Journal of Virological Methods Rapid production of ... - Science Direct

Journal of Virological Methods,

43 (1993) 0 1993 Elsevier Science Publishers B.V. All rights reserved / 0166-0934/93/$06.00

VIRMET

7-20

Journal of Virological Methods

01486

Rapid production of full-length, infectious geminivirus clones by abutting primer PCR (AbP-PCR) R.W. Briddon,

A.G. Prescott, P. Lunness, and P.G. Markham

L.C.L.

Chamberlin

Department of Virus Research, John Innes Institute, John Innes Centre for Plant Science Research, Norwich (UK) (Accepted

3 December

1992)

Summary The application of the polymerase chain reaction (PCR) method of DNA amplification for the isolation of full-length, infectious clones of geminiviruses is described. Non-overlapping, abutting 20-mer oligonucleotide primers were used to produce a linear product from the circular geminivirus genomic template. Clones of African cassava mosaic virus (ACMV) DNA A, obtained by this method, were infectious following mechanical inoculation (in the presence of ACMV DNA B) onto Nicotiana benthamiana. Normal ACMV symptoms resulted and typical geminate viral particles were detected by electron microscopy. The use of PCR for the detection and production of fulllength, infectious geminivirus clones is discussed. Polymerase chain reaction; Geminivirus; Abutting primer

Introduction The geminiviruses form a diverse group of plant-infecting viruses that are characterised by having a twinned isometric (geminate) particle (Bock et al., 1974) encapsidating a genome consisting of circular single-stranded DNA (Goodman, 1977). More than 30 plant diseases are now recognised as being Correspondence to: Centre

R.W. Briddon, Department of Virus Research, John for Plant Science Research, Colney Lane, Norwich NR4 7UH, UK.

Innes

Institute,

John

Innes

8

caused by members of this virus group (Brunt et al., 1990). A number of these viruses cause significant crop losses; for example, maize streak virus (MSV) and ACMV in Africa (Efron et al., 1989; Fargette et al., 1985) beet curly top virus (BCTV) in the United States of America (Fullerton, 1964) and tomato yellow leaf curl virus (TYLCV) in the Mediterranean region (Pilowsky and Cohen, 1990). The geminiviruses are transmitted either exclusively by the whitefly, Bemisia tabaci, to dicotyledonous plants (e.g., ACMV and TYLCV) or to either monocotyledonous or dicotyledonous plants by differing leafhopper species (e.g., MSV and BCTV, respectively). This dichotomy also extends to the genome arrangement of these viruses. The large group of whitefly-transmitted geminiviruses usually have a bipartite genome with the coat protein and all functions required for replication encoded on DNA A and genes required for movement of the virus within plants, encoded on DNA B (Davies and Stanley, 1989). However, one whitefly-transmitted virus (TYLCV) is reported to have a single genomic component which encodes all functions (Navot et al., 1991; Kheyr-Pour et al., 1992) and in this respect resembles the leafhoppertransmitted viruses (Davies and Stanley, 1989). In the investigations presented here, we demonstrate the use of a novel polymerase chain reaction-based technique of obtaining full-length, infectious geminivirus clones from nanogram quantities of total nucleic acids extracted from diseased plants.

Materials and Methods Primer design and synthesis Non-overlapping, abutting primers were designed to the coding region of the coat protein gene of the ACMV clone, pJSO92 (Stanley and Gay, 1983) as shown in Fig. 1. The primers were produced with 5’ GGG ends so as to produce %a1 restriction endonuclease sites (5’-CCCGGG-3’) when blunt-end ligated into SmaI linearised vectors. This required a single base change for each primer but did not alter the coding specificity of the coat protein open reading frame. Oligodeoxynucleotide primers were synthesised by standard phosphoramidate

Viral strand primer 5'-GGGGGCCTGGGCTGACACAC-3' 5'-TCTGTAAGGTGATTAGTGATGTGACGCGTGGGCCTGGGCTGACACACAGGGTCGG-3' 3'-AGACATTCCACTAATCACTACACTGCGCACCCGGACCCGACTGTGTGTCCCAGCC-5‘ 3'-CCACTAATCACTACACTGGG-5' Complementary strand primer Fig. 1. The viral and complementary strand abutting primers are shown aligned to the sequence of the coat protein gene of the Kenyan isolate of ACMV. Sequence changes introduced into the primers so as to produce 5’-‘GGG’ ends are shown highlighted.

