Preserved polyadenylated ribonucleic acid in dormant conidia of Neurospora crassa and new RNA synthesis during spore germination

EXPERIMENTALMYCOLOGY

11,

317-330(1987)

Preserved Polyadenylated Ribonucleic Acid in Dormant Co eurospora crassa and New RNA Synthesis during Spore Ge ROBERT BRAMBL,"

NORA PLESOFSKY-VIG,? PETERJ.RUSSELL$

JAMES R. HAMMETT,?:AND

*Plant Molecular Genetics Institute and Department of Plant Pathology and fDepartment of Genetics Cell Biology and Department of Plant Pathology, 495 Borlaug Hail, The University of Minnesota, Saint Minnesota 55108, and $Department of Biology, Reed College, Portland, Oregon 97202

and Paul,

Accepted for publication August 12. 1987 BRAMBL, R., PLESOFSKY-VIG, N., HAMMETT, 3. R., AND RUSSELL, P. 3. 1987. Preserved polyadenylated ribonucleic acid in dormant conidia of Neurospora crassa and new RNA synthesis during spore germination. Experimental Mycology 11, 317-330. Molecular hybridization with cloned cDNAs established that transcripts for specific proteins were present in the RNA of dormant conidiospores of Neurospora crassa. Among these were transcripts for the proteolipid subunit of the mitochondrial ATPase-ATP synthase; a larger proportion of the proteolipid transcripts was present in the nonpolyadenylated rather than the polyadenylated RNA fraction (relative to the pattern of germinated spores). The mRNA encoding the Neurospora M, 83.000 heat-shock protein was also preserved in the dormant spores; however, the transcript for the M, 30,000 beat-shock protein was not detectable. The polyadenylated RNA from dormant conidia was translatable in vitro; specific immunoprecipitation and electrophoresis assays showed that this preserved RNA included transcripts for nucleus-encoded subunit peptides of the mitochondrial enzymes cytochrome c oxidase and the ATPase-ATP synthase. As determined by molecular hybridizafon, conidia began to accumulate RNA for the ATPase proteolipid subunit immediately upon their suspension in incubation medium, with a doubling of the quantity of these specific transcripts by 15 min. Total RNA content of the spores dropped sharply during the first 30 min of germination, but by 45 min the amount of total RNA began to increase. Two inhibitors of RNA synthesis, lomofungin and proflavine-SO,, inhibited spore germination by about 90%. in addition to inhibiting incorporation of 13H]uridine into RNA. This inhibition of spore germination by proftavine-SO, required that the spores be exposed to the drug during the first 60 min after inoculation into nutrient medium; if the inhibitor was added after 60 min of incubation, the spores germinated with normal kinetics. Dormant conidia, therefore, contain a population of preserved mRNA, but the translation of this preserved mRNA is not sufficient for spore germination; new gene transcription appears to be required for the resumption of protein synthesis and germ tube emergence. c: 1987 Academic

Press, Inc.

DESCRIPTORS: Neurospora crassa; polyadenylated RNA; mRNA; conidia: spore germination; RNA synthesis; heat-shock proteins; ATPase; cytochrome c oxidase. INDEX

Dormant asexual spores of fungi, like quiescent cells of other types of organisms that undergo a similar phase of developmental arrest, contain a population of preserved, stable mRNAs. The cellular translation of these mRNAs is presumed to be responsible for the protein synthesis which occurs early in spore activation and which appears to be essential to spore germination (Lovett, 1976; Van Etten et nl., 1976; rambl et al., 1978). This preservation and

delayed utilization of latent mRNA has been regarded as an example of control of gene expression and cell develo the post-transcriptional level. Nevertheless, tke types of genetic Bnformation specified by the preserved mRNA in dormant spores have not yet been defined. Examples of identifiable mRNAs unique to conidia have not transcripts of nonsuch as trpC and ar 317 0147-5975187 $3.00 Copyright All rights

0 1987 by Academic Press, Inc. of reproduction in any fom reserved.

318

BRAMBL

ET AL.

