Polymerase chain reaction detection and assessment ...

Australasian Plant Pathology (I 997) 26: 155-1 64

Polymerase chain reaction detection and assessment of genetic variation in New South Wales isolates of passionfruit woodiness potyvirus Nemat SokhandanA,Michael R G i g s Band John W. Bowye+ *Department of Crop Sciences, The University of Sydney, New South Wales 2006 Australia BKey Centre for Biodiversity & Bioresources, School of Biological Sciences, Macquarie University, Sydney, New South Wales 2109 Australia

Abstract A reverse transcription polymerase chain reaction system was developed to detect passionfruit woodiness potyvirus (PWV) in leaves of Passifora species. PWV coat protein (CP) sequences which were amplified from New South Wales isolates and PWV strain K had identical Hinf I, Hue III,Rsa I and Cfo I restriction profiles. Some polymorphism was revealed by digestion with Alu I. PWV CP sequences from 20 out of 21 plants had identical Alu I patterns which differed from that of PWV-K. The remaining isolate produced A h I restriction fragments characteristic of both the common New South Wales isolates and of PWV-K, suggesting that the original plant contained a mixed infection. Three CP cDNA clones, each from an individual plant sample, and six clones from the sample which showed a mixed infection were sequenced. Analysis of the sequence data showed that these isolates from New South Wales had over 97% nucleotide sequence similarity and were closely related to PWV-K. There was 82-85% similarity in the deduced amino acid sequence to the previously described tip blight, severe and mild strains.

Introduction Potyviruses comprise the largest group of plant viruses and include many of major agricultural importance (Hollings and Brunt 1981). They have a single-stranded positive-sense RNA genome of approximately 10 kilobases (kb) which is translated as a single polypeptide and processed by proteolysis into a t least eight functional proteins Wechmann et al. 1992). Passionfruit woodiness potyvirus (PWV) has long been a threat to the passionfruit industry in New South Wales (Peasley and Fitzell 1981) and Queensland (Greber 1966).The use of PWV-tolerant hybrids and mild-strain protection has provided some control (Peasley and Fitzell198 l), but periodic infection of crops carrying the mild strain with cucumber mosaic cucurnovirus (CMV) causes a severe tip necrosis disease (Pares et al. 1985). Transformation of plants with various viral sequences offers new prospects for virus disease control (Lomonosoff 1995).There is some evidence that the level of coat protein-mediated protection against potyviruses is directly related to the degree of similaritybetween the nucleotide sequence of the Australasian Plant Pathology Vol. 26 ( 3 ) 1997

protecting coat protein and that of the challenging virus (Stark and Beachy 1989). PWV-K, a severe strain, is only distantly related to the other PWV strains known as PWV tip blight (PWV-TB), P W severe (PWV-S) and PWV mild (PWV-M) (Shukla et al. 1988; Gough and Shukla 1992) and thus may not be effective in protecting against these strains. We are interested in developing virus-mediated resistance to PWV and have begun with a study of the molecular variation in the coat protein of Australian isolates of the virus. Thls paper describes the amplification, cloning and sequence analysis of the PWV coat protein (CP) gene from Passifora species in New South Wales. The presence of CMV in the samples was also assessed. Methods

Passionfruit samples Leaves of commercial passionfruit and wild Passifora species were collected from the North Coast region of New South Wales (the main area of commercial production in Australia) and from the Sydney metropolitan area. Characteristicsof the samples are given in Table 1.

Double-stranded RNA extractions Doublestranded RNA (dsRNA) was purified from 5 g of passionfruit leaves and resuspended in 100 pL TE buffer according to the protocol described by Rizos et al. (1992).

