A vertically transmitted amalgavirus is present in
certain accessions of the bryophyte Physcomitrium
patens

Pol Vendrell-mir, Pierre-François Perroud, Fabian Haas, Rabea Meyberg, Florence Charlot, Stefan Rensing, Fabien Nogué, Josep Casacuberta

► To cite this version:

Pol Vendrell-mir, Pierre-François Perroud, Fabian Haas, Rabea Meyberg, Florence Charlot, et al.. A vertically transmitted amalgavirus is present in certain accessions of the bryophyte Physcomitrium patens. Plant Journal, 2021, 108 (6), pp.1786-1797. 10.1111/tpj.15545 . hal-03632720

HAL Id: hal-03632720

https://hal.inrae.fr/hal-03632720

Submitted on 4 Feb 2024

**HAL** is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire **HAL**, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# A vertically transmitted amalgavirus is present in certain accessions of the bryophyte Physcomitrium patens

Pol Vendrell-mir, Pierre-François Perroud, Fabian Haas, Rabea Meyberg, Florence Charlot, Stefan Rensing, Fabien Nogué, Josep Casacuberta

► To cite this version:

Pol Vendrell-mir, Pierre-François Perroud, Fabian Haas, Rabea Meyberg, Florence Charlot, et al.. A vertically transmitted amalgavirus is present in certain accessions of the bryophyte Physcomitrium patens. The Plant Journal, 2021, 108 (6), pp.1786-1797. 10.1111/tpj.15545. hal-03632720

HAL Id: hal-03632720

https://hal.inrae.fr/hal-03632720v1

Submitted on 4 Feb 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
The Plant Journal (2021) **108**, 1786–1797

doi: 10.1111/tpj.15545

# A vertically transmitted amalgavirus is present in certain accessions of the bryophyte *Physcomitrium patens*

Pol Vendrell-Mir<fnref n="1" />,† ID, Pierre-François Perroud<fnref n="2" />,† ID, Fabian B. Haas<fnref n="3" /> ID, Rabea Meyberg<fnref n="3" /> ID, Florence Charlot<fnref n="2" /> ID, Stefan A. Rensing<fnref n="3" />,<fnref n="4" /> ID, Fabien Nogue<fnref n="2" />,∗ ID and Josep M. Casacuberta<fnref n="1" />,∗ ID

Received 10 August 2021; accepted 16 October 2021; published online 5 November 2021.

*For correspondence (e-mail josep.casacuberta@cragenomica.es [JMC]; fabien.nogue@inrae.fr [FN]).

†These authors contributed equally to this work.

## SUMMARY

In the last few years, next-generation sequencing techniques have started to be used to identify new viruses infecting plants. This has allowed to rapidly increase our knowledge on viruses other than those causing symptoms in economically important crops. Here we used this approach to identify a virus infecting *Physcomitrium patens* that has the typical structure of the double-stranded RNA endogenous viruses of the Amalgaviridae family, which we named *Physcomitrium patens* amalgavirus 1, or PHPAV1. PHPAV1 is present only in certain accessions of *P. patens*, where its RNA can be detected throughout the cell cycle of the plant. Our analysis demonstrates that PHPAV1 can be vertically transmitted through both paternal and maternal germlines, in crosses between accessions that contain the virus with accessions that do not contain it. This work suggests that PHPAV1 can replicate in genomic backgrounds different from those that actually contain the virus and opens the door for future studies on virus-host coevolution.

**Keywords:** *Physcomitrium patens*, *Physcomitrella patens*, amalgavirus, vertical transmission, ribosomal framework.

## INTRODUCTION

Since the seminal works of Martinus Beijerinck and Dmitri Ivanovsky in the late 19th century that allowed the characterization of tobacco mosaic virus (Scholthof, 2004), thousands of viruses have been discovered in organisms across the three domains of life. Both DNA and RNA viruses infect plants. However, whereas unicellular chlorophyte algae seem to be infected mainly by large DNA viruses, RNA viruses and small DNA viruses seem to be the major classes of viruses infecting angiosperms (Mushegian et al., 2016).

Although most plant virus infections are asymptomatic in the wild (Roossinck, 2015), the vast majority of viruses have been discovered analyzing the lesions or symptoms they induce in economically important crops. Only recently, thanks to the development of next-generation sequencing (NGS) techniques, this has been complemented by a more systematic screen for the presence of viruses in wild and cultivated plants (Villamor et al., 2019).

As a consequence, there is still a clear bias in the databases towards viruses infecting angiosperms, with the only exception of the unicellular algae Chlorophyta, which have a relatively well-studied virome (Mushegian et al., 2016; Yamada et al., 2006). In particular, the knowledge on viruses infecting bryophytes, which include liverworts, mosses, and hornworts, is very limited, with only few viral related RNA-dependent RNA polymerase (RdRP) sequences described in some species (Mushegian et al., 2016).

*Physcomitrium patens*, formerly known as *Physcomitrella patens*, has been widely used as a model species to study plant evolution and development (Rensing et al., 2020). However, in spite of the wide range of tools and information available for this species, no viruses naturally infecting *P. patens* have been described to date. The only viral-related sequences described in *P. patens* are sequences likely acquired horizontally from nucleocytoplasmic large DNA virus relatives (NCLDVs) (Maumus

<footnote n="1">¹Centre for Research in Agricultural Genomics CSIC-IRTA-UAB-UB, Campus UAB, Edifici CRAG, Bellaterra, Barcelona 08193, Spain,</footnote>

<footnote n="2">²Institut Jean-Pierre Bourgin, INRAE, AgroParisTech, Université Paris-Saclay, Versailles 78000, France,</footnote>

<footnote n="3">³Plant Cell Biology, Department of Biology, University of Marburg, Marburg, Germany, and</footnote>

<footnote n="4">⁴BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany</footnote>
The Plant Journal (2021) **108**, 1786–1797

doi: 10.1111/tpj.15545

# A vertically transmitted amalgavirus is present in certain accessions of the bryophyte *Physcomitrium patens*

Pol Vendrell-Mir<fnref n="1" />,† ID, Pierre-François Perroud<fnref n="2" />,† ID, Fabian B. Haas<fnref n="3" /> ID, Rabea Meyberg<fnref n="3" /> ID, Florence Charlot<fnref n="2" /> ID, Stefan A. Rensing<fnref n="3" />,<fnref n="4" /> ID, Fabien Nogue<fnref n="2" />,∗ ID and Josep M. Casacuberta<fnref n="1" />,∗ ID

Received 10 August 2021; accepted 16 October 2021; published online 5 November 2021.

*For correspondence (e-mail josep.casacuberta@cragenomica.es [JMC]; fabien.nogue@inrae.fr [FN]).

†These authors contributed equally to this work.

## SUMMARY

In the last few years, next-generation sequencing techniques have started to be used to identify new viruses infecting plants. This has allowed to rapidly increase our knowledge on viruses other than those causing symptoms in economically important crops. Here we used this approach to identify a virus infecting *Physcomitrium patens* that has the typical structure of the double-stranded RNA endogenous viruses of the Amalgaviridae family, which we named *Physcomitrium patens* amalgavirus 1, or PHPAV1. PHPAV1 is present only in certain accessions of *P. patens*, where its RNA can be detected throughout the cell cycle of the plant. Our analysis demonstrates that PHPAV1 can be vertically transmitted through both paternal and maternal germlines, in crosses between accessions that contain the virus with accessions that do not contain it. This work suggests that PHPAV1 can replicate in genomic backgrounds different from those that actually contain the virus and opens the door for future studies on virus-host coevolution.

**Keywords:** *Physcomitrium patens*, *Physcomitrella patens*, amalgavirus, vertical transmission, ribosomal framework.

## INTRODUCTION

Since the seminal works of Martinus Beijerinck and Dmitri Ivanovsky in the late 19th century that allowed the characterization of tobacco mosaic virus (Scholthof, 2004), thousands of viruses have been discovered in organisms across the three domains of life. Both DNA and RNA viruses infect plants. However, whereas unicellular chlorophyte algae seem to be infected mainly by large DNA viruses, RNA viruses and small DNA viruses seem to be the major classes of viruses infecting angiosperms (Mushegian et al., 2016).

Although most plant virus infections are asymptomatic in the wild (Roossinck, 2015), the vast majority of viruses have been discovered analyzing the lesions or symptoms they induce in economically important crops. Only recently, thanks to the development of next-generation sequencing (NGS) techniques, this has been complemented by a more systematic screen for the presence of viruses in wild and cultivated plants (Villamor et al., 2019).

As a consequence, there is still a clear bias in the databases towards viruses infecting angiosperms, with the only exception of the unicellular algae Chlorophyta, which have a relatively well-studied virome (Mushegian et al., 2016; Yamada et al., 2006). In particular, the knowledge on viruses infecting bryophytes, which include liverworts, mosses, and hornworts, is very limited, with only few viral related RNA-dependent RNA polymerase (RdRP) sequences described in some species (Mushegian et al., 2016).

*Physcomitrium patens*, formerly known as *Physcomitrella patens*, has been widely used as a model species to study plant evolution and development (Rensing et al., 2020). However, in spite of the wide range of tools and information available for this species, no viruses naturally infecting *P. patens* have been described to date. The only viral-related sequences described in *P. patens* are sequences likely acquired horizontally from nucleocytoplasmic large DNA virus relatives (NCLDVs) (Maumus

<footnote n="1">¹Centre for Research in Agricultural Genomics CSIC-IRTA-UAB-UB, Campus UAB, Edifici CRAG, Bellaterra, Barcelona 08193, Spain,</footnote>

<footnote n="2">²Institut Jean-Pierre Bourgin, INRAE, AgroParisTech, Université Paris-Saclay, Versailles 78000, France,</footnote>

<footnote n="3">³Plant Cell Biology, Department of Biology, University of Marburg, Marburg, Germany, and</footnote>

<footnote n="4">⁴BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany</footnote>
et al., 2014). These sequences, which are remnants of past
infections, are transcribed in *P. patens* and it has been pro-
posed that they might protect gametes from viral infection
via small interfering RNA-mediated silencing (Lang et al.,
2018). Moreover, although no virus has been described to
infect *P. patens* in the wild, it has been shown that *P.
patens* can be infected in the laboratory with viruses such
as the tomato spotted wilt virus (Hühns et al., 2003). Here
we used the NGS-based approach described in (Gilbert
et al., 2019) to look for viral sequences in available *P.
patens* RNA sequencing (RNA-Seq) libraries (Haas et al.,
2020; Kamisugi et al., 2016; Perroud et al., 2018) and we
describe the first *P. patens*-associated virus, *P. patens*
amalgavirus 1 (PHPAV1).

RESULTS

Searching for a *P. patens* virus

As most viruses infecting plants are either single-stranded
RNA (ssRNA) or double-stranded RNA (dsRNA) viruses we
decided to take advantage of the large amount of RNA-Seq
data available for *P. patens* (Perroud et al., 2018) to screen
for the possible presence of viral sequences that may cor-
respond to *P. patens*-infecting viruses. We screened the
RNA-Seq libraries for sequences not mapping to the *P.
patens* reference genome. These sequences were then
assembled into a total of 184 184 contigs, corresponding
to 135 766 independent sequences, as some of them repre-
sented different isoforms of the same sequences. The
length of these contigs ranged from 200 nucleotides (nts)
to 12 470 nts. We then looked for sequence similarities of
these contigs to known viruses using different approaches.
We first used blastx to look for similarities between the
proteins potentially encoded by the RNA contigs and those
of the reference viral database (Refseq) from NCBI (on 23
November, 2018). Six contigs gave significant similarity
with annotated viruses: four to the same isolate of the
tobamovirus pepper mild mottle virus, one to another
tobamovirus (tropical soda apple mosaic virus [TSAMV]),
and one to RNA segment 3 of the bromovirus brome
mosaic virus (Table S1). However, in five of these cases,
the *P. patens* contigs were short (close to the lower thresh-
old of the contig size, i.e., 200 nt) and matched a small
fraction of the corresponding viral sequences (Table S1).
Moreover, in all cases the sequence obtained from *P.
patens* RNA-Seq data was almost identical to that of the
virus isolate reported (Table S1), even when these viruses
infect Solanaceae plants or cereals, which are phylogeneti-
cally very distant from *P. patens*. All this suggested that
the presence of these viral sequences was due to a possi-
ble contamination during library preparation or sequencing
processes rather than to the presence of viruses infecting
some of the *P. patens* samples. The contig showing simi-
larity to TSAMV had a size similar to that of the virus (6318

nt). The sequence of this contig was also almost identical
(99.8% identity) to that of the original virus isolated from
*Solanum viarum* (Adkins et al., 2007), suggesting that the
presence of the corresponding sequences in *P. patens*
RNA-Seq libraries may also be the result of a laboratory
contamination. Consistent with this possibility the reads
corresponding to the contig showing similarity to TSAMV
were present in only one out of the three replicates of the
two positive samples, which were obtained from pro-
tonema of the Gransden accession (Table S2). The pres-
ence of spurious reads in NGS samples used for viral
detection is a known problem (Cantalupo and Pipas, 2019).
The sources of contamination can be manifold, including
viruses contaminating the laboratory reagents (Naccache
et al., 2013) and human viruses present in the laboratory
personnel. In this respect, it is interesting to note that
plant-infecting viruses, including TSAMV, are frequently
found in the human oropharynx and gut as a result of
plant consumption (Aguado-García et al., 2020; Balique
et al., 2015), which may suggest a possible source of con-
tamination.

