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Spore germination and ribosomal activity in the rust fungi I. Comparison of a bean rust fungus and a culturable wheat rust fungus
Physiological
Plant Pathology
(1972)
2, 27-35
Spore germination and ribosomal activity in the rust fungi I. Comparison of a bean rust fungus and a culturable wheat rust fungus R. C. STAPLESand Boyce
Thompson
Institute,
Z. YANIV Yonkers,
New
York
10701,
U.S.A.
and W.
R. BUSHNELL
Cooperative Agriculture,
Rust Laboratory, Plant Science Research Division, Agricultural Research Service, University of Minnesota, St. Paul, Minnesota 55101, U.S.A.
(Acceptedfor
publication
August
U.S. Department
of
1971)
Ribosomes from uredospores of the as-yet-uncultured bean rust fungus (Uromycesphaseoli) and the culturable Australian wheat rust fungus race 126-ANZ-6,7 (Puccinia gaminis f. sp. tritici) were compared for transferase activity, capacity to bind polyuridylic acid and capacity to incorporate leucine into ribosomes in vivo. While the ribosome activities in bean rust uredospores declined before the germ tube was formed, these activities were maintained by ribosomes from the Australian wheat rust fungus. Mycelium derived from uredospores of the wheat rust fungus, and continuously subcultured for 4 years, had ribosomes that were three times more active in the transferase assay than ribosomes from uredospores.
INTRODUCTION The rusts are a group of parasitic fungi usually obligately dependent on living hosts for growth and completion of their life cycles, but several strains of two rust species, wheat stem rust and flax rust, have been cultured axenically from uredospores [I4, 171. While culture of the rusts is a remarkable achievement, the simple media employed have not been helpful in suggesting the transitions which occur when the sporeling begins to grow. Detailed studies with the bean rust fungus have shown that its uredospores germinate without cell division. Although the protein and RNA contents do not increase like they do when saprophytic conidia germinate [II], protein synthesis is required for germination to occur [.5], probably for formation of cell wall peptides [13]. We compared the properties of ribosomes from uredospores of the saprophytic Australian wheat rust fungus, race 126-ANZ-6,7, and the bean rust fungus which has not yet been cultured. We hoped to provide an insight into the transitions that occur as the sporeling develops into a mycelium. MATERIALS
AND
METHODS
Chemicals
[U-14C]Leucine (specific activity 273 mCi/mmol) and [U-14C]phenylalanine (specific 375 mCi/mmol) were purchased from New England Nuclear Corp., Boston, Mass., U.S.A. Poly[2J4C]uridylic acid, purchased from Miles Laboratories, Kankakee, Ill., U.S.A., had a molecular weight greater than 50,000 daltons activity
28
R. C. Staples, Z. Yaniv and W. R. Bushriell
(gel filtration), and had a specific activity of 0.843 mCi/mmol P. Polyuridylic acid was also purchased from Miles Laboratories, and had a molecular weight greater than 100,000 daltons. Yeast transfer ribonucleic acid (tRNA) was purchased from General Biochemicals Corp., Chagrin Falls, Ohio, U.S.A.
SPores Uredospores of the bean rust fungus [U. phaseoli (Pers.) Wint.] were grown and collected weekly from infected leaves of bean (Phaseolus vulgaris L. var. “Pinto”) plants grown in controlled environment chambers as described previously [II]. Dry uredospores were bottled and stored for periods up to 7 days at 4 “C. These dry spores were termed “resting spores”. Uredospores of the wheat rust fungus [P. graminis (Pers.) f. sp. tritici Eriks. & E. Henn., Australian race 126-ANZ-6,7] were grown and collected twice weekly from infected leaves of wheat (Triticum aestivum L. var. “Little Club”) plants grown in the glass-house. Dry uredospores were bottled and stored in the refrigerator. Mycelia Mycelial cultures of the Australian wheat rust fungus were produced from sterile uredospores on Evans’ peptone medium as described by Bushnell [Z]. The cultures used for these studies had been subcultured monthly for 4 years at St. Paul, Minn., U.S.A. For the present study, mycelial pieces about 2.6 mm wide were cut from colonies 1 month old and placed on media containing 3% glucose, Czapek’s minerals, 1.5% Difco agar, 0.4% Evans’ peptone and 0.4% Difco Casamino acids, technical grade. Four colonies were grown in each g-cm Petri dish in moist jars at 15 “C. Only those colonies that grew in diameter in the final 6 to 26 days before harvest were used. These colonies were shipped in ice-cooled boxes overnight to Yonkers, N.Y., U.S.A. and used the following day. Fresh weight was determined for all colonies at harvest. For dry weight determinations, 12 colonies from each harvest were dried at 93 to 96 “C for 2 days. Spore germination Resting bean rust uredospores were prepared for germination by floating them on water at 4 “C for 16 h as described previously [IO]. After hydration, 200 mg spores were dusted onto two baking dishes containing water as described previously [12]. The baking dishes had a total surface area of 2070 cm2. Germination was carried out in the dark at 20 “C. The agar slide technique of Schein [9] was used to determine germinability by counting at least 200 spores. Germ tubes longer than twice the spore diameter were measured using an ocular micrometer. It was not necessary to hydrate the wheat rust uredospores because germination occurred readily at all concentrations of spores tested up to 0.13 mg/cm2. Consequently, resting wheat rust spores were considered to be equivalent to hydrated bean rust spores, and both are referred to collectively as “nongerminated” spores. Transferuse
assays
Binding. Ribosomes were assayed for polyuridylic acid-directed dependent binding of phenylalanyl-tRNA using the modification by Nirenberg & Leder [7] described previously [19].
and transferase-I of the procedure
Spore
germination
and
ribosomal
activity
in rust
fungi.
