Allopolyploidy and the evolution of plant virus resistance
BMC Evolutionary Biology
Allopolyploidy and the evolution of plant virus resistance
John Gottula 0
Marc Fuchs 0
0 Department of Plant Pathology and Plant-Microbe Biology, Cornell University, New York State Agricultural Experiment Station , Geneva, NY 14456 , USA
Background: The relationship between allopolyploidy and plant virus resistance is poorly understood. To determine the relationship of plant evolutionary history and basal virus resistance, a panel of Nicotiana species from diverse geographic regions and ploidy levels was assessed for resistance to non-coevolved viruses from the genus Nepovirus, family Secoviridae. The heritability of resistance was tested in a panel of synthetic allopolyploids. Leaves of different positions on each inoculated plant were tested for virus presence and a subset of plants was re-inoculated and assessed for systemic recovery. Results: Depending on the host-virus combination, plants displayed immunity, susceptibility or intermediate levels of resistance. Synthetic allopolyploids showed an incompletely dominant resistance phenotype and manifested systemic recovery. Plant ploidy was weakly negatively correlated with virus resistance in Nicotiana species, but this trend did not hold when synthetic allopolyploids were taken into account. Furthermore, a relationship between resistance and geographical origin was observed. Conclusion: The gradients of resistance and virulence corresponded to a modified matching allele model of resistance. Intermediate resistance responses of allopolyploids corresponded with a model of multi-allelic additive resistance. The variable virus resistance of extant allopolyploids suggested that selection-based mechanisms surpass ploidy with respect to evolution of basal resistance to viruses.
Allopolyploidy; Grapevine fanleaf virus (GFLV); Nepovirus; Nicotiana; Resistance; Susceptibility; Tomato ringspot virus (ToRSV)
The ‘Red Queen Hypothesis’ suggests that coevolution
between hosts and pathogens or pests results in a ‘boom
and bust’ cycle where neither host nor its invader can
gain lasting supremacy . Allopolyploidy could provide
an opportunity for host species to outpace Red Queen
coevolution and achieve epochal gains in resistance such
as when two moderately-resistant diploids give rise to an
allotetraploid with a full complement of resistance genes.
This allopolyploid resistance hypothesis incorporates
resistance into models explaining heterosis [2,3], and has
been tested experimentally in multiple plant and animal
systems [4,5]. Allopolyploidization contributes to 2-4%
of speciation events in Angiosperms .
Viruses have challenged plants for millennia [7-9]. The
genus Nicotiana has been used as a model system for
studying plant-virus interactions and for investigating
genotypic and phenotypic changes that occur at and
after polyploidization . The genus Nicotiana has 76
recognized species, 35 of which are allotetraploids
arising from at least five independent interspecific
allopolyploidization events . The most likely diploid
progenitors of most Nicotiana allopolyploids have been
determined using nuclear and plastid DNA sequence
information [11-14]. While the majority of Nicotiana
allopolyploids retained their original chromosome number,
most species in section Suaveolentes underwent a
reduction in chromosome number. Genomic changes can occur
in the earliest generations following polyploidization
[15-18], and all well-studied Nicotiana allotetraploids
have undergone gene loss or conversion [12,13]. The main
center of diversity for Nicotiana is Bolivia and the natural
range of this genus extends throughout South America, to
the Western US, Australia and Africa . In particular,
N. tabacum and N. rustica likely originated in South
America, N. clevelandii and N. quadrivalvis are endemic
to the Western US, and all but one species of section
Suaveolentes are endemic to Australia .
Plant viruses are commonly characterized by their
experimental host ranges, sometimes incorporating
reactions on Nicotiana species in their descriptions .
The susceptibility status of N. tabacum is known for 541
plant viruses, and at least 29 Nicotiana species have
been used in virus host range studies . Members of
Nicotiana section Suaveolentes (such as N.
benthamiana) tend to have the widest experimental host ranges
[21-23] and N. benthamiana’s multi-pathogen
susceptibility makes it an important tool for phytopathology
research [10,24]. Although the biological basis of Nicotiana
nonhost resistance to viruses is unknown, a mutated form
of RNA-dependent RNA polymerase 1 in N. benthamiana
compromises its broad-spectrum antiviral resistance
response . Several dominant, strain-specific virus
resistance mechanisms have been described in Nicotiana
[26-28], and closely related viruses exhibit differential
capacities for Nicotiana systemic infection [27,28].
Interspecific hybridization can be a useful tool for
transferring resistance genes to crops species and for
investigating virus resistance [19,29,30]. Interspecific
(euploid) hybrids of Solanum tuberosum and S.
brevidens showed quantitative resistance to three diverse
potato viruses compared to S. tuberosum, which exhibited
high virus titers after inoculation . The broad-spectrum
virus resistance was quantitatively enhanced if the hybrid
contained additional copies of the S. brevidens genome or
if the plants were aneuploids missing an S. tuberosum
chromosome . Introgression of an alien chromosome
from N. africana into N. tabacum produced tolerance
(an amelioration of symptoms) to Potato virus Y in
N. tabacum, but did not confer the immunity exhibited by
N. africana per se . These data support the conclusion
that basal virus resistance is quantitatively controlled by
Nepoviruses are nematode-transmitted
polyhedralshaped viruses of the family Secoviridae . These
viruses, including Grapevine fanleaf virus (GFLV) and
Tomato ringspot virus (ToRSV), have single-stranded
bipartite RNA genomes in positive-sense orientation.
GFLV and ToRSV are present in most arable temperate
regions and cause severe economic losses to grapevine
and other crops [34,35]. Based on the distribution of their
highly specific nematode vectors, the likely origins of
GFLV and ToRSV are the Near East and Eastern North
America, respectively [36,37]. N. tabacum exhibits a
recovery reaction after infection of GFLV and ToRSV, and
salicylic acid (SA)-based resistance mechanisms appear
to be critical for recovery from ToRSV [38,39]. RNA
silencing mediates N. tabacum resistance [40,41] and
tolerance [25,42-44] to the nepoviruses Tomato black
ring virus and Tobacco ringspot virus. Although RNA
silencing- and SA-based mechanisms of nepovirus
resistance have been described, no nepovirus resistance
genes have been identified including in well-studied
Vitis spp. , and the diversity and heritability of
nepovirus resistance responses are unknown.
