JGV Papers in Press. Published May 15, 2015 as doi:10.1099/vir.0.000185
Journal of General Virology Visual monitoring of Cucumber mosaic virus infection in Nicotiana benthamiana following transmission by the aphid vector Myzus persicae --Manuscript Draft-Manuscript Number:
VIR-D-15-00216R2
Full Title:
Visual monitoring of Cucumber mosaic virus infection in Nicotiana benthamiana following transmission by the aphid vector Myzus persicae
Short Title:
Monitoring CMV infection post transmission
Article Type:
Standard
Section/Category:
Plant - RNA Viruses
Corresponding Author:
Jeremy R Thompson Cornell University College of Agriculture and Life Sciences Ithaca, NY UNITED STATES
First Author:
Bjoern Krenz
Order of Authors:
Bjoern Krenz Agathe Bronikowski Xiaoyun Lu Heiko Ziebell Jeremy R Thompson Keith L Perry
Abstract:
The single-stranded, positive-sense and tripartite RNA virus Cucumber mosaic virus (CMV) was used in this study as a method for monitoring the initial stages of virus infection following aphid transmission. The RNA2 of CMV was modified to incorporate, in a variety of arrangements, an open reading frame (ORF) encoding an enhanced green fluorescent protein (eGFP). The phenotypes of five engineered RNA2s were tested in Nicotiana tabacum, N. clevelandii and N. benthamiana. Only one construct (F4), in which the 2b ORF was truncated at the 3' end and fused in-frame with the eGFP ORF, was able to systemically infect N. benthamiana plants, express eGFP and be transmitted by the aphid Myzus persicae. The utility of this construct was demonstrated following infection as early as one day post transmission (dpt) continuing through to systemic infection. Comparisons of the inoculation sites in different petiole sections one to three dpt clearly showed that the onset of infection and eGFP expression always occurred in the epidermal or collenchymatous tissue just below the epidermis; an observation consistent with the rapid time frame characteristic of the non-persistent mode of aphid transmission.
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Krenz et al JGV 1
Visual monitoring of Cucumber mosaic virus infection in Nicotiana
2
benthamiana following transmission by the aphid vector Myzus persicae
3 4
Running title: Monitoring CMV infection post transmission
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Contents category: Plant RNA viruses
6 7
Bjoern Krenz1, 2, Agathe Bronikowski1,4, Xiaoyun Lu1, Heiko Ziebell1,3, Jeremy R.
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Thompson*1 and Keith L. Perry1.
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1 Plant
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Science, Cornell University, 334 Plant Science Building, Ithaca, NY 14853-5904
12
USA.
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2 Lehrstuhl
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Staudtstrasse 5, 91058 Erlangen, Germany
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3
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Messeweg 11/12, 38104 Braunschweig, Germany
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4
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Germany
Pathology and Plant-Microbe Biology Section, School of Integrative Plant
Biochemie, Department Biologie - Universität Erlangen-Nürnberg,
Julius Kühn-Institut · Institute for Epidemiology and Pathogen Diagnostics,
Institute for Microbiology, Universität Stuttgart, Allmandring 31 , 70569 Stuttgart,
19 20
Corresponding author: Jeremy R. Thompson [email protected]
21 22
Summary: 184 words
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Text: 5,198
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Figs: 6
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Tables: 1
26 1
Krenz et al JGV 27
28 29
Summary
30 31
The single-stranded, positive-sense and tripartite RNA virus Cucumber mosaic
32
virus (CMV) was used in this study as a method for monitoring the initial stages
33
of virus infection following aphid transmission. The RNA2 of CMV was modified
34
to incorporate, in a variety of arrangements, an open reading frame (ORF)
35
encoding an enhanced green fluorescent protein (eGFP). The phenotypes of five
36
engineered RNA2s were tested in Nicotiana tabacum, N. clevelandii and N.
37
benthamiana. Only one construct (F4), in which the 2b ORF was truncated at the
38
3’ end and fused in-frame with the eGFP ORF, was able to systemically infect N.
39
benthamiana plants, express eGFP and be transmitted by the aphid Myzus
40
persicae. The utility of this construct was demonstrated following infection as
41
early as one day post transmission (dpt) continuing through to systemic
42
infection. Comparisons of the inoculation sites in different petiole sections one
43
to three dpt clearly showed that the onset of infection and eGFP expression
44
always occurred in the epidermal or collenchymatous tissue just below the
45
epidermis; an observation consistent with the rapid time frame characteristic of
46
the non-persistent mode of aphid transmission.
47 48 49 50 2
Krenz et al JGV 51 52
Introduction
53
Many plant viruses have evolved not only with their host but also the vector that
54
transmits them. Understanding this three-way relationship is integral to developing
55
control strategies. Cucumber mosaic virus (CMV) has a worldwide distribution with one
56
of the broadest host ranges of any known virus species (for a recent review see
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(Jacquemond, 2012)). CMV has a tripartite single-stranded RNA genome, with each
58
genomic RNA packaged separately within icosahedral capsids. CMV genomic RNAs
59
1 and 2 encode the 1a and 2a proteins, respectively, which are involved in viral
60
replication. RNA2 also encodes a small protein called 2b, which is a suppressor of
61
RNA silencing (Pumplin & Voinnet, 2013; Wang et al, 2012), and plays a role in
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promoting cell-to-cell movement (Soards et al, 2002). RNA3 is also bicistronic,
63
encoding the movement protein (MP) and the virion capsid protein (CP). Natural
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transmission of CMV is mediated by at least 75 species of aphids (Palukaitis et al,
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1992) in a non-persistent, non-circulative mode characterized by short acquisition and
66
inoculation periods (from seconds to minutes), with the aphid rapidly losing the ability
67
to transmit.
68
Aphids are sap-sucking insects that have a highly specialized needle-like stylet
69
that can penetrate and feed on plant cells. The typical feeding behavior of an aphid
70
includes initial exploratory probes, during which it is assumed the insect ‘tastes’ the
71
plant to establish whether it is a host or non-host (Fereres & Moreno, 2009). Aphids
72
need more than a minute to penetrate deeper than the epidermis (Pirone & Harris,
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1977), the stylet advancing intercellularly before prolonged feeding is initiated by the
74
puncturing of a cell membrane. This behavior has been successfully monitored by
75
measuring the changes in electric potential that occur during feeding. For non-
76
persistent viruses, like CMV, an ingestion-salivation model for transmission is presently 3
Krenz et al JGV 77
the most accepted, whereby the aphid is capable of acquiring and inoculating virus
78
particles during the initial phases of probing (Martin et al, 1997; Powell, 2005).
79
Following the penetration of the stylet, the aphid secretes gel saliva which hardens,
80
enclosing the entire stylet in a protective sheath (Kimmins, 1986; Tjallingii & Esch,
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1993). Once the virus is transferred into a host plant cell it is assumed to begin
82
replication, spread to adjacent cells, and subsequently move systemically. Therefore,
83
establishing precisely in which cells these first colonizing events take place is of
84
particular interest.
85
The potential of CMV as a tool for heterologous expression was first
86
demonstrated using GFP in variety of arrangements within RNA3. For all constructs
87
GFP expression was limited to a few cells around the inoculation site (Canto et al,
88
1997). In a similar study, a diploid RNA3 was engineered whereby in one RNA3 the
89
MP was replaced by GFP, and in the other RNA3 the CP was replaced by a multiple
90
cloning site for the insertion of foreign genes. Although functional, movement and gene
91
expression was limited to the inoculated leaf, with recombination frequently restoring
92
RNA3 wild-type (Zhao et al, 2000). In another system, dependency on the CP for cell-
93
to-cell spread was circumvented by deleting the C-terminal 33 amino acids of the MP,
94
thereby allowing the CP open reading frame (ORF) to be completely replaced by a
95
heterologous protein (Fujiki et al, 2008). The above approaches all exploited the RNA4
96
subgenomic promoter located upstream of the CP. An alternative approach has been
97
to use the other CMV subgenomic promoter (for subgenomic RNA4A) located
98
upstream and driving transcription of an RNA encoding the 2b silencing suppressor.
99
One construct was engineered to contain a cloning site immediately upstream of the
100
2b start codon thereby entirely deleting the 2b ORF and truncating the 2a ORF. This
101
construct, named C2-H1, was used to efficiently express a human cytokine (Matsuo et
102
al, 2007) and fluorescent proteins (Takeshita et al, 2012). Another construct C2-A1 4
Krenz et al JGV 103
was used to insert both targets for gene silencing (Kanazawa et al, 2011; Otagaki et
104
al, 2006) and fluorescent proteins (Kanazawa et al, 2011; Takeshita et al, 2012). It also
105
was engineered to contain a cloning site, this time truncating the 2b ORF but leaving
106
the 2a ORF intact. The 2b ORF, although essential for normal functioning of the virus
107
can be truncated at the C-terminus and still have a close-to wild-type phenotype
108
(Lewsey et al, 2009); complete deletion of the 2b ORF (and the C-terminal region of
109
the 2a ORF) results in impaired infection or movement and a significant attenuation of
110
symptoms (Ding et al, 1995; Soards et al, 2002).
111
In this study our objective was to develop a means to identify the initial sites of
112
CMV inoculation by aphid feeding and to follow the spread of infection in the entire
113
plant. To do this we developed and tested a number of heterologous infectious clones
114
of CMV with an incorporated enhanced green fluorescent protein (eGFP) arranged in
115
a variety of configurations in order to empirically test each in planta for the desired
116
phenotype, i.e. stability of the construct and eGFP expression during all phases of
117
infection and transmission. We confirm the amenability of CMV to stable fluorescent
118
tagging of its 2b protein, both full-length and truncated, and demonstrate the biological
119
stability of these constructs through infection, from aphid transmission and systemic
120
spread in the plant to aphid acquisition. To our knowledge, this is the first
121
demonstration of a phenotypically near wild-type, fluorescently expressing virus that is
122
vector-transmissible. Using this tool, we also provide evidence to support the
123
hypothesis that the initial cells to be infected during aphid feeding are epidermal, an
124
observation consistent with the rapid time frame of transmission for non-persistently
125
transmitted viruses.