I
29
Polymeri
cw [‘“Cl Leucine incorporation Collodion membranes were dusted with spores and the membrane surface sprayed with water as described by Wynn & Gajdusek [l&j. Then 1 ml of water containing 5 @Zi [l*C]leucine was sprayed directly onto the membrane surface and the spores germinated at 20 “C for 1 h. The ribosomes were extracted by pulverizing 20 membranes (1 g spores) in liquid N, as before [12]. The ribosomes were collected by centrifiigation, resuspended, collected on Millipore filters (Millipore Filter Corp., type HA, Bedford, Mass., U.S.A.) and counted by liquid scintillation techniques. Binding
of polyuridylic
acid
Ribosomes washed with 0.25% sodium deoxycholate were incubated with poly [2-r4C]uridylic acid for 10 min at 30 “C. Aliquots containing 3 to 4 Ass0 units were layered on top of 10 to 30% sucrose density gradients prepared as described by Brakke & Van Pelt [I]. The gradients were centrifuged in a Spinco SW 41-Ti rotor (Beckman Instruments, Palo Alto, Calif., U.S.A.) for 75 min, and fractionated into 1 -ml fractions. The fractions were precipitated with cold 5% trichloroacetic acid, collected on Millipore filters (type HA) and counted in the scintillation counter. Quantitative
assays
The amount of ribosomes in suspension was estimated spectrophotometrically at 260 nm assuming that there was 110 pg/A,,,. Protein concentration was estimated by the method of Warburg & Christian [15]. RESULTS
Aspects of morphology Progress of germination. Fig. 1 (a). The Australian tubes were formed by 3 and all spores germinated of both species elongated the lengths shown were
Germination of the two species of rusts is compared in wheat rust spores germinated within 1 h, and all germ h. Bean rust spores required 2 h before germination began, in 4 h. Once germination was initiated, the germ tubes at nearly the same rate [Fig. 1 (b)]. Measurements beyond unreliable, but frequently reached 1000 pm in 24 h.
Axenic culture of the wheat rust fungus. The fungus produced dense, white colonies which increased in dry weight at rates of 0.21 to 0.26 mg/day The colonies were 5 to 8 mm in diameter at harvest, and had dead brown-to-black degenerate mycelia comprising about one-third of total the colonies. The colonies contained clusters of uredospores as well
mycelial (Table 1). centers of volume of as a few
30
R. C. Staples,
Z. Yaniv
and
W. R. Bushnell
teliospores, but very few spores were broken in the grinding procedure used for the extraction of the mycelia. The total fresh weight of samples (53 to 62 colonies) was 2 to 8 g. The dry weight of these colonies was about 23% of their fresh weight. 100 2
80
.-2 6 -2 & ”
60 40 20 0
7 I $ 2 E & CJ
400
200
0 Time
(h)
FIG.
1. Comparative germination of uredospores of bean rust and Australian wheat fungi. (a) Time-course of germination; (b) rate of germ tube elongation. Germination carried out on 1% agar at 20 “C, using a spore concentration of 0.1 mg/cms. O-O, Bean rust; l l , wheat rust, ANZ. 1
TABLE
Diameter
and weight
of mycelial
colonies of the Australian transferme activity Value
Observation No. of colonies Colony age (days) Colony diameter (mm) Fresh weight (mg) Dry weight (mg)
rust was
1 62 74 7.7 122 -
All media contained 3% glucose, Czapek’s minerals Evans peptone and 0.404 Difco Casamino acids, tech.
wheat
rust fungus
per sample 2 53 55 7.2 103 23.8 and
used in assays for
3 60 35 4.9 42 9.6
1.5%
Difco
agar
plus
0.4%
Ribosome activity during spore germination. Leucine incorporation in vivo was high with the ribosomes from bean rust uredospores just beginning to germinate (Table 2), but this activity declined dramatically after 20 h. In contrast, ribosomal activity in uredospores of the Australian wheat rust fungus more than doubled over a 20-h germination period. Transferase
activity during germination
Transferase-I from resting and some of its properties
spores of the bean rust fungus was purified previously described [20]. Transferase-I catalyzes the polyuridylic
Spore
germination
and
ribosomal
activity
in rust
fungi.
