Protoplast formation and regeneration from sporangia and encysted zoospores of Phytophthora infestans

Physiological and Molecular Plant Patholo

(1989) 34, 2 9 9-307

Protoplast formation and regeneration from sporangia and encysted zoospores of Phytophthora infestans ALISON M . CAMPBELLt, RICHARD P . MOON+, JAMES M . DUNCANt, SARAH-JANE GURR+

and JAMES R . KINGHORN+

t Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, and *, Plant Molecular Genetics Unit, Sir Harold Mitchell Building, Queens Terrace, St . Andrews, KY169TH, . .K U (Accepted for publication August 1988)

Protoplasts of Phytophthora injestans were formed from sporangia or encysted zoospores by digestion with NovoZym 234 in stabilizing osmotica . The highest yields, representing a conversion of up to 90 °-c of initial sporangia or cysts were obtained in KCl/CaCI 2 mixtures with osmolalities around 1 . 7 osmolals . Protoplast integrity was determined by autolysis in water, absence of fluorescence after Calcofluor treatment and protoplast fusion after treatment with polyethylene glycol . Protoplasts formed cell walls, as judged by fluorescence, within a few hours of transfer to suitable media containing sorbitol, and most had formed germ tubes within 24 h . The young germlings developed into macroscopic colonies in liquid or solid media after 3-4 days incubation . Regeneration frequencies of > 50 0/. were obtained, especially with protoplasts generated in KCl/CaCI

INTRODUCTION

The development of a successful gene-mediated transformation system is a prerequisite for molecular genetic experimentation on P . infestans, the cause of potato late blight . This approach should help to elucidate the mechanisms underlying pathogenicity and virulence in this important plant pathogen . An essential step in the development of a transformation system is the production of viable protoplasts . Protoplasts have been obtained in low numbers from mycelial mats of liquid cultures of P . cinnamomi, P . Parasitica [1, 2] and P . infestans [8], with protoplast regeneration in the last of these species . More recently, higher levels of protoplast formation and regeneration have been reported for P . parasitica [6] by NovoZym digestion of young mycelium harvested from cellulose film overlying solid medium . P . infestans produces large numbers of sporangia which can be harvested easily, free of contaminating material . They can be germinated directly or can be induced by low temperature to release large numbers of zoospores which encyst and then germinate . The availability of sporangia and encysted zoospores offers, therefore, alternative starting material which might be more amenable to protoplast technology, and subsequent molecular genetic studies, than mycelium . In this communication we report formation of protoplasts at high frequencies from these cell forms and subsequent regeneration into mycelial cells . 0885-5765/89/040299+9 $03 .00/0

© 1989 Academic Press Limited



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MATERIALS AND METHODS

Strains (ATCC 48720) was obtained under licence from the American Type Culture Collection and leaflet tests on R-gene differentials [7] confirmed it to be race 0 . Appropriate matings on V-8 agar showed it to be of mating type Al . Phytophthora infestans

Media Strains were maintained routinely on slopes of rye B agar [3] at 10 °C . For sporangia production, strains were grown in the dark at 17 ° C in 90 mm Petri dishes containing rough rye agar which is, essentially, the same as rye B agar except that rough rye contains comminuted rye grains . Pea broth extract medium (PBE) was prepared by boiling 125 g frozen garden peas in 700 ml distilled water for 20 min . The peas were removed by straining through muslin and the volume of the extract was adjusted to 1 litre . Glucose casein mineral medium (GCM) contained 20 g glucose, 5 g casein hydrolysate, 0 . 5 g KH 2 PO, 0 . 25 g MgSO,7H 2 O, 1 ml trace elements solution [4] and distilled water to 1 litre . Media were sterilized by autoclaving at 121 ° C for 15 min . Solid counterparts were prepared by adding 0 . 5 % Oxoid No . 1 agar .

Osmotica Various osmotica (see results section) were added to media prior to autoclaving. Osmolalities were measured by freezing point depression .

Counts Counts of sporangia, protoplasts and regenerated protoplasts were made at various stages and the results subjected to appropriate statistical analyses .

Microscopy Cell walls were stained with Calcofluor white PMS (American Cyanamid Co ., USA) added to suspensions of sporangia, zoospores, cysts and protoplasts to give a final concentration of 0 . 1 % w/v [5, 6] . Suspensions were examined by UV fluorescence microscopy after short periods of incubation at room temperature using an Olympus BH-2 microscope fitted with a U & V Excitation Dichroic Mirror Assembly with a Y495 supplementary barrier filter and illuminated with a high pressure mercury lamp .

