Australasian Plant Pathology (1 996) 25: 249-254

Genetic variation of turnip yellow mosaic tymovirus within single plants of Cardamin e lilacina

M.L.SkotnickiA, A.M. Mackenzie and AJ. Gibbs

Molecular Evolution and Systematics, Research School of Biological Sciences, Institute of Advanced Studies, Australian National University, Canberra, Australian Capital Territory 2601 Australia *Present Address: Photobioenergetics, Research School of Biological Sciences, Institute of Advanced Studies, Australian National University, Canberra, Australian Capital Territory 2601 Australia

Abstract

An RNA hybrid mismatch polymorphism (RHMP) method was used to investigate genomic variation of turnip yellow mosaic tymovirus (TYMV) within single plants of its wild perennial host Cardamine lilacina. Two probes covering portions of the three TYMV genes were used to examine the viral RNA isolated directly from single shoots of the host plant. Up to 16 separate shoots of each infected plant, including some which were attached to others by their perennial rhizomatous bases, were analysed. The RHMP results demonstrated that TYMV often varied within single plants, and even within single shoots, in both regions of the genome tested. More within-plant viral variation was observed when shoots were collected from sites with many infected plants than from sites where few plants were infected, suggesting that proximity to other infected plants may facilitate coinfection and recombination between TYMV isolates.

Introduction

Recently, genomic sequencing of single virus iso- lates has been combined with the use of RNA hybrid mismatch polymorphisms (RHMPs) to ana- lyse the natural genetic variation in populations of plant viruses in the wild (Skotnicki et al. 1993). Sev- eral different ssRNA viruses have been studied by these RNase protection methods (Kurath and Palukaitis 1990; Kurath et al. 1993; Rodriguez- Cerezo et al. 1991); the most extensive study re- ported is that of turnip yellow mosaic tymovirus (m, Matthews 1970) from an isolated location in the Australian alpine region (Skotnicki et al. 1993). For TYMV, it was found that of about 100 isolates tested by RHMPs, every one had some nucleotide differences in the 50% of its genome screened, but a 'master copy' genome could be derived from com- parison of several TYMV sequences @omingo et al. 1985; Steinhauer and Holland 1987).

The host plant of TYMV in Australia is a peren- nial, sward-forming brassica, Cardamine lilacina, which is only found in a few, small, late-snow patch regions of the Australian Alps (Guy and Gibbs 1985;

Nolan et al. 1996), and which is buried under snow for up to nine months of the year, with rhizomes containing TYMV sending out new shoots between spring snow melt and fresh snow fall in the autumn.

Therefore we have used the RHMP method to investigate the extent of within-plant variation of TYMV

Methods

Virus collection Samples of TYMV-infected plants of Cardamine lilacina were obtained from locations at Blue Lake, Mt. Twynam, Club Lake and Mt. Townsend in the Mt. Kosciusko alpine area of Australia (Skotnicki et al. 1993).

Distinct differences in leaf shape were observed in plants of C. lilacina, and although many of the swards were uniform and appeared to consist of single plants, others were obviously mixed. Therefore, for each virus isolate, plants were chosen with characteristic leaf morphologies which were easily distinguishable from surrounding plants. Up to 16 shoots were collected from each infected plant,

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and virions were isolated separately from each shoot to minimise the possibility of obtaining mixed samples. For some plants, two or more sampled shoots were joined at their rhizomatous base. For one plant, virions were isolated from four individual leaves from the same shoot.

A number of plants at Blue Lake were marked, and shoots were collected from six of these 2 months and 12 months after the initial sampling.

Virion and RNA isolation Virions and nucleic acid were isolated as previously described (Skotnicki et al. 1993). Briefly, sap was extracted from infected leaves of C. lilacina in a leaf grinder, mixed with ch1oroform:n-butanol, and centrifuged to remove debris. The virions were sedimented in an ultracentrifuge, resuspended in buffer and stored at 4OC. Approximately 100 pg viral nucleic acidlg leaf tissue was obtained after extraction from these virion preparations.

RHMP probe construction Two fragments of the TYMV-BL genome (Skotnicki et al. 1992) were used to construct complementary RNA probes (Figure 1). These fragments, hydrolysed from the full-length infectious TYMV-BL DNA clone pBL16 (Skotnicki et al. 1992), were inserted into plasmid pGEM7Z (Promega), so that transcription from the T7 pro- moter gave RNA complementary to the genomic RNA (Skotnicki et al. 1993). Standard methods de- scribed by Sambrook et al. (1989) were used. The cloned fragments and their orientations in the

TYMV-BL 1 5'

I

genome 0

probes

vectors were checked by DNA sequencing before transcription into RNA.

The transcription method used was that recom- mended by Promega (1991) using Pharmacia nucleotides and T7 RNA polymerase, and "S-UTP to label the negative-strand RNA probe. RNA was transcribed from approximately 1.5 pg plasmid DNA, linearised with an appropriate restriction endonuclease; inclusion of approximately 50 pCi 35S-UTP in the reaction gave sufTicient labelled RNA probe for use in over 100 hybridisationlcleavage reactions.

RHMP method The RNA hybrid mismatch polymorphism method was developed to allow rapid analysis of many different RNA samples (Myers et al. 1985; Skotnicki et al. 1993; Winter et al. 1985). Briefly, 1 pg viral RNA was mixed with radioactively-labelled RNA probe in hybridisation buffer. The samples were annealed, incubated with RNase T1 and RNase A, and the remaining dsRNA was precipitated (Chomczynski and Sacchi 1987) and pelleted by centrifugation. The RNA was electrophoresed through 5% polyacrylamidel7 M urea gels, and exposed to Amersham 35S-Hyerpaper for up to 1 week.

