Photostimulation of conidiation in mutants of Neurospora crassa

J. Photo&em.

Photobiol.

Photostimulation Neurospora Helga

B: Bid.,

9 (1991)

189-199

of conidiation

189

in mutants

of

crassa

Ninnemann

Insttiut fur Chemische Pfan.zenph@dogie 7400 Fiibingen (F.R.G.)

der Universitiit

!Fiibingen, Con-ensstrc@e

41,

(Received September 5, 1990; accepted October 31, 1990)

Keywords. Neurospcyra crassa, photoconidiation, blue light effect, nitrate reductase, flavoprotein, light-insensitive mutants.

Abstract Various mutants of Neu~~spora crassa were screened for light-stimulated conidiation which is a blue light effect and, at least in strain albino-band, is mediated by the flavoprotein nitrate reductase (NR). NH- mutants showed practically no photoconidiation under standard conditions. However, in fusion products of nit-l (diaphorase activity present, terminal activity missing) plus nit-3 (terminal activity present, diaphorase activity missing), NR activities and photoconidiation were partishy restored. Mutants with altered light sensitivities, such as white collar WC-1 and light-insensitive lis-2 and lis-3, had normal NR activities and their conidiation was promoted by light, whereas WC-2 and lis-1 responded only slightly. These two mutants showed low NR activities especially when grown on solid medium which might be the cause of their blindness. Experiments with NR- mutants indicated that nitrite reductase might also act as a blue light photoreceptor.

1. Introduction

In order to obtain an understanding of the photoregulated processes in Narospora, such as photosuppression of rhythmic conidiation, shifts of the phase of the circadian rhythm of conidiation, carotenogenesis, enhancement of conidiation by light (photoconidiation) and formation of protoperithecia, various mutants with known genetic and biochemical defects have been used (albino, poky, riboflavin-deficient (rib-), white collar (WC) and light-insensitive (lis strains) to search for information on the photoreceptors involved (l-71. In the search for the photoreceptor molecules of such light-stimulated processes, a flax&-mediated absorbance change of a b-type cytochrome in the mycelium of Neu~ospora, stra.inal-2, bd, has been discovered and interpreted as a reaction close to the primary photoact [8]. A correlation has been observed between photoconidiation of Neurospora and the absorbance increase at 423 and 557 nm induced by light in viva [2], and nitrate reductase (NR) has been proposed as a photoreceptor for Ekvier

Sequoia/Printed in The Netherlands

190

light-stimulated conidiation [ 31. (This light-stimulated conidiation must be distinguished from stress-induced conidia formation which starts, for example, when the hyphae creep up the glass wall of their growth dishes; this is independent of light.) We and others have also shown that light-induced phase shifts of conidial banding cannot be correlated with the A4 increase of the b-type cytochrome in vivo 12, 6). In addition, NR was not identified as the photoreceptor for carotenogenesis [9], which supports the concept that various photoresponses might be mediated by multiple photoreceptors or methods of signal transduction (see, for example, ref. 10). In this paper, we simunarize some of our observations on different Neurospcyra mutants with respect to their ability to use nitrate as a nitrogen source and to respond to light with enhanced conidiation.

2. Materials

and methods

2.1. Fhgal

strains Nmn-ospm-a crassa al-2, bd was derived from a cross performed by Professor Stuart Brody, University of California, San Diego. The nitrate nonutilizer (nit) and WC strains were obtained from the Fungal Genetics Stock Centre, Kansas, U.S.A. The lis strains were obtained from J. Paietta. To obtain stock solutions of conidia, mycelia were grown in 100 ml Erlenmeyer flasks on NOa- medium or Vogel’s medium (see below) in the dark; conidia were washed from these mycelia with distilled water in red safety light and were kept in water at 4 “C in the dark. Conidia density was adjusted to (l-2) x lo8 conidia ml- ‘. Cultures were inoculated under red safety light from these stock solutions which were discarded when older than 14 days.

