TYPE Original Research PUBLISHED 14 December 2022 DOI 10.3389/fpls.2022.1011565 Genome-wide identification OPEN ACCESS of Reverse Transcriptase EDITED BY Gennady I. Karlov, All-Russia Research Institute of domains of recently Agricultural Biotechnology, Russia REVIEWED BY inserted endogenous plant Katja R. Richert-Pöggeler, Julius Kühn-Institut, Germany Osamah Alisawi, pararetrovirus (Caulimoviridae) University of Kufa, Iraq *CORRESPONDENCE Carlos de Tomás and Carlos M. Vicient* Carlos M. Vicient carlos.vicient@cragenomica.es Structure and Evolution of Plant Genomes Group, Centre for Research in Agricultural Genomics, CSIC-IRTA-UAB-UB, Edifici CRAG, Bellaterra, Barcelona, Spain SPECIALTY SECTION This article was submitted to Plant Bioinformatics, a section of the journal Frontiers in Plant Science Endogenous viral elements (EVEs) are viral sequences that have been RECEIVED 04 August 2022 integrated into the nuclear chromosomes. Endogenous pararetrovirus (EPRV) ACCEPTED 15 November 2022 are a class of EVEs derived from DNA viruses of the family Caulimoviridae. PUBLISHED 14 December 2022 Previous works based on a limited number of genome assemblies CITATION demonstrated that EPRVs are abundant in plants and are present in several de Tomás C and Vicient CM (2022) Genome-wide identification of species. The availability of genome sequences has been immensely increased Reverse Transcriptase domains of in the recent years and we took advantage of these resources to have a more recently inserted endogenous plant pararetrovirus (Caulimoviridae). extensive view of the presence of EPRVs in plant genomes. We analyzed 278 Front. Plant Sci. 13:1011565. genome assemblies corresponding to 267 species (254 from Viridiplantae) doi: 10.3389/fpls.2022.1011565 using tBLASTn against a collection of conserved domains of the Reverse COPYRIGHT Transcriptases (RT) of Caulimoviridae. We concentrated our search on © 2022 de Tomás and Vicient. This is an open-access article distributed under complete and well-conserved RT domains with an uninterrupted ORF the terms of the Creative Commons comprising the genetic information for at least 300 amino acids. We Attribution License (CC BY). The use, obtained 11.527 sequences from the genomes of 202 species spanning the distribution or reproduction in other forums is permitted, provided the whole Tracheophyta clade. These elements were grouped in 57 clusters and original author(s) and the copyright classified in 13 genera, including a newly proposed genus we called owner(s) are credited and that the Wendovirus. Wendoviruses are characterized by the presence of four open original publication in this journal is cited, in accordance with accepted reading frames and two of them encode for aspartic proteinases. Comparing academic practice. No use, plant genomes, we observed important differences between the plant families distribution or reproduction is permitted which does not comply and genera in the number and type of EPRVs found. In general, florendoviruses with these terms. are the most abundant and widely distributed EPRVs. The presence of multiple identical RT domain sequences in some of the genomes suggests their recent amplification. KEYWORDS pararetrovirus, Reverse Transcriptase (RT), Caulimoviridae, endogenous, virus Abbreviations: EPRV, Endogenous pararetrovirus; RT, Reverse Transcriptase; EVEs, Endogenous viral elements; OUT, operational taxonomic unit. Frontiers in Plant Science 01 frontiersin.org de Tomás and Vicient 10.3389/fpls.2022.1011565 Introduction orthologous across several species, this gives a minimum estimate for the age of the virus that integrated into the Endogenous viral elements (EVEs) are viral sequences that genome (Aiewsakun and Katzourakis, 2015). have been integrated into the nuclear chromosomes, enabling Caulimoviridae is a family of double-stranded DNA (dsDNA) their vertical transmission and potential fixation in host viruses infecting plants that contain a reverse transcription stage populations (Feschotte and Gilbert, 2012). Viral integration in their replication cycle (International Committee on Taxonomy within eukaryotic genomes is a widely recognized phenomenon of Viruses, ICTV, https://ictv.global/). Although integration into described in many species thanks to the sequencing of whole the genome is not an essential part of their replication cycle, there genomes. Some of these EVEs are the consequence of a mandatory are much evidence of their presence as integrated forms among genome integration stage in the life cycle of reverse-transcribing genomes of the plant kingdom (Geering et al., 2014; Diop et al., viruses, such as retroviruses (Katzourakis and Gifford, 2010), but 2018) and they have been included as a new category in some for others, such as all plant viruses, hepadnaviruses or the SARS- repetitive DNA sequence databases like Repbase (Bao et al., 2015). CoV-2, the integration in the host genome is not part of the virus Caulimoviridae can be classified into 11 genera based on their life cycle and the mechanisms of integration are few well genome organization (number of open reading frames and the understood (Kojima et al., 2021; Zhang et al., 2021). arrangement of protein domains within them) and the The first described plant EVE was a Geminiviridae element morphology of their virus particles (ICTV, https://ictv.global/). (Bejarano et al., 1996). EVEs derived from Caulimoviridae are Some