VIRUS GENES 7:3,265-275, 1993 © Kluwer Academic Publishers, Manufactured in The Netherlands

Nucleotide Sequence of the Coat Protein Gene of Citrus Tristeza Virus: Comparison of Biologically Diverse Isolates Collected in Israel

MUNIR MAWASSI, RON GAFNY, AND MOSHE BAR-JOSEPH S. Tolkowsky Laboratory, A.R.O. The Volcani Center, Bet Dagan 50250, Israel

Received May I0, 1992 Accepted July 1, 1992

Requests for reprints should be addressed to M. Bar-Joseph, The S. Tolkowsky Laboratory, A.R.O. The Volcani Center, Bet Dagan 50250, Israel.

Key words: closteroviruses, capsid protein, cDNA, polymerase chain reaction, aphid transmission

Abstract

The sequences of the coat protein genes of four seedling yellows (SY) and four non-SY (NSY) of citrus tristeza virus (CTV) isolates, which were collected in Israel over a period of 30 years, were analyzed. Pairwise comparisons showed extensive similarities in the nucleotide and amino acid sequences of six isolates designated the VT group. This group consists of three NSY isolates that cause a very mild CTV reaction on the sensitive combination of sweet orange (SwO) grafted on sour orange (SO), and three SY isolates that cause severe SY and SwO/SO reactions. MT, a CTV isolate that is consistently nontransmitted by Aphis gossypii, was found to be different in two amino acids (Val 103 and Glu t 13) from each of the A. gossypii transmissible CTV isolates. Sequencing of the cDNA clones obtained from ST, a variably transmitted CTV isolate, showed extensive sequence variation among the tested clones. The sequence information indicates that the current CTV epidemics in Israel are caused by at least two CTV subspecies (VT and HT) displaying extensive differences in their coat pro- tein genes.

Introduction

Citrus tristeza virus (CTV) causes one of the most important diseases of citrus. CTV is a member of the Closterovirus group and has long flexous filamentous particles, 10-12 nm in diameter and 2000 nm in length. CTV virions contain a positive, single-stranded RNA genome, of 20 kb estimated size and a coat protein

266 MAWASSI, GAFNY, AND BAR-JOSEPH

subunit, which runs in NaDodSO4-PAGE as a single band with an estimated molecular weight (MW) of 25-28 kD (1,2). Recently, the coat protein gene of the T36 Florida CTV isolate (3) has been cloned, sequenced, and identified as an ORF of 669 bases with a calculated MW of 24909.

Numerous isolates of CTV have been described from various citrus-growing areas of the world (2-8). These isolates often differ considerably in symptomatol- ogy in various citrus hosts. In some cases the involvement of more than one virus species has been suggested (8). However, it is generally accepted that the wide range of the symptoms are produced by variants of a single virus (1). These include the commonest syndrome in which there is a decline of sweet orange (SwO; Citrus sinensis) on sour orange (SO; C. aurantium) rootstocks. Other severe strains produce a seedling yellows (SY) reaction in inoculated sour orange, lemon, and grapefruit seedlings. Many CTV isolates produce stem-pitting symp- toms in acid lime (C. aurantifolia), and other severe CTV strains cause stem pitting in grapefruits and sweet oranges. The biological diversity of CTV strains is also reflected in their vector transmissibility. The Meyer lemon (MT) isolate was found to be consistently nontransmissible by Aphis gossypii (2), whereas many recent CTV isolates from Israel (9), California (10), and Spain (4) were transmitted very efficiently by this aphid. Other isolates, e.g., the ST from Israel, showed a variable transmission (11).

The aim of this research was to analyze the coat protein gene of a collection of isolates differing in host reaction and aphid transmissibility in an attempt to associate biological function with the CTV coat protein nucleotide and amino acid sequence motifs.