9

chemistry on a Pharmacia Gene Assembler Plus oligonucleotide synthesiser. After detachment of the oligonucleotide from the support in 35% (v/v) ammonia at 55°C for 16 h, the samples were ethanol precipitated and resuspended in water. No further puriIication was used. Sources of total nucleic acid Samples of total nucleic acid were produced from ACMV-infected and healthy iVicotiana benthamiana plants by grinding approximately 0.5 g leaf tissue with sterile sand in the presence of 1 ml extraction buffer [ 10 mM TrisHCl (pH 7.0), 100 mM NaCl, 10 mM EDTA, 1% (w/v) SDS] at room temperature in a mortar and pestle. Samples were extracted three times with buffer (50 mM Tris-HCI [pH 8.31) saturated phenol, nucleic acids ethanol precipitated at -20°C overnight and redissolved in sterile distilled water. Nucleic acid content was estimated spectrophotometrically at 260 nm on a Hitachi V-3200 spectrophotometer. Infected leaf material was obtained from N. benthamiana plants mechanically inoculated with either the cloned genomic components of the Kenyan isolate of ACMV (Stanley, 1983) or with sap extract of cassava plants infected with an as yet uncharacterised isolate of ACMV originating from the vicinity of the village of Ogorocco, Nigeria. This isolate was collected by Dr. S.A. Shoyinka in 1990. Ampl@ation

conditions

PCR reactions were carried out essentially as described previously (Saiki et al., 1988). Amplifications were performed in volumes of 100 ,LL~ containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCI, 0.01% (w/v) gelatin, 200 ,LLMeach of dATP, dTTP, dGTP and dCTP (Pharmacia), 0.05 PM of each primer, 100 ng of total nucleic acid, 2.5 U of Taq polymerase I (Amplitaq, Perkin Elmer Cetus) and were overlayed with 100 ~1 of mineral oil (Sigma) to reduce evaporation. Reactions were carried out in a Techne PHC-3 Thermal Cycler programmed for 50 cycles of 40 s at 94”C, 1 min at 55°C and 4 min at 72”C, using the fastest available transition between each temperature. Isolation, cloning and analysis of amplification

products

Amplification products were separated on 1% agarose gels and isolated by freeze-squeeze (Thuring et al., 1975), following ethidium bromide staining. Gel isolated products were ligated into SmaI linearised Ml3 mp18 (Norrander et al., 1983) without further treatment. Cloning and transformation of Escherichia coli employed standard procedures (Sambrook et al., 1980). The nucleotide sequences of the coat protein genes of clones pPCR4(N), pPCRS(N) and pPCR6(N) were determined by dideoxynucleotide chain termination se uencing (Sanger et al., 1980) using Sequenase Version II (USB) and [(a- 39S]dATP (New England Nuclear) in the forward direction and

IO

the fmol Cycle Sequencing System (Promega) and [y-33P]rATP (New England Nuclear) in the reverse direction. Where necessary, specific primers were produced to extend the sequence. Inoculation

and analysis oj’plants

N. benthamiana seedlings were mechanically-inoculated with the linearised clones obtained by PCR, in the presence of a dimer of ACMV DNA B (Klinkenberg et al., 1989) as previously described (Stanley, 1983). Control inoculations were performed using ACMV DNA A (linearised from clone pCLVl.3A (Klinkenberg et al., 1989) using restriction endonuclease EcoRV) in the presence of a dimer of DNA B. Plants were screened daily for ACMV symptoms. Infected plants were examined by immunosorbent electron microscopy (Roberts and Harrison, 1979) using a polyclonal antiserum raised against a Kenyan isolate of ACMV (Stanley et al., 1985).

Results PCR amplfication

of ACMV

DNA A

The results of a typical amplification of ACMV DNA A from an extract of an infected plant is shown in Fig. 2. A band of approximately 2800 bp corresponding to full-length ACMV DNA A was amplified in all reactions

M

12345678

7000 2100820

-

Fig. 2. Ethidium bromide-stained agarose gel of samples amplified from N. henthamiana plants infected with the Kenyan clone of ACMV. Samples in lanes 1 to 5 were amplified from 100, 50, 150,200 and 250 ng of input nucleic acid, respectively. Samples in lanes 6 to 8 resulted from amplification of reaction mixtures containing no input DNA, 100 ng of input DNA but no primers and 100 ng of nucleic acids extracted from a healthy plant. The position of the full-length amplification product is indicated with an arrow and the sizes of co-electrophoresed markers (lane M) are given.

