Asexual Development Is Increased in Neurospora crassa cat ...ec.asm.org/content/2/4/798.full.pdf ·...

EUKARYOTIC CELL, Aug. 2003, p. 798–808 Vol. 2, No. 41535-9778/03/$08.00�0 DOI: 10.1128/EC.2.4.798–808.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Asexual Development Is Increased in Neurospora crassacat-3-Null Mutant Strains

Shaday Michan, Fernando Lledías,† and Wilhelm Hansberg*Instituto de Fisiología Celular, Universidad Nacional Autonoma de Mexico, Mexico, D.F., Mexico

Received 23 October 2002/Accepted 29 May 2003

We use asexual development of Neurospora crassa as a model system with which to determine the causes ofcell differentiation. Air exposure of a mycelial mat induces hyphal adhesion, and adherent hyphae grow aerialhyphae that, in turn, form conidia. Previous work indicated the development of a hyperoxidant state at the startof these morphogenetic transitions and a large increase in catalase activity during conidiation. Catalase 3(CAT-3) increases at the end of exponential growth and is induced by different stress conditions. Here weanalyzed the effects of cat-3-null strains on growth and asexual development. The lack of CAT-3 was notcompensated by other catalases, even under oxidative stress conditions, and cat-3RIP colonies were sensitive toH2O2, indicating that wild-type (Wt) resistance to external H2O2 was due to CAT-3. cat-3RIP colonies grown inthe dark produced high levels of carotenes as a consequence of oxidative stress. Light exacerbated oxidativestress and further increased carotene synthesis. In the cat-3RIP mutant strain, increased aeration in liquidcultures led to increased hyphal adhesion and protein oxidation. Compared to the Wt, the cat-3RIP mutantstrain produced six times more aerial hyphae and conidia in air-exposed mycelial mats, as a result of longerand more densely packed aerial hyphae. Protein oxidation in colonies was threefold higher and showed moreaerial hyphae and conidia in mutant strains than did the Wt. Results indicate that oxidative stress due to lackof CAT-3 induces carotene synthesis, hyphal adhesion, and more aerial hyphae and conidia.

We study cell differentiation by using the asexual life cycle ofNeurospora crassa as a model system. There is a wealth ofinformation about the genetics, biochemistry, physiology, andcellular and molecular biology of N. crassa, and its genome hasbeen recently sequenced by the Whitehead Institute/MIT Cen-ter for Genome Research (www-genome.wi.mit.edu) (10a).

A synchronous process of asexual spore (conidium) forma-tion is started when an aerated liquid culture is filtered and theresulting mycelial mat is exposed to air (35, 38). Filaments(hyphae) in direct contact with air adhere to each other within40 min, adherent mycelium starts growing aerial hyphae after2 h, and conidia are formed at the tips of the branched aerialhyphae after 8 to 9 h of air exposure (38). Thus, formation ofconidia from growing hyphae involves three morphogenetictransitions: growing hyphae to adherent mycelium; adherentmycelium to aerial hyphae; and aerial hyphae to conidia.

A hyperoxidant state develops at the start of these morpho-genetic transitions (14, 39–42) and during germination ofconidia (26). A hyperoxidant state is defined as an unstable,transient state in which reactive oxygen species surpass theantioxidant capacity of the cell (13, 15). The occurrence of ahyperoxidant state is indicated by oxidation of total proteinthat occurs at the start of the aforementioned morphogenetictransitions (39, 41). Glutamine synthetase and glutamate de-hydrogenase oxidation occurs during adhesion of hyphae, for-

mation of aerial hyphae, and return to the growth state (41,42). Catalase is modified during conidiation (15) and in vitrobecause of the reaction of singlet oxygen (1O2) with its heme(25). Also, during germination of conidia, total protein oxida-tion and catalase oxidation by 1O2 increases with light, a sourceof 1O2, or insufficient 1O2 quenching by carotenes (26).

