiviral nonanucleotide sequence (TAATATTAC) was detected in both components, and they shared a common region of 180 nt with 98.33% identity. Furthermore, upstream the TATA box, the putative Rep binding sites TGTAA and TGTAC were identified in both segments, strongly suggesting that they are cognate DNA segments. These putative core iteron sequences were observed in two tomato mottle leaf curl virus (ToMoLCV) sequences, whereas other ToMoLCV isolates and the closest begomoviruses contained other iteron-like sequences (Supplementary Fig. S1).
An SDT analysis [7] of the DNA-A showed the highest genome-wide identity of 81.65% with tomato golden leaf distortion virus (ToGLDV, HM357456), while the DNA-B shared 82.57% identity with tomato interveinal chlorosis virus-2 (ToICV2, MK087039) (Supplementary Fig. S2). Since the current species demarcation criterion of Begomovirus is 91% nucleotide identity of the DNA-A, we conclude that this isolate is a new member of the genus, and the name Tomato mottle leaf distortion virus is proposed. Although tomato mottle leaf distortion virus (ToMoLDiV) DNA-A sequence is closer to ToGLDV, it clustered in a phylogenetic tree with tomato mosaic severe dwarf virus (ToMSDV) (Fig. 2a). The DNA-B clustered with ToICV2 (Figure 2b) in the same branch with ToMSDV. ToMoLDiV is thus closely related to ToGLDV, ToMSDV, and ToICV2.