Journal of General Virology (1990), 71, 1443-1449. Pr&ted & Great Britain
1443
Cucumber mosaic virus satellite RNA (strain Y): analysis of sequences
which affect systemic necrosis on tomato
Martine Devic, I Martine Jaegle2t and David Baulcombe I*
1The Sainsbury Laboratory, Colney Lane, Norwich N R 4 7 U H and 2Department o f Molecular Genetics,
Institute o f Plant Science Research, Mar& Lane, Cambridge CB2 2JB, U.K.
The location o f a sequence within the Y satellite R N A
of cucumber mosaic virus (CMV) that confers the
ability to induce necrosis on tomato plants has been
analysed using chimeric satellite RNAs. These recombinant R N A molecules contained parts of the Y
(necrogenic) and Ra (benign) satellite R N A s and were
inoculated into t o m a t o plants together with C M V
helper virus. F r o m the composition o f the recombinant
satellite RNAs that induced necrosis it was concluded
that, of the nucleotides which differ between Y and Ra
satellite RNAs, those affecting necrosis are on the 3'
side o f nucleotide 259. The composition o f satellite
RNAs that failed to induce necrosis implies that at least
some of the necrogenic positions are on the 3' side of
nucleotide 311. The symptoms induced by mutated
forms o f Y and Ra satellite R N A s showed that
nucleotide spacing between positions 322 and 323 and
sequence identity at one or more o f nucleotides 318,
323 or 325 affects the necrogenic potential o f Y
satellite RNA. The effect of a frameshifting mutation
in Y satellite R N A and the location o f the necrogenic
sites relative to open reading frames in other satellite
RNAs suggested that necrosis is not caused by
polypeptides encoded in satellite RNA.
Introduction
Palukaitis, 1989). In the Y and B strains of CMV satellite
R N A these necrogenic sequences act independently of a
second symptom-inducing domain in the 5' part of the
molecule. These second domains affect the production of
a yellow mosaic symptom on tobacco (Y satellite R N A :
Devic et al., 1989; Masuta & Takanami, 1989) or a
chlorosis symptom on tomato (B satellite R N A : Kurath
& Palukaitis, 1989). In this paper, we describe the
necrogenic domain of the Y satellite R N A in more detail
and show that its action does not involve production of
satellite RNA-encoded polypeptides.
Satellite RNAs are small molecules which replicate
rapidly in plants in association with a specific helper
virus. The presence of the satellite R N A in a viral
inoculum often has an effect on the symptoms induced by
the helper virus (Francki, 1985). Cucumber mosaic virus
(CMV) satellite RNAs have been extensively studied
and, up to now, more than 25 isolates originating from
widely separated geographical areas have been characterized and sequenced either directly from R N A or from
cloned e D N A (Richards et al., 1978; Collmer et al., 1983 ;
Gordon & Symons, 1983; Avila-Rincon et al., 1986;
Kaper et al., 1986, 1988; Garcia-Arenal et al., 1987;
Hidaka et al., 1988; Jacquemond & Lauquin, 1988;
Devic et al., 1989; Masuta & Takanami, 1989). These
isolates can be classified in two groups (benign or
necrogenic) according to their ability to produce symptoms on tomato. The necrogenic satellite RNAs induce a
systemic necrosis on tomato. The benign satellite R N A s
do not induce necrosis, although they may induce
chlorotic or mosaic symptoms on tobacco or tomato. The
necrogenic capability is affected by nucleotides in the 3"
part of the satellite RNA- (Devic etal., 1989; Kurath &
Present address: Department of Cell Biology and Genetics,
Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam,
The Netherlands.
0000-9450 © 1990 SGM
Methods
Viral strains, satellite RNAs and eDNA clones. CMV-KIN was
obtained from Professor B. D. Harrison (Scottish Crop Research
Institute, Invergowrie, U.K.). The origin, cloning and nucleotide
sequenceof Y and Ra satelliteRNAs weredescribedpreviously(Devic
et al., 1989). In both instances the eDNA was cloned into a
transcription vector (Ahlquist & Janda, 1984) for the production of
RNA inocula. The recombinant cDNAs incorporatingparts of the Y
and Ra sequences were constructedby using shared cleavage sites for
NheI, AsuII and HgaI restrictionenzymes(Fig. 1). The identityof each
hybrid eDNA was checkedby sequencingthe eDNA directlyfrom the
plasmid DNA (Murphy & Kavanagh, 1988).