In studies on the activity of antioxidant enzymes during theasexual life cycle of N. crassa, large differences in catalasespecific activity were observed. There was a stepwise increasein catalase activity during the process that leads to formation ofconidia. In fact, conidia have 60 times more catalase activitythan hyphae growing in liquid medium (15, 30). Catalase 3(CAT-3) and CAT-1 constitute the main catalase activities andare differentially regulated during the N. crassa asexual lifecycle. CAT-3 activity increases during exponential growth andis induced by different stress conditions (30); CAT-1 increasesat the stationary growth phase and is accumulated in conidia(8, 30).

Most of the hydrogen peroxide (H2O2) in cells comes fromsuperoxide (O2

��) dismutation. O2�� arises mainly by electron

leakage from the respiratory chain and from the activity ofenzymes, such as NADPH oxidase and other oxidases. O2

�� isdismutated by O2

�� dismutase (SOD) to form H2O2 and di-oxygen (O2). There is a cytosolic, a mitochondrial, and usuallyan extracellular SOD. There is also a high redundancy ofenzymes for the disposition of H2O2: catalases, catalase/per-oxidases, peroxidases, and peroxiredoxins. When O2

�� andH2O2 disposal is insufficient, hydroxyl radical (HO�) is formedfrom H2O2 reduction by metal ions and 1O2 is generated byspontaneous dismutation of O2

�� , metal-catalyzed reaction ofO2

�� with H2O2, and decomposition of H2O2 by different com-pounds (reviewed in reference 27). Instead, in the presence of

* Corresponding author. Mailing address: Instituto de FisiologíaCelular, Universidad Nacional Autonoma de Mexico, Apartado Postal70-242, 04510 Mexico, D.F., Mexico. Phone: (5255) 5622 5655. Fax:(5255) 5622 5630. E-mail: [email protected].

† Present address: Departamento de Biología Molecular de Plantas,Instituto de Biotecnología, Universidad Nacional Autonoma de Mex-ico, 62250 Cuernavaca, Mor., Mexico

798

on June 15, 2018 by guesthttp://ec.asm

.org/D

ownloaded from

http://ec.asm.org/

SOD and catalase, O2�� and H2O2 are converted quantitatively

into water and O2. Thus, disposal of O2�� and H2O2 is vital to

avoid formation of highly reactive HO� and 1O2.Induction of antioxidant mechanisms is an expected conse-

quence of a hyperoxidant state and explains the increase incatalase activity in our model system. Many other microorgan-isms have more than one catalase, and in some of them, acatalase is related to cell differentiation (2, 10, 17, 19, 20, 28,31, 46). This does not imply that catalases are essential for celldifferentiation. Because of its importance for cell survival,there is ample redundancy in antioxidant mechanisms. Nulli-fication of different antioxidant enzymes is probably requiredto impair conidiation in N. crassa, and this will probably lead tocell death. However, if cell differentiation is a response to a hy-peroxidant state, nullification of an antioxidant enzyme shouldlead to increased oxidative stress and increased cell differen-tiation.

Here we analyzed the effect of cat-3 inactivation on asexualdevelopment. cat-3-null mutant strains tend to develop oxida-tive stress, measured by protein oxidation, and react by increas-ing carotenes levels and cell adhesion and development.

MATERIALS AND METHODS

N. crassa strains, plasmids, and growth conditions. Wild type (Wt) strains74-ORS23-1A and ORS-SL6-a and his-3 mutant strain 6103A were obtainedfrom the Fungal Genetics Stock Center. The his-3 mutant strain contains a singlemutation in a gene encoding a multifunctional enzyme for histidine biosynthesis.Plasmid pDE1, containing a truncated his-3 gene, was used to direct cat-3 inte-gration to this locus (9).

Liquid cultures were grown in Vogel’s minimal medium (VM) containing 1.5or 2% sucrose from an inoculum of 105 to 106 conidia/ml at an air/liquid ratioof 3:2, and incubated at 30°C with agitation at 200 or 250 rpm for 12 to 16 h. Forthe his-3 mutant, the growth medium was supplemented with 200 �g of L-histidine per ml.