containing target DNA, primers and enzyme (lanes 1 to 5). No specific product was produced in the absence of either primers or target DNA (lanes 7 and 8, respectively), nor from reactions which contained total nucleic acids extracted from a healthy plant (lane 6). Optimum input concentration was found to be between 50 and 150 ng of total nucleic acid extracted from infected plants in this case. The optimum concentration varied between individual extracts (results not shown). The yield of amplified viral DNA was estimated to be approximately 20 pg per 100 ~1 reaction, from an input of 100 ng of total nucleic acid. In addition to the full-length DNA A product, minor bands demonstrated to be ACMV DNA A specific (by Southern blotting and hybridisation, results not shown) were detected. The precise nature of these bands has not been investigated. Cloning and infectivity analysis Six clones, three for each ACMV isolate, containing potentially full-length ACMV DNA A amplified genomic components were chosen for further analysis. All three clones obtained from the Kenyan isolate (pPCRl(K), pPCR2(K) and pPCR3(K)) and two obtained from the Nigerian isolate (pPCRS(N) and pPCR6(N)) proved infectious to N. benthamiana by mechanical inoculation in the presence of a dimer ACMV DNA B (Table 1). The latent periods (the time between inoculation and symptom appearance) for the control DNA A clone (pCLV1.3A) and the PCR-derived clones pPCRl(K), pPCR2(K) and pPCR3(K) were identical (between 5 and 7 days). Symptom appearance with clones pPCRS(N) and pPCR6(N) were delayed 1 to 2 days over those of the controls. N. benthamiana plants infected with PCR-derived clones of the Kenyan isolate of ACMV showed symptoms typical for the Kenyan isolate (chlorotic lesions on the leaf surface and leaf curling (Stanley, 1983)) which were indistinguishable from control inoculated plants. Symptoms produced by the two clones obtained from the Nigerian ACMV isolate were less severe than the Kenyan isolate with the appearance of yellow chlorotic lesions on the leaf surfaces being delayed by up to 5 days. Initial symptoms TABLE I Infectivity of PCR-derived Clone no.

pCLVl.3A pPCRl(K) pPCR2(K) pPCR3(K) pPCR4(N) pPCRS(N) pPCR6(K)

clones Infectivity (plants infected/inoculated)

+ + + + + + +

DNA DNA DNA DNA DNA DNA DNA

B B B B B B B

1

2

3

IO/IO 919 lo/lo IO/IO O/IO 7110 7110

IO/l0

919 IO/l0 lo/lo IO/IO 019 S/IO S/l0

919 IO/IO IO/IO O/5 s/10 S/IO

12

B 1 2

3

C

123123

lin SC

Fig. 3. Southern blot analysis of DNA extracted from clones pCLVl.3A (A), pPCRS(N) (B) and pPCR6(N) either untreated (I), digested with MU (2) or digested DNA forms

N. benthamiana plants mechanically inoculated with (C) in the presence of ACMV DNA B. Samples were with Smal(3). The position of supercoiled and linear are shown.

were almost exclusively leaf curling. Southern blot analysis of restriction endonuclease digested nucleic acid extracts of N. benthamiana plants infected with the PCR-derived clones showed that the introduced SmaI site was maintained during infection (Fig. 3). In addition, analysis of infected leaf material by immunosorbent electron microscopy showed the presence of typical geminate particles for all five infectious clones (results not shown). The DNA sequence of the coat protein gene for the clones obtained from the Nigerian isolate are shown in Fig. 4 and the predicted amino acid sequence derived from this in Fig. 5. These sequences were compared to two published ACMV coat protein sequences, one from Kenya (ACMV-K, Stanley and Gay, 1983) and one from Nigeria (ACMV-N, Morris et al., 1990). The PCR-derived Nigerian clones were distinct from both ACMV-K and ACMV-N. The alignments show 11 nucleotide substitutions in ACMV-K when compared to all four Nigerian sequences; these result in 6 amino acid changes. ACMV-N contains 4 nucleotide sequence variations in comparison to both ACMV-K and the PCR-derived Nigerian clones; resulting in a single amino acid alteration in the coat protein. Eight nucleotide differences to both ACMV-K and ACMV-N

13

ACMV-K ACMV-N pPCR4 (N) pPCR5(N) pPCR6 (N)