The second approach followed was to look for similari-
ties of the obtained RNA contigs to viral RdRPs using
HMM profiles, a strategy recently used with success to
identify new RNA viruses in fungi (Gilbert et al., 2019).
Two contigs showed significant similarity to RdRP. One of
them corresponded to the contig showing sequence simi-
larity to TSAMV, which was already discarded as a possi-
ble contamination (see above). The second contig showing
similarity to RdRPs (TRINITY_DN26323_c4_g7_i1), which is
3597 nts long, contains a sequence giving significant simi-
larity to an RdRP HMM profile. A search for sequence simi-
larities showed that the nucleotide sequence of this contig
was not significantly similar to any sequence deposited in
public databases, including the *P. patens* genome, but that
the potentially encoded polypeptide did show significant
similarity with RdRPs from amalgaviruses and parti-
tiviruses (Figure 1a). The absence of nucleotide sequence
similarity to sequences deposited in the databases sug-
gested that the corresponding RNA-Seq reads present in
the *P. patens* libraries were not the result of a contamina-
tion with a known virus and that they may correspond to a
previously undetected *P. patens* virus. Moreover, the distri-
bution of the RNA-Seq reads corresponding to this contig
among the *P. patens* samples, with reads present in all
replicates of only few samples of similar tissues (Table S2),
also suggested that the presence of the reads was not the
result of a contamination and was consistent with the pres-
ence of a virus in the specimen being sequenced.

Characterization of the first *P. patens* amalgavirus

The viruses of the Amalgaviridae family are small dsRNA
viruses (around 3400 bp) containing two partially overlap-
ping ORFs (Figure 2a). ORF1 encodes a product whose
et al., 2014). These sequences, which are remnants of past
infections, are transcribed in *P. patens* and it has been pro-
posed that they might protect gametes from viral infection
via small interfering RNA-mediated silencing (Lang et al.,
2018). Moreover, although no virus has been described to
infect *P. patens* in the wild, it has been shown that *P.
patens* can be infected in the laboratory with viruses such
as the tomato spotted wilt virus (Hühns et al., 2003). Here
we used the NGS-based approach described in (Gilbert
et al., 2019) to look for viral sequences in available *P.
patens* RNA sequencing (RNA-Seq) libraries (Haas et al.,
2020; Kamisugi et al., 2016; Perroud et al., 2018) and we
describe the first *P. patens*-associated virus, *P. patens*
amalgavirus 1 (PHPAV1).

RESULTS

Searching for a *P. patens* virus

As most viruses infecting plants are either single-stranded
RNA (ssRNA) or double-stranded RNA (dsRNA) viruses we
decided to take advantage of the large amount of RNA-Seq
data available for *P. patens* (Perroud et al., 2018) to screen
for the possible presence of viral sequences that may cor-
respond to *P. patens*-infecting viruses. We screened the
RNA-Seq libraries for sequences not mapping to the *P.
patens* reference genome. These sequences were then
assembled into a total of 184 184 contigs, corresponding
to 135 766 independent sequences, as some of them repre-
sented different isoforms of the same sequences. The
length of these contigs ranged from 200 nucleotides (nts)
to 12 470 nts. We then looked for sequence similarities of
these contigs to known viruses using different approaches.
We first used blastx to look for similarities between the
proteins potentially encoded by the RNA contigs and those
of the reference viral database (Refseq) from NCBI (on 23
November, 2018). Six contigs gave significant similarity
with annotated viruses: four to the same isolate of the
tobamovirus pepper mild mottle virus, one to another
tobamovirus (tropical soda apple mosaic virus [TSAMV]),
and one to RNA segment 3 of the bromovirus brome
mosaic virus (Table S1). However, in five of these cases,
the *P. patens* contigs were short (close to the lower thresh-
old of the contig size, i.e., 200 nt) and matched a small
fraction of the corresponding viral sequences (Table S1).
Moreover, in all cases the sequence obtained from *P.
patens* RNA-Seq data was almost identical to that of the
virus isolate reported (Table S1), even when these viruses
infect Solanaceae plants or cereals, which are phylogeneti-
cally very distant from *P. patens*. All this suggested that
the presence of these viral sequences was due to a possi-
ble contamination during library preparation or sequencing
processes rather than to the presence of viruses infecting
some of the *P. patens* samples. The contig showing simi-
larity to TSAMV had a size similar to that of the virus (6318

nt). The sequence of this contig was also almost identical
(99.8% identity) to that of the original virus isolated from
*Solanum viarum* (Adkins et al., 2007), suggesting that the
presence of the corresponding sequences in *P. patens*
RNA-Seq libraries may also be the result of a laboratory
contamination. Consistent with this possibility the reads
corresponding to the contig showing similarity to TSAMV
were present in only one out of the three replicates of the
two positive samples, which were obtained from pro-
tonema of the Gransden accession (Table S2). The pres-
ence of spurious reads in NGS samples used for viral
detection is a known problem (Cantalupo and Pipas, 2019).
The sources of contamination can be manifold, including
viruses contaminating the laboratory reagents (Naccache
et al., 2013) and human viruses present in the laboratory
personnel. In this respect, it is interesting to note that
plant-infecting viruses, including TSAMV, are frequently
found in the human oropharynx and gut as a result of
plant consumption (Aguado-García et al., 2020; Balique
et al., 2015), which may suggest a possible source of con-
tamination.

The second approach followed was to look for similari-
ties of the obtained RNA contigs to viral RdRPs using
HMM profiles, a strategy recently used with success to
identify new RNA viruses in fungi (Gilbert et al., 2019).
Two contigs showed significant similarity to RdRP. One of
them corresponded to the contig showing sequence simi-
larity to TSAMV, which was already discarded as a possi-
ble contamination (see above). The second contig showing
similarity to RdRPs (TRINITY_DN26323_c4_g7_i1), which is
3597 nts long, contains a sequence giving significant simi-
larity to an RdRP HMM profile. A search for sequence simi-
larities showed that the nucleotide sequence of this contig
was not significantly similar to any sequence deposited in
public databases, including the *P. patens* genome, but that
the potentially encoded polypeptide did show significant
similarity with RdRPs from amalgaviruses and parti-
tiviruses (Figure 1a). The absence of nucleotide sequence
similarity to sequences deposited in the databases sug-
gested that the corresponding RNA-Seq reads present in
the *P. patens* libraries were not the result of a contamina-
tion with a known virus and that they may correspond to a
previously undetected *P. patens* virus. Moreover, the distri-
bution of the RNA-Seq reads corresponding to this contig
among the *P. patens* samples, with reads present in all
replicates of only few samples of similar tissues (Table S2),
also suggested that the presence of the reads was not the
result of a contamination and was consistent with the pres-
ence of a virus in the specimen being sequenced.

Characterization of the first *P. patens* amalgavirus

The viruses of the Amalgaviridae family are small dsRNA
viruses (around 3400 bp) containing two partially overlap-
ping ORFs (Figure 2a). ORF1 encodes a product whose
<figure><img src="image_2.png" /><figcaption>**Figure 1.** (a) Neighbor-joining phylogenetic unrooted tree of the RdRP encoded by PHPAV1 and the most similar sequences deposited in databases, including RdRPs from amalgaviruses and partitiviruses. The RdRP putative sequence of the *P. patens* PHPAV1 virus is labeled with an asterisk. RdRP sequences obtained from plant, fungus, oomycete, and animal viruses are shown in green, brown, red, and violet, respectively. (b) Neighbor-joining phylogenetic unrooted tree of the RdRP proteins encoded by PHPAV1 and representative viruses of the nine amalgavirus species and the single zybavirus species using the same color code used in (a).</figcaption></figure>
<figure><img src="image_2.png" /><figcaption>**Figure 1.** (a) Neighbor-joining phylogenetic unrooted tree of the RdRP encoded by PHPAV1 and the most similar sequences deposited in databases, including RdRPs from amalgaviruses and partitiviruses. The RdRP putative sequence of the *P. patens* PHPAV1 virus is labeled with an asterisk. RdRP sequences obtained from plant, fungus, oomycete, and animal viruses are shown in green, brown, red, and violet, respectively. (b) Neighbor-joining phylogenetic unrooted tree of the RdRP proteins encoded by PHPAV1 and representative viruses of the nine amalgavirus species and the single zybavirus species using the same color code used in (a).</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>**Figure 2.** (a) Schematic representation of the ORFs encoded by PHPAV1 compared with those of already characterized amalgaviruses. (b) Comparison of the putative ribosomal frameshift sequence of PHPAV1 with the consensus translation frameshift motif of amalgaviruses.</figcaption></figure>

function has not been established yet, although it has been proposed to function as a nucleocapsid protein (Krupovic et al., 2015) or a replication factory matrix-like protein (Iso-gai et al., 2011). ORF2 is encoded in the +1 frame with respect to ORF1 and it is supposed to be translated as part of an ORF1-ORF2 fusion protein by means of a specific ribosome frameshift (Martin et al., 2011; Nibert et al., 2016). The ORF2-encoded part of the ORF1-ORF2 fusion protein shows sequence similarity to partitivirus RdRPs. The Amalgaviridae family has been divided into two genera: amalgaviruses, which include nine viral species, all infecting plants, and zybaviruses, with only one viral species, infecting budding yeast. Apart from the different types of host, the viruses belonging to the two genera also differ in their genome size, which is smaller for the zyba-virus (3.1 kbp) than for the amalgaviruses (3.4–3.5 kbp) (Tzanetakis et al., 2021).

The sequence of contig TRINITY_DN26323_c4_g7_i1 is 3597 bp long (Data S1) and contains two overlapping ORFs, ORF1 and ORF2, with ORF2 being encoded in the +1 frame with respect to ORF1 (Figure 2a). The two ORFs are 1119 and 2601 nts long, respectively. They are flanked by a 5' UTR of 8 nts and a 3' UTR of 15 nts, and the two ORFs overlaps for 176 nts (positions 951 to 1127 in the sequence). Interestingly, the ORF1-ORF2 overlapping region contains the sequence UUU CGU that fits the consensus of the +1 ribosomal frameshift motif described for amalgaviruses (UUU CGN) (Nibert et al., 2016) (Figure 2b). As already mentioned, ORF2 shows high sequence similarity to RdRPs of amalgaviruses and partitiviruses, whereas ORF1 does not show significant similarities to proteins in public databases. Because of all the above, we propose that the sequence of contig TRINITY_DN26323_c4_g7_i1 corresponds to the complete RNA of a new virus of the

Amalgaviridae family. As this virus infects a plant and has a genome size similar to that of characterized amalgaviruses and bigger than that of the only zyba-virus characterized (Tzanetakis et al., 2021), we named this virus *P. patens* amalgavirus 1 (PHPAV1). Nevertheless, a phylogenetic analysis of the RdRP sequence of PHPAV1 and those of the nine amalgavirus species and the single zyba-virus species shows that PHPAV1 RdRP seems more closely related to that of the single zyba-virus described than to that of the characterized amalgaviruses (Figure 1b). A better sampling of the viruses of the Amalgaviridae family infecting plants and fungi will probably be needed to clarify this apparent contradiction. In any case, PHPAV1 constitutes not only the first virus of the Amalgaviridae family described in *P. patens* but also the first virus described infecting a bryophyte.

