Uptake of host DNA by the parasitic fungus Plasmodiophora brassicae

Physiological and Molecular Plant Pathology (1988) 33, 163-171

Uptake of host DNA by the parasitic fungus

Plasmodiophora brassicae T . BRYNGELSSON,

M.

GUSTAFSSON,

B.

GREEN

and C .

LIND

Department of Crop Genetics and Breeding, The Swedish University of Agricultural Sciences, S-268 00 Svalov/Sweden (Acceptedfor publication December 1987)

In situ hybridization, restriction analysis and blot hybridizations have revealed the presence of host DNA sequences in the resting spores and zoospores of Plasmodiophora brassicae . These host DNA sequences are incorporated into the parasite during each infection cycle in the form of high molecular weight DNA.

INTRODUCTION

is a eukaryotic, soil-borne fungus causing the clubroot disease on rape (Brassica napus) and other species of the family Brassicacae . The disease is economically important and it has been estimated that P . brassicae infest more than 10% of the land on which Brassica crops are cultivated [2] . Much of the difficulty in controlling the disease arises from the persistence of the resting spores in the soil and their resistance to chemical treatments . The life cycle of the pathogen includes two phases, the first occurring in root hairs of the host plant, and the second in living cells of the cortex and stele of the root . Infection of root hairs takes place when zoospores, released from germinating resting spores, encyst and penetrate the surface of the root hair [1] . The amoeboid stage develops within the host cell, where the initially uninucleate plasmodium undergoes mitotic divisions to become multinucleate . These multinucleate plasmodia then cleave to form zoosporangia . The secondary zoospores migrate to the base of the root hair, encyst and penetrate further into the root tissue, possibly after fusing in pairs . In the cortical cells a multinucleate stage is produced after which thick-walled resting spores differentiate (for details see [91) . Simultaneously, undifferentiated proliferation of root tissue takes place, leading to the development of the characteristic galls . The resting spores are liberated into the soil when the gall tissue decays (Fig . 1) . During the infection cycle, the naked amoeba and the plasmodium live inside the host cell creating the opportunity for interaction between parasite and host genomes, Plasmodiophora brassicae (Plasmodiophoromycetes)

MATERIALS AND METHODS Plant material

A breeding line of rape, BNHR 233, and a, white mustard cv . Trico, both obtained from SvalofAB, Svalov, Sweden, were used . The isolate of Plasmodiophora brassicae used, Pb l, Abbreviation used in text : BSA, bovine serum albumin . 0885--5765/88/050163+09 $03 .00/0

© 1988 Academic Press Limited

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Fro . 1 . Main root of Brassica napus infected by Plasmodiophora brassicae ; plasmodia i abovel and resting spores (below) . Scale bar= 10 gm .

was originally obtained from Dr Roland Jonsson, Svalof AB . It was multiplied on the susceptible line BNHR 233 and harvested galls were kept in a deep freeze at a temperature of - 20 ° C . For each test, inoculum was always taken from the same batch of galls . To prepare the inoculum, infected roots were macerated in a blender and then filtered through cheesecloth . The inoculum contained at least 10 7 spores ml - ' . Seedlings of the plant lines were dipped in a spore suspension for 5 min and then planted in a special greenhouse bench in a soil mixture consisting ofclay, soil and peat . The inoculated plants were grown at a temperature of 21 °C and a photoperiod of 16 h . After 6 weeks the plants were harvested .

