Nucleotide and amino acid variation in the coat protein coding region of turnip mosaic virus...

Physiological and Molecular Plant Pathology (1997) 51, 195–208

Nucleotide and amino acid variation in the coat protein

coding region of turnip mosaic virus isolates and

possible involvement in the interaction with the brassica

resistance gene TuRB01

P. L", K. P#, C. J$, A. G%, J. SC #, E. K"

and J. A. W*$

" Institute of Plant Genetics, Polish Academy of Sciences, PoznanU , Poland, # Institute of Plant Molecular Biology,Academy of Sciences of the Czech Republic, Cw eskeU Bude] jo�ice, Czech Republic, $Horticulture Research International,Wellesbourne, Warwick, CV35 9EF, U.K. and %Zeneca Agrochemicals, Jealott’s Hill Research Station, Bracknell,RG12 6EY, U.K.

(Accepted for publication August 1997)

The nucleotide and predicted amino acid sequences of the coat protein coding region of fourisolates (UK 1, CZE 1, CDN 1 and GK 1) and a putative mutant of one of these isolates(UK 1M) of turnip mosaic virus (TuMV) were determined. Comparisons between these andthose of previously sequenced isolates showed homologies of 88±6–99±9% in nucleotides and93±4–100% in amino acid sequences. Nucleotide substitutions were revealed at 180 differentpositions that give rise to 30 predicted amino acid changes. The coat protein coding regionsconsisted of 864 nucleotides, with the exception of GK 1 which had 861, having a deletion inamino acid position 44. This is the first recorded deletion in the coat protein coding region ofTuMV. Alignment of the predicted amino acid sequences of UK 1, CZE 1, CDN 1 and GK 1isolates with the previously sequenced CDN 2, CHN, JPN 1, JPN 2, JPN (31), L12396 and KORisolates revealed three distinct groups which, with a few exceptions, were correlated with thegeographical and}or host plant origin of isolates. The GK 1 isolate showed the lowest nucleotide(88±6–89±9%) and amino acid (93±4–96±2%) homologies with the other isolates, whereas theUK 1 isolate showed the highest nucleotide homology (89±7–99±9%) and CDN 1 showed thehighest amino acid homology (95±9–99%). The coat protein coding region of the putative mutantisolate (UK 1M) had a single nucleotide difference (G to A) from UK 1 at position 9 which didnot give rise to any change in the predicted amino acid sequence. UK 1M is able to infect Brassicanapus plants possessing a mapped resistance gene (TuRB01) that is effective against UK 1. Thephenotypes of most of the sequenced isolates on plants containing TuRB01 are known, and theseare consistent with the hypothesis that the single nucleotide substitution at position 9 of the coatprotein coding region conditions the interaction with this gene. All isolates tested that had theresidue G* gave no detectable replication in plants possessing TuRB01, whereas isolates that hadthe residue A* were either partially virulent on plants carrying TuRB01 causing local infectiononly, or were fully virulent resulting in full systemic infection. This hypothesis is unprecedentedin plant virus interactions and further experimentation utilizing full-length infectious clones ofTuMV will be necessary to test its validity. # 1997 Academic Press Limited

*Author to whom correspondence should be addressed: Dr John A. Walsh, Plant Pathology andMicrobiology Department, Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, U.K.

Abbreviations used in text : CP, coat protein; ELISA, enzyme-linked immunosorbent assay; NTR, non-translated region; NIb, nuclear inclusion protein b.

0885–5765}97}09019514 $25.00}0}pp970116 # 1997 Academic Press Limited

196 P. Lehmann et al.

INTRODUCTION

Turnip mosaic virus (TuMV) is the most important virus infecting cruciferous crops

and occurs in many parts of the world, including the temperate and tropical regions of

Africa, Asia, Australia, Europe, and North and South America [21].

The virus has a wide host range of over 318 plant species in 156 genera of 43 plant

families, including many cultivated and weed plants [6]. It is transmitted by at least

89 species of aphids [5] in the non-persistent manner. Considerable biological

variation in TuMV isolates has been observed, particularly in terms of their interaction

with different host plants and resistance genes [9, 11]. However, nothing is known

about the molecular basis of this variation.