ATGTCGAAGC ATGTCGAAGC ATGTCGAAGC ATGTCGAAGC ATGTCGAAGC

GACCAGGAGA GACCAGGAGA GACCAGGAGA GACCAGGAGA GACCAGGAGA

TATCATCATT TATCATCATT TATCATCATT TATCATCATT TATCATCATT

TCCACTCCAG TCCACTCCAG TCCACTCCAG TCCACTCCAG TCCACTCCAG

50 GATCCAAGGT GCTCGAAGGT GCTCCAAGGT GCTCCAAGGT GCTCCAAGGT

ACMV-K ACMV-N pPCR4(N) pPCR5 (N) pPCR6 (N)

51 TCGTCGAAGA TCGTCGAAGG TCGTCGAAGG TCGTCGAAGG TCGTCGAAGG

CTGAACTTCG CTGAACTTCG CTGAACTTCG CTGAACTTCG CTGAACTTCG

ACAGCCCATA ACAGCCCATA ACAGCCCATA ACAGCCCATA ACAGCCCATA

CAGGAACCGT CAGGAACCGT CAGGACCCGT CAGGAACCGT CAGGAACCGT

100 GCTACTGCCC GCTACTGCCC GCTACTGCCC GCTACTGCCC GCTACTGCCC

ACMV-K ACMV-N pPCR4(N) pPCR5(N) pPCR6 (N)

101 CCACTGTCCA CCACTGTCCA CCACTGTCCA CCACTGTCCA CCACTGTCCA

CGTCACAAAT CGTCACAAAT CGTCACAAGT CGTCACAAGT CGTCACAAGT

CGAAAACGGG CGAAAACGGG CGAAAACAGC CGAAAACGGC CGAAAACGGC

CCTGGGTGAA CCTGGATGAA CCTGGATGAA CCTGGATGAA CCTGGATGAA

150 CAGGCCCATG CAGGCCCATG CAGGCCCATG CAGGCCCATG CAGGCCCATG

ACMV-K ACMV-N pPCR4 (N) pPCR5(N) pPCR6(N)

151 TACAGAAAGC TACAGAAAGC TACAGAAAGC TACAGAAAGC TACAGAAGGC

CCACGATGTA CCATGATGTA CCATGATGTA CCATGATGTA CCATGATGTA

CAGGATGTAT CAGGATGTAT CAGGATGTAT CAGGATGTAT CAGGATGTAT

AGAAGCCCAG AGAAGCCCAG AGAAGCCCAG AGAAGCCCAG AGAAGCCCAG

200 ACATACCTAG ACATACCTAG ACATACCTAG ACATACCTAG ACATACCTAG

ACMV-K ACMV-N pPCR4(N) pPCRS(N) pPCR6 (N)

201 GGGCTGTGAA AGGCTGTGAA GGGTTGTGAA GGGTTGTGAA GGGTTGTGAA

GGc~CATGTA GGCCCATGTA GGCCCATGTA GGCCCATGTA GGcccATGTA

AGGTCCAGTC AGGTCCAGTC AGGTCCAGTC AGGTCCAGTC AGGTCCAGTC

GTTTGAGCAG GTTTGAGCAG GTTTGAGCAG GTTTGAGCAG GTTTGAGcAG

250 AGGGATGATG AGGGATGATG AGGGATGATG AGGGATGATG AGGGATGATG

ACMV-K ACMV-N pPCR4(N) pPCR5(N) pPCR6(N)

251 TGAAGCACCT TGAAGCACCT TGAAGCACCT TGAAGCACCT TGAAGCACCT

TGGTATCTGT TGGTATCTGT TGGGATCTGT TGGGATCTGT TGGGATCTGT

AAGGTGATTA AAGGTGATTA AAGGTGATTA AAGGTGATTA AAGGTGATTA

GTGATGTGAC GTGATGTGAC GTGATGTGAC GTGATGTGAC GTGATGTGAC

300 GCGTGGGCCT ACGTGGGCCT CCGGGGGCCT CCGGGGGCCT CCGGGGGCCT

ACMV-K ACMV-N pPCR4(N) pPCR5 (N) pPCR6 (N)