Isolation of resting spores and zoospores

Infected roots (200 g) were homogenized in 800 ml of ice-cold homogenizing buffer [0 . 25 M sucrose, 20 mm 3-N-morpholino-2-hydroxypropanesulphonic acid pH 7 . 5, 2 . 5 mm EDTA, 0 . 2% bovine serum albumin (BSA)], filtered through cheesecloth and passed through a 30 tm net . The filtrate was centrifuged at 50 g for 5 min to remove large particles and the supernatant then centrifuged at 2000 g for 5 min at 4 °C . The resulting pellet consisted of . three layers, each of which was analysed separately by light microscopy . The lower black layer consisted of soil particles, the intermediate white

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layer of intact and empty resting spores and the upper, brownish portion, of zoospores and resting spores . The brownish surface of the pellet was removed and the zoospores allowed to hatch by incubating them in tap water for 16 h at room temperature . After centrifugation at 2000g, the pellet was resuspended in homogenizing buffer and washed four to six times by repeated centrifugation . The final pellet was dispersed in tap water, layered onto a 30-60©-o sucrose density gradient and centrifuged at 10 500g for 10 min . The zoospores, which banded at approximately 50% sucrose, were removed and diluted with tap water . They were recovered after centrifugation at 2000g and immediately used for extraction of DNA . Resting spores were isolated from the white portion of the pellet . The material was repeatedly resuspended in homogenizing buffer and centrifuged until a completely white pellet was obtained . DNA was then extracted from this material, although in most experiments it was isolated from the total pellet, after four or five additional washes with tap water . The total pellet was a mixture of resting spores and zoospores . Isolation of DNA DNA was prepared from plant and fungal material essentially as described by Murray & Thompson [6] . The tissue was lyophilysed and then ground in liquid nitrogen before homogenizing in extraction buffer . The homogenate was heated to 60 © C and extracted with chloroform :isoamylalcohol (24 :1) before precipitating the nucleic acids with cetyltrimethylammonium bromide . The precipitate was purified in ethidium bromide-GO gradients, precipitated with ethanol and dissolved in 10 mm Tris pH 7 . 6, 1 mm EDTA . The DNA was finally freed from polysaccharides by centrifugation at 36 OOOg for 45 min at 4 ©C . Hybridization probes Total DNAs from B . napus and Sinapis alba were restricted with Hind III and separated on 2% agarose gels. The region with the 175 by monomeric band of the tandem repeat was excised, squashed and dissolved in 5 M sodium perchlorate at 60 © C [8] . The DNA was purified by hydroxyapatite chromatography and by ion exchange chromatography on DEAE-Sephacel [5] . Ribosomal DNA fragments were detected by filter hybridization to plasmid pHV294 [3], which contains a rDNA unit from barley in vector pAC184 . The probes were -i labelled by nick translation to a specific activity of approximately 5 x 10 7 ct min - I gg using a[ 32 P]dCTP [9] . Hybridization Restriction enzyme digestions were carried out as specified by the supplier (BoehringerMannheim) using 8 units °g -1 DNA . DNA fragments were separated by electrophoresis on agarose gels and transferred to Zeta-probe membranes (Bio-Rad) by electroblotting . The membranes were prehybridized at 69 © C for 5 h in 2 x SSC, 10 , SDS, 10 x Denhardt's solution and 0 . 1 mg ml -- ' of sonicated salmon sperm DNA . The hybridization was carried out by the addition of [ 32 P]-labelled probe and incubation for 16 h at 69 ©C for the tandem repeats and 68 ©C for the rDNA probe . The membranes were first washed with two changes of 1 x SSC, 1 % SDS, at room temperature and then twice

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1

2

1

2

3 4

5

6

FIG . 2 . Southern blot analysis of host and clubroot DNA using the monomeric unit of the Brassica napus tandem repeat as a probe . (a) Ethidium bromide stained 2°-;, agarose gel of Hindlil-digested clubroot DNA (1) and B. napus DNA (2) . The corresponding autoradiogram is shown in (b! . (c)

Southern blot ofHindl II-digested clubroot DNA isolated from resting spores (3) and zoospores (4) . (d) Southern blot of native (5) and Hind II I-digested (6) clubroot DNA separated on a I ° agarose gel .