TuMV is a member of the Poty�irus genus which is the largest genus of plant viruses

(180 members) and is now assigned to the Potyviridae family, recently reported to

contain 198 viruses [23]. Its genome consists of one molecule of single-stranded, positive

sense RNA of approximately 10 kb with a polyadenylated 3« end. This is expressed as

a large polyprotein which is subsequently cleaved by proteases to yield several

functional proteins. The coat protein (CP) coding region of viruses in the Potyviridae

is the most extensively characterized region of their genome [23], and CP amino acid

sequence homologies have been used to define distinct members of the potyvirus group

(sequence identities ranged from 38 to 71%; average 54%) and strains of individual

viruses (sequence identities of 90–99%; average 95%) [22]. As well as encapsidating

the viral nucleic acid, other functions assigned to it include: short distance movement,

vector transmissibility [1] and symptom expression of infected plants [19]. It has also

been shown that although the CP product is not required for genome amplification,

translation to a position between CP codons 138 and 189 is required [12].

Nucleotide sequences for the CP of a number of different isolates of TuMV have

been published in recent years [3, 10, 13, 14, 20]. In this paper we present the nucleotide

and predicted amino acid sequences of the CP of a putative mutant and four isolates

of TuMV representing the most common and widespread pathotypes occurring in

Europe, discuss homologies with other isolates and suggest that the CP RNA may have

a role in the interaction with the Brassica gene, TuRB01, which confers resistance to

some isolates of TuMV.

MATERIALS AND METHODS

Virus isolates

Where known, the pathotype, geographical and host plant origins and EMBL

database entries for sequences of the TuMV isolates UK 1, UK 1M, CDN 1, CDN 2,

CHN, GK 1, CZE 1, JPN 1, JPN 2, JPN (31) and an isolate of unknown origin, are

given in Table 1. The isolate UK 1M is thought to be a mutant of the UK 1 isolate and

its origin was described by Jenner and Walsh [9]. These isolates were propagated in

mustard (Brassica juncea) cv. Tendergreen. In the case of CHN, there were discrepancies

between the published sequence and the EMBL database entry, so we used the latter

in our comparisons.

Turnip mosaic virus coat protein variation 197

T 1Host plant, geographical origin, pathotype, citation and EMBL database accession for TuMV isolates

where known

TuMVisolate Plant origin

Geographicalorigin Pathotype Citation

EMBLaccession

UK 1 Brassica napus England 1 [26] X65978UK 1M B. napus England 3 [9] X81141CDN 1 B. napus Canada 4 [26] X81140CDN 2 B. napus Canada 3 [14] D10927CHN Raphanus sati�us China N}A [10] X52804CZE 1 B. oleracea Czech

Republic3 [9] YO9144

GK 1 Matthiola incana Greece 9 [26] YO9114JPN 1 R. sati�us Japan 7 [13] D83184JPN 2 R. sati�us Japan 7 [20] D88614JPN (31) R. sati�us Japan N}A [13] N}AKOR R. sati�us Korea N}A [3] X83968L12396 N}A N}A N}A N}A L12396

N}A¯not available.

F. 1. Nucleotide sequencing strategy of pUC18 plasmids containing the amplified coat proteinand 3« non-translated region (NTR) of TuMV; PUC1 and PUC2 universal primers withsequences corresponding to the pUC18 sequence; PS5–PS10, primers with sequences cor-responding to the CP coding region of the CDN 2 isolate of TuMV. The direction of sequencingis indicated by arrows. NIb, nuclear inclusion b protein. The HindIII and SalI restriction sitespresent in the primers P3S and P4S are shown. The position of the 5« end of the coat proteincoding region is marked (59), as is the 3« end (926).

Isolation of RNA

Total RNA was extracted from 500 mg of infected mustard leaves frozen in liquid

nitrogen. The leaves were powdered using a mortar and pestle before homogenization

in buffer: 25 m Tris-HCl (pH 7±5) containing 25 m MgCl#, 25 m KCl and 1%

SDS. This mixture was subsequently extracted twice with phenol, RNA was

precipitated in ethanol and resuspended in water.


cDNA synthesis and PCR

All amplification and sequencing primers were designed from the published sequence

of the CDN 2 isolate of TuMV [14]. Oligonucleotides were synthesized by the

phosphoramidite method [24] on an Applied Biosystems synthesiser (Applied

Biosystems, Foster City, U.S.A.). Viral RNA was reverse-transcribed following the

method given in the Cetus GeneAmp RNA kit (No. N808-0017; Perkin Elmer,

Norwalk, U.S.A.). The incubation mixtures (20 µl) contained 50 U of reverse

transcriptase (Moloney murine leukaemia virus), 20 U of RNase inhibitor, 2±5 µ

oligo(dT)"'

primer and 0±1 µg of total RNA. Incubations were performed at 42 °C for

15 min, and then the reverse transcriptase was inactivated by heating at 99 °C for