Hot-start PCR The method was based on that of Chou etal. (1992).The PCRreaction mix (79.5 pL of 50mMKCk lOmMTrispH9.0,0.1%TritonX-100, 1.25mM MgCl,, 4 pmol PWV coat protein reverse primer) was added to the cDNA reaction mix (20 pL) and incubated at 80°C on a Hybaid Omnigene therDesigning oligonucleotide primers The CP- mal cycler for 5 min. Then 0.5 pL of Promega Tag encoding region of PWV-K (Gough and Shukla DNA polymerase (5 UIpL) in storage buffer B was 1992) was aligned with the nucleotide sequences of added to the tube while holding the temperature at related potyviruses, including zucchini yellow 80°C, followed by denaturation at 94°C for 1 min, mosaic, watermelon mosaic, plum pox, bean common then 35 cycles of denaturation at 94°C for 30 sec, mosaic, lettuce mosaic, peanut stripe and South annealing at 60°C for 30 sec and extension at 72°C African passiflora viruses, extracted from Genebank for 1 min. An extension period of 5 min was applied and EMBL databases using DNASIS (Hitachi) soft- after the 35th cycle. A similar program, but with an ware. Primers were designed based on the RNA annealing temperature of 40°C for 30 sec and extencoding for the first six and last six amino acids of the sion at 72OC for 1.5 min was also used. coat protein. Restriction sites were included at the For ampMcationof CMV CP cDNA, 39.75 pL PCR 5' termini of both primers to facilitateduectional clon- reaction mix (as above) containing 5 pmol CMV ing into plasmid vectors. A start codon was incor- 3' primer was added to 10 pL cDNA reaction mix and porated into the upstream primer (PWVf) to facilitate incubated at 80°C on the thermal cycler for 5 min. expression of the coat protein gene. The upstream Then 1.25U of Promega Tag DNA polymerase was primer PWVf (5'tccggatccATGTCTGGCAAAG added to the tube while holding the temperature at ATAA AGAT3') correspondedto nucleotides 422 to 80°C.The initial denaturationwas performed at 94°C 440 of the PWV 70 clone which includesabout 1800 for 1 min, then 35 cycles of denaturation at 94°C for bp from the 3' end of PWV strain K CP cDNA (Gough 30 sec, annealing at 50°C for 30 sec and extension at and Shukla 1992). The downstream primer PWVr 72°C for 1.5min were applied. A final extension step (5'gaattcgagctcTTACTGCACAGGCCCCAT3') cor- at 72°C for 5 min was included. Another thermal responded to nucleotides 1242 to 1259 of PWV 70. cycling program using the 'hot-start' technique with Primers for the coat protein gene of CMV were 40°C as the annealing temperature and 3.9 secI0C also used in this study. They were designed by temperature ramp from 40°C to 72°C as suggested Rizos et al. (1992) and were gifts from Linda Gum by Rizos et al. (1992) was also applied. (NSW Agriculture). The upstream primer (5'GC'IT CTCCGCGAG3') corresponded to nucleotides Restriction endonuclease digestions The RT1149-1 161and the downstream primer (5'GCCGTA PCR products from PWV-infected passionfrut samAGCTGGATGGAC3')to nucleotides 1998-20 15of ples were separately digested with the restriction CMV-Q RNA3 @avies and Symons 1988). enzymes Hae III, Hinf I, CfoI, Rsa I orAlu I. RT-PCR product (19.5 pL) was added to a digestion mix Reverse transcription First strand cDNA synthe- composed of 2.5 pL Promega 10X restrictionbuffer B sis of the PWV CP and CMV CP genes was carried (0.6mMTris-Ha 0.6mMMgC4,5 mM NaC1, pH7.5) out according to the protocol described by Gillings or buffer C (1 mM Tris-HC1, 1 rnM MgCl,, 5 mM et al. (1993).Reverse transcription for the synthesis NaCl, pH 7.9) depending on the restriction enzyme. of PWV CP cDNA was performed in 20 pL. To BSA(0.5 pL at IpgJpL),RNAse(0.5 G a t 1pglpL) 15.75 pL of master mix (composed of 5 mM MgCl,, and 20 U of restriction endonucleasewere added to 50 mMKCk 10 mMTris pH 8.3,l mMof each (INTP, each restriction reaction mix. The reaction mix was 4 pmol PWVf, 20 unitsPmmega RNase inhibitor and incubated at 37°C for 2 h. 50 units Promega Moloney murine leukemia virus reverse transcriptase) 4.25 pL of denatured PWV Agarose gel electrophoresis dsRNA samples dsRNA was added. The first strand synthesis of were fractionated on 1% agarose gels cast and run CMV CP cDNA was carried out similarly using in TBE buffer (0.04 M boric acid, 0.04 M Tris-base 5 pmol CMV 5' primer in 10 pL of reverse transcrip- and 1 mM EDTA, pH 8.3). PCR products were tion reaction mix containing dsRNA at the same fractionatedon 1.5%agarose gels in TBE. Digests concentration as for PWV. of PCR products were separated on 3% Metaphor Australasian Plant Pathology Vol. 26 (3) 1997