in the same buffer for 1 h at the incubation temperature . After drying, the filters were exposed to X-ray film. In situ hybridization Isolated zoospores were squashed on glass slides, the cover slips were removed by the dry ice method and the DNA denatured in 70% formamide, 2 x SSC, pH 7 . 0, for 2 min at 70 °C . Prehybridization was performed for 1 h at 64 °C in 3 x SSC containing 4 µg ml - I of sonicated salmon sperm DNA . Nick translated, tritium-labelled probe (20000 ct min -I per slide) was added and allowed to hybridize at 64 °C for 16 h . Unhybridized material was removed by two washes in 2 x SSC at room temperature and two washes in 3 x SSC at 66 ° C for 1 h . The slides were coated with photographic emulsion (Ilford K .2) and exposed for approximately one week before developing . RESULTS

DNA, isolated from non-infected tissues of B. nap us and from spores of P. brassicae, was restricted with HindIIl and the fragments were separated on a 2% agarose gel [Fig . 2(a)] . A ladder pattern, typical for tandemly repeated sequences, is shown not only by

167 Host DNA uptake by Plasmodiophora the host but also by the parasite preparation . This was unexpected as tandemly repeated DNA sequences have not been reported to be present in fungi . The homology between the tandem repeat in B . napus and P. brassicae was analysed by Southern blot hybridization of the monomeric unit from rape, which has a size of 175 by [4] to restricted genomic DNA from B . napus and P. brassicae . Figure 2 (b) shows that the monomeric unit hybridized under stringent conditions to HindIII-digested DNA from both host and parasite . A number of control experiments were performed to test whether contamination of host DNA occurred during the preparation of DNA from P . brassicae. Spore preparations were examined for the presence of host cells by light microscopy and SEM, but no intact cells or cell debris could be detected . There was, however, the possibility that host DNA was released during spore preparation which became attached to the spore wall or membrane . To test this possibility, one-half of a purified spore suspension was treated with DNase I (300 .Lg m1 - ' for 1 h at 4'C in 0 . 01 M MgCl 2 , 0 . 25 M sucrose, 0 . 2% BSA, 0-05% cysteine, 0 . 02 M MOPS pH 7 . 4) before DNA extraction, while the other half was not treated . After Southern blot hybridization to the monomeric unit of the B . napus tandem repeat, the same pattern was seen in the restricted DNA from both treated and untreated spores . The ability of B . napus DNA to bind to the surface of clubroot spores was also tested by adding [ 32 P]-labelled rape DNA (100 µg ; 10 7 ct min - ') to 250 g of homogenized clubroot tissue before the fungal spores were isolated, and then measuring the level of radioactivity associated with the preparations, by liquid scintillation counting, through the steps of spore purification and extraction of the DNA . In the final spore suspension only 0 . 08°%0 of label was present, while in the purified DNA it was 0 . 05°-0 . The yield of DNA was 770 µg, including 50 ng of added rape DNA . Thus, the exogenous rape DNA represents 0 . 006°,0 of the total DNA preparation . The conclusion drawn from the results of these experiments is that the spore wall and membranes of P. hrassicae are essentially free from surface-associated host DNA . The preceding experiments showed that the DNA sequence from B . napus was most likely present within the resting spores of P. brassicae . This conclusion was supported by in situ hybridization using a tritium labelled monomer of the B. napus tandem repeat as a probe . Squash preparations were made of zoospores, which lack cell walls, and hybridization was carried out (Fig . 3) . Silver grains were distributed over the zoospores suggesting that the host DNA sequences were present inside the resting spore . However, the resolution of the light microscopic autoradiograph was not high enough to be able to draw any conclusions as to whether the DNA was bound to the surface or to the interior of the zoospore . The presence of host DNA inside the resting spore cell wall was also shown by Southern blot analysis of DNA from purified zoospores . Resting spores were allowed to germinate at room temperature and the zoospores were recovered from a 30-60°,;, sucrose gradient . DNA was extracted from the zoospores and from a fraction consisting of' resting spores and empty spore walls . These DNA fractions were compared by Southern blot hybridization to the B. napus tandem repeat . Figure 2 (c) demonstrates the presence of the repetitive rape DNA sequence in both resting spores and zoospores . Taken together the results of these experiments are not consistent with contamination, but strongly indicate that the rape DNA has been incorporated into the fungus in one way or another .