31
I
acid-directed and GTP-dependent binding of phenylalanyl-tRNA to ribosomes. This reaction was used to compare ribosomal activities at various stages of germination without the limitations imposed by the presence of unstable activating enzymes [IO]. Since studies with transferase-I have so far been confined to nongerminated spores, a brief characterization of this binding step in germinated bean rust spores is presented. TABLE In viva
incorporation Spore
Q One
of [WJeucine
into protein
2 by germinating
Exposure to [%Tjleucine during germination
stage
and non-germinating
Specific activitya of ribosomes (pmol/mg)
Ratio germinated non-germinated
Bean rust Non-germinated Germinated
Otolh 20 to 21 h
127 5
0.04
Wheat rust Non-germinated Germinated
Otolh 20 to 21 h
148 300
2.00
pmol
of [r*C]leucine
produced
405 cts/min
tq
FIG. 2. Comparison and germinated (Ouredospores only were
of used
to
on the counter.
protein/tube
the activity of transferase-I ) bean rust uredospores. in each 30-min assay.
l
uredospom
from non-germinated Ribosomes (300 yg)
(0 -0) from resting
Optimum requirements for binding. Concentrations of ribosomes, magnesium and enzyme required for optimum binding of phenylalanyl-tRNA to bean rust ribosomes were found to be essentially the same as those for ribosomes from nongerminated spores [ZO] except for the amount of transferase-I. Ribosomes from germinated spores required 100 [*g of transferase-I, while those from non-germinated spores required 20 pg. Not only do ribosomes from germinated spores require more transferase-I for optimal activity than non-germinated spores, but there is less of it in the germinated bean spore. In Fig. 2, it is shown that protein extracts from a gram of germinated rust spores are about half as active with ribosomes as the extracts from a gram of non-germinated spores.
32
R. C. Staples,
Z. Yaniv
and
W. R. Bushnell
Changes in transferase activity during germination. Transferase-I activity was followed during germination of the rust uredospores (Fig. 3). With bean rust, transferase-I activity of the ribosomes was highest in the hydrated, non-germinated spores only, then this activity declined rapidly. The fact that binding activity declines before the germ tube has even emerged clearly shows that inactivation of the ribosome occurs in bean rust uredospores while the germ tube is just beginning to elongate most rapidly [Fig. 1 (b)]. T ransferase-I activity of wheat rust ribosomes remained fully active for 20 h.
Time
(h)
FIG. 3. Transferase-I dependent binding of phenylalanyl-tRNA with polyuridylic acid by ribosomes from germinated rust uredospores. Bean rust spores were hydrated for 16 h at 4 “C, filtered and redispersed on water to initiate germination. Wheat rust spores were not hydrated. Concentration of ribosomes was calculated after purification by density gradient centrifugation, and 240 (*g of ribosomes was used in each assay. O-O, Wheat rust, ANZ; o---O, bean rust. TABLE
Polymerization
activity
of ribosomes from
Ribosome
a One (240
3
germinated
and non-germinated
Phenylalanine + polyuridylic acid bolbg)
incorporation” - polyuridylic (pmol/md
Bean rust Non-germinated Germinated
78 5
31 2
Wheat rust Non-germinated Germinated Mycelia
80 94 310
31 30 90
pmol of [l%]phenylalanine pg) were purified by density
produced gradient
550 cts/min centrifugation.
on
the
uredospores acid
counter.
Ribosomes
Polymerization. Ribosomes from non-germinated and germinated spores were assayed for their capacity to form polypeptides with and without polyuridylic acid as messenger RNA. Both with polyuridylic acid and without it, it was found that bean rust ribosome activity declined 94% (Table 3), and wheat rust ribosomes remained fully active.
Spore
germination
and
ribosomal
activity
in rust
fungi.
33
I
Wheat rust ribosomes from mycelia growing axenically polymerized about three times as much phenylalanine as ribosomes from uredospores (Table 3). Again the same changes were found in the presence or absence of polyuridylic acid.
Binding
of polyuridylic
acid
The capacity of ribosomes from bean rust uredospores to bind polyuridylic acid also declined during germination [Fig. 4(a), (c)l. Although germination reduced the capacity of these ribosomes to bind polyuridylic acid by 93%, the capacity to polymerize phenylalanyl-tRNA was reduced 94% (Table 3), suggesting that there is a dependence between the two processes. As expected, the capacity of ribosomes from wheat rust uredospores to bind polyuridylic acid did not decline during germination [Fig. 4(b), (d)]. I.0
Fraction
number
FIG. 4. Sucrose density gradient analysis of binding of polyuridylic acid by ribosomes from uredospores. (a) Hydrated, non-germinated uredospores of the bean rust fungus; (b) non-hydrated, resting uredospores of the Australian wheat rust fungus, race 126~ANZ-6,7; (c) uredospores of the bean rust fungus germinated 20 h; (d) uredospores of the Australian wheat rust fungus, race 126-ANZ-6,7, germinated 20 h. The monoribosomes are in fraction 4, polyribosomes in the heavier fractions. Centrifugation was from right to left.
