The Plant Journal (2014)
doi: 10.1111/tpj.12417
The NIa-Pro protein of Turnip mosaic virus improves growth
and reproduction of the aphid vector, Myzus persicae (green
peach aphid)
Clare L. Casteel1, Chunling Yang2, Ananya C. Nanduri1, Hannah N. De Jong1, Steven A. Whitham2 and Georg Jander1,*
1
Boyce Thompson Institute for Plant Research, Ithaca, NY 14853, USA, and
2
Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA 50011, USA
Received 1 September 2013; revised November 19 2013; accepted 10 December 2013.
*For correspondence (e-mail [email protected]).
SUMMARY
Many plant viruses depend on aphids and other phloem-feeding insects for transmission within and among
host plants. Thus, viruses may promote their own transmission by manipulating plant physiology to attract
aphids and increase aphid reproduction. Consistent with this hypothesis, Myzus persicae (green peach
aphids) prefer to settle on Nicotiana benthamiana infected with Turnip mosaic virus (TuMV) and fecundity
on virus-infected N. benthamiana and Arabidopsis thaliana (Arabidopsis) is higher than on uninfected controls. TuMV infection suppresses callose deposition, an important plant defense, and increases the amount
of free amino acids, the major source of nitrogen for aphids. To investigate the underlying molecular mechanisms of this phenomenon, 10 TuMV genes were over-expressed in plants to determine their effects on
aphid reproduction. Production of a single TuMV protein, nuclear inclusion a-protease domain (NIa-Pro),
increased M. persicae reproduction on both N. benthamiana and Arabidopsis. Similar to the effects that are
observed during TuMV infection, NIa-Pro expression alone increased aphid arrestment, suppressed callose
deposition and increased the abundance of free amino acids. Together, these results suggest a function for
the TuMV NIa-Pro protein in manipulating the physiology of host plants. By attracting aphid vectors
and promoting their reproduction, TuMV may influence plant–aphid interactions to promote its own
transmission.
Keywords: plant–insect interactions, plant defense, mutualism, Myzus persicae, Nicotiana benthamiana,
Arabidopsis thaliana, Turnip mosaic virus, Vector, Pathogen, aphid.
INTRODUCTION
For most plant viruses, transmission by phloem-feeding
insects, such as aphids, is an essential component of the
infection cycle (Ng and Perry, 2004). Thus, aphid feeding
behavior can affect pathogen success, and there is likely to
be strong evolutionary pressure for these viruses to manipulate plant–insect interactions to optimize their own transmission (Sisterson, 2008; Ingwell et al., 2012). Broadly,
insect-mediated virus transmission can be categorized as
persistent, semi-persistent, or non-persistent. Whereas persistent viruses are internalized and pass through the gut
and hemolymph to the salivary glands, semi-persistent
and non-persistent viruses attach transiently to the foregut
or mouthparts, respectively (Gray and Banerjee, 1999;
Blanc et al., 2011). These different transmission modes
suggest that there should also be diverse strategies that
viruses use to manipulate host plants and promote transfer
by insects (Mauck et al., 2012).
© 2013 The Authors
The Plant Journal © 2013 John Wiley & Sons Ltd
Evidence is accumulating that virus infections influence
plant resistance to insect herbivores in both natural and
agricultural systems (Eigenbrode et al., 2002; Belliure
et al., 2005; Mauck et al., 2010, 2012). Virus infection can
alter nutritional composition, defense responses, and other
host plant traits that affect the settling, feeding, and dispersal of insect vectors. In the case of non-persistently
transmitted viruses, which are acquired rapidly during initial aphid probing to reach the phloem (Hoh et al., 2010;
Martiniere et al., 2013), infected host plants are often less
suitable for insect growth than healthy controls (Sisterson,
2008; Bosque-Perez and Eigenbrode, 2011; Mauck et al.,
2012). Thus, it has been proposed that non-persistent
viruses can optimize their transmission by attracting insect
vectors using volatile cues, making the infected plants less
suitable for long-term feeding, and thereby promoting dispersal to new hosts (Eigenbrode et al., 2002; Ngumbi et al.,
1
2 Clare L. Casteel et al.
elevates the free amino acid content of the phloem, promotes aphid settling, and increases aphid reproduction.
Furthermore, the likely role of the viral protein NIa-Pro in
vector–plant ecology was identified by cloning 10 TuMV
genes, expressing each one individually in N. benthamiana, and assessing M. persicae performance and behavior,
and physiological changes in the host plants.
RESULTS
In initial experiments, we compared M. persicae fecundity
on uninfected and virus-infected N. benthamiana plants.
Similar to the findings of previously reported turnip experiments (Hodgson, 1981), infection with both wild type
TuMV and an engineered strain that expressed green fluorescent protein (GFP; TuMV–GFP) (Lellis et al., 2002)
increased M. persicae fecundity (Figure 1). In contrast, two
other viruses, Potato Virus X (PVX), which is not aphidtransmitted, and CMV, which is non-persistently transmitted by M. persicae, did not increase aphid reproduction
(Figure 1).
Association of non-persistent viruses with aphid stylets
is transient and virus attachment is thought to be most efficient immediately after the initiation of aphid feeding
(Mauck et al., 2012). However, reverse transcription polymerase chain reaction (RT-PCR) to detect TuMV RNA in
groups of five aphids (Figure S1a,b) or individual aphids
(Figure S1c) from well established colonies on TuMVinfected N. benthamiana showed that they carry the virus
with high frequency. Among individual aphids, 100% (10
out of 10) were found to be viruliferous by RT-PCR. Efficient transmission by an established aphid colony also is
demonstrated by the fact that 100% of uninfected plants
that were placed adjacent to an established M. persicae
colony on TuMV-infected N. benthamiana were infected
subsequently with TuMV (Figure S2). Given the similar
Aphid Fecundity
(Progeny/adult after 9 days)
2007; Sisterson, 2008; Werner et al., 2009; Mauck et al.,
2010, 2012; Bosque-Perez and Eigenbrode, 2011).