To impose oxidative stress, mycelium gown from an inoculum of 3 � 105

conidia/ml was harvested by filtration after 14 h of growth, washed briefly withfresh medium, and transferred to growth medium containing either 1 mM H2O2,30 mM CaCO3, or 5 mM paraquat. Mycelia were recovered after 0.5, 1, 3, and6 h, and catalase activity in cell extracts was determined.

Conidia were isolated from solid cultures with VM, supplemented with 1.5%sucrose and 1.5% agar, in Erlenmeyer flasks. Cultures were inoculated withconidia, and incubated for 3 days at 30°C in the dark and then for 2 days at 25°Cin the light. To grow colonies, petri dishes with 1.5% agar in VM supplementedwith 0.05% fructose, 0.05% glucose, and 2% sorbose (VSM) (6) were inoculatedwith 200 to 250 conidia and incubated at 30°C for 3 or 7 days. When colonieswere isolated, a cellophane sheet was layered onto solid cultures in petri dishesbefore plating of conidia. Cellophane was washed and autoclaved in distilledwater. Illumination of colonies in petri plates was done with a 500-W tungstenbulb at a distance of 50 cm (5 W/cm2) for 1 h.

Treatment with H2O2 was done in solid cultures inoculated with 250 conidiaand incubated at 30°C, and 2 days later, 10 ml of either 5, 10, 15, or 20 mM H2O2

was added to each culture. After a 10-min treatment, the H2O2 was discardedand incubation was continued for 2 more days. Colony counts were determinedand compared with those of untreated controls.

Hyphal adhesion was determined at different air/liquid ratios in 25-ml Erlen-meyer flasks containing 5, 10, 15, or 20 ml of a growth medium inoculated with3 � 105 conidia/ml and grown for 15 h at 30°C and 250 rpm. Cultures werephotographed, and mycelia were harvested by filtration to determine proteinoxidation. To measure the amount of aerial hyphae and conidia, liquid culturesinoculated with 106 conidia/ml were incubated for 16 h at 30°C and 200 rpm. Onehundred milliliters of culture was filtered, and the resulting mycelial mat insidepetri dishes was exposed to air for 24 h at room temperature. Aerial mycelia wererecovered with a spatula, vacuum dried, and weighed. To measure the height anddensity of aerial hyphae, we took advantage of the ability of aerial hyphae to stickto glass. Microscope slides standing on mycelial mats were removed after differ-ent times of aerial growth. The height and area covered by aerial hyphae weredetermined, conidiation was analyzed under a light microscope, and the protein

in detached aerial hyphae was measured. The height and density of aerial hyphaesticking to the slides was also determined by using 2.5-ml liquid cultures in 50-mlFalcon tubes with the slide in the tube only touching the culture surface. Fordetermination of conidial counts, 1 ml of sterile water was added to harvestedaerial mycelium and agitated in a Vortex mixer for 5 min, and free conidia werecounted with a Neubauer chamber. Conidia in colonies were determined in 20randomly picked colonies and counted as indicated. To show increased aerialhyphae, some mats were covered with darkly stained filter paper. Aerial hyphaegrew through the stained filter, and cultures were photographed after 24 h ofdevelopment.

Disruption of cat-3. Two primer oligonucleotides, each containing an EcoRIrestriction site (5�-CGCCGAATTCATGCGTGTCAACGCTCTT-3� and 5�CCCGAATTCTTACTCCTCATCATCGC-3�) were used to amplify the cat-3 cDNAsequence from plasmid pSM1 by PCR. The amplified 2-kb cat-3 cDNA sequencewas cloned by replacing the lacZ EcoRI fragment in plasmid pDE1 (9), yieldingpSM3. The truncated N. crassa his-3 gene in the plasmid was used to directintegration to this locus. Forty microliter of 1.25 � 1010 conidia/ml from his-3mutant strain FGSC 6103 was electroporated at 1.5 kV with circular pSM3 (500�g) (29). Histidine prototrophs were isolated and analyzed by DNA blot hybrid-ization to select transformants with the cat-3 sequence adjacent to the repairedhis-3 locus. After three cycles of single-colony isolation, a transformant wascrossed to the Wt strain to obtain his-3 prototrophs lacking the cat-3 transcriptthe and CAT-3 protein.