of these genera have been reported as EVEs in plant abundant in plants (Diop et al., 2018). EVEs derived from another genomes (Endogenous pararetrovirus, EPRVs) but, in addition, non-retroviral dsRNA, ssRNA, or ssDNA viruses have also been many of the EPRVs belong to a genus for which so far no episomal described as, for example, from Narnaviridae (Choi et al., 2021), counterparts have been described (Geering et al., 2014; Chen and Partitiviridae, Betarhabdovirinae and Betaflexiviridae Kishima, 2016; Diop et al., 2018). (Chiba et al., 2011). The genome integration mechanism of the The integration of an EVE into or near a gene can potentially EVEs remains largely uncharacterized and different mechanisms modify gene transcription or modify mRNA processing, for the integration were proposed. The most accepted theory is resulting in mutant phenotypes. Most of the described EPRVs that endogenization results from a non-homologous are inserted in intergenic regions and have no apparent recombination between virus and host genomes, usually in the deleterious effect on the host. However, there are examples of context of either a double-stranded DNA break repair or a EPRVs inserted inside genes with potential effects on gene transposon-mediated process (Richert-Pöggeler et al., 2021). expression as, for example, in the case of Vitis vinifera, which If EVEs are integrated into or near host genes, this will be has several EPRVs inserted in introns (Geering et al., 2014). generally detrimental, and they will be removed from host Most of the EPRVs are transcriptionally or translationally population by purifying selection. In the rare cases that the inactive because they are partial and/or comprise rearranged integration of an EVE is beneficial, it will be fixed in the host sequences and/or inactivating mutations. Often EPRVs form population by positive selection, in the same way that occurs clusters resulting from the simultaneous integration of several with other types of genomic elements like transposons (Catlin complete or partial copies in tandem or nested (Richert-Pöggeler and Josephs, 2022). However, most of the EVEs are neutral and et al., 2003). Infrequently, these integrated sequences are will become degraded due to the accumulation of disruptive transcriptionally active and the resulting RNAs can serve as mutations, insertions or deletions. Due to the random nature of precursors of extrachromosomal viral DNA and lead to systemic these mutations, it is possible to reconstruct the sequences of and vertically transmitted infections (Hohn et al., 2008; the infectious viruses based on the EVEs sequences, Gayral et al., 2008). Transcriptional activation can be driven by particularly for high copy number EVEs (Aiewsakun and viral promoters present within the integrated element or plant Katzourakis, 2015). In consequence, EVEs can be considered promoters in the vicinity of the EPRV sequence (Lockhart et al., as genomic “fossils” and be employed for investigating viral 2000; Kuriyama et al., 2020). On the other hand, EPRV derived origins and diversity and become the main tool for a new RNAs can also be inducers for RNA interference (RNAi) and gene emerging field called Paleovirology. Paleovirology is the study silencing mechanisms through the generation of small interfering of the ancient evolution of viruses through analyzing RNAs (siRNAs) (Bertsch et al., 2009; Ricciuti et al., 2021). endogenous viral elements in the host genomes (Etienne, RNA-directed DNA polymerase (Reverse Transcriptase, RT) 2017). Due to the increasing number of sequenced genomes, coding sequences are present in a wide variety of genetic numerous EVEs can be uncovered, and some of them are elements and contains a relatively well conserved central distinct from the currently known episomal viruses (Johnson, domain, allowing its use for phylogenetic analyses (Hansen 2019). Another important property of EVEs is that they can be and Heslop-Harrison, 2004) and for searches for homologues used to calibrate the timing of virus evolution. If an EVE is of, for example, EPRVs in genome sequences (Diop et al., 2018). Frontiers in Plant Science 02 frontiersin.org de Tomás and Vicient 10.3389/fpls.2022.1011565 Previous studies have examined the EPRVs diversity in plant removed from the analyses. RT sequences having higher genomes based on the limited number of genome sequences similarity with Caulimoviridae were used for further analyses available in each case (Geering et al., 2014; Diop et al., 2018) (Supplementary Data 4). Nowadays, the number of sequenced plant genomes have For cluster determination, the selected sequences from the increased significantly, and we decided to screen them for the genome assemblies were grouped using CD-HIT with a sequence presence of EPRVs, obtaining a broader picture of identity cut-off of 60% (Cluster60) or of 100% (Cluster100), a the distribution of these endogenous elements. We identified bandwidth of alignment of 20 and a length of sequence to skip of the major EPRV lineages and analyzed their distribution in the 10. One sequence was then selected to be representative of each different plant orders and genera. We also describe a new cluster60 (Supplementary Data 5). Only in the case of cluster60- possible genus of Caulimoviridae present only as EPRVs we 8 we selected two sequences because the sequences in this cluster called Wendovirus. were clearly divided in two