DISCUSSION The
incorporation
of rust while ribosomes from the decline in ribosome Australian
3
wheat
exogenous leucine by ribosomes from uredospores of the fungus doubled in activity during germination (Table 2), the bean rust uredospores became virtually inactive. Since activity could provide an important clue to host dependency,
34
R. C. Staples,
Z. Yaniv
and
W. R. Bushnell
detailed studies were made of ribosomes from these divergent species of rusts to delineate the differences at the molecular level. The loss of ribosome activity during germination of the bean rust fungus is apparently due to failure by the ribosomes to bind messenger RNA (Fig. 4). This change is accompanied by a loss of capacity to bind tRNA in response to transferase-I (Fig. 3), and suggests that ribosomes progressively lose the capacity to make protein. Whatever the mechanism, it seems obvious that ribosomes from the bean rust fungus suffer a loss of function during the earliest phases of spore germination. For example, transferase activity declined strikingly in 2 h (Fig. 3), yet the few germ tubes produced had just entered the linear phase of elongation [Fig. 1 (b)]. In contrast, ribosomes from the wheat rust fungus remain active throughout the period of germ tube extension (Fig. 3). A susceptible host plant must have properties which enable the bean rust fungus to maintain or revive its ribosomal functions. Differentiation of the germ tube seems to be a prerequisite both for successful parasitism [4] and for saprophytic growth [IS]. The germ tubes of uredospores studied here were produced on water or moist surfaces of collodion and were not differentiated. Studies with the bean rust fungus have shown that the important change upon differentiation is synthesis of,a template RNA [8], and that the onset of the decline of ribosome activity is the same whether or not the spores are differentiating. While the present studies deal only with non-differentiated germ tubes, the striking differences in ribosome activity found after 2 h of germination suggest that at least some of the changes expected in the transition to saprophytism have already occurred in advance of differentiation. The Australian race 126-ANZ-6,7 of the wheat stem rust fungus was cultured in 1966 by Williams et al. [17]. The transition from sporeling to mycelium requires nearly 7 days [6], and the mycelium develops rapidly once the transition to hyphal growth has occurred (Table 1). Further adaptation to the artificial medium occurs during the first few months of serial subculturings before a stable saprophytic culture is obtained ([3], Bushnell unpublished data). Such an adapted culture had ribosomes that were about three times more active in assays for activity of transferase-I than ribosomes from uredospores (Table 3). Obviously the complete transition to mycelial growth involves an increase in ribosomal activity as well as maintenance of ribosomal function. A more detailed study of the transition of the sporeling to mycelium will be required to establish these changes more definitively. This investigation was supported in part by a grant (GB-17003) from the National Science Foundation and by the Herman Frasch Foundation. Cooperative investigation: Boyce Thompson Institute; Crops Research Division, of Agriculture; and Department Agricultural Research Service, U.S. Department Scientific Journal Series Paper No. of Plant Pathology, University of Minnesota. 7720, Minnesota Agricultural Experiment Station. Mention of a trademark name, proprietary product or specific equipment does not constitute a guarantee or warranty by the U.S.D.A., nor imply its approval to the exclusion of other products also available.
Spore
germination
and
ribosomal
activity
in rust
fungi.
I
35
REFERENCES 1.
BRAKKE, M. K. & VAN PELT, N. (1970).
Linear-log sucrose gradients for estimating sedimentation of plant viruses and nucleic acids. Analytical Biochemistry 38, 56-64. BUSHNELL, W. R. (1968). In vitro development of an Australian isolate of Puccinia gruminis f. sp. trititi. Phytofiatholopy 58, 526-527. BUSHNELL, W. R. & STEWART, D. M. (1971). Development of American isolates of Puccinia gruminis f. sp. tritici on an artificial medium. Phytopatholop 61, 376-379. CHAKRAVARTI, B. P. (1966). Attempts to alter infection processes and aggressiveness of Puccinia graminis var. tritici. Phytopathology 56, 223-229. DIJNKLE, L. D., MAHESHWARI, R. & ALLEN, P. J. (1969). Infection structures from rust urediospores: Effect of RNA and protein synthesis inhibitors. Science, jvew 2’ork 163,481-482. KUHL, J. L., MACLEAN, D. J., SCOTT, K. J. & WILLIAMS, P. G. (1971). The axenic culture of Pucciniu species from uredospores : experiments on nutrition and variation. Canadian Journal of Botany 49, 201-209. NIRENBERG, M. & LEDER, P. (1964). RNA codewords and protein synthesis. The effect of trinucleotides upon the binding of sRNA to ribosomes. Science, Jvew York 145, 1399-1407. RAMAKRISHNAN,L. & STAPLES, R. C. (1970). Changes in ribonucleic acids during uredospore differentiation. Phytopatholopy 60, 1087-1091. SCHEIN, R. D. (1962). Storage viability of bean rust uredospores. Phytopatholopy 52, 653-657. STAPLES, R. C. (1968). Protein synthesis by uredospores of the bean rust fungus. Netherlands Journal of Plant Pathology 74 (sup& 1) 25-36. STAPLES, R. C.. SYAMANANDA, Comoarative biochemistrv , R., , KAO. V. & BLOCK. R. T. (1962). of obligately parasitic and saprophytic fungi. II. ’ Assimilation of C14-labeled substrates by germinating spores. Contributions. Boyce Thompson Institute for Plant Research 21, 345-362. STAPLES, R. C., YANIV, Z., RAMAKRISHNAN, L. & LIPETZ, J. (1971). Properties of ribosomes from germinating uredospores. In Morphological and Biochemical Events In Plant-Parasite Znteraction ed. by S. Akai & S. Ouchi, pp. 59-90. The Phytopathological Society of Japan, Tokyo. TROCHA, P. & DALY, J. M. (1970). Protein and ribonucleic acid synthesis during germination of uredospores. Plant Physiology, Lancaster 46, 520-526. TUREL, F. L. M. (1969). Saprophytic development of the flax rust Melampsora lini, race No. 3. Canadian Journal of Botany 47, 82 l-823. WARBURG, 0. & CHRISTIAN, W. (1942). Isolierung und Kristallisation des Garungsferments Enolase. Biochemische
2. 3. 4. 5. 6.
7. 8. 9. 10. 11.