Although experimental work has shed light on the
effect of allopolyploidy on pest resistance [4,5], very little
is currently known about how allopolyploidy could
impact evolution of plant virus resistance. The objective of
this research was to investigate the relationship between
allopolyploidy, geographical origin and genomic bases of
basal antiviral responses in Nicotiana. The
Nicotiananepovirus pathosystem is a logical choice to test basal
(nonspecific) antiviral responses because Nicotiana
species are generally inbreeding , nepovirus strains are
genetically stable  and these plants and viruses have
not coevolved. In this study, we tested the nepovirus
resistance status of Nicotiana and ascertained heritability
using synthetic allopolyploids. We also tested whether
the resistance is local or systemically acquired. The
central hypotheses were that greater or lesser basal
resistance could be explained by geography and ancestry, and
that allopolyploids exhibit greater levels of virus
resistance than diploids.
Test for virus presence
Twenty-four Nicotiana species and synthetic
allopolyploids of distinct geographic origins were evaluated for
their reaction to infection with GFLV strains GHu and
F13, and ToRSV strain AP (Table 1). Since GFLV-GHu
displays levels of virulence intermediate to that of
GFLV-F13 and ToRSV-AP in most Nicotiana species,
plants were primarily assessed for resistance to
GFLVGHu. Each plant-virus combination was sampled at
three or more time points except when a definite
resistance or susceptibility determination could be made
in the first or second apical leaf i.e. for
GFLV-F13inoculated 4×(N. sylvestris × N. tomentosiformis), 4×(N.
glutinosa × N. tabacum), 4×(N. sylvestris × N.otophora)
and 4×(N. rustica × N. tabacum) (sampled once), and
GFLV-GHu-inoculated 4×(N. glutinosa × N. tabacum) and
N. goodspeedii (sampled twice). All panels were surveyed
for virus presence in every plant [populations of four to 32
(median 17) plants], except for GFLV-F13-inoculated
2×(N. tabacum × N. benthamiana), where 23 plants in an
original population of 70 plants was sampled for virus
presence in apical leaves in a stratified sampling approach.
DAS-ELISA was used to determine virus presence or
absence for 2719 GFLV samples and 536 ToRSV samples
in 48 plant-virus combinations. DAS-ELISA absorbance
Table 1 Sources of Nicotiana species and synthetic allopolyploids used in this study
Nicotiana sp. Authority Accessiona Germplasm source PI number Originb
attenuata Torr. ex S. Watson N/A Bureau of Land Management W6 27220
tabacum cv. Xanthi L.
Speg. and Comes N/A
C. Rick, Univ Calif
Botanical Interests, Inc.;
PI number Hybrid accession reference ploidy
L. Burk; Prosser, WA
L. Burk; Prosser, WA
Cameron, UC Berkeley
amphihaploid G.B. Collins; Lexington, KY Kentucky State U.
aN/A: not available.
bSpecific origin of the accession is given, where known. Otherwise, the endemic range of the species according to  is listed.
cNCSU: North Carolina State University.
values had a bimodal distribution, which allowed a clear
delineation of virus-positive from virus-negative
samples. Infection frequencies at each leaf position in each
virus-host sample group were summed to calculate virus
incidence, which was the dependent variable in
correlation analyses. The trajectory of virus incidence among
leaf positions in given host-virus combinations were
evaluated to generate six discrete resistance categories.
Based on the spatial distribution of virus in host plants,
host-virus interactions were labeled ‘susceptible,’ ‘immune’
or one of four categories of recovery (‘early’, ‘intermediate’
and late’), or ‘delayed susceptibility’.
Virus-inoculated plants were checked regularly for
symptoms. The only instances of visible symptoms were for
GFLV-GHu on N. benthamiana, N. clevelandii, N.
goodspeedii and 2×(N. tabacum × N. benthamiana), and for
ToRSV-AP on N. benthamiana and 2×(N. tabacum × N.
benthamiana). GFLV-GHu symptoms on N. benthamiana
SW US Pinyon forest
SW US/ NW Mex
Ibanex Province, Bolivia
Bolivia, Ecuador, Peru
Bolivia, Ecuador or Peru
SW US or NW Mexico
and N. clevelandii were consistent with those previously
described , and included vein clearing on N.
benthamiana and amorphous ring-like mottling on N. clevelandii.
GFLV-GHu symptoms on N. goodspeedii included vein
clearing analogous to that observed for N. benthamiana.
GFLV-GHu symptoms on the 2×(N. tabacum × N.
benthamiana) were composed of non-necrotic
ringspots on the first or second leaf position. ToRSV-AP
symptoms on N. benthamiana were similar to those
previously described , and included stunting, severe
mottling, and necrosis from which the plant ultimately
recovered. ToRSV-AP caused mild mottling and slight
stunting on 2×(N. tabacum × N. benthamiana) and
symptoms were not observed on N. tabacum cv. Xanthi.
Inoculated leaf infection
DAS-ELISA revealed different frequencies of virus
infection in inoculated leaves (Figure 1). Some host-virus
combinations consistently produced absorbance values
below the virus detection threshold, which reflects
immunity or perhaps limited subliminal (single cell) infections.
4×(N. sylvestris × N. tomentosiformis), 4×(N. sylvestris × N.
otophora), 4×(N. glutinosa × N. tabacum) and N.
paniculata exhibited immunity to GFLV-F13 in inoculated leaves.
Some host-virus combinations resulted in less than 50%
inoculated leaf infection including GFLV-GHu-inoculated N.
obtusifolia (13%) and N. glauca (14%), and
GFLV-F13inoculated 4×(N. rustica × N. tabacum) (43%) and
4×(N. glutinosa × N. tabacum) (44%) (Additional file
1: Table S1). All other tested host-virus combinations
produced 50% or greater inoculated leaf infection (Additional
file 1: Table S1). Since GFLV-GHu always produced
infections in inoculated or apical leaves, and ToRSV-AP
inoculations always produced some frequency of infection in the
first apical leaf, there is no immunity within this Nicotiana
panel to these two viruses (Table 2).
