MPPOL

Introduction
Materials & Methods
Results
Discussion
References

Molecular Plant Pathology On-Line [http://www.bspp.org.uk/mppol/1997/1124pipe]

Telomere-associated restriction fragment length polymorphisms in Phytophthora infestans

1Pipe ND, 2Shaw, DS.

1bss215@bangor.ac.uk. 2d.s.shaw@bangor.ac.uk

University of Wales, Bangor, School of Biological Sciences, Bangor, Gwynedd, LL57 2UW, UK.



Corresponding
author


DS Shaw. University of Wales, Bangor, School of Biological Sciences, Bangor, Gwynedd, LL57 2UW, UK. Tel: +44 (0)1248 382541, Fax: +44 (0) 1248 370731, Email: d.s.shaw@bangor.ac.uk



ABSTRACT



The probe pLT11, containing the telomeric DNA repeat (TTTAGGG)n from Arabidopsis thaliana hybridized to genomic DNA from Phytophthora infestans digested with HindIII. Fragments hybridizing to the telomeric DNA probe were sensitive to digestion with BAL31 confirming that they were telomere-containing end fragments. Hybridization of the telomeric probe to HindIII-MspI genomic fragments from P. infestans produced more than 24 bands. End-fragments were highly polymorphic between and within two populations. Isolates characterised as identical by the multi-locus probe RG57 had differing end-fragment profiles. The use of end-fragment length polymorphisms for fingerprinting isolates of P. infestans is discussed.




INTRODUCTION



Populations of P. infestans have been characterized mainly with isozyme and restriction fragment length polymorphism (RFLP) markers Carter et al., 1991, Goodwin et al., 1992a, Drenth et al., 1993, Goodwin et al., 1994, 1995). The multilocus RFLP probe RG57 (Goodwin et al., 1992b) has provided the most useful fingerprints of nuclear DNA. While searching for more sensitive markers, we investigated the use of telomeric probes for the detection of end-fragments in digested nuclear DNA of P. infestans.

Telomeric DNA sequences located at chromosome termini are involved in chromosomal stability and replication (Blackburn, 1991). The relatively conserved nature of telomeric DNA repeats allows the use of heterologous DNA probes to identify telomeric sequences. For example the Arabidopsis thaliana telomeric repeat (TTTAGGG)n identified clones containing telomeric sequences in the smut fungus, Ustilago maydis; the telomeric repeat from U. maydis was subsequently identified as (TTAGGG) (Guzmán and Sánchez, 1994). The nuclease BAL31 sequentially degrades DNA from chromosomal termini and has been used to verify the telomeric location of fragments hybridizing to a heterologous telomeric DNA probe. Treatment of genomic DNA with BAL31 progressively shortened restriction fragments hybridizing to a telomeric probe before destroying the signal, indicating that the hybridization detected was due to end- or telomere-associated fragments (Kipling, 1995). BAL31 insensitivity may indicate the presence of homologous DNA sequences at a sub-terminal or internal location (Kipling, 1995).

In this study we show that P. infestans contains telomeric DNA sequences which hybridize to the telomeric repeat (TTTAGGG) from A. thaliana, and that telomere-associated end-fragments are highly polymorphic.



MATERIALS
AND METHODS



To assess polymorphisms revealed by the telomeric probe, two populations of P. infestans from fields with blighted potato crops in August, 1996 at Llanllechid, Gwynedd and 10 km distant, at Bethel, Gwynedd were screened. Samples of nine single-lesion isolates from Llanllechid and fourteen from Bethel were chosen from larger collections made at each site in 1996 (Table1). Isolates were maintained on rye A agar medium at 18 °C in the dark. Mycelium for DNA extractions was grown in pea broth containing 12.5 µg/ml rifamycin, 12.5 µg/ml ampicillin, and 25 µg/ml nystatin for 14 days at 18 °C. Genomic DNA was extracted using methods based on those described by Raeder and Broda (1985).