### Presence of PHPAV1 in different *P. patens* accessions

Analysis of the reads assembled corresponding to PHPAV1 showed that they were all from the sporophyte libraries analyzed. These libraries were the only ones obtained from the *P. patens* Reute accession (Table S2). This suggested that the virus may accumulate in sporophytes or that it may be present in the Reute accession but not in Gransden, which was the accession all the other libraries were obtained from. In order to analyze the pattern of accumulation of PHPAV1 in the Reute ecotype we performed qRT-PCR analyses using RNAs obtained from different tissues and organs of the Reute accession. As shown in Figure 3, the viral RNA was detected in all tissues analyzed and not only in sporophytes.

In order to analyze the presence of PHPAV1 in *P. patens* accessions we performed qRT-PCR analysis on RNA obtained from gametophores of eight different *P. patens*
<figure><img src="image_3.png" /><figcaption>**Figure 2.** (a) Schematic representation of the ORFs encoded by PHPAV1 compared with those of already characterized amalgaviruses. (b) Comparison of the putative ribosomal frameshift sequence of PHPAV1 with the consensus translation frameshift motif of amalgaviruses.</figcaption></figure>

function has not been established yet, although it has been proposed to function as a nucleocapsid protein (Krupovic et al., 2015) or a replication factory matrix-like protein (Iso-gai et al., 2011). ORF2 is encoded in the +1 frame with respect to ORF1 and it is supposed to be translated as part of an ORF1-ORF2 fusion protein by means of a specific ribosome frameshift (Martin et al., 2011; Nibert et al., 2016). The ORF2-encoded part of the ORF1-ORF2 fusion protein shows sequence similarity to partitivirus RdRPs. The Amalgaviridae family has been divided into two genera: amalgaviruses, which include nine viral species, all infecting plants, and zybaviruses, with only one viral species, infecting budding yeast. Apart from the different types of host, the viruses belonging to the two genera also differ in their genome size, which is smaller for the zyba-virus (3.1 kbp) than for the amalgaviruses (3.4–3.5 kbp) (Tzanetakis et al., 2021).

The sequence of contig TRINITY_DN26323_c4_g7_i1 is 3597 bp long (Data S1) and contains two overlapping ORFs, ORF1 and ORF2, with ORF2 being encoded in the +1 frame with respect to ORF1 (Figure 2a). The two ORFs are 1119 and 2601 nts long, respectively. They are flanked by a 5' UTR of 8 nts and a 3' UTR of 15 nts, and the two ORFs overlaps for 176 nts (positions 951 to 1127 in the sequence). Interestingly, the ORF1-ORF2 overlapping region contains the sequence UUU CGU that fits the consensus of the +1 ribosomal frameshift motif described for amalgaviruses (UUU CGN) (Nibert et al., 2016) (Figure 2b). As already mentioned, ORF2 shows high sequence similarity to RdRPs of amalgaviruses and partitiviruses, whereas ORF1 does not show significant similarities to proteins in public databases. Because of all the above, we propose that the sequence of contig TRINITY_DN26323_c4_g7_i1 corresponds to the complete RNA of a new virus of the

Amalgaviridae family. As this virus infects a plant and has a genome size similar to that of characterized amalgaviruses and bigger than that of the only zyba-virus characterized (Tzanetakis et al., 2021), we named this virus *P. patens* amalgavirus 1 (PHPAV1). Nevertheless, a phylogenetic analysis of the RdRP sequence of PHPAV1 and those of the nine amalgavirus species and the single zyba-virus species shows that PHPAV1 RdRP seems more closely related to that of the single zyba-virus described than to that of the characterized amalgaviruses (Figure 1b). A better sampling of the viruses of the Amalgaviridae family infecting plants and fungi will probably be needed to clarify this apparent contradiction. In any case, PHPAV1 constitutes not only the first virus of the Amalgaviridae family described in *P. patens* but also the first virus described infecting a bryophyte.

### Presence of PHPAV1 in different *P. patens* accessions

Analysis of the reads assembled corresponding to PHPAV1 showed that they were all from the sporophyte libraries analyzed. These libraries were the only ones obtained from the *P. patens* Reute accession (Table S2). This suggested that the virus may accumulate in sporophytes or that it may be present in the Reute accession but not in Grasden, which was the accession all the other libraries were obtained from. In order to analyze the pattern of accumulation of PHPAV1 in the Reute ecotype we performed qRT-PCR analyses using RNAs obtained from different tissues and organs of the Reute accession. As shown in Figure 3, the viral RNA was detected in all tissues analyzed and not only in sporophytes.

In order to analyze the presence of PHPAV1 in *P. patens* accessions we performed qRT-PCR analysis on RNA obtained from gametophores of eight different *P. patens*
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Quantification of PHPAV1 RNA in different tissues of *P. patens* Reute. The levels of viral RNA are given relative to the mRNA levels of the gene encoding the 60S ribosomal subunit.</figcaption></figure>

accessions: Gransden, Reute, Villersexel, Kaskaskia, Wisconsin, Uppsala, Trondheim, and Lviv, which have different geographical origins (Figure 4a). We confirmed the presence of the viral RNA in Reute and its absence in Gransden, as the analysis of the RNA-Seq data already suggested. Moreover, we detected the virus in two additional accessions, Kaskaskia and Lviv, whereas the virus was not detected in the remaining four accessions, Villersexel, Wisconsin, Uppsala, and Trondheim (Figure 4b). The presence of the virus does not correlate with any symptom, although the phenotypic differences between the accessions make it difficult to rule out minor phenotypic effects. Sequencing of the 308-bp PCR products indicated that the sequence of the virus present in different accessions is not identical. Indeed, we detected three different

base pairs in the sequence obtained from Kaskaskia and one different base pair in the sequence obtained from Lviv with respect to the sequence of the virus present in Reute. RNA-Seq data from *P. patens* Kaskaskia and Villersexel have been recently reported (Haas et al., 2020; Kamisugi et al., 2016). We did not detect reads corresponding to PHPAV1 in the Villersexel libraries, in line with the qRT-PCR results discussed above, which suggests that PHPAV1 is not present in this accession. On the other hand, the RNA-Seq data from the Kaskaskia accession gave us the opportunity to deduce the complete sequence of the virus present in this accession. Here we detected a total of 19 different base pairs over the entire 3597 bp of the virus sequence compared to the sequence obtained from Reute samples, including the one already detected based on the
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Quantification of PHPAV1 RNA in different tissues of *P. patens* Reute. The levels of viral RNA are given relative to the mRNA levels of the gene encoding the 60S ribosomal subunit.</figcaption></figure>

accessions: Gransden, Reute, Villersexel, Kaskaskia, Wisconsin, Uppsala, Trondheim, and Lviv, which have different geographical origins (Figure 4a). We confirmed the presence of the viral RNA in Reute and its absence in Gransden, as the analysis of the RNA-Seq data already suggested. Moreover, we detected the virus in two additional accessions, Kaskaskia and Lviv, whereas the virus was not detected in the remaining four accessions, Villersexel, Wisconsin, Uppsala, and Trondheim (Figure 4b). The presence of the virus does not correlate with any symptom, although the phenotypic differences between the accessions make it difficult to rule out minor phenotypic effects. Sequencing of the 308-bp PCR products indicated that the sequence of the virus present in different accessions is not identical. Indeed, we detected three different

base pairs in the sequence obtained from Kaskaskia and one different base pair in the sequence obtained from Lviv with respect to the sequence of the virus present in Reute. RNA-Seq data from *P. patens* Kaskaskia and Villersexel have been recently reported (Haas et al., 2020; Kamisugi et al., 2016). We did not detect reads corresponding to PHPAV1 in the Villersexel libraries, in line with the qRT-PCR results discussed above, which suggests that PHPAV1 is not present in this accession. On the other hand, the RNA-Seq data from the Kaskaskia accession gave us the opportunity to deduce the complete sequence of the virus present in this accession. Here we detected a total of 19 different base pairs over the entire 3597 bp of the virus sequence compared to the sequence obtained from Reute samples, including the one already detected based on the
<figure><img src="image_5.png" /><figcaption>**Figure 4.** Presence of PHPAV1 in different *P. patens* accessions. (a) Geographical distribution of the *P. patens* accessions analyzed. The names of the accessions containing the virus are shown in red and those not containing the virus are shown in black. (b) Quantification of the PHPAV1 levels in gametophores of different *P. patens* accessions. The viral RNA levels are given relative to the mRNA levels of the gene encoding the 60S ribosomal subunit.</figcaption></figure>

PCR analysis mentioned above (Table S3). These nucleo-
tide variations are outside the motif potentially linked to
the translation frameshift allowing the translation of the
ORF1-ORF2 fusion protein and would result in only five

amino acid changes in the encoded polypeptides, two in
ORF1 and three in ORF2 (Table S3). Interestingly, sequenc-
ing of the PCR products from the three accessions that
contain the virus, as well as of the Reute and Kaskaskia
<figure><img src="image_5.png" /><figcaption>**Figure 4.** Presence of PHPAV1 in different *P. patens* accessions. (a) Geographical distribution of the *P. patens* accessions analyzed. The names of the accessions containing the virus are shown in red and those not containing the virus are shown in black. (b) Quantification of the PHPAV1 levels in gametophores of different *P. patens* accessions. The viral RNA levels are given relative to the mRNA levels of the gene encoding the 60S ribosomal subunit.</figcaption></figure>

PCR analysis mentioned above (Table S3). These nucleo-
tide variations are outside the motif potentially linked to
the translation frameshift allowing the translation of the
ORF1-ORF2 fusion protein and would result in only five

amino acid changes in the encoded polypeptides, two in
ORF1 and three in ORF2 (Table S3). Interestingly, sequenc-
ing of the PCR products from the three accessions that
contain the virus, as well as of the Reute and Kaskaskia
RNA-Seq reads available, did not reveal any variability of
the virus sequence within each of these ecotypes.

## PHPAV1 vertical transmission

Amalgaviruses are endogenous viruses thought to be
transmitted vertically (Martin et al., 2011). We analyzed the
transmission of the virus by means of crosses between
accessions containing or lacking the virus. In order to ana-
lyze its possible transmission through parental and mater-
nal gametes, we performed two different crosses. For the
first one, designed to assess the potential transmission of
the virus through the male germline, we took advantage of
the reduced male fertility of the Gransden accession (Mey-
berg et al., 2020) to cross it with the virus-containing Kas-
kaskia accession. We used a Kaskaskia line expressing a
YFP (Ka-YFP; Methods S1) to ensure selecting sporophytes
resulting from the cross. In the second cross, a Reute male
sterile Ppccdc39 strain (Meyberg et al., 2020), containing
the virus, was crossed with Villersexel-mCherry (Vx-red)
which does not contain the virus, acting as male progeni-
tor. As shown in Figure 5, the virus could be detected in a
pool of 10 individual plants resulting from a single sporo-
phyte from each of the two crosses, indicating that the

virus can be transmitted by both male (Figure 5a) and
female (Figure 5b) reproductive organs, antheridia and
archegonia. The sequence of the PCR products allowed
identifying the characteristic nucleotide differences of the
PHPAV1 sequence from Reute and Kaskaskia accessions,
confirming the origin of the identified sequences. Interest-
ingly, whereas the 10 individual plants resulting from the
Villersexel × Reute cross all showed the presence of the
virus (Figure 5e), only a fraction (6/10) of the plants result-
ing from the Kaskaskia × Gransden cross seems to contain
PHPAV1 (Figure 5d). As all the 10 plants come from the
same fertilization event, differences in the presence of the
virus should be due to a post-fertilization loss of the virus
in some plants. Whether this loss is a stochastic event dur-
ing cell division or it is due to genetic differences between
Kaskaskia and Gransden accessions affecting viral mainte-
nance remains to be analyzed. Moreover, as the virus pre-
sent in the Kaskaskia accession is not identical to the one
present in the Reute accession, the differences in the
prevalence of the virus in the offspring could also be due
to differences in the persistence of the virus itself. In order
to start investigating this aspect, we designed a cross
between Kaskaskia (Ka-YFP) acting as paternal progenitor

<figure><img src="image_6.png" /><figcaption>**Figure 5.** Detection of the PHPAV1 RNA in crosses of different accessions of *P. patens*. (a and d) Crosses between Kaskaskia acting as male and Gransden as female. (b and e) Crosses between Kaskaskia acting as male and Reute as female. (c and f) Crosses between Villersexel acting as male and Reute as female. (a, b, and c) Quantification of the RNA of the virus in a pool of 10 plants resulting from a single sporophyte obtained from each cross. (d, e, and f) Quantification of the virus in each of the 10 independent plants. The viral RNA levels are given relative to the mRNA levels of the gene encoding the 60S ribosomal subunit.</figcaption></figure>
RNA-Seq reads available, did not reveal any variability of
the virus sequence within each of these ecotypes.