groups. The cluster representative sequences were aligned with the representative sequences of episomal or endogenous Materials and methods Caulimoviridae (Supplementary Data 1) using MEGA-X (Kumar et al., 2018). The resulting alignment was then used to Discovery and analyses of recently build a phylogenetic reconstruction using the maximum inserted endogenous Caulimoviridae likelihood (ML) method and 500 bootstrap replicates using MEGA-X. The resulting tree was then used as a reference to We built a library containing an assortment of 182 RT central classify the EPRV-RTs found in the genome assemblies. domain amino acid sequences (Supplementary Data 1). This The minimum ages of the integration events reported in this collection includes one sequence from Retroviridae, 14 from Ty3/ study were inferred by identifying the most distantly related pair Gypsy LTR retrotransposons of the six most abundant genera in of host species sharing a particular cluster of EPRVs and plants (Athila, CRM, Galadriel, Ogre, Reina, Retand and Tekay), applying the estimated species divergence dates in TimeTree 104 from the eleven genera of Caulimoviridae (Badnavirus, (http://www.timetree.org/) (Kumar et al., 2017). Caulimovirus, Vaccinivirus, Soymovirus, Cavemovirus, Potential ORFs were predicted using ORF Finder (https:// Solendovirus, Dioscovirus, Rosadnavirus, Tungrovirus, Petuvirus www.ncbi.nlm.nih.gov/orffinder/) and the presence of Pfam and Ruflodivirus), and 63 from six groups of exclusively domains in their encoded polypeptides was confirmed using endogenous Caulimoviridae (Florendovirus, Xendovirus, MOTIF Search (https://www.genome.jp/tools/motif/). Yendovirus, Zendovirus, Gymnendovirus and Fernendovirus) (hereafter referred to as operational taxonomic units (OTUs) following the nomenclature proposed by Diop et al., 2018. For Results further analyses, we selected ten sequences representatives of the Caulimoviridae groups (Supplementary Data 2). Distribution of genomic sequences We selected 278 genome assemblies corresponding to 267 encoding Reverse Transcriptase domains species (Supplementary Data 3): two from Bacteria, one from of recently inserted endogenous Chromista, two from Protozoa, 13 from Animal, six from Fungi pararetroviruses (Caulimoviridae) and 254 from Plantae kingdom. Plantae kingdom’s genomes include three Rodophyta, seven Chlorophyta, three Bryophyta, The objective of the work was to determine the presence of one Marchantiophyta and 240 Tracheophyta genomes. sequences encoding complete conserved RT domains Tracheophyta includes one Lycopodiopsida, four Pinopsida, 35 corresponding to endogenous pararetrovirus (Caulimoviridae) Liliopsida (11 families) and 200 Magnoliopsida (46 families) within a collection of publicly available genome sequence genomes. The genomes outside the Plantae kingdom were used assemblies from plant species and using some non-plant genome as negative controls. assemblies as negative controls. To identify them, we used a custom We compared the ten RT sequences with the 278 genome designed tBLASTn-based discovery pipeline, using as a probe a assemblies using tBLASTn with default parameters (except –e collection of 10 representative RT sequences of the different option set to 1e−10). Only the hits with at least 300 amino acid Caulimoviridae genera and OTUs (Supplementary Data 2). To residues and no stop codons nor frameshifts were selected for give priority to the recently inserted copies, we only select sequences further analysis. To avoid the inclusion in the selection of encoding RT domains of at least 300 amino acids that contain tandem duplications, we removed a hit if it was located less uninterrupted reading frames. Frequently EPRVs are inserted in than 1500 bp to another (Supplementary Data 3). For each tandemly arranged structures. To remove these duplications, when genome assembly, the selected set of RT sequences were a RT coding region was located less than 1500 bp of another we clustered with the 182 RT selected reference domains and only kept one of them. Due to their high sequence similarity, this those having higher similarity with retrotransposons were first selection also contained RT sequences from Ty3/gypsy LTR- Frontiers in Plant Science 03 frontiersin.org de Tomás and Vicient 10.3389/fpls.2022.1011565 retrotansposons (Metaviridae). To remove them, EPRVs were representatives of all Caulimoviridae genera and OTUs confirmed by phylogenetic analyses. They were aligned with RT (Supplementary Data 1). Our phylogenetic analysis clustered sequences of representative Caulimoviridae and LTR together all the previous known sequences corresponding to the retrotransposons (Supplementary Data 1). Those sequences same genera and OTU of the Caulimoviridae, confirming the showing higher similarity with the Metaviridae than with robustness of the analysis (Figure 1). This phylogenetic Caulimoviridae were removed. Finally, we obtained 11.527 RT- reconstruction allowed us to determine the diversity and EPRV sequences (Supplementary Data 4). nature of our collection of RT-EPRV sequences (Table 2). None of the analyzed genomes outside Plantae Kingdom They were separated into 13 phyla. 