Results

The RHMP banding patterns obtained with isolates from single plants in all locations clearly showed

Figure 1 The genome of TYMV-BL, showing the three genes encoded (replicase (RP), overlapping protein (OP), and virion protein (VP)), and the two regions B and E used as RNA probes in RHMP experiments.

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that both between-plant and within-plant virus variation occurred (Figure 2 and Table 1). However, whether the virus isolates differed in either of the genomic regions probed appeared to be independ- ent of the plant or location from which they were collected.

More than 60% of the isolates gave indistin- guishable RHMP patterns between shoots from the same plant. About 10% gave significantly different RHMP patterns (and thus viral genomic sequences) with viral RNA fiom different shoots (Figure 2). This variation was definitely between shoots from the same plant, rather than from mixed plant popula- tions, as the different RHMP patterns occurred between shoots joined at their bases (Table 2). Also, when checked by RAPD analysis, which has been

used to distinguish different plants of C. lilacina (Nolan et al. 1996), all of the sampled shoots gave identical RAPD patterns and were thus from single plants.

More within-plant viral variation was observed when shoots were collected from sites with many infected plants (such as plants A to G) than from sites where few plants were infected (plant J), sug- gesting that proximity to other infected plants may facilitate coinfection and recombination between TYMV isolates, or that there was less selection for particular genotypes at such sites.

Table 2 also shows the results obtained when four leaves were tested from the same shoot. In this case, one of the two probes detected sequence variation even within individual leaves.

Figure 2 RHMP patterns obtained with probe E and TYMV isolates from sixteen shoots each of two different plants of Cardamine lilacina from Blue Lake. The banding patterns obtained are quite different for the two plants (on left and right gels), and differences are clearly visible between some shoots of each plant, although there is much more variation within one plant (right gel) than the other. Size markers (75-250 bp) are included for comparison of the two gels, at left and right. Arrows above gel lanes indicate shoots with some clearly different RHMP bands (ie. different viral genomes).

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Table 1 RHMP patterns obtained from TYMV in single plants of Cardaminelilacina

Plant Source No. of shoots No. of shoots with No. of shoots with tested same RHMP bands different RHMP bands

1-5 bands >5 bands Probe B Probe E Probe B Probe E Probe B Probe E

ABL: Blue Lake; CL: Club Lake; TO: Mt. Townsend; TW: Mt. Twynam.

Table 2 RaMP patterns obtained fmm TYMV in joined shoots of Cardamine lilacha

Plant Source No. ofjoined No. of shoots with No. of shoots with shoots tested same RHMP bands different RHMP bands

1-5 bands >5 bands Probe B Probe E Probe B Probe E Probe B Probe E

- -

ABL: Blue Lake; CL: Club Lake; TO: Mt. Townsend; TW: Mt. Twynam. BFor plant E, three joined shoots showed no variation in the regions tested, but four leaves from another shoot (*) did show some variation between TYMV isolates from the different leaves.

It was originally planned to test whether TYMV could vary genetically within a year, by resampling marked plants on three occasions aver this time. The results from these tests showed that in two of six plants screened three times, alterations to the RHMP pattern were detectable for one of the two probes used; in both plants only a few RHMP band differences were observed. However, of necessity different shoots were collected on each occasion, and so it cannot necessarily be concluded from these results that the variation observed had occurred within the year rather than having already

been present at the beginning of the year but not sampled at that time.

Discussion

The RHMP method has enabled investigation of genetic variation in a plant virus within single host plants in the wild. Overall, the results showed that TYMV could vary quite extensively within a single plant, and even within leaves of a single shoot. Only about 15% of the TYMV genome was screened by

Australasian Plant Pathology Vol. 25 (4) 1996

the two probes used, and these did not cover the most variable region of the genome at the 5' end (Skotnicki et al. 1993). Thus, ifthe whole genome of these isolates had been compared it is probable that much more sequence variation would have been detected.

Other studies of genetic variation in tymoviruses (and other plant viruses) have involved the use of RHMPs on isolates from whole plants, or sequencing of isolates from different geographical locations. Both of these methods revealed extensive genetic variation between isolates of two different tymoviruses (Skotnicki et al. 1993; Skotnicki et al. 1996), but gave no indication of whether variation could be detected between different shoots of the same host plant.

It was not possible to ascertain whether variation of the TYMV genome had occurred within one year, as there was already extensive within-plant varia- tion, and of necessity different shoots were sampled at the different times. However, the majority of iso- lates did not vary sufficiently in this time for altera- tions in RHMP patterns to be detected. It is interest- ing to note that variation in the TYMV genome was detected in four leaves from a single shoot; this indicates that either variants were present in the rhizome at the beginning of the growing season, or that variation occurred during growth of the shoot, with separation of the different TYMV variants as the leaves developed.

The RHMP method has enabled the extent of variation to be analysed for a virus with a single- stranded RNA genome within its wild host plant. It would now be possible to obtain an estimation of the extent of in vivo genome variation with time, by using this method on a single species of infectious RNA transcribed in vitro from a full-length clone of TYMV (Skotnicki et al. l992), inoculated to glass- house-grown plants of C. lilacina. Experiments are also under way to test for recombination between two TYMV isolates when they are coinfected and passaged sequentially through host plants; the RHMP method will facilitate detection of recombinants as well as mutants which may arise.

Acknowledgement

Plants were collected under NSW Parks and Wildlife Permit AN?.

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