Production and regeneration of protoplasts from sporangia Ten ml of osmoticum, usually 1 rs KCl, was poured over the surface of 10 day-old cultures, and sporangia were harvested by rubbing the surface gently with a glass rod . The suspension was filtered through 53 pm mesh microfilament nylon cloth to remove pieces of medium, sporangiophores and hyphae and the sporangia concentrated by centrifugation (10 min, 600 g) at room temperature . The resultant pellets were resuspended in 10 ml of various osmotica containing 20 mg ml -1 NovoZym 234 (batch PPM 1961), Novo BioLabs, Denmark) and incubated at 25 °C with gentle reciprocating shaking for 24 h . Suspensions were subsequently centrifuged (10 min, 40 g) and



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extensively washed with osmoticum without enzyme and then transferred to various regeneration media based either on PBE or GCM and incubated at 17 ° C . Production and regeneration of protoplasts from zoospores

Zoospores were obtained by flooding rough rye agar cultures with 5-10 ml sterile distilled water at 4 °C for 2 . 5-3 h and were passed through 20 µm mesh monofilament nylon cloth to remove sporangia . They were encysted by shaking vigorously using a vortex mixer for 1 min, centrifuged (15 min, 10000 g) and washed twice in osmoticum . They were resuspended in osmotica containing 50 mg ml -1 of NovoZym 234 and incubated at 25 ° C with gentle shaking for 1 . 5 h . The suspension was centrifuged and washed twice as for sporangial protoplasts and then diluted in PBE plus various osmotic regeneration media as before .

RESULTS Production and regeneration of protoplasts from sporangia

About 5 x 105 sporangia were obtained per Petri dish (Fig . la) . The first signs of digestion by the NovoZym were at the points of attachment and at the apical papillum,

FIG . 1 . Stages in the formation of sporangial protoplasts and their subsequent regeneration : (a) Sporangial suspension before treatment . (b) After 12 h treatment with NovoZym 234. (Note protoplast emerging from papillum end of the sporangium .) (d) Macroscopic colony after 4 days. (c) After 24 h treatment with NovoZym 234 .



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Time in Novo Zym 234 + IM KCl (h ) FIG . 2 . Time course of the formation of protoplasts (Q-Q) from sporangia (I-e) incubated in 1 m KCI with 20 pg ml - ' NovoZym 234 . Protoplasts were allowed to regenerate in PBE+1 m Sorbitol (Q-Q) . Vertical bars represent the standard errors of the mean .

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CaC1. 2 (m) FIG . 3 . Effect of osmoticum on release and regeneration of sporangial protoplasts of P . infestans . The sporangia were treated for 24 h at 25 °C with 20 mg ml - ' NovoZym 234 in 1 m KCI or KCl/CaCl 2 mixtures as osmotica. The KCI concentration in the mixtures were adjusted so that all osmotica had the same osmolality as 1 m KCl . The numbers of protoplasts ml - ' produced after 24 h are shown ( •- -- •) . The percentage regeneration of these protoplasts after 2 days at 17 °C in pea broth and glucose casein mineral medium (GCM) are also shown . The pea broth contained the following amendments : 0. 5 m sorbitol (0-0), 1 . 0 m sorbitol (0-0), 1 . 5 m sorbitol ([I-EI), 2. 0 m sorbitol (M-/) ; and 0. 86 m KCI +0 . 1 m CaCl2 (A-A) . GCM (A-A) .



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CaCl2 (m) FIG . 4 . Effect of CaC1 2 on production and regeneration of sporangial protoplasts of P. infestans . The sporangia were treated for 24 h at 25 ° C with 20 mg ml - ' NovoZym 234 in osmotica containing various concentrations of CaCl 2 with the osmolality kept constant by the addition of KCl . The numbers of protoplasts ml - ' produced after 24 h are shown (0---0) . The percentage regeneration of these protoplasts after 2 days at 17 °C in pea broth + sorbitol + CaC12 are also shown : regeneration in pea broth+ l M sorbitol alone (0- 0), plus 0 . 1 M CaCl 2 (0-0), 0 . 2 M CaCl2 0 .4 M CaC1 2 (0-/) .