Northern blotting. Leaf samples were homogenizedin 50 mM-TrisHCI pH 9.0, 100 mM-NaCl, I0 mM-EDTA, 2% SDS, 0.1 mg/ml
Proteinase K (5 ml/g tissue). After two extractions with phenol-
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1444
M. Devic, M. Jaegle and D. Baulcombe
chloroform, nucleic acids were precipitated with ethanol. Aliquots of
each sample (4 ~tg) were fractionated by electrophoresis in a
formaldehyde-agarose gel (Maniatis et al., 1982). The gel was blotted
onto a nitrocellulose membrane. The membrane was hybridized with a
cDNA insert from I 17N satellite RNA or a cDNA clone of CMV RNA
3 (Baulcombe et al., 1986). The cDNA inserts were annealed with
random hexanucleotides and radiolabelled as described by Feinberg &
Vogelstein (1984).
Site-directed mutagenesis. Mutations were introduced using the
Muta-Gene M13 in vitro mutagenesis kit (Bio-Rad). The following
oligonucleotides were used in these experiments: dTCAGCATAACCTAAGTCTTAGC for Y1, dCTGGCATGGCATTATAGGCTTTAGC for Ral, dTAGACATTCACGGAGATCAGCATAGCATAAGCCTTAGCTTCTCCC for Ra2, dGGAGACTGGCATGGCATAGGCTTTAGCTTCTCCC for Ra3 and dTTCACGGAGACTGGCATAGCATA for Y2. The mutant Y8 was created differently.
The plasmid containing the Y satellite cDNA was linearized at the
BstXI site (position 153). After treatment with T4 polymerase to create
blunt ends, the plasmid was recircularized by ligation and used to
transform MC1022 cells. The identity of the plasmid in the transformed
cells was analysed by sequencing. We selected one clone containing a
mutated Y satellite cDNA insert, pY8, with a deletion of G at position
153.
Assessment of the biological activity o f the clones. RNA was
synthesized in vitro and inoculated into plants in the presence of the
helper virus RNA as previously described (Devic et al., 1989). Each
experiment included control plants inoculated either with buffer alone
or CMV RNA without satellite RNA. The host plant species were
tobacco (Nicotiana tabacum cv. Samsun NN) and tomato (Lycopersicon
esculentum cv. Ailsa Craig). For each transcript, six to 12 plants were
inoculated, in at least two independent experiments. After 2 to 5 weeks,
the symptoms were noted and individual plants assayed for the
presence of satellite RNA by Northern blotting. Each type of progeny
satellite RNA was cloned as cDNA and sequenced (Devic et al., 1989).
Unless stated otherwise, the progeny satellite RNA was identical to the
inoculum.
Sequence comparison. To avoid confusion, all the nucleotide positions
including those in Ra and mutant satellite RNAs refer to the
homologous sites in Y satellite RNA (Fig. 3).
Results
Location of necrogenic sequences
Several constructions of hybrid R N A molecules containing different amounts of Y satellite R N A in an Ra
background were created in order to locate precisely the
necrogenic domain of the Y satellite RNA. These
recombinant molecules were obtained by ligation of the
5' part of the Y satellite c D N A to the 3' part of the Ra
satellite c D N A or vice versa. In each construction, the 5'
fragment included the Pr promoter so that the constructions could be transcribed in vitro. The in vitro transcripts
of these recombinant molecules were assayed on six
tobacco plants and six tomato plants. The symptoms
were noted after 2 to 5 weeks (Fig. 1).