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FIG . 3 . In situ hybridization of the monomeric unit of the Brassica napes tandem repeat to zoospores of Plasmodiophora brassicae.

The incorporation of host DNA sequences could be the result of an active process occurring during each infection cycle or it could happen as a rare event during the coevolution of host and parasite . These two possibilities were tested by infecting susceptible cultivars of B . napes and S. alba with the same isolate of P . brassicae . The diverged version of the tandem repeat present in S . alba does not cross hybridize to the B . napus repeat under conditions of high stringency [4], making it possible to detect sequences which are picked up during the infection cycle . Fungal DNA was isolated from the infected roots of both B . napus and S . alba cultivars and hybridized at high stringency to the tandem repeats from infected plants of each . The result of one such hybridization is shown in Fig . 4(a) and (b) and demonstrates that the fungus had taken up host-specific DNA sequences during the infection process . The fungal uptake of plant DNA could be limited to the tandemly repeated sequence family described above or it could include other types of DNA sequence . A heterologous rDNA probe from barley, pHV294 [3], was used in Southern blot hybridizations to restricted DNA from host and parasite [Fig . 4(c)] . The blot shows that mustard-specific rDNA was taken up by clubroot originating from infected roots of S . alba and rape-specific rDNA when galls from B . napus was used as source for P . brassicae . Similar results were obtained when a cDNA clone for the napin gene was used as a probe (data not shown) . Thus, it is likely that all or most sequences present in the host genome are capable of being taken up by P . brassicae . The DNA is taken up as high molecular weight entities, as shown in Fig . 2 (d), where the B. napus tandem repeat was hybridized to native clubroot DNA . Hybridization occurs over the whole lane indicating the presence of tandem repeats on DNA fragments of all sizes .

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Host DNA uptake by Plasmodiophora (c)

5 6 7 8 2 3 4 Southern blot hybridization using the Brassica napus tandem repeat as probe (a) to Hindi II-digested clubroot DNA isolated from infected roots of B . napus (I) and Sinapis alba (2) . In (b), Hindill-digested clubroot DNA from infected roots of S. alba (3) and B. napus (4) was probed with the monomeric unit of the S . alba tandem repeat . In (c), a heterologous rDNA probe was hybridized to BamHI-digested DNA from B . napus (5), clubroot DNA from infected galls of B . napu.s (6) and S . alba (7) and DNA from S. alba (8) . 1

FIG . 4.

DISCUSSION

When DNAs from the parasitic fungus Plasmodiophora brassicae and its host Brassica napus were analysed by restriction analysis and Southern blot hybridization (Fig . 2) we discovered the presence of a tandemly repeated sequence in the fungus which is very similar to one present in the host . It is unlikely that two such similar or identical tandem repeats have been created in two such distantly related species and, having regard to the unusual life cycle of the fungus, which involves proliferation and sexual recombination within host cells, it is more likely that there has been an exchange of DNA sequences between host and parasite . Of primary interest is whether the foreign sequence was taken up by the fungus during the coevolution of the host-parasite system or if uptake occurs regularly during each infection cycle . The latter possibility was tested by infecting a susceptible variety of Sinapis alba with an isolate of P . brassicae known to contain the rape-specific tandem repeat . The new resting spores, produced in the roots of S . alba, were lacking the B. napus tandem repeat but contained a diverged version of the repeat, specific for S . alba (Fig . 4) . The conclusion is that P. brassicae takes up host-specific sequences during each infection cycle . That the uptake is not limited to tandemly repeated DNA was shown by Southern blot hybridizations to other probes . As the DNA is taken up as high molecular weight