126 127
Results
128
Infectivity and eGFP expression of F-constructs in Nicotiana spp. 5
Krenz et al JGV 129
In total, five different CMV RNA2 constructs with altered 2b regions were engineered
130
and named F1 to F5 (Fig.1). The F1 construct contained the complete 2b ORF fused
131
in-frame with the eGFP ORF to produce an RNA2 of 3770 nt. The F2 construct fused
132
the full-length 2a ORF in-frame with the eGFP ORF resulting in a C-terminus (89 nt)
133
truncation of the 2b ORF and a resulting RNA2 of 3679 nt. The F3 construct has the
134
2b ORF deleted and an eGFP ORF substituted and under the control of the 2b
135
promoter; this resulted in a truncated 2a ORF, yielding an RNA2 of 3438 nt. The F4
136
construct, was similar to the F2 construct in that it contained a C-terminus (90 nt)
137
truncation of the 2b ORF and a full-length 2a ORF, but in contrast to F2 the truncated
138
2b ORF was fused in-frame with eGFP to give a full-length RNA2 of 3683 nt. Finally,
139
the F5 construct organization corresponded to the F4 construct but without eGFP. This
140
latter construct was engineered as a reference for the effects of eGFP insertion in those
141
constructs with a truncated 2b. Nicotiana tabacum, N. clevelandii and N. benthamiana
142
plants were mechanically inoculated with capped transcripts derived from RNA1
143
(pFny109), RNA3 (pFny309), and either the wild-type RNA2 clone (pFny209) or one
144
of the F-constructs, and monitored for virus symptoms and local and systemic eGFP
145
expression at 2, 7, 11, 14 and 21 days post inoculation (dpi). Each F-construct was
146
inoculated onto 3 plants (2 leaves each) and the experiment was repeated either three
147
or four times (Table 1).
148
N. tabacum. Twenty-five percent (three of twelve) of the plants inoculated with
149
the F1 construct showed symptoms similar to CMV wild-type infection but with no
150
visible eGFP fluorescence in newly emerging leaves 7 dpi. However, at 2 dpi F1
151
construct-inoculated leaves showed patches (approximately 10 cells in diameter) of
152
eGFP fluorescence (data not shown), which did not expand over time. Half of the plants
153
inoculated with the F5 construct showed symptoms similar to CMV wild-type infection.
154
F2, F3 and F4 constructs did not systemically infect N. tabacum. (Table 1) 6
Krenz et al JGV 155
N. clevelandii. The F5 construct was able to systemically infect, causing severe
156
symptoms with necrosis. All other constructs were non-infectious, except for one plant
157
out of a total of nine inoculated with the F3 construct that developed a systemic
158
infection and an associated eGFP signal (Table 1).
159
mechanically from this infected N. clevelandii to additional plants of this host led to
160
infection but loss of fluorescence.
Attempts to transmit virus
161
N. benthamiana. Plants were systemically infected by all F-constructs (F1-F5),
162
whereas only F1 and F4 construct-inoculated plants had eGFP signals in both
163
inoculated and newly emerged leaf tissue. The symptom phenotype observed for F-
164
constructs varied (Table 1). For F1, F4 and F5 they were similar to wild-type CMV. In
165
F2 and F3 plants symptoms were highly attenuated. F1 and F4 construct-inoculated
166
leaves showed patches (approximately 10 cells in diameter) of eGFP fluorescence at
167
2dpi which subsequently expanded and in some cases moved along major veins (Fig.
168
2a-b). At 14dpi F4 construct-inoculated plants showed systemic symptoms with eGFP
169
fluorescence being evenly distributed in young leaf tissue and in roots and flower buds
170
(not shown). After 14 to 21 dpi newly emerging leaves contained typical virus-
171
symptomatic areas that lacked eGFP fluorescence.
172 173
In N. benthamiana, F4 RNA is packaged into virions, whereas F1 RNA is not.
174
Plants inoculated with the F1 construct were systemically infected, with eGFP-
175
expression observed in the upper leaves. Sap inoculations using F1-infected, eGFP-
176
expressing leaf material as inoculum resulted in a systemic infection, but without eGFP
177
expression in six of six plants (total for two independent experiments).
178
hypothesized that an RNA2 with additional eGFP sequences may not be packaged
179
due to its size or other limitations. To test this, we harvested eGFP expressing,
We
7
Krenz et al JGV 180
systemically infected leaves at 7dpi and analyzed RNAs extracted from purified virions
181
versus total RNAs obtained directly from the leaves for the presence of the eGFP
182
sequences. RT-PCR analysis of F4-infected leaves revealed that eGFP sequences
183
could be recovered from virions and total RNA extracts (Fig. 3). By contrast, in F1-
184
infected plants eGFP sequences could only be recovered from the total RNA extracts;
185
eGFP sequences were not detected among the virion RNAs, suggesting that the F1
186
RNA2 is capable of systemic spread, but is not packaged into virions (Fig. 3).
187 188
F4 RNA can be stably transmitted by aphids
189
To test transmissibility of the F1 and F4 RNAs, aphids were fed on eGFP expressing
190
areas of systemically infected leaf tissue in order to acquire the virus, and then
191
transferred to healthy N. benthamiana plants (3 plants per construct in three separate
192
experiments). Local and systemic eGFP signals were checked at 2, 4 and 7 days post-
193
transmission (dpt). None of the plants (zero of nine) incubated with aphids that had fed
194
on F1 construct infected plants developed eGFP signals, while all plants inoculated by
195
aphids that had fed on F4 construct infected plants developed eGFP signals (nine of
196
nine plants) in the inoculated (Fig. 2c-d) and systemic (Fig. 2e-f) leaves. Repeated
197
aphid transmission, as described above, every 14 days showed F4 RNA to be stably
198
transmitted over the course of five passaging experiments.
199 200
Epidermal cells are the first to be infected upon transmission
201
N. benthamiana petioles (10 experiments, 2-3 petioles/experiment) and leaves (2
202
experiments, 1 plant/experiment, 4-5 leaves/plant) were incubated with aphids
203
viruliferous for the F4 construct. Petioles were examined for eGFP signals at 1, 2 and
204
3 dpt (Figs 4 and 5). In the first two experiments petioles were examined after 3 dpt.
205
Five sites showed eGFP expression with signals observed in cells from the epidermis 8
Krenz et al JGV 206
to the vascular tissue (Fig. 4g; 5c, f). Sections were subsequently stained with fuchsin
207
(Fig. 4h) to visualize stylet sheaths, and therefore correlate aphid feeding sites with
208
eGFP signals. Both single (Fig. 4a-c) and branched stylet sheaths (Fig. 4d-i) were
209
observed, the latter probably being the result of intracellular exploratory probes by a
210
single aphid. Experiments were repeated and examined for eGFP signals at 2 dpt (Fig.
211
4d-f; Fig. 5b, e) and at 1 dpt (Fig. 4a-c; Fig. 5a, d) to identify the initial cell(s) that were
212
infected by F4. At 1dpt, eGFP expression was limited to the epidermal and neighboring
213
collenchymatous cells in the cross-sections of 10 F4-inoculated petioles and leaves
214
(Fig. 4a, b; Fig. 5a, d and g-l).
215 216
Discussion
217
Here we report the development of a CMV reporter system and demonstrate its
218
applicability for use in monitoring the initial stages of virus infection following vector
219
transmission. The reported competency of CMV constructs exploiting the 4A
220
subgenomic promoter for the expression of foreign genes (Kanazawa et al, 2011;
221
Matsuo et al, 2007; Otagaki et al, 2006) in combination with the fact that the replication
222
encoding RNAs will be the first to be translated upon infection, (versus the structural
223
proteins encoded on RNA3), led us to pursue a similar design.
224
Specifically, we engineered four eGFP-containing constructs in the RNA2 of
225
Fny-CMV. Two of these, F3 and F4, were comparable to the GFP derivatives of the Y-
226
CMV constructs C2-H1 and C2-A1 (Kanazawa et al, 2011; Takeshita et al, 2012),
227
respectively, except that the latter contained additionally engineered cloning sites. Of
228
the four Fny-CMV constructs tested in this study, all were able to infect N.
229
benthamiana, two (F1 and F4) expressed eGFP, and one (F4) was aphid transmissible.
230
There was no difference in eGFP expression in local F1 or F4 inoculated leaves, eGFP
231
expression occurring throughout expanding leaves with a gradual decline in expression 9
Krenz et al JGV 232
after 14 dpi. The reason for the F1 and F4 phenotypes’ decreased eGFP expression
233
is most likely due to the absence of any selection pressure to maintain the eGFP ORF;
234
RT-PCR analysis of inoculated and systemically infected tissue 21 dpi confirmed a loss
235
in the integrity of the eGFP ORF in all constructs tested (Fig. 6). Such reversions due
236
to recombination are a well-documented phenomenon in heterologous virus
237
expression systems (Chapman et al, 1992; Culver, 1996; Dolja et al, 1993).
238
Transmissibility or its absence could be explained by the physical limitations of virion
239
assembly imposed by genome segment length. F4 RNA2 is 87 nt shorter than that of
240
F1 and 633 nt longer than the wild type RNA2. Full-length F1 RNA2 could be detected
241
in eGFP expressing islands in inoculated leaves, but not in virus particles isolated from
242
those islands, whereas full-length F4 RNA2 was found in both eGFP islands and
243
extracted virions (Fig. 3). We cannot exclude the possibility that virions from F1 and F4
244
infected plants were less stable and that the virion purification method employed here
245
could be too harsh to isolate intact virus particles, however the results of the aphid
246
transmission experiments support the hypothesis that F1 RNA2 encapsidation is
247
hindered, perhaps due to a size limitation, a phenomenon that has been demonstrated
248
for plant RNA viruses both in vivo (Qu & Morris, 1997) and in vitro (Cadena-Nava et al,
249
2012).
250
The transmission phenotypes of F2 and F3 were not evaluated because eGFP
251
expression was not consistently observed despite the development of systemic
252
infection in N. benthamiana. This was presumably due to a loss in the integrity of the
253
eGFP ORF by recombination early in infection as was observed at later timepoints for
254
all F genotypes (Fig. 6). Unlike the other constructs designed, both the F2 and F3 are
255
predicted to have altered 2a proteins; the 2a of F2 is fused in-frame to eGFP, and the
256
C-terminal region of the 2a in F3 is truncated, an effect already shown reduce virus
257
accumulation and attenuate symptoms in N. glutinosa (Du et al, 2008). The F4 10
Krenz et al JGV 258
genotype is almost identical (save for an extra 4 nt) to that of F2 except that the eGFP
259
ORF is fused to the 2b instead, supporting the idea that the essential role of the 2a
260
ORF in virus replication is encumbered when fused to eGFP (Margolin, 2000), in
261
particular at the C terminus where a number a highly conserved polymerase motifs are
262
located (Koonin, 1991).
263
Comparable phenotypes were observed in F1, F4 and wild-type infected plants,
264
indicating that the presence of eGFP in F1 and F4 does not influence symptoms. In a
265
detailed study of a CMV 2b GFP fusion protein there was no change observed in 2b
266
functionality (Gonzalez et al, 2010). Functional mapping of the 2b ORF suggests that
267
the 2b protein N terminus is critical for symptom induction; mutations of the first 17 N-
268
terminal residues in this domain, two nuclear localization sequences or a putative
269
phosphorylation sequence resulted in asymptomatic infections. However, a deletion of
270
the 16 C-terminal residues of the 2b protein had little effect on symptoms in Arabidopsis
271
thaliana when compared to wild-type but caused an increase in symptom severity in
272
N. benthamiana (Lewsey et al, 2009). A similar slight increase in symptom severity
273
(leaf distortion) was observed in this study for those F-constructs with a truncated 2b
274
(F1, F4 and F5) both in N. benthamiana and N. tabacum. This phenomenon was also
275
demonstrated with engineered constructs in an analogous genotype of a CMV isolate
276
from subgroup II (results not shown).