The first set of constructions using the NheI site was
used previously (Devic et al., 1989) to demonstrate that
i
1
~
| 2
/
Y
NheI
Ra
Ra
2 19
Nhei
Y
~
/
~\\\\\\\\\\\~\\\'~1
I_
II
i
3
~
| 4
Y
~ ~
Yellow mosaic
Attenuation
Attenuation
Necrosis
Yellow mosaic
Attenuation
AsulI Ra
5
Ra
L
III
Symptom
Tobacco
Tomato
9
AsulI Y
~\\~\\\\~1 Attenuation
I- 5
y
HgaI Ra
| ~\\\\\\\\\\\\\\\\\\\\\\\\\~\\\\\~
Yellow mosaic
/
311
/6
Ra
HgaI Y
~\\'~1 Not tested
L
Necrosis
Attenuation
Not tested
Fig. 1. Symptoms induced by transcripts of recombinant satellite
cDNAs. Reciprocal pairs of constructions are grouped by set (I, II and
III). The hatched and the black boxes symbolize respectively the Y and
Ra satellite sequences. Six tobacco and six tomato plants were
inoculated in this experiment. In both host species, the infectivity was
100 K and all plants contained satellite RNA. 'Attenuation' indicates
that the symptoms of the virus were still visible on the inoculated leaves
but not on the systemic leaves.
nucleotides affecting yellow mosaic symptoms on
tobacco are on the 5' side, and necrogenic sequences are
on the 3' side, of the NheI site at nucleotide 219 (Fig. 1,
set I). Experiments with the second set of constructions,
produced using an AsuII site (Fig. 1, set II), showed that
the necrogenic sequences are within 111 nucleotides of
the 3' end of the satellite R N A ; the product of
construction 4 was capable of inducing necrosis on
tomato but that of construction 3 attenuated the viral
symptoms. In set III (Fig. 1) construction 6 was not
made, owing to the presence of a second HgaI site in the
Ra satellite c D N A sequence. The product of construction 5 did not induce necrosis. This suggests that, of the
nucleotides that differ in the sequences of the Y and Ra
satellite RNAs, those affecting necrosis on tomato are on
the 3' side of position 259 (AsuII site). The result with
construction 5 (Fig. 1) shows that at least some of those
necrogenic differences are located on the 3' side of
position 311 (HgaI).
As a control, the constructions were inoculated with
CMV to tobacco to confirm that the specificity of
symptom induction of the 5' domain remained unchanged (Fig. 1). In each instance, the yellow mosaic
symptom was induced by satellite RNAs containing the
5' sequence of Y satellite RNA.
The presence of satellite R N A in the infected leaves
was verified by Northern blot analysis (Fig. 2) in which
R N A from infected plants was probed for satellite R N A
or CMV R N A sequences. The satellite R N A probe
detected monomer satellite R N A most strongly. In some
samples, a dimeric form was also detected. The CMV
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Necrogenie sequences in C M V satellite R N A
1445
Comparison of satellite R N A sequences
(a)
1
2
1
3
2
4
3
5
4
6
5
7
6
8
7
9
8
9
RNA
1 and 2
RNA 3
RNA 4
Fig. 2. Northern blot analysis of progeny satellite RNA from tomato
plants infected with transcripts of recombinant clones. The gets were
loaded with 4 ~tgof total nucleic acids. The blots were hybridizedwith
probes specificfor either satellite RNA (a) or CMV RNA (b). Plants
were inoculated with buffer (lane 1), CMV RNA (lane 2) and CMV
R N A together with in vitro transcripts of Y satellite clone (lane 3), Ra
satellite (lane 4), construction 1 (lane 5), construction 2 (lane 6),
construction3 (lane7), construction4 (lane 8) or construction5 (lane9).
R N A probe detected R N A 3 and R N A 4 strongly. R N A
1 and R N A 2 comigrated and hybridized weakly with
this probe. R N A from mock-inoculated plants (lane 1)
contained no satellite R N A (Fig. 2a) or CMV R N A (Fig.
2b). The CMV-inoculated plants (Fig. 2, lane 2)
accumulated large amounts of CMV R N A but no
satellite RNA. This confirmed that the CMV inoculum
was not contaminated by endogenous satellite RNA.
Every plant inoculated with in vitro transcripts of
recombinant c D N A molecules accumulated satellite
R N A (Fig. 2a, lanes 3 to 9) and CMV RNA. There was
less viral R N A (to a variable extent) in plants accumulating satellite R N A than in plants inoculated with CMV
R N A without satellite R N A (Fig, 2b, lanes 3 to 9). Fig.