2.2. Media Liquid or solid media (usually with 1.2% agar) contained the salts of Vogel’s medium [ 111 plus 1% glucose. In NOa- medium the only nitrogen source was 50 mM NOa-; in Vogel’s medium (VM) the original 25 mM NH,NOa was supplied; in NH,+ medium 25 mM NH&l was used as nitrogen source. The media were freshly prepared for each experiment. Liquid cultures were grown in 2 1 Erlenmeyer flasks using 300 ml medium at 30 “C in the dark (rotary shaker, 80 rev nun-‘). Solid cultures were grown in large Petri dishes (4=20 cm). They were inoculated with a conidia-soaked filter paper (4 = 5 mm) placed at the margin of the dish in red safety light. The dishes were wrapped in dark cloth and kept in a 30 “C culture room. 2.3. Irradiation Irradiation of solid cultures was performed about 48 h after inoculation (for al-2, bd; other strains were irradiated when their growth front had proceeded one-third to one-half through the Petri dish (between 29 and 53 h)). At the start of the irradiation the positions of the growth front and the conidia already formed in the dark were marked on the reverse of the dish.

191

Irradiation was performed in a laminar flow bank with two light tubes (Osram Universal White L 65 W/25) resulting in 1.5-3 W rnm2. 2.4. Protoplasts Protoplasts of mutants nit-l (diaphorase activity present, terminal activity missing) and nit-3 (terminal activity present, diaphorase activity missing) were prepared using 1% glucuronidase at 30 “C for 30-60 min and fused with 30% polyethylene glycol and 50 mM CaCl, at pH 10 in 0.6 M sorbitol [ 12 1. Hybrid fusion products nit 1 + 3 with their NR complement were selected in NOs- medium. NR activities were determined according to Nicholas and Nason [ 131 and Scheideler and Ninnemann [ 141. Photoconidiation (i.e. conidia formation in the light above that in the dark) was recorded photographically or by drawing. 3. Results Our early experiments have shown that photoconidiation of Neurospora albino-band (NR+) occurs preferentially in partially starved mycelia (precultured for 16 h with gentle shaking at 30 “C in liquid medium with nitrate as sole nitrogen source and starved in distilled water for at least 2 h) (Pig. 1, see also Pig. 2 in ref. 2). Photoconidiation is not observed or is greatly delayed in mycelia precultured in liquid medium containing ammonium chloride or ammonium nitrate and then starved in water [2]. Since NR synthesis and activity are induced by nitrate and repressed by ammonium ions [ 151, NR activity is present in mycelia grown in nitrate medium and absent or low in mycelia grown in media containing ammonium or ammonium nitrate.

Fig. 1. Photoconidiation of Neurospora CT-usu strain al-2, bd after starvation. Race tubes with arg-n-dnimalmedium (arginine, 0.5 g 1-l; NaNOa, 25 mM; glucose, 0.3 g 1-r) were inoculated with mycelia grown for 16 h in NOa- medium (50 mM) and starved without substratesfor 3 h in the dark. Half of the mycelia were irradiated for 10 min with 30 W me2 (three lower race tubes); the other mycelia were kept as dark controls (three upper race tubes). Inoculation sites are located at the right ends of the tubes. The race tubes were kept in the dark at 30 “C. The arrow indicates the light-induced 5rst band of conidia.

192

Also mycelia grown on solid nitrate medium without intentionally induced starvation show a pronounced stimulation of conidiation by light (Pig. 2), whereas conidiation in the dark controls is low. Mycelia grown on media with NH&l as the only nitrogen source conidiate profusely in the dark, but conidiation is not (or only slightly) enhanced by light (Pig. 3). However, on solid media the availability of nutrients and the total and partial NR activities are decreased in comparison with those in liquid media so that cultures on solid media may automatically be exposed to a certain starvation stress (Table 1). In the NR- mutants nit-l, nit-2 (missing an NR structural gene) and nit-3 which are unable to reduce nitrate with NADPH as substrate, but have NADPH-,cytochrome c (diaphorase) activity, no NR activity and reduced methyl viologen --, nitrate activity respectively, photostimulation of conidiation is absent under our standard conditions (see Section 2). No stimulation of conidiation is observed in any of the three nit mutants grown on ammonium chloride or ammonium nitrate and irradiated for 2 or 4 h, whereas carotenogenesis is greatly enhanced (Pig. 4). With non-physiological light conditions, e.g. for 24 h or longer, some conidia are light induced (in nit-l more than in nit-3 and n&2), a result previously observed with race tube

Fig. 2. Photoconidiation of Net~rosporu crcusa al-2, bd on nitrate medium. The medium was solidified with 1.2% agsr. A piece of filter paper was soaked with conidia and placed at the margin of the culture plate. The cultures were grown at 30 “C in the dark until the front of the mycelia had grown over one-half of the dish (approximately 48 h). One plate was kept as a dark control (top); the other was irradiated for 2 h with 3 W mm2 (bottom dish) after the position of the growth front had been marked on the glass dish (right photograph; dots mark conidia already present in the dark. Plates were photographed 24 h after irradiation.