GGGCTGACAC GGGCTGACAC GGGCTGACAC GGGCTGACAC GGGCTGACAC

ACAGGGTCGG ACAGAGTCGG ACAGAGTCGG ACAGAGTCGG ACAGAGTCGG

AAAGAGGTTT AAAGAGGTTT AAAGAGGTTT AAAGAGGTTG AAAGAGGTTG

TGTATCAAGT TGTATCAAGT TGTATCAAGT TGTATCAAGT TGTATCAAGT

350 CCATTTACAT CCATTTACAT CCATTTACAT CCATTTACAT CCATTTACAT


351 TCTTGGTAAG TCTTGGTAAG TCTTGGTAAG TCTTGGTAAG TCTTGGTAAG

ATCTGGCTGG ATCTGGATGG ATCTGGATGG ATCTGGATGG ATCTGGATGG

ATGAAACTAT ATGAAAATAT ATGAAAATAT ATGAAAATAT ATGAAAATAT

TAAGAAGCAA TAAGAAGCAG TAAGAAGCAG TAAGAAGCAG TAAGAAGCAG

400 AATCACACTA AATCACACCA AATCACACGA AATCACACGA AATCACACGA


401 ATAATGTGAT ATAATGTGAT ATAATGTGAT ATAATGTGAT ATAATGTGAT

TTTTTACCTG GTTTTACCTG GTTTTACCTG GTTTTACCTG GTTTTAccTG

CTTAGGGATA CTTAGGGATA CTTAGGGATA CTTAAGGATA CTTAGGGATA

GAAGGCCGTA GAAGGCCTTA GAAGGCCTTA GAAGGCCTTA GAAGGCCTTA

450 TGGCAATGCG TGGCAATRCG CGGCAATACG TGGCAATACG TGGCAATACG

1

14 ACMV-K ACMV-N pPCR4(N) pPCR5 (N) pPCR6 (N)

451 CCCCAAGACT CCCCAAGACT CCCCAAGACT CCCCAAGACT CCCCAAGACT

TCGGGCAGAT TTGGGCAGAT TTGGGCAGAT TTGGGCAGAT TTGGGCATAT

ATTTAACATG ATTTAACATG ATTTAACATG ATTTAACATG ATTTAACATG

TTTGATAATG TTTGATAATG TTTGATAATG TTTGATAATG TTTGATAATG

500 AGCCCAGTAC AGCCCAGTAC AGCCCAGTAC AGCCCAGTAC AGCCCAGTAC


501 TGCAACAATT TGCAACAATC TGCAACAATT TGCAACAATT TGCAACAATT

AAGAACGATT GAGAACGATT AAGAACGATT AAGAACGATT AAGAACGATT

TGAGGGATAG TGAGGGATAG TGAGGGATAG TGAGGGATAG TGAGGGATAG

GTTTCAGGTG GTTTCAGGTG GTTTCAGGTT GTTTCAGGT3 GTTTCAGGTT

550 TTGAGGAAAT TTGAGGAAAT TTGAGGAAAT TTGAGGAAAT TTGAGGAAAT

ACMV-K ACMV-N pPCR4(N) pPCRS(N) pPCR6(N)