2(a) shows the variation in migration of the progeny
satellite R N A due to the presence or absence of the 35
extra bases of the Y satellite R N A , which suggests that
satellite RNAs were replicated without undergoing gross
changes in size. This was confirmed by sequence analysis
of the progeny satellite RNA.
Fig. 3 shows an alignment of several satellite R N A
sequences between position 259 (AsulI site) and the 3'
end. The upper part of the figure shows the necrogenic
satellite RNAs together with the consensus sequence of
positions where all the necrogenic satellite RNAs are
identical. The necrogenic satellite R N A s are identical at
8 6 ~ of positions in the region shown.
In the lower part of the figure, the benign satellite
RNAs are divided into two groups according to their
resemblance to the necrogenic consensus sequence.
Group A comprises four sequences, R, 1, OY2 and WL1,
each of which differs from the necrogenic consensus in
fewer than six positions (marked by asterisks in Fig. 3).
This group of benign satellite R N A s is identical at 85%
of nucleotides on the 3' side of nucleotide 259. The
satellite R N A sequences in group B (74.5~ identical) are
more heterogeneous than those in group A and have
more differences (between six and 19 positions, marked
by asterisks in Fig. 3) from the necrogenic consensus,
including additional nucleotides at position 323. Taken
together, the benign satellite RNAs of groups A and B
are identical at only 67.5 % of the nucleotides.
Within the region of 111 nucleotides in the sequence
alignment of the 25 satellite RNAs, no single position
shows complete correlation with the ability to induce
necrosis. Positions 318, 323 and 325 show the best
correlation; in all necrogenic satellite R N A s these
positions are respectively G, U and C whereas in all the
benign satellite RNAs of group B they are A, G and U
and are interrupted by extra nucleotides between
positions 322 and 323. The benign satellite R N A s of
group A resemble necrogenic or benign satellite R N A s of
group B at these positions.
Point mutations in the necrogenic domain
When we began our studies, the 1 satellite R N A was
the only known member of group A. Between nucleotide
259 (AsulI site) and the 3' end there are only five
differences between Y and 1 satellite RNAs and, of
those, two are not conserved in the necrogenic consensus
(Fig. 3). The other differences are three nucleotides at
positions 318,323 and 325. In order to test whether these
nucleotides are important for the induction of necrosis,
the Y satellite R N A sequence was altered by sitedirected mutagenesis at the c D N A level in these
positions (318 G -~ A, 323 U ~ G and 325 C --, U) to
create Y1 (Fig. 4).
The in vitro transcripts of the mutated satellite c D N A
were tested with CMV in 12 tomato plants (Table 1) and
as a control in tobacco plants. After 10 days, tobacco
inoculated with the Y1 transcript produced a bright
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1446
M. Devic, M. Jaegle and D. Baulcombe
3' E n d
AsulI
318
259
Y UUCGA~G~ ACACUCUGUUAGGUGGUAUC
II7N .............................
c AGUC
GUGGAUGACG
n
D
seq 10
ch 20
x15
Yn
x2n
x7
x12
Necrotic
---u . . . . . . . . . . . . . . . . . . . . . . . . .
G
Group B
G
,
UUCGAAAGAA
*
AAACUCUGU.
...........
B1 . . . . . . . . . . .
S .........
Q ...........
B2 . . . . . . . . . . .
B3 . . . . . . . . . . .
WL2 ...........
E ...........
CUUA..,UG~
369
UAUGCUGAUC
UCCGUGAAUG
UCUAUACAUU
CCUCUACAGG
. . . . . . . . . . . . . . . . . .
G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
•. . . . . . .
. . . . . . . . . . . . . . . . . .
G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
- . . . . . . . . .
c .......
. .
u
AGUCAUGACG
U
V
C
G
~
CACGCAGGGA
GAGGCUAAGG
CUUA.
A
. .UGC
. . . . . . . . . . . . . . . . . . . . . .
~ . . . .*. . . . . .
G-~..-U
..........
GUGG
. . . . . . . . . . . . . . . . . . . . . .
U--A
G-U
. . . . . . . . . . . . . . . . . . . . .