Materials and Methods

Virus strains and ELISA

The origins and collection dates of eight isolates and two subclones of CTV are summarized in Table 1. All CTV isolates were propagated in graft inoculated acid lime (C. aurantifolia) and allimau citrus (C. macrophylla) plants. The CTV titers in the new flush of allimau plants, 3 months postinoculation, were analyzed by ELISA (12).

Virus, ssRNA, and dsRNA isolation

CTV particles were partially purified from 30 g of allimau citrus bark according to Bar-Joseph et al. (13), and the viral RNA was extracted with phenol/chloro- form and ethanol precipitated. The RNA pellet was washed twice with 75% etha- nol, dried, and resuspended in water. Total dsRNA was extracted (14) from the bark of allimau citrus experimentally infected with the isolate VT.

COMPARISON OF THE COAT PROTEIN IN CTV STRAINS 267

Table I. Some biological characteristics of citrus tristeza virus isolates collected in Israel

Collection Host reaction Isolate/ ...... ELISA -- Transmissibility subclone Date Source" OD/405 Sw/SO SY by A. gossypii Ref.

Ach-T 1984 N1 0.75 Mild - + 26 HT 1978 NI 0.95 Severe - + 15 ST 1960 ND 1.04 Mild - - / + 11,15 ST4 1975 AT 0.81 ND - + Mik-T 1978 N1 0.73 Mild - + 15 MT 1979 IB 1.27 ND + - 2,15 VT 1970 NI 0.84 Severe + + 9 VTa 1975 AT 0.62 ND ND + Hed-T 1982 NI 1.13 Severe + + 15 Mor-T 1986 NI 0,96 Severe + + 26

aNl = natural infection; AT = subcloned by transmission with A. gossypii; IB = introduced through infected budwood; ND = not determined.

cDNA synthesis

C T V - R N A was h e a t e d for 10 min at 60°C wi th 0.2 i~g p r i m e r de s igna t ed oC, complemen ta ry to the 3' o f the CTV CP gene (5' C A G A T C A A C G T G T G T T G A 3'), and coo l ed on ice. The c D N A r e a c t i o n was p e r f o r m e d in a 20 ~1 mix tu re con- ta ining T a q p o l y m e r a s e buf fe r (P romega ) , 5 m M d i th io th re i to l d N T P s (0.5 m M each) , 40 U R N a s e inh ib i to r (Boehr inge r ) , and 25 U A M V r e v e r s e t r a n s c r i p t a s e (Promega) . The r e a c t i o n was ca r r i ed out fo r 60 min at 42°C.

Polymerase chain reaction (PCR)

The c D N A was ampl i f ied in a 100 txl r e a c t i o n mix tu re con ta in ing Taq D N A po ly - m e r a s e buf fe r (P romega ) , 10% d i m e t h y l su i fox ide D M S O , d N T P s (0.3 m M each) , 10 m M d i th io th re i to l , 0 .1% Tr i ton x-100, 60 pmol p r i m e r oC, 60 pmol p r i m e r des igna t ed oS , wh ich has the 5' s e q u e n c e o f the coa t p ro te in gene (5' A T G G A C - G A C G A A A C A A A G 3') , and 2.5 U Taq D N A p o l y m e r a s e (Promega) . The c D N A was ampl i f ied for 35 c y c l e s o f I min at 96°C, 2 rain at 48°C, and 3 min at 72°C. The coa t p r o t e i n gene f rom s o m e s t ra ins was ampl i f ied unde r the s a m e c y c l e cond i t i ons us ing the Ven t D N A p o l y m e r a s e (Bio labs) a cco rd ing to the manufac - t u r e r ' s i n s t ruc t ions . Then 10% of P C R p r o d u c t s were t e s ted in 1% aga rose gels .

