Uredospore wall digestion during germination independent of macromolecular synthesis

PhpsologiGal Plant Pathology (1976) 9, 265-272

Uredospore wall digestion during germination of macromolecular synthesis

independent

SAMUEL L. HESS, PAUL J. ALLEN and HOPE LESTER Department of Botany, University of Wisconsin, Madison 53706, U.S.A. (Acceptidfor publication August 1976)

Dissolution of the wall in the germ pore region of uredospores of Puccinia graminis tritici is completed within 30 min when spores are incubated in suspension in a medium containing a germination stimulant. The spores are immediately permeable to L-leucine, uracil and phosphorus, but impermeable to erotic acid. Uracil and phosphorus are incorporated into a macromolecule, which in the case of phosphorus was shown not to be RNA. L-Leucine is incorporated into protein from the first few minutes of incubation. Cycloheximide inhibits the incorporation of leucine without interference with wall dissolution. Methyl cis-ferulate, on the other hand, prevents wall dissolution but has no effect on L-leucine incorporation. Wall dissolution, the first observable phase of germination, does not therefore depend on RNA or protein synthesis, and inhibition of this step in germination by methyl cis-ferulate does not result from an inhibition of protein synthesis.

INTRODUCTION

The first step in development of rust uredospores is controlled by an endogenous inhibitor which blocks the breakdown of the spore wall to form a pore through which the germ tube can emerge [S]. In Puccinia graminis tritici, the inhibitor is methyl cis-ferulate (MF) [I]. U n d er conditions favorable to germination, removal of the inhibitor permits dissolution of the germ pore plug to occur in less than 30 min [a. To determine whether this phase of germination is under nuclear or cytoplasmic control, it is important to establish definitively whether any steps in macromolecular synthesis are a prerequisite to wall digestion. Preliminary evidence from inhibitors [S] indicated that they were not. Additional evidence on the fate of added precursors and on the time relations between synthesis and wall digestion was needed and is presented in this paper. MATERIALS

AND

METHODS

Uredospores of Puccinia graminis Pers. var. tritici Eriks. & E. Henn. race 56 were obtained as previously described [2]. L-[(U) -l*C]leucine (285 mCi/mmol) , Omnifluor, carrier-free [32P]04 and Aquasol were obtained from New England Nuclear. [G-l*C]orotic acid (60.8 mCi/mmol) was obtained from Nuclear Chicago. [2-l*C]uracil (52.5 mCi/mmol) was obtained from Amersham Searle. oxyethylated tertiary octylphenol polymethylene polymer (Triton WR 1339, TN) was obtained from Ruger Chemical Co. Methyl cl-ferulate (MF) was prepared from trans-ferulic acid as previously described [I, S].