Despite the ecological and agricultural importance of tritrophic interactions between viruses, vectors, and hosts,
few studies have addressed the molecular mechanisms
through which viruses mediate changes in host plant resistance to insect vectors. In the case of the non-persistently
transmitted Cucumber mosaic virus (CMV), the virally
encoded 2b silencing suppressor protein disrupts jasmonate-mediated defense responses in Arabidopsis thaliana
(Arabidopsis; Lewsey et al., 2010). Myzus persicae (green
peach aphid), an important CMV vector, reproduces more
rapidly on CMV-infected tobacco (Nicotiana tabacum) than
on uninfected controls. In addition, plants infected with a
CMV 2b deletion mutant are more aphid resistant, a finding that suggests that this protein is required for enhanced
aphid performance (Ziebell et al., 2011). In another threeway interaction, Tomato yellow leaf curl China virus, a
whitefly-transmitted virus, suppresses plant defenses
using the bC1 protein of the viral beta-satellite DNA,
thereby benefiting the insect vector. (Zhang et al., 2012).
Turnip mosaic virus (TuMV, a member of the Potyviridae) is a monopartite positive-strand RNA virus that
infects hundreds of dicot plant species (Walsh and
Jenner, 2002). TuMV is transmitted by M. persicae and
more than 80 other aphid species (Shattuck, 1992),
making it one of the most damaging viruses for vegetable crops worldwide (Tomlinson, 1987). Unlike most other
non-persistently transmitted viruses, TuMV and Potato
virus Y, both members of the Potyviridae, can make
infected host plants more suitable for M. persicae reproduction (Hodge and Powell, 2008; Kersch-Becker and Thaler, 2013). In wild populations of Arabidopsis, about 20%
of the plants have been found to be infected with TuMV
(Pagan et al., 2010), showing that Arabidopsis is a natural
host for this virus. M. persicae, one of the most important insect vectors for TuMV, readily feeds on Arabidopsis
in greenhouses and in nature (Bush et al., 2006; Harvey
et al., 2007).
Due to its ability to systemically infect two well studied
model plants, Arabidopsis and Nicotiana benthamiana
(Sanchez et al., 1998; Martın Martın et al., 1999; Bombarely
et al., 2012), TuMV has become a model for studying potyvirus–host interactions (Walsh and Jenner, 2002). As with
other potyviruses, TuMV codes for 11 proteins, 10 of which
are translated as a single polyprotein before cleavage by
the P1, HC-Pro, and NIa proteases (Mavankal and Rhoads,
1991; Verchot et al., 1991; Chung et al., 2008). To provide
further insight into the molecular biology of plant–virus–
vector interactions, we investigated how TuMV infection of
Arabidopsis and N. benthamiana affects the reproductive
success of its aphid vector, M. persicae, and the role of
specific viral proteins in vector–plant ecology. Our results
show that TuMV infection suppresses host plant defenses,
16
14
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10
8
6
4
2
0
C
B
A
AD
D
Mock TuMV- TuMV PVX CMV
GFP WT
18
11
16
14
32
Figure 1. Aphids reproduce better on TuMV-infected plants.
Number of progeny produced by a single aphid after 9 days on mock-inoculated or virus-infected N. benthamiana. WT = wild type, GFP = green fluorescent protein, TuMV = Turnip mosaic virus, CMV = Cucumber mosaic
virus, PVX = Potato virus X. Numbers under the X-axis labels indicate sample sizes. A–DDifferent letters indicate significant differences (P < 0.05) by
analysis of variance (ANOVA) and Tukey’s HSD post-hoc test.
© 2013 The Authors
The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12417
Plant–aphid–virus interactions 3
alate aphids were more abundant in colonies on virusinfected N. benthamiana than on control plants
(Figure 2h).
In choice tests, both apterous and alate M. persicae settled on TuMV-infected N. benthamiana in preference to on
uninfected controls (Figure 3). A similar preference for
TuMV-infected plants in complete darkness shows that
visual cues were not involved in host plant choice by apterous aphids (Figure 3). Alate aphids, however, did not differentiate between TuMV-infected and uninfected plants
that were kept in complete darkness (Figure 3), suggesting
either less efficient orientation in the dark or greater use of
visual cues in host plant choice.
To determine whether there is a general suppression of
insect defenses in TuMV-infected plants, e.g. by inhibition
of jasmonate-mediated defenses, we conducted experiments with two chewing herbivores, the crucifer-feeding
specialist Pieris rapae (white cabbage butterfly) and the
generalist Trichoplusia ni (cabbage looper). P. rapae
weight gain on TuMV-inoculated plants was significantly reduced compared with mock-inoculated controls
(Figure 4), this result suggested changes in tissue consumption due to either defense induction or altered
phagostimulant content. T. ni growth, on the other hand,
was not affected significantly (Figure 4).
20
*
15
10
*
10
5
0
5
8
19
0
thaliana
80
benthamiana
80
*
60
*
60
6
40
20
20
0
17
100
100
40
18
12
0
8
11
(e)
(f)
B
100
Aphid Weight (mg)
benthamiana
80
60
A
A
40
20
0
8
11
16
(g)
12
10
8
6
4
2
0
3
2.5
2
1.5
1
0.5
0
*
4
7
(h)
*
22
26
Number of Alates
(b) Nicotiana
Percent Aphid Survival
(c) Arabidopsis (d) Nicotiana
15
thaliana
Aphid Fecundity
(Progeny/adult after 2 days)
Aphid Fecundity
(Progeny/adult after 9 days)
(a) Arabidopsis
Aphid Development
(% adult aphids after 6 days)
effects of wild type TuMV and TuMV–GFP on M. persicae
reproduction (Figure 1), all further experiments to characterize this interaction were conducted with TuMV–GFP.
This approach facilitated tracking of the virus infection and
made it possible to quantify aphid growth on leaves that
were infected, but which did not yet show visible virus
infection symptoms.
