Evolutionary and ecological links between plant and fungal viruses
Abstract
| Contents | ||
|---|---|---|
| Summary | 86 | |
| I. | Introduction | 86 |
| II. | Lineages shared by plant and fungal viruses | 87 |
| III. | Virus transmission between plants and fungi | 90 |
| IV. | Additional plant virus families identified in fungi by metagenomics | 91 |
| Acknowledgements | 91 | |
| References | 91 | |
Summary
Plants and microorganisms have been interacting in both positive and negative ways for millions of years. They are also frequently infected with viruses that can have positive or negative impacts. A majority of virus families with members that infect fungi have counterparts that infect plants, and in some cases the phylogenetic analyses of these virus families indicate transmission between the plant and fungal kingdoms. These similarities reflect the host relationships; fungi are evolutionarily more closely related to animals than to plants but share very few viral signatures with animal viruses. The details of several of these interactions are described, and the evolutionary implications of viral cross-kingdom interactions and horizontal gene transfer are proposed.
I. Introduction
Plants and fungi have been interacting for at least 400 Myr and have established complex mutualistic symbiotic relationships that are often critical to the health of both partners (Rodriguez & Redman, 2008; Selosse et al., 2015), as well as antagonistic relationships that comprise a large part of the field of plant pathology. Viruses are very common among members of both kingdoms, and many virus families are shared by plants and fungi. A significant number of these shared virus families have members that are integrated into plant and fungal genomes (Liu et al., 2010; Chiba et al., 2011), implying very ancient relationships. By contrast, although fungi also interact extensively with animals and are closer to animals phylogenetically (within Opisthokonta), very few fungal viruses have been identified that are related to animal viruses (Table 1). Since wild plants are almost always colonized by endophytic, parasitic and mycorrhizal fungi (Rodriguez et al., 2009), there have been ample opportunities for transmission between plants and fungi. Opportunities for transmission between animals and fungi may also occur, but only a few have been described. In one example, an animal-like virus was found in a plant pathogenic fungus (Liu et al., 2009). Overall, more research has been done on plant-interacting fungi, probably because they have a greater impact on humans. Crop plants have both positive and negative interactions with fungi that affect agriculture, while fungal interactions with humans and other animals, other than yeasts, are poorly studied and often fall into the category of neglected tropical diseases.
| ICTV-recognized familiesa | Genome | Other hostsb |
|---|---|---|
| Alfaflexiviridae | (+) ssRNA | Plants |
| Barnaviridae | (+) ssRNA | None |
| Chrysoviridae | dsRNA | Plants |
| Endornaviridae | (+) ssRNA | Plants, oomycetes |
| Gammaflexiviridae | (+) ssRNA | None |
| Hypoviridae | (+) ssRNA | None |
| Megabirnaviridae | dsRNA | None |
| Narnaviridae | (+) ssRNA | Plants, algae |
| Partitiviridae | dsRNA | Plants |
| Quadriviridae | dsRNA | None |
| Reoviridae | dsRNA | Plants, vertebrates, invertebrates |
| Totiviridae | dsRNA | Plants, protists |
| Unclassified familiesc | ||
| Amalgaviridae | dsRNA | Plants |
| Bunyaviridae | (−) ssRNA | Plants, vertebrates |
| Fusariviridae | (+) ssRNA | None |
| Geminiviridae | ssDNA | Plants |
| Hepaviridae | (+) ss RNA | Vertebrates |
| Ourmiavirus d | dsRNA | Plants, invertebrates |
| Rhabdoviridae | (−) ssRNA | Plants, vertebrates |
- ss, single stranded; ds, double-stranded
- a Families recognized as fungal viruses by the International Committee for the Taxonomy of Viruses (ICTV, 2017).
- b Hosts in other kingdoms that are infected by shared virus families. This does not include more distantly related viruses.
- c Fungal viruses have been identified that are related to these families but have not been formally placed; viruses identified only through metagenomics are not included in this table.
- d The ourmiaviruses have not been classified beyond the genus level.
Virus types shared among plants and fungi include those with double-stranded (ds) RNA genomes, single-stranded (ss) RNA genomes of either plus (+) or minus (−) polarity (genome polarity of ssRNA viruses refers to whether the genomic RNA is the coding strand (+), or the anticoding strand (−), and affects the infection process; (−) sense viruses must carry a polymerase in the virus particle) and ssDNA genomes. No reverse-transcribing viruses have been found in extant fungi, although endogenous retroelements are common and suggest a past history with these viruses (Ghabrial et al., 2015; Roossinck & Bazán, 2017). The most common viruses characterized from fungi are those with dsRNA genomes, but recent metagenomic studies are providing evidence of a broader range of virus types (Marzano et al., 2016; Mu et al., 2018).