## PHPAV1 vertical transmission

Amalgaviruses are endogenous viruses thought to be
transmitted vertically (Martin et al., 2011). We analyzed the
transmission of the virus by means of crosses between
accessions containing or lacking the virus. In order to ana-
lyze its possible transmission through parental and mater-
nal gametes, we performed two different crosses. For the
first one, designed to assess the potential transmission of
the virus through the male germline, we took advantage of
the reduced male fertility of the Gransden accession (Mey-
berg et al., 2020) to cross it with the virus-containing Kas-
kaskia accession. We used a Kaskaskia line expressing a
YFP (Ka-YFP; Methods S1) to ensure selecting sporophytes
resulting from the cross. In the second cross, a Reute male
sterile Ppccdc39 strain (Meyberg et al., 2020), containing
the virus, was crossed with Villersexel-mCherry (Vx-red)
which does not contain the virus, acting as male progeni-
tor. As shown in Figure 5, the virus could be detected in a
pool of 10 individual plants resulting from a single sporo-
phyte from each of the two crosses, indicating that the

virus can be transmitted by both male (Figure 5a) and
female (Figure 5b) reproductive organs, antheridia and
archegonia. The sequence of the PCR products allowed
identifying the characteristic nucleotide differences of the
PHPAV1 sequence from Reute and Kaskaskia accessions,
confirming the origin of the identified sequences. Interest-
ingly, whereas the 10 individual plants resulting from the
Villersexel × Reute cross all showed the presence of the
virus (Figure 5e), only a fraction (6/10) of the plants result-
ing from the Kaskaskia × Gransden cross seems to contain
PHPAV1 (Figure 5d). As all the 10 plants come from the
same fertilization event, differences in the presence of the
virus should be due to a post-fertilization loss of the virus
in some plants. Whether this loss is a stochastic event dur-
ing cell division or it is due to genetic differences between
Kaskaskia and Gransden accessions affecting viral mainte-
nance remains to be analyzed. Moreover, as the virus pre-
sent in the Kaskaskia accession is not identical to the one
present in the Reute accession, the differences in the
prevalence of the virus in the offspring could also be due
to differences in the persistence of the virus itself. In order
to start investigating this aspect, we designed a cross
between Kaskaskia (Ka-YFP) acting as paternal progenitor

<figure><img src="image_6.png" /><figcaption>**Figure 5.** Detection of the PHPAV1 RNA in crosses of different accessions of *P. patens*. (a and d) Crosses between Kaskaskia acting as male and Gransden as female. (b and e) Crosses between Kaskaskia acting as male and Reute as female. (c and f) Crosses between Villersexel acting as male and Reute as female. (a, b, and c) Quantification of the RNA of the virus in a pool of 10 plants resulting from a single sporophyte obtained from each cross. (d, e, and f) Quantification of the virus in each of the 10 independent plants. The viral RNA levels are given relative to the mRNA levels of the gene encoding the 60S ribosomal subunit.</figcaption></figure>
and Reute (*Ppccdc39*) acting as maternal progenitor. As shown in Figure 5c, the virus could be detected in a pool of 10 individual plants resulting from a single sporophyte from the cross, and also in each of the 10 individual plants resulting from this cross (Figure 5f). The analysis of the sequence of the virus present in these 10 individual plants showed that only the Reute virus was detected.

DISCUSSION

Amalgaviridae is a recently reported family of dsRNA
viruses, described as an amalgam between viruses of the
Totiviridae and Partitiviridae families, with a structure
more related to the former but encoding proteins phyloge-
netically related to the latter (Martin et al., 2011). The
Amalgaviridae family has been recently divided in two
genera: amalgaviruses, including nine viral species infect-
ing plants, and zybaviruses, including only one species
that infects budding yeast (Tzanetakis et al., 2021).
Whereas most amalgaviruses reported are plant viruses
(Goh et al., 2018; Martin et al., 2011; Park et al., 2018;
Sabanadzovic et al., 2009; Zhan et al., 2019), viruses
described as amalgaviruses and present in fungi have also
been reported (Koloniuk et al., 2015), and different
sequences potentially related to amalgaviruses and zyba-
viruses obtained from plant, fungi, oomycetes, and ani-
mals have been deposited in public databases. The host
species and the size of PHPAV1, which is more similar to
that of the viruses classified as amalgaviruses, prompted
us to tentatively classify PHPAV1 as an amalgavirus. How-
ever, the RdRP sequence of PHPAV1 seems more closely
related to that of the fungus-infecting virus that defines the
zybavirus genus than to that of the nine plant-infecting
viruses of the amalgavirus genus. Moreover, phylogenetic
analysis of the PHPAV1 sequence together with the most
similar sequences deposited in databases shows that
PHPAV1 falls into a not well-defined group of sequences of
viruses from fungi, oomycetes, and animals outside the
two main groups, of which one contains most plant viral
sequences and the other contains most fungal viral
sequences. The apparent lack of consistency of this phy-
logeny with that of the species these viruses infect could
simply be the result of a partial sampling. Amalgaviruses
have only been described recently and the fact that in most
cases these endogenous viruses do not seem to lead to
lesions or symptoms makes their discovery more difficult.
Therefore, it is very plausible that the picture we have at
present is incomplete and this could lead to apparently
incongruent phylogenetic relationships when comparing
these sequences. Indeed, whereas no amalgaviruses have
been described in bryophytes, sequences related to parti-
tivirus RdRPs, which are close relatives of amalgaviruses,
have been detected in different bryophytes (Mushegian
et al., 2016). Alternatively, although amalgaviruses are
thought to be transmitted vertically, the close proximity in

nature of many of the species these viruses infect could
support their horizontal transfer. A more in depth sampling
and careful analysis would be required to answer this open
question.

The viruses of the Amalgaviridae family are vertically
transmitted endogenous viruses. As they are dsRNA virus
and they do not integrate into the genome of their hosts,
the dsRNA needs to be present throughout the life cycle of
the host. We show here that PHPAV1 is present in all tis-
sues tested, which are representative of the *P. patens* life-
cycle, including in the tissues and organs that will give rise
to the germline. In addition, we show that PHPAV1 is trans-
mitted through protoplasts, as the transgenic lines *Ka-YFP*
and *Ppccdc39* were obtained by regenerating single trans-
genic protoplasts obtained from Kaskaskia and Reute
plants, respectively.

However, PHPAV1 is only present in certain *P. patens*
accessions. Among the eight accessions tested, PHPAV1
appears to be present in only three, Reute, Kaskaskia, and
Lviv, and to be absent from the other five, Gransden, Viller-
sexel, Wisconsin, Uppsala, and Trondheim. This distribution
of the virus does not fit the geographical distribution of *P.
patens* accessions, with the virus present in geographically
distant accessions and absent from closer ones. For exam-
ple, the virus is present in one of the two accessions from
the USA, Kaskaskia, and is present in Reute but not in Viller-
sexel in spite of these two accessions being from close geo-
graphical origins in central Europe. This distribution could
be the result of recent independent infections of the acces-
sions that contain the virus or the recent loss in some acces-
sions of the virus, which could have infected a precursor
individual of all *P. patens* accessions.

The results presented here show that the virus can be
transmitted through both paternal and maternal germlines
and that the virus can be detected in the offspring of
crosses between accessions, even when only one of the
parental accessions (either the parental or the maternal
progenitor) contains the virus. This suggests that although
the virus is only present in certain accessions, it can repli-
cate and be maintained in other *P. patens* genetic back-
grounds. However, it is interesting to note that in the cross
between Kaskaskia acting as the male strain and Gransden
acting as the female strain, only six out of the 10 plants
resulting from a single sporophyte showed detectable
levels of PHPAV1, suggesting that the virus was not main-
tained at a detectable level in all of them. This absence of
PHPAV1 could simply be the result of a stochastic loss of
the virus due to an uneven distribution of the virus present
at low level in the progenitor cell. But this result could also
indicate that certain genetic backgrounds, resulting from
the recombination of Gransden and Kaskaskia genomes,
may not allow the maintenance of the virus. In other
words, this result may suggest that one or several genetic
factors, variable among *P. patens* accessions, may restrict
and Reute (*Ppccdc39*) acting as maternal progenitor. As shown in Figure 5c, the virus could be detected in a pool of 10 individual plants resulting from a single sporophyte from the cross, and also in each of the 10 individual plants resulting from this cross (Figure 5f). The analysis of the sequence of the virus present in these 10 individual plants showed that only the Reute virus was detected.

DISCUSSION

Amalgaviridae is a recently reported family of dsRNA
viruses, described as an amalgam between viruses of the
Totiviridae and Partitiviridae families, with a structure
more related to the former but encoding proteins phyloge-
netically related to the latter (Martin et al., 2011). The
Amalgaviridae family has been recently divided in two
genera: amalgaviruses, including nine viral species infect-
ing plants, and zybaviruses, including only one species
that infects budding yeast (Tzanetakis et al., 2021).
Whereas most amalgaviruses reported are plant viruses
(Goh et al., 2018; Martin et al., 2011; Park et al., 2018;
Sabanadzovic et al., 2009; Zhan et al., 2019), viruses
described as amalgaviruses and present in fungi have also
been reported (Koloniuk et al., 2015), and different
sequences potentially related to amalgaviruses and zyba-
viruses obtained from plant, fungi, oomycetes, and ani-
mals have been deposited in public databases. The host
species and the size of PHPAV1, which is more similar to
that of the viruses classified as amalgaviruses, prompted
us to tentatively classify PHPAV1 as an amalgavirus. How-
ever, the RdRP sequence of PHPAV1 seems more closely
related to that of the fungus-infecting virus that defines the
zybavirus genus than to that of the nine plant-infecting
viruses of the amalgavirus genus. Moreover, phylogenetic
analysis of the PHPAV1 sequence together with the most
similar sequences deposited in databases shows that
PHPAV1 falls into a not well-defined group of sequences of
viruses from fungi, oomycetes, and animals outside the
two main groups, of which one contains most plant viral
sequences and the other contains most fungal viral
sequences. The apparent lack of consistency of this phy-
logeny with that of the species these viruses infect could
simply be the result of a partial sampling. Amalgaviruses
have only been described recently and the fact that in most
cases these endogenous viruses do not seem to lead to
lesions or symptoms makes their discovery more difficult.
Therefore, it is very plausible that the picture we have at
present is incomplete and this could lead to apparently
incongruent phylogenetic relationships when comparing
these sequences. Indeed, whereas no amalgaviruses have
been described in bryophytes, sequences related to parti-
tivirus RdRPs, which are close relatives of amalgaviruses,
have been detected in different bryophytes (Mushegian
et al., 2016). Alternatively, although amalgaviruses are
thought to be transmitted vertically, the close proximity in

nature of many of the species these viruses infect could
support their horizontal transfer. A more in depth sampling
and careful analysis would be required to answer this open
question.

The viruses of the Amalgaviridae family are vertically
transmitted endogenous viruses. As they are dsRNA virus
and they do not integrate into the genome of their hosts,
the dsRNA needs to be present throughout the life cycle of
the host. We show here that PHPAV1 is present in all tis-
sues tested, which are representative of the *P. patens* life-
cycle, including in the tissues and organs that will give rise
to the germline. In addition, we show that PHPAV1 is trans-
mitted through protoplasts, as the transgenic lines *Ka-YFP*
and *Ppccdc39* were obtained by regenerating single trans-
genic protoplasts obtained from Kaskaskia and Reute
plants, respectively.