Cloning

The P C R p r o d u c t s we re s e p a r a t e d in 1% aga rose gel and e lu t ed by Q i a e x get ex t r ac t i on ki t (Qiagen) . T h e c D N A p r o d u c e d b y ampl i f i ca t ion wi th Ven t D N A

268 MAWASSI, GAFNY, AND BAR-JOSEPH

polymerase was ligated to EcoR V restricted pBluescript vector (Stratagene). The cDNA produced with Taq DNA polymerase was ligated directly to pCR1000 vector (Invitrogene). The vectors were transformed into E. coli JM101 cells, and white colonies containing the CTV coat protein insert were screened and selected by PCR amplification of tooth picked colonies rapidly prepared by boiling.

Sequencing

The DNA for the sequencing reaction was prepared by plasmid midi preparation kit columns (Qiagen). Then 4 ~g DNA were denaturated by heating for 5 min at 85°C in 200 mM NaOH, and precipated by K-acetate and ethanol. The sequencing reactions were performed by the Sequenase version 2 (USB) kit according to the manufacturer's instructions using 35S-dATP for labelling. The products were run on 6% hydrolink (AT Biochem) gels containing 45% urea.

Results

Biological characteristics of CTV isolates

Table 1 summarizes the collection time and origin of eight CTV isolates that were used for analysis. All CTV isolates were continuously maintained in grafted acid lime or allimau seedlings. In addition, two aphid transmitted subclones designated ST4, which was obtained from ST, and a subclone VTa, which was obtained by aphid transmission of the VT strain, were also analyzed.

All tested isolates had high ELISA titers in allimau citrus, but differed consider- ably in their effects on the CTV-sensitive combination of SwO/SO (Table 1). Four of the CTV isolates induced an SY reaction on SO or lemon seedlings. One of the isolates, designated MT, was nontransmissible by A. gossypii (2).

Sequence analysis

The nucteotide sequences of the coat protein gene of Israeli CTV isolates were analyzed using cDNA amplified by PCR. The PCR products from all the virus strains gave the same size of about 670 bp in 1% agarose gel. The DNA bands were extracted from the agarose and cloned either into pCR1000 vector or pBlue- script vector restricted by EcoR V as described. The identity of the clones was confirmed by PCR, using the specific primers oC and oS and by hybridization with total RNA prepared from healthy and CTV-infected allimau citrus. Plasmids from selected clones that reacted specifically with CTV RNA were sequenced. The sequencing was done at least for two different clones for every strain. The total of 669 nucleotides of the coat protein genes of eight Israeli CTV isolates

COMPARISON OF THE COAT PROTEIN IN CTV STRAINS 269

and two subclones are shown in Fig. l, and the results of their pairwise compari- son are shown in the upper right half of Table 2.

The resulting proteins have calculated MWs ranging between 23,891 and 24,985. Six CTV isolates--Ach-T, Mik-T, VT, VTa, Heal-T, and Mor-T (the VT group)--were found to have differences in 1-6 bases only. The ST-C1 isolate and an aphid-transmitted subclone ST4 were also similar to the VT group and were different in only six to nine nucleotides. One of the local isolates, HT, and the introduced isolate, MT, showed significant differences, having 47-53 and 47-54 nucleotide differences, respectively (Table 2). MT differed in 47 nucleo- tides from HT. Twenty-one base changes, however, were common to MT and HT, but different from the VT group. In positions 58 and 343 four and five isolates had G and T replacements for A and C, respectively. The sequences of the coat protein cDNAs constructed on ssRNA and dsRNA templates from VT isolate were identical (not shown). Hybridization experiments using the CTV coat pro- tein clones (Mawassi et al., in preparation) showed that the coat protein cDNA did not hybridize with the CTV dsRNA fragment of 0.8 kbp, which previously was considered as the CTV coat protein coding sequence (15).

The amino acid (a.a.) sequence was highly conserved among CTV isolates of the VT group, as shown in Fig. 2 and in the pairwise comparison in Table 2 (lower left). Two isolates, Mor-T and VT, had identical amino acid sequences, whereas Ach-T, Hed-T, and Mik-T differed from VT by 1, 2, and 4 a.a., respec- tively. The aphid-transmitted subclone ST4 differed in 4, 10, 12, and 13 a.a. from four clones designated ST-C1, ST-C2, ST-C3, and ST-C4, respectively, which were derived from the variably aphid-transmitted isolate ST (Fig. 3), whereas VTa differed only in 2 a.a. from VT.