5 min. Two amplification primers at the concentration of 0±5–1±0 µ, 2±5 U of

AmpliTaq DNA polymerase and PCR buffer were added to the reverse transcriptase

mixture to give a final volume of 100 µl. PCR was then performed for 1 min at 94 °C,

1 min at 55 °C and 2 min at 72 °C, for 35 cycles. PCR products were analysed on 1±5%

(w}v) agarose gels.

The amplification primer P3S contained an oligo d(T)"(

tail and the primer P4S had

a sequence corresponding to the 3«-end of the NIb gene of TuMV. Additional

nucleotides were added to the primers to provide SalI and HindIII restriction sites as

shown below for subsequent cloning (restriction sites are in bold) :

P3S 5«-GGCCACGCGTCGACTAGTACTCGAGTTTTTTTTTTTTTTTTT-3«P4S 5«-ATCAAGCTTCAGGCAATCTTTGAGGATTAT-3«

Cloning and sequence analysis

All the cloning steps were conducted using standard protocols. The PCR fragments of

predicted length, 1135 bp, were purified from agarose gels and ligated into pUC18

plasmids (Amersham International, Bucks., U.K.). The presence and orientation of

each cloned fragment was confirmed by restriction analysis. Two independent clones,

each containing a full-length CP gene and a 3« non-translated region, were sequenced

for each virus isolate (UK 1, UK 1M, CZE 1, CDN 1 and GK 1) using the strategy

shown in Fig. 1. Double-stranded DNA templates were prepared as described in the

method given in the Promega Magic Miniprep kit (Promega, Madison, U.S.A.).

Denatured plasmids were sequenced directly using the Sequenase version 2±0 DNA

sequencing kit (U.S. Biochemical, Cleveland, U.S.A.).

Sequence data, multiple alignment and phylogenetic relationships were analysed

using the PC-GENE programme package version 6±50 (IntelliGenetics, Mountain

View, U.S.A.).

Interaction of TuMV isolates with plants containing the resistance gene TuRB01

The phenotypes of the isolates UK 1, UK 1M, CDN 1, GK 1, CZE 1, CDN 2, JPN 1,

and JPN 2, were determined on oilseed rape plants (Brassica napus), containing the gene

TuRB01 conferring an extreme form of resistance to some isolates of TuMV [Walsh,

unpublished] and on another oilseed rape line (R4) [9], containing a single dominant

resistance gene [26] with virtually identical specificity to TuMV isolates as TuRB01


[9]. We were not able to obtain the isolates CHN, JPN (31), KOR and L12396 to

determine their phenotypes.

RESULTS

The nucleotide sequences of the CP coding region of TuMV isolates UK 1, UK 1M,

CZE 1, CDN 1 and GK 1 are presented in Fig. 2. In all isolates the CP consisted of 864

nucleotides with the exception of GK 1, which had 861 due to a triplet deletion at

position 129–131. The CP coding regions of all isolates were terminated with the UGA

stop codon, except that of GK 1, which was terminated by the codon UAA (Fig. 2).

There was only one nucleotide difference between the UK 1 and UK 1M isolates. This

was at position 9 and it did not change the predicted amino acid (Fig. 3). Comparison

of the nucleotide sequence of the CP of CDN 1 with that published for CDN 2 showed

98% homology, with 17 substitutions, of which three led to differences in predicted

amino acids. This demonstrates that CDN 1 and CDN 2 are distinct isolates.

Comparisons between the nucleotides of the CPs of the sequenced isolates and those of

previously sequenced isolates (after alignment of the predicted amino acid sequences)

revealed homologies of 88±6–99±9% (Table 2). The GK 1 isolate showed the lowest

homology with other isolates (88±6–89±9%) and UK 1 showed the highest (89±7–

99±9%). Most of the substitutions were in the region close to the 5« end rather than

the central and 3« terminal regions (Fig. 2). Differences in the first and second

positions of codons that lead to amino acid changes represented 25% of substitutions.

Six of these that coded for leucine involved CTN to TTPu substitutions and did not

change the predicted amino acid. The highest homologies of the GK 1 sequence with

any other potyvirus CP that we could find were 68% (plum pox virus, lettuce mosaic

virus and sweet potato G virus) and 69% (tulip mosaic virus).