266

S. L. Hess, P. J. Allen and H. Lester

RNA synthesis For experiments with r4C, 170 mg of spores were suspended in 85 ml of 0.04% TN in potassium-calcium phosphate buffer ( lop2 M Kf, lo-4 M Gas+) at pH 7.0 [4] with lOA M nonyl alcohol (NA) and incubated at 20 “C on a reciprocal shaker at 150 cycles/min. For experiments with s2P, the same medium was used except the buffer was sodium-calcium borate (10-z M Na+, 10m4M Caz+) at pH 7.5. Immediately following suspension of the spores in the buffered medium, 8 PCi of [14C]oro& acid or [14C]uracil or 500 l&i of 32P, were added to the suspension. Uptake of the labelled compounds was determined by removal of 20 ml from the suspension at 15 min intervals beginning with a control removed at 0 time and extending to incubation periods up to 60 min. Each sample was removed and pipetted into 5 ml of a 1 mg/ml solution, respectively, of cold erotic acid, uracil or sodium phosphate buffer (0.1 M, pH 7.5). The spores were collected by filtration on Whatman no. 1 filter paper and washed with 10 ml of 0.04% TN. They were then ruptured in a Braun cell homogenizer for 1.5 min with 20 g glass beads and 14 ml of 0.014 M Tris buffer at pH 7.8 containing 0.07 1 M NaCl, O-071 M EDTA and 0.14% bentonite. The resulting homogenate was centrifuged at 12 000 g for 10 min, the supernatant removed, its volume recorded and 3-O ml mixed with 9.5 ml Aquasol. Radioactivity of each sample was determined by counting in a Model 3385 Packard liquid scintillation spectrometer. The ct/min were converted to d/min after determining the efficiency of the instrument. Incorporation of labelled phosphorus or uracil into RNA was determined by incubating 200 mg spores in 100 ml sodium-borate buffer containing 250 $i [Wluracil or 500 PCi 32P. After 60 min of incubation, the spores were removed by filtration and disrupted as described above. A 9-O ml sample of the resulting homogenate was mixed with 1.0 ml of 10% sodium dodecyl sulfate (SDS) to give a final concentration of 1 y0 SDS, O-1y0 bentonite, 0.05 M NaCl, 0.05 M EDTA and 0.01 M Tris buffer. This was stirred for 30 min at room temperature with 5 ml of buffersaturated phenol containing O*1o/o 8-hydroxyquinoline [8]. The RNA was then extracted by the phenol method of Ingle et al. [7]. The pellet derived from the ethanol precipitate was redissolved in 2-O ml of 0.025 M Tris buffer, pH 8.1, containing O-025 M NaCl. Further purification of RNA was achieved by selective precipitation with cetyltrimethylammonium bromide (CTAB) [3]. The final CTAB precipitate was resuspended in 1.0 ml of O-1 M sodium acetate buffer, pH 5.2, the optical density at 260 nm recorded and the radioactivity determined by counting the aliquot with Aquasol. To determine the uptake of [14C]uracil or s2P for these same samples, the remainder of the original homogenate was centrifuged at 12 000 g for 10 min, the supernatant removed and its volume recorded. Three ml samples were counted with 9.5 ml Aquasol in a liquid scintillation spectrometer as described above. Electrophoresisof RNA An aliquot containing 80 to 100 pg RNA was precipitated with 3 vol. of 95% ethanol, the precipitate collected after centrifugation and dissolved in 100 ~1 of 8% sucrose in 0.001 M Na, EDTA, pH 6.2. A 50 ~1 sample was layered on each

Uredospore wall digestion during germination

267

6 x 100 m m gel of 2.0% acrylamide-I -0% agarose, pH 8.2 [IO]. The RNA was fractionated on the gel by electrophoresis at 2 mA/tube for I.5 h at 5 “C. The gels were removed from the tubes and scanned at 260 nm. After recording the A,,,, the gels were sliced into 0.8 m m segments, which were then dissolved in 0.7 ml H,O, at 60 “C overnight and the radioactivity measured. RNA concentrations were determined by the orcinol method [I] or by absorbance at 260 nm. Protein concentrations were determined by the Lowry method PI* Protein synthesis Uptake and incorporation of r..-[Wlleucine was measured following procedures similar to those used for RNA precursors, with the following modifications or additions: the NA of the basic medium was in some experiments replaced with MF at a concentration which completely inhibited pore plug digestion (6 x 1Om7M) ; or the basic medium containing NA, or with NA replaced by MF, was supplemented with 50 pg/ml cycloheximide (CH). At the beginning of each incubation period, 4 l&i of L-[14C]leucine was added to each flask. Samples were removed and added to unlabelled L-leucine, and the spores then separated from the incubation mixture, washed, homogenized and centrifuged following the procedures used with RNA precursors. Uptake was determined by mixing 3.0 ml of the supernatant with Aquasol and counting as previously described. To distinguish L-[r4C]leucine which had been incorporated into protein from free L-[Wlleucine, 1.0 ml of the supernatant was treated with an equal volume of hot 20% trichloroacetic acid (TCA) and the precipitate collected on a glass fibre filter. The filter was washed 3 times with 10 ml of 20% TCA, dried and the ct/min determined with IO.0 ml toluene-based Omnifluor in the liquid scintillation spectrometer. D/min were determined as above. RESULTS