30 of the clusters contain RT- EPRV sequences and among the genomes of the were associated with sequences of Caulimoviridae with Plantae kingdom, we did not find RT-EPRVs in Chlorophyta, episomal forms: 10 Petuvirus, 5 Dioscovirus, 5 Soymovirus, 5 Rodophyta, Bryophyta or Marchantiophyta. Among the Tungrovirus, 2 Badnavirus, 2 Caulimovirus and 1 Solendovirus. Tracheophyta species, we did not find RT-EPRVs in the class We did not find any representative of the genera Cavemovirus, Lycopodiopsida (Selaginella moellendorffii) but we found RT- Rosadnavirus or Vaccinivirus, and neither from the recently EPRVs in genomes of all Tracheophyta classes (Pinopsida, proposed genera Ruflodivirus. This result suggests that the virus Liliopsida and Magnoliopsida), confirming previous results species of these genera do not carry out endogenization, at least (Gong and Han, 2018). All the four Pinopsida genomes not recently or as frequently, or they only do it in a small range analyzed contain RT-EPRV sequences (between 4 and 46). We of species whose complete genomic sequence is not yet available. included 35 genomes of species of the class Liliopsida and we Of the rest, 20 clusters corresponded to OTUs from which only found RT-EPRV sequences in 22 of them (63%) (between 1 and endogenous forms have been found: 11 Florendovirus, 3 63). Finally, we found RT-EPRV sequences in 180 of the 201 Xendovirus, 3 Yendovirus, 3 Zendovirus and 1 Gymnendovirus. Magnaliopsida genomes (88%) (between 1 and 1186). As we will describe later in detail, the remaining 6 clusters were When comparing the results with the genomes of species associated with each other, forming a new OTU we called belonging to the same genus, or varieties of the same species, the Wendovirus (Figure 1). results obtained are, in general, similar. For example, the We observed important differences between genera for both genomes of the two species of Kalanchoe contain 20 and 24, the number of RT- EPRV sequences and the diversity of species the two of Vitis contain 24 and 29 and the three of Solanum in which they were found (Table 1). Florendovirus are clearly the between 29 and 35. However, this is not always the case, and we most abundant followed by Petuvirus, Solendovirus and can observe important differences in the number of RT-EPRVs Zendovirus. However, whereas Florendovirus is present in in species of the same genus. For example, in the genera Arachis genomes of 40 families of species, Petuvirus is present in 14 (between 56 and 473), Prunus (between 3 and 144), Rosa and Solendovirus and Zendovirus in only two. Interestingly, (between 76 and 340), Citrus (between 63 and 306) and although we only detected 80 RT-EPRV sequences Nicotiana (between 12 and 130). Some of these differences can corresponding Badnavirus, they present a wide distribution (3 be due to differences in the quality of the genome assemblies. For Classes, 10 Orders and 11 Families). On the opposite, example, the presence of undetermined nucleotides can give rise Gymnendovirus are only present in Pinopsida. to a reduction in the number of RT-EPRVs we detected. If we look at the different classes of plants, we observed However, there are cases in which the best quality genome is important differences. Pinopsida only contains Gymnendovirus. the one with the least number of sequences. For example, we Magnolids contains Badnavirus, Petuvirus, Solendovirus, included three species of the genera Arabidopsis and the genome Tungrovirus, Florendovirus and Yendovirus. Liliopsida contains with the least number of sequences is the one with the best Badnavirus, Dioscovirus, Florendovirus and Yendovirus. Finally, quality (Arabidopsis thaliana). All these results suggest that in Magnaliopsida contains all the genera except Gymnendovirus. some of the species there have been very recent integrations If we look at the distribution of the clusters in the different of EPRVs. plant species, we observed a wide diversity (Table 2). Some of them are exclusively present in one class. For example, Gymnendovirus-1 is only present in Pinopsida, Tungrovirus-3 Classification of the RT-EPRVs present in is only present in Magnolids, Badnavirus-2, Dioscovirus-2 and -5 plant genomes and Yendovirus-1 are only present in Liliopsida, and many clusters are only present in Magnaliopsida. On the opposite, To provide a classification, RT-EPRV sequences with at least Badnavirus-1, Florendovirus-1 and Florendovirus-3 are present 60% amino acid identity to each other were grouped, yielding a in Magnolids, Liliopsida and Magnoliopsida. Looking at more total of 57 clusters. The total number of sequences and genomes detail, 31 of the 57 clusters are present in genomes of only one represented in each cluster varies greatly (Table 1). We family of plants, whereas two are present in genomes of more performed a phylogenetic analysis using representative than 20 plant families (both florendovirus). These differences of sequences of each cluster (Supplementary Data 5) and distribution are reflected in the Maximum Age Value (Table 1), Frontiers in Plant Science 04 frontiersin.org de Tomás and Vicient 10.3389/fpls.2022.1011565 FIGURE 1 Phylogenetic relationships within the episomal and endogenous Caulimoviridae. Phylogram obtained from a maximum likelihood analysis with protein sequence data from RT conserved domains using 500 bootstrap replications. The size of the point indicated the bootstrap support of the tree branch. Known episomal and endogenous pararetrovirus are shown in grey and small letters. New endogenous Clusters60 are shown in bold letters. The color of the branch indicates the genus of