However, PHPAV1 is only present in certain *P. patens*
accessions. Among the eight accessions tested, PHPAV1
appears to be present in only three, Reute, Kaskaskia, and
Lviv, and to be absent from the other five, Gransden, Viller-
sexel, Wisconsin, Uppsala, and Trondheim. This distribution
of the virus does not fit the geographical distribution of *P.
patens* accessions, with the virus present in geographically
distant accessions and absent from closer ones. For exam-
ple, the virus is present in one of the two accessions from
the USA, Kaskaskia, and is present in Reute but not in Viller-
sexel in spite of these two accessions being from close geo-
graphical origins in central Europe. This distribution could
be the result of recent independent infections of the acces-
sions that contain the virus or the recent loss in some acces-
sions of the virus, which could have infected a precursor
individual of all *P. patens* accessions.

The results presented here show that the virus can be
transmitted through both paternal and maternal germlines
and that the virus can be detected in the offspring of
crosses between accessions, even when only one of the
parental accessions (either the parental or the maternal
progenitor) contains the virus. This suggests that although
the virus is only present in certain accessions, it can repli-
cate and be maintained in other *P. patens* genetic back-
grounds. However, it is interesting to note that in the cross
between Kaskaskia acting as the male strain and Gransden
acting as the female strain, only six out of the 10 plants
resulting from a single sporophyte showed detectable
levels of PHPAV1, suggesting that the virus was not main-
tained at a detectable level in all of them. This absence of
PHPAV1 could simply be the result of a stochastic loss of
the virus due to an uneven distribution of the virus present
at low level in the progenitor cell. But this result could also
indicate that certain genetic backgrounds, resulting from
the recombination of Gransden and Kaskaskia genomes,
may not allow the maintenance of the virus. In other
words, this result may suggest that one or several genetic
factors, variable among *P. patens* accessions, may restrict
the proliferation of the virus. This would explain why the virus can be present in a particular *P. patens* accession and absent from another accession located geographically close and potentially interfertile.

Finally, our results also show that in a cross between two accessions containing the virus, the offspring does not necessarily inherit the virus from both parents. Indeed, in the cross analyzed here between Kaskaskia acting as paternal progenitor and Reute acting as maternal progenitor, the 10 plants resulting from a single sporophyte did contain the virus, but in all cases the virus was inherited from the maternal progenitor. This result could suggest that viral transfer from the maternal and from the paternal progenitor is not equally efficient, but it may also point to an effect on viral persistence of viral variants, as the sequence of the virus present in Kaskaskia and Reute accessions is not identical. It is interesting to note that, whereas the sequence of the virus from different accessions is different, we have not detected any variability of the virus within each of the accessions. RNA viruses are often characterized by a high sequence variability known as quasispecies-like structure (Holland et al., 1982). However, it has been suggested that endogenous persistent viruses may be much less variable (Safari and Roossinck, 2014). Although there is almost no population study of persistent viruses in plants, a recent report of two persistent endogenous viruses from spinach (*Spinacia oleracea*), a partitivirus and an amalgavirus, showed that they present very low sequence diversity (Samarfard et al., 2020). Similarly, the analysis of archeological samples has shown that a maize (*Zea mays*) endogenous persistent virus belonging to the family Chrysoviridae has undergone only about 3% divergence after 1000 years of maize cultivation (Peyambari et al., 2018). The reasons for the low variability of endogenous persistent viruses are not fully understood, but may be related to their stamped machine mode of replication, which they share with most dsRNA viruses, and their particular lifestyle (Safari and Roossinck, 2014). Indeed, their long coexistence with their hosts and not being targeted by the host silencing mechanisms makes a quasispecies-like structure much less advantageous. The absence of sequence variability of the viral sequences within a single accession, together with the sequence differences found in PHPAV1 from different accessions, may suggest an accession-specific evolution and adaptation.

Most infections of amalgaviruses in plants are asymptomatic. Although in some cases the infected plants did show symptoms, the correlation between the symptoms and the presence of the virus could not be confirmed (Zhan et al., 2019). In any case, the absence of symptoms does not imply that the infections have no effect on the host. It has been shown recently that the infection of tomato (*Solanum lycopersicum*) by Southern tomato virus, which belongs to the amalgavirus family, results in changes of

expression of micro-RNAs that could modify the response of the host to different stresses (Elvira-González et al., 2020). In fact, different reports point to beneficial effects of persistent viral infections in plants, including protection against acute infections of more harmful viruses, regulation of nodulation, better tolerance to stress, and increased plant height and fruit production (Takahashi et al., 2019).

Eukaryotic, and in particular, plant genomes contain integrated sequences from endogenous viruses. For example, plant genomes frequently contain sequences of caulimoviruses (Geering et al., 2014), and it has been shown that the *P. patens* genome contains NCLDV sequences (Lang et al., 2018; Maumus et al., 2014). These integrated viral sequences may protect the plant against viral infections, as proposed for the integrated NCLDV in *P. patens* (Lang et al., 2018), and may also contribute to the evolution of the genome acting as sources of novel genetic material (Geering et al., 2014). As amalgaviruses do not integrate in the genome of their hosts, a direct role in host genome evolution is less obvious to imagine. However, their potential beneficial effects remain a very attractive field for future study. The report here of an amalgavirus infecting only some *P. patens* accessions provides an interesting model to study the potential function of these viruses and their adaptation to their hosts.

## EXPERIMENTAL PROCEDURES

### Libraries used

We checked for the presence of PHPAV1 in Gransden and Reute accessions using the RNA-Seq data available in the first release of the *P. patens* gene atlas (Lang et al., 2018) and in Kaskaskia and Villersexel accessions using RNA-Seq data deposited in NCBI SRA in BioProjects PRJNA601618 and PRJNA602303 (Haas et al., 2020) and libraries SRX031156 and SRX031155 (Kamisugi et al., 2016).

### Contig formation and virus identification

All reads were quality-trimmed using bbduk (Bushnell, 2014). We mapped all the reads to the *P. patens* genome (including the plastid genomes) (Lang et al., 2018) using STAR (Dobin et al., 2013) with the following flag to select the unmapped reads: `-outSAMunmapped`. Then, we searched for all these reads that did not align to the reference genome using the samtools-1.12 package (Li et al., 2009) with the flag (-f 4). The selected reads from all libraries were pooled to be assembled using Trinity (Grabherr et al., 2011) with default parameters.

Two different approaches were followed to identify viral sequences in the assembled sequences. First, we used blastx to search for similarities between the putatively encoded polypeptides and viral proteins deposited at the reference viral database from NCBI Refseq (Brister et al., 2015) with an e-value cutoff of 0.05 (done on 23 November, 2018). Second, we searched for RdRP signatures in all the polypeptides potentially encoded by the assembled sequences using HMMscan (version 3.1b2), based on hidden Markov models (HMMs), in an approach similar to the one described by Gilbert et al. and using the same e-value cutoff of 10 (Gilbert et al., 2019). We used the HMM profiles from the
the proliferation of the virus. This would explain why the virus can be present in a particular *P. patens* accession and absent from another accession located geographically close and potentially interfertile.

Finally, our results also show that in a cross between two accessions containing the virus, the offspring does not necessarily inherit the virus from both parents. Indeed, in the cross analyzed here between Kaskaskia acting as paternal progenitor and Reute acting as maternal progenitor, the 10 plants resulting from a single sporophyte did contain the virus, but in all cases the virus was inherited from the maternal progenitor. This result could suggest that viral transfer from the maternal and from the paternal progenitor is not equally efficient, but it may also point to an effect on viral persistence of viral variants, as the sequence of the virus present in Kaskaskia and Reute accessions is not identical. It is interesting to note that, whereas the sequence of the virus from different accessions is different, we have not detected any variability of the virus within each of the accessions. RNA viruses are often characterized by a high sequence variability known as quasispecies-like structure (Holland et al., 1982). However, it has been suggested that endogenous persistent viruses may be much less variable (Safari and Roossinck, 2014). Although there is almost no population study of persistent viruses in plants, a recent report of two persistent endogenous viruses from spinach (*Spinacia oleracea*), a partitivirus and an amalgavirus, showed that they present very low sequence diversity (Samarfard et al., 2020). Similarly, the analysis of archeological samples has shown that a maize (*Zea mays*) endogenous persistent virus belonging to the family Chrysoviridae has undergone only about 3% divergence after 1000 years of maize cultivation (Peyambari et al., 2018). The reasons for the low variability of endogenous persistent viruses are not fully understood, but may be related to their stamped machine mode of replication, which they share with most dsRNA viruses, and their particular lifestyle (Safari and Roossinck, 2014). Indeed, their long coexistence with their hosts and not being targeted by the host silencing mechanisms makes a quasispecies-like structure much less advantageous. The absence of sequence variability of the viral sequences within a single accession, together with the sequence differences found in PHPAV1 from different accessions, may suggest an accession-specific evolution and adaptation.

Most infections of amalgaviruses in plants are asymptomatic. Although in some cases the infected plants did show symptoms, the correlation between the symptoms and the presence of the virus could not be confirmed (Zhan et al., 2019). In any case, the absence of symptoms does not imply that the infections have no effect on the host. It has been shown recently that the infection of tomato (*Solanum lycopersicum*) by Southern tomato virus, which belongs to the amalgavirus family, results in changes of

expression of micro-RNAs that could modify the response of the host to different stresses (Elvira-González et al., 2020). In fact, different reports point to beneficial effects of persistent viral infections in plants, including protection against acute infections of more harmful viruses, regulation of nodulation, better tolerance to stress, and increased plant height and fruit production (Takahashi et al., 2019).

Eukaryotic, and in particular, plant genomes contain integrated sequences from endogenous viruses. For example, plant genomes frequently contain sequences of caulimoviruses (Geering et al., 2014), and it has been shown that the *P. patens* genome contains NCLDV sequences (Lang et al., 2018; Maumus et al., 2014). These integrated viral sequences may protect the plant against viral infections, as proposed for the integrated NCLDV in *P. patens* (Lang et al., 2018), and may also contribute to the evolution of the genome acting as sources of novel genetic material (Geering et al., 2014). As amalgaviruses do not integrate in the genome of their hosts, a direct role in host genome evolution is less obvious to imagine. However, their potential beneficial effects remain a very attractive field for future study. The report here of an amalgavirus infecting only some *P. patens* accessions provides an interesting model to study the potential function of these viruses and their adaptation to their hosts.

## EXPERIMENTAL PROCEDURES

### Libraries used

We checked for the presence of PHPAV1 in Gransden and Reute accessions using the RNA-Seq data available in the first release of the *P. patens* gene atlas (Lang et al., 2018) and in Kaskaskia and Villersexel accessions using RNA-Seq data deposited in NCBI SRA in BioProjects PRJNA601618 and PRJNA602303 (Haas et al., 2020) and libraries SRX031156 and SRX031155 (Kamisugi et al., 2016).

### Contig formation and virus identification

All reads were quality-trimmed using bbduk (Bushnell, 2014). We mapped all the reads to the *P. patens* genome (including the plastid genomes) (Lang et al., 2018) using STAR (Dobin et al., 2013) with the following flag to select the unmapped reads: `-outSAM unmapped`. Then, we searched for all these reads that did not align to the reference genome using the samtools-1.12 package (Li et al., 2009) with the flag (-f 4). The selected reads from all libraries were pooled to be assembled using Trinity (Grabherr et al., 2011) with default parameters.

Two different approaches were followed to identify viral sequences in the assembled sequences. First, we used blastx to search for similarities between the putatively encoded polypeptides and viral proteins deposited at the reference viral database from NCBI Refseq (Brister et al., 2015) with an e-value cutoff of 0.05 (done on 23 November, 2018). Second, we searched for RdRP signatures in all the polypeptides potentially encoded by the assembled sequences using HMMscan (version 3.1b2), based on hidden Markov models (HMMs), in an approach similar to the one described by Gilbert et al. and using the same e-value cutoff of 10 (Gilbert et al., 2019). We used the HMM profiles from the
following PFAM accessions: PF00680, PF00978, PF02123, PF05183, PF07925, and PF17501.