and protoplasts emerged after 12 h, mostly through the dissolved papilla (Fig . lb) . More were released during the period 12-24 h by further dissolution of the sporangial wall, giving eventual protoplast yields of some 25-90 % (generally about 70 %) of the sporangia treated (Fig . 2) . Protoplasts were uniform in appearance (Fig . lc) and size, about 19±0 . 8 gm in 1 M KCl, increasing in diameter with decreasing KCl concentration and eventually bursting below 0 . 6 M KCl . At KCl concentrations above 2 . 0 M the morphology of the protoplasts was distorted . Release of viable protoplasts was best in salt solutions such as NaCl, KCI and KCl/CaCl 2 mixtures in the range equivalent to 0 . 6-1 . 6 M KCI in osmolality and was less satisfactory in organic osmotica such as sorbitol and mannitol . In contrast with this last finding, up to 95 0/,o of sporangial protoplasts began to regenerate sporelings within 1-3 days of transfer to PBE containing 1 M sorbitol as osmoticum, while a much smaller number regenerated in media with NaCl or KCI as osmotica (Fig . 3) . Here too, there appeared to be an interaction between the osmoticum used in the release of protoplasts and that used in the regeneration to sporelings . For example, protoplasts produced in KC1 or NaCl regenerated poorly in PBE-sorbitol, while protoplasts produced in KCl-CaCl 2 mixtures regenerated well in the same medium (Fig . 3), but addition of CaCl2 to the regeneration medium did not improve sporeling regeneration (Fig . 4) . Freshly prepared protoplasts lysed almost immediately when transferred to water



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FIG . 5 . An unregenerated and a regenerated protoplast two days after transfer to PBE+1 M sorbitol and staining with Calcofluor . The photographs were taken in combined UV and transmitted light and to cope with the different luminosities of fluorescing and non-fluorescing bodies they have been printed twice with different exposure times . The unregenerated protoplast is similar in appearance to freshly prepared protoplasts stained with Calcofluor .

and remains of cell walls could not be seen . Fusion of protoplasts was observed when polyethylene glycol 4000, 20 % w/v was added to the suspensions . There was no fluorescence when freshly prepared protoplasts were stained with Calcofluor, in marked contrast to the fluorescence from untreated sporangia, but within 3 h of transfer to a regeneration medium fluorescence was detected in most cases . Initially the fluorescence was weak, but within a few days most protoplasts were fluorescing as intensely as the original sporangial preparations (Fig . 5) . Production and regeneration of protoplasts from zoospore cysts

Protoplasts were produced from zoospore cysts after a relatively short immersion for 1 . 5 h in NovoZym as compared to 24 h for sporangia . Best yields were obtained using KCl/CaCl2 mixtures as osmotica, with high levels of regeneration, > 90% in some treatments, when the protoplasts were transferred to PBE+O . 8 M or 1 M sorbitol (Fig . 6) . Motile zoospores did not fluoresce in Calcofluor but did so within 1 min after encystment, and increased over the next 2 min (Fig . 7a, b) . Freshly prepared zoospore protoplasts lysed immediately on transfer to water . They did not fluoresce in Calcofluor (Fig . 7c), except rarely where they had adhered to each other during centrifugation



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CaCL2 (m) FIG . 6 . Production and regeneration of zoospore protoplasts of P . infestans. Zoospore cysts were treated for 1Q h at 25 °C with 50 mg ml- ' NovoZym 234 in osmotica containing CaCI 2 with the osmolality kept constant by the addition of KCl . The numbers of protoplasts present after 11 h are shown (40 ---~) . The protoplasts were assessed for percentage regeneration after 2 days at 17 ° C in pea broth with 0. 8 M sorbitol (ED-EI), or I M sorbitol (0-0 ) .