AND

CONCLUSIONS

No uptake of [14C]orotic acid was observed. During a 30 min incubation of spores with this precursor, radioactivity of the homogenate did not rise significantly above the control level of 13 d/min pg RNA, obtained from spores removed and homogenized immediately after introduction of the labelled erotic acid. Thus the previously reported failure to obtain measurable incorporation of erotic acid [6] could have resulted and, in view of the data below, did result from failure of this precursor to enter the spores. In contrast to erotic acid, both uracil and phosphorus were taken up rapidly by uredospores. Uptake of [14C]uracil and s2P were observed at the earliest samplings and continued at an appreciable rate over the 45 to 60 min periods of exposure (Fig. 1). Uptake occurred at the same rate in spores inhibited by MF and in those germinating in the presence of NA. Fractionation of the homogenate to separate RNA from other constituents after a 60 min incubation gave a recovery of about 40% of the total RNA in the phenol extract and in the subsequent CTAR precipitate (Table 1). The phenol extract was slightly enriched in s2P/pg RNA, but most of the labelled phosphorus was lost on further purification. Only 0.06% of the labelled phosphorus of the original homogenate appeared in the CTAB precipitate (Table 2).

S. L. Hess, P. J. Allen and H. Lester

263

Time (mid FIG. 1. Uptake of ssP and [Z-r*C]uracil by suspended urcdospores of P. g. r&Z. The spores were incubated in suspension in calcium-sodium borate buffer (10-s M Na+, 10-4 M Gas+), pH 7.5, with 094% Triton WR 1339 and IO-4 M a-nonyl alcohol. (0) ssP uptake; (0) [2J*C] uracil uptake.

- 0.40

- 0.30

0.60

- 0.20

O-30

- o-10

0.00 2

0

4

6

8

f 3 54 0 x P w a E 3.

IO

Distance migrated km) FIG. 2. Fractionation of RNA from the CTAB precipitate by gel electrophoresis. The gel 50 pg RNA in 50 $ was composed of 2.0% acrylamide and 1.0% agarose. Approximately were applied to each tube and electrophoresis carried out at 2 mA/tube for 1.5 h at 4 “C. (-)

O.D.;

(- - - -) @lOl

=P,

269

Uredospore wall digestion during germination TABLE I RNA” content of variousfractionsjkm P. g. tritici ureahsporesafter incubationfor I h in suspension Fraction

mg/200 mg spores

o/o total

0/0 spore weight

Homogenateb Aqueous phenol ETOH precipitate CTAB precipitate

2.53fO.40 189+0*14 1~10+0*10 0*94+0*14

100 43 44 37

0.55 0.55 0.47

(dRNA determined by the orcinol method [II]. b Protein content of the spore homogenate was 4.8 + 0.4% as determined method [9].

1.26

by the Lowry

Upon fractionation by acrylamide-agarose gel electrophoresis of 50.0 pg of the CTAB precipitate (with 8.3 ct/min pg) none of the label migrated with any of the RNA fractions, but remained in the first O-8 to 1.6 m m of the gel, where no substance absorbing at 260 nm occurred (Fig. 2). Thus the small amount of radioactivity which appeared in the CTAB precipitate was incorporated into some compound other than RNA. TABLE 2 Incorporation of a2P and ~C]uracil into RNA fracttim of uredosporesof P. g. tritici incubated in mpensionb in the presenceof labeled precursors