agarose (FMC BioProducts). Gels were run at 100 V for 90 min and stained with ethidium bromide for 30 min. The nucleic acid bands were visualized on a short-wave (254 nm) UV transilluminator and photographed using Polaroid Qpe 667 positive film (Sambrook et al. 1989). Purification of RT-PCR products, ligation into the pGEM-T vector and transformation of Escherichia coli RT-PCR products, amplified using 60°C as the annealing temperature, were purified using Amicon EZ enzyme remover columns (Amicon Inc., Beverly, MA United States of America). The amplified PWV CP cDNA uas ligated into the pGEM-T vector (Promega) and used to transform cells of Escherichia coli JM109 according to the manufacturer's protocol. Screening of transformed bacterial cells by PCR The method was based on that of Saris et al. (1990). PCRs contained 50 mM KCl, 10 mM Tris pH9.070.1%TritonX-100, 2 mMMgCl,, 0.2 mMof each dNTP,4 pmol of PWV CP forward and reverse primers, and 0.75 units of Tag DNA polymerase per 30 pL. White, light blue or dark blue colonies resulting from transformations were suspended in 30 pL aliquots of the mix using sterile toothpicks and then patched on Luria Broth (LB) plates containing 100 pg/mL ampicillin to preserve the screened colony. The reaction mix was incubated in the thermal cycler at 94°C for 5 min to lyse the bacteria and denature the plasmid, followed by 35 cycles of denaturationat 94OC for 30 sec, annealing at 60°Cfor 30 sec, and extension at 72°C for 1 min, then a cycle of 72°C for 5 min. The PCRproducts (10 pL aliquots) were separated on 1.5% agarose gels. Preparation of template for DNA sequencing Bacterial colonies which were positive for the CP gene (860 bp product) were inoculated into 3 mL LB containing 100 pgmL ampicillin and incubated in a shakingwatehath at 37°C overnight. Plasmid DNA was extracted from each culture using the Promega W m d D N A purification system The extracted DNA was ethanol-precipitated by adding 2.5 volumes cold (-20°C) ethanol and 0.1 volume 3 M sodium acetate pH 5.2 and storing at -80°C for 15 min, followed by centrifugation at 13000g for 30 min. The DNA pellet was washed with 70% ethanol, dried and resuspended in 20 pL sterile deionised H,O. Sequencing of coat protein PCR products PWV CP clones were sequenced by the DNA Sequencing and Synthesis Facility of Westmead Hospital and the Sydney University & Prince Alfred Macroanalysis Centre using Perkin Elmer sequencer Australasian Plant Pathology Vol. 26 (3) 1997

