Nitrogen starvation or glucose limitation induces conidiation in constantly shaken liquid cultures of Neurospora crassa

Müller, B. T.

Russo, V. E.A.

English

Kansas State University

Fungal Genetics Reports Volume 36 Article 11 Nitrogen starvation or glucose limitation induces conidiation in constantly shaken liquid cultures of Neurospora crassa. B. T. Müller V. E.A. Russo Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation Müller, B. T., and V.E. Russo (1989) "Nitrogen starvation or glucose limitation induces conidiation in constantly shaken liquid cultures of Neurospora crassa.," Fungal Genetics Reports: Vol. 36, Article 11. https://doi.org/10.4148/1941-4765.1508 This Regular Paper is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. Nitrogen starvation or glucose limitation induces conidiation in constantly shaken liquid cultures of Neurospora crassa. Abstract Nitrogen starvation or glucose limitation induces conidiation in constantly shaken liquid cultures of Neurospora crassa. This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol36/iss1/11 Müller, B.T. and V.R.A. RussoNitrogen starvation or glucoselimitation induces conidiation inconstantly shaken liquid culturesIt is a general belief that in a liquidshaker culture, wild-type Neurospora crassa ex-hibits  only vegetative growth without producingconidia or protoperithecia (Berlin, V. and C.Yanofsky 1985. Mol. Cell. Biol. 5:849-855).  Itis well known that some liquid media supportproduction of macroconidia in static cultures(Weiss, B. and G. Turian 1966. J. Gen. Microbiol.of Neurospora crassa          44:407-418).  Here we report the occurrence ofconidial differentiation in constantly agitatedliquid cultures of  Neurospora crassa upon starvation for nitrogen or limitation forglucose.For our experiments we used Neurospora crassa wild type strain STa.  A modifiedVogel's medium (Russo, V.E.A. 1988. J. Photochem. Photobiol.  2:59-65) with 2% glucoseinstead of sucrose was employed as a standard growth medium; the pH was 6.8.  Ammoniumnitrate was replaced by NH4Cl or NaNO3 to provide only the reduced or oxidized form ofnitrogen. 250 ml Erlenmeyer  flasks containing 75 ml liquid medium were inoculated with 2x 10^5 conidia/ml and grown in the dark in an incubator shaker (New Brunswick Scientific)at 150 rpm, 34°c.  In a first series of experiments we grew the fungus for 24 h in a setof media differing in the amount and type of nitrogen sources and differing in glucoseconcentrations.  Using the conditions listed in Table 1, we obtained different growthbehavior of hyphae and conidiation or no conidiation at all.  The cultures with conidiawere orange, those without were white. Hyphae not only differ in the absence or presenceof the conidiophores, but also in their overall macroscopic and microscopic structures.For instance, in medium containing 50 mM NH4Cl and 2% glucose, the fungus forms more orless separate hyphae with only short branches coming out from long hyphae, thus producinga homogenous distribution of the mycelium in the medium.  On the other hand, thereplacement of NH4Cl with NaNO3 (50 mM) causes the fungus to grow as a uniform bigagglomerate.  Hyphae of the agglomerate appear to have intertwined branches which producea three-dimensional net-like structure.  From the data presented in Table 1 we concludethat conidiation is induced within 24 h if the nitrogen is supplied at low concentration(2 fl).  Another way to induce conidiation is to limit glucose in the presence of NH4Cl.Under these conditions, there appears to be no correlation between conidiation and pH ofthe medium or dry weights of the cultures.Table 1. Effect of nitrogen source and glucose concentration on growth behavior, colorand conidiation of hyphae (average of 2 experiments each with four replicates).Nitrogen Glucose    Growth Color Conidia    mg dry weight    pH**Source Behavior per 75 ml afterculture 24 h1    50 mM NH4Cl     2%        S            w        no        300 ± 15 3 . 22       2  mM  NH4Cl        2%            A                  do           yes            76  ±   2       5.73      50  mM  NH4Cl        2%            A                  w            no            190  ±  25       6.32  mM  NaNO3                      A,AS*              po           yes            75  ±   350  mM  NH4Cl                      MSA                po           yes           110  ±   56      50  mM  NaNO3                      AS                 w            no             75  ±   3A = one big agglomerateS = no agglomeration, separate growth of hyphaeAS = between A and SMSA=  many small agglomeratesw = whitepo = pale orangedo = deep orange* Sometimes A, sometimes AS** standard error of the mean was ± 0.1 pHTo test how fast the conidia are produced under agitation conditions, we grew hyphaein standard medium with 50 mM NH4Cl for 16 h.  After that time, cultures had reachedapproximately the mid exponential growth phase.  Cultures were carefully harvested ontosterile, 7 cm Schleicher and Schüll filter paper disks with a Büchner funnel under redsafety light, washed twice with sterile, prewarmed (34°C) double distilled water andresuspended in fresh standard medium without any nitrogen source.  Hyphae were neverallowed to dry during filtration.  After transfer, the hyphae formed a very loose clump,probably as a result of the filtration procedure.  Cultures were incubated further andexamined for the presence of conidia at different times after transfer.  Pieces of themycelium were taken from the edge of the agglomerate and the percentage of hyphae thatwere septated into conidia was estimated using a light microscope. Pictures were takenfrom the hyphae (Figure 1).  As controls we transferred hyphae to mediumcontaining either NH4Cl or NaNO3, or NaCl (each 50 mM) and analysed the cultures after12 h.  The results are presented in Table 2.  