In four CTV isolates--HT, MT, VT, and Mor-T--Asn in position 20 was re- placed by Asp, and in isolates HT, ST-C1, ST4, Mik-T, and MT, Pro in position If5 was replaced by Ser.

The a.a. Ile in position 103 was replaced in the nontransmissible isolates MT and ST-C2,C3 by Val and Met, respectively. The MT isolate differed in two amino acids (Vat 103 and Glu 1t3) from each of the A. gossypii transmissible CTV isolates.

Discussion

Extensive biological diversity among CTV isolates collected in Israel over the last 30 years was previously reported (t6). Serological assays using polyclonal antibodies failed to differentiate among distinct CTV strains (2), whereas recent studies from Florida, using a monoclonal antibody line designated MCA-13, showed differences between mild and severe CTV isolates (17). Differences in the coat protein among some Spanish isolates and between the Spanish and those from Florida were also indicated by proteolytic digestion (7). The involvement of the coat protein gene in pathogenicity was recently demonstrated in plants

270 MAWASSI, GAFNY, AND BAR-JOSEPH

i0 20 30 40 50 60 70

1 ATC-C4~GACGAA CAAAGAAATTGAAGAAC2u%AAACAAGGAAACGAAAGAAGGCGACAATGTTGTTGCAGCGGAGTCT

2 ........................................... A ........... -G .......... T--C ......

3 .............................................................................

4 ..............................................................................

5 ............................ - .................................................

6 ........................................................ -G .......... T--T ......

7 ....................................................... -G ....................

8 .............................................................................

9 ..............................................................................

i0 ........................................................ -G . . . . . . . . . . . . . . . . . . . .

80 90 100 110 120 130 140 150

1 TCTTTCGGTTCTGTAAACTTACACATCGATCC GACTCTGATAGCGATGAACGATGTGCGTCGGTTGGGTACCCAACAG

2 ..... A .... C ........................ T ..... A .................. A .... A ...........

3 ..............................................................................

4 ...................................................... T .......................

5 .................................. C ..........................................

6 ......... -C ................................................. A ................

7 ..............................................................................

8 ..............................................................................

9 ..............................................................................

i0 ..............................................................................

160 170 180 190 200 210 220 230

1 AATGCCGCTTTGAACAGAGATTTGTTTC TTACTTTGAA GAGAAGTATCCTAATTTGTC TGATAAAGATAAGGACTTT

2 --C--T .............. C--A--C ............. G----C ....... C--AC ....................

3 ...................................................................... T .......

4 ..............................................................................

5 ..............................................................................

6 ..... T ........ T ................ -C ....... G ........... -C--AC----C ........ A ......

7 ..............................................................................

8 ..............................................................................

9 .............................................. T ...............................

i0 .............................................................................

240 250 260 270 280 290 300 310

I CACTTAGCTA~TGTTGTATCGTTTAGCGGTTAAGAGTTCATCATTGC2%AAGCGATGACGACACTACGGGTATAACG

2 ---A ......................... A ................. A .............. -G-C ............

3 ..............................................................................

4 ..............................................................................

5 ............................................................................ A-

6 ................. A ........... A ................. A ....... -C--T--T--C ..... -G-G---

7 ............................................................................

8 ..............................................................................

9 ..............................................................................

i0 ..............................................................................

320 330 340 350 360 370 380

1 TACACTC~GTCGAAGTGGATTTGCCTGACAAACTTTGGACTGACGTCGTGTTrAATTCTAA~TATCGGT

2 .............. T--T ............ T ..... G ........................ -C ........... T---

3 .............................. T ...............................................

4 ..... A ........................ T ...............................................

5 .............................. T ...............................................