model 373 A, version 1.2.1 and ABI PRISM 377 version 2.1.1, respectively. The complete CP gene was sequenced for six clones from sample NB5 and one clone from each of samples CL 1, CL2 and SD 1 (Table 1). Analysis of PWV CP cDNA sequencing data Sequence data were analysed using the Australian National Genomic Information Services (ANGIS) packages. The GCG package was used to align sequence data for each clone. The sequencing data were translated into amino acid sequencesusing the translationprogram in the GCG package. The translated sequences were aligned using 'pileup' (Feng and Doolittle 1987)and the percentages of different amino acids in each sequence (&stance) were calculated using the neighbour-joining method of Saitou and Nei (1987). Then a phylogram was generated using PROTDIST in the PHYLIP package pelsenstein 1993)and the TREEVIEW program. The sequence data for the previously characterised strains, PWV-M, PWV-S and PWV-TB (SwissProt, accession nos P32574, P32575 and P32576, respectively) ( Shukla et al. 1988 ) and also for two related potyviruses, SAPV (South African passiflora potyvirus, S51666) and PStV (peanut stripe potyvirus, 22 1700) were extracted from the relevant databases and included in the analysis. Results DsRNA analyses Agarose gel electrophoresis showed that 19 out of 33 passionfruit samples exhibited a dsRNA band of ca. 10 kb, consistent with the expected size of the PWV replicative form. Five samples exhibited dsRNA bands with sizes correspondingto the replicative forms of CMV (Valverde et al. 1990; Pares et al. 1992). Four samples contained dsRNAs consistent with infections with both PWVand CMV (Table 1). Examples of these results are shown in Figure 1 and the complete data are summarised in Table 1. RT-PCR products The application of PCR using PWV CP primers and 48OC as the annealingtemperature resulted in the amplification of many nonspecific bands. The use of' hot-start' PCR with either 40°C or 60°C as the annealing temperature led to the synthesis of a single 860 bp product. Application of RT-PCR to the dsRNA preparations from 33 passionfruit samples resulted in amplification, from 2 1 of the

Table 1 Summary of passionfruit samples, disease symptoms, dsRNA profiles of leaf extracts, PWV and CMVcoat protein gene RT-PCR data andAlu I restrictionpattern of the PWV CP cDNA Sample Location Newrybar Newrybar Newrybar Newrybar Newrybar Newrybar Newrybar Newrybar Burringbar Bumngbar Burringbar Burringbar EG5 Burringbar Q(1 ClothiesCk Q98% similarity)to strain K. The relationship among the New South Wales isolates seemed to be independent of their CP A h I patterns. For example, isolate PWVCLl with the Alu I pattern different from that of PWV-K had 98.3% similarity to PWV-K, whereas the clone PWVNB5c4 with an Alu I pattern similar to that of PWV-K had 97.6% similarityto PWV-K (Figure 4).

Figure 3 Electrophoreticpatterns on 1.5%agarose ofAlu I digests of PWV coat protein PCR products from two New South Wales samples and the reference strain PWV-K. Lane 1,1 pg 100bp ladder (F'harrnacia); lane 2, isolate NE33;lane 3, isolate NB5; lane 4, PWV-K. Fragments with sizes of 79,37 and 9 bp are not visible on this gel. The band of about 540 bp is partially digested DNA.

The results from the alignment of the deduced amino acid sequence data for 9 isolates of PWV from New South Wales, and of strains K TB, S and M are summarised in Figure 5. Discussion

dsRNA analysis is regarded as a fast, simple, relatively inexpensive, and reliable indicator of RNA virus-infection of plants (Valverde et al. 1990).The technique can also be used to distinguish different viruses and strains of the same virus (Valverde et al. 1990).However, we found that RT-PCR is a more sensitive and reliable technique especially for detection of CMV in passionfruit. Our dsRNA analyses (Table 1) showed that only five samples had dsRNA profiles consistent with infection by CMV (Valverde et al. 1990; Pares et al. 1992).However, 13 samples in which CMV dsRNAs could not be detected gave positive results in RT-PCR with CMV coat protein primers (Rizos et al. 1992).Digestion of the PCRproducts with Msp I gave a restriction pattern consistent with that of CMV Msp I-group 1 Nzos et al. 1992),confirming the presence of CMV subgroup 1 in these samples. dsRNA extracts from symptomatic passionfruit leaves had a band with a size of approximately 10 kb (Figure 1) which corresponds to the dsRNA expected for a potyvirus (Rechmann et al. 1992). An RT-PCR product expected for the PWV coat protein gene was amplified from all samples exhibiting ~s dsRNA band. Extracts from two isolates (NB4 and CL2) did not exhibit this dsRNA on 1%agarose, but a DNA fragment of expected size was detected by RT-PCRusing PWV CP primers. The lack of dsRNA bands in some of the samples from which PWV and1 or CMV CP cDNA were subsequently amplified may possibly be attributed, in the case of CMV, to a low yield of dsRNA from the mfected tissue (Valverde et al. 1990) and/or to the effect of host from which the dsRNA is extracted @odds et al. 1987). RT-PCR has been used successfully for the detection of a range of RNA plant viruses (Henson and French 1993). Our application of RT-PCR to detect PWV in infected passionfruit leaves further supports the value of this assay in plant virus diagnosis. We thought that the use of 60°C as the annealing temperature might have resulted in selective amplification of the CP gene of isolates in which the N and C terminal sequences had the highest level of similarity to the primers we had used. To test for such Australasian Plant Pathology Vol. 26 (3) 1997