The number of progeny produced on TuMV-infected
Arabidopsis and N. benthamiana was significantly higher
than on mock-inoculated plants (Figure 2a,b), and twice as
many aphids were in the adult stage on TuMV-infected
plants after 6 days (Figure 2c,d). Adult aphids transferred
onto N. benthamiana, which is a less suitable host than
Arabidopsis, showed a higher survival rate on TuMVinfected plants than on controls (Figure 2e). Among the
surviving aphids, weight gain was more rapid on virusinfected plants (Figure 2f). Aphids did not have to complete their entire life cycle on TuMV-infected plants to
experience reproductive benefits; adult M. persicae that
moved onto infected N. benthamiana produced more
progeny over 48 h than those that moved onto mockinfected controls (Figure 2g). Alate aphids, which can
move further to new host plants, often are produced in
response to crowding (Debarro, 1992). Consistent with a
more rapid population growth on TuMV-infected plants,
40
30
*
20
10
8
0
10
Figure 2. TuMV infection improves aphid performance on Arabidopsis and N. benthamiana.
Effects of TuMV infection on M. persicae biology. Number of progeny produced by a single aphid after 9 days on TuMV-infected or mock-inoculated Arabidopsis (a), and N. benthamiana (b). The percentage of aphids in the adult stage after 6 days of development on TuMV-infected or mock-inoculated Arabidopsis (c),
and N. benthamiana (d).
(e) Aphid survival after 1 week on healthy, TuMV-infected, or mock-inoculated N. benthamiana.
(f) Weight of aphids after 4 days on N. benthamiana with and without TuMV infection.
(g) Number of progeny produced by a single aphid after 2 days on TuMV-infected or mock-inoculated N. benthamiana.
(h) Number of alates produced on leaves of mock-inoculated or TuMV-infected N. benthamiana after 3 weeks. *P < 0.05, Student’s two-tailed t-test. A,BDifferent
letters on bars indicate significant differences, P < 0.05, by analysis of variance (ANOVA) and Tukey’s HSD post-hoc test. Numbers in bars indicate sample sizes;
mean standard error (SE) for all data.
© 2013 The Authors
The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12417
4 Clare L. Casteel et al.
(a) Light
Aphid Settlement
(% aphids after
24 h)
Apterous
Alate
100
80
*
60
*
40
20
0
(b) Dark
Mock
TuMV
Mock
TuMV
TuMV
Mock
TuMV
Aphid Settlement
(% aphids after
24 h)
100
*
80
60
40
20
0
Mock
Figure 3. Aphids prefer TuMV-infected plants.
M. persicae arrestment on TuMV-infected N. benthamiana growing under
(a) continuous light, or (b) continuous dark conditions. Groups of 10 aphids
were allowed to disperse onto pairs of a mock-inoculated and TuMVinfected plants, and were then counted after 24 h (mean standard error
(SE) of N = 24, *P < 0.05, chi-squared test).
Caterpillar Weight
After 7 days (mg)
10
*
8
6
4
2
29
51
18
19
Mock
TuMV
Mock
TuMV
0
P. rapae
T. ni
Figure 4. Lepidopteran growth is not improved by TuMV.
Weight of P. rapae and T. ni caterpillars feeding on leaves of mock-inoculated or TuMV-infected plants (mean standard error (SE), numbers in bars
indicate sample sizes, *P < 0.05, Student’s two-tailed t-test).
Because TuMV infection enhances aphid fecundity
and development, we investigated whether one or more
individual TuMV proteins can elicit this response through
transient expression in N. benthamiana (Figure 5a; confirmation of gene expression by RT-PCR is shown in Figure
S3). The fecundity of M. persicae was unaffected on plants
that transiently expressed six of the 10 tested TuMV genes,
was decreased significantly by the expression of HC-Pro,
6K1, and VPg, and was increased significantly by NIa-Pro
expression (Figure 5a). Both transient NIa-Pro expression
in N. benthamiana (Figure 5b) and expression in stable
transgenic Arabidopsis (Figure 5c) increased M. persicae
fecundity in a manner similar to that in actual TuMV infec-
tion. The settling preference of apterous aphids on virusinfected plants (Figure 3) also was recapitulated on plants
that expressed NIa-Pro after 1 and 24 h (Figure 5d). In these
experiments, expression of NIa-Pro mRNA in transgenic
plants was comparable with levels of viral RNA in TuMVinfected plants (Figure S4). However, this transcript abundance may not reflect NIa-Pro protein abundance, which
would be influenced by the different translation modes of
the two expression systems and was not quantified in this
experiment. To confirm that actual accumulation of NIa-Pro
protein can influence aphid reproduction, the experiment
was repeated with a FLAG-tagged version of the protein,
which also increases aphid fecundity (Figure S5).
Two likely virus-induced changes in the host plants that
could promote aphid growth are improved nutritional
content and reduced defenses. As aphids primarily take up
nitrogen in the form of free amino acids, we examined the
free amino acid content of mock-inoculated and TuMVinfected leaves of Arabidopsis and N. benthamiana.
Free amino acid content was significantly higher in
virus-infected leaves than in mock-inoculated controls
(Figure 6a). Although most amino acids increased in virusinfected leaves, changes in specific amino acids differed
quantitatively between host plants (Figure S5), and may
reflect underlying differences in the phloem transport of
amino acids in these two species. Free amino acid
content also was increased in NIa-Pro-expressing leaves
(Figures 6b and S6), suggesting that such changes in the
NIa-Pro-expressing plants could improve aphid performance.