277
The use of fluorescent proteins to tag virus components and therefore track
278
infection in the host has been extensively exploited both in animal and plant systems.
279
The only previous case of a fluorescently tagged engineered virus that was stably
280
transmitted via an insect vector is the phloem-limited Potato leafroll virus which was
281
engineered to express GFP (Nurkiyanova et al, 2000). Aphids that fed on extracts from
282
infected protoplasts were able to transmit the virus, but subsequent cell-to-cell
283
movement from the inoculated cell was found to be defective; progeny that moved 11
Krenz et al JGV 284
systemically were found to have de novo deletions annulling the function of the GFP
285
ORF (Nurkiyanova et al, 2000). The CMV (F4) genotype reported here provides a
286
unique tool in the identification of the initial host cells inoculated by the insect vector
287
upon transmission. Prior to the appearance of symptoms, systemic infection was
288
observed as early as 4 dpt in the form of eGFP signals in major veins (Fig. 2a).
289
However, because of the large surface area the use of whole plants for the
290
identification of the primary inoculated cells proved impracticable. Therefore, in order
291
to monitor the early onset of infection and to simplify the identification of the primary
292
inoculated cells, detached petioles were used in aphid transmission experiments.
293
Comparison of petioles with an infection site at 1, 2 and 3 dpt clearly showed that the
294
onset of eGFP expression always occurred in the epidermal cells or collenchymatous
295
cells just below the epidermis; observations in experiments with leaves were consistent
296
with these results (Fig. 5m-o). Fluorescence was at times difficult to discriminate in
297
epidermal cells (Fig. 4d-f and 5m-o) possibly due to the physical effects of hand-
298
sectioning which could slightly deform cells at the edge of the tissues. Nevertheless,
299
in some sections fluorescence in the epidermis could be unambiguous (Fig. 5k-l) or
300
present but notably weaker than the underlying collenchyma (Fig. 5g-i). At these early
301
stages eGFP expression was never observed in phloem or surrounding cells, although
302
autofluorescence in xylem tissue was consistent in both infected and uninfected
303
tissues. Therefore, our results indicate that aphid transmission of F4 occurs at the
304
earliest time point of plant penetration by the aphid during a period of initial exploratory
305
probing, as proposed previously by Martin et al., (1997).
306 307
Methods
308
Plant growth conditions
12
Krenz et al JGV 309
N. tabacum, N. clevelandii and N. benthamiana seeds were grown in a greenhouse
310
with a 16/8h (light/day) photoperiod at 22±3°C.
311
F-constructs
312
Restriction endonucleases and DNA-modifying enzymes were used as recommended
313
by the manufacturers. Plasmids pFny 209 (Rizzo & Palukaitis, 1990) and pPRLVeGFP
314
(Taliansky et al., 2004) were the templates for the F-constructs and enhanced GFP
315
(eGFP),
316
mutagenesis (Liu et al, 2002) involving the insertion of Pfu polymerase (Stratagene,
317
La Jolla, CA, USA) generated PCR fusion products containing primer-generated
318
mutations into pFny209 linearized by NcoI and PstI. For the F1 to F4 constructs, which
319
were to contain eGFP, three separate PCR fragments (5’NcoI +virus; eGFP;
320
virus+PstI3’) were initially generated and then fused stepwise. Each virus fragment (5’
321
and
322
eGFP+virus+PstI3’), and then both virus/eGFP fragments were joined into one final
323
fusion fragment. For the F5 construct two PCR products (5’NcoI+virus, virus+PstI3’)
324
that introduced the desired deletion in the 2b gene were fused into a single fragment.
325
All engineered constructs were verified by DNA sequencing (Genomics Facility,
326
Institute of Biotechnology, Cornell University). Details of the primers used for each pre-
327
fusion PCR fragment ((5’NcoI+virus)(eGFP)(virus+PstI3’)) for each F-construct were
328
as follows:
329
F1: (5’NcoI+virus) - forward primer (L) (5’-TCGTCACCATGGCTGAGTTTGCCT-3’)
330
(corresponding to Fny CMV RNA2 (Genbank accession number D00355) nts 1846-
331
1869) and the reverse primer (f1)(corresponding to Fny 209 nts 2731-2748 and the
332
first 17 nt of eGFP); (eGFP) - forward primer (complementary to f1) and reverse primer
333
(eGFPstopRNA2) ( 5’-ACGAGCTGTACAAGTAATGAAACCTCCCCTTCCGCAT-3’)
334
(corresponding to the last 17 nt of eGFP, stop codon TGA and Fny RNA2 nts 275213
respectively.
3’)
was
first
All
F-constructs
individually
fused
were
with
engineered
eGFP
by
PCR-mediated
(5’NcoI+virus+eGFP
and
Krenz et al JGV 335
2768); (virus+PstI3’) - forward primer (complementary to the reverse primer
336
(eGFPstopRNA2))
337
AGTCGACCTGCAGTGGTCTCCTTTTGGA–3’) (corresponding to Fny 209 nts 3036-
338
3050 and sequence AGTCGACCTGCAG containing the PstI site).
339
F2: (5’NcoI+virus) - forward primer (L) and the reverse primer (f2) (5’–
340
TCGCCCTTGCTCACCATGACTCGGGTAACTCCGCCA-3’) (corresponding to Fny
341
209 nt 2639-2657 and the first 17 nt of eGFP; (eGFP) - Forward primer
342
(complementary to the reverse primer f2) and reverse primer (eGFPstopRNA2);
343
(virus+PstI3’) – as for F1.
344
F3: (5’NcoI+virus) - forward primer (L) and the reverse primer (f3) (5’ –
345
TCGCCCTTGCTCACCATGATAGAAATTCTTTCGCTGTTT- 3’) (corresponding to
346
Fny 209 nts 2403-2418, sequence GATAGA (introducing two stop codons so that the
347
2a gene will end at nt 2417) and the first 17 nt of eGFP; (eGFP) - Forward primer
348
(complementary to the reverse primer f3) and reverse primer (eGFPstopRNA2);
349
(virus+PstI3’) – as for F1.
350
F4: (5’NcoI+virus) - forward primer (L) and the reverse primer (f4) (5’-
351
TCGCCCTTGCTCACCATCTCAGACTCGGGTAACTCCGCCA-3’) (corresponding to
352
Fny 209 nts 2639-2661 and the first 17 nt of eGFP); (eGFP) - forward primer
353
(complementary to the reverse primer f4) and reverse primer (eGFPstopRNA2);
354
(virus+PstI3’) – as for F1.
355
F5: (5’NcoI+virus) - forward primer (L) and the reverse primer F5 (5’–
356
ATGCGGAAGGGGAGGTTTCACTCAGACTCGGGTAACTCCGCCA-3’)
357
(corresponding to Fny 209 nts 2639-2661 and the first 20nt of RNA2 3’UTR);
358
(virus+PstI3’) - forward primer (complementary to the reverse primer f5) and reverse
359
primer (RNA2PstI).
and
the
reverse
primer
(RNA2PstI)
(5’–
360 14
Krenz et al JGV 361
Virion purification and RT-PCR analysis
362
Virions was purified from infected N. benthamiana plants according to the methods of
363
(Lot et al, 1972) and resuspended in 5 mM sodium borate, 0.5 mM EDTA, pH 9.0.
364
Concentrations were determined spectrophotometrically assuming an extinction
365
coefficient for CMV of 5.0 ml mg−1 cm−1 at a wavelength of 260 nm (Francki et al, 1966).
366
Total RNA was extracted using the RNeasy® Plant Mini Kit (Qiagen, Valencia, CA) and
367
subjected to reverse-transcription polymerase chain reaction (RT-PCR) using M-MLV
368
(Promega, Madison, WI) and GoTaq® DNA Polymerase (Promega), with primer
369
sequences designed to amplify from upstream of the 2b coding region (forward 5’-
370
CTATTATTCAGATCGTCGTC-3’) to the 3’ end of the eGFP ORF (reverse 5’-
371
CTTGTACAGCTCGTCCATGC -3’). The resulting amplification products were
372
checked on a non-denaturing 1% agarose gel and sequenced.
373 374
Aphid transmission
375
Non-viruliferous and wingless adults of the aphid Myzus persicae were reared
376
on healthy cucumber seedlings in a growth chamber with a 16/8h (light/day)
377
photoperiod at 22±3°C. Aphids were placed in sealed plastic dishes and starved for 24
378
h at 4°C. Before feeding the dishes were returned to room temperature for 1 h. Starved
379
aphids were transferred with a brush to virus-infected plants and left to feed for 5 min
380
during which their activity was monitored under a dissecting microscope. Feeding
381
aphids were then transferred to either petioles or leaves (detached and non-detached)
382
of healthy N. benthamiana plants and left overnight. The following day the plants were
383
sprayed with the insecticide Orthene (Valent, Walnut Creek, CA, USA) at a
384
concentration of 0.4 gL-1 and then returned to the greenhouse for another two weeks.
385
For microscopic imaging, petioles were hand-sectioned with a razor blade and
386
examined under a dissecting or fluorescence microscope (detailed below). 15
Krenz et al JGV 387 388
Transcript and sap inoculations
389
Infectious viral RNA from Fny CMV and the F-constructs (F1-F5) were
390
reconstituted by mixing equal amounts of in vitro transcripts, synthesized using the T7
391
mMessage mMachine kit (Ambion, Life Technologies, Grand Island, NY, USA), from
392
full-length linearized cDNA clones (in parentheses) encoding RNA1 (pFny109) and
393
RNA3 (pFny309) in combination with either RNA2 (pFny209) or one of the F-
394
constructs. Equal volumes of transcripts were combined and gently rubbed onto
395
carborundum-dusted leaves of plants at a three-to-five leaf stage. Mechanically
396
inoculated leaves were examined for eGFP expression one week post-inoculation
397
under an Olympus SZX-12 Stereo Microscope (Olympus Corporation, Melville, NY,
398
USA). Virus symptoms were scored at 7 and 14 days post-inoculation (dpi). Younger
399
leaves were analyzed for systemic infection at two weeks post-inoculation. Infection of
400
the inoculated plants was confirmed serologically using CMV ImmunoStrip ®Tests
401
(Agdia, Elkhart, IN, USA). All CMV RNA combinations were inoculated onto 3 plants (2
402
leaves each) in three or four separate experiments (Table 1).