193

Fig. 3. Stimulation of conidlation of strain al-2, bd grown on NOa- medium as a function of irradiation time (a), and failure of photoconidiation on NH,+ medium (b). Irradiation with 3 W mm2 (from left to right): none (top left), 30 and 60 min (top row), 2 h and 4 h (bottom row).

cultures in our laboratory [ 161. Whether this result is due to a residual NR activity or is independent of NR activity and can be accounted for by the presence of nitrite reductase (NIR, see below) is yet to be established, e.g. by testing NIR- mutants such as nit-4 or nit-6. Hybrid strains nit 1 + 3 (from fusion of protoplasts of mutants nit-l and nit-3 with polyethylene glycol [ 171; selected and cultivated in nitrate medium (Fig. 5)) respond to light with enhanced conidiation (Fig. 6). However, conidiation of the nit 1 + 1 and nit 3 + 3 control fusion products is light insensitive. Conidiation of mutants nit-l and nit-3 is stimulated by 2 h

1

500-3500 2500-4800 3000-4900 300-900 400-2500 500-2500

1go-250 300-500 100-200 140-180 90-l 70

1000-l 100 1000-l 100

340-480 500-1200 500-I 000

WC-I WC-2

1% 1 III-2 lis-3

20-50 loo-160 100-240

50-140 15-180

80-350

200-l 000 300-700 200-600 200-300 600-l 700 400-1000

400-850 1 IO-480 220-440 400-600 250-450

1

Diaphorase (NADPH + Cyt c)

110-530

Terminal (IvIVH-~NO~-)~

Range of NR activities from at least 6-12 experiments for each mutant given In units per milligram protein per ten minute test period. unit = 1 nmol. NOa- formed or 1 nmol cytochrome c (Cyt c) reduced. “16 h NO,- medium, 30 “C. ‘48-55 h NO,- medium, 30 “C. ‘NADPH, reduced nlcotinamide adenine dinucleotide phosphate. %VH, methyl viologen.

al-2, bd

120-320

*

Diaphorase (NADPH + Cyt G)

Total (NADPH + NO,-)’

)”

Total (NADPH -NO,-)e

Terminal (MVH + NO:, -

On solid mediumb

In liquid medium*

NR activities from mycelia grown

600-l 000

Neurvspvra strain

Total and partial NR activities of different mutants of Newrvsporu massa

TABLE

4. Effect of irradiation on NR- mutants nit-l, nit-2 and nit-3. Culture and irradiation conditions as described in Fig. 2. Top dishes, dark controls; bottom dishes, irradiated. Left, nit-l; middle, nit-Z; right: nit-3.

Fig.

Fig. 5. Heterofusion product nit 1+ 3 from NR- mutants nit-l and nit-3 after chemical fusion. The fusion products were grown in NOs- medium where only M-complemented mycelia could grow (nit 1 + 3, middle Erlenmeyer flask); fusions of nit-l with nit-l (right) or nit-3 with nit3 (left) could not grow under these conditions.

irradiation when the mycelia are grown on solid media with lo-25 mM nitrite as sole nitrogen source. NIR may act as a photoreceptor in these cases, but this has yet to be proved. With 5 mM nitrite in the medium, carotenogenesis is induced by light, but conidiation only slightly. The white collar mutants WC-1 and WC-2 behave qualitatively differently with respect to photoconidiation; 2 h of light results in distinctly enhanced conidiation in WC-1 grown on nitrate medium (Fig. 7, left), but only in slightly stimulated hyphal branching and conidiation in WC-2 (Fig. 7, right). NR activities of WC-l in liquid and on solid nitrate medium are comparable

196

Fig. 6. Photoconidiation of heterofusion product nit 1 + 3. Culture conditions and starvation ss in Fig. 1. Irradiation time, 5 h with 3 W m-‘. Top race tube strain al-2, bd, photoconidiation of control; middle, failure of fusion product nit 3 + 3 to photoconidiate; bottom, photoconidiation of NF&complemented nit 1 + 3 mycelium.