Figure 1 Infection frequencies in inoculated and apical leaves of populations of plants tested for resistance to Grapevine fanleaf virus
(GFLV) strains GHu and F13, and Tomato ringspot virus (ToRSV) strain AP. The percent virus incidence is indicated for inoculated and apical
leaves. Asterisks (*) after plant names indicate that the inoculated leaves in the plant-virus combination were not tested.
Wide crosses (synthetic allopolyploids)
4x(N. sylvestris x N. otophora)
4x(N. sylvestris x N. tomentosiformis)
4x(N. glutinosa x N. tabacum)
2x(N. tabacum x N. benthamiana)
4x(N. quadrivalvis x N. tabacum)
4x(N. rustica x N. tabacum)
4x(N. debneyi x N. clevelandii)
aSpecies and synthetic allopolyploids are referenced by their sections within the genus Nicotiana, primary location of origin, and their haploid chromosome
numbers according to .
bCategories of resistance (1, most resistant, through 6, most susceptible) are indicated for each virus-host combination tested.
cBoxes without resistance ratings represent virus-host combination not tested.
High resistance interactions
Virus-host combinations yielding no detectable virus in
inoculated leaves (and apical leaves) were designated as
immune (category 1). Immunity was observed for N.
paniculata, 4×(N. sylvestris × N. tomentosiformis), 4×(N.
sylvestris × N. otophora) and 4×(N. glutinosa × N. tabacum)
inoculated with GFLV-F13 (Figure 1; Table 2).
GFLV-F13inoculated N. tabacum did not produce apical leaf infection,
but whether this plant is immune (category 1) or possesses
early recovery (category 2) to GFLV-F13 is uncertain
because inoculated leaves were not tested. All tested members
of section Tomentosae, N. debneyi and 4×(N. rustica × N.
tabacum) exhibited early recovery (category 2) to
GFLVF13 (Table 2). N. obtusifolia, N. glauca, N. sylvestris, N.
kawakamii, N. tabacum and N. tomentosiformis exhibited early
recovery after GFLV-GHu inoculation. Early recovery was
not observed for these species in response to inoculation
with ToRSV-AP (Table 2).
Moderate resistance interactions
Late recovery (category 3) was the most frequent
hostvirus interaction phenomenon observed in this test
panel, and was seen for all virus isolates tested. All
tested members of section Tomentosae, 4×(N. rustica ×
N. tabacum), N. tabacum and resynthesized
allopolyploids involving possible N. tabacum progenitor species
[4×(N. sylvestris × N. tomentosiformis) and 4×(N.
sylvestris × N. otophora)] showed late recovery to ToRSV-AP
(Table 2). 2×(N. tabacum × N. benthamiana) and 4×(N.
quadrivalvis × N. tabacum) showed late recovery to
GFLV-F13, and 4x(N. quadrivalvis × N. tabacum),
4×(N. sylvestris × N. otophora), 4×(N. sylvestris × N.
tomentosiformis), 4×(N. glutinosa x N. tabacum), N.
glutinosa, N. paniculata and N. setchelii showed late recovery
to GFLV-GHu (Table 2). Intermediate recovery (category
4), characterized by fluctuation of virus incidence
over three or more leaf axes (typically between 33% and
67%, Additional file 1: Table S1), was observed in
GFLVGHu-inoculated N. debneyi, N. rustica, 4×(N. rustica × N.
tabacum) and 2×(N. tabacum × N. benthamiana), and in
GFLV-F13-inoculated 4×(N. debneyi × N. clevelandii)
Low or no resistance interactions
Delayed susceptibility (category 5) was observed only in
response to GFLV-GHu inoculation of N. otophora,
N. suaveolens, and 4×(N. debneyi × N. clevelandii) (Figure 1;
Table 2). Plants were designated as susceptible (category 6)
when 100% of the plants became infected and virus was
present in all tested leaves. N. benthamiana and N.
clevelandii were susceptible to GFLV-F13 and GFLV-GHu,
as expected , N. goodspeedii and N. attenuata were
susceptible to GFLV-GHu, and N. benthamiana and
4×(N. quadrivalvis × N. tabacum) were susceptible to
ToRSV-AP (Figure 1; Table 2).
Additive resistance phenomena in synthetic
Incompletely dominant virus resistance was observed in
synthetic Nicotiana allopolyploids. Whereas N. tabacum
showed high resistance to GFLV-GHu, ToRSV-AP and
GFLV-F13, and N. benthamiana was fully susceptible to
all three virus strains, 2×(N. tabacum × N. benthamiana)
exhibited delayed susceptibility to GFLV-GHu,
intermediate recovery to ToRSV-AP, and late recovery to
GFLV-F13 (Figure 2; Table 2). Whereas N. clevelandii
was fully susceptible to all viruses tested, and N. debneyi
exhibited early recovery to GFLV-F13 and intermediate
recovery to GFLV-GHu, 4×(N. debneyi × N. clevelandii)
exhibited intermediate recovery to GFLV-F13 and
delayed susceptibility to GFLV-GHu (Figure 2, Table 2).
The 4×(N. rustica × N. tabacum) response to GFLV-GHu
was not categorically different than the response of
N. rustica (both category 4), but the synthetic
allopolyploid showed consistently lower incidence of infection in
apical leaves (23-40%) compared to N. rustica (40-80%),
which could reflect the contribution of N. tabacum
(category 2) to resistance (Figure 1; Additional file 1:
Table S1). The intermediate virus resistance observed
across Nicotiana lineages (Figure 3) suggests quantitative
resistance is not due to a single gene with dosage effects,
but due to multiple genes with dosage effects.