403
Sap inoculations were carried out mechanically by grinding virus infected tissue
404
in 0.1 M phosphate buffer (pH 7.0) and rubbing onto healthy N. benthamiana leaves
405
dusted with carborundum. Mock inoculations were done using 0.1 M phosphate buffer
406
(pH 7.0) alone. Inoculated plants were maintained in the greenhouse (as above) and
407
inspected for symptom development and eGFP expression at 2, 7, 11, 14 and 21 dpi.
408
Infection of the inoculated plants was confirmed serologically
409 410
Microscopy
411
GFP expression was investigated using either an Olympus SZX-12 Stereo
412
Microscope equipped with DFPLFL 0.5X PF, DFPLAPO 1.2X and DFPLFL 1.6X PF 16
Krenz et al JGV 413
objectives coupled with 10X eyepieces (Zoom range to 108X) and two parfocal
414
objectives (0.5X and 1.6X) (filter Ex 470/40, Em LP 500) or a Leica DM5500
415
Epifluorescence Microscope (filter BP450/490; 500; BP500-550) (Leica Microsystems
416
Inc., Buffalo Grove, IL, USA). Aphid stylet sheaths were stained with fuchsin as
417
described by Brennan et al., (2001). Areas on the petioles where aphids had been
418
feeding for 24 h were thinly hand-sectioned using a razor blade. Sections were fixed
419
in 70% aqueous ethanol and then immersed in an acid fuchsin solution [10 parts 70%
420
ethanol and 1 part 0.2% acid fuchsin in ethanol:glacial acetic acid (1:1, v/v)]. Stylet
421
sheaths in these samples appeared bright orange under an epifluorescence
422
microscope Leica DM5500 (filter Ex 515–560, DM 580) (Leica). Images were
423
processed using the software LAS-AF (Leica). Samples were also visualized under UV
424
light (filter Ex 340–380, DM 400) for optimal autofluorescence in cells around the stylet
425
sheaths.
426 427
Acknowledgements
428
Thanks to Mamta Srivastava of the Plant Cell Imaging Center (PCIC) of the Boyce
429
Thompson Institute (BTI) at Cornell University Campus for assistance with the
430
microscopy work. Thanks also to two anonymous reviewers for their useful
431
suggestions in drafting this manuscript.
432 433 434
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437
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Krenz et al JGV 438
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Kalinina, N. O., Carr, J. P., Palukaitis, P. & Canto, T. (2010) Cucumber mosaic virus
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Liu, S. J., He, X. H., Park, G., Josefsson, C. & Perry, K. L. (2002) A conserved capsid
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Martin, B., Collar, J. L., Tjallingii, W. F. & Fereres, A. (1997) Intracellular ingestion
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Matsuo, K., Hong, J. S., Tabayashi, N., Ito, A., Masuta, C. & Matsumura, T. (2007)
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Zhao, Y., Hammond, J., Tousignant, M. E. & Hammond, R. W. (2000) Development
563
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564
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565 566 567 568 569 570
Figure legends
571
Fig. 1 Schematic organization of CMV Fny RNA2 and derived F-constructs (F1 to F5).
572
Open reading frames (ORFs) are indicated by shaded rectangles, primer annealing
573
sites (for RT-PCR analysis by thin arrows, nucleotide length (nt) of individual RNA by
574
numbers in brackets and restriction sites of NcoI and PstI by dotted lines. F1 contains
575
the complete 2b ORF fused in-frame with the eGFP ORF. F2 has the full-length 2a
576
ORF in-frame fused with the eGFP ORF causing a C-terminus truncation of the 2b
577
ORF. F3 contains the 2b ORF deleted and an eGFP ORF substituted and under the
578
control of the 2b promoter; this resulted in a truncated 2a ORF. F4 contains a full-length
579
2a ORF and a C-terminus truncation of the 2b ORF fused in-frame with eGFP. F5
580
corresponds exactly to the F4 construct but without eGFP.
581 582
Fig. 2. eGFP expression of F4-infected N. benthamiana. (a), eGFP expression in a
583
mechanically inoculated leaf at 4 days post-inoculation (dpi); and (b). 6 dpi. (c) Onset 23
Krenz et al JGV 584
of eGFP expression in an infected leaf 4 days after aphid transmission, abaxial surface
585
of the leaf is shown. (d) Same leaf as in (c) two days later. Asterisk indicates the same
586
position in both photos, the putative infection site. (e) Youngest leaf of plant shown in
587
(c) and (d). Note the weak but visible eGFP signals along major veins already after 4
588
dpi. (f) Same leaf as in (e) 14 dpi. eGFP expression is now nearly equally distributed
589
throughout the leaf. Size bars = 1cm.
590 591
Fig. 3. Detection and characterization of eGFP sequences from purified virion RNAs
592
(encapsidated) and from total plant RNAs (unencapsidated + encapsidated) by RT-
593
PCR. Amplified fragments generated from RNAs extracted from F1-, F4- and wild-type
594
Fny-CMV purified virus particles and from leaf tissue originating from infected N.
595
benthamiana plants (7dpi) were separated by agarose gel electrophoresis. In vitro
596
transcripts served as templates for the positive control. Forward and reverse primers
597
were designed to anneal upstream of the 5’ end of the 2b open reading frame (ORF)
598
and the 3’ end of the eGFP ORF, respectively. Controls were RNAs extracted from
599
mock-inoculated plants (M) and water (H2O). Expected RT-PCR fragment sizes were
600
1151 bp and 1063 base pairs (bp) for F1 and F4, respectively. L – DNA size ladder.
601 602
Fig. 4. Cellular localization of eGFP (a, d, g) in petiole sections of F4-infected N.
603
benthamiana plants one to three days after aphid transmission and the visualization of
604
feeding-associated stylet sheaths by fuchsin staining (b, e, h). (a) and (b) one day post
605
transmission (dpt). (d) and (e) two dpt. (g) and (h) three dpt. (c, f, i) show the original
606
eGFP images with the putative positions of the stylet sheath as identified in B, D and
607
F, marked with a long dotted line. Short dotted line – vasculature edge (v). Solid line – 24
Krenz et al JGV 608
epidermis (ep). Collenchyma – co. Size bar = 100µm. Filters: GFP (BP450/490; 500;
609
BP500-550), Fuchsin (Ex 515–560, DM 580).
610 611
Fig. 5. Cellular localization of eGFP in petiole and leaf sections of F4-infected N.
612
benthamiana plants one to three days after aphid transmission. Petiole sections in
613
brightfield (bf) light are shown in (a), one day post transmission (dpt); (b), two dpt and
614
(c), three dpt. (d) to (f), show eGFP expression in the corresponding petiole sections
615
of (a) to (c). A magnification of the area delineated by the white rectangle in (a) and (d)
616
is shown in panels (g) to (i). (g), (j), (m), brightfield (bf); (h), (k), (n) eGFP expression
617
(a false red color is shown for improved contrast); (i), (l), (o), merged. Petioles in (a) to
618
(i) were detached from the leaf during the inoculation experiment. Petiole and leaf in
619
(j) to (o) were intact (attached to the plant) until harvested for processing. (g-o) one
620
dpt. Size bar = 100µm.
621 622
Fig 6. eGFP ORF reduction in F-constructs three weeks post inoculation. (a) PCR
623
amplification of infected plants three weeks post inoculation. Agarose gel (1%)
624
electrophoresis of RT-PCR products derived from pooling RNA extracted from N.
625
benthamiana inoculated (i) and systemically (s) infected leaves of three plants at three
626
weeks post inoculation with Fny-CMV transcripts generated from constructs 109
627
(RNA1) and 309 (RNA3) in combination with one F-construct (RNA2) (F1, F2, F3, F4).
628
PCR products amplified from plasmid constructs (Lanes marked - O) are also included
629
for comparison: F-constructs (F1-F5) and wild-type 209 (RNA2). Nucleotide sizes in
630
base pairs are shown above each arrow next to the DNA size ladder (L). Primers used
631
were
forward
5’-AGAGTTCAGGGTTGAGCGTGTAA-3’
and
reverse
5’25
Krenz et al JGV 632
TGGTCTCCTTTTGGAGGCCCCACA-3’ annealing to nucleotide positions 2372-2394
633
and 3050-3027 of Fny-CMV RNA2 (Acc. No. NC_002035), respectively (primer
634
locations are shown in Fig 6(b)). PCR products derived from systemic leaves were
635
cloned into the pGEM-T vector (Promega) and their sequences obtained. (b) PCR
636
amplification of infected N. benthamiana plants three weeks post inoculation reveals
637
deletions in RNA2 derivatives. Two clones from each F-construct (F1, F2, F3 and F4)
638
were sequenced. The deletions observed are indicated by the opaque white box with
639
the dotted border. In all cases, except F4, both clones were identical in sequence. The
640
arrows show the location of the primers used to PCR amplify the deleted region.
641 642 643 644 645 646 647 648 649 650 651 652 26
Krenz et al JGV N. tabacum
N. clevelandii
N. benthamiana
virus #
Systemic symptoms
GFP
#
Systemic symptoms
GFP
#
Systemic symptoms
GFP
F1
3/12
stunting; leaf distortion; mosaic
I
0/9
none
-
8/12
stunting; leaf distortion; mosaic
I/S
F2
0/9
none
-
0/9
none
-
4/12
mild leaf curling to asymptomatic
-
F3
0/9
none
-
1/9
stunting; mosaic; systemic necrotic spots
I/S
8/12
very mild mosaic
I*
F4
0/9
none
-
0/9
none
-
7/12
stunting; leaf distortion; mosaic
I/S
F5
6/12
stunting; leaf distortion; mosaic
n/a
8/9
stunting; mosaic; systemic necrotic spots
n/a
7/12
stunting; leaf distortion; mosaic
n/a
wt
12/12
stunting; leaf distortion; mosaic
n/a
9/9
stunting; mosaic; systemic necrotic spots
n/a
12/12
stunting; leaf distortion; mosaic
n/a
653 654 655 656 657 658 659 660 661 662 663 664
Table 1. Phenotypes of virus infection in Nicotiana tabacum, N. clevelandii and N.
665
benthamiana plants mechanically inoculated with a mix of capped transcripts derived
666
from Fny-CMV RNA1,2,3 (wild-type (wt)) or one of the F-constructs with RNA1 and 3
667
(F1-5). Fractions represent plants (N=9-12) showing systemic viral symptoms 7-14 27
Krenz et al JGV 668
days post inoculation in three to four independent experiments. GFP column –
669
fluorescence in inoculated (I) and systemic (S) leaves. * - eGFP signals erratic in
670
inoculated leaves; ranging from none (4 plants) to weak (3 plants) to strong (1 plant).
671
n/a – not applicable.