6 - .......... A--T--T ........ A---T .............................. -C ........... T---

7 ..............................................................................

8 ..............................................................................

9 ..............................................................................

I0 ..............................................................................

Fig. 1. Comparison of 669 nucleotide sequence of the citrus tristeza virus coat protein gene among seedling yellows (SY) and non-SY CTV isolates collected in Israel over 30 years. Isolates: (1) Ach-T, (2) HT, (3) ST, (4) ST4, (5) Mik-T, (6) MT, (7) VT, (8) VTa, (9) Hed-T, (10) Mor-T. * Isolates causing SY reaction.

C O M P A R I S O N O F T H E C O A T P R O T E I N 1N CTV S T R A I N S 271

400 410 420 430 440 450 460

1 AACCGTACTAACGCCCTTCGAGTCT~AGGACTAACGATGCCCTTTATTTAGC~TAGACAGAATCC4?2~T

2 ...................... T ........ A ................ -C .... T .... C ...............

3 ........................ A ............................... -C ....................

4 ....................... A ................................ C ....................

5 ..............................................................................

6 ................................ A .................... G- -T .............. C ..... C

7 ..............................................................................

8 ..............................................................................

9 ..............................................................................

10 ................. C ............................................................

480 490 500 510 520 530 540

1 T TGAG~ATGC~CGTC CGCTAGATGCAGC, CaATTC CGC4~CCK?4~TATCATTAC CTGTGTGC, AGATTTC TTGACC GGA

2 .......... GC ............. -C ........................... T ....... G ........... T--G

3 ............. A ........... G ....................................................

4 ............. A ............................... A ................................

5 ..............................................................................

6 .......... GT ......... T .......... A ..... A ........... C ...........................

7 ..............................................................................

8 ..............................................................................

9 ..............................................................................

i0 ..............................................................................

550 560 570 580 590 600 610 620

1 GC TGC4ZTTGACTGATTTAGAGTGTGCTGTGTACATACAAGCTAAAGAACAATTGTTGAAGAAGC GAGCa?4~CTGATGAA

2 ..... T .............. A ................................ A ....................... T

3 ..............................................................................

4 ...............................................................................

5 ..............................................................................

6 ..... A .............. A ..... A .................... G ..... A--A .................... G

7 ..............................................................................

8 ..............................................................................

9 ..................................................... A ........................

i0 ..............................................................................

630 640 650 660 699

1 GTCGTAGTTACCAA~C-CTTC.~:-C~AAATTCAACACACGTTGA

2 ........... T ....................................

3 ................................................

4 ................................................

5 ................................. C ..............

6 ........... T ....................................

7 ................................................

8 ................................. C ..............

9 ................................................

i0 ................................................

Fig,, 1 (cont inued)

Table 2. N u m b e r of n u c l e o t i d e ~ and a m i n o ac id b d i f f e rences a m o n g c i t rus t r i s t eza v i rus i so la t e s

A c h - T H T ST-C 1 ST4 Mik-T MT VT VTa H e d - T Mor-T

Ach-T - - 48 6 7 4 49 1 I 2 2

HT 14 - - 52 53 50 47 47 49 48 48

ST-C1 6 18 - - 5 8 53 7 7 8 8

ST4 6 18 4 - - 9 54 8 8 9 9

Mik-T 3 15 7 7 - - 51 5 3 6 6

MT 8 10 12 12 9 - - 48 50 49 49

VT 1 13 7 7 4 7 - - 2 3 l

VTa t 15 7 7 2 9 2 - - 3 3

Hed-T I 14 7 7 4 9 2 2 - - 4 Mor-T 1 13 7 7 4 7 0 2 2 - -

a U p p e r r ight ha l f o f tab le . b L o w e r left ha l f o f table .

272 MAWASSI, GAFNY, AND BARJOSEPH

60

1 ) M~DETKKLKNK~q<ETI~GDNVVAAES SFGSVNLHIDPTL~ L

2 ) .................. D ........ S ........... T .... -Q-S ...........