551 TTCATGCCAC TTCATGCCAC TTCATGCCAC TTCATGCCAC TTCATGCCAC

TGTTGTTGGT TGTTATTGGT TGTTATTGGT TGTTATTGGT TGTTATTGGT

GGTCTATATT GGTCCATCTG GGTCCATCGG GGTCCATCGG GGTCCATCGG

GCATGAAGGA GCATGAAGGA GCATGFiAGGA GCATGAAGGA GCATGAAGGA

600 GCAGGCGTTG GCAGGCGTTG GCAGGCGTTG GCAGGCGTTG GCAGGCGTTG


601 GTGAAAAGGT GTGAAAAGGT GTGAAAAGGT GTGAAAAGGT GTGAAAAGGT

TTTACAGGTT TTTACAGGTT TTTACAGGTT TTTACAGGTT TTTACAGGTT

GAATCATCAC GAATCATCAC GAATCATCAC GAATCATCAC GAATCATCAC

GTGACATACA GTGACATACA GTGACATATA GTGACATATA GTGACATATA

650 ATCATCAGGA ATCATCAGGA ATCATCAGGA ATCATCAGGA ATCATCAGGA


651 GGCAGGGAAG GGCAGGGAAG GGCAGGGAAG GGCAGGGAAG GGCAGGGAAG

TATGAGAATC TACGAGAATC TACGAGAATC TACGAGAATC TACGAGAATC

ACACAGAGAA ACACAGAGAA ACACAGAGAA ACACAGAGAA ACACAGAGAA

TGCTTTGCTT TGCTTTGCTT TGCTTTGCTT TGCTTTGCTT TGCTTTGCTT

100 CTGTACATGG TTGTACATGG TTGTACATGG TTGTACATGG TTGTACATGG


701 CATGTACTCA CATGTACTCA CATGTACGCA CATGTACGCA CATGTACGCA

TGCCTCCAAT TGCCTCCAAT TGCCTCCAAT TGCCTCCAAT TGCCTCCAAT

CCTGTATATG CCTGTATATG CCTGTATATG CCTGTATATG CCTGTATATG

CGACGTTGAA CTACGTTGAA CGACGTTGAA CGACGTTGAA CGACGTTGAA

150 AATACGTATA AATACGTATA AATACGTATA AATACGTATA AATACGTATA


751 TACTTCTATG TACTTCTATG TACTTCTATG TACTTCTATG TACTTCTATG

ACAGTATTGG ACAGTATTGG ACAGTATTGG ACAGTATTGG ACAGTATTGG

CAATTAA CAATTAA CAATTAA CAATTAA CAATTAA

-T Fig,4.Nucleotide sequence of the coat protein genesofACMV DNA A clones derived fromtheNigerian isolate by Ab-PrimerPCR. The sequences of the published Kenyan and Nigerian isolates areshown for comparison. Areasof sequence variation arehighlighted. Primerdetermined sequences areunderlined.

were common in all 3 PCR-derived clones; one of these was primer-determined. These nucleotide substitutions effected 3 amino acid changes. Finally each PCR-derived clone proved to contain sequence alterations in comparison to each other. pPCR4, 5 and 6 contained 3, 1 and 2 nucleotide substitutions, respectively: resulting in single amino acid changes in pPCR4(N) and pPCRS(N) and 2 amino acid changes in pPCR6(N).

15

50

1


MSKRPGDIII MSKRPGDIII MSKRPGDIII MSKRPGDIII MSKRPGDIII

STPGSKVRRR STPGSKVRRR STPGSKVRRR STPGSKVRRR STPGSKVRRR

LNFDSPYRTR LNFDSPYRTR LNFDSPYRTR LNFDSPYRNR LNFDSPYRNR

ATAPTVHVTS ATAPTVHVTS ATAPTVHVTS ATAPTVHVTS ATAPTVHVTS

RKRAWMNRPM RKRAWMNRPM RKQPWMNRPM RKRPWMNRPM RKRPWMNRPM 100

51 ACMV-K ACMV-N pPCR4(N) pPCR5(N) pPCR6(N)

YRKPTMYRMY YRKPMMYRMY YRKPMMYRMY YRKPMMYRMY YRRPMMYRMY

RSPDIPRGCE RSPDIPRGCE RSPDIPRGCE RSPDIPRGCE RSPDIPRGCE

GPCKVQSFEQ GPCKVQSFEQ GPCKVQSFEQ GPCKVQSFEQ GPCKVQSFEQ

RDDVKHLGIC RDDVKHLGIC RDDVKHLGIC RDDVKHLGIC RDDVKHLGIC

KVISDVTRGP KVISDVTRGP KVISDVTRGP KVISDVTRGP KVISDVTRGP


101 GLTHRVGKRF GLTHRVGKRF GLTHRVGKRF GLTHRVGKRL GLTHRVGKRL

CIKSIYILGK CIKSIYILGK CIKSIYILGK CIKSIYILGK CIKSIYILGK

IWMDENIKKQ IWMDENIKKQ IWMDENIKKQ IWMDENIKKQ IWMDENIKKQ

NHTNNVMFYL NHTNNVMFYL NHTNNVMFYL NHTNNVMFYL NHTNNVMFYL

150 LRDRRPYGNA LRDRRPYGNT LRDRRPYGNT LKDRRPYGNT LRDRRPYGNT


151 PQDFGQIFNM PQDFGQIFNM PQDFGQIFNM PQDFGQIFNM PQDFGHIFNM

FDNEPSTATI FDNEPSTATI FDNEPSTATI FDNEPSTATI FDNEPSTATI

KNDLRDRFQV ENDLRDRFQV KNDLRDRFQV KNDLRDRFQV KNDLRDRFQV

LRKFHATVIG LRKFHATVIG LRKFHATVIG LRKFHATVIG LRKFHATVIG

200 GLYCMKEQAL GPSGMKEQAL GPSGMKEQAL GPSGMKEQAL GPSGMKEQAL


201 VKRFYRLNHH VKRFYRLNHH VKRFYRLNHH VKRFYRLNHH VKRFYRLNHH

VTYNHQEAGK VTYNHQEAGK VTYNHQEAGK VTYNHQEAGK VTYNHQEAGK

YENHTENALL YENHTENALL YENHTENALL YENHTENALL YENHTENALL

LYMACTHASN LYMACTHASN LYMACTHASN LYMACTHASN LYMACTHASN

250 PVYATLKIRI PVYATLKIRI PVYATLKIRI PVYATLKIRI PVYATLKIRI


251 YEYDSIGN YFYDSIGN YPYDSIGN YFYDSIGN YBYDSIGN

Fig.5.Predicted aminoacidsequences ofthecoatproteins ofclones pPCR4(N),pPCRS(N)and@X6(N) aligned tothose ofthepublished Kenyanand Nigerian isolates. Sequencedifferences totheKenyanisolate areshown highlighted.