To test the alternate hypothesis, that TuMV infection
compromises plant defenses, we measured two well characterized plant responses to aphid feeding: (1) expression
of CYP81F2, which encodes a cytochrome P450 that contributes to 4-methoxyindol-3-yl-methylglucosinolate synthesis and aphid defense in Arabidopsis and other
crucifers (Kim and Jander, 2007; Clay et al., 2009; De Vos
and Jander, 2009; Pfalz et al., 2009); and (2) callose accumulation, which is observed as a response to aphid feeding in many plant species (Dreyer and Campbell, 1987;
Walling, 2000; Botha and Matsiliza, 2004; Meihls et al.,
2013). Consistent with a suppression of aphid-specific
plant defense responses by TuMV, aphid-induced CYP81F2
expression was reduced significantly by TuMV infection
(Figure 7a). We also measured callose accumulation in
TuMV-infected and mock-inoculated plants, with and without M. persicae infestation. Aphid feeding increased callose deposition in both Arabidopsis (Figure 8a,b) and
N. benthamiana (Figure 8c). However, this induction was
reduced significantly in TuMV-infected plants (Figure 8a–
c). CYP81F2 expression and callose deposition also was
measured in NIa-Pro-expressing Arabidopsis leaves. NIaPro expression reduced aphid-induced callose deposition
by more the 50% compared with control Arabidopsis
© 2013 The Authors
The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12417
(a)
2.5
2
1.5
*
0.5
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0
10
22
Nicotiana
benthamiana
C
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BC
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8
A
AB
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*
*
25
10
(c)
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Arabidopsis
thaliana
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28 55 21 22
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(d)
100
Aphid Preference
(% aphids choosing)
1
(b)
Aphid Fecundity
(Progeny/adult after 12 days)
Nicotiana
benthamiana
*
Aphid Fecundity
(Progeny/adult after 9 days)
Relative Aphid Fecundity
(Progeny/ adult after 12 days (+/–SE)
Plant–aphid–virus interactions 5
80
13
22
Nicotiana
benthamiana
1 h 100 24 h
*
80
60
60
40
40
20
20
0
*
24 24
0
24 24
Figure 5. Effects of single TuMV protein expression on M. persicae fecundity.
(a) Relative change in M. persicae fecundity on Agrobacterium-infiltrated N. benthamiana plants that transiently express 10 TuMV proteins from the cauliflower
mosaic virus 35S promoter compared with empty vector (EV) control plants (mean standard error (SE), P < 0.05, Dunnett’s test). Buffer = control plants infiltrated with the MgCl2 infiltration buffer and no Agrobacterium.
(b) Number of progeny produced by aphids on N. benthamiana expressing NIa-Pro, the EV control, TuMV-infected plants and mock-inoculated controls
(mean SE, A–Cletters represent significant differences of analysis of variance (ANOVA) and Tukey’s HSD post-hoc test).
(c) Number of progeny produced by aphids on Arabidopsis expressing NIa-Pro, the EV control, TuMV-infected plants and mock-inoculated controls (mean SE,
A–C
letters represent significant differences by ANOVA and LSD post-hoc tests).
(d) Aphid settling preference after 1 and 24 h for leaves of N. benthamiana expressing NIa-Pro or the EV control (mean SE, *P < 0.05, chi-squared test) under
dark conditions. Numbers in bars represent sample sizes.
(Figure 8a) and was consistent with what was observed in
experiments with the whole virus. However, aphid-induced
expression of CYP81F2 was not inhibited in plants
that expressed NIa-Pro constitutively in Arabidopsis
(Figure 7b).
DISCUSSION
Our results suggest that the NIa-Pro protein contributes to
the improved M. persicae growth and the increased settling that is observed on TuMV-infected plants (Figure 5).
In addition to being the main protease that cleaves the
TuMV polyprotein, NIa-Pro probably has other functions,
including non-specific DNase activity (Anindya and Savithri, 2004; Rajama€ki and Valkonen, 2009). During potyvirus
infection of plant cells, NIa-Pro can be transported to the
nucleus as a fusion with VPg, which is adjacent to NIa-Pro
in the viral polyprotein and has a nuclear localization signal (Schaad et al., 1996). Thus it has been hypothesized
that, once inside the nucleus, NIa-Pro and/or VPg interferes
with host defenses (Riechmann et al., 1992; Anindya
and Savithri, 2004; Beauchemin et al., 2007; Rajama€ki and
Valkonen, 2009). However, as we observed an increase in
aphid performance on plants that expressed NIa-Pro alone
(Figure 5), it is likely that transport to the nucleus is not
required for suppression of plant defenses against aphids.
Aphids and other phloem-feeding insects obtain nitrogen primarily in the form of free amino acids, which constitute only about 2% of the plant nitrogen content (Lam
et al., 2003; Lemaitre et al., 2008). As phloem free amino
acid content closely tracks cytosolic free amino acid content (Riens et al., 1991; Winter et al., 1992; Lohaus et al.,
1994), the measurement of amino acids in whole leaves is
a good proxy for the amino acids that the aphids are able
to acquire from the phloem. In response to both TuMV
infection and NIa-Pro expression, there were significant
increases in foliar free amino acid content (Figure 6).
Although specific changes in individual amino acids differed among host plants and between TuMV and NIa-Pro
expression (Figure S6), the cumulative effect of increased
amino acids may be more important to aphids. Buchnera
aphidicola, obligate bacterial endosymbionts of aphids,
can synthesize essential amino acids that are deficient in
© 2013 The Authors
The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12417
6 Clare L. Casteel et al.
(a)
Free Amino Acids
(pmol/mg of tissue)
Nicotiana
benthamiana
20000
Arabidopsis
thaliana
*
16000
*
12000
8000
4000
0
Nicotiana
benthamiana
Free Amino Acids
(pmol/mg of tissue)
(b)
8000
Arabidopsis
thaliana
*
6000
*
4000
2000
0
Figure 6. TuMV and NIa-Pro effect on amino acid content.
(a) Free amino acid changes in leaves of mock-inoculated or TuMV-infected
plants (mean standard error (SE) of N = 6).
(b) Free amino acid changes in leaves of plants that expressed NIa-Pro or
the empty vector (EV), (mean SE of N = 6) *P < 0.05, Student’s two-tailed
t-test.