Fraction Homogenate Aqueous phenol ETOH precipitate CTAB precipitate

pmol 3sPc/pg RNA0 220+80x lo-l2 369 + 80 x lo-l2 27.7 + 3.6 x lo-l2 0.43 z 0.02 x lo-l2

o/o recovery saPd 100 51 4 046

pm01 [2-W]uraciP/ pg RNA= 121+4x 10-o 365*98x lo-* 6.3* 1.1 x 10-O 4*4+ 1.2 x 10-s

o/o recovery [r*C]uracild 100 96 1.2 O-6

a RNA was determined by the or&o1 method [II]. b 200 mg of spores were suspended in 100 ml of incubation medium consisting of 10” M sodium-10-d M calcium borate buffer, pH 7.5, with 0.04% Triton W R 1339 and lo-” M n-nonyl alcohol. O One half mCi of carrier-free [32P]0, was added to the medium. d Radioactivity as o/0 of that in the original spore homogenate. e One quarter mCi of [2-W]uracil was added to the incubation medium.

The results with labelled uracil were essentially similar (Table 2). The CTAB precipitate recovered after incubation with 250 PCi [2-r4C]uracil contained only 0.6% of the radioactivity of the homogenate, but because of the low level of activity (0.5 ct/min pg), no zones of radioactivity were detected after gel electrophoresis. These experiments show that even though uracil and phosphorus penetrate the spore during the first hour of germination, no detectable incorporation into RNA occurs. This fact was, however, evident only after the final stages of purification by gel electrophoresis, when contaminating 32P was finally removed. Previous reports of RNA synthesis based on the incorporation of labelled precursors may therefore be misleading, since our results show some incorporation into a non-RNA component which contaminates the phenol and CTAB fractions.

270

S. L. Hess, P. J. Allen and H. Lester

Uptake of labelled r.-leucine by uredospores was demonstrated for all experimental conditions (Fig. 3). Uptake of L-[14C]leucine was evident in the first few minutes of incubation. It continued for the duration of the experiment, but the internal level did not reach that of the external solution. Neither MF nor CH reduced the rate of leucine penetration into the spore. CH appears if anything to have increased the rate of uptake. 30

Time (min)

FIG. 3. Uptake of L-r*Cjleucine by suspended uredosporesof P. g. fritin'. The spores were suspended in calcium-potassium phosphate buffer ( 10ez M Kf, 10-a M Ca*+), pH 7.0, with 0*04% Triton WR 1339. In addition the suspension contained: (0) 10-d M n-nonyl alcohol, (A) 6 x 10-T M methyl &-ferulate, ( l ) lo+ M n-nonyl alcohol with 50 pg/ml cycloheximide, (A) 6 x 10-T M methyl cis-ferulate with 50 pg/ml cycloheximide.

Incorporation of L-[r4C]leucine into protein began within a few minutes after the start of incubation. Although the initial rate of incorporation was low E1.2,131 it increased with time (Fig. 4). CH at 50 kg/ml inhibited incorporation almost completely beginning during the first few minutes of incubation. CR therefore penetrates the uredospore rapidly and there is not an initial period when protein synthesis escapes the action of this inhibitor. Figure 4 shows that the onset and progress of protein synthesis occur similarly in the presence or absence of M.F, and, as reported previously [6], have reached comparable levels after 30 min incubation. During this interval, dissolution of the germ pore plug progresses unabated in the presence of CH and in the absence of protein synthesis. The failure of the germ tube to emerge and grow in the presence of CH indicated that the later phases of germination, unlike wall digestion, are dependent upon new protein synthesis. The results presented above show that during the entire period of germ pore plug dissolution (30 min) there is no new RNA synthesized but L-leucine is taken up and incorporated into protein. This incorporation is prevented by CH from the beginning, but not at any time by MF. It is therefore concluded that the dissolving of the spore wall in the region of the germ pore does not require new synthesis of either nucleic

Uredospore

wall digestion

271

during germination

acids or protein and must depend upon the activity of enzymes which are already present in the dormant spore. These findings conf%m previous indications from

Time (mid

FIG. 4. Incorporation of L-[U-14C]leucine into tricbloroacetic acid insoluble protein by suspended uredospores of P. g. &i&i.