Resistance profiles of allopolyploids and their progenitors
We tested two natural allopolyploids (N. clevelandii and
N. tabacum) and the closest relatives of their known
progenitors for GFLV-GHu resistance. The closest extant
diploid progenitors of N. clevelandii are N. obtusifolia
(maternal genome donor) and N. attenuata (paternal
genome donor) . While N. obtusifolia exhibited an
early recovery phenotype (category 2), both N.
clevelandii and N. attenuata showed complete susceptibility
(Figure 1; Table 2). N. tabacum, its representative
maternal genome donor (N. sylvestris), and one possible
representative paternal genome donor (N. tomentosiformis) each
exhibited early recovery (category 2). N. otophora, another
representative of N. tabacum’s possible paternal genome
donors exhibited delayed susceptibility (category 5) to
GFLV-GHu. Resynthesized allopolyploids corresponding
to either N. tabacum ancestry scenario exhibited late
recovery phenotypes (category 3) to GFLV-GHu with low
Figure 2 Effect of synthetic Nicotiana allopolyploids on resistance to Grapevine fanleaf virus (GFLV) strains F13 (left panels) and GHu
(right panels). N. tabacum, N. benthamiana and the 2x(N. tabacum x N. benthamiana) amphihaploid (upper panels); and N. debneyi, N. clevelandii
and 4x(N. debneyi x N. clevelandii) allopolyploid (lower panels) were tested for additive resistance.
Figure 3 Grapevine fanleaf virus strain GHu (GFLV-GHu) resistance categories superimposed on a Nicotiana phylogenetic tree modified
from Clarkson et al.  (curved lines), containing sections (abbreviated in black lettering) with allopolyploid ancestries as established
by Clarkson et al.  and Kelly et al.  (solid straight lines). Shading surrounding sections denote the resistance category of representative
species tested for GFLV-GHu resistance: blue (category 2, early recovery), purple (categories 3 and 4, late or intermediate recovery), or red (categories 5
and 6, delayed or full susceptibility). Representative Nicotiana species (sections) used in this study are N. paniculata (Paniculatae, ‘Pan’), N. rustica (Rusticae,
‘Rus’), N. obtusifolia (Trigonophyllae, ‘Tri’), N. benthamiana, N. debneyi, N. suaveolens and N. goodspeedii (Suaveolentes, ‘Sua’), N. clevelandii (Polydicliae, ‘Pol’),
N. glauca (Noctiflorae, ‘Noc’), N. sylvestris (Sylvestres ‘Syl’), N. tabacum (Nicotiana ‘Nic’), N. glutinosa (Undulatae, ‘Und’) N. attenuata (Petunioides, ‘Pet’),
(Tomentosae, ‘Tom’) including N. kawakamii, N. otophora, N. setchelii and N. tomentosiformis. Members of Tomentosae and Suaveolentes exhibited
different GFLV-GHu resistance profiles and are accordingly dually or triply colored. Descent of synthetic allopolyploids used in this study (white letters)
is indicated by dashed lines: 4x(N. sylvestris x N. tomentosiformis) (‘sxt’), 4x(N. rustica x N. tabacum) (‘rxt’), 4x(N. glutinosa x N. tabacum) (‘gxt’),
2x(N. tabacum x N. benthamiana) (‘txb’), 4x(N. quadrivalvis x N. tabacum) (‘qxt’) and 4x(N. debneyi x N. clevelandii) (‘dxc’).
virus incidence levels (Figure 1; Additional file 1: Table S1).
Thus, N. tabacum exhibits an early recovery
phenotype similar to that of its maternal genome donor and
of N. tomentosiformis, but less resistance than that of
N. otophora or representative resynthesized allopolyploids.
Additionally, species of section Suaveolentes showed low
or occasionally moderate resistance to GFLV-GHu, while
its most closely related proposed paternal genome donor
(N. sylvestris)  showed high resistance (early recovery)
(Table 2). While neoallopolyploids showed intermediate
GFLV-GHu resistance characteristics, extant
allopolyploids did not show intermediate GFLV-GHu resistance
characteristics (Figure 3).
Systemic recovery was tested in apical leaves of
GFLVresistant (categories 1 or 3) synthetic allopolyploids 4×
(N. sylvestris × N. tomentosiformis), 4×(N. glutinosa ×
N. tabacum), and 4×(N. sylvestris × N. otophora) (Table 3).
Resistance was induced with GFLV-GHu or GFLV-F13,
and one upper, apical leaf of each recovered plant was
reinoculated with GFLV-GHu and tested for virus presence.
Notably, plants that showed inoculated leaf
susceptibility to GFLV-GHu lost this susceptibility in the apical leaf
of the recovered plant, no matter whether the resistance
was induced with GFLV-F13 or GFLV-GHu (Table 3).
GFLV-GHu was occasionally detected in the apical
inoculated leaf of GFLV-GHu-recovered plants encompassing
Table 3 Systemic recovery from Grapevine fanleaf virus
(GFLV) strains F13 and GHu
4x(N. sylvestris x
aGFLV-GHu was inoculated to the fourth apical leaf following induction of
resistance (resistance response against the virus in the 1st inoculation
bPlants (n = 9 to 29) were characterized as having systemic recovery if GFLV
was undetectable at five days post-inoculation.
two of 21 plants in 4×(N. sylvestris × N. tomentosiformis)
and one of nine plants in 4×(N. sylvestris × N. otophora).
Of the plants that did not acquire systemic recovery, the
possibility of the originally-inoculated GFLV-GHu infected
these apical leaves cannot be excluded given that late
recovery does not bar the virus from infecting the fourth leaf
position, albeit at a low incidence.
Relationship between host geographic origin and
Australian and North American accessions generally
displayed greater levels of susceptibility than South
American accessions to all virus strains tested (Table 2).
The Australian species N. benthamiana and the North
American species N. clevelandii were fully susceptible to
all viruses tested, and Australian species N. debneyi,
N. suaveolens and N. goodspeedii, and North American
species N. attenuata and N. quadrivalvis displayed lower
levels of resistance than South American species to
GFLVGHu individually or in hybrid backgrounds (Table 2).