Alignment of the predicted amino acid sequences of the CPs of the isolates UK 1,

CZE 1, CDN 1, GK 1, CDN 2, CHN, JPN 1, JPN 2, JPN (31), KOR, and L12396

revealed amino acid homologies of 93±4–99±7% (Table 2). Again the GK 1 isolate had

the lowest homology with other isolates (93±4–96±2%). The highest homology of GK 1

with any other potyvirus that we were able to find was 71% with plum pox virus. The

CDN 1 isolate had the highest homology with other isolates (95±5–99%). There were

35 differences in predicted amino acid sequence between isolates and these occurred in

32 positions. Of these, 19 (in 17 positions) were in the N-terminal region (positions

1–53), 15 (in 14 positions) were in the core region (positions 54–269) and one was in

the C-terminal region (positions 270–288). In comparison with the fully sequenced

isolate, CDN 2, the newly sequenced isolates, UK 1 and CDN 1, showed high amino

acid homology, whereas the GK 1 isolate had lower homology and the CZE 1 isolate

had an intermediate homology (Table 2). At positions 43 and 50 of the GK 1 isolate,

where differences with CDN 2 and some other isolates were seen, the amino acids were

identical with those of the KOR, JPN 1, JPN 2, and CHN isolates, and at positions 20

and 79, where further differences with CDN 2 and other isolates were seen, they were

identical to those of JPN 2 and CHN. The GK 1 isolate had unique amino acids at

positions 2, 31, 45, 108, 112, and 164 in comparison with other sequenced isolates of

TuMV and the only recorded deletion at position 44. Of the other isolates sequenced

by us, only CZE 1 had a unique amino acid (position 84). Phylogenetic analysis of the


F. 2. For legend see opposite.

CP amino acid sequences clustered the isolates into three groups (Fig. 4). Group 1

comprised isolates predominantly from Europe and North America (there was only one

exception, the Japanese isolate JPN (31)). Group 2 consisted of exclusively Asian

isolates, and group 3 contained the Greek isolate (GK 1) only.


F. 2. Comparison of the nucleotide sequences of the coat protein coding regions of the CDN 1,UK 1, UK 1M, CZE 1, and GK 1 isolates of TuMV with that of the CDN 2 isolate. Thetranslation stop codon for the single viral open reading frame is at positions 865–867. (*) Identicalnucleotides ; ([) similar nucleotides.

The DAG triplet (Asp-Ala-Gly) of the N-terminal domain of the coat protein of

TuMV isolates, which is involved in aphid transmission of potyviruses [1], is present

in UK 1, CDN 1, GK 1, L12396, JPN 1, and CHN, whereas CDN 2, CZE 1 and KOR

had Asp-Ala-Asp, JPN 2 had Asp-Thr-Gly and JPN (31) had Asp-Ala-Asn.

The UK 1M isolate of TuMV exhibited an altered phenotype on plants with the

gene TuRB01 and plants of the line R4 compared with UK 1. Unlike UK 1, which

gave no detectable infection on such plants, UK 1M induced a severe systemic necrotic

infection of all plants ; this was lethal in some plants. When the nucleotide sequences


F. 3. For legend see opposite.

and phenotypes of CZE 1, CDN 1, CDN 2, GK 1, JPN 1, and JPN 2 on plants

possessing TuRB01 and R4 plants was compared with UK 1 and UK 1M (Table 3),

it became evident that all isolates with G at position 9 (UK1, JPN 1, and JPN 2) were

unable to infect plants with TuRB01 and R4 plants. All other isolates had A at position


F. 3. Coat protein amino acid sequence alignment of TuMV isolates showing wheredifferences occur.

T 2Percentage coat protein amino acid homology (abo�e diagonal) and nucleic acid homology (below diagonal)