dulans, have been identified in the MATERIALS AND METHODS poly(A)+ RNA fraction of dormant spores Growth, harvesting, and germination of (Yelton et al., 1983). It is possible that a conidia. Conidia were obtained from culportion of this spore RNA persists vestigially through dormancy after synthesis tures of a wild-type strain of N. crassa (74and translation during sporulation and is OR23-IA) grown on a vegetable juice agar subsequently degraded upon germination. medium as described (Brambl, 1975). NonAlternatively, translation of some of the hydrated conidia were collected by partipreserved mRNAs could be essential to re- tioning the hydrophobic conidia into an sumption of normal metabolism during inert isoparaffinic hydrocarbon fluid and spore germination, as proposed for the sub- subsequent collection by filtration, as deunits of the ATPase-ATP synthase of Bo- scribed in detail elsewhere (Bonnen and tryodiplodia theobromae (Wenzler and Brambl, 1983). This method allowed longBrambl, 1981). Protein synthesis on cytoterm storage of conidia at -60°C without plasmic ribosomes clearly seems required loss of viability. Conidia were germinated for spore germination (Lovett, 1976; in minimal medium (Vogel, 1956) with 2% Brambl et al., 1978), but it is not known sucrose and 7 x lo6 or 7 x lo7 conidia/ml. whether the preserved transcripts are suffi- The flasks were shaken in a rotary incucient for this protein synthesis or whether bator (1.50 rpm) at 30°C. de nova RNA synthesis additionally is reRNA extraction. For isolation of RNA from activated conidia, the culture flasks quired. We have begun to examine some of these were rapidly cooled to 0°C by swirling questions by analysis of the poly(A) + RNA them in granulated dry ice and the spores fraction of dormant and germinating co- were collected by filtration at 4°C. The nidia, or asexual spores, of the fungus Neuspores were disrupted in a CO,-cooled homogenizer rospora crassa. Results presented in this Braun MSK mechanical paper show that the preserved mRNA (Brambl, 1975) with silanized glass beads in (Mirkes, 1974; Bonnen and Brambl, 1983) 8 M guanidine-HCl (pH 7.6), 5 mM dithioencodes proteins such as the subunit pep- threitol, 50 mM Tris-HCl (pH 7.6) tides of two mitochondrial enzymes and (Chirgwin et al., 1979). After cell breakage, one (but not all) of the major heat-shock sodium sarkosyl was added to a concentraproteins of Neurospora. These preserved tion of OS%, and the aqueous extract was transcripts, one of which appears to have a separated from the beads and cell debris by shorter 3’-polyadenylate tract than the centrifugation. This supernatant fluid was counterpart mRNA of germinated spores, extracted with water-saturated chloromay not be solely responsible for synthesis form-butanol (4:1, v/v). RNA was precipiof their encoded proteins early in germinatated with 0.5 vol of ethanol and 0.025 vol tion, however, since we also found that the of 1 M acetic acid and was reprecipitated spores are capable of new gene transcripseveral times from 6 M guanidine-HCI. tion within the first few minutes after sus- The final RNA was solubilized with 10 mM pension in a germination medium. We show Tris-HCl (pH 7.6) and 0.1 mM EDTA and here that chemical inhibition of de nova precipitated with ethanol. For preparation RNA synthesis blocks both protein syn- of RNA used for in vitro translation, the thesis and spore germination, thus indiRNA was extracted with 0.1 M Tris-HCl cating that the preserved mRNA is insuffi(pH 8.8), 1% sodium sarkosyl, 0.05 M socient to direct the early, essential protein dium acetate, 1.O% sodium pyrophosphate, synthesis of spore germination. 0.05 M NaCl, 5 mM disodium EDTA, and

PRESERVED

POLY(A)+

RNA IN Neurosporo

0.5 mgiml heparin. The beads were removed from the slurry by filtration through a coarse filter. The resulting aqueous extract was shaken with an equal volume of chloroform:isoamyl alcohol (24: 1, v/v), and the two layers were separated by centrifugation. This extraction was repeated (usually four times) until no precipitate was found at the interface. In preparing RNA used to compare the mRNAs of dormant and germinated spores in hybridization experiments, the RNA was extracted from the cell brei initially with chloroform:isoamyl alcohol, followed by an extraction with buffer-saturated phenol that contained 0.1% 8-hydroxyquinoline. Typically, from about 4 g fresh wt of conidia, we obtained 50 mg of total RNA. Isolation of poly(A)+ RNA. The poly(A)+ RNA fraction was prepared with oligodeoxythymidyiic acid [oligo(dT)]-cellulose chromatography (Aviv and Leder, 1972). The column resin was obtained from Collaborative Research (Bedford, MA). The RNA sample was heated to 68°C for 2 min and rapidly cooled in an ice-water bath just before application to the column. The two major column fractions (unbound and bound material that absorbed strongly at 254 nm) were each combined with 0.1 vol of 2 A4 sodium acetate (pH 5.5) and 2 vol of ethanol for 12-h precipitation at - 20°C. 1~ vitro translation of mRNA, immunoprecipitation, and peptide electrophoresis. The rabbit reticulocyte translation system was obtained from Bethesda Research Laboratories (Bethesda, MD), and the proteins were radiolabeled with L-[3,4,5-3H]leucine (140 CilmM; New England Nuclear, 3oston, MA). Similar results were obtained by use of a wheat embryo in vitro translation system prepared as described previously (Wenzler and Brambl, 1978). The antisera to a mixture of subunits of cytochrome c oxidase and to subunits of the Pi-ATPase were prepared and used as de-

CONIDIA

319

scribed elsewhere (Jack1 and Sebald, 1975; Brambl, 1980; Wenzler and and the enzyme-IgCl complexes were isolated with protein A-Sepharose (Werner and Machleidt, 1978). The peptides of the in vitro-radiolabeled immu s were mixed with immunopre e same enzymes obtained from mite a of Neurospora cells that had been radiolabeled in vivo with L-[i4C]leucine (340 mCi/ mM), and they were subjected to electrophoresis in 12.5 or 15% polyacryla gels containing Na dodecyl sulfate a viously described (Brambl and 1976). The gels were sliced into I-mm segments for radioactivity analysis by liquid scintillation spectrometry. Detection of mRNAs for specific genes. Dot-blot and Northern blot ~ybrid~~a~~o~ assays were performed as described in detail elsewhere (Plesofsky-Vig an 1987) with cloned cDNAs prepar major heat-shock proteins (hsp) of hTeupospora (of I& 83,000 and 30,000), w have recently described (Plesofsky Brambl, 1987), and with a cloned AV48, for the proteolipid subunit of the Neurospora mitochondrial ATPase (Viebrock et al., 1982). Equal amounts of RNA (2.5 and 10 pg) from each cell treat~e~t were applied under light vacuum to nitrocellulose filters, and replicate filters were