The nucleotide sequence data reported here appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under accession numbers X683 18, X68319 and X68320 for clones pPCR6(N), pPCRS(N) and pPCR4(N), respectively.

Discussion The usefulness of PCR as a method of producing full-length, infectious geminivirus clones from small tissue samples was investigated in order to assess

16

its potential to replace the standard, time-consuming protocols. As geminiviruses characteristically have single-stranded, circular DNA genomes the use of non-overlapping, abutting primers is essential to allow the production of full-length, linear products from these templates. However, it is also likely that the double-stranded, supercoiled form of the viral genome, involved in viral DNA replication (Townsend et al., 1986), acts as a template for amplification during the PCR reaction. The inclusion of partial blunt-end restriction enzyme recognition sites at the 5’ end of the primers (in this case S’GGG to yield a SmaI site) allows the recovery of cloned amplification products from appropriately digested vectors. ACMV was chosen for these studies due to the ease with which this virus is mechanically inoculable as cloned DNA, thereby facilitating the infectivity screening of resulting clones. The amplification of ACMV DNA A molecules required as little as 50 ng of total nucleic acids from an infected leaf, demonstrating that the direct isolation of clones from small samples of original field-collected material is possible. The three PCR-derived clones of the Kenyan ACMV isolate selected for analysis proved infectious when coinoculated with cloned ACMV B, producing characteristic ACMV symptoms on N. benthamiana test plants. In all respects, these clones behaved in an identical manner to the control (Kenyan) ACMV DNA A clone (Table 1). The symptoms produced by the infectious clones amplified from the Nigerian ACMV isolate, when mechanically inoculated to N. benthamiunu, were identical to the symptoms produced by sap inoculation of this isolate directly from cassava to N. benthumiunu. In comparison to the symptom characteristics of the Kenyan isolate, those of the Nigerian clones and isolate were delayed, less severe and showed much less chlorosis during the first week of infection. Previously characterised cloned genomic components of a Nigerian ACMV isolate, which were derived from an unrelated source, were also shown to have milder symptoms than the Kenyan isolate when inoculated to N. benthumiunu and were infectious as pseudorecombinants with the Kenyan clones (Stanley et al., 1985). Sequence comparisons show the PCR-derived Nigerian clones to be similar to, but distinct from the previously characterised Nigerian ACMV isolate (ACMV-N, Morris et al., 1990). ACMV-N was isolated from material derived from a single, infected cassava seedling provided by Dr. H.W. Rossell, IITA, Ibadan, Nigeria (Sequeira and Harrison, 1982). The exact geographical origin of the isolate is not stated in the original reference dated 1982. The PCRderived clones were isolated from material originally collected at Ogorocco in 1990 by Dr. S.A. Shoyinka. Thus the two Nigerian isolates were collected at different times and probably from different locations. A study of isolate variation has been reported for the whitefly-transmitted geminivirus, bean golden mosaic virus (BGMV) in the Dominican Republic (Gilbertson et al., 1991). The results of PCR analysis of a number of geographically distinct isolates showed 95-98% nucleotide sequence identity over approximately 200 nucleotides of a hypervariable region of DNA B. This compares to over 98%