ACCC
AC .........
G ........
.
-G---GUAU-
AG---O
. . . . . . . . . . . . . . . . . . . . . .
C-U
A--G
........
**
CACGCAGGGA
GAAGCUAA~A
. . . . . . . . . . . . . . . .
G .....
.
****
*
*
CAOGCCAGUC
- ~ - - ~ - ~
.....
~
UCC~UG,AAUG
UGA
. . . . .
U--G
U~UAAACAUU
. . . .
u, -G---Gum-
AG
G . . . . . . . . . . . . . . . . . . . . . .
• *
*
***3
*
*
UGA
. . . . . . . . . . . . . . . .
-G---GUAU-
AG---U
. . . . . . . . . . . . . . . .
G . . . . . . . . . . . . . . . . . . . . . .
• *
*
.3.*
*
*
CGA
. . . . . . . . . . . . .
*
~----
c.......
c.......
c. . . . . . .
c.......
c.......
u
U
u
u
u
-G---GUAU-G---GUm-G---GUm-G---GUm-G---GUAU-
AG---U
. . . . . . . . . . . . . . . .
G ......
UGA
........
G . . . .
U--U.-
UGA
........
~ . . . . . . .
G . . . .
A=ACUCUGU
=
A---GUG---=C
U .........
U .........
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . .
•*
......
C*
****
. . . . . . .
*
*
G . . . . . . . . . . . . . . . . . . . . .
G . . . . . . . . . . .
U . . . . . . . .
**
*
****
*
*
UGA
........
AG
. . . . . . . . . . . . . . . . . . . .
G . . . . . . . . . . . . . . . . . . . . . .
UGA
. . . . . . . . . . . . . . . .
AG
. . . . . . . . . . . . . . . . . . .
G .............
• *
U . . . . . . . .
*
*
UGA
. . . . . .
----
UGA
. . . . . . . . . . . . . . . .
*
. . . . . . . . . . . . . . . . . . . . . .
===GA---GACG
CACGCAGGGA
*
-
U ....
CCUA=A=GGU =AUGC==UC
GA=GCUAAAA
C-U
. . . .
CCUCCACAGG
O--O.-
A
•
. . . . . . . .
***
CCUAUAAGGU
C . . . . . . .
AG---U
ACCC
kD .........
A . . . . . . . .
CA
c.......
AG
CCUC.ACAGG
A . . . . . . .
C A C G C A G G G A G A G G C U = A G = CUUA... = G = = A = G C U G A U C U C C = U G = A U G U C U A = = C A U U C C U = = A C A G G A C C C
====AUGACG
GUUGACGACG
.......
--U
UCUAU.CAUU
GUGG
****
G-U
UCCGUGAAUG
. . . . . . . . . . . . . . . . . . . . . . . . .
AAGUGUAUCC
.......
UACGCUGAUC
GUGG
x2c ...........c.......u -G---GUAU- - - G - - U
Benign B
GAAGCUAAGG
325
•. . . . . . . . .
U ........
A. . . . . . . . . . . . . . . . . . . . . . . . . . . .
G AGUC . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A.- . . . . . AU ..........
.............................
G AGUC . . . . . . . . . A ........
G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
- . . . . . . AU ..........
.............................
G AGUC . . . . . . . . . A ........
G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
•. . . . . . .
A-G ........
.............................
G AGUC . . . . . . . . . A ........
- . . . . . . . . . . . . . . . . . .
A. . . . . . . . . . . . . . . . . .
A . . . . . . . . . G AGUC . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
- . . . . . . . . .
G ........
.............................
G AGUC . . . . . . . . . A ........
G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
-. . . . . . . . .
G ........
.............................
G AGUC . . . . . . . . . A ........
G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
-. . . . . . . . .
G ........
.............................
G AGUC . . . . . . . . . A ........
=tJc=~,~c.~ ACaCUCUGO= A~O~OAV= - - = A U G A C G C A C = C A G G G A GA---GCUAAGG C U U A . . . U G C U A U G C U G A U C U C C G U G A A U G U C U A = = C A U U CC-' 'CAGG A C C C
Group A
R UUCGAAAGAA
ACACUCUGUU
AGGUGGUAUG
1 ............................