were a~toradiographed, and the radioactivity of the filters was scintillation spectrometry ; hybrrdr dioactivity was found to increase linearly with the increase in RNA bound to t filter (a four-fold increase for each). ‘I measured radioactivity (dpm) for each RNA sequence of a ~arti~~la~ cell treat’ Abbreviations used: IgG, immunoglobulin (G; hsp, heat-shock protein; pNcHsp, probe encoding Neuw~pora heat-shock protein.

320

BRAMBL

n-rent was multiplied by 0.1% of the estimated RNA content of the cells, as described in detail elsewhere (Plesofsky-Vig and Brambl, 1987). Quantification

of cellular

RNA content. of Neurospora

Because the RNA content cells would be expected to change during spore germination, we determined the original RNA content of each preparation. Purified rabbit globin RNA (5 kg) was added to N. crassa cell homogenates (containing approximately 1 g of spores) from which RNA was isolated. Isolated N. crassa RNA, in parallel with a known amount of pure globin mRNA as standard, was applied to a nitrocellulose filter and was hybridized to a P-globin probe, pRB 1.95 [containing the 5’ portion and flanking sequences of the pl gene for the rabbit pglobin (Lacy et al., 1979)]. The resultant measure of efficiency of RNA extraction was used to calculate the original RNA content of the cell homogenate (Table 1). Use of the inhibitors 3’-deoxyadenosine (cordycepin), lomofungin, and profavine-

ET AL.

and its concentration was determined (Chaykin, 1966). Radioactivity was determined by liquid scintillation spectrometry. RESULTS

Identification of ATPase proteolipid transcripts in dormant and germinated conidia. To test for preservation of specific transcripts in dormant conidia of Neurospora, we employed molecular hybridiza-

tion with a cloned cDNA for the proteolipid subunit of the Neurospora mitochondrial ATPase (Viebrock et al., 1982). The RNA preparations of dormant conidia and of germinated spores or young mycelial cells (360 min) were subfractionated by oligo(dT)-cellulose chromatography into fractions enriched or depleted in polyadenylated RNA. Quantitative dot-blot assay of these fractions (Fig. 1) with the proteolipid cDNA showed that these transcripts were present in the RNA obtained from dormant spores but, surprisingly, that a large proportion of the proteolipid transcripts was present in the nonbinding fraction rather than in the polyadenylated RNA fraction. By contrast, in the RNA fractions from the germinated spores, a much larger proportion of the proteolipid transcripts was found in the polyadenylated RNA fraction. These results suggest that the 3’polyadenylate segments of these transcripts may be somewhat shorter in dormant spores than in germinated spores.

SO,. Lomofungin was a gift of G. B. Whitfield (Upjohn Co.). Conidia were incubated in liquid medium with either 3’-deoxyadenosine (4.0 mM), lomofungin (20 pg/ml), or proflavine-SO, (15 kg/ml). Conidia were incubated for the specified periods and then radiolabeled for 60 min with [5-3H]uridine or [18-3H]adenine (1.5 t&i/ml, sp act 14 CilmM). The RNA was extracted and fractionated by affinity chromatography. Identification of heat-shock transcripts Conidial protein synthesis in the pres- preserved in dormant conidia. Our recent ence of these three inhibitors was meastudies have shown that the ungerminated conidia of Neurospora synthesize heatsured by the addition of L-[4,5-3H]lysine (1.25 t.Ki/ml, sp act 53 CilmM) to the me- shock proteins within minutes after suspension of the dormant spores in an incubation dium for a 60-min interval at each point, The total soluble protein was extracted by medium at the heat-shock temperature of 45°C (Plesofsky-Vig and Brambl, 1985a). grinding the conidia with 0.01 M Tris-HCl (pH 8.0) in a mortar and pestle, followed by This rapid protein synthetic response of accentrifugation and precipitation of the solu- tivated spores, which other work (Bonnen bilized proteins with trichloroacetic acid and Brambl, 1983) led us to expect to be (10% final concentration). The precipitated transcriptionally inactive, suggested to us protein was suspended in grinding buffer, earlier (Plesofsky-Vig and Brambl,