(b)
7
B
6
5
4
C
3
2
1
0
A
A
Relative Transcript Abundance
(Fold Change)
Relative Transcript Abundance
(Fold Change)
(a)
7
6
B
5
B
4
3
2
A
A
1
0
Figure 7. CYP81F2 expression is involved in virus enhanced aphid fecundity. Relative CYP81F2 expression was measured by quantitative RT-PCR in
(a) mock-inoculated plants and TuMV-inoculated, with or without aphid
feeding for 48 h and (b) in leaves of plants expressing NIa-Pro or the empty
vector with and without M. persicae feeding for 24 h. Mean standard
error (SE) of N = 4. A–CDifferent letters indicate significant differences by
analysis of variance (ANOVA) and Tukey’s HSD post-hoc test, arbitrary units,
with expression in mock-inoculated plants set to 1.
the aphid diet (Hansen and Moran, 2011; Macdonald et al.,
2012). Although increased phloem amino acid content can
improve the nutrient content of the plant, there must be
additional factors that limit aphid growth. For instance,
increased uptake of amino acids by M. persicae on the
Arabidopsis ant1 amino acid transporter mutant did not
increase aphid reproduction and suggests that available
nitrogen did not limit aphid growth in this experiment
(Hunt et al., 2006).
Virus-mediated changes in plant defense against aphids
are also important for the ecology of this interaction. Callose deposition in response to aphid feeding was reduced
by both TuMV infection and transgenic NIa-Pro expression
(Figure 8). However, although aphid induction of CYP81F2
mRNA was reduced in TuMV-infected plants compared
with wild type, this effect was not observed in Arabidopsis
plants that stably expressed NIa-Pro (Figure 7). Thus,
although we cannot rule out limitations of the experimental design, it is possible that NIa-Pro contributes to
some, but not all, of the plant defense suppression that is
mediated by TuMV.
Despite a decrease in plant defenses and an increase in
free amino acid content, P. rapae grows less well on TuMVinfected Arabidopsis. However, P. rapae is a crucifer-feeding specialist that has co-opted glucosinolates and perhaps
other plant defenses as attractive signals, and caterpillars
grow less well on Arabidopsis plants in which glucosinolate-mediated defenses are inhibited (Barth and Jander,
2006). Thus, if crucifer-specific defenses are reduced in
TuMV-infected plants, P. rapae might grow less well.
However, it should be noted that CYP81F2, which catalyzes the conversion of indole-3-yl-methylglucosinolate
to 4-hydroxy-indole-3-yl-methylglucosinolate to provide
defense against M. persicae (Kim and Jander, 2007; Pfalz
et al., 2011), does not affect resistance against lepidopteran
herbivores (Pfalz et al., 2009). Free amino acids, which constitute only a small portion of the total nitrogen in plant
leaves (Lam et al., 2003; Lemaitre et al., 2008), are likely to
be less important for caterpillars that consume the entire
leaf than for aphids that feed specifically from the phloem.
Most viruses express proteins that inhibit one or more
components of RNA silencing, a common anti-viral
defense mechanisms in plants and other eukaryotes
(Csorba et al., 2009; Fraile and Garcıa-Arenal, 2010). Previous studies with CMV, a non-persistently transmitted plant
virus, showed that infection increased M. persicae survival
on tobacco (Lewsey et al., 2010). Whereas the CMV 2b
silencing suppressor protein was implicated in enhancing
aphid performance (Ziebell et al., 2011), TuMV HC-Pro
reduced aphid reproduction (Figure 5). These differing
effects could be the result of functional differences
between the two proteins. Whereas CMV 2b has a dual
mode of silencing inhibition, sequestering siRNAs and
interacting with AGO1 to prevent RISC (RNA-induced
© 2013 The Authors
The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12417
Plant–aphid–virus interactions 7
(a)
400
Callose spots/mm2
D
Arabidopsis thaliana
300
B
200
C
C
A
100
A
A
A
0
Mock
Mock
Aphid
TuMV
TuMV
Aphid
EV
EV Aphid NIa-Pro
NIa-Pro
Aphid
(c)
(b)
Mock
Mock
Aphid
silencing complex) assembly, HC-Pro only prevents RISC
assembly (Roth et al., 2004; Csorba et al., 2009; Burgyan
and Havelda, 2011). Our observation of increased aphid
resistance in plants that express TuMV HC-Pro (Figure 5) is
consistent with a previous report showing that plants that
express Tobacco mosaic virus HC-Pro are significantly
more resistant to multiple unrelated pathogens (Pruss
et al., 2004).
TuMV is somewhat unusual among non-persistently
transmitted viruses, which generally make plants less suitable as feeding sites for their aphid vectors (reviewed by
Mauck et al., 2012). Aphids that land on a plant rapidly
acquire non-persistently transmitted viruses during their
initial probing to reach the phloem (Hoh et al., 2010;
Martiniere et al., 2013) and, if the feeding site is not suitable, move elsewhere and transmit the viruses to a new
host. Our observation that M. persicae from well established colonies on TuMV-infected plants are viruliferous
(Figure S1) and transmit viruses to neighboring plants (Figure S2) suggests that TuMV, unlike many other non-persistently transmitted viruses (Sisterson, 2008; Mauck et al.,
2012), is acquired efficiently not only during initial aphid
probing to reach the phloem but also during stable feeding. This difference in virus uptake may make it advantageous for TuMV, to promote long-term feeding on host
plants and thereby a more rapid increase in the aphid population. Future research will determine whether other potyviruses, e.g. Potato virus Y (Kersch-Becker and Thaler,
2013), follow a similar strategy in promoting aphid growth
by means of the NIa-Pro protein.
M. persicae behavior on TuMV-infected plants may influence the dynamics of virus transmission in the field. It was
TuMV
TuMV
Aphid
Callose spots/mm2
Figure 8. TuMV and NIa-Pro reduce callose
accumulation.
(a) Callose deposition in Arabidopsis plants that
were mock-inoculated, TuMV-infected, expressing NIa-Pro, or transformed with the empty vector (EV), with and without M. persicae
infestation (mean standard error (SE) of
N = 4–6, A–Ddifferent letters indicate significant
differences by analysis of variance (ANOVA) and
Tukey HSD post-hoc test).