Caulimoviridae; Bad, Badnavirus; Dio, Dioscovirus; Yen, yendovirus; Tun, tungrovirus; Zen, zendovirus; Vac, vaccinivirus; Ros, rosadnavirus; Flo, florendovirus; Gym1 and Gym2, gymnendovirus1 and 2; Pet, petuvirus; Fer, fernendovirus; Cav, cavemovirus; Sol, solendovirus; Cau, caulimovirus; Ruf, ruflodivirus; Soy, soymovirus; Xen, xendovirus; and Wen, wendovirus. which depends on the maximum phylogenetic distance between Another four groups also correspond to the same genome, with a the species present in the cluster. total of 1014 sequences (962 are Solendovirus, 31 are Florendovirus and 21 Yendovirus). In total, we found 1183 RT- EPRV sequences in this genome and more than 81% are present Very recent EPRV amplification in in the Cluster100 selection. This is a very clear indication of a plant genomes relatively recent proliferation of EPRVs in the pepper genome. Next, we perform a phylogenetic analysis of representatives The above results suggest that, at least in some species, there of each Cluster-100 and from the described OTUs from has been a recent amplification in the number of EPRV Caulimoviridae (Figure 2). The sequences of some of the sequences inserted in their genomes. To try to delve further clusters100 are very similar and, probably, they correspond to into this aspect, we decided to select those cases in which 100% the same virus. This is the case of clusters100-1 and -26 identical RT-EPRV sequences were present in 10 or more copies (Solendovirus of Capsicum annuum), clusters100-11 and -13 in the same genome. Using this highly restrictive criterion, (Petuvirus of Atalantia buxifolia) and Clusters100-5 and -6 we detected 31 clusters grouping a total of 1534 sequences (Petuvirus of Citrus medica). The sequences of clusters100-12 (Table 3). These clusters (clusters100) involve 19 genomes. and -16 (Florendovirus of Fortunella hindsii) and of clusters100- Only one corresponds to a Liliopsida (Hordeum vulgare) and 19 and -24 (Florendovirus of Atalantia buxifolia) are also near the remaining 18 are genomic sequences of Magnaliophyta. identical. The sequences of the Clusters100-20 and 29, that Nine EPRV OTUs are represented in the Clusters100 correspond to two different but closely related species including Caulimovirus, Dioscovirus, Florendovirus, Petuvirus, (Nicotiana tabacum and Nicotiana sylvestris), are also almost Solendovirus, Tungrovirus, Yendovirus, Zendovirus and the identical, which suggests that they could come from the same newly proposed Wendovirus. virus capable of infecting both species. Figure 2 also shows that Cluster100-10 is particularly noteworthy as it includes 951 some of the endogenous sequences grouped in Clusters100 are sequences present in the genome of pepper (Capsicum annuum). very similar to the sequences of episomal virus. For example, the Frontiers in Plant Science 05 frontiersin.org Frontiers in Plant Science de Tomás and Vicient TABLE 1 Cluster60 statistics. Cluster Cluster N. EPRV-RT-seqs N.Classes N.Orders N.Families N.Genus N.Species A B Max.Age (MY) BADNAVIRUS 80 3 10 11 12 13 Badnavirus-01 20 75 2 9 10 12 12 Dioscorea Amborella 191 Badnavirus-02 43 5 1 2 2 2 2 Phalaenopsis Musa 117 CAULIMOVIRUS 38 1 4 4 6 9 Caulimovirus-01 28 36 1 3 3 5 8 Helianthus Arabidopsis 118 Caulimovirus-02 52 2 1 1 1 1 1 Gossypium Gossypium 0 DIOSCOVIRUS 144 2 5 5 7 9 Dioscovirus-01 23 49 1 3 3 4 5 Cynara Cajanus 118 Dioscovirus-02 25 43 1 1 1 1 2 Dioscorea Dioscorea 0 Dioscovirus-03 31 24 1 1 1 2 2 Glycine Vigna 23 Dioscovirus-04 34 16 1 1 1 1 1 Macadamia Macadamia 0 Dioscovirus-05 35 12 1 1 1 1 2 Dioscorea Dioscorea 0 PETUVIRUS 1693 2 14 16 47 66 Petuvirus-01 1 1202 1 5 5 10 19 Arachis Citrus 108 Petuvirus-02 14 131 1 9 9 16 18 Amborella Helianthus 191 Petuvirus-03 15 129 1 3 4 6 9 Coffea Gossypium 118 06 Petuvirus-04 19 78 1 1 1 11 13 Brassica Rorippa 27 Petuvirus-05 22 52 1 1 1 1 1 Ipomoea Ipomoea 0 Petuvirus-06 27 39 1 1 1 7 9 Arachis Cicer 59 Petuvirus-07 30 24 1 3 3 4 6 Populus Gossypium 108 Petuvirus-08 33 18 1 1 1 3 8 Citrus Atalantia 18 Petuvirus-09 36 12 1 2 2 2 2 Durio Macadamia 123 Petuvirus-10 39 8 1 1 1 1 1 Eucalyptus Eucalyptus 0 SOLENDOVIRUS 1124 1 2 2 5 8 Solendovirus-01 3 1124 1 2 2 5 8 Nymphaea Nicotiana 179 SOYMOVIRUS 454 1 5 6 12 14 Soymovirus-01 6 391 1 1 1 1 3 Arachis Arachis 0 Soymovirus-02 24 49 1 4 5 6 6 Lactuca Cleome 118 Soymovirus-03 42 6 1 1 1 1 1 Chenopodium Chenopodium 0 10.3389/fpls.2022.1011565 Soymovirus-04 44 5 1 1 1 3 3 Brassica Cakile 13 Soymovirus-05 48 3 1 1 1 1 1 Medicago Medicago 0 TUNGROVIRUS 308 2 5 5 10 32 Tungrovirus-01 8 251 1 3 3 10 29 Prunus Vitis 117 frontiersin.org Tungrovirus-02 29 32 1 1 1 1 1 Lindenbergia Lindenbergia 0 (Continued) Frontiers in Plant Science de Tomás and Vicient TABLE 1 Continued Cluster Cluster N. EPRV-RT-seqs N.Classes N.Orders N.Families N.Genus N.Species A B Max.Age (MY) Tungrovirus-03 38 9 1 1 1 1 1 Cinnamomum Cinnamomum 0 Tungrovirus-04 46 4 1 1 1 1 2 Malus Malus 0 Tungrovirus-05 54 2 1 1 1 1 1 Citrus Citrus 0 FLORENDOVIRUS 6162 3 29 40 91 151 Florendovirus-01 0 3207 2 27 34 70 114 Asparagus Amborella 191 Florendovirus-02 2 1188 1 6 8 21 35 Brassica Nicotiana 118 Florendovirus-03 4 949 2 21 27 38 47 Asparagus Amborella 191 Florendovirus-04 7 317 1 2 2 2 3 Coffea Lindenbergia 77 Florendovirus-05 12 133 1 1 1 3 5 Arachis Lotus 59 Florendovirus-06 13 132 1 2 2 5 8 Lindenbergia Nicotiana 79 Florendovirus-07 16 120 1 8 9 13 18 Amborella Brassica 191 Florendovirus-08 18 79 1 2 2 7 8 Glycine Manihot 