PRESERVED

POLY(A)+

RNA IN Neurospora CQNIDIA

321

TABLE 1 Specific RNA Content of Conidiospores Changes with Time of Spore Incubation” Source of RNA, mg total spore RNA content,b and +g RNA assayed Spore, dormant (12.57 mg) 10 lJ% 2.5 I% Average (10 )*g)’ 15 min (IO.28 mg) 10 IJS 2.5 pg Average (10 p,g) 30 min (9.31 mg) 10 I% 2.5 I% Average (10 pg) 45 min (12.27 mg) 10 I% 2.5 I-G Average (10 kg)

dpm ( - bkgd) pNcHsp-83

pNcHsp-30

Adjusted dpm ( x 0.1% RNA) AV48

pNcHsp-83

pNcHsp-30

AV48

1951 780

850 393

133 38

2,452 980 3,186

1068 494 1522

167 48 180

2829 636

653 337

390 93

2,908 654 2,762

671 346 1028

401 96 393

2924 a41

724 816

1060 269

2,722 789 2,939

677 760 1857

9&7 250 994

5758 2696

735 435

1731 567

7,065 3,308 10,149

902 534 :519

2124 930 2922

u The RNA was extracted directly from dormant spores or from spores incubated 15, 30, and 45 min before harvest. Equal amounts of each RNA (10 and 2.5 kg) were applied in replicate to nitrocellulose filters which were subsequently hybridized in parallel with four radiolabeled probes: pNcHsp-83 and pNcHsp-30, which encode heat-shock sequences: AV48, which contains cDNA for the entire proteolipid subunit of ?he mitochondrial ATPase-ATP synthase of Neurospora: and pRB 1.95, which contains the 5’ portion of the B-1 gene for rabbit P-globin. The nitrocellulose filters were autoradiographed; the spots of RNA were cut out for counting by scintillation spectrometry, and background (bkgd) counts were subtracted. The filter-bound radioactivity represents the concentration of specific RNA present in 10 pg of RNA applied to the filter, and to convert this radioactivity to a figure that would reflect the original cellular RNA content, radioactivity was multiplied by 0.1% of the estimated total RNA of each preparation (10 pg/lO mg). The total RNA was determined by dividing the final yield of RNA for the preparation by its own extraction efficiency, which was determined by hybridization of the B-giobin sequence to the globin mRNA add to the Nezrrospom cell extract, compared with hybridization to a known amount of globin mRNA. b The RNA content is milligrams of RNA per gram of spore fresh weight at inoculation. c These normalized values are the averages of the DPM measurements for 10 pg of RNA and for 2.5 pg of RNA (the iatter was multiplied by 4 to give a lo-kg RNA equivalent).

1985a,b) that the dormant conidia may pre- detectable RNA encoding hsp30 in the dorserve mRNAs for the heat-shock proteins mant spores (in either the binding or nonthat become translated selectively upon ex- binding fractions of the affinity chromatogposure to heat shock. We isolated and raphy fractionation); but, in contrast, we characterized cloned cDNAs for the Neufound that RNA for hsp83 is preserved in conidia of Neur rospora heat shock proteins of M, 83,000 dormant and 30,000 (Plesofsky-Vig and Brambl, enriched in the poly(A)+ 1987), and in the present study we used The hsp83 of Neuvospova strongly induced by exp these probes to determine if the compleis mentary transcripts were preserved in the shock temperatures, scribed in cells at lower dormant conidia of Neurospora. As shown in Fig. 2, we found no autoradiographically sence of heat shock, but t

322

BFCAMBL

MYdIal Poly (A)+

ET AL.

RNA

Poly (A) -

Total

1

-1631

2

.Spore Poly

(A)+

Poly

(. .:’

,,

b

,,

RNA (A)-

Total -517

b

2. Northern blot hybridization analysis for transcripts of genes for two heat-shock proteins of Neurosporu, hsp83 (A) and hsp30 (B), in dormant and activated spores. The plasmids pNcHsp-83 and pNcHsp-30 were radiolabeled with 32P via nick translation for hybridization to RNAs separated by electrophoresis and transferred to nitrocellulose. The following RNA fractions were subjected to electrophoresis in both (A) and (B): lane 1, 6 pg poly(A)+ RNA from dormant spores; lane 2, 43 kg poly(A)- RNA from dormant spores; lane 3, 41 pg of total RNA from germinated spores incubated at 30°C; and lane 4,34 pg total RNA of germinated spores incubated at the heatshock temperature of 45°C for 30 min. The positions of DNA molecular-weight markers of 1631 and 517 b are indicated, and the band of radioactivity in the region of the gel below the mRNA for hsp83 in late A4 is due to the intense radioactivity of the marker originally in the adjacent electrophoresis gel lane. FIG.

FIG. 1. Dot-blot hybridization analysis of transcripts for ATPase proteolipid subunit in dormant and germinated spores (mycelial cells). The cloned cDNA (AV48), containing the proteolipid subunit sequence and radiolabeled with 32P via nick translation, was hybridized with total and fractionated RNA applied to the nitrocellulose filters in the following quantities in RNA, poly(A)+ rows 1 and 2, respectively: Mycelial RNA: 3.6 and 0.9 kg; poly(A)- RNA: 100 and 2.5 p.g; unfractionated or total RNA: 100 and 25 pg. Spore RNA, poly(A)+ RNA: 20 and 5 pg; poly(A)- RNA: 100 and 25 kg; unfractionated or total RNA: 100 and 25 pg. Carrier tRNA from Saccharomyces (200 kg) was included in each sample applied to this filter.

to be transcribed only under heat-shock conditions (Fig. 2; Plesofsky-Vig and Brambl, 1987). These results identify directly an additional type of transcript present in the poly(A)+ RNA fraction of dormant Neurospora conidia, and they show that the early response of activated conidia to heat shock must depend not upon translation of preserved mRNA but

solely upon de IZOVOgene transcription synthesis of hsp30.