3) ..........................................................

4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L ...............

5) ..........................................................

6) .................. D ......................... -Q .............

7 ) ................... D ........................................

8) ............................................................

9) ..........................................................

I0) ................... D ........................................

120

1 ) F LTLKEKYPNLSDKDKDF}~.AMMLYRLAVKS SSLQSDDDTTGI TYTREGVEVDLPDKLWT

2 ) ..... G-H---P ....... I ................... A .............. S-E---

3) .............. -M ...................................... S .....

4) ...................................................... S .....

5) ........................................... K .......... S .....

6) ..... G ..... P .............................. V ......... E-S .....

7) ............................................................

8) ............................................................

9 ) ....... F ....................................................

i0) ............................................................

180

1 ) DVVFNSKG I GNRTNALRVWGRTNDALYLAF CRQNRNLSYAGRPLDAGI PAGYHYLCADFL

2) ....................................... G ....................

3) .................. R .......... L .......... E---G ...............

4 ) .................. R .......... L .......... E .......... N ........

5) ............................................................

6 ) ....................................... G ....................

7) ............................................................

8) ............................................................

9) ............................................................

i0) ............................................................

223

1 ) TGAGLTDLECAVY I QAKEQLLKKRGADEVVVTNVRQLGKFNTR

2 ) ........................... D ...............

3) ...........................................

4) ...........................................

5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L---

6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8) ....................................... L---

9) ...........................................

I0) ...........................................

Fig. 2. Comparison o f amino acid sequences of the citrus tristeza virus coat protein gene among seedling ye l lows (SY) and non-SY CTV isolates collected in Israel. Isolate numbers are the same as in Fig. 1. * Isolates causing SY reaction.

infected with tobacco mosaic virus (TMV). Mutants of TMV with changes in the coat protein gene were found to become localized by the N' gene of Nicotiana sylvestris plants or to escape the action of the N' gene (18,19). Pairwise compari- son showed extensive similarities among the nucleotide and amino acid sequences of six isolates designated the VT group and consisting of three N S Y isolates (Ach-T, Mik-T, and ST), causing a very mild CTV reaction on the sensitive combination of sweet orange (SwO) grafted on sour orange (SO) and three SY isolates (Hed-T, VT, and Mot-T) that cause severe SY and SwO/SO collapse reactions (Figs. I and 2). These results indicate that the SY and SwO/SO reac- tions are not directly associated with a specific coat protein gene sequence variant.

Considerable differences were observed between the VT group and HT, a local NSY isolate. HT was found to be related to MT, an SY isolate originating from

COMPARISON OF THE COAT PROTEIN IN CTV STRAINS 273

6O 1 ) ~DETKKLKNKNKE TEEGDDWAAE S SF GSVNLHI DPTL IA~VRRLGTQQNAAI/~DL

2 ) ............................................ L ...............

3) ............................................................

4 ) ........................................ T .... -Q--A ..........

5) ................................... V .... T .... -Q .............

6 ) ........................................ T .... -Q .............

7) ............................................. -Q .............

120

1 ) FLTLKEKYPNLSDKDKDFHLAMMLYRLAVKSS SLQSDDDTTGITYTREGVEVDLPDKLWT

2) ...................................................... S .....

3) ............... M ...................................... S .....

4 ) ......................................... -M ........... S .....

5) ......................................... -M--G ........ S .....

6) ..... G---S ......... I .................................. S .....

7) ..... G ..... P .............................. V ......... E-S .....

180

1 ) DVVFNSKG I GNRTNALRVWGRTNDALYLAF CRQNRNLSYAGRPLDAG I PAGY HYLCAD FL

2 ) .................. R .......... L .......... E .......... N ........

3) .................. R .......... L .......... E---G ...............

4 ) ....................................... GE ...................

5) ....................................... G ....................

6) ............................. S---D ..... G ....................

7) ....................................... G ....................