possible discrimination an annealingtemperature of 40°C was used for all samples in which PWV was detectedby PCR with annealingtemperature of 60°C. Restriction analysis of the RT-PCR products from both PCR programs showed the same restriction patterns (data not shown), indicatinga high level of homology in the 5' and 3' CP regions of all detected isolates. Restriction analysis of the PWV CP cDNA using Alu I endonuclease showed two banding patterns among the New South Wales isolates. All except one had anAlu I pattern different from that of PWV strain K. Isolate NJ35 (Table 1) had a combination of the strain K pattern together with that of the other 20 New South Wales isolates . According to the nucleotide sequence of PWV-K, the four predicted

1t'

Alu I sites in its coat protein cDNA should produce fragments of 3 11,279,229,37 and 9 bp. In the New South Wales isolates the presence of an additional Alu I site in the CP cDNA resulted in the cleavage of the 279 bp fragment into fragmentsof 200 and 79 bp (Figure 3). The combination of the two Alu I patterns in isolate NB5 suggested the source plant was infected with two PWV variants, which was supported by sequencing clones of each Alu I profile group. This finding parallels other evidence of genetic variation in RNA viruses withm single plants (Skotnicki et al. 1996). Coat protein sequence data have been used as a basis to distinguish potyviruses (Shukla and Ward 1989), but there is no absolute sequence identity level that can be regarded as the definitive cut-off

PWV-NB5c6 PWV-NB5c5 PWV-K PWV-CL2 PWV-SD 1

I

I-, I

I

-

PWV-TB PWV-M PStV SAPV

0.00

5.08

10.16

I

15.24

20.32

25.40

30.48 I

Figure 4 A phylogram based on deduced coat protein amino acid sequences of PWV isolates and clones from the North Coast region of New South Wales and from Sydney, of the PWV strains tip blight (TB),mild (M), severe (S) and K, and of the potyiruses peanut stripe virus and South African passiflora virus. IsolatesPWV-CL1, PWV-CL2, PWV-SD1 and PWV-NB5 are from samples CL1, CL2, SD1 and NJ35, respectively (Table 1).Six clones from the isolate PWV-NB5 are shown as PWVNBScl, PWVNI35c2, etc. The divisions on the horizontal scale represent the distances (in %) among the different clones, isolates, strains and viruses. The accession numbers in Genbank for PWVCL1, PWVSD 1 and PWVNBSc5 are U67 149, U67 150 and U67 151, respectively. Australasian Plant Pathology Vol. 26 (3) 1997