of TuMV isolates

UK 1UK1M CDN 2 CDN 1

JPN(31) CZE 1 L12396 KOR JPN 1 JPN 2 CHN GK 1

UK 1 — 100±0 99±7 98±6 97±6 97±9 96±9 94±8 95±8 94±8 94±1 95±8UK 1M 99±9 — 99±7 98±6 97±6 97±9 96±9 94±8 95±8 94±8 94±1 95±8CDN 2 97±8 97±9 — 99±0 98±6 98±3 97±2 95±1 96±2 95±1 94±4 95±1CDN 1 98±2 98±3 98±0 — 98±6 98±6 98±3 95±5 97±2 96±2 95±5 96±2JPN (31) 98±0 97±9 96±8 96±9 — 99±0 98±3 95±1 97±9 95±5 94±8 94±8CZE 1 93±5 93±7 93±4 92±8 93±3 — 97±6 95±5 97±2 95±5 94±8 94±8L12396 97±1 97±0 95±8 96±0 97±7 92±4 — 95±1 96±9 95±1 94±4 94±4KOR 93±4 93±5 93±7 93±5 93±3 91±5 92±8 — 96±9 95±8 96±2 93±4JPN 1 96±8 96±7 95±4 95±7 97±8 92±3 96±7 94±1 — 97±6 96±9 95±1JPN 2 90±3 90±2 89±6 90±1 90±2 88±7 90±1 94±1 91±4 — 98±6 95±5CHN 89±7 89±6 89±3 89±5 90±8 88±6 89±5 94±2 90±8 98±7 — 94±8GK 1 89±7 89±9 89±3 89±5 88±9 89±6 88±6 89±2 89±0 89±2 89±0 —


F. 4. Computer-drawn phylogenetic tree derived from the predicted amino acid sequencehomologies of TuMV isolates.

T 3Nucleotide sequences of the N-terminal ends of the coat proteins of TuMV isolates, amino acids at position3, and phenotypes obtained on Brassica napus plants with the TuMV resistance gene TuRB01 or plants

of the R4 B. napus differential line (where known) for sequenced isolates

TuMVisolate(pathotypewhereknown) Nucleotide sequence positions 1–21

Aminoacidat

position3

Phenotypeson B. napuslines N-o-1(possesses

TuRB01) andRf [predictedphenotype]

UK 1(1) 5«-GCA GGT GAG ACG CTT GAT GCA-3« E 0JPN 1(7) . . . . . . . . G . . . . . . . . . . . . E 0JPN 2(7) . . . . . . . . G . . . . . . . . . A . C E 0CHN . . . . . . . . G . . . . . . . . C . . C E [0]KOR . . . . . . . . A . . . . . . . . C . . C E [}

N}R}R

N]

UK 1M(3) . . . . . . . . A . . . . . . . . . . . . E N

GK 1(9) . . . AA . . . A . . A . . . . . . . . T E RN

CZE 1(3) . . . . . C . . A . . . . . . . . C . . . E n

CDN 1(4) . . . . . . . . A . . . . . . . . . . . . E CDN 2(3) . . . . . . . . A . . . . . . . . C . . . E

N

0¯No symptoms and no virus detected; N

¯ systemic infection where local symptomswere necrotic ; ¯ systemic infection (without necrosis) ; R

N¯ local necrotic infection and

no systemic spread.


9 and were able to partially (GK 1) or fully (UK 1M, CZE 1, CDN 1, CDN 2)

overcome these resistances.

DISCUSSION

As in other potyviruses, the N-terminal domain of the CP of the TuMV isolates was

found to be the most variable region of the CP. This region and the P1 and P3 proteins

are the most variable regions of potyviruses [23]. Our prediction of one amino acid

difference between the 11 isolates in the C-terminal 19 residues was one less than that

reported by Shukla et al. [23] when comparing CDN 2, CHN and JPN 2. This was

because we used the CDN 2 sequence reported by Nicolas and Laliberte! [14], which

differs from that reported by Tremblay et al. [25] and used by Shukla et al. [23], due

to what was believed to be a cloning artefact. The homologies between the amino acid

sequences of the different TuMV isolates (93±4–99±7%) are within the range reported

for other potyviruses (90–99%) [22]. The isolate that had the lowest homology with

other isolates (GK 1; 93±4–96±2%) had much lower homology with other potyviruses

(maximum 71%), confirming that all isolates were TuMV. This was also evident from

comparisons between the 3« non-translated regions of these isolates which had identities

of 93±3–99±5% [15], well within the range reported by Frenkel et al. [7] (83–99%) for

strains of other potyviruses. The UK 1 and CDN 1 isolates with the highest nucleotide

and amino acid homologies, respectively, with other sequenced TuMV isolates may be

the best models for a consensus sequence of the TuMV CP. With 288 (or 287 in the case

of GK 1) amino acids, the CP of TuMV is within the size range of other potyviruses

(251–332 amino acids). The deletion in the GK 1 isolate is the first reported for the CP

of TuMV. Of the 13 CP amino acids that were conserved amongst 26 aphid-

transmitted and 5 mite- and fungus-transmitted potyviruses and the further 42 that

were conserved amongst the 26 aphid-transmitted potyviruses [23], all of the former

except one (position 61 of TuMV CP) and all of the latter except two (positions 135

and 181 of TuMV CP) were conserved amongst the TuMV isolates.