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entities [Fig . 2(d)], it is likely that all DNA sequences in the host genome can be taken up by a population of plasmodia . The presence of host specific DNA sequences in the DNA preparations from P . brassicae could be explained by contamination with host DNA . Resting spores, which were used as the source for clubroot DNA, were isolated from decaying roots of B . napus and it is possible that rape DNA from dead host cells binds to the cell wall or membrane of the resting spore . This possibility was tested by challenging crude resting spore preparations with radioactively labelled rape DNA but only negligible amounts (0 . 08°(,), insufficient to explain our results, bound . In another control, the purified resting spores were treated with DNase before DNA extraction, but this treatment did not remove the tandem repeat originating from B . napus . Thus, the conclusion is that host DNA is present inside the resting spore or zoospore . That the exogeneous DNA is actually present within the zoospore was shown by in situ hybridization to purified zoospores and Southern blot hybridization to zoospore DNA . These experiments indicate that the host DNA must have been incorporated in the fungus before the secondary plasmodium cleaves to form resting spores . It is important to emphasize that, during the last part of the infection cycle, secondary plasmodia develop into resting spores . The zoospores are located in these resting spores . Thus, the incorporated host DNA must have passed through the primary membrane of the fungus in order to reach the zoospores. The mechanism by which host DNA is incorporated into the fungal cells and its intracellular location is unknown . Endocytosis is one possible uptake mechanism . Dictyostelium discoideum, another eukaryote, but a member of the myxomycetes, is a phagocytozing species which is normally grown on a lawn of Klebsiella aerogenes in the laboratory . DNA sequences homologous to the Klebsiella genome should be found in D . discoideum if endocytosis were a possible mechanism . However, hybridization of K. aerogenes plasmid DNA against D . discoideum nuclear DNA has failed to show any homology [7] . The function of the exogeneous DNA is unknown, and nothing similar has been reported before . This DNA is probably not expressed as it is exchanged during each infection cycle, e .g . from rape DNA to white mustard DNA . It could function as reserve nutrients for the resting spores, but it is difficult to explain why it is stored in a high molecular weight form . One would expect the exogeneous DNA to be at least partially degraded . The continuous uptake of host DNA could be of importance for the evolution of P. brassicae . Pieces of host DNA may occasionally be incorporated into the fungal chromosomes and contribute to a better adaptation of the species . We wish to thank Christer Hallden and Gunnel Karlsson for helping to prepare the in situ hybridization . This work was supported by grants from the Swedish Council for Forestry and Agricultural Research (SJFR) and from the Swedish Foundation for Research on Oil Crops (SSO) . REFERENCES 1 . AIST, J . R . & WILLIAMS, P . H . (1971) . The cytology and kinetics of cabbage root hair penetration by

Plasmodiophora brassicae. Canadian Journal of Botany 52, 1441-1449 .

171 Host DNA uptake by Plasmodiophora 2 . BuczAcKi, S . T . (1983) . Plasmodiophora. An inter-relationship between biological and practical problems. In Zoosporic Plant Pathogens, Ed . by S . T . Buczacki, pp . 161-191 . Academic Press, London . 3 . GERLACH, W . L . & BEDBROOK, J . R . (1979) . Cloning and characterization of ribosomal RNA genes from wheat and barley . Nucleic Acids Research 7, 1869-1885 . 4. HALLDEN, C ., BRYNGELSSON, T ., SALL, T . & GUSTAFSSON, M . (1987) . Distribution and evolution of a tandemly repeated DNA sequence in the family Brassicaceae . Journal of Molecular Evolution 25, 318-323 . 5 . MANIATIS, 'l' ., FRITSCH, E . F . & SAMBROOK, J . (1982) . Molecular Cloning . A Laboratory Manual . Cold Spring Harbor Laboratory, New York, U .S .A . 6.

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