(b) Images of Arabidopsis callose deposition,
detected by aniline blue staining.
(c) Callose deposition in mock-inoculated and
TuMV-infected N. benthamiana, with and without M. persicae infestation (mean SE of
N = 4–5, A,Bdifferent letters indicate significant
differences by ANOVA and Tukey’s HSD post-hoc
test).
120
B
100
Nicotiana
benthamiana
80
60
A
40
A
A
20
0
Mock
Aphid
TuMV TuMV
Aphid
recently demonstrated that altered volatile profiles can
attract aphids to CMV-infected plants (Mauck et al., 2010).
Similarly, our results show that M. persicae prefer to settle
on both TuMV-infected and NIa-Pro-producing plants
(Figures 3 and 5d). The absence of host plant preference
by alate aphids in the dark (Figure 3) may indicate greater
use of visual cues by alate than by apterous M. persicae.
Because TuMV-infected plants are more suitable for aphid
reproduction (Figure 1), more aphids will be available for
virus transmission, including larger numbers of alates (Figure 2h) that facilitate more widespread dispersal (Debarro,
1992). Thus, by promoting aphid settling and reproduction,
TuMV probably also benefits itself by making more aphids
available for subsequent virus transmission to new host
plants.
Together, our results provide insight into the molecular
mechanisms that underlie an important ecological interaction. By demonstrating increased M. persicae growth on
TuMV-infected plants and showing that this phenotype can
be recapitulated by expression of a single virus protein,
NIa-Pro, we provide evidence that TuMV infection alters
plant defense against aphids. As potyviruses are among
the most widely distributed plant viruses, and plants in
natural settings are frequently infected, virus-mediated
suppression of plant defenses may play an important role
in plant–aphid interactions.
EXPERIMENTAL PROCEDURES
Plants and growth conditions
N. benthamiana seeds were obtained from Peter Moffett (Universite de Sherbrooke, Quebec, Canada). Arabidopsis seeds were
© 2013 The Authors
The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12417
8 Clare L. Casteel et al.
obtained from the Arabidopsis Biological Resource Center
(www.arabidopsis.org). All plants were grown in Cornell mix [by
weight 56% peat moss, 35% vermiculite, 4% lime, 4% Osmocoat
slow-release fertilizer (Scotts, Marysville, OH, USA, http://
www.scotts.com), and 1% Unimix (Scotts)] in 20 9 40-cm nursery
flats in Conviron growth chambers. The light intensity in the
growth chambers was 200 mmol m 2 sec 1 at 23°C with 50% relative humidity and a 16 h:8 h day:night cycle. The same growth
conditions were used in all subsequent experiments. Plants used
for all experiments were 1 month old, unless otherwise noted. All
experiments were conducted at least two times, with varying
numbers of biological replicates per treatment per experiment.
TuMV infection
TuMV–GFP was propagated from infectious clone p35TuMVGFP
(Lellis et al., 2002). To prepare inoculum, TuMV–GFP-infected
N. benthamiana leaves were weighed, ground in four volumes of
20 mM sodium phosphate buffer (pH 7.2), filtered through Organza
Mesh cloth, and frozen in aliquots at 80°C. For inoculations, one
leaf from each plant was dusted with carborundum (Sigma, St.
Louis, MO, USA, http://www.sigmaaldrich.com) and rub-inoculated with TuMV–GFP leaf extract using a cotton-stick applicator.
A corresponding set of control plants was dusted with carborundum and mock-inoculated with a cotton-stick applicator that was
soaked in uninfected N. benthamiana leaf extract, prepared in the
same manner as the virus-infected extract. One week after inoculation, an ultraviolet (UV) light lamp (UV Products, Upland, CA,
Blak Ray model B 100AP) was used to identify fully infected leaves
that contained the TuMV–GFP virus. All plants were 2–3 weeks old
at the time of infection. Inoculations with other viruses (wild type
TuMV, CMV, and PVX) were performed with the same methods.
Insects
Aphid experiments were conducted with a tobacco-adapted red
strain of M. persicae that was obtained from Stewart Gray (USDA
Biological Integrated Pest Management Research Laboratory,
Ithaca, NY, USA, http://www.ars.usda.gov). Aphids were reared on
tobacco (Nicotiana tabacum) with a 16 h/8 h photoperiod at 24°C
/19°C (150 mmol m 2 sec 1; 50% relative humidity). P. rapae were
from a colony maintained by the Jander laboratory, which is descended from 20 insects collected in the wild on the Cornell University campus in July, 2008. Trichoplusia ni eggs were purchased
from Benzon Research (Carlisle, PA, USA, http://www.benzon
research.com).
Virus transmission studies
Two groups of five M. persicae individuals (apterous and alates,
separately) were fed on N. benthamiana N. benthamiana infected
with wild type TuMV for 1 week. RNA was extracted from pooled
aphid tissue and cDNA was synthesized. The presence of TuMV
was verified by RT-PCR with primers for the NIa-Pro gene. In a separate experiment, wild-type TuMV-infected N. benthamiana plants
were each infested with 10 apterous aphids. After 48 h, five aphids
per plant were collected and the viruliferous status was verified as
above. To assess infection frequency in individual aphids, aphids
were fed on N. benthamiana infected with wild-type TuMV and
uninfected control plants. After 48 h, 10 single aphids were
collected from infected plants and the viruliferous status was verified by RT-PCR as above. Control aphids were collected from
N. benthamiana plants that were not infected with TuMV.
To assess transmission to neighboring plants, aphid colonies
were established on N. benthamiana that had been infected with
wild type TuMV for 2 weeks. Three days later, when the aphids
had become established, two plants that were infected with TuMV
and infested with aphids, were placed in an arena surrounded by
eight healthy plants. Aphids from the viruliferous colonies were
allowed to move freely among plants for 2 weeks. At the end of
the experiment the apical leaves of the healthy plants were collected for infection verification. RNA was extracted from plant tissue and cDNA synthesized as below. TuMV infection was verified
by RT-PCR with primers for the NIa-Pro gene (Table S1).