Vegetative mycelia were frozen in liquid nitrogen and stored at �70°C untilused. Total RNA was isolated with TRIZOL (GIBCO, BRL) in accordance withthe instructions of the manufacturer. For Northern blot assays, 10 �g of RNA perlane was loaded onto a 0.7% agarose gel containing formaldehyde, run at 60 V,transferred to nylon membranes (Hybond-N; Amersham Pharmacia Biotech),and hybridized with a cat-3 probe. Genomic DNA was isolated as described byVollmer and Yanofsky (45). For Southern blot assays, 5 �g of DNA was digestedwith 20 U of MscI, electrophoresed on an agarose gel, transferred to Hybond-XLmembrane (Amersham RPN 203 S), fixed to the membrane with UV light (UVStratalinker 1800; Stratagene), and hybridized with a cat-3 or his-3 probe. Ra-dioactivity was detected by autoradiography with Kodak Biomax MR film.

Sexual crosses were performed on synthetic cross medium as described byDavis and De Serres (6).

Protein isolation. Mycelia were harvested by filtration and resuspended in 1 mlof acetone, agitated for 15 s in a Vortex mixer, and centrifuged for 5 min at14,000 � g. Acetone was eliminated, and the precipitate was dried by evapora-tion. One hundred milligrams of the dry pellet was resuspended in 300 �l of20 mM HEPES, pH 7.2, containing 1 mM phenylmethylsulfonyl fluoride, 1 mMdithiothreitol, and 0.1 mM desferrioxamine B mesylate. Hyphae were broken byagitation with 100 mg of glass beads (710 to 1,180 �m) in a Vortex mixer for 30min at 4°C. Protein was determined by the method of Bradford or Lowry et al.

For detection of secreted CAT-3 in liquid medium after 16 h growth, myceliawere separated by filtration and 1 liter of growth medium was dialyzed againstdistilled water, concentrated in an Amicon YM100 to 1 ml, precipitated with 2volumes of acetone, and resuspended in 300 �l of HEPES-phenylmethylsulfonylfluoride-dithiothreitol-desferrioxamine B mesylate buffer.

CAT-3 activity and immunodetection. Catalase (hydrogen peroxide:hydrogenperoxide oxidoreductase; EC 1.11.1.6) activity was either measured by determin-ing the initial rate of O2 production with a Clark microelectrode or detected ingels after polyacrylamide gel electrophoresis (PAGE) (25). For determination ofcatalase activity, PAGE under nondenaturing conditions was used, usually withloading of 50 U of catalase activity or 30 �g of protein in each lane. Minigels of8 by 9 cm and 0.75 cm thick with 8% polyacrylamide and 0.2% bisacrylamidewere made in accordance with the Laemmli procedure but without sodiumdodecyl sulfate and �-mercaptoethanol and without boiling of the samples. ForCAT-3 immunodetection, denaturing conditions were used. After PAGE, gelswere immediately used for immunodetection or stained for catalase activity.Two-dimensional (2-D) PAGE was done as described before (25). For immu-nodetection, proteins were electrotransferred to nitrocellulose filters (GibcoBRL) at 100 V for 1 h with a Mighty Small Transfer unit (Hoefer) and the bufferdescribed by Towbin et al. (43). Filters were blocked with 3% skim milk inphosphate-buffered saline–Tween 20 (0.03%) buffer at room temperature andthen incubated with rabbit sera containing polyclonal anti-CAT-3 or anti-CAT-1antibodies diluted as appropriate in phosphate-buffered saline–Tween 20–0.1%skim milk. Antibodies were detected with anti-rabbit immunoglobulin G coupledto horseradish peroxidase and developed with 4-chloro-1-naphthol as the sub-strate.