Fig. 7. Photoconidiation of Neu~spora crassa strains white collar WC-1 (left) and WC-2 (right). Experimental conditions as in Fig. 2. Top, dark controls; bottom, irradiated cultures.

with those of strain al-2, bd; those of WC-2 on solid medium is somewhat lower (see Table 1). With regard to light-insensitive (lis) mutants isolated and described by Paietta and Sargent [ 101, dark controls of lis-1 do not form conidia on nitrate

J?ig. 8. Light effects on Neurosporu crassa light-insensitive (lis) strains. Culture conditions and irradiation as in F’ig. 2. Top dishes, dark controls; bottom dishes, irradiated mycelia. Left, lis-1; middle, lis-2; right, lis-3.

TABLE

2

Conidiation and light

response of various Neurosporu

crassa

strains as a function of nitrogen source

strain

Nitrogen source

NADPH + NOB NR activity

Conidiation in the dark

Additional photoconidiation

al-2, bd al-2, bd nit- 1 nit-2 nit-3 nit 1+ 3 WC-1 WC-2 lis-1 lis-2 lis-3

NH,+ NOBNH,NOB NH4N03 NH,N03 NOsNOsNOsNOBNOsNOs-

none +++ Diaphorase None Terminal

++ + + +

-

+c+> + ++ -

-4+> +(+) ++

++ +

++ ++

Additional the dark.

photoconidiation

+(+) +++ +++ ++ +++ ++ registers

conidia formation

++ -I(+) -

-I(+) -

in the light on top of that found in

medium. This strain exhibits no photoconidiation under standard conditions (24 h of light; Fig. 8, left); a small band of light-induced conidia is observed after 6-8 h of light. Mutant lis-2 conidiates well in the dark and can be

198

stimulated to form additional conidia in the light (Fig. 8, middle). In lis-3, definite photoconidiation is caused by 2 or 4 h of light (Fig. 8, right). The total and partial NR activities of G-2 and lis-3 in liquid and on solid nitrate media are comparable with those of strain albino-band; those of the “blind” lis-1 are lower after culture in liquid and especially on solid nitrate medium (Table 1). The data are summarized in Table 2. 4. Discussion Newrosporucrassa al-2, bd is a carotenoid-less, rhythmically conidiating mutant. The photoreceptor for blue light-induced photoconidiation is supposedly a flavoprotein; many observations point towards the flavin of NR 13, 181. Photoconidiation was achieved in mycelia of Neurospora crassa strain al-2, bd grown on nitrate medium under our conditions with and without intentionally induced starvation (Figs. l-3). On medium without any nitrogen source, no photoconidiation was observed; on medium containing only NH&l or VM with NH4N03, practically no photoconidiation occurred (Fig. 3). In the NR- mutants nit-l, nit-2 and nit-3, which grow very poorly in or on nitrate medium and were therefore cultivated on VM, 2-4 h of light did not enhance conidia formation (Fig. 4). However, in the somatic fusion products nit 1 -I-3, total NR activity and light sensitivity for photoconidiation were partially restored (Fig. 6) [ 171. Irradiation of nit-l and nit-3, grown on NOz- medium (lo-25 mM), resulted in pronounced photoconidiation after 2 h of light. Prolonged irradiation (24 h or continuous light) of mutants nit-l, nit-2 and nit-3 grown on VM allowed some light-stimulated conidiation. However, it is conceivable that this prolonged irradiation might act as stress rather than as a pure light signal for enhanced conidiation. Thus distinct photoconidiation occurred under physiological conditions only in strains with total NR activity. Continuous irradiation forced some conidiation. Although the two WC mutants and the three lis mutants had normal or substantial NR activities, only WC-l, lis-2 and lis-3 showed distinct light-stimulated conidiation, whereas light affected conidia formation of WC2 and lis-1 only slightly (Figs. 7 and 8); these mutants behaved as if they were blind to light-inducing conidiation. It is not yet known whether the lower than normal NR activities of WC-2 and lis-1, especially when grown on solid NOs- medium, caused this blindness or whether these mutants have a defect in their signal transduction chain. Quantum yield measurements have demonstrated that the flavin bound to NR can act as a photoreceptor and primary light-induced reductant for the cytochrome b reduction monitored as AA [18]. The involvement of the molybdenum cofactor of NR in the signal transduction for photoconidiation was demonstrated for mycelia grown with vanadate or tungstate instead of molybdate; they formed an inactive NR and could not translate the light signal into light-induced conidiation [ 191. These results will be presented more extensively in a separate publication.