II. Lineages shared by plant and fungal viruses
1. The mitoviruses
The mitoviruses are members of the family Narnaviridae, some of the simplest viruses known, comprising an RNA genome that expresses only one protein, an RNA-dependent RNA polymerase (RdRp). They are widespread and common in filamentous fungi; for example, a recent virome analysis of the plant pathogenic fungus Sclerotinia sclerotiorum found 25 different mitoviruses in this single fungal species (Mu et al., 2018).
The mitoviruses do not encode a coat protein and are found as naked RNA (Hillman & Cai, 2013). They are unique, in that they infect the mitochondria, although the related narnaviruses infect the cytoplasm of fungi and many invertebrates (Shi et al., 2016). Mitoviruses have some similarity to bacterial leviviruses, with four of the RdRp domains conserved (Esteban et al., 1992), but it is unclear if this involves a true common ancestor or reflects convergent adaptation to a prokaryotic environment. Until recently, viruses that infect the mitochondria had only been found in filamentous fungi. However, many vascular plants contain mitovirus-like sequences in their nuclear genome. Bruenn et al. (2015) searched for mitovirus complete or partial genomes in plant mitochondria and found 175 mitovirus sequences in 90 different plant species, many transcriptionally active. In many cases, mitovirus sequences were also found in the nuclear genome of the same plant species that apparently were transferred from the mitochondria. Mitochondrial–nuclear gene transfers are common (Adams & Palmer, 2003), but the synteny in these regions between the plant mitochondrial and nuclear genomes is high, implying a recent transfer. There is no evidence that any of the nuclear versions of mitovirus sequences are transcriptionally active. Plant mitochondria with mitovirus sequences are found in all groups of vascular plants that have sequenced mitochondrial genomes (Bruenn et al., 2015). The recently published gymnosperm mitogenomes from Ginkgo biloba and Welwitschia mirabilis (Guo et al., 2017) also contained elements of mitoviruses (M. J. Roossinck, unpublished data). The mitochondrial genome of the carnivorous plant Utricularia reniformis contains seven mitovirus-related partial RdRp sequences; three of these are transcribed (Silva et al., 2017). A recent study looking for mitoviruses in plants using transcript analysis found numerous transcripts with complete mitovirus-like genomes in several plant species. Analyses of these transcripts support the idea that they are indeed mitoviruses of plants, rather than sequences integrated into the plant mitochondrial genomes. They were found in a wide range of plants, some of which diverged > 30 Ma (Nibert et al., 2018).
Where did the mitoviruses come from? It was suggested some years ago that they originated in plants and acted as antifungal elements, because many have hypovirulence characteristics in plant pathogenic fungi (Shackelton & Holmes, 2008). On the other hand, an origin in mitochondria may be more plausible given the relationships between the RdRps of mitoviruses and bacteriophage in the Leviviridae family. In addition, all of the integrated plant mitochondrial sequences are very similar, and hence are likely monophyletic (Bruenn et al., 2015). The congruence between the phylogenies of the recently described putative plant mitoviruses and those of the plant hosts implies an ancient origin, but it does not shed light on the ultimate origin of mitoviruses or transmission among plants and fungi (Nibert et al., 2018).
Fungal mitochondria use a different genetic code from plant mitochondria; in particular, UGA codes for tryptophan (Trp), rather than a stop codon, in most fungal mitochondria, and fungal mitoviruses use UGA for Trp as well, whereas the mitovirus sequences in plant mitochondria do not contain the UGA for Trp. Hence, either way the transfer happened, there had to be adaptation for this change. In some fungal mitoviruses there are few or no UGA codons, however, and this correlates with the use of UGA in the host fungal mitochondria, which varies considerably (Nibert, 2017). Given the overall plasticity of RNA genomes, this adaptation does not seem to constitute a large barrier.