101 Florendovirus-09 41 7 1 1 2 2 2 Capsicum Nicotiana 24 Florendovirus-10 47 4 1 1 1 2 2 Cucumis Momordica 48 Florendovirus-11 51 2 2 2 2 2 2 Asparagus Prunus 160 GYMNENDOVIRUS 95 1 1 1 2 3 Gymnendovirus-1-1 17 95 1 1 1 2 2 Pinus Picea 130 07 WENDOVIRUS 282 1 7 7 10 17 Wendovirus-01 9 200 1 1 1 4 11 Citrus Atalantia 18 Wendovirus-02 21 70 1 2 2 3 3 Helianthus Coffea 101 Wendovirus-03 40 7 1 2 2 3 4 Citrus Solanum 118 Wendovirus-04 49 3 1 1 1 1 1 Lindenbergia Lindenbergia 0 Wendovirus-05 55 1 1 1 1 1 1 Olea Olea 0 Wendovirus-06 56 1 1 1 1 1 1 Portulaca Portulaca 0 XENDOVIRUS 65 1 6 6 8 10 Xendovirus-01 26 41 1 4 4 6 8 Vaccinium Rosa 118 Xendovirus-02 32 19 1 1 1 1 1 Olea Olea 0 Xendovirus-03 45 5 1 1 1 1 1 Ipomoea Ipomoea 0 YENDOVIRUS 334 2 6 7 17 23 Yendovirus-01 10 190 1 1 1 9 11 Oryza Eleusine 47 10.3389/fpls.2022.1011565 Yendovirus-02 11 142 2 5 5 8 12 Dioscorea Solanum 160 Yendovirus-03 50 3 2 2 2 2 2 Ananas Nymphaea 179 ZENDOVIRUS 781 1 2 2 5 19 frontiersin.org Zendovirus-01 5 768 1 1 1 4 18 Fragaria Rubus 41 Zendovirus-02 37 11 1 1 1 2 4 Fragaria Rosa 31 Zendovirus-03 53 2 1 1 1 1 1 Pistacia Pistacia 0 Frontiers in Plant Science de Tomás and Vicient TABLE 2 Distribution of Cluster60 in plant families. 08 10.3389/fpls.2022.1011565 Numbers are the average number of RT-EPRV sequences per genome of each cluster 60. The genomes are grouped according to the plant families. BADN, Badnavirus; CAUL, Caulimovirus; SL, Solendovirus; G, Gymnendovirus. frontiersin.org de Tomás and Vicient 10.3389/fpls.2022.1011565 TABLE 3 Cluster 100 with 10 or more copies. Cluster 100% Num. Seq. Genome EPRV group 1 951 Capsicum annuum Solendovirus-01 2 77 Lotus japonicus Florendovirus-01 3 53 Citrus maxima Petuvirus-01 4 43 Hydrangea quercifolia Florendovirus-01 5 27 Citrus medica Petuvirus-01 6 26 Citrus medica Petuvirus-01 7 24 Salvia splendens Florendovirus-03 8 22 Ipomoea triloba Petuvirus-05 9 21 Capsicum annuum Yendovirus-02 10 20 Capsicum annuum Florendovirus-03 11 20 Atalantia buxifolia Petuvirus-01 12 19 Fortunella hindsii Florendovirus-02 13 19 Atalantia buxifolia Petuvirus-01 14 16 Helianthus annuus Wendovirus-02 15 16 Ipomoea triloba Dioscovirus-01 16 14 Fortunella hindsii Florendovirus-02 17 13 Lactuca sativa Florendovirus-03 18 12 Castanea dentata Florendovirus-01 19 12 Atalantia buxifolia Florendovirus-02 20 12 Nicotiana tabacum Solendovirus-01 21 12 Lindenbergia philippensis Tungrovirus-02 22 11 Lactuca sativa Caulimovirus-01 23 11 Lotus japonicus Florendovirus-01 24 11 Atalantia buxifolia Florendovirus-02 25 11 Capsicum annuum Florendovirus-03 26 11 Capsicum annuum Solendovirus-01 27 10 Fragaria nilgerrensis Florendovirus-01 28 10 Arachis hypogaea Florendovirus-01 29 10 Nicotiana sylvestris Solendovirus-01 30 10 Hordeum vulgare Yendovirus-01 31 10 Rosa chinensis Zendovirus-01 RT sequence of the citrus blight associated virus is highly similar Supplementary Data 6). The structure was very similar in all to the sequences of cluster100-3, -5 and -6, all of them belonging of them, with four partially overlapping ORFs. Comparisons to genomes of the genus Citrus, and the sequence of the tobacco with protein motif databases allowed us to find different vein clearing virus is similar to clusters100-20 and -29, belonging conserved domains (Supplementary Data 6). The ORF1 to genomes of the genus Nicotiana. encodes for a zinc finger motif, which is typical of the Caulimoviridae coat proteins. The ORF2 encodes for a movement protein and an aspartic proteinase. The ORF3 Wendovirus, a new group encodes a second aspartic proteinase, the RT and the of Caulimoviridae RNAseH. Finally, the ORF4 encodes a protein without significant homologies to other reference proteins and without Six of the Cluster60 and one of the Cluster100 correspond to a known protein domains but that is well-conserved in all the new group of endogenous Caulimoviridae with distinctive wendovirus elements. The most noticeable aspect of these characteristics that, following the nomenclature proposed by Diop structures is the presence of two aspartic proteinase domains et al. (2018) (Zendovirus, Xendovirus and Yendovirus), we have instead of one, as usual. They are located close to each other, but called them Wendovirus (Supplementary Data 4 and Table 3). in two different ORFs (2 and 3). In the case of the HelAnn-006 We were able to reconstruct the structure of the Wendovirus element (Wendovirus2 cluster), although the domains and their for seven genomes corresponding to Cluster60 (Figure 3; order are conserved, the ORF2 is shorter and the ORF3 is Frontiers in Plant Science 09 frontiersin.org de Tomás and Vicient 10.3389/fpls.2022.1011565 FIGURE 2 Phylogenetic relationships of representative sequences of the Cluster100. Representative sequences of the RT-EPRV Cluster100 (in red) were aligned with RT sequences of pararetroviral elements (in black), and a phylogenetic tree was constructed using the NJ method and 1000 bootstrap replications. divided in two. When compared to databases, the highest (Diop et al., 2018) and one is shown here for first time, we similarities of these two aspartic proteinase domains are with called Wendovirus. Contrary to what has been observed in other members of Caulimoviridae. plant viruses as Geminivirus or Nanovirus (Nino Barreat and Katzourakis, 2021), EVEs from Caulimoviridae are exclusively present in plants. Recently integrated RT-EPRVs are present in Discussion genomes