12.
13. 14. 15. 16. 17. 18. 19.
20.
Plant Pathology
(1972)
2, 27-35
Spore germination and ribosomal activity in the rust fungi I. Comparison of a bean rust fungus and a culturable wheat rust fungus R. C. STAPLESand Boyce
Thompson
Institute,
Z. YANIV Yonkers,
New
York
10701,
U.S.A.
and W.
R. BUSHNELL
Cooperative Agriculture,
Rust Laboratory, Plant Science Research Division, Agricultural Research Service, University of Minnesota, St. Paul, Minnesota 55101, U.S.A.
(Acceptedfor
publication
August
U.S. Department
of
1971)
Ribosomes from uredospores of the as-yet-uncultured bean rust fungus (Uromycesphaseoli) and the culturable Australian wheat rust fungus race 126-ANZ-6,7 (Puccinia gaminis f. sp. tritici) were compared for transferase activity, capacity to bind polyuridylic acid and capacity to incorporate leucine into ribosomes in vivo. While the ribosome activities in bean rust uredospores declined before the germ tube was formed, these activities were maintained by ribosomes from the Australian wheat rust fungus. Mycelium derived from uredospores of the wheat rust fungus, and continuously subcultured for 4 years, had ribosomes that were three times more active in the transferase assay than ribosomes from uredospores.
INTRODUCTION The rusts are a group of parasitic fungi usually obligately dependent on living hosts for growth and completion of their life cycles, but several strains of two rust species, wheat stem rust and flax rust, have been cultured axenically from uredospores [I4, 171. While culture of the rusts is a remarkable achievement, the simple media employed have not been helpful in suggesting the transitions which occur when the sporeling begins to grow. Detailed studies with the bean rust fungus have shown that its uredospores germinate without cell division. Although the protein and RNA contents do not increase like they do when saprophytic conidia germinate [II], protein synthesis is required for germination to occur [.5], probably for formation of cell wall peptides [13]. We compared the properties of ribosomes from uredospores of the saprophytic Australian wheat rust fungus, race 126-ANZ-6,7, and the bean rust fungus which has not yet been cultured. We hoped to provide an insight into the transitions that occur as the sporeling develops into a mycelium. MATERIALS
AND
METHODS
Chemicals
[U-14C]Leucine (specific activity 273 mCi/mmol) and [U-14C]phenylalanine (specific 375 mCi/mmol) were purchased from New England Nuclear Corp., Boston, Mass., U.S.A. Poly[2J4C]uridylic acid, purchased from Miles Laboratories, Kankakee, Ill., U.S.A., had a molecular weight greater than 50,000 daltons activity
28
R. C. Staples, Z. Yaniv and W. R. Bushriell
(gel filtration), and had a specific activity of 0.843 mCi/mmol P. Polyuridylic acid was also purchased from Miles Laboratories, and had a molecular weight greater than 100,000 daltons. Yeast transfer ribonucleic acid (tRNA) was purchased from General Biochemicals Corp., Chagrin Falls, Ohio, U.S.A.
SPores Uredospores of the bean rust fungus [U. phaseoli (Pers.) Wint.] were grown and collected weekly from infected leaves of bean (Phaseolus vulgaris L. var. “Pinto”) plants grown in controlled environment chambers as described previously [II]. Dry uredospores were bottled and stored for periods up to 7 days at 4 “C. These dry spores were termed “resting spores”. Uredospores of the wheat rust fungus [P. graminis (Pers.) f. sp. tritici Eriks. & E. Henn., Australian race 126-ANZ-6,7] were grown and collected twice weekly from infected leaves of wheat (Triticum aestivum L. var. “Little Club”) plants grown in the glass-house. Dry uredospores were bottled and stored in the refrigerator. Mycelia Mycelial cultures of the Australian wheat rust fungus were produced from sterile uredospores on Evans’ peptone medium as described by Bushnell [Z]. The cultures used for these studies had been subcultured monthly for 4 years at St. Paul, Minn., U.S.A. For the present study, mycelial pieces about 2.6 mm wide were cut from colonies 1 month old and placed on media containing 3% glucose, Czapek’s minerals, 1.5% Difco agar, 0.4% Evans’ peptone and 0.4% Difco Casamino acids, technical grade. Four colonies were grown in each g-cm Petri dish in moist jars at 15 “C. Only those colonies that grew in diameter in the final 6 to 26 days before harvest were used. These colonies were shipped in ice-cooled boxes overnight to Yonkers, N.Y., U.S.A. and used the following day. Fresh weight was determined for all colonies at harvest. For dry weight determinations, 12 colonies from each harvest were dried at 93 to 96 “C for 2 days. Spore germination Resting bean rust uredospores were prepared for germination by floating them on water at 4 “C for 16 h as described previously [IO]. After hydration, 200 mg spores were dusted onto two baking dishes containing water as described previously [12]. The baking dishes had a total surface area of 2070 cm2. Germination was carried out in the dark at 20 “C. The agar slide technique of Schein [9] was used to determine germinability by counting at least 200 spores. Germ tubes longer than twice the spore diameter were measured using an ocular micrometer. It was not necessary to hydrate the wheat rust uredospores because germination occurred readily at all concentrations of spores tested up to 0.13 mg/cm2. Consequently, resting wheat rust spores were considered to be equivalent to hydrated bean rust spores, and both are referred to collectively as “nongerminated” spores. Transferuse
assays
Binding. Ribosomes were assayed for polyuridylic acid-directed dependent binding of phenylalanyl-tRNA using the modification by Nirenberg & Leder [7] described previously [19].
and transferase-I of the procedure
Spore
germination
and
ribosomal
activity
in rust
fungi.