Exceptions to these geography-based resistance trends
included the N. debneyi (Australia) early recovery
response to GFLV-F13, the N. obtusifolia (North America)
early recovery response to GFLV-GHu, and the N.
otophora (South America) delayed susceptibility response to
GFLV-GHu. Overall, origin had a significant (P < 0.0001)
and moderate correlation for GFLV-GHu incidence when
hybrids were excluded from the analysis (r = 0.683) and a
weaker correlation (r = 0.5422, P < 0.0001) when hybrids
were included, with South American species showing
greater resistance than Australian species, which in turn
showed greater resistance than species from the Southwest
US. Because the effect of section cannot be separated from
the effect of origin (Table 2), the effect of origin on virus
resistance could reflect phylogenetic factors.
Limited relationship between host ploidy level and
There was a weak association between ploidy level and
virus susceptibility. For example, n = 12 diploids from
section Tomentosae generally displayed greater levels of
resistance than n = 16-24 allopolyploids of section
Suaveolentes, and similar levels of resistance to N. tabacum
and N. rustica (n = 24) (Table 2). The correlation
between GFLV-GHu incidence and chromosome number
was low (r = −0.036) and nonsignificant (P = 0.2597) when
hybrids were included in the analysis, and low (r = −0.286)
but significant (P < 0.0001) when hybrids were excluded,
indicating that increasing ploidy is weakly negatively
related to GFLV-GHu virus incidence among extant
Nicotiana species. These results indicate that increasing ploidy is
correlated with slightly greater virus susceptibility, but that
the trend is abolished when synthetic allopolyploids are
taken into account.
Other trends in virus resistance
Members of section Tomentosae produced higher
inoculated leaf infection rates (75-100%) for GFLV-GHu than
for GFLV-F13 (44-67%) (Additional file 1: Table S1).
Every tested member of section Tomentosae produced
an early recovery phenotype for GFLV-F13 and a late
recovery phenotype for ToRSV-AP (Table 2). Members of
section Tomentosae showed variability in response to
GFLV-GHu, where N. kawakamii and N. tomentosiformis
exhibited early recovery, N. setchelii displayed late
recovery, and N. otophora showed delayed susceptibility
(Table 2). The delayed susceptibility of N. otophora
to GFLV-GHu was masked in the 4×(N. sylvestris ×
N. otophora) synthetic allopolyploid, which reflected the
early recovery of N. sylvestris to GFLV-GHu (Table 2).
Early recovery was also observed for N. tabacum
inoculated with GFLV-GHu, a species believed to have evolved
from a N. sylvestris × N. otophora or N. sylvestris ×
N. tomentosiformis hybridization event . Members of
section Suaveolentes exhibited intermediate or low
resistance to the nepovirus strains tested, except for N. debneyi,
which displayed early recovery after inoculation with
GFLV-F13 (category 2) (Table 2).
N. tabacum and its corresponding resynthesized
allopolyploids [4×(N. sylvestris × N. otophora) and 4×(N.
sylvestris × N. tomentosiformis)] exhibited high or moderate
virus resistance phenotypes for each virus tested (Table 2).
Both resynthesized allopolyploids are immune to
GFLVF13, and N. tabacum also displays high resistance to this
virus. N. tabacum and its resynthesized allopolyploids
showed late recovery to ToRSV-AP, though N. tabacum
frequently had lower frequencies of infection at any given
leaf position than its corresponding neoallopolyploids
(Figure 1; Additional file 1: Table S1). The response of
N. tabacum and the synthetic allopolyploids 4×(N.
sylvestris × N. otophora) and 4×(N. sylvestris × N.
tomentosiformis) to GFLV-GHu were similar in terms of inoculated
leaf infection, but N. tabacum showed early recovery
whereas the neoallopolyploids showed late recovery,
though the overall apical virus incidence levels were
similar (Additional file 1: Table S1). The recovery responses
of N. tabacum to GFLV and ToRSV inoculation confirm
previous reports [38,39].
Synthetic polyploids formed from resistant and
susceptible species frequently displayed resistance in the
moderate categories (Figure 3). 2×(N. tabacum × N.
benthamiana) and 4×(N. debneyi × N. clevelandii)
exhibited intermediate resistance phenotypes after
inoculation with GFLV-GHu and GFLV-F13 compared to their
parents (Figure 2; Table 2). The same was true for the
2×(N. benthamiana × N. tabacum) response to ToRSV-AP
(Table 2). An intermediate level of apical leaf infection was
also seen in the 4×(N. rustica × N. tabacum) response to
GFLV-GHu (Figure 1).
ToRSV-AP typically produced equal or greater
categorical ratings than GFLV-GHu, and GFLV-GHu always
produced equal or higher category ratings than
GFLVF13 (Table 2). An exception to this virulence trend was
that N. otophora and 4×(N. rustica × N. tabacum)
showed lower resistance (higher category ratings) to
GFLV-GHu than to ToRSV-AP (Table 2). Virulence
differences between GFLV-F13 and GFLV-GHu were
highly apparent in synthetic allopolyploid plants with
resistant and susceptible parents, including 2×(N.
tabacum × N. benthamiana), 4×(N. rustica × N. tabacum),
and 4×(N. debneyi x N. clevelandii) (Table 2; Figure 2).
There was a significant (P < 0.0001) but weak (r = 0.406)
correlation between virus composition and infection
frequencies across plant genotypes (species or synthetic
According to individual components of χ2 in the
contingency table that compared observed and expected
virus incidence frequencies for each virus at each leaf
position, there is a higher virus incidence in the first
apical leaf than expected for ToRSV-AP; conversely,
there is less virus incidence in the first apical leaf than
expected for GFLV-F13 (data not shown). Expected
and observed apical virus incidence values are similar
for GFLV-GHu. These results suggest that ToRSV-AP
displays higher virulence and GFLV-F13 displays lower
virulence than GFLV-GHu in this panel of Nicotiana
A spectrum of plant resistance and viral virulence was
observed in the present Nicotiana-nepovirus panel.
Changes in virus incidence were characterized using
DAS-ELISA on multiple leaves of large samples of plants
(Figure 1; Additional file 1: Table S1) and used to distill
six categories of host resistance (immunity, susceptibility,
and four categories of recovery) from which Nicotiana
species, synthetic allopolyploids and viruses were
compared. While all host-virus combinations exhibiting low
leaf inoculation frequencies (<50%) exhibited early
recovery, this phenotype was frequently associated with a high
infection frequency (>50%) in inoculated leaves (Figure 1).