Carotenoid extraction and determination. Three-day-old colonies (n � 250)growing over cellophane on solid cultures were illuminated for 1 h with intenselight (5 W/cm2) and immediately recovered with a spatula. Colonies were resus-

VOL. 2, 2003 OXIDATIVE STRESS AND CELL DIFFERENTIATION 799


.org/D

ownloaded from

http://ec.asm.org/

pended in 400 �l of 20 mM HEPES, pH 7.2, and broken by agitation in anEppendorf tube with 300 mg of glass beads at the Vortex mixer’s maximal speedat 4°C for 30 min. Carotenoids were extracted from 400 �l of cell extract,containing 4 mg of protein, with 400 �l of butanol-chloroform (1:3 vol/vol). Themixture was stirred for 2 min in a Vortex mixer at full speed and thereaftercentrifuged in a microcentrifuge. The butanol-chloroform phase, containing

FIG. 1. cat-3-null strains lack cat-3 transcript and CAT-3 protein.(A) Catalase activity in gels after PAGE under nondenaturing condi-tions of 30 �g of protein extract from the Wt and cat-351 and cat-360

mutant strains. (B) Autoradiography of total RNA (10 �g) isolatedfrom growing Wt or cat-351 or cat-360 mutant mycelia hybridized witha radioactive cat-3 probe. (C) Ethidium bromide staining of the gelused in panel B to evaluate RNA loading. (D and E) Immunodetectionof CAT-3 and CAT-1 in Wt and cat-351 and cat-360 mutant cell ex-tracts. Polyclonal antibodies that specifically recognize CAT-3 or CAT-1 were used and developed with a peroxidase-bound anti-rabbit anti-body. (F to H) Catalase activity in gels after 2-D PAGE of Wt (F) orcat-351 (G) or cat-360 (H) mutant cell extracts. (I) Southern blot anal-ysis of DNAs from Wt and cat-360 mutant cells digested with MscI andhybridized with a radioactive cat-3 probe; phage � DNA digested withHindIII was used as molecular weight (MW) standards.

FIG. 2. Lack of CAT-3 activity in a cat-3-null mutant strain is notcompensated for by other catalases. Wt (white bars) and cat-360 mu-tant (dark bars) mycelia grown for 14 h were transferred to freshmedium containing 1 mM H2O2 (A), 30 mM CaCO3 (B), or 5 mMparaquat (C). After 0.5, 1, 3, or 6 h of treatment, catalase activity wasdetermined in cell extracts. The results shown are averages of threeindependent experiments.

800 MICHAN ET AL. EUKARYOT. CELL


.org/D

ownloaded from

http://ec.asm.org/

most of the carotenoids, was recovered and diluted 50 times with butanol-chloroform, and spectra were run in a Beckman spectrometer.

Carbonyl content in total protein. Liquid-grown mycelium was harvested byfiltration and dried with acetone. Protein was extracted as mentioned above, andcarbonyl content was determined as described by Ahn et al. (1), but the extrac-tion with butanol-chloroform was repeated six times to ensure the elimination ofall lipids (carotenes). Colonies (n � 150) on cellophane-overlaid solid cultureswith VSM were grown for 3 or 7 days in the dark at 30°C. Colonies were recov-ered with a spatula and dried with acetone. Protein was extracted from five col-onies in 300 �l of buffer, and carbonyl content was determined as described above.

RESULTS

Disruption of cat-3. During meiosis in N. crassa, chromo-somal sequences that are repeated in the haploid genome aremutated through a process called repeat-induced point muta-tion (RIP). In this process, methylation of cytosines generateC-to-T and G-to-A transitions that inactivate every repeatedsequence (4). RIP has been used with great success to generatenull mutants of N. crassa by introducing a copy of the gene to

be disrupted into the genome and crossing the transformedstrain.

A 2-kb fragment of cat-3 cDNA was cloned into a plasmidcontaining an N-terminally truncated his-3 gene and used totransform an N. crassa histidine auxotroph containing a pointmutation in the his-3 locus. Histidine prototrophs were isolatedand analyzed by DNA blot hybridization to select transfor-mants containing cat-3 sequence adjacent to the repaired his-3locus. After three cycles of single-colony isolation, a transfor-mant was crossed to the Wt strain to obtain his-3 prototrophswith no cat-3 transcript or CAT-3 protein. Twenty of the 42randomly picked progeny colonies were CAT-3 defective, in-dicating 1:1 segregation. The phenotype of two of them, cat-351

and cat-360, mating type A, was characterized in more detailand compared to the Wt strain.