for

Translation of specific transcripts in the population of preserved 3’-polyadenylated that the poly(A)+ RNA. To establish RNA from the dormant conidia of Neurosfunctional as a transpora was potentially latable mRNA, we translated this RNA in vitro and analyzed the translation products

by electrophoresis after specific immunoprecipitation with antibodies to the nucleus-encoded subunit peptides of the mitochondrial enzymes cytochrome c oxidase

PRESERVED

POLY(A)’

RNA IN Neurospora

and the ATPase-ATP synthase. Secondarily, we wished to determine if the mRNAs for subunits of enzymes which we know to be assembled and functional in the earliest phases of spore germination (Stade and Brambl, 1981a,b; Wenzler and Brambl, 1981) also were preserved in the dormant cells, since it has not been shown whether metabolic reactivation is solely dependent upon function of preserved proteins oralternatively-upon translation of the preserved mRNA. In Fig. 3a are shown the results of an electrophoresis of the [3H]leucine-labeled ire vitro translation products of the poly(A)+ RNA isolated from dormant conidia of Neurospora and precipitated with

I

, 10

, 20

30

40

/ 50

,I 60

Gel Slice Number

, 70

! JO, 00

CONIDIA

323

an antiserum to a mixture of the seven subunits of the purifie of this organism ( translation products were coelect resed with authentic subunits of cytochrome c oxidase isolated by imm~~o~recipitation from mitochrondria of [r”@]leucine-labeled Neurospora cells s e four electrophoretic bands betwe and 7.5 (of approximately 10,000) correspond to the fo ally synthesized subunits 4, 5, cytochrome c oxidase (W 1979). As expected, the pep sized in vitro have slightly sl phoretic mobilities than the a units because of t

1

I

I

1

IO

20

30

40

GO-

50

-lo

,”

80

Gel Slice Number

3. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the ~3H~leucine-labeled (0) polypeptides derived from in vitro translation products of poly(A)+ RNA from dormant spores and isolated by immunoprecipitation with antisera to the subunit peptides of (a) cytochrome c oxidase or of (b) F,-ATPase of Neurospora. These same antisera also were used to obtain specific immunoprecipitates from detergent-lysed mitochondria of Neurospora labeled in vivo with [14C]leu.tine (0) for coelectrophoresis with the in vitro-synthesized peptides. Electrophoretic migration was from left to right in 15% (a) or 12.5% (b) polyacrylamide gels, and the closed blocks indicate the position of the tracking dye (cytochrome c) at the termination of the electrophoresis. Background counts have been subtracted from the data in this figure; each sample was counted for at least 5 min. FIG.

m-

324

BRAMBL

minal leader sequences that have not been removed from these presumed precursor peptides by the normal mitochondrial membrane processing system (Hay et al., 1984). The single electrophoretic band at slices 30-36 (of approximately M, 26,000) is a consistent, unidentified product of the immunoprecipitation assay of the in vitro translation mixture; the peptide was precipitated by any of several independently prepared antisera to the subunits of cytochrome c oxidase, but not by other antisera. A similar experiment was performed with an antiserum prepared against the F,ATPase of Neurosporu (Jack1 and Sebald, 1975). This antiserum would be expected to precipitate approximately eight cytoplasmically synthesized peptides of the enzyme’s F, component, of which the two largest (M, 58,000 and 56,000) are diagnostic (Wenzler and Brambl, 1981). As shown in Fig. 3b, the antiserum to the F,ATPase precipitated, from the in vitro translation mixture, a group of peptides whose electrophoretic mobilities are very similar to, but slightly slower than those of the [14C]leucine-labeled subunits of the authentic enzyme. Nearly identical peptides were immunoprecipitated from translation mixtures of mRNA from germinated spores. In other experiments, these in vitro-synthesized precursor peptides of both cytochrome c oxidase and ATPase have been shown to be converted to the size of the mature subunits by incubation of the translation mixture products with physiologically active mitochondria (R. Brambl, unpublished data). Accumulation of RNA mination. We extracted

early in spore ger-

the total RNA of dormant and germinated Neurospora conidia at 15, 30, and 45 min of incubation to measure changes in specific transcripts and to determine whether the cellular RNA content changed early in spore germination. Earlier pulse-labeling studies sug-

ET AL.