Moderate or high leaf inoculation frequencies (≥50%)
were associated with an entire range of resistance and
susceptibility phenotypes (category 2 through category 6)
(Figure 1). Within individual plant genotypes, ToRSV-AP
generally produced higher susceptibility ratings than
GFLV-GHu, and GFLV-GHu always produced an equal
or greater susceptibility rating than GFLV-F13 (Table 2),
and the correlation between virus identity and virus
incidence ratings were significant. The spectra of quantitative
resistance displayed by Nicotiana accessions and virulence
among nepoviruses suggest the role of multiple interacting
alleles from Nicotiana accessions and nepoviruses in the
determination of the ultimate infection outcomes. Similar
plant genotype by virus genotype interactions were
observed in a panel of 21 Arabidopsis accessions challenged
with three Cucumber mosaic virus isolates .
The full susceptibility seen for 4×(N. quadrivalvis ×
N. tabacum) and delayed susceptibility of the 2×(N.
tabacum × N. benthamiana) responses to ToRSV
raises the interesting possibility that N. quadrivalvis and
N. benthamiana may possess a dominant ToRSV
susceptibility factor in N. tabacum backgrounds. The
observation of ringspot symptoms on the GFLV-GHu-inoculated
2×(N. tabacum x N. benthamiana) amphihaploid
suggests that the vein clearing symptomology typical of
N. benthamiana infection  is a recessive trait. Similarly,
while ToRSV-AP produced necrosis on N. benthamiana,
necrosis was not observed on the 2×(N. tabacum ×
N. benthamiana) amphihaploid or on N. tabacum. The
absence of N. tabacum-ToRSV necrotic ringspot
symptoms was unexpected given previous reports [39,50]. The
lack of hypersensitive responses observed in this host
panel is consistent with the lack of involvement of a
specific gene-for-gene recognition system in Nicotiana-GFLV
and Nicotiana-ToRSV interactions. This lack of
hypersensitive response and the absence of coevolutionary
history between Nicotiana and GFLV or ToRSV
supports the idea that resistance or susceptibility is due to
the interaction of broad-spectrum immune responses
and virulence factors .
Most plants in the host panel used in this study
recovered from virus infection after infection was initially
established in inoculated leaves. Recovery from virus
infection can be controlled by simple or complex host
plant genetics, and can be countered by effective
pathogen virulence factors [30,52,53]. Host plant and
pathogen genotype determined the level of plant recovery to
GFLV (Figure 2). Compatibility between host and viral
components is a prerequisite for infection in the
matching allele model [54,55]. The partial resistance
phenotypes observed in this study do not fit with the strict
bimodality of the matching allele concept. However, a
modified matching allele model that allows for partial
compatibility and limited infection [1,55] (Figure 4)
could explain the range of resistance and virulence
observed in the Nicotiana-nepovirus interactions observed
The intermediate resistance responses of Nicotiana
neoallopolyploids are congruent with the additive
resistance hypothesis proposed by Fritz et al. . By applying
the modified matching allele model to the additive
resistance hypothesis, we theorize that susceptible parents
contribute susceptibility alleles and resistant parents
contribute resistance alleles, and their neoallopolyploids
contain novel combinations of resistance and
susceptibility factors (Figure 4). Neoallopolyploids would possess
Figure 4 Pictographic description of the modified matching allele model applied to the additive resistance hypothesis. Resistant and
susceptible parents (e.g. diploid progenitors of an allopolyploid) carry unique complements of resistance factors (blue) and susceptibility factors
(red). The allopolyploid plant would maintain a mix of resistance and susceptibility factors from each parent (fixed heterozygosity), and also
would be expected to exhibit unique (nonadditive) expression profiles of resistance and susceptibility factors.
a greater number of matching alleles than their more
resistant parent, but the dosage of resistance factors would
be reduced compared to the resistant parent.
Furthermore, non-additive gene expression, which is commonly
observed in allopolyploids and other hybrids [57-59],
could modify expression of resistance and susceptibility
alleles (Figure 4).
While the identities of the Nicotiana’s nepovirus
resistance alleles are unknown, re-inoculation experiments
(Table 3) show that the resistance signal is translocated
to result in systemic recovery. Because the N. tabacum
ToRSV resistance response appears to be SA-mediated
, susceptibility alleles conferred by N. benthamiana
in the 2×(N. tabacum × N. benthamiana) hybrid could
allow ToRSV to quantitatively inhibit SA biosynthesis,
affect conversion of SA to an alternate derivative, or vitiate
downstream SA-activated resistance responses [60,61].
Similarly, null or ineffective RNA silencing alleles present
in susceptible backgrounds could conceivably compromise
RNA silencing-mediated virus resistance in hybrids .
These hypotheses are consistent with Fraser’s model of
virus resistance , which postulates that the effects of
resistance alleles are proportional to their dosage and
levels of influence on resistance pathways.
Although animal allopolyploids frequently show
dominant parasite susceptibility [4,5,63], Nicotiana
neoallopolyploids exhibit virus resistance greater than one but
not both of their parents (Figure 3). In cases where both
parents were either resistant or susceptible, the
neoallopolyploid displayed a resistance response similar to their
parents, and thus there was no inherent penalty or
benefit from hybridization or genome duplication (Figure 3).
Contrary to the model that neoallopolyploid plants could
face a depression of innate immunity , our findings
suggest that allopolyploidization itself did not penalize
Nicotiana for virus resistance.
‘Revolutionary changes’ that accompany polyploidy
can be distinguished from ‘evolutionary changes,’ which
follow allopolyploidization [65,66]. The maintenance of
virus resistance in N. tabacum contrasts with the
apparent loss of virus resistance in section Polydicliae, which
did not maintain partial virus resistance imparted by its
likely maternal genome donor (N. obtusifolia) (Figure 3).