The apparent geographical clustering of most of the Asian isolates and most of the

European plus New World isolates based on CP amino acid homologies suggests a

degree of genetic isolation and divergent evolution between these two regions. This is

also implied by the pathotypes of the isolates, where known (Table 1). The isolates

UK 1, CZE 1, CDN 2 and CDN 1 in group 1 belong to a number of different

pathotypes (1, 3, 3 and 4, respectively), JPN 1 and JPN 2 belong to pathotype 7 and

GK 1 belongs to pathotype 9 [9]. The presence of one Asian isolate in the European

cluster means that either this isolation has not been complete, or there has been parallel

evolution. The host plant origin (where known) of these isolates implies that the

clustering obtained could possibly be due to the plant genetic background. All isolates

in group 1 (for which the host is known) with the exception of JPN (31), came from

brassica crop species [25, 26], all isolates in group 2 came from Raphanus sati�us (radish)

as did JPN (31) [10, 18, 20], and the GK 1 isolate representing group 3 came from

Matthiola incana (stock) [26]. The very wide host range of TuMV (widest of all

potyviruses in terms of plant genera and families) [23] and the widespread movement

of plant germplasm suggest that the JPN (31) isolate is a relatively recent introduction

from abroad and favours a geographical explanation for clustering. Nucleotide


sequences have been found to assign tomato yellow leaf curl virus isolates to

specific groups that were country-specific [4].

In regions of the CP where the amino acid sequences of the Asian isolates differed

from those of the European and North American isolates, the GK 1 isolate has affinities

with the former (positions 20, 43, 50, and 79) and the latter (positions 34, and 53).

This, combined with the fact that this isolate had the lowest homologies with other

isolates, makes it tempting to speculate that it may represent an ancestral type from

which the other sequenced isolates, all of which (where the original host is described)

were from cultivated crops, have evolved. Although GK 1 infects B. juncea systemically,

it only systemically infects one of the B. napus differentials from the European

pathotyping system, and even this line is infected weakly [9]. This suggests that it is not

well adapted to cultivated brassicas. Just one other isolate belonging to the same

pathotype (9) as GK 1 has been found so far [9], and this originated from Cheiranthus

cheiri (wallflower). Many other isolates have been collected from Greece and all came

from cultivated brassicas. Although no sequence information is available on these

isolates, the phenotypes of most of them on the B. napus differentials (pathotype 1) were

very different to GK 1 and identical to some isolates in group 1 of Fig. 4 [9]. The

evolution of viruses is considered to result from point mutation, recombination of

subgenomic fragments, overprinting of existing genes, and replication slippage.

Sequence data on the UK 1, UK 1M, CDN 1, CDN 2, CHN, KOR, and L12396

isolates provided no evidence of recombination in the CP and the 3« non-translated

regions [17]. However, recombination was detected or suspected in 18 out of 109

potyvirus isolates tested. Sano et al. [20] suggested RNA recombination may have

occurred between JPN 2 and a plant cellular sequence. Utilizing the sequence data of

CDN 2, Hancock et al. [8] found evidence of replication slippage in the CP and 5«terminus regions of the TuMV genome.

The predicted amino acid sequences of the DAG triplets of all the sequenced isolates

suggested that the isolates CDN 2, CZE 1, JPN 2, JPN (31) and KOR would not be

aphid transmissible, whereas UK 1, CDN 1, GK 1, L12396, JPN 1 and CHN should

be aphid transmissible. The UK 1 isolate of TuMV is known to be aphid transmissible

[26]. Sako [18] reported that JPN 1 was aphid transmissible, whereas JPN (31) had

been aphid transmissible but was no longer. No biological data on the aphid

transmissibility of CDN 1, CDN 2, CZE 1, GK 1, JPN 2, CHN, KOR, and L12396 are

available.