gested that RNA synthesis in these spores accelerated only after rates of protein synthesis were accelerating rapidly (Bonnen and Brambl, 1983). To determine accurately the efficiencies of extraction, we included a known amount of mRNA for rabbit B-globin in the RNA preparations for analysis with a homologous cloned sequence for B-globin (Plesofsky-Vig and Brambl, 1987). Equal amounts of the spore RNAs were dotted onto nitrocellulose filters and were hybridized in parallel with three radiolabeled probes (in addition to the B-globin sequence), one of which contained cDNA for the proteolipid subunit of the mitochondrial ATPase (AV48), and the other two contained cDNAs that encode the Neurospora heat-shock proteins hsp83 (pNcHsp-83) and hsp30 (pNcHsp-30). Through the hybridized dot blots, we found three different patterns for these RNAs in dormant and germinating spores (Fig. 4 and Table 1). hsp30 RNA was not present in these spores in detectable amounts, as shown also by the Northern analysis of Fig. 2. However, the RNA for hsp83, which is a normal cellular protein as well as a heat-shock protein, was preserved in dormant spores. The level of hsp83 RNA did not change during the first 30 min of germination, but after 30 min it began to increase, showing a three-fold increase between 30 and 45 min of germination. In contrast, RNA for the ATPase proteolipid subunit increased by the earliest time point assayed. In comparison to dormant spores, there was a greater than two-fold increase in proteolipid RNA by 15 min after spore suspension, and this increase continued at a similar rate during each of the subsequent 15-min time intervals. Table 1 shows the hybridized radioactivity for each of the assayed transcripts; these figures were adjusted for the cellular content of RNA in the spore extracts. We adjusted the radioactivity measurements to avoid bias that would result from a de-

PRESERVED 0 1

15

POLY(A)+ 30

RNA IN Neurosporo CQNIDIA

45

“.

NcHSP30 2

‘. .

325

and the later increase of the hspX3 R As shown by the Northern analysis of Fig. 2, transcripts for hsp30 are not present in the dormant spores (the rad~oact~~~t~ shown in Table 1 is due to nonspecifi bridization), and the data of Table 1 strate no increase in cellular conce of the hsp30 RNA during spore i~c~~at~~~ at 30°C.

1 Pmteolipid 2

FIG. 4. Dot-blot hybridization analysis of RNA from dormant conidiospores and from spores incubated at 30°C for 15, 30, or 45 min before cell harvest and RNA extraction. Ten micrograms (row 1) and 2.5 pg (row 2) of RNA were applied in replicate to nitrocellulose filters and were hybridized in parallel with four nick-translated hybridization probes: pNcHsp-83 heatand pNcHsp-30, which encode Neurospora shock sequences; AV48, which contains cDNA for the proteolipid subunit of the mitochondrial ATPase-ATP and pRB 1.95, which consynthase of Neuuospoua; tains the 5’ portion of the B-1 gene for rabbit l3globin. Additional experimental details are provided in Table 1.

crease in other spore RNAs and a consequent inflation of undiminished RNA sequences. We found that the total RNA content of the spores dropped sharply during the first 30 min of germination but the level of RNA increased by 45 min. The quantitative data of Table 1 demonstrate the early increase of the proteolipid mRNA over the quantity preserved in the dormant spores

Conidial germination and RNA and protein synthesis in presence of inhi~itQ~s. 3’Deoxyadenosine (cordycepin) has been shown to be a specific inhibitor of the addition of poly(A) sequences to era1 mammalian and plant c and Perry, 1974; Walbot et ai. mett and Katterman, this inhibitor in the Ne cubation medium, and inhibition of the incorpora adenine into poly(A)+ RNA. were labeled between 120 and germination, there was a decrease from unit o 20,700 cpm/A,,, treated cells to 2045 cp of treated cells. With the radiolabel applied during the 300- to 360-min interval of germination, continued treatment of the spores with the inhibitor yield inhibition (94%) of inc [3H]adenine into poly(A) + duction from 61,600 to 3327 of RNA. Nevertheless, this hi hibition of [3H]adenine in of poly(A)+ RNA synthes upon rate or extent of germ tube emergence in comparison to that of ~~trea~ed cells (both had germinated to 95% by 340 mm). Protein synthesis during spore germination, as measured by ~~cor~orat~~~ of ccl [3H]lysine into acid-insoluble similarly was almost unaffect deoxyadenosine, with the extract from the treated cells yielding 7.6 x IO4 cpm/A2s0 unit of protein and those from the control cells yielding 7.9 x IO4 cpm/A,, unn. Lomofungin has been shown to be an ef-

326

BRAMBL

fective inhibitor of RNA synthesis in yeast (Paveletich et al., 1974). In Neurospora conidia, we found that lomofungin inhibited at least 90% of the incorporation of [3H]uridine into both poly(A)+ RNA and nonpolyadenylated RNA. Strikingly, in these conidia this drug (at 20 kg/ml of spore incubation medium) inhibited spore germ tube emergence by 85 to 90%, even after 480 min of incubation with this inhibitor. (Normally, the population of conidia attains 90 to 95% germination by 300 min of incubation.) Protein synthesis during the earliest phases of germination would be expected to be most dependent upon the preserved mRNA and thus independent of drug-sensitive de nova synthesis of RNA. However, we found that the rates of protein synthesis during the first 120 min of spore incubation (as measured by incorporation of [3Hllysine into acid-insoluble material) were reduced by lomofungin to less than 50% of those of the untreated cells (0.6 x lo4 cpm/AzsO unit of protein in comparison to 1.6 x lo4 cpm/A,sO unit), and after 120 min of incubation, rates of protein synthesis in the treated cells dropped further as a result of even more severe inhibition (0.4 x lo4 cpmlAzsO unit vs 3.0 x lo4 cpm/A,,, unit by 480 min). We obtained a similar pattern of inhibition with proflavine-SO,, a substance determined previously (Hollomon, 1970) to be an effective inhibitor of RNA synthesis in Neurospora. The proflavine-SO, (at 1.5 pg/ml of spore incubation medium) sharply inhibited germ tube emergence only if present in the spore incubation medium during the first 60 min after inoculation (e.g., 20% germination in the cells treated at 30 min vs 90% in the untreated control spores); if the inhibitor was added after 60 min of incubation, the spores germinated with normal kinetics (94% germination of spores treated with proflavineSO, at 120 min of incubation). If proflavine-SO4 was present throughout spore