I
29
Polymeri
cw [‘“Cl Leucine incorporation Collodion membranes were dusted with spores and the membrane surface sprayed with water as described by Wynn & Gajdusek [l&j. Then 1 ml of water containing 5 @Zi [l*C]leucine was sprayed directly onto the membrane surface and the spores germinated at 20 “C for 1 h. The ribosomes were extracted by pulverizing 20 membranes (1 g spores) in liquid N, as before [12]. The ribosomes were collected by centrifiigation, resuspended, collected on Millipore filters (Millipore Filter Corp., type HA, Bedford, Mass., U.S.A.) and counted by liquid scintillation techniques. Binding
of polyuridylic
acid
Ribosomes washed with 0.25% sodium deoxycholate were incubated with poly [2-r4C]uridylic acid for 10 min at 30 “C. Aliquots containing 3 to 4 Ass0 units were layered on top of 10 to 30% sucrose density gradients prepared as described by Brakke & Van Pelt [I]. The gradients were centrifuged in a Spinco SW 41-Ti rotor (Beckman Instruments, Palo Alto, Calif., U.S.A.) for 75 min, and fractionated into 1 -ml fractions. The fractions were precipitated with cold 5% trichloroacetic acid, collected on Millipore filters (type HA) and counted in the scintillation counter. Quantitative
assays
The amount of ribosomes in suspension was estimated spectrophotometrically at 260 nm assuming that there was 110 pg/A,,,. Protein concentration was estimated by the method of Warburg & Christian [15]. RESULTS
Aspects of morphology Progress of germination. Fig. 1 (a). The Australian tubes were formed by 3 and all spores germinated of both species elongated the lengths shown were
Germination of the two species of rusts is compared in wheat rust spores germinated within 1 h, and all germ h. Bean rust spores required 2 h before germination began, in 4 h. Once germination was initiated, the germ tubes at nearly the same rate [Fig. 1 (b)]. Measurements beyond unreliable, but frequently reached 1000 pm in 24 h.
Axenic culture of the wheat rust fungus. The fungus produced dense, white colonies which increased in dry weight at rates of 0.21 to 0.26 mg/day The colonies were 5 to 8 mm in diameter at harvest, and had dead brown-to-black degenerate mycelia comprising about one-third of total the colonies. The colonies contained clusters of uredospores as well
mycelial (Table 1). centers of volume of as a few
30
R. C. Staples,
Z. Yaniv
and
W. R. Bushnell
teliospores, but very few spores were broken in the grinding procedure used for the extraction of the mycelia. The total fresh weight of samples (53 to 62 colonies) was 2 to 8 g. The dry weight of these colonies was about 23% of their fresh weight. 100 2
80
.-2 6 -2 & ”
60 40 20 0
7 I $ 2 E & CJ
400
200
0 Time
(h)
FIG.
1. Comparative germination of uredospores of bean rust and Australian wheat fungi. (a) Time-course of germination; (b) rate of germ tube elongation. Germination carried out on 1% agar at 20 “C, using a spore concentration of 0.1 mg/cms. O-O, Bean rust; l l , wheat rust, ANZ. 1
TABLE
Diameter
and weight
of mycelial
colonies of the Australian transferme activity Value
Observation No. of colonies Colony age (days) Colony diameter (mm) Fresh weight (mg) Dry weight (mg)
rust was
1 62 74 7.7 122 -
All media contained 3% glucose, Czapek’s minerals Evans peptone and 0.404 Difco Casamino acids, tech.
wheat
rust fungus
per sample 2 53 55 7.2 103 23.8 and
used in assays for
3 60 35 4.9 42 9.6
1.5%
Difco
agar
plus
0.4%
Ribosome activity during spore germination. Leucine incorporation in vivo was high with the ribosomes from bean rust uredospores just beginning to germinate (Table 2), but this activity declined dramatically after 20 h. In contrast, ribosomal activity in uredospores of the Australian wheat rust fungus more than doubled over a 20-h germination period. Transferase
activity during germination
Transferase-I from resting and some of its properties
spores of the bean rust fungus was purified previously described [20]. Transferase-I catalyzes the polyuridylic
Spore
germination
and
ribosomal
activity
in rust
fungi.