Aphid fecundity, development and alate bioassays
To assess the effect of TuMV infection on aphid fecundity, one
apterous adult aphid was placed in a 1.5 cm diameter clip cage on
a fully infected or mock-inoculated N. benthamiana or Arabidopsis
leaf. Infection is not uniform in the host plant, thus only fully
infected leaves were used in subsequent experiments. After 24 h,
all aphids except one nymph were removed. The single nymph
was allowed to develop and progeny were counted after
9–12 days (depending on the experiment) to determine fecundity.
To measure aphid development rate, cages with aphids were set
up as in the fecundity experiments, but five apterous adults were
added to each cage. After 24 h, all insects, except 10 nymphs,
were removed from each cage. After 6 days, the number of aphids
in the adult stage was recorded in each cage. For measuring alate
production, a single nymph was allowed to develop and reproduce on mock- and TuMV-inoculated leaves. After 3 weeks, the
number of alates in the colony was counted. Each complete experiment was repeated 2–3 times with at least 10 repetitions (cages)
per treatment.
Aphid and caterpillar weight gain bioassays
Groups of 10 1-day-old adult aphids were confined on the leaves
of 3-week-old TuMV- or mock-inoculated plants in 1.5 cm diameter clip cages. After 4 days, five adults from each plant were collected. For experiments with P. rapae and T. ni, neonate
caterpillars were confined on the leaves of 3-week-old TuMV- or
mock-inoculated plants using 100 ml volume mesh-covered cups.
Caterpillars were allowed to feed on plants for 7 days before harvesting. Aphids and caterpillars were lyophilized and dry weight
was determined using a precision balance.
Aphid preference bioassays
For aphid preference experiments, two N. benthamiana plants (a
TuMV-infected and a mock-inoculated plant) were placed in a
30 cm diameter experimental arena, with their positions in the
arena randomly assigned. In one experiment, a microcentrifuge
tube containing 10 apterous aphids was placed at the height of
the base of the plants (10 cm) equidistant from the two test plants.
After 1 h or 24 h, the number of aphids on each plant was
counted. This study was conducted on two consecutive days with
12 replications per day. Another experiment was performed as
above, but with 10 alate aphids in each microcentrifuge tube. This
complete set of experiments was performed under constant light
in one set of experiments and under constant dark in another set
of experiments.
Amino acid assays
For analysis of leaf free amino acids, ~100 mg of plant tissue was
collected, weighed, placed in 2 ml microcentrifuge tubes with two
3-mm steel beads, and frozen in liquid nitrogen. Tissue was
ground to fine powder using a Harbil model 5G-HD paint shaker
© 2013 The Authors
The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12417
Plant–aphid–virus interactions 9
(Fluid Management, Wheeling, IL, USA, http://www.fluidman.
com). Ground tissue was extracted with 20 mM HCl (10 ll mg 1 of
tissue), the extracts were centrifuged (3800 g for 20 min at RT),
and the supernatant was saved for analysis.
Amino acids were derivatized using an AccQ-Fluor reagent kit
(Waters, Milford, MA, USA, http://www.waters.com). For derivatization, 2.5 ll extracts were mixed with 17.5 ll borate buffer, and
the reaction was initiated by the addition of 5 ll 6-aminoquinolylN-hydroxysuccinimidyl carbamate reagent, followed by immediate mixing and incubation for 10 min at 55°C. L-Norleucine was
used as an internal standard. From each sample, 10 ll were
injected onto a Nova-Pak C18 column using a Waters 2695 pump
system, and the data were recorded using Waters Empower Software. Amino acid derivatives were detected using a Waters model
2475 fluorescence detector with an excitation wavelength of
250 nm and an emission wavelength of 395 nm. Solvent A, AccQTag Eluent A, was premixed from Waters; Solvent B was acetonitrile:water (60:40). The gradient used was 0–0.01 min, 100% A;
0.01–0.5 min, linear gradient to 3% B; 0.5–12 min, linear gradient
to 5% B; 12–15 min, linear gradient to 8% B; 15–45 min, 35% B;
45–49 min, linear gradient to 35% B; 50–60 min, 100% B. The flow
rate was 1.0 ml min 1.
Callose staining
Leaves were collected from host plants 24 h after infestation with
25 M. persicae adults. For virus experiments, aphids were added
to 4-week-old plants, 7 days after virus inoculation. For transient
expression aphids were added 2 days after infiltration. For visualization of callose, leaves were cleared with 95% ethanol overnight
and stained with 150 mM K2P04 (pH 9.5), 0.01% aniline blue for 2 h
(Koch and Slusarenko, 1990). The leaves were examined for UV
fluorescence using a Leica fluorescence stereoscope (365 nm excitation, 396 nm chromatic beam splitter, 420 nm barrier filter) and
the number of callose spots was quantified manually.
RNA isolation
Arabidopsis and N. benthamiana leaves infected with TuMV, were
harvested, frozen in liquid nitrogen, and ground to a fine powder
as above. Total RNA was extracted from frozen tissue samples
using the SV Total RNA Isolation system with on-column DNase
treatment (Promega, Madison, WI, USA, http://www.promega.
com). RNA integrity was verified using a 1.2% formaldehyde agarose gel (Sambrook et al., 1989). After RNA extraction and DNase
treatment, 1 lg of total RNA was reverse transcribed with SMART
MMLV reverse transcriptase (Clontech, Mountain View, CA, USA,
http://www.clontech.com) using oligo-dT12–18 as a primer.
Quantitative real-time PCR
Arabidopsis plants were inoculated with TuMV, and 14 adult
aphids were added to mock-inoculated and TuMV-infected plants.
Insects were removed and tissue was collected into liquid nitrogen. Plant RNA was isolated using an SV Total RNA Isolation kit
(Promega). Four-week-old Arabidopsis stably expressing the
empty vector pMDC32 or NIa-Pro in pMDC32 were used for transgenic plant experiments. Fourteen adult aphids were added as
above and tissue was collected after 24 h after the initiation of
aphid feeding.