ET AL.

incubation, the inhibitor reduced incorporation of E3H]uridine into RNA by 92% at 300 min (0.21 x lo5 cpm/A,, unit vs 2.63 x lo5 cpm/A,, unit), germ tube emergence was inhibited 89%, and incorporation of [3H]lysine into protein of treated spores was 52% of that of the control cells (1.52 x lo5 and 3.16 x lo* cpm/A,, unit, respectively) . DISCUSSION

The dormant conidia of Neurospora contain a population of latent or preserved mRNA (Mirkes, 1974; Bonnen and Brambl, 1983), like dormant spores of several other fungi (reviewed in Van Etten et al., 1976; Brambl et al., 1978). The discovery of this mRNA in fungal spores, as well as in cells of other types of organisms exhibiting similar phases of dormancy and developmental arrest, led to the view that development likely was interrupted at the level of translation, that resumption of metabolism and development was regulated post-transcriptionally, and that the preserved mRNA likely encoded the genetic information required for the dormant cells to resume metabolic activity (Brambl et al., 1978). This implication of a central role for the preserved mRNA in germination has been based upon (a) the physical isolation of polyribosomes and poly(A)+ RNA from the dormant spores; (b) the observation that one of the first measurable metabolic activities of spore germination is a resumption of rapid rates of protein synthesis in the apparent absence of RNA synthesis or in the presence of very low rates of RNA synthesis; (c) the finding that both spore germination and protein synthesis are sensitive to the protein synthesis inhibitor cycloheximide; and (d) the apparent insensitivity of spore germination to chemical inhibitors of RNA synthesis, such as actinomycin D (reviewed in Lovett, 1976; Van Etten et al., 1976; Brambl et al., 1978). Although the preserved mRNA and its

PRESERVED

POLY(A)+

RNA IN Neurospora CONIDIA

translation are assumed to be essential to spore germination, few data have been reported describing the genetic information encoded by this mRNA or what peptides it specified. By in vitro translation, it has been shown that in spores of the fungus Botryodiplodia theobromae the preserved mRNA, in comparison to that of germinated spores, encodes both common and distinct groups of peptides (Wenzler and Brambl, 1978). Similar stage-specific differences in mRNA information content are indicated by in vitro translation of mRNA extracted from dormant ascospores of Saccharomyces cerevisiae (Kurtz and Lindquist, 1984; 1986), and molecular hybridization techniques have been used to show that dormant conidia of A. nidulans accumulate a number of poly(A)+ RNA sequences that are unique to that stage of development (Timberlake, 1980). Some of these poly(A) + RNAs found only in the conidia are transcribed from genes that are physically clustered within the Aspergillus genome, thus implying a coordination of gene expression for developmental stagespecific poly(A)+ RNAs (Gwynne et al., 1984). In the present study we isolated the poly(A)+ RNA fraction from dormant conidia of Neurospora, and by use of an in vitro translation system and molecular hybridization with cloned cDNA probles we demonstrated the presence of specific transcripts in the population of preserved mRNA of these spores. We have identified specific transcripts for subunits of cytochrome c oxidase and the ATPase-ATP synthase and for a major heat-shock protein, hsp83, whose mRNA normally is present in growing cells under non-heatshock conditions. In contrast, transcripts for hsp30, another major heat-shock protein irn Neurospora, were not found in the RNA of the dormant spores. The mRNAs for both of these heat-shock proteins are rapidly induced in the activated spores

327

upon exposure to elevated temperature (Plesofsky-Vig and Brambl, 1987). We learned that mRNA for one ATPase subunits (the proteolipid subu 12) was present in both the poly(A)and poly(A)+ RNA fractions of dormant spores, although it was greatly enriched in the poly(A)+ RNA fraction of germinated spores. This finding suggests that the extent of polyadenylation of a specific transcript may change during spore activat~~~ and germination. Our finding that spore germination and protein synthesis are insensitive to the polyadenylation inbi~itor 3’-deoxyadenosine makes it seem ~~iikely, however, that adenylation or supple tary adenylation of either preexisti newly synthesized mRNA is requir translation in vivo sf the mRNA of activated spores. Based upon t translatability, other mRNA tory enzyme subunits also w the poly(A)+ RNA fraction of dormant spores (as were transcripts for a protein). Previous studies have not clearly s that new mRNA synthesis is requir fungal spore germination. ~ollomo~ showed that proflavine simultaneously inhibited RNA synthesis and germination of Neurospora conidia, but other workers reported that germination oft much less sensitive to this RNA synthesis (Inoue and RNA synthesis, but not zoosp tion, was sensitive to actin Blastocladiella and Allomyces

emersonii ( arbuscula

1972). Similarly, inhibition thesis in ascospores of S. ethidium bromide did not block germination (Rosseau and Halvorson, I Results of our experiment chemical inhibitors of RNA synthesis, lomofungin and proflavine.+, show that in the presence of either inh or ~e~r~$~~ra conidiospore germination is blocke