2. The ourmiaviruses
The ourmiaviruses were discovered in melons growing in the Urmia region of Iran (Lisa et al., 1988). When the sequence of the genomes of three ourmiaviruses was determined, the putative RdRp had striking similarity to the RdRp of members of the Narnaviridae family. However, the ourmiaviruses have two additional RNAs that encode a movement protein (a protein that facilitates movement of viruses between plant cells by changing the size exclusion limit of the plasmodesmata; Lucas, 2006), most closely related to plant tombusviruses, and a coat protein (CP) distantly related to icosahedral viral CPs of plants and other hosts (Rastgou et al., 2009). Hence, the ourmiaviruses seem to have arisen from a narnavirus that acquired additional coding capacity to function in plants.
The links between the fungal narna- and mitoviruses, with the bacteriophage levivirus and the ourmiaviruses in this lineage, were recently reviewed (Dolja & Koonin, 2018). This lineage includes a number of invertebrate viruses as well. The ancestral base of the tree is in the branch containing the (+) sense RNA phage in the Leviviridae family. The addition of plant mitoviruses does not really affect this analysis, but we can add plants to the mitovirus hosts (Fig. 1).
Based on a newly discovered mitovirus whose RdRp is closer to ourmiaviruses than to other mitoviruses, Hrabáková et al. propose a new group of ourmiamycoviruses that includes several fungal viruses. This study places the mitovirus node basal to the node of the two sister groups containing plant-infecting ourmiaviruses and the fungal ourmiamycoviruses (Hrabáková et al., 2017). Like other mitoviruses, the ourmiamycoviruses are found in the mitochondria and contain only an RdRp gene. Hence, this group may represent an evolutionary intermediate between the fungal or plant mitovirus group and the plant ourmiaviruses that escaped the mitochondria to take up residence in the cytoplasm and acquired the genes for a CP and a movement protein that allows virus movement between plant cells through the plasmodesmata.
3. Persistent viruses
Several families of viruses in plants have persistent lifestyles: they are exclusively transmitted vertically; they do not move between plant cells but are distributed throughout the plants via cell division; they generally have low titres; and they do not have detectable negative impacts on their hosts, and some are clearly beneficial (Roossinck, 2010, 2012a, 2014, 2015). These virus families, Amalgaviridae, Chrysoviridae, Endornaviridae, Partitiviridae and Totiviridae, have numerous members that also infect plant-interacting fungi; indeed, several families were originally identified in fungi (Roossinck, 2015) (Fig. 2). The amalgaviruses were originally described in plants (Liu & Chen, 2009; Sabanadzovic et al., 2009), but they have recently been found in yeast and entomopathogenic fungi (Koloniuk et al., 2015; Depierreux et al., 2016; Pyle et al., 2017). In this case, the link between plant and fungal viruses exists even in an animal-infecting fungus.
The chrysoviruses were first identified in fungi. Their genomes consist of three or four dsRNAs that encode an RdRp, a CP and at least one other protein, called either P3 or p98, of unknown function. The totiviruses encode only an RdRp and a CP and are predominantly characterized from fungi, but they are often found in metagenomic studies of plant viruses (Roossinck, 2012b; Chen et al., 2016), and it seems very likely that they also infect plants in a persistent manner. In some cases, integrated totivirus-like sequences are found in plant genomes (Liu et al., 2010; Chiba et al., 2011). Totiviruses are also found in protists (Goodman et al., 2011) and perhaps in insects (Zhai et al., 2010).
Phylogenetic analyses of the chrysoviruses, the endornaviruses and the partitiviruses show that the viral relationships are not congruent with the host relationships. In all cases, clades are found that have both plant and fungal viruses, implying extensively horizontal transfer between plants and fungi (Roossinck, 2010, 2015; Roossinck et al., 2011).
Like the Narnaviridae, the members of the Endornaviridae family do not have CPs, although they encode a number of other putative proteins. They have been found in plants, fungi and oomycetes. The endornaviruses are ssRNA viruses, with the RdRp most closely related to plant closteroviruses, although they are often referred to as dsRNA viruses because the replicative form is what is found in infected tissues (Roossinck et al., 2011). The endornaviruses were first identified in plants and appear to have slow rates of evolution. For example, there are two related viruses in rice (Oryza sativa alphaendornavirus) and in the progenitor of japonica rice cultivars (Oryza rufipogon alphaendornavirus). These two viruses presumably diverged c. 10 000 yr ago at the time of rice domestication (Molina et al., 2011), and the viral nucleotide sequences have diverged by only c. 24% during that time (Safari & Roossinck, 2018). This rate (c. 2.5 × 10−5 substitutions per year) is much slower than the calculated evolutionary rate for acute ssRNA viruses of 10−3 to 10−4 substitutions per year (Pagán & Holmes, 2010). The low titres of these viruses imply slower rates of replication, which could be responsible for this difference. In addition, in the stable form of the virus the replicative form is double stranded. Single-stranded DNA genomes have higher rates of evolution than ds genomes (Gago et al., 2009), and evolutionary rates of RNA genomes also may differ depending on whether they are ss or ds (Safari & Roossinck, 2014). Although these differences have been attributed to polymerase fidelity, they may also be due to post-transcriptional damage to ss molecules.