17

identity, (over 777 bases of a highly conserved open reading frame) between the 3 PCR-derived clones and ACMV-N. Thus the level of sequence variation found between the four Nigerian ACMV clones is consistent with that found for the Dominican Republic BGMV clones. A major concern in any PCR-based protocol is the fidelity of DNA copies produced by Tuq DNA polymerase I, due to its lack of 3’-5’ exonuclease activity (Saiki et al., 1988; Eckert and Kunkel, 1991). The reaction conditions described in this paper were chosen so as to minimise infidelity, as suggested by Bloch (199 1). In addition, recent improvements in the rate of error production have been achieved by either genetic modification of Tuq polymerase I or the selection of polymerases from other thermophilic organisms (Cariello et al., 1991). Error production by Taq polymerase lacking 3’-5’ exonuclease activity did not prove to be significant for the amplification of full-length ACMV DNA A clones, as five out of six clones obtained were infectious and produced symptoms characteristic of the isolates from which they were derived. The sequence variability of pPCR4(N), pPCRS(N) and pPCR6(N) may either be derived from natural variation within a virus population or from the infidelity of Tuq polymerase I. It is impossible to determine which (or how many) of the nucleotide differences observed were already present in the virus population used for PCR and which were caused by Tuq intidelity. An estimation of the error rate in this experiment is best calculated from a comparison of ACMV-N with the sequences of the three PCR-derived clones, (this is not the optimal comparison as ACMV-N derives from a different source) There are 38 nucleotide differences within 2331 bp of PCR-derived coat protein sequence compared to ACMV-N, equating to a variation of 1.6%. Of these, 30 differences (i.e., 10 nucleotides) are common to all three PCR clones and are therefore most likely to be isolate determined. Of the 8 alterations in sequence not common to all three clones, 7 result in changes in the amino acid sequence, (some of which are functionally conservative). Thus the estimate of error rate is 8 mutations in 233 1 nucleotides or l/7,300 per nucleotide per cycle which is similar to that reported for Taq polymerase I (reviewed in Eckert and Kunkel, 1991). One of the 6 PCR-derived clones, pPCR4(N), proved to be non-infectious when inoculated onto N. benthamiana. The sequence variability of pPCR4(N) detected within the coat protein open reading frame cannot be the cause of its lack of infectivity, as the coat protein of ACMV is not required for this purpose (Etessami et al., 1989). The causal mutation(s) must therefore lie in another part of the genomic component. Any deleterious effects of Tag polymerase I infidelity can be minimised by ensuring that multiple clones are isolated and that these are screened for normal biological activity. Mutations in the DNA of PCR-generated geminivirus clones which do not alter the biology of the virus can be assumed to be within the bounds of normal sequence variability in geminiviruses. The experiments described in this paper relied upon primers designed to the known sequence of ACMV (Stanley and Gay, 1983). These primers have also

been used to amplify full-length DNA genomic components of tobacco leaf curl virus and Pseuderantheum yellow vein virus (R.W. Briddon and P.G. Markham, unpublished results). The highly conserved nature of the coat protein of the whitefly-transmitted viruses makes this an ideal priming site for this sub-group of geminiviruses. Conserved sequences within the Cl and C2 open reading frames of the monocotyledon-infecting geminiviruses have proved useful in typing these viruses by a PCR-based method (Rybicki and Hughes, 1990) and may be suitable for allowing amplification of the whole genome of this sub-group. This technique also makes possible the cloning of the DNA B genomes of the whitefly-transmitted, bipartite geminiviruses using sequence information gained from the DNA A genome. Primers designed to the common region, a region of approximately 200 bp homologous between the A and B genomic components (Stanley and Gay, 1983), allows amplification and cloning of both components. Abutting primers designed to the common region of Asystasia golden mosaic virus successfully amplified both genomic components of this virus (J. Bird and R.W. Briddon unpublished results). The technique of AbP-PCR has wider applications, including the isolation of sub-genomic viral DNAs. In the case of the geminiviruses, sub-genomic DNAs occur as a small proportion of total viral DNA in infected plant tissue (Stanley and Townsend, 1985) and have proven difficult to isolate by normal procedures. Fig. 2 shows that small DNA fragments are amplified by AbPCR, these have proven to be virus-specific and may represent sub-genomic DNAs. Recent results suggest that geminivirus sub-genomic DNAs can ameliorate the symptoms caused by supra-infection with the parental virus (Stanley et al., 1990) and may prove useful for crop protection. Other small, circular DNA viruses may also be amenable to cloning by this technique; any small circular DNA molecules within the range capable of amplification by thermostable polymerases may be amplified by Ab-PCR provided that some sequence data is available to allow suitable primers to be designed.

Acknowledgements The authors thank Dr. S.A. Shoyinka for providing the Ogorocco isolate of ACMV, L. Chernak (ILS) for useful information on Tuq polymerase and Prof. J.W. Davies and Dr. J. Stanley for critical reading of the manuscript. This work was funded by the Overseas Development Administration through the Natural Resources Institute under contract number EMC X0127. Viruses were held and manipulated in accordance with the Plant Health (Great Britain) Order 1987 (SI No. 1758), under licences issued by the Ministry of Agriculture, Fisheries and Food nos. PHF 1185B/36(111) and PHF 1185B/15(111).

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