28
Figure 1 Click here to download Figure: Fig1 JGV.TIF
Figure 2 Click here to download Figure: CMV Fig 2 JGV.tif
Figure 3 Click here to download Figure: Fig3.tif
Figure 4 Click here to download Figure: Fig4a.tif
Figure 5 Click here to download Figure: Fig 5.tif
Figure 6 Click here to download Figure: Fig 6.tif
Journal of General Virology Visual monitoring of Cucumber mosaic virus infection in Nicotiana benthamiana following transmission by the aphid vector Myzus persicae --Manuscript Draft-Manuscript Number:
VIR-D-15-00216R2
Full Title:
Visual monitoring of Cucumber mosaic virus infection in Nicotiana benthamiana following transmission by the aphid vector Myzus persicae
Short Title:
Monitoring CMV infection post transmission
Article Type:
Standard
Section/Category:
Plant - RNA Viruses
Corresponding Author:
Jeremy R Thompson Cornell University College of Agriculture and Life Sciences Ithaca, NY UNITED STATES
First Author:
Bjoern Krenz
Order of Authors:
Bjoern Krenz Agathe Bronikowski Xiaoyun Lu Heiko Ziebell Jeremy R Thompson Keith L Perry
Abstract:
The single-stranded, positive-sense and tripartite RNA virus Cucumber mosaic virus (CMV) was used in this study as a method for monitoring the initial stages of virus infection following aphid transmission. The RNA2 of CMV was modified to incorporate, in a variety of arrangements, an open reading frame (ORF) encoding an enhanced green fluorescent protein (eGFP). The phenotypes of five engineered RNA2s were tested in Nicotiana tabacum, N. clevelandii and N. benthamiana. Only one construct (F4), in which the 2b ORF was truncated at the 3' end and fused in-frame with the eGFP ORF, was able to systemically infect N. benthamiana plants, express eGFP and be transmitted by the aphid Myzus persicae. The utility of this construct was demonstrated following infection as early as one day post transmission (dpt) continuing through to systemic infection. Comparisons of the inoculation sites in different petiole sections one to three dpt clearly showed that the onset of infection and eGFP expression always occurred in the epidermal or collenchymatous tissue just below the epidermis; an observation consistent with the rapid time frame characteristic of the non-persistent mode of aphid transmission.
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Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Krenz JGV_v22.docx
Krenz et al JGV 1
Visual monitoring of Cucumber mosaic virus infection in Nicotiana
2
benthamiana following transmission by the aphid vector Myzus persicae
3 4
Running title: Monitoring CMV infection post transmission
5
Contents category: Plant RNA viruses
6 7
Bjoern Krenz1, 2, Agathe Bronikowski1,4, Xiaoyun Lu1, Heiko Ziebell1,3, Jeremy R.
8
Thompson*1 and Keith L. Perry1.
9 10
1 Plant
11
Science, Cornell University, 334 Plant Science Building, Ithaca, NY 14853-5904
12
USA.
13
2 Lehrstuhl
14
Staudtstrasse 5, 91058 Erlangen, Germany
15
3
16
Messeweg 11/12, 38104 Braunschweig, Germany
17
4
18
Germany
Pathology and Plant-Microbe Biology Section, School of Integrative Plant
Biochemie, Department Biologie - Universität Erlangen-Nürnberg,
Julius Kühn-Institut · Institute for Epidemiology and Pathogen Diagnostics,
Institute for Microbiology, Universität Stuttgart, Allmandring 31 , 70569 Stuttgart,
19 20
Corresponding author: Jeremy R. Thompson [email protected]
21 22
Summary: 184 words
23
Text: 5,198
24
Figs: 6
25
Tables: 1
26 1
Krenz et al JGV 27
28 29
Summary
30 31
The single-stranded, positive-sense and tripartite RNA virus Cucumber mosaic
32
virus (CMV) was used in this study as a method for monitoring the initial stages
33
of virus infection following aphid transmission. The RNA2 of CMV was modified
34
to incorporate, in a variety of arrangements, an open reading frame (ORF)
35
encoding an enhanced green fluorescent protein (eGFP). The phenotypes of five
36
engineered RNA2s were tested in Nicotiana tabacum, N. clevelandii and N.
37
benthamiana. Only one construct (F4), in which the 2b ORF was truncated at the
38
3’ end and fused in-frame with the eGFP ORF, was able to systemically infect N.
39
benthamiana plants, express eGFP and be transmitted by the aphid Myzus
40
persicae. The utility of this construct was demonstrated following infection as
41
early as one day post transmission (dpt) continuing through to systemic
42
infection. Comparisons of the inoculation sites in different petiole sections one
43
to three dpt clearly showed that the onset of infection and eGFP expression
44
always occurred in the epidermal or collenchymatous tissue just below the
45
epidermis; an observation consistent with the rapid time frame characteristic of
46
the non-persistent mode of aphid transmission.
47 48 49 50 2
Krenz et al JGV 51 52
Introduction
53
Many plant viruses have evolved not only with their host but also the vector that
54
transmits them. Understanding this three-way relationship is integral to developing
55
control strategies. Cucumber mosaic virus (CMV) has a worldwide distribution with one
56
of the broadest host ranges of any known virus species (for a recent review see
57
(Jacquemond, 2012)). CMV has a tripartite single-stranded RNA genome, with each
58
genomic RNA packaged separately within icosahedral capsids. CMV genomic RNAs
59
1 and 2 encode the 1a and 2a proteins, respectively, which are involved in viral
60
replication. RNA2 also encodes a small protein called 2b, which is a suppressor of
61
RNA silencing (Pumplin & Voinnet, 2013; Wang et al, 2012), and plays a role in
62
promoting cell-to-cell movement (Soards et al, 2002). RNA3 is also bicistronic,
63
encoding the movement protein (MP) and the virion capsid protein (CP). Natural
64
transmission of CMV is mediated by at least 75 species of aphids (Palukaitis et al,
65
1992) in a non-persistent, non-circulative mode characterized by short acquisition and
66
inoculation periods (from seconds to minutes), with the aphid rapidly losing the ability
67
to transmit.
68
Aphids are sap-sucking insects that have a highly specialized needle-like stylet
69
that can penetrate and feed on plant cells. The typical feeding behavior of an aphid
70
includes initial exploratory probes, during which it is assumed the insect ‘tastes’ the
71
plant to establish whether it is a host or non-host (Fereres & Moreno, 2009). Aphids
72
need more than a minute to penetrate deeper than the epidermis (Pirone & Harris,
73
1977), the stylet advancing intercellularly before prolonged feeding is initiated by the
74
puncturing of a cell membrane. This behavior has been successfully monitored by
75
measuring the changes in electric potential that occur during feeding. For non-
76
persistent viruses, like CMV, an ingestion-salivation model for transmission is presently 3
Krenz et al JGV 77
the most accepted, whereby the aphid is capable of acquiring and inoculating virus
78
particles during the initial phases of probing (Martin et al, 1997; Powell, 2005).
79
Following the penetration of the stylet, the aphid secretes gel saliva which hardens,
80
enclosing the entire stylet in a protective sheath (Kimmins, 1986; Tjallingii & Esch,
81
1993). Once the virus is transferred into a host plant cell it is assumed to begin
82
replication, spread to adjacent cells, and subsequently move systemically. Therefore,
83
establishing precisely in which cells these first colonizing events take place is of
84
particular interest.
85
The potential of CMV as a tool for heterologous expression was first
86
demonstrated using GFP in variety of arrangements within RNA3. For all constructs
87
GFP expression was limited to a few cells around the inoculation site (Canto et al,
88
1997). In a similar study, a diploid RNA3 was engineered whereby in one RNA3 the
89
MP was replaced by GFP, and in the other RNA3 the CP was replaced by a multiple
90
cloning site for the insertion of foreign genes. Although functional, movement and gene
91
expression was limited to the inoculated leaf, with recombination frequently restoring
92
RNA3 wild-type (Zhao et al, 2000). In another system, dependency on the CP for cell-
93
to-cell spread was circumvented by deleting the C-terminal 33 amino acids of the MP,
94
thereby allowing the CP open reading frame (ORF) to be completely replaced by a
95
heterologous protein (Fujiki et al, 2008). The above approaches all exploited the RNA4
96
subgenomic promoter located upstream of the CP. An alternative approach has been
97
to use the other CMV subgenomic promoter (for subgenomic RNA4A) located
98
upstream and driving transcription of an RNA encoding the 2b silencing suppressor.
99
One construct was engineered to contain a cloning site immediately upstream of the
100
2b start codon thereby entirely deleting the 2b ORF and truncating the 2a ORF. This
101
construct, named C2-H1, was used to efficiently express a human cytokine (Matsuo et
102
al, 2007) and fluorescent proteins (Takeshita et al, 2012). Another construct C2-A1 4
Krenz et al JGV 103
was used to insert both targets for gene silencing (Kanazawa et al, 2011; Otagaki et
104
al, 2006) and fluorescent proteins (Kanazawa et al, 2011; Takeshita et al, 2012). It also
105
was engineered to contain a cloning site, this time truncating the 2b ORF but leaving
106
the 2a ORF intact. The 2b ORF, although essential for normal functioning of the virus
107
can be truncated at the C-terminus and still have a close-to wild-type phenotype
108
(Lewsey et al, 2009); complete deletion of the 2b ORF (and the C-terminal region of
109
the 2a ORF) results in impaired infection or movement and a significant attenuation of
110
symptoms (Ding et al, 1995; Soards et al, 2002).
111
In this study our objective was to develop a means to identify the initial sites of
112
CMV inoculation by aphid feeding and to follow the spread of infection in the entire
113
plant. To do this we developed and tested a number of heterologous infectious clones
114
of CMV with an incorporated enhanced green fluorescent protein (eGFP) arranged in
115
a variety of configurations in order to empirically test each in planta for the desired
116
phenotype, i.e. stability of the construct and eGFP expression during all phases of
117
infection and transmission. We confirm the amenability of CMV to stable fluorescent
118
tagging of its 2b protein, both full-length and truncated, and demonstrate the biological
119
stability of these constructs through infection, from aphid transmission and systemic
120
spread in the plant to aphid acquisition. To our knowledge, this is the first
121
demonstration of a phenotypically near wild-type, fluorescently expressing virus that is
122
vector-transmissible. Using this tool, we also provide evidence to support the
123
hypothesis that the initial cells to be infected during aphid feeding are epidermal, an
124
observation consistent with the rapid time frame of transmission for non-persistently
125
transmitted viruses.