of Lycopodiopsida, Pinopsida, Liliopsida and Magnoliopsida, but not necessary in all the genomes of these Endogenous viral elements (EVEs) are viral sequences groups. For example, they are not present in the genomes of integrated in host genomes that are inherited as host DNA Arabidopsis thaliana, Zea mays, Triticum aestivum, Phaseolus sequences (Holmes, 2011). Some of the EVEs, are derived from vulgaris, Theobroma cacao or Spinacia oleracea. They are also viruses in which integration into the genome is part of their absent in the Selaginella moellendorffii (Marchantiophyta) and in replication cycle, for example, mammalian retroviruses. However, Rhodophyta, Chlorophyta or Bryophyta. many viruses in which integration into the genomic DNA is not a We have found that, in some cases, the integration events can part of their normal replication cycle can also be found as EVEs, as be considered very recent. Once in the genome, the EPRV is the case of the endogenous Caulimoviridae (Endogenous sequences begin to accumulate point random mutations, so, if the Pararetrovirus, EPRVs). The presence of EPRVs has been sequences are identical that means that they probably integrated described in the genomes of different plant species (Hohn et al., recently in the genome. We have found multiple sequences 2008). In this work we have focused on determining the presence of encoding identical RT domains in different species being the most EPRV sequences relatively recently integrated, based on the extreme case Capsicum annuum in whose genome we found up to selection of elements with complete and conserved RT domains. 951 sequences encoding identical RT domains. Recent genome Based on the RT domain sequence similarity we detected integrations of Caulimoviridae sequences have been described in 11.527 sequences distributed in 57 clusters corresponding to 13 some species, such as banana (Gayral et al., 2010). It is interesting to OTUs. Twelve of these groups had already been described note that, in some cases, these identical RT sequences correspond to Frontiers in Plant Science 10 frontiersin.org de Tomás and Vicient 10.3389/fpls.2022.1011565 FIGURE 3 Schematic representation of wendovirus endogenous pararetrovirus. A scaled linear view of the genome organization of Wendovirus. The name of the sequences is the same as in Supplementary Data 4. Grey arrows mark open reading frames and colored regions within ORFs are conserved protein domains: blue, zinc finger typically present in the coat proteins; green, Movement Protein; yellow, Aspartic Proteinase; red, Reverse Transcriptase; pink, RNaseH. groups that have only been detected as endogenous forms and the mechanisms of amplification or reduction of the (Florendovirus, Yendovirus, Zendovirus, Wendovirus) suggesting integrated sequences. First, Caulimoviridae integration that probably at least some of them may have their requires the presence of viruses that are infectious for the corresponding episomal virus species that have not been species and that the defense mechanisms of the plant are not yet identified. able to eliminate, or not completely. Second, the main The distribution of the different clusters of EPRVs between integration mechanism is thought to involve illegitimate species shows a great diversity. Some clusters are present recombination, which requires the existence of DNA exclusively in certain plants as, for example, Gymnendovirus in double-strand breaks and subsequent repair mechanisms Pinopsida, Zendovirus1 in the tribus Potentilleae and Roseae, (Richert-Pöggeler et al., 2021). Furthermore, to be Soymovirus1 in the genus Arachis or Wendovirus1, only present transmitted, integration must occur in reproductive cells. in Rutaceae. In other cases, such as Florendovirus1 and 3, the Third, once integrated, EPRVs, copies are inactivated by distribution is very wide, including Lilipsida and Magnoliopsida. sequence degeneration or fragmentation, or by the insertion In general, the distribution of the different groups of EPRVs is of transposable elements, and subjected to epigenetic silencing consistent with the phylogeny, but not always. For example, (reviewed by Richert-Pöggeler et al., 2021). All these processes Petuvirus2 are present in Amborella trichopoda and in eight lead to the degeneration of the coding sequences. Finally, it has Magnoliopsida orders, Florendovirus7 are present in Amborella also been proposed that once integrated, the sequences can be trichopoda and in seven Magnoliopsida orders and Solendovirus1 amplified, and different mechanisms have been suggested such are present in Nymphaea colorata and in Solanaceae. A possible as transposition like retroelements, rolling circle amplification, explanation for these species distributions is the horizontal unequal meiotic crossing-over of tandem arrays, or ectopic transmission of the virus between species. There are data recombination between EPRV clusters on non-homologous suggesting multiple viral jumps between different animal chromosomes (reviewed by Richert-Pöggeler et al., 2021). species in Hepadnavirus (Dill et al., 2016), and previous data Variations in any of these processes together with the time also suggests such horizontal transfers can occur for EPRVs in elapsed since the last event of integration could explain the plants (Diop et al., 2018; Gong and Han, 2018). observed differences in the number of EPRVs in the analyzed We have detected