## Phylogenetic analyses

We took the best 50 hits from the blastx search done (19 April, 2021) using the nucleotide sequence of PHPAV1 against the non-redundant protein sequences from the NCBI database to build the phylogenetic tree. All these sequences were aligned using Mafft (Katoh and Standley, 2013) (with --auto and -adjust direction parameters) and trimmed with TrimAL (Capella-Gutiérrez et al., 2009) (with -automated1 mode). We inferred an approximately maximum-likelihood phylogenetic tree using the FastTree program (Price et al., 2010) with default parameters and visualized it using iTol (Letunic and Bork, 2019).

## Virus variability among different accessions

The reads corresponding to PHPAV1 from different accessions were aligned to the Reute PHPAV1 sequence using bowtie2 (Langmead and Salzberg, 2013) (with: —threads 8 —local —no-unal -k 2).

## Plant material and culture

We used wild-type *P. patens* accessions of different origins: Gransden (Gd) pedigree Gd_JP_St Louis (Haas et al., 2020) and Reute-K1 (Re) (Hiss et al., 2017), which are routinely used in laboratory settings, and Viller sexel-K3 (Vx), Kaskaskia (Ka), Lviv (Lv), Trondheim-K2 (Td), and Uppsala-K1 (Up), which were obtained from the International Moss Stock Center (https://www.moss-stock-center.org/). In addition, we used the Wisconsin (Wi) accession, which has been recently isolated from the wild (Haas et al., 2020). In order to facilitate the isolation of crossed plants, we used transgenic *P. patens* expressing a fluorescent protein. Vx-red has been established in a Viller sexel-K3 background and accumulates mCherry (Perroud et al., 2011). *Re-mCherry* is a Reute-K1 trans determinant that accumulates mCherry (Perroud et al., 2019). Ka-YFP has been established in the Kaskaskia ecotype and accumulates eYFP (Methods S1). For crossing analyses, a male sterile Re-mutant, Ppccdc39, was used (Meyberg et al., 2020).

If not mentioned otherwise, mosses were grown at 24°C with a cycle of 16 h of light (quantum irradiance of 60-80 µmol m⁻² s⁻¹ light) and 8 h of darkness on plates containing BCDA medium (Cove et al., 2009). Harvested samples were systematically flash-frozen in liquid N₂ and stored at -80°C until further processing. For all comparisons between accessions as well as for the progeny analyses, gametophores were harvested from plants grown for 1 month.

Protonema Reute samples were harvested from a 6-day-old entrained culture grown on solid BCDA medium overlayed with cel-lophane. Reute juvenile (asexual) gametophore samples were har-vested from a 1-month-old culture grown on BCD medium (Cove et al., 2009). Adult Reute gametophore (bearing gametangia) sam-ples were harvested from culture grown for 1 month on BCD med-ium and subsequently transferred to 15°C with a cycle of 8 h of light (30 µmol m⁻² s⁻¹) and 16 h of darkness for 2 weeks (Hohe et al., 2002). The presence of gametangia was visually confirmed prior to harvesting. The sporophyte samples were obtained from culture grown initially under the adult gametophore regime but watered after the 2 weeks at 15°C to facilitate fertilization. After sub-sequent growth in the same conditions, green and brown spor-o-phyte samples (Hiss et al., 2017) were collected after 5-7 weeks.

Crossing experiments were performed by coculture of two par-ental strains as previously described (Perroud et al., 2011). Mature sporophytes (brown) displaying fluorescence developed on a gametophore devoid of fluorescence were manually isolated and

stored at least for a week in the dark at 4°C before performing the germination assay. For each tested sporophyte, 300 to 400 spores were germinated (Perroud et al., 2019) and the predicted 1:1 seg-regation of the fluorescent marker was confirmed to ensure cross-ing prior to further work. Then, 10 sporelings were isolated per crossed sporophyte and their tissues were amplified to perform subsequent analyses.

## RNA extraction and cDNA library preparation

RNA extraction and DNase treatment were done using the Maxwell® RSC Plant Kit (Promega, Ref #AS1500) following the manufacturer's instructions. RNA was resuspended in 30 µl of sterile water. We used 200 ng of total RNA for first-strand cDNA synthesis using a 5'-modified oligo-dT primer (Casacuberta et al., 1995) and a specific primer complementary to the PHPAV1 sequence (oxPV63: 5'-CCCACCTCCCTTCACAGACGATC-3') with SuperScript™ III reverse transcriptase (Thermo Fisher). As a con-trol we performed a reaction with the same amount of RNA (200 ng) without adding the SuperScript™ III reverse transcriptase.

## qRT-PCR

Quantitative real-time PCRs were performed in 96-well plates using a Roche LightCycler II instrument. We used SYBR Green I Master Mix (Roche Applied Science) with primers (oxPV64: 5'-CAAAAGCCTATT CCTCTGCATGG-3', oxPV65: 5'-CTGAGAGAAACTGTCCCGTAACT-3') at 1 µM and 40 ng of cDNA obtained from the reverse transcription to conduct the qRT-PCR analysis. Each sam-ple was run in triplicate with negative reverse transcriptase for each sample and negative controls (H₂O). The following PCR con-ditions were used: 95°C for 5 min, followed by 95°C for 10 sec, 56°C for 10 sec, and 72°C for 10 sec. For normalization we used the relatively highly expressed gene encoding the 60S ribosomal subunit as previously described (Le Bail et al., 2013). The PCR products were purified using the Macherey-Nagel Nucleospin® Gel and PCR cleanup kit (Ref #740609.50) and sequenced.

## ACKNOWLEDGEMENTS

This work was supported by grants from the Spanish Ministerio de Economía, Industria y Competitividad (AGL2016-78992-R/FEDER) and Ministerio de Ciencia e Innovación (PID2019-106374RB-100/AEI/10.13039/501100011033) to JMC. The IJPB benefits from the support of the LabEx Saclay Plant Sciences (SPS) (ANR-10-LABX-0040-SPS). We thank Prof. Mitsuyasu Hasebe for the generous gift of the plasmid p35S-loxP-BSD. We are grateful to Leonie Verhage for providing us with the *P. patens* ecotypes Lviv, Trondheim-K2, and Uppsala-K1. We thank Jean-Luc Gallois and Juan José López-Moya for helpful discussions.

## CONFLICT OF INTEREST

The authors have no conflict of interest to declare.

## DATA AVAILABILITY STATEMENT

All relevant data can be found within the manuscript and its supporting materials. The complete sequence of PHPAV1 can be found at GenBank under accession number BK059361.

## SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article.
following PFAM accessions: PF00680, PF00978, PF02123, PF05183, PF07925, and PF17501.

## Phylogenetic analyses

We took the best 50 hits from the blastx search done (19 April, 2021) using the nucleotide sequence of PHPAV1 against the non-redundant protein sequences from the NCBI database to build the phylogenetic tree. All these sequences were aligned using Mafft (Katoh and Standley, 2013) (with --auto and -adjust direction parameters) and trimmed with TrimAL (Capella-Gutiérrez et al., 2009) (with -automated1 mode). We inferred an approximately maximum-likelihood phylogenetic tree using the FastTree program (Price et al., 2010) with default parameters and visualized it using iTol (Letunic and Bork, 2019).

## Virus variability among different accessions

The reads corresponding to PHPAV1 from different accessions were aligned to the Reute PHPAV1 sequence using bowtie2 (Langmead and Salzberg, 2013) (with: —threads 8 —local —no-unal -k 2).

## Plant material and culture

We used wild-type *P. patens* accessions of different origins: Gransden (Gd) pedigree Gd_JP_St Louis (Haas et al., 2020) and Reute-K1 (Re) (Hiss et al., 2017), which are routinely used in laboratory settings, and Viller sexel-K3 (Vx), Kaskaskia (Ka), Lviv (Lv), Trondheim-K2 (Td), and Uppsala-K1 (Up), which were obtained from the International Moss Stock Center (https://www.moss-stock-center.org/). In addition, we used the Wisconsin (Wi) accession, which has been recently isolated from the wild (Haas et al., 2020). In order to facilitate the isolation of crossed plants, we used transgenic *P. patens* expressing a fluorescent protein. Vx-red has been established in a Viller sexel-K3 background and accumulates mCherry (Perroud et al., 2011). *Re-mCherry* is a Reute-K1 transfortant that accumulates mCherry (Perroud et al., 2019). Ka-YFP has been established in the Kaskaskia ecotype and accumulates eYFP (Methods S1). For crossing analyses, a male sterile Re-mutant, Ppccdc39, was used (Meyberg et al., 2020).

If not mentioned otherwise, mosses were grown at 24°C with a cycle of 16 h of light (quantum irradiance of 60-80 µmol m⁻² s⁻¹ light) and 8 h of darkness on plates containing BCDA medium (Cove et al., 2009). Harvested samples were systematically flash-frozen in liquid N₂ and stored at -80°C until further processing. For all comparisons between accessions as well as for the progeny analyses, gametophores were harvested from plants grown for 1 month.

Protonema Reute samples were harvested from a 6-day-old entrained culture grown on solid BCDA medium overlayed with cellophane. Reute juvenile (asexual) gametophore samples were harvested from a 1-month-old culture grown on BCD medium (Cove et al., 2009). Adult Reute gametophore (bearing gametangia) samples were harvested from culture grown for 1 month on BCD medium and subsequently transferred to 15°C with a cycle of 8 h of light (30 µmol m⁻² s⁻¹) and 16 h of darkness for 2 weeks (Hohe et al., 2002). The presence of gametangia was visually confirmed prior to harvesting. The sporophyte samples were obtained from culture grown initially under the adult gametophore regime but watered after the 2 weeks at 15°C to facilitate fertilization. After subsequent growth in the same conditions, green and brown sporophyte samples (Hiss et al., 2017) were collected after 5-7 weeks.

Crossing experiments were performed by coculture of two parental strains as previously described (Perroud et al., 2011). Mature sporophytes (brown) displaying fluorescence developed on a gametophore devoid of fluorescence were manually isolated and

stored at least for a week in the dark at 4°C before performing the germination assay. For each tested sporophyte, 300 to 400 spores were germinated (Perroud et al., 2019) and the predicted 1:1 segregation of the fluorescent marker was confirmed to ensure crossing prior to further work. Then, 10 sporelings were isolated per crossed sporophyte and their tissues were amplified to perform subsequent analyses.

## RNA extraction and cDNA library preparation

RNA extraction and DNase treatment were done using the Maxwell® RSC Plant Kit (Promega, Ref #AS1500) following the manufacturer's instructions. RNA was resuspended in 30 µl of sterile water. We used 200 ng of total RNA for first-strand cDNA synthesis using a 5'-modified oligo-dT primer (Casacuberta et al., 1995) and a specific primer complementary to the PHPAV1 sequence (oxPV63: 5'-CCCACCTCCCTTCACAGACGATC-3') with SuperScript™ III reverse transcriptase (Thermo Fisher). As a control we performed a reaction with the same amount of RNA (200 ng) without adding the SuperScript™ III reverse transcriptase.

## qRT-PCR

Quantitative real-time PCRs were performed in 96-well plates using a Roche LightCycler II instrument. We used SYBR Green I Master Mix (Roche Applied Science) with primers (oxPV64: 5'-CAAAAGCCTATT CCTCTGCATGG-3', oxPV65: 5'-CTGAGAGAAACTGTCCCGTAACT-3') at 1 µM and 40 ng of cDNA obtained from the reverse transcription to conduct the qRT-PCR analysis. Each sample was run in triplicate with negative reverse transcriptase for each sample and negative controls (H₂O). The following PCR conditions were used: 95°C for 5 min, followed by 95°C for 10 sec, 56°C for 10 sec, and 72°C for 10 sec. For normalization we used the relatively highly expressed gene encoding the 60S ribosomal subunit as previously described (Le Bail et al., 2013). The PCR products were purified using the Macherey-Nagel Nucleospin® Gel and PCR cleanup kit (Ref #740609.50) and sequenced.

## ACKNOWLEDGEMENTS

This work was supported by grants from the Spanish Ministerio de Economía, Industria y Competitividad (AGL2016-78992-R/FEDER) and Ministerio de Ciencia e Innovación (PID2019-106374RB-100/AEI/10.13039/501100011033) to JMC. The IJPB benefits from the support of the LabEx Saclay Plant Sciences (SPS) (ANR-10-LABX-0040-SPS). We thank Prof. Mitsuyasu Hasebe for the generous gift of the plasmid p35S-loxP-BSD. We are grateful to Leonie Verhage for providing us with the *P. patens* ecotypes Lviv, Trondheim-K2, and Uppsala-K1. We thank Jean-Luc Gallois and Juan José López-Moya for helpful discussions.