The spores were incubated in suspension for the indicated

time periods in calcium-potassium phosphate buffer (10-s Y K+, lo-” M Cast), pH 7.0, with 0.04% Triton

WR 1339. In addition

the suspensions contained:

(0) 10d4 M n-nonyl alcohol,

(A) 6x 10-T M methyl cis-ferulate, (0) 10M4M n-nonyl alcohol with 50 pg/ml cycloheximide (A) 6 x 10-r M methyl cis-ferulate with 50 w/ml

cycloheximide.

inhibitor studies [S, 61, but indicate that reports of early RNA synthesis probably resulted from incomplete purification of RNA [II’]. This work was supported in part by grant GB-43231 from the NSF and by funds from the Research

Committee

of the Graduate

School, University

of Wisconsin.

REFERENCES 1. ALLEN, P. J. (1972). Specificity of the &isomers of inhibitors of uredospore germination in the rust mngi. Proceedingsof the National Academy of Sciences,U.S.A. 69, 3497-3500. 2. ALLEN, P. J., STRANGE, R. N. & ELNAGHY, M. A. (1971). Properties of germination inhibitors from stem rust uredospores. Phytopathology 61, 1382-l 389. 3. BELLAMY, A. R. & RALPH, R. K. (1968). Recovery and purification of nucleic acids by means of cetyltrhnethylammonium bromide. In Methods in Enqwwlogy, Ed. by L. Grossman & K. Moldave, Vol. 12, Part B, pp. 156-160. Academic Press, New York. 4. DUNKLE, L. D. & ALLEN, P. J. (1971). Infection structure differentiation by wheat stem rust uredospores in suspension. Phytopathology61,649-652. 5. DUNKLE, L. D., MAHESHWARI, R. & ALLEN, P. J. (1969). Infection structures from rust uredospores: effect of RNA and protein synthesis inhibitors. Science163, 481-482. 6. HESS, S. L., ALLEN, P. J., NELSON, D. & LESTER, H. (1975). Mode of action of methyl cis-ferulate, the self-inhibitor of stem rust uredospore germination. Physiological Plant Pathology 5, 107-l 12. 7. INGLE, J., KEY, J. L. & HOLM, R. E. (1965). Demonstration and characterization of DNA-like RNA in excised plant tissue. Journal of Molecular Biology 11, 730-746. 8. KIRBY, K. S. (1968). Isolation of nucleic acids with phenolic solvents. In Methods in Enzymology, Ed. by L. Grossman & K. Moldave, Vol. 12, Part B, pp. 87-99. Academic Press, New York.

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9. Loway, 0. H., ROSEBROUGH,N. J., FARR, A. L. & RANDALL, R. J. (I 95 1). Protein measurement with the Fohn phenol reagent. Journal of Biological f%m&y 193, 265-275. 10. PEACOCK, A. C. & DINGMAN, C. W. (1988). Molecular weight estimation and separation of ribonucleic acid by electrophoresis on agarose-acrylamide composite gels. Biochemist 7, 668-674. 11. SCHNEIDER, W. C. (1957). Determination of nucleic acids in tissues by pentose analysis. In Methods in Enzymology, Ed. by S. P. Colowick & N. 0. Kaplan, Vol. 3, pp. 680434. Academic Press, New York. 12. STAPLES,R. C., SYAMANANDA, R., Lo, V. & BLOCK, R. J. (1962). Comparative biochemistry of obligately parasitic and saprophytic fungi. II. Assimilation of r%-labelled substrates by germinating spores. Contributionsfrom Boyce lhmjson Institui 21, 345-362. 13. TRCJCHA,P. & DALY, J. M. (1970). Protein and ribonucleic acid synthesis during germination of uredospores. Plant Ptpiohgy 46, 520-526.