No CAT-3 activity (Fig. 1A) or cat-3 transcript (Fig. 1B) wasdetected in either mutant stain. In addition, no CAT-3 antigenwas detected with polyclonal antibodies directed against puri-

FIG. 3. cat-3-null mutant strains are sensitive to H2O2. Wt or cat-351 or cat-360 mutant conidia (n � 250) were plated on VSM, and after 2 days,colonies were covered with an H2O2 solution. After a 10-min treatment, the H2O2 solution was discarded and the plates were incubated for another2 days. Thereafter, colonies were counted and compared to untreated controls. (A) Plates of Wt and mutant strain colonies after treatment with20 mM H2O2. (B) Percentages of colonies that survived H2O2 treatment at the indicated concentrations.



.org/D

ownloaded from

http://ec.asm.org/

fied CAT-3 (Fig. 1D). As a control, CAT-1 antigen was de-tected in similar amounts in the Wt and CAT-3 mutant strains(Fig. 1E). To further confirm the absence of CAT-3 and pos-sible mutant peptides with catalase activity, 2-D PAGE wasperformed. Only CAT-1 was detected in cat-351 (Fig. 1G) andcat-360 mutant strains (Fig. 1H).

To prove that the lack of CAT-3 was due to RIP, the cat-360

mutant and Wt DNAs were digested with different restrictionenzymes that are sensitive to DNA methylation and analyzedby DNA blot hybridization with cat-3 as the probe. With MscI,cat-3 hybridized to a DNA fragment from cat-360 with a highermolecular weight, consistent with loss of the single MscI site inthe gene (Fig. 1I).

CAT-3 loss is not compensated for by other catalases, andmutant strains are H2O2 sensitive. Because N. crassa hasthree monofunctional catalases and one catalase/peroxidase(31a), increasing the activity of another catalase could com-pensate for the lack of CAT-3 in the mutant strains. Previousexperiments indicated that H2O2, CaCO3, and particularlyparaquat induced cat-3 transcript and CAT-3 activity levels(30). We assayed total catalase activity in liquid cultures sub-jected to these oxidative stress conditions: in cat-3RIP strains,with or without stress, total catalase activity remained at sim-ilar levels (Fig. 2A to C); in the Wt strain, 6 h of stress led toa twofold increase in catalase activity under the first two con-ditions (Fig. 2A and B) and a seven- to eightfold increase withparaquat (Fig. 2C). These results indicate that the lack ofCAT-3 was not compensated for by other catalases.

Other H2O2-disposing enzymes such as peroxiredoxins and/or peroxidases could also have a compensating effect. To an-alyze the H2O2 sensitivity of the cat-3RIP strains, 20 mM H2O2

was added to 2-day-old colonies grown in petri dishes. H2O2

was eliminated after 10 min, and colony survival was analyzed2 days later. cat-3RIP strains did not resist this treatment, whilethe Wt strain seemed unaffected (Fig. 3A). Similar assays with5 to 20 mM H2O2 concentrations showed a dose-responseeffect. At 10 mM H2O2, only 25 to 30% of the cat-3 coloniessurvived, compared to more than 90% of the Wt colonies (Fig.3B).

CAT-3 is secreted. CAT-3 has an N-terminal signal peptidethat is processed (30) and probably used for enzyme secretion,as was found for homologous catalases (3, 12). To find out ifCAT-3 is secreted, we stained colonies in petri dishes forcatalase activity. A large halo of catalase activity observed inWt colonies was absent or strongly reduced in the cat-360 strain(Fig. 4A), suggesting that CAT-3 activity diffuses out of colo-nies.