126 127
Results
128
Infectivity and eGFP expression of F-constructs in Nicotiana spp. 5
Krenz et al JGV 129
In total, five different CMV RNA2 constructs with altered 2b regions were engineered
130
and named F1 to F5 (Fig.1). The F1 construct contained the complete 2b ORF fused
131
in-frame with the eGFP ORF to produce an RNA2 of 3770 nt. The F2 construct fused
132
the full-length 2a ORF in-frame with the eGFP ORF resulting in a C-terminus (89 nt)
133
truncation of the 2b ORF and a resulting RNA2 of 3679 nt. The F3 construct has the
134
2b ORF deleted and an eGFP ORF substituted and under the control of the 2b
135
promoter; this resulted in a truncated 2a ORF, yielding an RNA2 of 3438 nt. The F4
136
construct, was similar to the F2 construct in that it contained a C-terminus (90 nt)
137
truncation of the 2b ORF and a full-length 2a ORF, but in contrast to F2 the truncated
138
2b ORF was fused in-frame with eGFP to give a full-length RNA2 of 3683 nt. Finally,
139
the F5 construct organization corresponded to the F4 construct but without eGFP. This
140
latter construct was engineered as a reference for the effects of eGFP insertion in those
141
constructs with a truncated 2b. Nicotiana tabacum, N. clevelandii and N. benthamiana
142
plants were mechanically inoculated with capped transcripts derived from RNA1
143
(pFny109), RNA3 (pFny309), and either the wild-type RNA2 clone (pFny209) or one
144
of the F-constructs, and monitored for virus symptoms and local and systemic eGFP
145
expression at 2, 7, 11, 14 and 21 days post inoculation (dpi). Each F-construct was
146
inoculated onto 3 plants (2 leaves each) and the experiment was repeated either three
147
or four times (Table 1).
148
N. tabacum. Twenty-five percent (three of twelve) of the plants inoculated with
149
the F1 construct showed symptoms similar to CMV wild-type infection but with no
150
visible eGFP fluorescence in newly emerging leaves 7 dpi. However, at 2 dpi F1
151
construct-inoculated leaves showed patches (approximately 10 cells in diameter) of
152
eGFP fluorescence (data not shown), which did not expand over time. Half of the plants
153
inoculated with the F5 construct showed symptoms similar to CMV wild-type infection.
154
F2, F3 and F4 constructs did not systemically infect N. tabacum. (Table 1) 6
Krenz et al JGV 155
N. clevelandii. The F5 construct was able to systemically infect, causing severe
156
symptoms with necrosis. All other constructs were non-infectious, except for one plant
157
out of a total of nine inoculated with the F3 construct that developed a systemic
158
infection and an associated eGFP signal (Table 1).
159
mechanically from this infected N. clevelandii to additional plants of this host led to
160
infection but loss of fluorescence.
Attempts to transmit virus
161
N. benthamiana. Plants were systemically infected by all F-constructs (F1-F5),
162
whereas only F1 and F4 construct-inoculated plants had eGFP signals in both
163
inoculated and newly emerged leaf tissue. The symptom phenotype observed for F-
164
constructs varied (Table 1). For F1, F4 and F5 they were similar to wild-type CMV. In
165
F2 and F3 plants symptoms were highly attenuated. F1 and F4 construct-inoculated
166
leaves showed patches (approximately 10 cells in diameter) of eGFP fluorescence at
167
2dpi which subsequently expanded and in some cases moved along major veins (Fig.
168
2a-b). At 14dpi F4 construct-inoculated plants showed systemic symptoms with eGFP
169
fluorescence being evenly distributed in young leaf tissue and in roots and flower buds
170
(not shown). After 14 to 21 dpi newly emerging leaves contained typical virus-
171
symptomatic areas that lacked eGFP fluorescence.
172 173
In N. benthamiana, F4 RNA is packaged into virions, whereas F1 RNA is not.
174
Plants inoculated with the F1 construct were systemically infected, with eGFP-
175
expression observed in the upper leaves. Sap inoculations using F1-infected, eGFP-
176
expressing leaf material as inoculum resulted in a systemic infection, but without eGFP
177
expression in six of six plants (total for two independent experiments).
178
hypothesized that an RNA2 with additional eGFP sequences may not be packaged
179
due to its size or other limitations. To test this, we harvested eGFP expressing,
We
7
Krenz et al JGV 180
systemically infected leaves at 7dpi and analyzed RNAs extracted from purified virions
181
versus total RNAs obtained directly from the leaves for the presence of the eGFP
182
sequences. RT-PCR analysis of F4-infected leaves revealed that eGFP sequences
183
could be recovered from virions and total RNA extracts (Fig. 3). By contrast, in F1-
184
infected plants eGFP sequences could only be recovered from the total RNA extracts;
185
eGFP sequences were not detected among the virion RNAs, suggesting that the F1
186
RNA2 is capable of systemic spread, but is not packaged into virions (Fig. 3).
187 188
F4 RNA can be stably transmitted by aphids
189
To test transmissibility of the F1 and F4 RNAs, aphids were fed on eGFP expressing
190
areas of systemically infected leaf tissue in order to acquire the virus, and then
191
transferred to healthy N. benthamiana plants (3 plants per construct in three separate
192
experiments). Local and systemic eGFP signals were checked at 2, 4 and 7 days post-
193
transmission (dpt). None of the plants (zero of nine) incubated with aphids that had fed
194
on F1 construct infected plants developed eGFP signals, while all plants inoculated by
195
aphids that had fed on F4 construct infected plants developed eGFP signals (nine of
196
nine plants) in the inoculated (Fig. 2c-d) and systemic (Fig. 2e-f) leaves. Repeated
197
aphid transmission, as described above, every 14 days showed F4 RNA to be stably
198
transmitted over the course of five passaging experiments.
199 200
Epidermal cells are the first to be infected upon transmission
201
N. benthamiana petioles (10 experiments, 2-3 petioles/experiment) and leaves (2
202
experiments, 1 plant/experiment, 4-5 leaves/plant) were incubated with aphids
203
viruliferous for the F4 construct. Petioles were examined for eGFP signals at 1, 2 and
204
3 dpt (Figs 4 and 5). In the first two experiments petioles were examined after 3 dpt.
205
Five sites showed eGFP expression with signals observed in cells from the epidermis 8
Krenz et al JGV 206
to the vascular tissue (Fig. 4g; 5c, f). Sections were subsequently stained with fuchsin
207
(Fig. 4h) to visualize stylet sheaths, and therefore correlate aphid feeding sites with
208
eGFP signals. Both single (Fig. 4a-c) and branched stylet sheaths (Fig. 4d-i) were
209
observed, the latter probably being the result of intracellular exploratory probes by a
210
single aphid. Experiments were repeated and examined for eGFP signals at 2 dpt (Fig.
211
4d-f; Fig. 5b, e) and at 1 dpt (Fig. 4a-c; Fig. 5a, d) to identify the initial cell(s) that were
212
infected by F4. At 1dpt, eGFP expression was limited to the epidermal and neighboring
213
collenchymatous cells in the cross-sections of 10 F4-inoculated petioles and leaves
214
(Fig. 4a, b; Fig. 5a, d and g-l).
215 216
Discussion
217
Here we report the development of a CMV reporter system and demonstrate its
218
applicability for use in monitoring the initial stages of virus infection following vector
219
transmission. The reported competency of CMV constructs exploiting the 4A
220
subgenomic promoter for the expression of foreign genes (Kanazawa et al, 2011;
221
Matsuo et al, 2007; Otagaki et al, 2006) in combination with the fact that the replication
222
encoding RNAs will be the first to be translated upon infection, (versus the structural
223
proteins encoded on RNA3), led us to pursue a similar design.
224
Specifically, we engineered four eGFP-containing constructs in the RNA2 of
225
Fny-CMV. Two of these, F3 and F4, were comparable to the GFP derivatives of the Y-
226
CMV constructs C2-H1 and C2-A1 (Kanazawa et al, 2011; Takeshita et al, 2012),
227
respectively, except that the latter contained additionally engineered cloning sites. Of
228
the four Fny-CMV constructs tested in this study, all were able to infect N.
229
benthamiana, two (F1 and F4) expressed eGFP, and one (F4) was aphid transmissible.
230
There was no difference in eGFP expression in local F1 or F4 inoculated leaves, eGFP
231
expression occurring throughout expanding leaves with a gradual decline in expression 9
Krenz et al JGV 232
after 14 dpi. The reason for the F1 and F4 phenotypes’ decreased eGFP expression
233
is most likely due to the absence of any selection pressure to maintain the eGFP ORF;
234
RT-PCR analysis of inoculated and systemically infected tissue 21 dpi confirmed a loss
235
in the integrity of the eGFP ORF in all constructs tested (Fig. 6). Such reversions due
236
to recombination are a well-documented phenomenon in heterologous virus
237
expression systems (Chapman et al, 1992; Culver, 1996; Dolja et al, 1993).
238
Transmissibility or its absence could be explained by the physical limitations of virion
239
assembly imposed by genome segment length. F4 RNA2 is 87 nt shorter than that of
240
F1 and 633 nt longer than the wild type RNA2. Full-length F1 RNA2 could be detected
241
in eGFP expressing islands in inoculated leaves, but not in virus particles isolated from
242
those islands, whereas full-length F4 RNA2 was found in both eGFP islands and
243
extracted virions (Fig. 3). We cannot exclude the possibility that virions from F1 and F4
244
infected plants were less stable and that the virion purification method employed here
245
could be too harsh to isolate intact virus particles, however the results of the aphid
246
transmission experiments support the hypothesis that F1 RNA2 encapsidation is
247
hindered, perhaps due to a size limitation, a phenomenon that has been demonstrated
248
for plant RNA viruses both in vivo (Qu & Morris, 1997) and in vitro (Cadena-Nava et al,
249
2012).
250
The transmission phenotypes of F2 and F3 were not evaluated because eGFP
251
expression was not consistently observed despite the development of systemic
252
infection in N. benthamiana. This was presumably due to a loss in the integrity of the
253
eGFP ORF by recombination early in infection as was observed at later timepoints for
254
all F genotypes (Fig. 6). Unlike the other constructs designed, both the F2 and F3 are
255
predicted to have altered 2a proteins; the 2a of F2 is fused in-frame to eGFP, and the
256
C-terminal region of the 2a in F3 is truncated, an effect already shown reduce virus
257
accumulation and attenuate symptoms in N. glutinosa (Du et al, 2008). The F4 10
Krenz et al JGV 258
genotype is almost identical (save for an extra 4 nt) to that of F2 except that the eGFP
259
ORF is fused to the 2b instead, supporting the idea that the essential role of the 2a
260
ORF in virus replication is encumbered when fused to eGFP (Margolin, 2000), in
261
particular at the C terminus where a number a highly conserved polymerase motifs are
262
located (Koonin, 1991).