## CONFLICT OF INTEREST

The authors have no conflict of interest to declare.

## DATA AVAILABILITY STATEMENT

All relevant data can be found within the manuscript and its supporting materials. The complete sequence of PHPAV1 can be found at GenBank under accession number BK059361.

## SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article.
**Table S1.** Sequence similarity of the selected contigs with the corresponding viruses.

**Table S2.** Reads mapping to the contigs corresponding to PHPAV1 and TSAMV in a subset of the analyzed gene atlas (Perroud et al., 2018) samples.

**Table S3.** Single-nucleotide polymorphisms detected between the reference PHPAV1 sequence from Reute and the viral reads detected in Kaskaskia.

**Methods S1.** Information regarding the development of the Ka-YFP line.

**Data S1.** Sequence of contig TRINITY_DN26323_c4_g7_i1, which corresponds to PHPAV1.

## REFERENCES

Adkins, S., Kamenova, I., Rosskopf, E. & Lewandowski, D. (2007) Identification and characterization of a novel tobamovirus from tropical soda apple in Florida. *Plant Disease, 91*, 287–293.

Aguado-Garcia, Y., Taboada, B., Morán, P., Rivera-Gutiérrez, X., Serrano-Vázquez, A., Ísa, P. et al. (2020) Tobamoviruses can be frequently present in the oropharynx and gut of infants during their first year of life. *Scientific Reports, 12*, 13595.

Bail, A.L., Scholz, S. & Kost, B. (2013) Evaluation of reference genes for RT qPCR analyses of structure-specific and hormone regulated gene expression in Physcomitrella patens gametophytes. *PLoS One, 8*, e070998.

Balique, F., Lecoq, H., Raoult, H. & Colson, P. (2015) Can plant viruses cross the kingdom border and be pathogenic to humans? *Viruses, 7*, 2074–2098.

Brister, J., Ako-Adjei, D., Bao, Y. & Blinkova, O. (2015) NCBI viral genomes resource. *Nucleic Acids Research, 43*, D571–D577.

Bushnell, B. (2014) *BBMap: a fast, accurate, splice-aware aligner. No. LBNL-7065E*. Berkeley, CA: Ernest Orlando Lawrence Berkeley National Laboratory.

Cantalupo, P. & Pipas, J. (2019) Detecting viral sequences in NGS data. *Current Opinion in Virology, 39*, 41–48.

Capella-Gutiérrez, S., Silla-Martinez, J.M. & Gabaldón, T. (2009) trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. *Bioinformatics, 25*, 1972–1973.

Casacuberta, J.M., Vernhettes, S. & Grandbastien, M.A. (1995) Sequence variability within the tobacco retrotransposon Tnt1 population. *EMBO Journal, 14*, 2670–2678.

Cove, D.J., Perroud, P.F., Charron, A.J., McDaniel, S.F., Khandelwal, A. & Quatrano, R.S. (2009) Culturing the moss Physcomitrella patens. *Cold Spring Harbor Protocols, 4*, pdb.prot5136.

Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S. et al. (2013) STAR: Ultrafast universal RNA-seq aligner. *Bioinformatics, 29*, 15–21.

Elvira-González, L., Medina, V., Rubio, L. & Galipienso, L. (2020) The persistent southern tomato virus modifies miRNA expression without inducing symptoms and cell ultra-structural changes. *European Journal of Plant Pathology, 156*, 615–622.

Geering, A., Maumus, F., Copetti, D., Choisne, N., Zwickl, D. J., Zytnicki, M. et al. (2014) Endogenous florendoviruses are major components of plant genomes and hallmarks of virus evolution. *Nature Communications, 5*, 5269.

Gilbert, K.B., Holcomb, E.E., Allscheid, R.L. & Carrington, J.C. (2019) Hiding in plain sight: new virus genomes discovered via a systematic analysis of fungal public transcriptomes. *PLoS One, 14*, e0219207.

Goh, C., Park, D., Lee, J., Sebastiani, F. & Hahn, Y. (2018) Identification of a novel plant amalgavirus (Amalgavirus, Amalgaviridae) genome sequence in Cistus incanus. *Acta Virologica, 62*, 122–128.

Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J. Z., Thompson, D. A., Amit, I. et al. (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. *Nature Biotechnology, 29*, 644–652.

Haas, F.B., Fernandez-Pozo, N., Meyberg, R., Perroud, P.F., Göttig, M., Stingl, N. et al. (2020) Single nucleotide polymorphism charting of P. patens reveals accumulation of somatic mutations during in vitro culture on the scale of natural variation by selfing. *Frontiers in Plant Science, 11*, 813.

Hiss, M., Meyberg, R., Westermann, J., Haas, F.B., Schneider, L., Schallenberg-Rüdinger, M. et al. (2017) Sexual reproduction, sporophyte development and molecular variation in the model moss Physcomitrella patens: introducing the ecotype Reute. *The Plant Journal, 90*, 606–620.

Hohe, A., Rensing, S., Mildner, M., Lang, D. & Reski, R. (2002) Day length and temperature strongly influence sexual reproduction and expression of a novel MADS-box gene in the Moss Physcomitrella patens. *Plant Biology, 4*, 595–602.

Holland, J., Spindler, K., Horodyski, F., Grabau, E., Nichol, S. & VandePol, S. (1982) Rapid evolution of RNA genomes. *Science, 215*, 1577–1585.

Hühns, S., Bauer, C., Buhlmann, S., Heinze, C., von Bargen, S., Paape, M. et al. (2003) Tomato spotted wilt virus (TSWV) infection of Physcomitrella patens gametophores. *Plant Cell, Tissue and Organ Culture, 75*, 183–187.

Isogai, M., Nakamura, T., Ishii, K., Watanabe, M., Yamagishi, N. & Yoshi- kawa, N. (2011) Histochemical detection of Blueberry latent virus in high- bush blueberry plant. *Journal of General Plant Pathology, 77*, 304–306.

Kamisugi, Y., Whitaker, J.W. & Cuming, A.C. (2016) The transcriptional response to DNA-double-strand breaks in Physcomitrella patens. *PLoS One, 11*(8), e0161204.

Katoh, K. & Standley, D.M. (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. *Molecular Biology and Evolution, 30*, 772–780.

Koloniuk, I., Hrabáková, L. & Petrzik, K. (2015) Molecular characterization of a novel amalgavirus from the entomopathogenic fungus Beauveria bassiana. *Archives of Virology, 160*, 1585–1588.

Krupovic, M., Dolja, V.V. & Koonin, E.V. (2015) Plant viruses of the Amalgaviridae family evolved via recombination between viruses with double-stranded and negative-strand RNA genomes. *Biology Direct, 10*, 1–7.

Lang, D., Ullrich, K.K., Murat, F., Fuchs, J., Jenkins, J., Haas, F. B. et al. (2018) The Physcomitrella patens chromosome-scale assembly reveals moss genome structure and evolution. *The Plant Journal, 93*, 515–533.

Langmead, B. & Salzberg, S. (2013) Bowtie2. *Nature Methods, 9*, 357–359.

Letunic, I. & Bork, P. (2019) Interactive tree of life (iTOL) v4: recent updates and new developments. *Nucleic Acids Research, 47*, W256–W259.

Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N. et al. (2009) The sequence alignment/map format and SAMtools. *Bioinformatics, 25*, 2078–2079.

Martin, R.R., Zhou, J. & Tzanetakis, I.E. (2011) Blueberry latent virus: an amalgam of the Partitiviridae and Totiviridae. *Virus Research, 155*, 175–180.

Maumus, F., Epert, A., Nogué, F. & Blanc, G. (2014) Plant genomes enclose footprints of past infections by giant virus relatives. *Nature Communications, 5*, 4268.

Meyberg, R., Perroud, P.F., Haas, F.B., Schneider, L., Heimerl, T., Renzaglia, K.S. et al. (2020) Characterisation of evolutionarily conserved key players affecting eukaryotic flagellar motility and fertility using a moss model. *New Phytologist, 227*, 440–454.

Mushegian, A., Shipunov, A. & Elena, S. (2016) Changes in the composition of the RNA virome mark evolutionary transitions in green plants. *BMC Biology, 14*, 68.

Naccache, S., Greninger, A., Lee, D., Coffey, L., Phan, T., Rein-Weston, A. et al. (2013) The perils of pathogen discovery: origin of a novel parvovirus-like hybrid genome traced to nucleic acid extraction spin columns. *Journal of Virology, 87*, 11966–11977.

Nibert, M.L., Pyle, J.D. & Firth, A.E. (2016) A +1 ribosomal frameshifting motif prevalent among plant amalgaviruses. *Virology, 498*, 201–208.

Park, D., Goh, C., Kim, H. & Hahn, Y. (2018) Identification of two novel Amalgaviruses in the common Eelgrass (Zostera marina) and in silico analysis of the Amalgavirus +1 programmed ribosomal frameshifting sites. *The Plant Pathology Journal, 34*, 150–156.

Perroud, P.F., Cove, D.J., Quatrano, R.S. & McDaniel, S.F. (2011) An experimental method to facilitate the identification of hybrid sporophytes in the moss Physcomitrella patens using fluorescent tagged lines. *New Phytologist, 191*, 301–306.

Perroud, P.F., Haas, F.B., Hiss, M., Ullrich, K. K., Alboresi, A., Amirebrahimi, M. et al. (2018) The Physcomitrella patens gene atlas project: large-scale RNA-seq based expression data. *The Plant Journal, 95*, 168–182.

Perroud, P.F., Meyberg, R. & Rensing, S.A. (2019) Physcomitrella patens Reute mCherry as a tool for efficient crossing within and between ecotypes. *Plant Biology, 21*, 143–149.

Peyambari, M., Warner, S., Stoler, N., Rainer, D. & Roossinck, M. (2018) A 1,000-year-old RNA virus. *Journal of Virology, 93*, e01188–e1218.
**Table S1.** Sequence similarity of the selected contigs with the corresponding viruses.

**Table S2.** Reads mapping to the contigs corresponding to PHPAV1 and TSAMV in a subset of the analyzed gene atlas (Perroud et al., 2018) samples.

**Table S3.** Single-nucleotide polymorphisms detected between the reference PHPAV1 sequence from Reute and the viral reads detected in Kaskaskia.

**Methods S1.** Information regarding the development of the Ka-YFP line.

**Data S1.** Sequence of contig TRINITY_DN26323_c4_g7_i1, which corresponds to PHPAV1.

## REFERENCES

Adkins, S., Kamenova, I., Rosskopf, E. & Lewandowski, D. (2007) Identification and characterization of a novel tobamovirus from tropical soda apple in Florida. *Plant Disease, 91*, 287–293.

Aguado-Garcia, Y., Taboada, B., Morán, P., Rivera-Gutiérrez, X., Serrano-Vázquez, A., Ísa, P. et al. (2020) Tobamoviruses can be frequently present in the oropharynx and gut of infants during their first year of life. *Scientific Reports, 12*, 13595.

Bail, A.L., Scholz, S. & Kost, B. (2013) Evaluation of reference genes for RT qPCR analyses of structure-specific and hormone regulated gene expression in Physcomitrella patens gametophytes. *PLoS One, 8*, e070998.

Balique, F., Lecoq, H., Raoult, H. & Colson, P. (2015) Can plant viruses cross the kingdom border and be pathogenic to humans? *Viruses, 7*, 2074–2098.

Brister, J., Ako-Adjei, D., Bao, Y. & Blinkova, O. (2015) NCBI viral genomes resource. *Nucleic Acids Research, 43*, D571–D577.

Bushnell, B. (2014) *BBMap: a fast, accurate, splice-aware aligner. No. LBNL-7065E*. Berkeley, CA: Ernest Orlando Lawrence Berkeley National Laboratory.

Cantalupo, P. & Pipas, J. (2019) Detecting viral sequences in NGS data. *Current Opinion in Virology, 39*, 41–48.

Capella-Gutiérrez, S., Silla-Martinez, J.M. & Gabaldón, T. (2009) trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. *Bioinformatics, 25*, 1972–1973.