~c
OY2 ............................
Gc
WL1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
c
Benign A tmcc,aAac,;m ACaCUCUGUU A~U~GU~U=
Ra
AGUC
CACGCAGGGA
323
. . . . .
CUAC
C . . . . . . . . .
. . . . . .
. . . . .
U--U.-
. . . . .
C ....
U .......
U .........
AUU
.........
CUAC
C . . . . . . . . .
....
A
---UA
........
U .........
CUAC
~
ACCC
........
........
U .........
.........
C . . . . . . . . . . . . . . . . . . .
UCC==G=AUG U=UA==CAU= CC===CAGG
ACCC
Fig. 3. A l i g n m e n t of several satellite R N A s e q u e n c e s b e t w e e n position 259 (AsulI) a n d the 3' end. The sequences of the satellite R N A s
h a v e b e e n previously d e s c r i b e d by D e v i c et al. (1989) for Y a n d Ra, J a c q u e m o n d & L a u q u i n (1988) for I 1 7 N a n d R, R i c h a r d s et al.
(1978) for n, K a p e r et al. (1988) for D, seq 10, c h 20, x 15, x2n, x7, x 12 a n d x2c, K a p e r et al. (1986) for Yn, H i d a k a et al. (I 988) for E a n d
OY2, G a r c i a - A r e n a l et al. (1987) for G, B1, B2, B3, WL1 a n d W L 2 , C o l l m e r et al. (1983) for 1, G o r d o n & S y m o n s (1983) for Q and
A v i l a - R i n c o n et al. (1986) for S satellite R N A . I d e n t i c a l nucleotides are i n d i c a t e d by a dash, g a p s by a dot and non-consensus
nucleotides by a double dash. The asterisks m a r k the nucleotides in b e n i g n satellite R N A s w h i c h differ from the n e c r o g e n i c consensus.
yellow mosaic showing that the mutated R N A replicated
in plants. However, even after 5 weeks, tomato plants
inoculated with CMV and the Y1 satellite R N A did not
show necrosis (Table 1). With the control, a non-mutated
Y satellite transcript, the tomato plants died from the
systemic necrosis after 2 weeks. Northern blot analysis of
R N A extracted from tomato plants 15 days after
infection showed that there were approximately equal
amounts of progeny R N A in plants infected with Y or Y1
transcripts (data not shown). The sequence of the
progeny of Y1 mutant corresponded to the c D N A
sequence. These results therefore confirm that at least
one of the nucleotides at positions 318, 323 and 325 is
involved in induction of necrosis.
In order to evaluate whether identity at these positions
is sufficient to distinguish a necrogenic from a benign
satellite R N A , a second mutation was created in the
benign Ra satellite R N A . The natural form of Ra
satellite R N A is identical to the 1 satellite R N A at
positions 318, 323 a n d 325 but different at other
positions. The mutant Ral, created by site-directed
309
318
323
325
334
Y
gaagcuaaC~cUua...UgCUaugcUGAu
Ra
gaagcuaaAAcCuaUAAGgUCaugcCAGu
Y1
gaagcuaaGAcUua...GgUUaugcUGAu
Ral
gaagcuaaAGcCuaUAAUgCUaugcCAGu
Ra2
gaagcuaaGGcUua...UgCUaugcUGAu
Ra3
gaagcuaaAGcCua...UgCCaugcCAGu
Y2
gaagcuaaGGcUua...UgCUaugcCAGu
Necrogenic consensus
GA-
GCUAAGGCUUA.
. . UGCUAUGCUGAU
Fig. 4. A l i g n m e n t of progeny satellite R N A sequences b e t w e e n 309
and 334. L o w e r case letters are nucleotides c o m m o n to Y a n d R a
satellite R N A s . L i g h t u p p e r case letters represent nucleotides specific
to the R a satellite R N A . Bold u p p e r case letters c o r r e s p o n d to
nucleotides specific to the Y satellite R N A . The n e c r o g e n i c consensus
is from Fig. 3. T h e asterisk m a r k s the p o s i t i o n of the fourth m u t a t i o n in
t h e progeny from p l a n t s infected w i t h t r a n s c r i p t s of the R a l
construction.