To confirm CAT-3 secretion, the liquid medium in which Wtor mutant strains grew for 16 h was dialyzed, concentrated, andanalyzed for CAT-3 activity and protein. CAT-3 activity (Fig.4B) and immunodetected CAT-3 protein (Fig. 4C) were re-covered from the growth medium of the Wt strain but not fromthat of the cat-3RIP mutant strains. These results demonstratethat CAT-3 is secreted.

cat-3 mutants show increased carotene content in the darkand after exposure to a pulse of light. Colonies of cat-3-nullmutants were more pigmented than the Wt. Pigmentation inN. crassa is mainly due to carotenes. Carotene synthesis isinduced by oxidative stress and by light. Carotene content wasmeasured in 3-day-old colonies grown in the dark or grown inthe dark and then illuminated for 1 h at 5 W/cm2. In the dark,almost no carotenes were detected in Wt colonies; however,the carotene level was 7.7-fold higher in the cat-360 mutantstrain (Fig. 5), denoting increased oxidative stress in thatstrain. Upon illumination, the total carotene content increased4.8- and 2.3-fold in the Wt and mutant strains, respectively.

FIG. 4. CAT-3 is secreted. Panels: A, plates with 4-day-old Wt orcat-360 mutant colonies stained for catalase activity; B, CAT-3 activity;C, immunodetection of CAT-3. Purified CAT-3 (lane 1) and totalprotein from the Wt (lane 2) and cat-351 (lane 3) and cat-360 (lane 4)mutant growth media after PAGE under nondenaturing conditions areshown. After transfer to nitrocellulose filters, CAT-3-specific poly-clonal antibodies were detected by the activity of peroxidase-boundanti-rabbit antibodies.



.org/D

ownloaded from

http://ec.asm.org/

Even under these conditions, the cat-360 mutant strain still had3.7 times more carotenes than did the Wt (Fig. 5).

Increased hyphal adhesion and protein oxidation in thecat-3 mutant. Hyphal adhesion is dependent on air and is thefirst morphogenetic step toward the conidiation process. Aerialhyphae in a mycelial mat develop only from a layer of adherenthyphae (38). Hyphal adhesion can also be observed in liquidcultures under oxidative stress conditions, such as increasedaeration (i.e., an increase in the air-to-liquid ratio). To analyzehyphal adhesion in cat-3RIP mutant strains, liquid medium wasinoculated with conidia and distributed among 25-ml Erlen-meyer flasks at different air-to-liquid ratios. Adhesion of Wthyphae was minimal at the highest air-to-liquid ratio. In thecat-360 mutant strain, adhesion was observed at all ratios andincreased considerably with aeration from many small aggre-gates to a single clump (Fig. 6A). Under these conditions, thefinal biomasses of all stains were similar (data not shown).

To confirm that hyphal adhesion observed was related tooxidative stress, protein oxidation was measured in total my-celial extracts. Protein oxidation, measured as carbonyl contentin total protein, increased with hyphal adhesion and aeration inboth strains but was consistently higher in the cat-360 mutantstrain (Fig. 6B).

cat-3 mutants show increased development of aerial hyphaeand formation of conidia. The second and third steps in theconidiation process are development of aerial hyphae and for-mation of conidia at the tips of aerial hyphae. The amount ofaerial hyphae and conidia was determined in air-exposed my-celial mats. Aerial hyphae and conidia were more abundant inthe cat-360 mutant strain than in the Wt (Fig. 7A and B). Infact, the mutant strain produced six times more aerial hyphaeand conidia than did the Wt (Fig. 7C).

Aerial hyphae and conidia were also more abundant in7-day-old colonies of the cat-360 mutant strain than in those ofthe Wt (Fig. 8A and B). To confirm that this trait was relatedto oxidative stress, protein oxidation was determined in colo-nies. After 3 days of growth in petri dishes, Wt and cat-360

mutant colonies had similar amounts of carbonyls in their totalprotein. Four days later, protein oxidation was lower in Wtcolonies and twofold higher in cat-360 colonies (Fig. 8C), indi-cating oxidative stress in mutant colonies.