Similarly, members of Suaveolentes exhibited high degrees
of virus susceptibility despite the resistance of their
paternal genome donor’s closest relative (N. sylvestris). Low
virus resistance in sections Polydicliae and Suaveolentes
suggests genetic drift and/or selection conferred a loss of
virus resistance inherited by neoallopolyploids. Nicotiana
neoallopolyploids show gene loss and neofunctionalization
[12,16,18]. Since favorable alleles have a lower chance of
becoming fixed in allopolyploids than diploids , drift
could have resulted in losses of innate immunity alleles in
the Polydicliae and Suaveolentes lineages (Figure 5).
Figure 5 Model of changes in quantitative innate virus resistance from a moderately resistant progenitor exhibiting fixed
heterozygosity for resistance genes (e.g. a neoallopolyploid). Random divergence of the allopolyploid progeny leads to several possible
lineages containing different resistance or susceptibility phenotypes whose existence depends on drift and pathogen pressure. High pathogen
pressure would select for the loss of susceptibility factors and maintenance and gain of resistance factors (trajectory 1, top row). Moderate or
irregular pathogen pressure would maintain an equilibrium of resistance and susceptibility factors within the plant population (trajectory 2,
middle row). Low pathogen pressure would remove the selective advantage of maintaining resistance factors, and could result in the loss of
resistance factors and the maintenance of susceptibility factors (trajectory 3, bottom row).
The correlation of higher levels of virus resistance in
South American Nicotiana species than North American
and Australian species suggest that geographic
influences had a major effect on the efficacy of antiviral
resistance responses. Alternatively, because plant taxon
(section) is frequently inseparable from origin, there is a
possibility that phylogeny rather than origin could
account for virus resistance. In either scenario, long-term
biota-specific interactions would be critical factors to
select for improved virus resistance. Existing virus
resistance alleles could be maintained or enhanced if virus
challengers perennially recur (trajectories 1 or 2), or
virus resistance alleles could be lost if virus challenges
diminish (trajectory 3) (Figure 5). Comparison of several
allopolyploids used in these virus resistance experiments
suggest that N. tabacum, endemic to the relatively large
and competitive biome of the Eastern Andes has
followed trajectory 1, while members of sections
Suaveolentes and Polydicliae, endemic to the relatively isolated
biota of Australia and Southwest US, respectively, have
followed trajectories 2 or 3 (Figure 5). Because N. debneyi
and N. benthamiana are monophyletic , but N. debneyi
shows greater antiviral resistance than N. benthamiana
(Table 2), random or selection-based processes may have
driven divergence of innate immune functions within this
allopolyploid lineage. The sister allopolyploids N.
clevelandii and N. quadrivalvis have similarly diverged for
herbivory resistance responses .
Changes in immune function due to allopolyploidy
could precipitate changes in challenging pathogens, and
prompt a Red Queen-type evolutionary response between
the plant and pathogen . Ineffective innate immune
systems could allow otherwise ill adapted viruses to acquire
more effective virulence factors and erode
quantitative resistance [68,69]. An allopolyploid that can endure
colonization by a pathogen or pest and that permits
pathogen adaptation to an otherwise resistant host progenitor is
referred to as a hybrid bridge . Rather than escaping
virus infection, allopolyploids could furnish another niche
for viruses to expand their host ranges.
The Red Queen Hypothesis explains how resistance and
virulence temporally change in parasitic relationships.
Allopolyploidy might represent an opportunity for plant
hosts to break the Red Queen cycle of coevolution by
gaining a new complement of dominant resistance
factors, but the potential for allopolyploids to experience
an epochal gain in innate immune function may be
compromised by the inheritance of susceptibility alleles or
genetic dysfunctionality caused by hybridization. The
Nicotiana-nepovirus interaction sheds light on potential
dynamics of how allopolyploidy may affect innate
immunity. Based on a detailed survey of the interaction of
non-coevolved plant and virus species, it appears that
host and pathogen genotypes contain multiple alleles that
interact in a quantitative fashion to determine the level of
resistance or susceptibility. Synthetic allopolyploids
faithfully display additive virus resistance characteristics that
correspond to modified matching allele interactions
(Figure 4). Virus resistance/susceptibility factors change
in allopolyploid progeny due to classical drift and
selection (Figure 5). These changes raise the interesting
possibility that moderately resistant allopolyploids could
provide a hybrid bridge, which could result in a new Red
Queen cycle of coevolution.
Seventeen Nicotiana species and seven synthetic
allopolyploids (Table 1) were assayed for nepovirus resistance.
With the exception of 2×(N. tabacum × N. benthamiana),
an infertile amphihaploid, all genetic materials were
selffertile. The synthetic allopolyploids exhibited no
obvious phenotypic segregation. Seeds of 2×(N. tabacum ×
N. benthamiana)  were a gift from Dr. G.B. Collins’s
research program (University of Kentucky, Lexington,
KY). Seeds of N. benthamiana, N. tabacum cv. Xanthi
and N. clevelandii were from Drs. D. Gonsalves and
R. Provvidenti (Cornell University New York State
Agricultural Experiment Station, Geneva, NY). Seeds of
N. rustica, N. glauca, N. glutinosa and N. sylvestris were
obtained from commercial sources (Table 1). All of the
other Nicotiana seeds were provided by the United States
Nicotiana Germplasm Collection maintained at North
Carolina State University (Raleigh, NC). Seedlings were
grown in four-inch pots containing soilless potting
media. Plants were grown in a greenhouse maintained
at 24-26°C supplemented with high pressure sodium
lamps for an 18 hour light/ 8 hour dark photoperiod,
and watered daily or every other day as needed, and
Virus strains and inoculation procedure
GFLV strain F13 from France [72,73] and strain GHu
from Hungary [47,74] were isolated from infected
grapevines, and ToRSV strain AP was isolated from an
infected apricot tree in New York State . GFLV and
ToRSV strains were maintained in N. benthamiana. Virus
inoculum was prepared by mechanically inoculating
N. benthamiana and storing infected tissue at −80°C
until inoculation of the host panel. Infected N.
benthamiana tissue was ground 1:10 (w:v) in inoculation buffer
(15 mM Na2HPO4 and 35 mM KH2PO4 pH 7.0) using a
steel grinding set in a tissue lyser (Qiagen, Valencia, CA)
and inoculated to three corundum-dusted leaves of each
test plant with a ceramic pestle. Panels of four to 32
(median 17) plants per virus-host combination (Additional
file 1: Table S1) were selected for uniformity in size and
mechanically inoculated when they had 4–5 leaves and
were approximately 3 cm in height. All plants were rinsed
with water five to ten minutes after inoculation.