Casacuberta, J.M., Vernhettes, S. & Grandbastien, M.A. (1995) Sequence variability within the tobacco retrotransposon Tnt1 population. *EMBO Journal, 14*, 2670–2678.

Cove, D.J., Perroud, P.F., Charron, A.J., McDaniel, S.F., Khandelwal, A. & Quatrano, R.S. (2009) Culturing the moss Physcomitrella patens. *Cold Spring Harbor Protocols, 4*, pdb.prot5136.

Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S. et al. (2013) STAR: Ultrafast universal RNA-seq aligner. *Bioinformatics, 29*, 15–21.

Elvira-González, L., Medina, V., Rubio, L. & Galipienso, L. (2020) The persistent southern tomato virus modifies miRNA expression without inducing symptoms and cell ultra-structural changes. *European Journal of Plant Pathology, 156*, 615–622.

Geering, A., Maumus, F., Copetti, D., Choisne, N., Zwickl, D. J., Zytnicki, M. et al. (2014) Endogenous florendoviruses are major components of plant genomes and hallmarks of virus evolution. *Nature Communications, 5*, 5269.

Gilbert, K.B., Holcomb, E.E., Allscheid, R.L. & Carrington, J.C. (2019) Hiding in plain sight: new virus genomes discovered via a systematic analysis of fungal public transcriptomes. *PLoS One, 14*, e0219207.

Goh, C., Park, D., Lee, J., Sebastiani, F. & Hahn, Y. (2018) Identification of a novel plant amalgavirus (Amalgavirus, Amalgaviridae) genome sequence in Cistus incanus. *Acta Virologica, 62*, 122–128.

Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J. Z., Thompson, D. A., Amit, I. et al. (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. *Nature Biotechnology, 29*, 644–652.

Haas, F.B., Fernandez-Pozo, N., Meyberg, R., Perroud, P.F., Göttig, M., Stingl, N. et al. (2020) Single nucleotide polymorphism charting of P. patens reveals accumulation of somatic mutations during in vitro culture on the scale of natural variation by selfing. *Frontiers in Plant Science, 11*, 813.

Hiss, M., Meyberg, R., Westermann, J., Haas, F.B., Schneider, L., Schallenberg-Rüdinger, M. et al. (2017) Sexual reproduction, sporophyte development and molecular variation in the model moss Physcomitrella patens: introducing the ecotype Reute. *The Plant Journal, 90*, 606–620.

Hohe, A., Rensing, S., Mildner, M., Lang, D. & Reski, R. (2002) Day length and temperature strongly influence sexual reproduction and expression of a novel MADS-box gene in the Moss Physcomitrella patens. *Plant Biology, 4*, 595–602.

Holland, J., Spindler, K., Horodyski, F., Grabau, E., Nichol, S. & VandePol, S. (1982) Rapid evolution of RNA genomes. *Science, 215*, 1577–1585.

Hühns, S., Bauer, C., Buhlmann, S., Heinze, C., von Bargen, S., Paape, M. et al. (2003) Tomato spotted wilt virus (TSWV) infection of Physcomitrella patens gametophores. *Plant Cell, Tissue and Organ Culture, 75*, 183–187.

Isogai, M., Nakamura, T., Ishii, K., Watanabe, M., Yamagishi, N. & Yoshi-kawa, N. (2011) Histochemical detection of Blueberry latent virus in high-bush blueberry plant. *Journal of General Plant Pathology, 77*, 304–306.

Kamisugi, Y., Whitaker, J.W. & Cuming, A.C. (2016) The transcriptional response to DNA-double-strand breaks in Physcomitrella patens. *PLoS One, 11*(8), e0161204.

Katoh, K. & Standley, D.M. (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. *Molecular Biology and Evolution, 30*, 772–780.

Koloniuk, I., Hrabáková, L. & Petrzik, K. (2015) Molecular characterization of a novel amalgavirus from the entomopathogenic fungus Beauveria bassiana. *Archives of Virology, 160*, 1585–1588.

Krupovic, M., Dolja, V.V. & Koonin, E.V. (2015) Plant viruses of the Amalgaviridae family evolved via recombination between viruses with double-stranded and negative-strand RNA genomes. *Biology Direct, 10*, 1–7.

Lang, D., Ullrich, K.K., Murat, F., Fuchs, J., Jenkins, J., Haas, F. B. et al. (2018) The Physcomitrella patens chromosome-scale assembly reveals moss genome structure and evolution. *The Plant Journal, 93*, 515–533.

Langmead, B. & Salzberg, S. (2013) Bowtie2. *Nature Methods, 9*, 357–359.

Letunic, I. & Bork, P. (2019) Interactive tree of life (iTOL) v4: recent updates and new developments. *Nucleic Acids Research, 47*, W256–W259.

Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N. et al. (2009) The sequence alignment/map format and SAMtools. *Bioinformatics, 25*, 2078–2079.

Martin, R.R., Zhou, J. & Tzanetakis, I.E. (2011) Blueberry latent virus: an amalgam of the Partitiviridae and Totiviridae. *Virus Research, 155*, 175–180.

Maumus, F., Epert, A., Nogué, F. & Blanc, G. (2014) Plant genomes enclose footprints of past infections by giant virus relatives. *Nature Communications, 5*, 4268.

Meyberg, R., Perroud, P.F., Haas, F.B., Schneider, L., Heimerl, T., Renzaglia, K.S. et al. (2020) Characterisation of evolutionarily conserved key players affecting eukaryotic flagellar motility and fertility using a moss model. *New Phytologist, 227*, 440–454.

Mushegian, A., Shipunov, A. & Elena, S. (2016) Changes in the composition of the RNA virome mark evolutionary transitions in green plants. *BMC Biology, 14*, 68.

Naccache, S., Greninger, A., Lee, D., Coffey, L., Phan, T., Rein-Weston, A. et al. (2013) The perils of pathogen discovery: origin of a novel parvovirus-like hybrid genome traced to nucleic acid extraction spin columns. *Journal of Virology, 87*, 11966–11977.

Nibert, M.L., Pyle, J.D. & Firth, A.E. (2016) A +1 ribosomal frameshifting motif prevalent among plant amalgaviruses. *Virology, 498*, 201–208.

Park, D., Goh, C., Kim, H. & Hahn, Y. (2018) Identification of two novel Amalgaviruses in the common Eelgrass (Zostera marina) and in silico analysis of the Amalgavirus +1 programmed ribosomal frameshifting sites. *The Plant Pathology Journal, 34*, 150–156.

Perroud, P.F., Cove, D.J., Quatrano, R.S. & McDaniel, S.F. (2011) An experimental method to facilitate the identification of hybrid sporophytes in the moss Physcomitrella patens using fluorescent tagged lines. *New Phytologist, 191*, 301–306.

Perroud, P.F., Haas, F.B., Hiss, M., Ullrich, K. K., Alboresi, A., Amirebrahimi, M. et al. (2018) The Physcomitrella patens gene atlas project: large-scale RNA-seq based expression data. *The Plant Journal, 95*, 168–182.

Perroud, P.F., Meyberg, R. & Rensing, S.A. (2019) Physcomitrella patens Reute mCherry as a tool for efficient crossing within and between ecotypes. *Plant Biology, 21*, 143–149.

Peyambari, M., Warner, S., Stoler, N., Rainer, D. & Roossinck, M. (2018) A 1,000-year-old RNA virus. *Journal of Virology, 93*, e01188–e1218.
Price, M.N., Dehal, P.S. & Arkin, A.P. (2010) FastTree 2—approximately maximum-likelihood trees for large alignments. *PLoS One*, 5, e9490.

Rensing, S.A., Goffinet, B., Meyberg, R., Wu, S.Z. & Bezanilla, M. (2020) The moss *Physcomitrium (Physcomitrella) patens*: a model organism for non-seed plants. *Plant Cell*, **32**, 1361–1376.

Roossinck, M. (2015) Plants, viruses and the environment: ecology and mutualism. *Virology*, **479–480**, 271–277.

Sabanadzovic, S., Valverde, R., Brown, J., Martin, R. & Tzanetakis, I. (2009) Southern tomato virus: the link between the families Totiviridae and Par-titiviridae. *Virus Research*, **140**, 130–137.

Safari, M. & Roossinck, M. (2014) How does the genome structure and life-style of a virus affect its population variation? *Current Opinion in Virol-ogy*, **9**, 39–44.

Samarfard, S., McTaggart, A., Sharman, M., Bejerman, N. & Dietzgen, R. (2020) Viromes of ten alfalfa plants in Australia reveal diverse known viruses and a novel RNA virus. *Pathogens*, **9**, 214.

Scholthof, K.-B. (2004) Tobacco mosaic virus: a model system for plant biol-ogy. *Annual Review of Phytopathology*, **42**, 13–34.

Takahashi, H., Fukuhara, T., Kitazawa, H. & Kormelink, R. (2019) Virus latency and the impact on plants. *Frontiers in Microbiology*, **10**, 2764.

Tzanetakis, I., Sabanadzovic, S. & Valverde, R. (2021) Amalgaviruses (Amal-gaviridae). *Encyclopedia of Virology*, **3**, 154–157.

Villamor, D.E.V., Ho, T., Al Rwahnih, M., Martin, R.R. & Tzanetakis, I.E. (2019) High throughput sequencing for plant virus detection and discov-ery. *Phytopathology*, **109**, 716–725.

Yamada, T., Onimatsu, H. & Etten, J.L.V. (2006) Chlorella viruses. *Advances in Virus Research*, **66**, 293–336.

Zhan, B., Cao, M., Wang, K., Wang, X. & Zhou, X. (2019) Detection and char-acterization of Cucumis melo Cryptic Virus, Cucumis melo Amalgavirus 1, and Melon Necrotic Spot Virus in Cucumis melo. *Viruses*, **11**, 81.
Price, M.N., Dehal, P.S. & Arkin, A.P. (2010) FastTree 2—approximately
maximum-likelihood trees for large alignments. *PLoS One*, **5**, e9490.

Rensing, S.A., Goffinet, B., Meyberg, R., Wu, S.Z. & Bezanilla, M. (2020) The moss *Physcomitrium (Physcomitrella) patens*: a model organism for non-seed plants. *Plant Cell*, **32**, 1361–1376.

Roossinck, M. (2015) Plants, viruses and the environment: ecology and mutualism. *Virology*, **479–480**, 271–277.

Sabanadzovic, S., Valverde, R., Brown, J., Martin, R. & Tzanetakis, I. (2009) Southern tomato virus: the link between the families Totiviridae and Par-titiviridae. *Virus Research*, **140**, 130–137.

Safari, M. & Roossinck, M. (2014) How does the genome structure and life-
style of a virus affect its population variation? *Current Opinion in Virol-
ogy*, **9**, 39–44.

Samarfard, S., McTaggart, A., Sharman, M., Bejerman, N. & Dietzgen, R. (2020) Viromes of ten alfalfa plants in Australia reveal diverse known viruses and a novel RNA virus. *Pathogens*, **9**, 214.

Scholthof, K.-B. (2004) Tobacco mosaic virus: a model system for plant biol-
ogy. *Annual Review of Phytopathology*, **42**, 13–34.

Takahashi, H., Fukuhara, T., Kitazawa, H. & Kormelink, R. (2019) Virus latency and the impact on plants. *Frontiers in Microbiology*, **10**, 2764.

Tzanetakis, I., Sabanadzovic, S. & Valverde, R. (2021) Amalgaviruses (Amal-gaviridae). *Encyclopedia of Virology*, **3**, 154–157.

Villamor, D.E.V., Ho, T., Al Rwahnih, M., Martin, R.R. & Tzanetakis, I.E. (2019) High throughput sequencing for plant virus detection and discovery. *Phytopathology*, **109**, 716–725.

Yamada, T., Onimatsu, H. & Etten, J.L.V. (2006) Chlorella viruses. *Advances in Virus Research*, **66**, 293–336.

Zhan, B., Cao, M., Wang, K., Wang, X. & Zhou, X. (2019) Detection and char-
acterization of Cucumis melo Cryptic Virus, Cucumis melo Amalgavirus
1, and Melon Necrotic Spot Virus in Cucumis melo. *Viruses*, **11**, 81.