263
Comparable phenotypes were observed in F1, F4 and wild-type infected plants,
264
indicating that the presence of eGFP in F1 and F4 does not influence symptoms. In a
265
detailed study of a CMV 2b GFP fusion protein there was no change observed in 2b
266
functionality (Gonzalez et al, 2010). Functional mapping of the 2b ORF suggests that
267
the 2b protein N terminus is critical for symptom induction; mutations of the first 17 N-
268
terminal residues in this domain, two nuclear localization sequences or a putative
269
phosphorylation sequence resulted in asymptomatic infections. However, a deletion of
270
the 16 C-terminal residues of the 2b protein had little effect on symptoms in Arabidopsis
271
thaliana when compared to wild-type but caused an increase in symptom severity in
272
N. benthamiana (Lewsey et al, 2009). A similar slight increase in symptom severity
273
(leaf distortion) was observed in this study for those F-constructs with a truncated 2b
274
(F1, F4 and F5) both in N. benthamiana and N. tabacum. This phenomenon was also
275
demonstrated with engineered constructs in an analogous genotype of a CMV isolate
276
from subgroup II (results not shown).
277
The use of fluorescent proteins to tag virus components and therefore track
278
infection in the host has been extensively exploited both in animal and plant systems.
279
The only previous case of a fluorescently tagged engineered virus that was stably
280
transmitted via an insect vector is the phloem-limited Potato leafroll virus which was
281
engineered to express GFP (Nurkiyanova et al, 2000). Aphids that fed on extracts from
282
infected protoplasts were able to transmit the virus, but subsequent cell-to-cell
283
movement from the inoculated cell was found to be defective; progeny that moved 11
Krenz et al JGV 284
systemically were found to have de novo deletions annulling the function of the GFP
285
ORF (Nurkiyanova et al, 2000). The CMV (F4) genotype reported here provides a
286
unique tool in the identification of the initial host cells inoculated by the insect vector
287
upon transmission. Prior to the appearance of symptoms, systemic infection was
288
observed as early as 4 dpt in the form of eGFP signals in major veins (Fig. 2a).
289
However, because of the large surface area the use of whole plants for the
290
identification of the primary inoculated cells proved impracticable. Therefore, in order
291
to monitor the early onset of infection and to simplify the identification of the primary
292
inoculated cells, detached petioles were used in aphid transmission experiments.
293
Comparison of petioles with an infection site at 1, 2 and 3 dpt clearly showed that the
294
onset of eGFP expression always occurred in the epidermal cells or collenchymatous
295
cells just below the epidermis; observations in experiments with leaves were consistent
296
with these results (Fig. 5m-o). Fluorescence was at times difficult to discriminate in
297
epidermal cells (Fig. 4d-f and 5m-o) possibly due to the physical effects of hand-
298
sectioning which could slightly deform cells at the edge of the tissues. Nevertheless,
299
in some sections fluorescence in the epidermis could be unambiguous (Fig. 5k-l) or
300
present but notably weaker than the underlying collenchyma (Fig. 5g-i). At these early
301
stages eGFP expression was never observed in phloem or surrounding cells, although
302
autofluorescence in xylem tissue was consistent in both infected and uninfected
303
tissues. Therefore, our results indicate that aphid transmission of F4 occurs at the
304
earliest time point of plant penetration by the aphid during a period of initial exploratory
305
probing, as proposed previously by Martin et al., (1997).
306 307
Methods
308
Plant growth conditions
12
Krenz et al JGV 309
N. tabacum, N. clevelandii and N. benthamiana seeds were grown in a greenhouse
310
with a 16/8h (light/day) photoperiod at 22±3°C.
311
F-constructs
312
Restriction endonucleases and DNA-modifying enzymes were used as recommended
313
by the manufacturers. Plasmids pFny 209 (Rizzo & Palukaitis, 1990) and pPRLVeGFP
314
(Taliansky et al., 2004) were the templates for the F-constructs and enhanced GFP
315
(eGFP),
316
mutagenesis (Liu et al, 2002) involving the insertion of Pfu polymerase (Stratagene,
317
La Jolla, CA, USA) generated PCR fusion products containing primer-generated
318
mutations into pFny209 linearized by NcoI and PstI. For the F1 to F4 constructs, which
319
were to contain eGFP, three separate PCR fragments (5’NcoI +virus; eGFP;
320
virus+PstI3’) were initially generated and then fused stepwise. Each virus fragment (5’
321
and
322
eGFP+virus+PstI3’), and then both virus/eGFP fragments were joined into one final
323
fusion fragment. For the F5 construct two PCR products (5’NcoI+virus, virus+PstI3’)
324
that introduced the desired deletion in the 2b gene were fused into a single fragment.
325
All engineered constructs were verified by DNA sequencing (Genomics Facility,
326
Institute of Biotechnology, Cornell University). Details of the primers used for each pre-
327
fusion PCR fragment ((5’NcoI+virus)(eGFP)(virus+PstI3’)) for each F-construct were
328
as follows:
329
F1: (5’NcoI+virus) - forward primer (L) (5’-TCGTCACCATGGCTGAGTTTGCCT-3’)
330
(corresponding to Fny CMV RNA2 (Genbank accession number D00355) nts 1846-
331
1869) and the reverse primer (f1)(corresponding to Fny 209 nts 2731-2748 and the
332
first 17 nt of eGFP); (eGFP) - forward primer (complementary to f1) and reverse primer
333
(eGFPstopRNA2) ( 5’-ACGAGCTGTACAAGTAATGAAACCTCCCCTTCCGCAT-3’)
334
(corresponding to the last 17 nt of eGFP, stop codon TGA and Fny RNA2 nts 275213
respectively.
3’)
was
first
All
F-constructs
individually
fused
were
with
engineered
eGFP
by
PCR-mediated
(5’NcoI+virus+eGFP
and
Krenz et al JGV 335
2768); (virus+PstI3’) - forward primer (complementary to the reverse primer
336
(eGFPstopRNA2))
337
AGTCGACCTGCAGTGGTCTCCTTTTGGA–3’) (corresponding to Fny 209 nts 3036-
338
3050 and sequence AGTCGACCTGCAG containing the PstI site).
339
F2: (5’NcoI+virus) - forward primer (L) and the reverse primer (f2) (5’–
340
TCGCCCTTGCTCACCATGACTCGGGTAACTCCGCCA-3’) (corresponding to Fny
341
209 nt 2639-2657 and the first 17 nt of eGFP; (eGFP) - Forward primer
342
(complementary to the reverse primer f2) and reverse primer (eGFPstopRNA2);
343
(virus+PstI3’) – as for F1.
344
F3: (5’NcoI+virus) - forward primer (L) and the reverse primer (f3) (5’ –
345
TCGCCCTTGCTCACCATGATAGAAATTCTTTCGCTGTTT- 3’) (corresponding to
346
Fny 209 nts 2403-2418, sequence GATAGA (introducing two stop codons so that the
347
2a gene will end at nt 2417) and the first 17 nt of eGFP; (eGFP) - Forward primer
348
(complementary to the reverse primer f3) and reverse primer (eGFPstopRNA2);
349
(virus+PstI3’) – as for F1.
350
F4: (5’NcoI+virus) - forward primer (L) and the reverse primer (f4) (5’-
351
TCGCCCTTGCTCACCATCTCAGACTCGGGTAACTCCGCCA-3’) (corresponding to
352
Fny 209 nts 2639-2661 and the first 17 nt of eGFP); (eGFP) - forward primer
353
(complementary to the reverse primer f4) and reverse primer (eGFPstopRNA2);
354
(virus+PstI3’) – as for F1.
355
F5: (5’NcoI+virus) - forward primer (L) and the reverse primer F5 (5’–
356
ATGCGGAAGGGGAGGTTTCACTCAGACTCGGGTAACTCCGCCA-3’)
357
(corresponding to Fny 209 nts 2639-2661 and the first 20nt of RNA2 3’UTR);
358
(virus+PstI3’) - forward primer (complementary to the reverse primer f5) and reverse
359
primer (RNA2PstI).
and
the
reverse
primer
(RNA2PstI)
(5’–
360 14
Krenz et al JGV 361
Virion purification and RT-PCR analysis
362
Virions was purified from infected N. benthamiana plants according to the methods of
363
(Lot et al, 1972) and resuspended in 5 mM sodium borate, 0.5 mM EDTA, pH 9.0.
364
Concentrations were determined spectrophotometrically assuming an extinction
365
coefficient for CMV of 5.0 ml mg−1 cm−1 at a wavelength of 260 nm (Francki et al, 1966).
366
Total RNA was extracted using the RNeasy® Plant Mini Kit (Qiagen, Valencia, CA) and
367
subjected to reverse-transcription polymerase chain reaction (RT-PCR) using M-MLV
368
(Promega, Madison, WI) and GoTaq® DNA Polymerase (Promega), with primer
369
sequences designed to amplify from upstream of the 2b coding region (forward 5’-
370
CTATTATTCAGATCGTCGTC-3’) to the 3’ end of the eGFP ORF (reverse 5’-
371
CTTGTACAGCTCGTCCATGC -3’). The resulting amplification products were
372
checked on a non-denaturing 1% agarose gel and sequenced.
373 374
Aphid transmission
375
Non-viruliferous and wingless adults of the aphid Myzus persicae were reared
376
on healthy cucumber seedlings in a growth chamber with a 16/8h (light/day)
377
photoperiod at 22±3°C. Aphids were placed in sealed plastic dishes and starved for 24
378
h at 4°C. Before feeding the dishes were returned to room temperature for 1 h. Starved
379
aphids were transferred with a brush to virus-infected plants and left to feed for 5 min
380
during which their activity was monitored under a dissecting microscope. Feeding
381
aphids were then transferred to either petioles or leaves (detached and non-detached)
382
of healthy N. benthamiana plants and left overnight. The following day the plants were
383
sprayed with the insecticide Orthene (Valent, Walnut Creek, CA, USA) at a
384
concentration of 0.4 gL-1 and then returned to the greenhouse for another two weeks.
385
For microscopic imaging, petioles were hand-sectioned with a razor blade and
386
examined under a dissecting or fluorescence microscope (detailed below). 15
Krenz et al JGV 387 388
Transcript and sap inoculations
389
Infectious viral RNA from Fny CMV and the F-constructs (F1-F5) were
390
reconstituted by mixing equal amounts of in vitro transcripts, synthesized using the T7
391
mMessage mMachine kit (Ambion, Life Technologies, Grand Island, NY, USA), from
392
full-length linearized cDNA clones (in parentheses) encoding RNA1 (pFny109) and
393
RNA3 (pFny309) in combination with either RNA2 (pFny209) or one of the F-
394
constructs. Equal volumes of transcripts were combined and gently rubbed onto
395
carborundum-dusted leaves of plants at a three-to-five leaf stage. Mechanically
396
inoculated leaves were examined for eGFP expression one week post-inoculation
397
under an Olympus SZX-12 Stereo Microscope (Olympus Corporation, Melville, NY,
398
USA). Virus symptoms were scored at 7 and 14 days post-inoculation (dpi). Younger
399
leaves were analyzed for systemic infection at two weeks post-inoculation. Infection of
400
the inoculated plants was confirmed serologically using CMV ImmunoStrip ®Tests
401
(Agdia, Elkhart, IN, USA). All CMV RNA combinations were inoculated onto 3 plants (2
402
leaves each) in three or four separate experiments (Table 1).