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Necrogenic sequences in CMV satellite RNA
Table 1. Symptoms induced by mutant satellite RNAs
Satellite
Type of
symptoms
on tomato
Y
Ra
Y1
Y2
Ra 1
Ra2
Ra3
Y8
Necrosis
Attenuation~
Attenuation~
CMV symptoms
Attenuation3~
Necrosis
Necrosis
Necrosis
27
ORF I
27
ORF IIA'
Number of plants
1447
60
Aminoacids
ORF lib
Necrosis
With symptoms* With progeny RNAt
3' YWT
12
12
12
12
12
12
3
11
12
12
12
2§
12
12
311
lI
* Symptoms were noted 2 weeks after infection.
t Determined by Northern blotting.
:~Viral symptomswere not produced on systemicallyinfected leaves.
§ The progeny was approximately 2~ of the level of non-mutated
satellite RNA in the control plant.
IIThe progeny was approximately 20% of the level of non-mutated
satellite RNA in the control plant.
mutagenesis of the c D N A is identical to the Y satellite at
these three positions (Fig. 4). Transcripts of R a l
attenuated C M V symptoms on tobacco, as predicted for
an efficiently replicating R N A , but failed to induce
necrosis on tomato (Table 1) even 5 weeks after
inoculation. The progeny satellite of R a l retained the
three mutated sites but had acquired a fourth mutation
(C ~ U at position 326; Fig. 4) which corresponds to the
Y satellite R N A sequence. Therefore, in order to convert
Ra into a necrogenic satellite R N A , more changes are
required in addition to those at nucleotides 318, 323 and
325. Further constructions were designed to test the
effect of changing the spacing between positions 322 and
323 which, as described above, was apparently correlated with the necrosis induction by most strains of
satellite R N A .
In mutant Ra2, the sequence from 309 to 334 of the Y
satellite R N A was substituted into the corresponding
region of the Ra satellite R N A by site-directed mutagenesis. In effect, this modified 12 nucleotides in the Ra
satellite sequence including the three corresponding to
318, 323 and 325 of the Y sequence (Fig. 4). The
modification also changed the spacing between positions
322 and 323. In vitro transcripts of Ra2 c D N A induced
systemic necrosis on 12 tomato plants when inoculated
with CMV (Table 1). This result showed that, of the
sequence differences between Y and Ra satellite R N A s ,
those necessary for necrogenesis are between nucleotides
309 and 334. These sites are indicated on Fig. 3 with
asterisks.
In mutant Ra3 the three nucleotides corresponding to
positions 318, 323 and 325 were mutated as in Ra2. In
addition the spacing between positions 322 and 323 was
modified to resemble that in the Y satellite R N A (Fig. 4).
Frameshifl
Necrosis
3'Mutant
Fig. 5. Schematic representation of ORFs in the wild-typeY satellite
RNA (YWT) and in the mutated satellite RNA pY8. ORF IIA' and
ORF IIB are not in the same frame. ORF liB extends from nucleotide
151 to 333. The two arrows mark the position of nucleotides 318 to 325
on the two satellite RNAs. The black triangle indicates the position of
the frameshift.
Tomato plants that accumulated C M V and the transcripts of Ra3, as judged by Northern blot analysis (data
not shown), showed systemic necrosis (Table 1). It is
concluded therefore that the crucial feature of the Y
satellite R N A that affects the induction of necrosis is the
identity of at least one of the three nucleotides 318, 323
and 325 in an appropriate spacing. However, the
mutations introduced into the Ra3 construction had an
effect on the satellite R N A accumulation in addition to
an influence on symptom induction: only three of 12
tomato plants accumulated satellite R N A and showed
necrosis (Table 1). In these plants the satellite R N A was
approximately fivefold less abundant than in plants
inoculated with non-mutated satellite R N A (data not
shown).