31
I
acid-directed and GTP-dependent binding of phenylalanyl-tRNA to ribosomes. This reaction was used to compare ribosomal activities at various stages of germination without the limitations imposed by the presence of unstable activating enzymes [IO]. Since studies with transferase-I have so far been confined to nongerminated spores, a brief characterization of this binding step in germinated bean rust spores is presented. TABLE In viva
incorporation Spore
Q One
of [WJeucine
into protein
2 by germinating
Exposure to [%Tjleucine during germination
stage
and non-germinating
Specific activitya of ribosomes (pmol/mg)
Ratio germinated non-germinated
Bean rust Non-germinated Germinated
Otolh 20 to 21 h
127 5
0.04
Wheat rust Non-germinated Germinated
Otolh 20 to 21 h
148 300
2.00
pmol
of [r*C]leucine
produced
405 cts/min
tq
FIG. 2. Comparison and germinated (Ouredospores only were
of used
to
on the counter.
protein/tube
the activity of transferase-I ) bean rust uredospores. in each 30-min assay.
l
uredospom
from non-germinated Ribosomes (300 yg)
(0 -0) from resting
Optimum requirements for binding. Concentrations of ribosomes, magnesium and enzyme required for optimum binding of phenylalanyl-tRNA to bean rust ribosomes were found to be essentially the same as those for ribosomes from nongerminated spores [ZO] except for the amount of transferase-I. Ribosomes from germinated spores required 100 [*g of transferase-I, while those from non-germinated spores required 20 pg. Not only do ribosomes from germinated spores require more transferase-I for optimal activity than non-germinated spores, but there is less of it in the germinated bean spore. In Fig. 2, it is shown that protein extracts from a gram of germinated rust spores are about half as active with ribosomes as the extracts from a gram of non-germinated spores.
32
R. C. Staples,
Z. Yaniv
and
W. R. Bushnell
Changes in transferase activity during germination. Transferase-I activity was followed during germination of the rust uredospores (Fig. 3). With bean rust, transferase-I activity of the ribosomes was highest in the hydrated, non-germinated spores only, then this activity declined rapidly. The fact that binding activity declines before the germ tube has even emerged clearly shows that inactivation of the ribosome occurs in bean rust uredospores while the germ tube is just beginning to elongate most rapidly [Fig. 1 (b)]. T ransferase-I activity of wheat rust ribosomes remained fully active for 20 h.
Time
(h)
FIG. 3. Transferase-I dependent binding of phenylalanyl-tRNA with polyuridylic acid by ribosomes from germinated rust uredospores. Bean rust spores were hydrated for 16 h at 4 “C, filtered and redispersed on water to initiate germination. Wheat rust spores were not hydrated. Concentration of ribosomes was calculated after purification by density gradient centrifugation, and 240 (*g of ribosomes was used in each assay. O-O, Wheat rust, ANZ; o---O, bean rust. TABLE
Polymerization
activity
of ribosomes from
Ribosome
a One (240
3
germinated
and non-germinated
Phenylalanine + polyuridylic acid bolbg)
incorporation” - polyuridylic (pmol/md
Bean rust Non-germinated Germinated
78 5
31 2
Wheat rust Non-germinated Germinated Mycelia
80 94 310
31 30 90
pmol of [l%]phenylalanine pg) were purified by density
produced gradient
550 cts/min centrifugation.
on
the
uredospores acid
counter.
Ribosomes
Polymerization. Ribosomes from non-germinated and germinated spores were assayed for their capacity to form polypeptides with and without polyuridylic acid as messenger RNA. Both with polyuridylic acid and without it, it was found that bean rust ribosome activity declined 94% (Table 3), and wheat rust ribosomes remained fully active.
Spore
germination
and
ribosomal
activity
in rust
fungi.
33
I
Wheat rust ribosomes from mycelia growing axenically polymerized about three times as much phenylalanine as ribosomes from uredospores (Table 3). Again the same changes were found in the presence or absence of polyuridylic acid.
Binding
of polyuridylic
acid
The capacity of ribosomes from bean rust uredospores to bind polyuridylic acid also declined during germination [Fig. 4(a), (c)l. Although germination reduced the capacity of these ribosomes to bind polyuridylic acid by 93%, the capacity to polymerize phenylalanyl-tRNA was reduced 94% (Table 3), suggesting that there is a dependence between the two processes. As expected, the capacity of ribosomes from wheat rust uredospores to bind polyuridylic acid did not decline during germination [Fig. 4(b), (d)]. I.0
Fraction
number
FIG. 4. Sucrose density gradient analysis of binding of polyuridylic acid by ribosomes from uredospores. (a) Hydrated, non-germinated uredospores of the bean rust fungus; (b) non-hydrated, resting uredospores of the Australian wheat rust fungus, race 126~ANZ-6,7; (c) uredospores of the bean rust fungus germinated 20 h; (d) uredospores of the Australian wheat rust fungus, race 126-ANZ-6,7, germinated 20 h. The monoribosomes are in fraction 4, polyribosomes in the heavier fractions. Centrifugation was from right to left.