Transcript abundance of CYP81F2 was analyzed with quantitative real-time RT-PCR (qRT-PCR), with eEF1-a (elongation factor 1alpha, At5 g60390) as an internal standard. eEF1-a was identified
from publicly available microarray data as constitutively
expressed after herbivory and stable expression was verified
across samples using qRT-PCR (Primers; Table S1). Gene-specific
primers used for qRT-PCR were designed using Primer-Blast
(http://www.ncbi.nlm.nih.gov/tools/primer-blast/) with the following criteria: melting temperature of 60°C, PCR amplicon lengths
of 90–150 bp yielding primer sequences with lengths of 18–24
nucleotides with an optimum at 21 nucleotides, and guanine–
cytosine contents of 40–60% (Table S1). Reactions were carried
out using 5 ll of the SYBR Green PCR master mix (Applied Biosystems, Grand Island, NY, USA, http://www.lifetechnologies.com),
with 800 nM of primer, in the ABI 7900HT instrument (Applied Biosystems). The PCR was initiated by incubation at 95°C for 10 min
to activate the enzyme. Then the following cycle was repeated 40
times: 95°C for 15 sec, 60°C for 15 sec, and 72°C for 15 sec. The CT
values were quantified and analyzed according to the standard
curve method.
DNA manipulations for genetic constructs
PCR products were amplified by gene-specific primers for each
TuMV gene (Table S1) flanked by the attB1 and attB2 universal
primers and individually cloned using the Gateway cloning kit
(XXXclonF and XXXclonR; Table S1; Invitrogen, Carlsbad, CA,
USA, http://www.lifetechnologies.com), following the instructions
of the manufacturer. First, the PCR products were cloned into
pDONR207 vector using BP clonase and then they were re-cloned
into pMDC32 destination vector with 35S promoter (Curtis and
Grossniklaus, 2003), which harbors the double 35S promoter,
using LR clonase (Invitrogen). For westerns, NIa-Pro was re-cloned
into pGWB511 destination vector with 35S promoter and a FLAG
tag (Nakagawa et al., 2007).
Transient expression of TuMV proteins
Recombinant Agrobacterium tumefaciens strain GV3101 cultures
that contained each of the 10 TuMV gene constructs individually
(primers, Table S1), were grown overnight at 28°C in Luria-Bertani
(LB) medium that contained the appropriate antibiotics (pMDC32,
kanamycin; pDONR207, gentamicin). Cells were pelleted at
2800 g, resuspended in infiltration medium (10 mM MgCl2), and
incubated for 2 h at room temperature. Resuspended cells were
infiltrated into the underside of 4-week-old N. benthamiana leaves
at an OD600 = 0.2 with a 1-ml needleless syringe. As a control, the
p35S: EV (empty vector) construct was also infiltrated into a separate set of plants for each experiment. The plants were incubated
in the growth chambers used above for 24 h, after which cages
were added to the underside of infiltrated leaves and fecundity or
preference experiments were performed as described above.
Because not all experiments could be performed at the same time,
fecundity is expressed relative to the EV control that was included
in each experiment. At the end of the experiments, leaf tissue was
collected from three leaves for each construct and the EV. RNA
was isolated and cDNA synthesized as described previously.
Using the gene-specific primers from cloning (Table S1), expression of each TuMV gene was verified by PCR, and no-reverse
transcriptase controls were used to verify that the PCR template
was derived from RNA (Figure S3).
Arabidopsis transformation and aphid bioassays with
transgenic plants
The transformation vectors harboring p35S:NIa-Pro or the p35S:
EV constructs were introduced into Agrobacterium and transferred
into wild-type Arabidopsis plants by floral dip transformation
(Clough and Bent, 1998). Positive transgenic lines were screened
on kanamycin-containing Murashige and Skoog (MS) agar plates
© 2013 The Authors
The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12417
10 Clare L. Casteel et al.
(Murashige and Skoog, 1962) and then confirmed by reverse-transcription PCR. Single leaves of 4-week-old Arabidopsis transformed with p35S: EV or p35S:NIa-Pro were used in fecundity,
amino acid and callose experiments as described above.
Statistical analysis
For fecundity, development, alate production, and free amino acid
experiments t-tests were used to determine significant differences
from controls. Analysis of variance (ANOVA) was used to determine
if there were comparisons among multiple samples. Post-hoc multiple comparison analyses were conducted using Least Significant
Difference (LSD) or Tukey’s post-hoc tests. For preference
chi-squared tests were performed. All statistical analyses were
conducted using JMP analysis software, version 9 (JMP 9.0; SAS,
Cary, NC, USA, http://www.jmp.com).
ACKNOWLEDGEMENTS
This research was supported by US National Science Foundation
award IOS-1121788 to GJ and SAW, United States Department of
Agriculture award 2010-65105-20558 to GJ, United States Department of Agriculture award 2013-2013-03265 to CLC, an American
Society for Plant Biologists Summer Undergraduate Research
Fellowship to HND, Binational Agricultural Research and Development Agency award US-4165-08C to SAW, and the Iowa State
University Plant Sciences Institute Virus–Insect Interactions group.
We thank Neha Pandya for assistance with aphid bioassays.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article.
Figure S1. Confirmation of TuMV-carrying aphids by RT-PCR.
Figure S2. Transfer of TuMV by established M. persicae colonies.
Figure S3. Confirmation of transient expression of 10 TuMV genes
in N. benthamiana
Figure S4. Expression of native and FLAG-tagged NIa-Pro in transgenic plants, and effects on aphid fecundity.
Figure S5. Changes in individual free amino acid in leaves of
mock-inoculated or TuMV-infected plants.
Figure S6. Changes in individual free amino acid in plants
expressing NIa-Pro or the empty vector
Table S1. Primers used in this study.
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© 2013 The Authors
The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12417