328

BRAMBL

incorporation of radiolabeled precursors into RNA and protein. Proflavine-SO, is maximally effective in inhibiting germination if it is present during the first 30 min of spore incubation; if added after 60 min, the inhibitor is relatively ineffective. It seems likely that this inhibition is due to an inhibition of mRNA synthesis rather than synthesis of other types of RNA, since the dormant spores contain large quantities of active ribosomes and tRNA (Bonnen and Brambl, 1983; R. Brambl, unpublished data). We believe that these are the clearest demonstrations available that new RNA synthesis is required for fungal spore germination. Cycloheximide-sensitive protein synthesis has been shown to be required for germination of spores of several organisms (reviewed in Van Etten et nl., 1976), and, if Neurosporu conidia are typical, it now seems likely that the preserved mRNA is not sufficient to direct this requisite protein synthesis of spore germination, and new transcription must provide at least as essential contribution. Our experiments reported here show that new transcription of specific genes in Neurospora conidiospores can begin during the first 15 min after suspension of the cells in an incubation medium, and it is this inhibitor-sensitive transcription during the first 60 min of germination that may be required for subsequent protein synthesis and germ tube emergence. We earlier reported that dormant conidiospores became transcriptionally active at heat-shock temperatures within 5 min of suspension in liquid medium (PlesofskyVig and Brambl, 1987). It therefore seemed possible that the spores might become rapidly activated for transcription during normal germination at 30°C and results of the present study indicate that germinating Neurospora spores initiate transcription earlier than was previously thought. The mRNA for the ATPase proteolipid subunit increased more than twofold by the earliest

ET AL.

time point assayed, 15 min after spore suspension at 30°C. Interestingly, in our earlier study of heat-shocked spores (PlesofskyVig and Brambl, 1987), we found that although proteolipid mRNA decreased by 15 min after spore suspension at 45°C there was an initial increase in this normal mRNA (at least twofold) by 5 min of heat shock. This evidence points to a readiness of dormant spores to begin transcribing the ATPase proteolipid gene immediately upon spore activation, an induction that precedes the rapid repressive effect of heat shock. In contrast, germinating spores do not appear to transcribe the gene for hsp83 until after 30 min of activation (at 30°C). Therefore, the time at which transcription is initiated by activated spores depends on the RNA being assayed. Results of this present study indicate that, in contrast to earlier expectations (Van Etten et al., 1976; Brambl et al., 1978; Bonnen and Brambl, 1983), new mRNA synthesis probably is essential for conidial germination in Neurospora and possibly for spores of other fungi. Previously, it has been assumed that the population of preserved mRNA in the dormant spores is responsible for the protein synthesis in the earliest phases of germination. Therefore, of what value to the germinating spore is the population of preserved mRNA? Is translation of a portion of this mRNA required for spore germination or is none of it used? Is the preserved mRNA a remnant of the sporulation stage that is irrelevant to germination? The transcripts for the ATPase proteolipid subunit are preserved in the dormant spores, but they probably are not required for assembly and function of this enzyme complex. Not only is the complete enzyme itself preserved, but new transcripts for this subunit peptide normally are made within minutes of spore suspension in the germination medium. As we report here, immediately after initiation of spore incubation there is a sharp

PRESERVED

POLY(A)+

RNA IN Neurospora

decrease in the amount of RNA in the activated spores, and this decrease is not reversed until after 30 min of germination. This decrease in RNA content may include a stage-specific degradation of some RNAs that are preserved in the dormant spores and which are not needed for germination. A major subset of transcripts preserved in spores of S. cerevisiue is degraded (or at least becomes untranslatable in vitro) early in spore germination (Kurtz and Lindquist, 1986). Nevertheless, one or more RNA species synthesized late in sporulation of . emersonii has been shown to be required for subsequent spore germ tube emergence (Jaworski and Harrison, 1986). Therefore, in organisms that exhibit a phase of developmental arrest and cell dormancy, the central role assumed for the preserved mRNA in directing resumption of cell development may not exist. Most of this latent mRNA may be of no informational value to the cell beyond the last stages of sporulation, although a small proportion may be preserved in dormancy to be translated as an essential accompaniment to the translation of the mRNA newly synthesized at the outset of spore germination. ACKNOWLEDGMENTS This research was supported by research grants to staff at the University of Minnesota from the National Institute of General Medical Sciences (GM 19398) and from the USDA Competitive Research Grants Office (SS-CRCR-l-1655) and to staff at Reed College from the National Science Foundation (BMS 74-18162), the National Institute of General Medical Sciences (GM 32922), and the Alfred P. Sloan Foundation (Postdoctoral Fellowship Award to J.R.H.). REFERENCES Avrv, H., AND LEDER, P. 1972. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Puoc. Nat/. Acad. Sci., USA 69: 1408-1412. BONNEN, A., AND BRAMBL, R. 1983. Germination physiology of Neurospora crassa conidia. Exp. Mycol. I: 197-207.

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329

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