The endornavirus genome is a large RNA that encodes a polyprotein of c. 4500 amino acids. A number of domains are found in this protein, but they vary significantly among the viruses in terms of the presence and absence and probable origins. The only domain in common to all is the RdRp at the carboxy terminus (Fig. 3). A phylogenetic analysis of the RdRps of endornaviruses indicates horizontal transfers between plants, fungi and oomycetes (Roossinck et al., 2011).
A metagenomic study of RNA viruses in orchids and their fungal endophytes from Western Australia found three endorna-like viruses that contained two open reading frames. However, their relationships with other endornaviruses and with each other are very distant and may be artefactual (the nucleotide similarities are much higher than the amino acid similarities, a strong indication of artefacts) (Ong et al., 2016).
However the endornavirus genomes arose, they have clearly had numerous interactions between fungi and plants, events that most likely occurred during endophytic interactions. Their occurrence in plant-interacting oomycetes (Hacker et al., 2005) supports their origin in plants. The exchange of material between plant cells and colonizing fungi has been thoroughly studied in the infections of Magnaporthe oryzae during rice blast disease (Kankanala et al., 2009), and it seems clear that both virus particles and large RNA molecules could occasionally be transferred among plants and colonizing fungi.
The partitiviruses are simple dsRNA viruses that infect plants, fungi and protists. Their genomes consist of two RNAs encapsidated in small icosahedral particles and encoding an RdRp and a CP. They are very common in both plants and fungi, although they have been poorly studied, probably because they have not been associated with any disease. Some are clearly beneficial to their plant hosts. For example, white clover cryptic virus suppresses nodulation in legumes when adequate nitrogen is present in the soil (Nakatsukasa-Akune et al., 2005). A number of partitivirus-like sequences have been identified in plant and fungal genomes, and some of these are transcriptionally active (Liu et al., 2010; Chiba et al., 2011). At this time, no plants that have active cytoplasmic partitivirus infections have been found with genomic sequences. This implies that, if the virus is providing a function for the plant and the viral genes are expressed from the plant genome, the cytoplasmic version may no longer be necessary.
Phylogenetic analyses of the RdRp of partitiviruses show different groups; some are specific to plants or to fungi, but at least two groups have a mixture of plants and fungi (Li et al., 2009; Roossinck, 2010; Nibert et al., 2014). However, the CP genes are clearly not monophyletic, and few of the characterized partitivirus CP genes are related, although they group more closely with their hosts than the RdRp genes (M. J. Roossinck, unpublished data). This suggests that the RdRp core gene acquired CP genes at various times and from different sources.
The recent characterization of numerous mitovirus-like entities in a variety of plants may add the Mitoviridae family to the list of plant persistent viruses that share members with fungi (Nibert et al., 2018).
III. Virus transmission between plants and fungi
Although there is ample evidence for horizontal transmission of fungal viruses based on phylogenetics and field studies, experimental transmission has been limited to anastomoses that occur between closely related fungi. Suppression of host self-recognition can increase transmission experimentally (Wu et al., 2017). Recently, new evidence has shown that cross-kingdom virus transmission can occur experimentally and appears to occur in nature.
Looking for potential lichen viruses, a metagenomic study identified a number of sequences related to a plant rhabdovirus, ivy latent cytorhabdovirus, and an ilarvirus, apple mosaic virus (ApMV). Further lichen isolates were tested with an enzyme-linked immunosorbent assay, which detects the viral coat protein, and a number also tested positive for ApMV. Using specific primers, the ApMV CP gene was recovered from six different lichen species found in different locations. The virus could be transmitted mechanically to cucumber (Petrzik et al., 2014). This surprising result indicated that at least one and probably two different plant viruses were infecting the lichen.