403
Sap inoculations were carried out mechanically by grinding virus infected tissue
404
in 0.1 M phosphate buffer (pH 7.0) and rubbing onto healthy N. benthamiana leaves
405
dusted with carborundum. Mock inoculations were done using 0.1 M phosphate buffer
406
(pH 7.0) alone. Inoculated plants were maintained in the greenhouse (as above) and
407
inspected for symptom development and eGFP expression at 2, 7, 11, 14 and 21 dpi.
408
Infection of the inoculated plants was confirmed serologically
409 410
Microscopy
411
GFP expression was investigated using either an Olympus SZX-12 Stereo
412
Microscope equipped with DFPLFL 0.5X PF, DFPLAPO 1.2X and DFPLFL 1.6X PF 16
Krenz et al JGV 413
objectives coupled with 10X eyepieces (Zoom range to 108X) and two parfocal
414
objectives (0.5X and 1.6X) (filter Ex 470/40, Em LP 500) or a Leica DM5500
415
Epifluorescence Microscope (filter BP450/490; 500; BP500-550) (Leica Microsystems
416
Inc., Buffalo Grove, IL, USA). Aphid stylet sheaths were stained with fuchsin as
417
described by Brennan et al., (2001). Areas on the petioles where aphids had been
418
feeding for 24 h were thinly hand-sectioned using a razor blade. Sections were fixed
419
in 70% aqueous ethanol and then immersed in an acid fuchsin solution [10 parts 70%
420
ethanol and 1 part 0.2% acid fuchsin in ethanol:glacial acetic acid (1:1, v/v)]. Stylet
421
sheaths in these samples appeared bright orange under an epifluorescence
422
microscope Leica DM5500 (filter Ex 515–560, DM 580) (Leica). Images were
423
processed using the software LAS-AF (Leica). Samples were also visualized under UV
424
light (filter Ex 340–380, DM 400) for optimal autofluorescence in cells around the stylet
425
sheaths.
426 427
Acknowledgements
428
Thanks to Mamta Srivastava of the Plant Cell Imaging Center (PCIC) of the Boyce
429
Thompson Institute (BTI) at Cornell University Campus for assistance with the
430
microscopy work. Thanks also to two anonymous reviewers for their useful
431
suggestions in drafting this manuscript.
432 433 434
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560
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563
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564
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565 566 567 568 569 570
Figure legends
571
Fig. 1 Schematic organization of CMV Fny RNA2 and derived F-constructs (F1 to F5).
572
Open reading frames (ORFs) are indicated by shaded rectangles, primer annealing
573
sites (for RT-PCR analysis by thin arrows, nucleotide length (nt) of individual RNA by
574
numbers in brackets and restriction sites of NcoI and PstI by dotted lines. F1 contains
575
the complete 2b ORF fused in-frame with the eGFP ORF. F2 has the full-length 2a
576
ORF in-frame fused with the eGFP ORF causing a C-terminus truncation of the 2b
577
ORF. F3 contains the 2b ORF deleted and an eGFP ORF substituted and under the
578
control of the 2b promoter; this resulted in a truncated 2a ORF. F4 contains a full-length
579
2a ORF and a C-terminus truncation of the 2b ORF fused in-frame with eGFP. F5
580
corresponds exactly to the F4 construct but without eGFP.
581 582
Fig. 2. eGFP expression of F4-infected N. benthamiana. (a), eGFP expression in a
583
mechanically inoculated leaf at 4 days post-inoculation (dpi); and (b). 6 dpi. (c) Onset 23
Krenz et al JGV 584
of eGFP expression in an infected leaf 4 days after aphid transmission, abaxial surface
585
of the leaf is shown. (d) Same leaf as in (c) two days later. Asterisk indicates the same
586
position in both photos, the putative infection site. (e) Youngest leaf of plant shown in
587
(c) and (d). Note the weak but visible eGFP signals along major veins already after 4
588
dpi. (f) Same leaf as in (e) 14 dpi. eGFP expression is now nearly equally distributed
589
throughout the leaf. Size bars = 1cm.
590 591
Fig. 3. Detection and characterization of eGFP sequences from purified virion RNAs
592
(encapsidated) and from total plant RNAs (unencapsidated + encapsidated) by RT-
593
PCR. Amplified fragments generated from RNAs extracted from F1-, F4- and wild-type
594
Fny-CMV purified virus particles and from leaf tissue originating from infected N.
595
benthamiana plants (7dpi) were separated by agarose gel electrophoresis. In vitro
596
transcripts served as templates for the positive control. Forward and reverse primers
597
were designed to anneal upstream of the 5’ end of the 2b open reading frame (ORF)
598
and the 3’ end of the eGFP ORF, respectively. Controls were RNAs extracted from
599
mock-inoculated plants (M) and water (H2O). Expected RT-PCR fragment sizes were
600
1151 bp and 1063 base pairs (bp) for F1 and F4, respectively. L – DNA size ladder.
601 602
Fig. 4. Cellular localization of eGFP (a, d, g) in petiole sections of F4-infected N.
603
benthamiana plants one to three days after aphid transmission and the visualization of
604
feeding-associated stylet sheaths by fuchsin staining (b, e, h). (a) and (b) one day post
605
transmission (dpt). (d) and (e) two dpt. (g) and (h) three dpt. (c, f, i) show the original
606
eGFP images with the putative positions of the stylet sheath as identified in B, D and
607
F, marked with a long dotted line. Short dotted line – vasculature edge (v). Solid line – 24
Krenz et al JGV 608
epidermis (ep). Collenchyma – co. Size bar = 100µm. Filters: GFP (BP450/490; 500;
609
BP500-550), Fuchsin (Ex 515–560, DM 580).
610 611
Fig. 5. Cellular localization of eGFP in petiole and leaf sections of F4-infected N.
612
benthamiana plants one to three days after aphid transmission. Petiole sections in
613
brightfield (bf) light are shown in (a), one day post transmission (dpt); (b), two dpt and
614
(c), three dpt. (d) to (f), show eGFP expression in the corresponding petiole sections
615
of (a) to (c). A magnification of the area delineated by the white rectangle in (a) and (d)
616
is shown in panels (g) to (i). (g), (j), (m), brightfield (bf); (h), (k), (n) eGFP expression
617
(a false red color is shown for improved contrast); (i), (l), (o), merged. Petioles in (a) to
618
(i) were detached from the leaf during the inoculation experiment. Petiole and leaf in
619
(j) to (o) were intact (attached to the plant) until harvested for processing. (g-o) one
620
dpt. Size bar = 100µm.
621 622
Fig 6. eGFP ORF reduction in F-constructs three weeks post inoculation. (a) PCR
623
amplification of infected plants three weeks post inoculation. Agarose gel (1%)
624
electrophoresis of RT-PCR products derived from pooling RNA extracted from N.
625
benthamiana inoculated (i) and systemically (s) infected leaves of three plants at three
626
weeks post inoculation with Fny-CMV transcripts generated from constructs 109
627
(RNA1) and 309 (RNA3) in combination with one F-construct (RNA2) (F1, F2, F3, F4).
628
PCR products amplified from plasmid constructs (Lanes marked - O) are also included
629
for comparison: F-constructs (F1-F5) and wild-type 209 (RNA2). Nucleotide sizes in
630
base pairs are shown above each arrow next to the DNA size ladder (L). Primers used
631
were
forward
5’-AGAGTTCAGGGTTGAGCGTGTAA-3’
and
reverse
5’25
Krenz et al JGV 632
TGGTCTCCTTTTGGAGGCCCCACA-3’ annealing to nucleotide positions 2372-2394
633
and 3050-3027 of Fny-CMV RNA2 (Acc. No. NC_002035), respectively (primer
634
locations are shown in Fig 6(b)). PCR products derived from systemic leaves were
635
cloned into the pGEM-T vector (Promega) and their sequences obtained. (b) PCR
636
amplification of infected N. benthamiana plants three weeks post inoculation reveals
637
deletions in RNA2 derivatives. Two clones from each F-construct (F1, F2, F3 and F4)
638
were sequenced. The deletions observed are indicated by the opaque white box with
639
the dotted border. In all cases, except F4, both clones were identical in sequence. The
640
arrows show the location of the primers used to PCR amplify the deleted region.
641 642 643 644 645 646 647 648 649 650 651 652 26
Krenz et al JGV N. tabacum
N. clevelandii
N. benthamiana
virus #
Systemic symptoms
GFP
#
Systemic symptoms
GFP
#
Systemic symptoms
GFP
F1
3/12
stunting; leaf distortion; mosaic
I
0/9
none
-
8/12
stunting; leaf distortion; mosaic
I/S
F2
0/9
none
-
0/9
none
-
4/12
mild leaf curling to asymptomatic
-
F3
0/9
none
-
1/9
stunting; mosaic; systemic necrotic spots
I/S
8/12
very mild mosaic
I*
F4
0/9
none
-
0/9
none
-
7/12
stunting; leaf distortion; mosaic
I/S
F5
6/12
stunting; leaf distortion; mosaic
n/a
8/9
stunting; mosaic; systemic necrotic spots
n/a
7/12
stunting; leaf distortion; mosaic
n/a
wt
12/12
stunting; leaf distortion; mosaic
n/a
9/9
stunting; mosaic; systemic necrotic spots
n/a
12/12
stunting; leaf distortion; mosaic
n/a
653 654 655 656 657 658 659 660 661 662 663 664
Table 1. Phenotypes of virus infection in Nicotiana tabacum, N. clevelandii and N.
665
benthamiana plants mechanically inoculated with a mix of capped transcripts derived
666
from Fny-CMV RNA1,2,3 (wild-type (wt)) or one of the F-constructs with RNA1 and 3
667
(F1-5). Fractions represent plants (N=9-12) showing systemic viral symptoms 7-14 27
Krenz et al JGV 668
days post inoculation in three to four independent experiments. GFP column –
669
fluorescence in inoculated (I) and systemic (S) leaves. * - eGFP signals erratic in
670
inoculated leaves; ranging from none (4 plants) to weak (3 plants) to strong (1 plant).
671
n/a – not applicable.
28
Figure 1 Click here to download Figure: Fig1 JGV.TIF
Figure 2 Click here to download Figure: CMV Fig 2 JGV.tif
Figure 3 Click here to download Figure: Fig3.tif
Figure 4 Click here to download Figure: Fig4a.tif
Figure 5 Click here to download Figure: Fig 5.tif
Figure 6 Click here to download Figure: Fig 6.tif