DISCUSSION The
incorporation
of rust while ribosomes from the decline in ribosome Australian
3
wheat
exogenous leucine by ribosomes from uredospores of the fungus doubled in activity during germination (Table 2), the bean rust uredospores became virtually inactive. Since activity could provide an important clue to host dependency,
34
R. C. Staples,
Z. Yaniv
and
W. R. Bushnell
detailed studies were made of ribosomes from these divergent species of rusts to delineate the differences at the molecular level. The loss of ribosome activity during germination of the bean rust fungus is apparently due to failure by the ribosomes to bind messenger RNA (Fig. 4). This change is accompanied by a loss of capacity to bind tRNA in response to transferase-I (Fig. 3), and suggests that ribosomes progressively lose the capacity to make protein. Whatever the mechanism, it seems obvious that ribosomes from the bean rust fungus suffer a loss of function during the earliest phases of spore germination. For example, transferase activity declined strikingly in 2 h (Fig. 3), yet the few germ tubes produced had just entered the linear phase of elongation [Fig. 1 (b)]. In contrast, ribosomes from the wheat rust fungus remain active throughout the period of germ tube extension (Fig. 3). A susceptible host plant must have properties which enable the bean rust fungus to maintain or revive its ribosomal functions. Differentiation of the germ tube seems to be a prerequisite both for successful parasitism [4] and for saprophytic growth [IS]. The germ tubes of uredospores studied here were produced on water or moist surfaces of collodion and were not differentiated. Studies with the bean rust fungus have shown that the important change upon differentiation is synthesis of,a template RNA [8], and that the onset of the decline of ribosome activity is the same whether or not the spores are differentiating. While the present studies deal only with non-differentiated germ tubes, the striking differences in ribosome activity found after 2 h of germination suggest that at least some of the changes expected in the transition to saprophytism have already occurred in advance of differentiation. The Australian race 126-ANZ-6,7 of the wheat stem rust fungus was cultured in 1966 by Williams et al. [17]. The transition from sporeling to mycelium requires nearly 7 days [6], and the mycelium develops rapidly once the transition to hyphal growth has occurred (Table 1). Further adaptation to the artificial medium occurs during the first few months of serial subculturings before a stable saprophytic culture is obtained ([3], Bushnell unpublished data). Such an adapted culture had ribosomes that were about three times more active in assays for activity of transferase-I than ribosomes from uredospores (Table 3). Obviously the complete transition to mycelial growth involves an increase in ribosomal activity as well as maintenance of ribosomal function. A more detailed study of the transition of the sporeling to mycelium will be required to establish these changes more definitively. This investigation was supported in part by a grant (GB-17003) from the National Science Foundation and by the Herman Frasch Foundation. Cooperative investigation: Boyce Thompson Institute; Crops Research Division, of Agriculture; and Department Agricultural Research Service, U.S. Department Scientific Journal Series Paper No. of Plant Pathology, University of Minnesota. 7720, Minnesota Agricultural Experiment Station. Mention of a trademark name, proprietary product or specific equipment does not constitute a guarantee or warranty by the U.S.D.A., nor imply its approval to the exclusion of other products also available.
Spore
germination
and
ribosomal
activity
in rust
fungi.
I
35
REFERENCES 1.
BRAKKE, M. K. & VAN PELT, N. (1970).
Linear-log sucrose gradients for estimating sedimentation of plant viruses and nucleic acids. Analytical Biochemistry 38, 56-64. BUSHNELL, W. R. (1968). In vitro development of an Australian isolate of Puccinia gruminis f. sp. trititi. Phytofiatholopy 58, 526-527. BUSHNELL, W. R. & STEWART, D. M. (1971). Development of American isolates of Puccinia gruminis f. sp. tritici on an artificial medium. Phytopatholop 61, 376-379. CHAKRAVARTI, B. P. (1966). Attempts to alter infection processes and aggressiveness of Puccinia graminis var. tritici. Phytopathology 56, 223-229. DIJNKLE, L. D., MAHESHWARI, R. & ALLEN, P. J. (1969). Infection structures from rust urediospores: Effect of RNA and protein synthesis inhibitors. Science, jvew 2’ork 163,481-482. KUHL, J. L., MACLEAN, D. J., SCOTT, K. J. & WILLIAMS, P. G. (1971). The axenic culture of Pucciniu species from uredospores : experiments on nutrition and variation. Canadian Journal of Botany 49, 201-209. NIRENBERG, M. & LEDER, P. (1964). RNA codewords and protein synthesis. The effect of trinucleotides upon the binding of sRNA to ribosomes. Science, Jvew York 145, 1399-1407. RAMAKRISHNAN,L. & STAPLES, R. C. (1970). Changes in ribonucleic acids during uredospore differentiation. Phytopatholopy 60, 1087-1091. SCHEIN, R. D. (1962). Storage viability of bean rust uredospores. Phytopatholopy 52, 653-657. STAPLES, R. C. (1968). Protein synthesis by uredospores of the bean rust fungus. Netherlands Journal of Plant Pathology 74 (sup& 1) 25-36. STAPLES, R. C.. SYAMANANDA, Comoarative biochemistrv , R., , KAO. V. & BLOCK. R. T. (1962). of obligately parasitic and saprophytic fungi. II. ’ Assimilation of C14-labeled substrates by germinating spores. Contributions. Boyce Thompson Institute for Plant Research 21, 345-362. STAPLES, R. C., YANIV, Z., RAMAKRISHNAN, L. & LIPETZ, J. (1971). Properties of ribosomes from germinating uredospores. In Morphological and Biochemical Events In Plant-Parasite Znteraction ed. by S. Akai & S. Ouchi, pp. 59-90. The Phytopathological Society of Japan, Tokyo. TROCHA, P. & DALY, J. M. (1970). Protein and ribonucleic acid synthesis during germination of uredospores. Plant Physiology, Lancaster 46, 520-526. TUREL, F. L. M. (1969). Saprophytic development of the flax rust Melampsora lini, race No. 3. Canadian Journal of Botany 47, 82 l-823. WARBURG, 0. & CHRISTIAN, W. (1942). Isolierung und Kristallisation des Garungsferments Enolase. Biochemische
2. 3. 4. 5. 6.
7. 8. 9. 10. 11.
12.
13. 14. 15. 16. 17. 18. 19.
20.