differences in the number of EPRVs in genomes. Nor can we rule out that the different quality of the the different genomes. Sometimes the differences are also genome assemblies may also affect. observed comparing the genomes of species of the same We have identified a new putative genus of the genus or varieties of the same species. The number of EPRVs Caulimoviridae, tentatively named ‘Wendovirus’. Wendovirus observed results from the combination of the virus integration genomes are about 7,7 Kb long and are present in the Frontiers in Plant Science 11 frontiersin.org de Tomás and Vicient 10.3389/fpls.2022.1011565 genomes of different Magnaliopsida species, especially in FEDER), Ministerio de Ciencia e Innovació n (PID2019- Rutaceae and in sunflower. Our phylogenetic analysis shows 106374RB-I00/AEI/10.13039/501100011033), by “ERDF A way of that wendovirus are related to Xendovirus and Soymovirus. They making Europe” and by the CERCA Programme of the Generalitat contain four ORFs that encode the typical protein domains in de Catalunya. We also acknowledge financial support from the Caulimoviridae: Zinc-Finger, Movement Protein, Aspartic Spanish Ministerio de Economı́a y Competitividad through the Proteinase, Reverse Transcriptase and RNAseH. A remarkable “Severo Ochoa Programme for Centres of Excellence in R&D” feature of wendovirus is the presence of two protease coding 2016-2019 (SEV-2015-0533) and CEX2019-000902-S funded by domains located in two different ORFs (Figure 3). Although both MCIN/AEI/10.13039/501100011033. encode aspartyl proteases, the domains are different (PF13975 in ORF2 and PF00077 in ORF3), so the hypothesis that their origin Acknowledgments was a genomic duplication can be discarded. When compared to protein bases, all these described domains, including the two We thank all groups members whose comments contributed aspartic proteinase domains, show the greatest similarities to this study. against other members of Caulimoviridae. Therefore, it seems to be ruled out that the second proteinase domain could come from some other families of viruses. Recombination between Conflict of interest EPRV fragments has been observed (Chabannes and Iskra- Caruana, 2013) and many viruses have modularly acquired The authors declare that the research was conducted in the domains and ORFs (Smyshlyaev et al., 2013; Koonin et al., absence of any commercial or financial relationships that could 2015). Encapsidation of genomes (or genome fragments) of be construed as a potential conflict of interest. different species of Caulimoviriridae in the same capsid can lead to recombination and formation of chimeric genomes. Virus-like particles (VLPs) containing host RNAs were found Publisher’s note to be produced during agroinfiltration of cucumber necrosis virus, some of them corresponding to retrotransposon or All claims expressed in this article are solely those of the retrotransposon-like RNA sequences (Ghoshal et al., 2015). authors and do not necessarily represent those of their affiliated On the other hand, template switching between two RNA organizations, or those of the publisher, the editors and the molecules during reverse transcription has been shown for reviewers. Any product that may be evaluated in this article, or retroviruses, LTR retrotransposons and is proposed for claim that may be made by its manufacturer, is not guaranteed Caulimoviridae (Froissart et al., 2005; Tromas et al., 2014; or endorsed by the publisher. Sanchez et al., 2017; Richert-Pöggeler et al., 2021). Such an acquisition of ORFs likely contributed to the evolution of the Wendovirus, although the possible functions of this second Supplementary material proteinase domain remain unknown. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ Data availability statement fpls.2022.1011565/full#supplementary-material SUPPLEMENTARY DATA SHEET 1 The original contributions presented in the study are RT domain sequences of Caulimoviridae, retrovirus and LTR- included in the article/Supplementary Material. Further retrotransposons used in this study. inquiries can be directed to the corresponding author. SUPPLEMENTARY DATA SHEET 2 Caulimoviridae RT sequences used in the tBLASTn analyses. Author contributions SUPPLEMENTARY DATA SHEET 3 Genome assemblies used in the tBLASTn analyses. All authors contributed equally to the design and processing data. All authors have read and approved the final manuscript. SUPPLEMENTARY DATA SHEET 4 RT-EPRV sequences found. SUPPLEMENTARY DATA SHEET 5 Funding Representative RT-EPRV sequences for the clusters60 used in the phylogenetic analysis. This work was funded by grants from the Spanish Ministerio de SUPPLEMENTARY DATA SHEET 6 Economı́a, Industria y Competitividad (AGL2016-78992-R/ Wendovirus sequences and polypeptides encoded. Frontiers in Plant Science 12 frontiersin.org de Tomás and Vicient 10.3389/fpls.2022.1011565 References Aiewsakun, P., and Katzourakis, A. (2015). Endogenous viruses: Connecting Hansen, C., and Heslop-Harrison, J. S. (2004). Sequences and phylogenies of recent and ancient viral evolution. Virology 479-480, 26–37. doi: 10.1016/ plant pararetroviruses, viruses, and transposable elements. Adv. Bot. Res. 41, 165– j.virol.2015.02.011 193. doi: 10.1016/S0065-2296(04)41004-0 Bao, W., Kojima, K. K., and Kohany, O. (2015). Repbase update, a database of Hohn, T., Richert-Pöggeler, K. 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