Two cloned telomeric DNA probes were used in this study: the 400 bp EcoRI-HindIII fragment of pLT11 (Vershinin et al., 1997) which contains the repeated telomeric sequence from A. thaliana (Richards and Ausubel, 1988) and the 262 bp EcoRI fragment of pNOM502 from Cladosporium fulvum (Coleman et al., 1993). PCR primers LR7-R (5'-AGATCTTGGTGGTAGTA-3') and LR12 (5'-GACTTAGAGGCGTTCAG-3') (Vilgalys and Hester, 1990) were used to amplify a 1.6 kb region of the 25S rRNA sub-unit from P. infestans for use as a non-telomeric control probe. The multilocus fingerprinting probe, RG57 (Goodwin et al., 1992b) was also used to characterise isolates. This 1.2 kb probe was amplified from P. infestans genomic DNA using PCR primers RG57P1F (5'-ACCATGCAGCTGAATTGCCAT-3') and RG57P1R (5'-CTCTCATAACCGACGTTTTC-3'), designed from the RG57 sequence (Drenth and Govers, unpublished data). DNA probes were gel-purified using Wizard PCR columns (Promega) then random-prime labelled with DIG-11-dUTP (Boehringer) allowing non-radioactive detection of DIG-labelled hybrids with the luminescent substrate CSPD (Boehringer).

To identify RFLPs in P. infestans, 8 µg genomic DNA was digested overnight, according to the manufacturer's instructions (Promega) with BamHI or HindIII and was also double-digested with BamHI or HindIII and EcoRI, EcoRV, KpnI, DraI, CfoI orMspI. A 1kb ladder was used as a size marker (Gibco BRL). Digested DNA was electrophoresed on a 0.8 % agarose gel for 16 h before being transferred to Hybond N+ (Amersham) using a vacuum blotter (Pharmacia). Filters were incubated in 100 ml prehybridization solution (2 % blocking reagent (Boehringer), 5 X SSC, 0.1 % N-lauroylsarcosine, 0.2 % SDS) for 60 min at 55 °C before hybridization in 100 ml of prehybridization solution containing 25 ng DIG-labelled probe DNA at 55 °C overnight. To remove unbound probe, filters were washed for 2 X 15 min in 2 X SSC, 0.1 % SDS at room temperature and then for 4 X 15 min in 0.1 X SSC, 0.1 % SDS at 55 °C. DNA fingerprints were detected using the chemiluminescent substrate, CSPD, according to the manufacturer's instructions (Boehringer) and by exposure to X-ray film for 3 h.

To determine if hybridizing fragments were telomeric, genomic DNA (80 µg) from P. infestans was digested for varying times up to 2 h using 20 units BAL31 nuclease (Gibco BRL) in 20 mM Tris HCl, pH 8.0, 12 mM MgCl2, 12 mM CaCl2, 600 mM NaCl (Coleman et al., 1993). Aliquots of 8 µg were removed at 0, 0.5, 1.5, 5, 10, 20, 40, 60, 90 and 120 min; digestion was stopped by adding EGTA, pH 8.0, to a final concentration of 20 mM. After a phenol/chloroform extraction, aliquots were precipitated with 0.1 vol. 3 M sodium acetate, pH 5.2 and 2.5 vol. 100 % ethanol. DNA pellets were washed in 70 % ethanol, then resuspended in 20 ml TE buffer (10 mM Tris HCl, pH 8.0, 1 mM EDTA, pH 8.0) and double-digested overnight with HindIII-MspI. After electrophoresis, DNA was transferred to Hybond N+ and hybridized to DIG-labelled probes.



RESULTS



The telomeric DNA probe from C. fulvum, pNOM502, failed to hybridize to genomic DNA from P. infestans digested with BamHI or HindIII at temperatures of both 68 °C and 55 °C. The telomeric DNA probe from A. thaliana, pLT11 hybridized to the same blots at 55 °C but not at 68 °C. To identify the best combination of restriction enzymes for demonstrating RFLPs, double-digests of HindIII or BamHI were performed with EcoRI, EcoRV, KpnI, DraI, CfoI and MspI (data not shown). The HindIII-MspI combination of restriction enzymes produced the clearest separation of fragments. The number of bands, including those co-migrating, was estimated to be at least 24 ( Figure 1). Smaller fragments showed smearing, typical of telomeric sequences of variable length.



Figure 1


Figure 2


Genomic DNA was digested with BAL31 for varying times up to 120 min before double-digestion with HindIII-MspI ( Figure 1). A small reduction in the length of the smaller end-fragments was apparent during digestion. The hybridization signal was progressively sensitive to digestion with BAL31 until it was undetected after 40 min. The 25S rDNA probe from P. infestans, hybridized to several bands, none of which showed a reduction in size due to the BAL31 treatment. ( Figure 2).

Within the collection from Llanllechid, six isolates showed one RG57 fingerprint and three isolates shared another ( Figure 3).