when a small band of fluorescence was present at the junction between them . They began to fluoresce within an hour of transfer to regeneration media (Fig . 7d) and later produced germ tubes . DISCUSSION NovoZym 234 has a number of enzyme activities, the most important of which are 1, 3-a-glucanase, 1, 3-,B-glucanase, laminarinase, xylanase, chitinase and protease (Nova Biolabs) . It has been used to digest the mycelium of Phytophthora parasitica [6], but chlamydospores, oospores and zoosporangia of this species were resistant . In our studies oogonia and oospores but not attached antheridia of P . infestans were also resistant to the enzyme (unpublished observation), but sporangial walls were removed after incubation in the enzyme for extended periods ; in our studies 24 h incubation gave the best results . The removal of the papilla and the surrounding area of the sporangial wall appeared to be an important step for protoplast production, especially if incubation in the enzyme was combined with gentle agitation . Some protoplasts may have been lost during the extended periods of incubation in the enzyme and osmoticum, but yields and regeneration rates were good . The best osmotica for the formation of protoplasts from both sporangia and zoospores were mineral salts, in particular mixtures of KCl and CaC1 2 and the yields



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Fin . 7 . Staining with calcofluor of various stages in the formation and regeneration of zoospore cyst protoplasts . (a) A zoospore at the moment of encystment . (b) The same zoospore about 3 min later . Note strong fluorescence . (c)i & (c)ii Zoospore cyst protoplasts immediately after NovoZym treatment . Note the intensely fluorescing sporangia, one of which contains zoospores . This photograph was taken in combined UV and transmitted light, and to cope with different luminosities it has been printed twice with different exposure times . (d) Protoplasts from the same batch as (c) but 1 h later, by which time they were fluorescing intensely .



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and subsequent regeneration were comparable with the best obtained with mycelium [6] . The beneficial effect of CaC1 2 has been observed previously but the optimum osmolality at 1 . 7 is higher than that found for mycelial mats (0 . 7-1 . 0) [6] . Three lines of evidence confirm the formation of protoplasts ; rapid lysis in hypotonic solutions, lack of fluorescence in Calcofluor solutions, and fusion in the presence of polyethylene glycol . Protoplasts produced walls when transferred to a suitable regeneration medium in which organic osmotica were better than mineral salts . In contrast to other reports [6], the presence of calcium ions was not beneficial to regeneration, although protoplasts released in osmotica containing calcium ions subsequently regenerated better . The ability to produce large numbers of protoplasts from sporangia and zoospores means that preparations can be obtained from agar plates by relatively simple procedures . The use of mycelium from young cultures in an exponential growth phase can therefore be avoided [6] . Furthermore, protoplasts can be produced from sporulating lesions on stems and leaves (unpublished results), an approach which may he of use with related, but obligate oomycetous fungi, such as the downy mildews . We thank Agriculture Food Research Council for a Link Award which enabled a collaborative approach between St Andrews University and the Scottish Crop Research Institute to be taken . One of us (R . M .) acknowledges the support of the Science and Engineering Research Council through an SERC-CASE studentship . We also thank Dr A . C . Newton (SCRI) for his helpful advice throughout .

REFERENCES 1 . BARTNICKI-GARCIA, S . & LIPPMANN, E . (1966) . Liberation of protoplasts from the mycelium of Phytophthora . Journal of General Microbiology 42, 411-416 . 2 . BARTNICKI-GARCIA, S . & LIPPMANN, E . (1967) . Enzymic digestion and glucan structure of hyphal walls of Phytophthora cinnamoni. Biochimica et Biophysica Acta 136, 533-543 . 3 . CATEN, C . E . & JINKS, J . L . (1968) . Spontaneous variability of single isolates of Phytophthora infestans . I . Cultural variation . Canadian Journal of Botany 46, 329-348 . 4 . ELLIOTT, G . C . (1972) . Calcium chloride and growth and reproduction of Phytophthora cactorum . Transactions of the British Mycological Society 58, 169-172 . 5 . HESS, D . & LEIPOLDT, G . (1979) . Regeneration of shoots and roots from isolated mesophyll protoplasts of Vemesia strumosa . Biochemie and Physiologie der Pflanzen 174, 411-417 . 6 . JAHNKE, K .-D ., LEIPOLDT, G . & PRELL, H . H . (1987) . Studies on the preparation and viability of Phytophthora parasitica spheroplasts . Transactions of the British Mycological Society 89, 213-220 . 7 . MALCOLMSON, J . F. (1969) . Factors involved in resistance to blight [Phytophthora infestans (Mont .) de Barry] in potatoes and assessment of resistance using detached leaves . Annals of Applied Biology 64, 461-468 . 8 . PESTI, M . & FERENCZY, L. (1979) . Formation and regeneration of protoplasts from Phytophthora infestans . Acta Phytopathologica Academia & Scientiarum Hungaricae 14, 1-5 .