A similar effect of mutation on satellite R N A
accumulation was observed with mutant Y2 (Table 1)
which was designed to test the effect of modifications to
sequences in Y satellite R N A located close to but not
within the region from 318 to 325. Tobacco plants
inoculated with C M V and the transcripts of Y2 normally
showed viral symptoms and very occasionally limited
yellow mosaic symptoms. T o m a t o plants inoculated
similarly showed viral symptoms rather than effects of
satellite R N A (Table 1). However, the absence or mild
nature of satellite RNA-induced symptoms m a y be a
consequence of the impaired ability of these mutant
R N A s to accumulate in infected plants; in most
inoculated plants, satellite R N A did not accumulate and
in the two plants in which it did, there was approximately 50-fold less Y2 R N A than in comparable plants
inoculated with Y satellite R N A (data not shown).
Are polypeptides encoded by the Y satellite RNA
necessary for the induction of symptoms ?
The necrogenic domain identified above is within a short
open reading frame (ORF liB; K a p e r et al., 1988) (Fig.
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1448
M. Devic, M. Jaegle and D. Baulcombe
5), but a frameshift mutation in ORF IIB had no
detectable effect. When the mutated transcripts were
inoculated with CMV to tomato plants (mutant pY8 in
Table 1), necrotic symptoms were induced 2 to 3 weeks
post-inoculation at the same time as symptoms on plants
inoculated with CMV and Y satellite RNA. As the
sequence of the progeny satellite R N A was identical to
that of the inoculum R N A (data not shown), the result
implies that polypeptides are not involved in the
induction of necrosis on tomato and that the effect must
involve a direct interaction of the satellite R N A with
host and/or viral components.
Discussion
In this paper, we have identified discrete features of the
Y satellite R N A within a 'necrosis-inducing' domain
which affects the ability of the R N A to induce a systemic
necrosis on tomato. The active domain requires the
presence of at least one of the following nucleotides in the
spacing of the Y satellite R N A : G at position 318, U at
position 323 and/or C at position 325. Similar results
have been obtained by Masuta & Takanami (1989) based
on analysis of the Y satellite R N A and the benign T73
satellite RNA, However because the T73 satellite R N A
has the same spacing as the Y satellite R N A between the
nucleotides 318 and 325, their study did not identify the
importance of nucleotide spacing.
Nucleotides outside the region from 318 to 325 may
also affect the necrogenesis on tomato as indicated by the
benign R satellite R N A (Jacquemond & Lauquin, 1988)
which is identical to the Y satellite R N A in that region. It
is likely that the feature of R satellite R N A which affects
necrogenicity is nucleotide 328 (Fig. 3). This is the only
difference between the R satellite R N A and the
consensus of necrogenic satellite RNAs in the 3' part of
the molecule (Fig. 3).
Results with a frameshift mutant (Y8) in ORF IIB,
which spans the necrogenic nucleotides, suggest that
polypeptides encoded by the satellite R N A (Hidaka et
al., 1988) do not play a role in necrotic symptom
induction but the possibility remains that there is
translational reinitiation at position 253, on the 3' side of
the mutation in Y8. However other necrogenic satellite
RNAs do not have an ORF which extends through
nucleotides 318 to 325 (Kaper et al., 1988). Analysis of
their sequences shows that they are all identical around
the positions implicated in necrogenesis (Fig. 3) and are
therefore likely to induce necrotic symptoms by the same
mechanism as Y satellite RNA. Presumably, the
necrogenic nucleotides form an R N A structure that
interacts with a second molecule either from the host or
the helper virus, or both, to trigger symptom production.
The secondary structure model of the Y satellite R N A
(Hidaka et al., 1988) is consistent with this idea, as the
necrosis-inducing domain is in a region that is highly
sensitive to nuclease attack and therefore accessible for
intermolecular interactions. We are now attempting to
exploit genetic variation in the host plant (White &
Kaper, 1987) and helper virus (Palukaitis, 1988; Masuta
et al., 1988) as means of identifying other components
that participate in these intermolecular interactions.
We thank Bgrbel K6hm for carrying out relevant experiments that
are not described here and Christopher Davies and Fr6d6ric Boccard
for helpful comments on the manuscript. We acknowledge the support
of the Agricultural Genetics Company to the Institute of Plant Science
Research and the Gatsby Foundation to the Sainsbury Laboratory. Use
of imported strains of CMV was licensed by MAFF (licence PHF
1162/71).
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