Introduction
In the past decade the concept of plant viruses as strictly disease-causing entities
has been challenged. While the most well-studied and obvious interactions between
plants and viruses are related to disease, there are several examples of mutualistic
relationships between plants and viruses, both indirect and direct. These mutualistic
interactions have not been fully explored, and many questions remain unanswered. One
problem is the lack of knowledge of plant viruses in nature. Metagenomic surveys have
estimated that only a small fraction of virus species are known. Additionally, globalization
has led to the increased movement of plant material and virus movement. As viruses
move from one area to another, new potential hosts offer the possibility of new interactions,
both negative and positive.
Beneficial plant-virus interactions
Viruses have been associated with plant disease since they were first described in
1898 (Beijerinck, 1898), but in recent years viruses with positive impacts on the
plant hosts they are associated with also have been described. Negative interactions
are mostly studied as disease symptoms such as stunting or necrosis, and the vast
majority of virus research has focused on the disease aspect of these interactions.
Beneficial interactions involve environmental protection to the host plant, protection
against other pathogens, or control of plant responses to nutritional needs (reviewed
in Roossinck, 2011). Plant viruses confer drought and cold tolerance to plants as
conditional mutualists: the plant is harmed by the viruses under normal conditions,
but benefited under extreme conditions. This was demonstrated for several different
viruses and plant hosts (Xu et al., 2008). Mild strains of plant viruses protect plants
from more severe isolates, a phenomenon known as cross-protection (Fraser, 1998) that
led to the initial generation of virus-induced pathogen protection in transgenic plants.
Endogenous pararetroviral elements in plants can confer resistance to exogenous viruses
(Staginnus et al., 2007). The coat protein gene of a persistent virus in white clover
affects the development of nodules under varying nitrogen levels, and this could be
transferred to other legumes (Nakatsukasa-Akune et al., 2005). Curvularia thermal
tolerance virus is a mycovirus that infects a plant fungal endophyte, Curvularia protuberata.
When both virus and fungus are present in hot springs panic grass (Dichanthelium lanuginosum)
the holobiont is able to grow in soil temperatures up to 65°C (Márquez et al., 2007).
Many more examples of mutualistic viruses can be found in other hosts (Roossinck,
2011). In addition, viruses are important in population control of their hosts, and
marine viruses are probably extremely important to the movement of carbon and trace
elements in the microbiome of the oceans (Danovaro et al., 2011).
Plant virus ecology and evolution
The existence of plant-virus mutualistic relationships should not be surprising when
one considers the numerous examples of mutualistic relationships between plants or
animals and other microbes. Despite examples, there has been very little focus on
exploring mutualistic relationships among plants and viruses. Viruses are also involved
in the complex interactions between plants and insects, and can alter insect feeding
behavior, fecundity, and ability to invade new territory (reviewed in Roossinck, 2013).
Further complicating our understanding of plant-virus interactions is the role globalization
has on the relationships between viruses and their plant hosts. Viruses are not stationary,
and their movement geographically and between host species can have drastic effects
on the ecology of a given area. Climate change can alter the behavior of many virus
vectors, promoting the spread of viral distribution across a larger geographic area
(Lebouvier et al., 2011). A prime example of the impact of viruses on plant species
balance is the well-studied beneficial effect the luteoviruses Barley yellow dwarf
virus and Cereal yellow dwarf virus had on the invasive annual grasses in California
grasslands (Malmstrom et al., 2005).
Using the estimation that over 20,000 microbes have invaded the United States (Pimentel
et al., 2005) as a measure of virus movement worldwide, it is reasonable to suggest
that this significant movement of viruses gives the opportunity of a virus jumping
from one plant host species to another, which in turn leads to new plant-virus interactions.
Metagenomic surveys can be useful for nations who are interested in protecting their
crops against invasive diseases (MacDiarmid et al., 2013). A working knowledge of
the geographic location, host range, and potential effects of plant viruses can assist
such nations in developing effective policies that distinguish those viruses that
will have negative economic impacts from viruses which are benign or even beneficial.
Viruses impact the evolution of plants at many levels, and plants clearly affect the
populations of viruses that infect them. There are examples of specific interactions
between plants and viruses, such as the silencing suppression genes found in many
RNA viruses. While not as prevalent as in animals or bacteria, there have been instances
of horizontal gene exchange from viruses to plants. Repeat sequences of geminiviruses
have been found in Nicotiana spp. (Bejarano et al., 1996; Ashby et al., 1997), and
pararetroviruses are frequently found integrated into plants (Hohn et al., 2008).