199

Acknowledgment The financial support of the Deutsche Forschungsgemeinschaft is gratefuLly acknowledged. References 1 R. D. Brain, D. 0. Woodwards and W. R. Briggs, Correlative studies of light sensitivity and cytochrome content in Neurospora crassa, Carnegie Inst. Washington Publ., 76 (1977) 295-299. 2 E. Klemm and H. Ninnemann, Correlation between absorbance change and a physiological response induced by blue light in Neurospora, Photo&em. Photobid., 28 (1978) 227-230. 3 E. Klemm and H. Ninnemann, Nitrate reductase - a key enzyme in blue light-promoted conidiation and absorbance change of Neurospora, Photochem. Photobid., 29 (1979) 629-632. 4 R. W. Harding and R. V. Turner, Photoregulation of the carotenoid biosynthetic pathway in albino and white collar mutants of Neurospora crassa, Plant Physiol., 68 (1981) 745-749. 5 J. Paietta and M. C. Sargent, Photoreception in Neurosporu crassa: correlation of reduced light sensitivity with flavin deficiency, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 5573-5577. 6 J. Paietta and M. C. Sargent, Blue light responses in nitrate reductase mutants of Neurospora crassa, Photo&em. Photobid., 35 (1982) 853-855. 7 F. Degli-Innocenti and V. Russo, Isolation of new white collar mutants of Neurosporu cr~.~.~aand studies on their behavior in the blue light-induced formation of protoperithecia, J. Bacterid., 159 (1984) 757-761. 8 V. Muf~oz, S. Brody and W. L. Butler, Photoreceptor pigment for blue right responses in NaLrospora crassa, Biochem. Biophys. Res. Commun., 58 (1974) 322-327. 9 T. A. Belozerskaya, S. S. Burikhunov, E. K. Chernyshova, M. S. Kritsky and N. P. Lvov, Does nitrate reductase play a key role in photoinduction of carotenoid synthesis lnNeurosporu crassa? Neurospora News& 29 (1982) 14-15. 10 J. Paietta and M. C. Sargent, Isolation and characterization of light-insensitive mutants of Neurospora crassa, Genetics, 104 (1983) 11-12. 11 H. J. Vogel, Distribution of lyslne pathways among fungi: evolutionary implications, Am. Nat., 98 (1964) 435-446. 12 W. A. Keller and G. Melchers, The effect of high pH and calcium on tobacco leaf protoplast fusion, Z. Naturforsch. Teil C, 28 (1973) 737-741. 13 D. J. D. Nicholas and A. Nason, in S. P. Colowick and N. 0. Kaplan (eds.), Methods in Enzymology, Vol. 3, Academic Press, New York, 1957, p. 983. 14 L. Scheideler and H. Ninnemann, Nitrate reductase activity test: phenazine methosulfate-ferricyanide stop reagent replaces postassay treatment, AnaZ. Biochern., 154 (1986) 29-33. 15 G. J. Sorger and J. Davies, Regulation of nitrate reductase of Neurospora at the level of transcription and translation, Biochem. J., 134 (1973) 673-685. 16 S. Mohren, Zur Licht-stimulierten Konidienbildung bei Neurosporu-Mutanten, Master’s Thesis, Unlversitit Tiibingen, 1980. 17 H. Ninnemann, Somatic hybridization ofNeurospora protoplasts, Plant Physiol., 65 (1980) Suppl. abstr. 22. 18 B. Pottiez, G. Krippahl and H. Ninnemann, Quantum requirement for photoreactivation of inactivated nitrate reductase of Neurosporu crassa strain al-2, bd, Bot. Actu, 101 (1988) 301305. 19 H. Ninnemann, Participation of pterin-molybdenum cofactor in the regulation of nitrate reductase activity and signal transduction during light-stimulated conidiation of Neurospora crossa, in W. R. UlIrlch, C. Rigano, A. Fuggi and P. J. Aparicio (eds.), Inorganic Nitrogen in Plants and Microorganisms, Springer, Berlin, 1990, pp, 190-195.