Hypothesizing that transmission between plants and fungi is part of the evolutionary history of numerous virus families, Nerva et al. (2017) used purified virus particles from the fungus Penicillium aurantiogriseum that is infected with five different mycoviruses to inoculate plant protoplasts. Two of the viruses, Penicillium aurantiogriseum totivirus 1 and Penicillium aurantiogriseum partitivirus 1, were able to replicate in protoplasts derived from Nicotiana benthamiana and Nicotiana tabacum (Nerva et al., 2017).
The interest in mycoviruses for biotechnology has been fuelled by the discovery of numerous ‘hypoviruses’, viruses that reduce the disease symptoms of plant pathogenic fungi. A screening of the potato pathogen Rhizoctonia solani for viruses uncovered a well-known plant virus, cucumber mosaic virus (CMV). The virus infection was stable during culture of the fungus in the laboratory. Total RNA from infected fungal culture was used to successfully infect N. benthamiana plants by mechanical inoculation. A closely related strain of CMV, Fny-CMV, was used to infect R. solani protoplasts, which were then regenerated and maintained the CMV infection. Finally, an uninfected strain of R. solani was used to colonize CMV-infected plants, followed by recovery of the fungus in culture. The fungus was again stably infected with CMV, indicating that it acquired the virus during the colonization of the plants. Testing the reverse transmission, uninfected potato plants were colonized with virus-infected R. solani. The plants did not develop symptoms typical of CMV infection, but the virus was recovered from plant leaf tissue (Andika et al., 2017). These studies demonstrate that transmission of viruses between plants and fungi can occur and indeed does occur in nature. Transmission of persistent viruses between plants and fungi may occur more frequently than thought. If a virus moves from a fungus to a plant, it can only become established as a plant persistent virus if it infects the germ-line cells and then be vertically transmitted in future plant generations. Hence, most of these transmission events would go unnoticed. However, transmission in the other direction, from a plant to a fungus, may have a better chance of becoming established as a persistent virus in the fungus. Opportunities for transfer are probably ample during fungal colonization of plants, including damage to cell walls that would allow viruses to move. The most detailed studies about transfer of materials across plants and fungi come from the work of Valent and colleagues on the plant pathogen Magnaporthe oryzae, where the fungus grows through the plant plasmodesmata, inducing the formation of complex structures in the plant cells that facilitate transfer of proteins and a variety of effector molecules in both directions (Kankanala et al., 2009).
IV. Additional plant virus families identified in fungi by metagenomics
Our knowledge about fungal viruses is biased towards those with RNA genomes, partly because most of the early discoveries of fungal viruses had RNA genomes, and it became very popular to use dsRNA as a marker for fungal virus infection (Morris & Dodds, 1979). Many families of the characterized fungal viruses have hosts that belong to the plant kingdom (Table 1). In addition to these families, recent metagenomic studies have added other families from the plant virus group: Benyviridae, Ophioviridae, Tymoviridae and Virgaviridae (Marzano et al., 2016; Mu et al., 2018). As the age of discovery continues, undoubtedly there will be more. These close ties between plant and fungal viruses probably reflect the intimate associations that have occurred between the plant and fungal kingdoms since the beginning of terrestrial flora (Selosse et al., 2015). The evidence for horizontal gene transfer among plant and fungal viruses, especially in the Reoviridae family, which may also incorporate domains from reoviruses of other kingdoms (Liu et al., 2012, 2017), reveals further interactions.
Timelines for virus evolution are usually fraught with subjective artefacts, but tools are improving, and the continued accumulation of metagenomic data is changing the whole picture of viromes (Simmonds et al., 2017; Dolja & Koonin, 2018). It is thought that relationships between plants and fungi allowed plants to emerge on land at least 400 Ma (Selosse et al., 2015). The first vertebrate animals did not emerge on land for another 30 Myr, and there is no evidence that fungi were involved in this process. Are the remarkable relationships between plant and fungal viruses reflective of the much longer associations of their hosts? Another common feature of plants and fungi is the cell wall, absent in animals. Acute plant viruses have developed mechanisms to move through the cell wall (Hong & Ju, 2017), whereas persistent plant viruses and fungal viruses, as far as is known, have not. As we gain a new understanding of the evolutionary history of horizontal transfer of viral genes among plants and fungi and place timelines on these events, we may be able to use these events to infer the timelines of plant and fungal interactions.
Acknowledgements
This work was supported by the Huck Institute of Life Sciences and the College of Agricultural Science, Penn State University.