The possibility that the UK 1 isolate could comprise a mixture of the UK 1 and

UK 1M genotypes where the latter is omnipresent has been considered, but this seems

extremely unlikely for a number of reasons. If the UK 1M genotype was always

present, then on every occasion when the R4 plant line, or plants containing TuRB01,

were inoculated, they would become infected. Although the UK 1 isolate has been seen

to give the altered phenotype on these resistant plants more than once, in the vast

majority of tests this was not the case. Also, 34 single lesion (clonal) isolates have been

prepared from the UK 1 isolate on tobacco plants and when these were subsequently

inoculated to R4 plants none caused infection. The single nucleotide difference

between the CPs of the UK 1 and UK 1M isolates might have indicated that the

dramatic change of phenotype seen on plants containing the TuRB01 resistance gene

and plants of the line R4 was due to another mutation in one or more of the other


coding regions of the viral genome. However, the relatively frequent occurrence of this

phenotypic change in a number of TuMV isolates from different parts of the world

suggests that it may involve a small change in the viral genome. This, combined with

the perfect correlation between the nucleotides at position 9 of the CP and the

interaction with TuRB01 and the gene in the plant line R4, strongly suggests that this

nucleotide is the virulence}avirulence determinant for these genes. The fact that this

nucleotide change does not give rise to any change in the predicted amino acid from

this codon indicates that any interaction with the resistance gene or its product must

be at the RNA level. Ultimate proof for this hypothesis will be dependent upon the

production of a full-length infectious clone which can have the base at position 9

substituted and phenotypes verified on R4 plants and plants containing TuRB01.

There is an example of a single nucleotide change giving rise to a change in amino acid

of the CP of tobacco mosaic virus, and being responsible for the induction of severe

symptoms in Nicotiana tabacum [2]. A single nucleotide substitution in the αa gene of

barley stripe mosaic virus (BSMV) also gave rise to an amino acid substitution

enabling an isolate that was normally non-pathogenic to oats to infect oats [27].

Whether it was the change in the viral RNA or the concomitant amino acid changes

that induced these two phenotypic switches is not known. A point substitution in the

5« non-coding region of BSMV RNA allowed the type strain of this virus to undergo

long-range systemic movement in Nicotiana benthamiana which the wild type was

incapable of [16]. This is a precedent for single nucleotides conditioning movement of

viruses in plants. Our hypothesis of a single nucleotide determining complete

virulence}avirulence is perhaps without precedent in plant viruses.

This research was funded by the European Community (project no. ERBCIPACT

923007), MAFF and BBSRC, U.K. The authors wish to thank P. E. Kyriakopoulou

for providing TuMV GK 1, V. Shattuck for CDN 1, M. Fortin for CDN 2, N. Sako for

JPN 1, and Y. Sano for JPN 2.

REFERENCES

1. Atreya CD, Raccah B, Pirone TP. 1990. A point mutation in the coat protein abolishes aphidtransmissibility of a potyvirus. Virology 178 : 161–165.

2. Banerjee N, Wang J-Y, Zaitlin M. 1995. A single nucleotide change in the coat protein of tobaccomosaic virus is involved in the induction of severe chlorosis. Virology 207 : 234–239.

3. Choi GS, Choi JK. 1993. Nucleotide sequence of coat protein gene of turnip mosaic virus (cqs strain).Korean Journal of Plant Pathology 9 : 256–262.

4. Deng D, McGrath PF, Robinson DJ, Harrison BD. 1994. Detection and differentiation of whitefly-transmitted geminiviruses in plants and vector insects by the polymerase chain reaction withdegenerate primers. Annals of Applied Biology 125 : 327–336.

5. Edwardson JR, Christie RG. 1986. Turnip mosaic virus. In: Viruses infecting forage legumes.Volume II. Florida Agricultural Experiment Station Monograph 14. University of Florida, Gainesville,438–453.

6. Edwardson JR, Christie RG. 1991. The potyvirus group. Volumes I–IV. Florida AgriculturalExperiment Station Monograph 16. University of Florida, Gainesville.

7. Frenkel MJ, Ward CW, Shukla DD. 1989. The use of 3« non-coding nucleotide sequences in thetaxonomy of potyviruses : application to watermelon mosaic virus 2 and soybean mosaic virus–N.Journal of General Virology 70 : 2775–2783.

8. Hancock JM, Chaleeprom W, Chaleeprom W, Dale J, Gibbs A. 1995. Replication slippage in theevolution of potyviruses. Journal of General Virology 76 : 3229–3232.

9. Jenner CE, Walsh JA. 1996. Pathotypic variation in turnip mosaic virus with special reference toEuropean isolates. Plant Pathology 45 : 848–856.