between species and strains (Shukla et al. 1994). The coat protein of virus strains may differ by only a small number of amino acid substitutions (Matthews 1985;VanRegenmortel1982). For example there is a high sequence identity (98%) in the coat protein of two strains of tobacco etch virus (Allison et al. 1985).On the other hand, Goughand Shukla (1992) found that strain K of PWV shares only 8546% amino acid sequence similarity with the PWV strains known as tip blight, mild and severe, but the authors concluded that PWV-K could be considered a distantly related strain of PWV, The analysis of 17 strains of eight distinct potyviruses showed that the sequence homology between strains of the one virus ranged from 90-99% (average 95%) (Shukla and Ward 1989). Our analysis revealed approximately 98% deduced amino acid sequence similaritybetween PWV-K and PWV-CL2, -SDl, XL1, -NB5c3, -NB5c4,-NB5~2 and-NBScl, and 99% similarity between strain K and PWV-NB5c6 and -NB5c5 (Figure 4). Accordingly, the isolates from the North Coast region of New South Wales and from Sydney and PWV-K can be considered as isolates of the same strain. Among other potyviruses, South African Passiflora virus and peanut stripe virus have 65-66% and 75-77% deduced amino acid similarity to New South Wales isolates of PWV, respectively. The alignment of the deduced amino acid sequencedata of the isolates from this study, and of strains S, TB, M and K revealed many substitutions in the coat protein (Figure 5). Unique single substitutions may be due to Tag polymerase nucleotide misinsertion errors which occur, on average, at a frequency of between and (Echols and Goodman 1991). It is likely, however, that the multiple substitutions in isolates CL2 and SD 1, at positions 28,33,138,253 and 266 are natural since they are shared by independent isolates. Similarly, isolate CL1 and clones NB5c3, NB5c4, NB5c2 and NB5cl Figure 5 Alignment of the deduced amino acid sequences of the CP of New South Wales isolates and clones of P W , strainsPWV-K, -S, -TB and -M, showing the positions where variation occurs. The positions are based on the deduced amino acid sequence of strain K (Gough and Shukla 1992).The first six and last six amino acids deducedhmRT-PCR products from the New South Wales isolates were not included in the alignment since they were included in the primers used to ampllfy the CP gene. Australasian Plant Pathology Vol. 26 (3) 1997

have identical substitutionsat positions 13 and 253 and form a cluster in the phylogram (Figure 4). The high level of similarity (>96.5%) in the coat protein of New South Wales isolates of PWV and the variation in the host plant symptoms indicate there is no correlation between the coat protein andvimlence of PWV. This conclusion is supported by the fact that strains such as PWV-K and PWV-S, which both cause severe symptoms, have coat proteins which are distantly related (Figure 4). However, the issue is complicated by the coinfection of most of the New South Wales samples with CMV (Table 1) and the possibility that they were also infected with a rhabdovirus (Pares et al. 1983) andlor passiflora latent carlavirus (Pares et al. 1997). Dual infection with PWV and CMV was correlated with tip necrosis and die-back in some samples, suggesting disease severity may, at least partly, be correlated with such infection rather than a severe strain of PWV. This is in agreement with the conclusion of Pares et al. ( 1985)that neither PWV nor CMV alone causes tip die-back in passionfruit. Our failure to detect either virus in five of seven samples showing mosaic symptoms points to possible infection of them by the rhabdovirus and /or carlavirus. The assessment of variation in the coat protein encoding region of different isolates of a virus is significant in terms of establishing a pathogenderived protection, based on the coat protein, against the virus. It was shown that there is a direct relationship between the degree of protection conferred by the CP of a virus and the level of similarity in the nucleotide sequence of this coat protein and that of the challenge virus (Stark and Beachy 1989). Since our restriction and sequence analyses revealed a high level of homology (>97%) among different isolatesof PWV from New South Wales, it is likely that resistance derived from the CP gene of any of these isolates should protect passionfruit vines against infection with any of them. In contrast the CP gene of these isolates might not protect passionfruit against the distantly related strains, PWV-TB, -M and -S.

Acknowledgements We would like to thank Keith Gough for the PWV70 clone, Ross Fitzell for assistance in collecting the passionfruit samples and Linda Gunn for CMV dsRNA extracted from capsicum and CMV primers. The work was supported in part by the Horticultural Australasian Plant Pathology Vol. 26 (3) 1997

Research and Development Corporation (Project FR 523) and the Australian Passionfruit Industry Association.

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