223

1 ) TGAGLTD LE CAVY I QAKEQLLKKRGAD EVVVTNVRQLGKFNTR

2) ...........................................

3) ...........................................

4) ......... R .................................

5) ......... R .................................

6 ) ................ R ..........................

7) ...........................................

Fig. 3. Comparison of amino acid sequences of the citrus tristeza virus coat protein gene among cDNA clones obtained from (1) VT, (2) ST4, (3) ST-C1, (4) ST-C2, (5) ST-C3, (6) ST-C4, and (7) MT.

China (2), and to T36, an SY isolate previously sequenced in Florida (3). MT, a CTV isolate that is consistently nontransmitted by A. gossypii (2), was found to be different in two amino acids (Val 103 and Glu 113) from each of the A. gossypii transmissible CTV isolates.

Comparisons of the predicted amino acid sequences of cDNA clones derived from ST and ST4 (Fig. 3) suggest that the ST isolate is composed of at least two coat protein variants, represented by (a) ST-C1 and ST4, and (b) ST-C2, ST-C3, and ST-C4. Such extensive variation within a single source was probably the result of super or coinfection of the original nontransmissible ST isolate (9) by a second transmissible CTV strain.

In the potyvirus group both the coat protein (20) and a nonstructural protein, the helper component (21), are involved in aphid transmission. Preliminary tests did not indicate the involvement of a nonstructural helper factor in CTV transmis- sion (Raccah and Bar-Joseph, unpublished). Further studies will be needed to determine which of the a.a. changes found among the ST variants and in MT affect their transmission by A. gossypii.

Expression of the MT coat protein in transgenic citrus is expected to result in plants expressing coat protein mediated cross protection (22) and a reduced risk

274 MAWASSI, GAFNY, AND BAR-JOSEPH

m

1 -

0

" 1 - -

- 2

- 3 - -

1 0 0 2 0 0

, i i i I | T " | " i ' J i""""|''"I'"~|'l | i | i J | | I 21

. .......... o

-2

I I I I i I 1 1 1 I I J I I I I I I I ! I I -3 1 O0 2 0 0

Fig. 4. The hydropathy plot of the coat protein of the VT isolate of citrus tristeza virus (24).

of CTV transmission by A. gossypii. Regions of conserved amino acids (Fig. 2) were observed among the tested isolates. Additional isolates will have to be compared in order to locate regions with strict conservat ion, e.g., like the RNA binding motifs previously observed in T M V (23),

The hydropathy plot (24) of the coat protein of the VT isolate is presented in Fig. 4. The N-terminal amino acids 1-27 form a hydrophyllic domain. It was noted (3) that this domain is proteolyt ical ly digested, both in planta and during virus purification. We predict , therefore, that this domain is exposed on the sur- face of the CTV particles. The evolut ionary relationships of the eight CTV iso- lates were not calculated and will have to await further characterizat ion of other local isolates and additional regions of the CTV genome.

The clustering relationships of the nucleotide sequences, as illustrated in Fig. 5, indicate that the coat protein gene of different isolates with diverse biological

I - t

MT e HT Mik-T Mor-T e VT e Ach-T

l .... -7[_ ST-C | Hed-T e

Fig. 5. A tree showing the clustering relationships between the nucleotide sequences of the coat protein gene of citrus tristeza virus isolates collected in Israel. The vertical branch lengths are propor- tional to the similarities between the sequences. The dendogram was produced by the UWGCG program PILEUP (25). * Isolates causing SY reaction.

COMPARISON OF THE COAT PROTEIN IN CTV STRAINS 275

properties might be rather similar and that current CTV epidemics in Israel (2) probably originated from at least two CTV subspecies (VT and HT) having exten- sive differences in the coat protein gene.

Acknowledgments

The authors thank Rose Gofman, D. Shalitin and J. Ben-Shalom for their skillful technical help. This study was supported by grants from BARD, GIARA, the Biotechnology Center, ARO, and the Citrus Marketing Board.

References

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