10. Kong L-J, Fang R-X, Chen Z-H, Mang K-Q. 1990. Molecular cloning and nucleotide sequence ofcoat protein gene of turnip mosaic virus. Nucleic Acids Research 18 : 5555.

11. Liu XP, Lu WC, Liu YK, Wei SQ, Xu JB, Liu JB, Liu ZR, Zhang HJ, Li JL, Ke GL, Yao WY,Cai YS, Wu FY, Cao SC, Li YH, Xie SD, Liu BX, Zhang CL. 1996. Occurrence and straindifferentiation of turnip mosaic potyvirus and sources of resistance in Chinese cabbage in China. ActaHorticulturae 407 : 431–440.

12. Mahajan S, Dolja VV, Carrington JC. 1996. Roles of the sequence encoding tobacco etch virus capsidprotein in genome amplification: requirements for the translation process and a cis-active element.Journal of Virology 70 : 4370–4379.

13. Nakashima H, Sako N, John K, Hori K, Nonaka F. 1991. Nucleotide sequences of the coat proteingenes of aphid transmissible and non-transmissible isolates of turnip mosaic virus. Annals of thePhytopathological Society of Japan 57 : 549–557.

14. Nicolas O, Laliberte! J-F. 1992. The complete nucleotide sequence of turnip mosaic potyvirus RNA.Journal of General Virology 73 : 2785–2793.

15. Petrzik K, Lehmann P. 1996. Classification of turnip mosaic virus isolates according to the 3«-untranslated region. Acta Virologica 40 : 151–155.

16. Petty ITD, Edwards MC, Jackson AO. 1990. Systemic movement of an RNA plant virus determinedby a point substitution in a 5« leader sequence. Proceedings National Academy of Sciences U.S.A. 87 :8894–8897.

17. Revers F, Le Gall O, Candresse T, Le Romancer M, Dunez J. 1996. Frequent occurrence ofrecombinant potyvirus isolates. Journal of General Virology 77 : 1953–1965.

18. Sako N. 1980. Loss of aphid transmissibility of turnip mosaic virus. Phytopathology 70 : 647–649.19. Salomon R. 1992. Proteolytic cleavage of the N-terminal region of potyvirus coat protein and its

relation to host recovery and vector transmission. In: Barnett OW, ed. Poty�irus Taxonomy. Archi�es ofVirology (Supplement 5) : 75–76.

20. Sano Y, van der Vlugt R, de Haan P, Takahashi A, Kawakami M, Goldbach R, Kojima M.1992. On the variability of the 3« terminal sequence of the turnip mosaic virus genome. Archi�es ofVirology 126 : 231–238.

21. Shattuck VI. 1992. The biology, epidemiology and control of turnip mosaic virus. In: Janick J, ed. PlantBreeding Re�iews 14. John Wiley & Sons, New York, U.S.A., 199–238.

22. Shukla DD, Ward CW. 1988. Amino acid sequence homology of coat proteins as a basis foridentification and classification of the potyvirus group. Journal of General Virology 69 : 2703–2710.

23. Shukla DD, Ward CW, Brunt AA. 1994. The Poty�iridae. CAB International, Wallingford, U.K.24. Sinha ND, Biernat J, McManus J, Ko$ ster H. 1984. Polymer support oligonucleotide synthesis

XVIII: use of β-cyanoethyl-N,N-dialkylamino-}N-morpholino phosphoramidite of deoxynucleosidesfor the synthesis of DNA fragments simplifying deprotection and isolation of the final product. NucleicAcids Research 12 : 4539–4557.

25. Tremblay MF, Nicolas O, Sinha RC, Lazure C, Laliberte! JF. 1990. Sequence of the 3«-terminalregion of turnip mosaic virus RNA and the capsid protein gene. Journal of General Virology 71 :2769–2772.

26. Walsh JA. 1989. Genetic control of immunity to turnip mosaic virus in winter oilseed rape (Brassicanapus ssp. oleifera) and the effect of foreign isolates of the virus. Annals of Applied Biology 115 : 89–99.

27. Weiland JJ, Edwards MC. 1996. A single nucleotide substitution in the αa gene confers oatpathogenicity to barley stripe mosaic virus strain ND18. Molecular Plant – Microbe Interactions 9 : 62–67.

Nucleotide and amino acid variation in the coat protein coding region of turnip mosaic virus...

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