Figure 3


Figure 4


End-fragment fingerprints allowed these six isolates to be sub-divided into five end-fragment patterns; the three other isolates shared the same end-fragment pattern ( Figure 4). In all, six unique restriction patterns (A to F, Table 1) were distinguished within the nine isolates. Similarly within the population from Bethel, five RG57 fingerprints were detected from a sample of 14 isolates and end-fragment fingerprints allowed further sub-division of all but one of these five fingerprints. In all, 11 unique restriction patterns (E-P,Table 1) were distinguished within the 14 isolates. Only two isolates with the same end-fragment pattern had different RG57 fingerprints.



DISCUSSION



A variety of telomeric tandem-repeat sequences exist among the true fungi (Kipling, 1995) although (TTAGGG)n is found in many filamentous fungi. We were unable to show hybridization of a probe containing this repeat to genomic DNA from P. infestans even at 55 °C. However, a probe with the telomeric repeat from Arabidopsis (TTTAGGG)n hybridized at 55 °C but not at 68 °C to BAL31-sensitive sequences of P. infestans genomic DNA. This suggests that the telomeric repeat of P. infestans is closer to (TTTAGGG)n than to (TTAGGG)n. Chromosome-sized DNA molecules from Phytophthora capsici separated by pulse-field gel electrophoresis (PFGE) have been shown to hybridize to a telomeric repeat from A. thaliana (Mena et al., 1994). It is not surprising that the probe from C. fulvum failed to hybridize in this present study as it is now generally accepted that Phytophthora is unrelated to the true fungi (Förster et al., 1990).

HindIII-MspI-digested genomic DNA from P. infestans resulted in the best separation of end-fragments. Smaller hybridizing fragments exhibited a characteristic smearing characteristic of variable-length telomeric DNA sequences. This is due to the natural variability in the number of telomeric repeats within a population of nuclei (Kipling, 1995). Digestion with BAL31 is expected to progressively shorten chromosomal DNA from the end of the telomere (Kipling, 1995). The BAL31 sensitivity of the signal produced by hybridization of the A. thaliana telomeric probe to genomic DNA from P. infestans and the related reduction in length detected towards the end of digestion indicates that fragments hybridizing to the probe include the telomere. Fragments hybridizing to the 25S rDNA probe from P. infestans did not change in size during the BAL31 digestion, indicating that BAL31-sensitivity was not a property of all genomic sequences.

Telomeric probes have identified RFLPs in C. fulvum (Coleman et al., 1993) and Beauveria bassiana (Viaud et al., 1996). Additionally, estimates of chromosome number may be made by counting the number of hybridizing RFLP fragments (chromosome ends). The chromosome number predicted by counts of telomere-associated fragments in C. fulvum (Coleman et al., 1993) and B. bassiana (Viaud et al., 1996) was consistent with band number as revealed by PFGE. Allowing for some co-migration of bands, the number of HindIII-MspI end-fragments in the blots was at least 24. In a diploid fungus, if end-restriction sites were homozygous, the number of chromosomes would be at least 12; if some sites were heterozygous, n <=12. This is consistent with cytological counts of chromosomes which estimate n = 8-10 (Sansome and Brasier, 1975) and n = 8-12 (Whittaker et al., 1991). Co-migration here may be under-estimated and within variable populations, heterozygosity of end-fragments may be substantial. It is therefore not possible to make more than a crude estimate of chromosome number by this method. Further refinement of digestion and separation could reduce co-migration of bands on the gel and increase the accuracy of end-fragment counts to give maximum and minimum possible chromosome numbers.

We have shown that the end-fragments of digested genomic DNA from P. infestans are polymorphic and provide a muli-locus fingerprint for the detection of variation within and between populations of the late-blight fungus. In a collection of 23 isolates from Gwynedd, the multilocus probe RG57 distinguished 6 fingerprints; four of these were further sub-divided giving a total of 16 end-fragment fingerprints. It is clear that end-fragment polymorphism offers greater sensitivity in detecting variants in P. infestans than the RG57 fingerprinting probe.

ACKNOWLEDGEMENTS

The technical assistance of Melissa Gatley is gratefully acknowledged. Thanks to Dallice Mills, Oregon State University (in whose laboratory this work was initiated), Richard Oliver, Carlsberg Laboratory (for the C. fulvum clone, pNOM502), Eric Richards, Washington University, St Louis (for the original A. thaliana telomere clone), Pat Heslop-Harrison, John Innes Centre, (for the A. thaliana clone, pLT11) and AndrJ Drenth and Francine Govers, Wageningen Agricultural University (for the RG57 sequence). Funding was provided by BBSRC and the Wain Travel Fund.



REFERENCES



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