Sampling and virus tests
Apical leaf positions were defined by counting nodes on
the whorl upwards from the highest inoculated leaf.
Apical leaves were collected at time points sufficient
to detect cumulative virus infection: nine to 18 dpi for
position one, 17 to 28 dpi for position two, and 26–
60 dpi for position three. In plants where intermediate
resistance phenotypes were observed, additional
collections were made at 41 to 57 dpi for position four.
Inoculated leaves were collected and processed between 21 and
Plant tissue was collected from inoculated plants
and processed for virus detection via double
antibodysandwich enzyme-linked immunosorbent assay
(DASELISA). Fresh tissue was ground in 1:10 (w:v) in 25 mM
sodium phosphate buffer using a semi-automated HOMEX
6 tissue homogenizer and mesh grinding bags (Bioreba,
Reinach, Switzerland). DAS-ELISAs for GFLV and ToRSV
were carried out in Nunc MaxiSorp® flat-bottom 96 well
polystyrene microtiter plates (Fisher Scientific, Pittsburgh,
PA) according to the manufacturer’s protocol (Bioreba).
Absorbance (OD405nm) was measured after two hours of
substrate incubation using a BioTek Synergy2 plate reader
and Gen5 software was used to calculate blank-subtracted
absorbance (Biotek, Winooski, VT). Each ELISA plate
contained positive and negative checks, and the validity of
each assay was ascertained before data was processed.
Samples were considered positive if their absorbance
values were greater than two times the mean absorbance
values of negative controls.
Evaluation of infection phenotypes
Virus symptoms were monitored daily on inoculated
and apical plant leaves. Leaf samples that were positive
or negative in DAS-ELISA for GFLV or ToRSV in each
inoculation group were counted and converted into
percent infection at each leaf position. Six resistance
categories were assigned based on the infection outcome in
inoculated leaves and in successive apical leaves.
Virushost combinations that yielded no detectable virus in the
inoculated leaf (and apical leaves) were designated as
‘immune’ (category 1). ‘Early recovery’ (category 2) was
defined as any level of inoculated leaf infection (10% to
100%) but the virus was rarely or infrequently (<10%)
detected in the first apical leaf. ‘Late recovery’ (category 3)
was defined at 10% to 100% infection in the first or second
apical leaf position but a decline in virus incidence at
higher leaf axes. ‘Intermediate recovery’ (category 4)
was defined as 20% to 80% infection frequencies in all
leaf axes, and no clear pattern of reduction or expansion
of virus incidence in successively higher axes. ‘Delayed
susceptibility’ (category 5) was defined as a steady
increase in virus incidence at successively higher apical leaf
axes until the highest tested position contained >75%
frequency of virus infection. ‘Full susceptibility’ (category 6)
was defined as virus incidence in 100% of apical leaves.
The inoculated leaf was tested to discern immunity from
Tests for systemic recovery
A subset of the host panel exhibiting recovery from
inoculation with GFLV strains F13 or GHu [4x(N. sylvestris ×
N. tomentosiformis), 4×(N. glutinosa × N. tabacum) and
4×(N. sylvestris × N. otophora)] was re-inoculated with
GFLV-GHu in the fourth leaf position 34 days after the
original inoculation. Re-inoculated leaves were tested for
GFLV incidence at five dpi by DAS-ELISA. Negative values
were interpreted as systemic recovery and positive values
were interpreted as a lack of systemic recovery.
Statistics were computed on JMP version 10.0 (SAS
Institute, Cary, NC). A score of one was assigned for each
leaf infected in the first three apical leaf positions, and the
sum of these scores among the samples at each leaf
position is referred to as virus incidence. Each plant
inoculated with a given virus was considered a replicate.
Contingency analyses were used to compute Pearson’s
correlations (r) and contingency tables. Correlation analyses
were made for species origin (South America, California,
Australia or synthetic), ploidy (x = 12 to 48), and virus
inoculum (GFLV-F13, GFLV-GHu or ToRSV-AP) with
respect to virus incidence at each leaf position. Origin and
virus inoculum was considered as categorical variables,
ploidy as continuous and virus incidence as ordinal data.
A contingency analysis for section was not included due
to the limited instances in which multiple species were
sampled within a section. Correlation analyses were
conducted where synthetic allopolyploids were either included
or excluded in the data set.
Additional file 1: Table S1. Plant responses to Grapevine fanleaf virus
(GFLV) strains F13 and GHu, and Tomato ringspot virus (ToRSV) strain AP. The
data set supporting the results of the article is available in the Dryad Digital
Repository in a Microsoft Word Document, doi:10.5061/dryad.3543v .
The authors declare they have no competing interests.
JG conceived the research, carried out virus inoculations and tests, analyzed the
data and drafted the manuscript. SS and JG performed statistical analyses. RL, SS
and MF critically revised the manuscript. RL provided much of the plant material
and MF participated in the study design. All authors approved the final
We thank the United States Nicotiana Germplasm Collection and researchers
at the University of Kentucky for providing Nicotiana seeds. We thank Larissa
Osterbaan, Drs. John Hart and Christophe Ritzenthaler for helpful discussions,
and Dr. Lisa Earle and Ben Orcheski for improving the manuscript. We appreciate
the technical assistance provided by Yen-Mei Cheung, Pat Marsella-Herrick and
David MacUmber. J. Gottula was supported by a USDA-NIFA-AFRI predoctoral
fellowship, M. Fuchs by USDA-NIFA as well as Nolan and Kaplan Funds, S. Saito
by California Grape and Tree Fruit League and R. Lewis by N.C. State University.
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