Sequences from cytoplasmic RNA viruses are found in plant genomes (Liu et al., 2010;
Chiba et al., 2011). There is also evidence that Closterovirus genes have integrated
into the mitochondria of grapevines, Vitis vinifera (Goremykin et al., 2009). Viruses
have been said to be responsible for a large amount of genetic flow in several different
systems (Bock, 2010; Liu et al., 2010; Wu and Zhang, 2011). This in turn would increase
the genetic plasticity of the hosts offering the opportunity for novel interactions
to take place.
We don't know what is out there
In the past decade there have been a few metagenomic type surveys exploring plant
virus biodiversity in wild plants, insects, and a few other environments (Wren et
al., 2006; Roossinck et al., 2010; Ng et al., 2011; Roossinck, 2012). Some of these
studies have used a more ecological approach, “ecogenomics,” that looks at the viral
populations in individuals rather than in the entire environment that is typical of
metagnomic studies (Roossinck et al., 2010). The most surprising result is that we
know very little of the size and diversity of plant virus families. These surveys
have revealed that the true diversity of virus species is much larger than earlier
estimates, with the discovery of new virus isolates, species, families, and even higher
level virus groups (Labonté and Suttle, 2013). An additional surprising result is
that viruses in wild plants do not cause any visible symptoms. With the knowledge
of how little we know of the biodiversity of viruses, new techniques, methods, and
questions need to be developed in order to detect and identify these new viruses.
In addition, the full extent of plant-virus interactions cannot be fully studied until
we have a better understanding of the ecology of plant viruses. While the metagenomic
surveys are a start, there are still many challenges ahead.
The viruses found using metagenomic sequencing data can be described in three different
ways: (1) Known-knowns: virus species or isolates that are already known to be in
the environment being surveyed; (2) Unknown-knowns: new virus species or isolates
of a known family, or known viruses that have not been found previously in the surveyed
environment and; (3) Unknown-unknowns: viruses that are completely novel and share
little to no sequence similarity with other known viruses. Sequencing data for each
instance can be analyzed differently based on the questions being addressed. The removal
of non-viral sequences from the sample either before or after sequencing will, of
course, increase the chances of identifying viruses within a metagenomic sequence
dataset, so care should be taken in both sample preparation before sequencing and
manipulation of sequence data after sequencing. Methods to enrich for plant viral-specific
sequences include isolation of virus-like particles (Muthukumar et al., 2009), enrichment
for double-stranded RNA (Roossinck et al., 2010), and the use of siRNAs (Kreuze et
al., 2009). All of these methods have strengths and weaknesses, but the use of double-stranded
RNA has given the deepest analyses so far. For known-knowns and unknown-knowns, screening
of the sequence dataset for the presence of known viruses can drastically reduce the
amount of time needed for analysis and as such detection and identification of viruses
(Stobbe et al., 2013).
The large amount of sequencing data that shares little or no nucleotide similarity
with known sequences in curated databases such as GenBank suggests that there are
still many unknown microbes that have yet to be described, with unknown-unknown viruses
likely to be prevalent among them. These unknown sequences continue to be difficult
to identify and will require new and novel methods to assign to a taxon. There have
been significant efforts to describe these unknown-unknowns in different environments,
with new analysis methods tailored for virus discovery either by sequence similarity
or by clustering genes (Williamson et al., 2008; Kristensen et al., 2010; Wu et al.,
2010; Ames et al., 2013; Labonté and Suttle, 2013). Protein sequences expressed by
viral-specific genes, such as the RNA dependent RNA polymerase (RdRp), can be used
to detect both unknown-knowns and unknown-unknowns (Kristensen et al., 2010).
Unknown-unknown viruses share little or no sequence similarity with known viruses,
so quality de novo assembly is essential as genome mapping or BLAST assisted assembly
is not an option. Assembly programs tailored for virus de novo assembly have been
created, and modifications to assembly processes have been used to generate fully
assembled genomes of extremely low titer microbes (Albertsen et al., 2013). Additionally,
new exciting sequencing platforms, such as the Oxford Nanopore, offer the ability
to sequence an entire viral genome in a single read (Schneider and Dekker, 2012).
This ability removes the need for assembly altogether. However, even with a perfectly
assembled genome, the need to identify the genome is still there. By clustering the
unknown sequences, the biodiversity of a given environment can be estimated, and this
has led to the discovery of new families of single stranded DNA viruses (Labonté and
Suttle, 2013).
Conclusions
There is still much to be discovered on the topic of plant-microbe interactions, and
of plant-virus interactions in particular. Metagenomics offers us a unique tool to
elucidate the current state of viruses in plants and the role viruses play in these
interactions. When these studies are done on individual plant samples rather than
pooled samples from a larger environment, a system known as “ecogenomics” (Roossinck
et al., 2010), they provide meaningful data for deeper ecological analyses of the
distribution of viruses and potential host- or environmental-specific interactions.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.