Fully septated conidia could be observedfrom 4 h after transfer (Figure 1b).  Hyphae had white color at this stage but becamepigmented during prolonged starvation, as the formation of conidia progressed.  After 12h incubation, the mycelial edge (about 3-5 mm) appeared very orange.  In the controlmedia with NH4Cl or NaN03 the mycelia lacked conidia and pigment.  The addition of NaClhad the same effect as nitrogen starvation, an indication that the observed inhibition ofconidiation by NH4Cl and NaN03 was not due to high concentration of Na^+ or Cl^-.  Itshould be noted that under constant agitation in liquid medium, the formed conidia do notentirely separate from each other, but produce chains and arbusculete structures.  Thereason for this is unknown.Figure 1.  Photomicrographs of hyphae from the edge of the mycelial agglomerate in thekinetics experiment.a) Hyphae from 16 h culture just before transferb)  Hyphae 4 h after transfer to nitrogen-free standard mediumc) Hyphae 12 h after transfer to nitrogen-free standard mediumd) Hyphae 12 h after to standard medium with NH4Cl  (50 mM) as nitrogen sourceTable 2.   Kinetics of condia production upon nitrogen starvation.  Hyphae grown 16 h in reductionstandard medium with 50 mM NH4Cl were transferred to standard medium without nitrogen.Estimation of the percent hyphae septated into conidia was done on two pieces obtainedfrom the edge of the agglomerate (average of two experiments each with two replicates).hours after addition % of hyphae      pH mg dry weighttransfer  to                       septated per 75 mlnitrogen- into culture**free medium conidia*0       -         0      4.5    1032             -                 0           5.8        1364             -                20           5.6        1568               -                  80            5.6          1966            -               5 0          5 . 6        1 8 31 2                -                     9 0             5 . 6           2 1 0  * standard error of the mean was about 20%** standard error of the mean was about 5%12       50 mM NaCl             90           5.5        21312       50 mM NH4Cl             0           3.9        31212       50 mM NaNO3             0           6.9        298Our experiments indicate that starvation for nitrogen or limitation for glucoseinduces formation of conidia in the ascomycete  Neurospora crassa. Using similar stepdownexperiments it was already shown that nitrogen regulates another morphogenetic process,the formation of protoperithecia (Sommer et al. 1987. Planta 170:205-208).  So, nitrogenstarvation plays a key role in triggering both the asexual and sexual life cycles ofNeurospora.Acknowledgements:  We thank Uta Marchfelder for typing and Niketan Pandit forcritically reading the manuscript. B.T.M. is an fellow of the Studienstiftung desdeutschen Volkes.  This work was partially supported by the DeutscheForschungsgemeinschaft - - - Max-Planck Institut für molekulare Genetik, D-1000 Berlin33, Federal Republic of Germany.Parekh. T. and H.S. Chhatpar Physiological responses of organisms to par-ticular stresses are well understood in only aRegulation of glucose-6-phosphate few cases (Bachofen, R. 1986 Bxperientia 42:1179-1182). Most of the salt-stress in nature is duedehydrogenase under salt-stress to sodium salts, particularly NaCl (Strongonov,B.P. 1973.condition in Aspergillus  sydowii Structure and function of plant cellsi n  saline habitats. Halsted  Press, New York).Little information is available regarding theeffect of salt on the regulation of enzymes in marine fungal  systems. In our studiesefforts have been made to understand the salt-mediated regulation of glucose-6-phosphatedehydrogenase (G6PDH) in halotolerant  Aspergillus  sydowii.Aspergillus sydowii was isolated from salt pans. The growth conditions employedwere the same as described earlier  (Karlekar et al.  1985 J. Biosci 9:197-201) except that casemino  acid and yeast extract were replaced by asparagine (1.0%).  Zn²+was added tothe synthetic medium as ZnSO4 at the desired concentration. Zinc deficient, zincsuboptimal, zinc optimal and zinc supraoptimal conditions indicate no addition, additionof 0.1 mg, 1 mg and 10 mg per 100 ml of ZnS04 to the above growth medium, respectively.Methods for the preparation of cell-free extract, assay of G6PDH  and protein were thesame as described earlier (Savant et al.  1982 Experientia 38:310-311).  Mycelial ash wasprepared by heat drying the mycelia  at 8OO°C  for 5 hours.Earlier studies with A. sydowii grown in the presence of 2M NaCl showed significant-ly higher levels of G6PDH compared with control cultures. Km was found to decrease whileVmax increased when NaCl was added to the growth medium (Parekh and Chhatpar 1986 In:Contemporary themes in biochemistry (Kon et al. eds.)  ICSU Press Cambridge pg. 334-335).Further studies on the in vitro effect of NaCl on kinetic constants of G6PDH incell-free extracts of a 2M NaCl grown culture showed a decrease in Vmax  without a changein Km values, suggesting a non-competitive type of inhibition of G6PDH  by NaCl (Table 1).Various possibilities for regulation of G6PDH are; (a) Higher Na+ accumulation in theculture grown in the presence of 2M NaCl condition might be responsible for increasingthe activity of G6PDH; (b) A different pattern of accumulation of other electrolytes mayalso be responsible for altering enzyme activity; and/or (c) The control culture may besynthesizing or accumulating inhibitor(s) of G6PDH.

Nitrogen starvation or glucose limitation induces conidiation in constantly shaken liquid cultures of Neurospora crassa.

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Nitrogen starvation or glucose limitation induces conidiation in constantly shaken liquid cultures of Neurospora crassa.

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