Counting of aerial hyphal initials per unit of area was notpossible because aerial hyphae rapidly form a syncytium. Toshow an increase in aerial hyphae per unit of area, mycelialmats were covered with dark-purple-stained filter paperthrough which aerial hyphae can grow. In the cat-360 mutant,the whole paper was covered with aerial hyphae, while in theWt, there was some uncovered space (Fig. 9A). Edges of themycelial mat are more air exposed and develop more aerialhyphae, particularly in the cat-360 mutant strain (Fig. 9A). Theheight of aerial hyphae was measured by placing standing mi-croscope slides on mycelial mats or holding them on the sur-face of liquid cultures. Aerial hyphae of the cat-360 mutantstrain grew to a greater height than did those of the Wt andconidiated later in both systems (Fig. 9B and C). Compared tothe Wt strain, the density of aerial hyphae that adhered to theslide was 2.75 times higher in the mutant in mycelial mats and1.75 times higher in standing liquid cultures (Fig. 9D). Theprotein content per unit of dry weight of aerial hyphae was198.5 6.7 �g/mg in both strains. There was no indication ofprecocious development of aerial hyphae: aerial hyphae startto grow 2 h after air exposure of mycelial mats and 4 h instanding liquid cultures.

FIG. 5. Carotene contents of Wt and cat-360 mutant colonies. Spectra of isolated carotenes from cell extracts containing 4 mg of protein.Three-day-old Wt and cat-360 colonies, grown in the dark on cellophane-covered solid medium, were illuminated for 1 h with intense light (5W/cm2), and carotenes were immediately extracted (Wt � L and cat-360 � L). Control plates were also illuminated but wrapped with aluminumfoil (Wt � D and cat-360 � D). The results of an experiment representative of the four independent experiments performed are shown.



.org/D

ownloaded from

http://ec.asm.org/

DISCUSSION

Here we present evidence indicating that cat-3-null strainstend to develop oxidative stress (increased protein oxidation)and react by increasing antioxidant levels (carotenes) and de-veloping large amounts of differentiated cell structures (adher-ent hyphae, aerial mycelium, and conidia). These results are inaccordance with the O2 avoidance theory of cell differentiation(13, 15).

The lack of CAT-3 in mutant strains was not compensatedfor by other catalase activities, even under oxidative stressconditions, in contrast to what is observed in Xanthomonascampestris (44). In N. crassa, CAT-1 is expressed mainly innongrowing cells (8, 30), CAT-4 is presumably restricted toperoxisomes, and CAT-2 is expressed in lysing hyphae (31a).Besides catalases, three peroxiredoxins, a glutathione peroxi-dase, cytochrome c peroxidase, and probably other peroxidasesare found in the N. crassa genome (www.genome.wi.mit.edu).However, these enzymes are efficient only at low concentrations

of H2O2; millimolar concentrations of H2O2 can only be dis-posed of by catalases (18, 32). Thus, despite such a high re-dundancy of H2O2-detoxifying enzymes, other enzymes cannotcompensate for loss of CAT-3 activity because these enzymesare much less efficient, are expressed in other cells, or have adifferent intracellular localization. This may explain why thelack of CAT-3 had a great impact on the antioxidant capabilityand cell differentiation of N. crassa.

The susceptibility of cat-3RIP mutant strains was made evi-dent by treatment of colonies with H2O2. Wt colonies werehardly affected by 20 mM H2O2, while cat-3RIP mutant strainsdid not survive this treatment. CAT-3 was shown to be secretedfrom Wt colonies and from hyphae in liquid cultures. Theprotein has a signal peptide for secretion that is processed (30).However, CAT-3 detected in the growth medium representsonly a few percent (3%) of the total catalase activity in cellextracts (data not shown). Thus, loss of CAT-3 into the me-dium is low and most of the activity in mycelial cell extracts isintracellular or bound to the cell wall.

Because lack of CAT-3 was not compensated for enzymati-cally, other antioxidants are expected to be induced in cat-3-null strains. Carotenes are antioxidants that are especiallyeffective at quenching 1O2 (36). In N. crassa, carotenes areinduced by light through the WC1/WC2 pathway but carotene

FIG. 6. Adhesion of hyphae and carbonyl content of mycelia grownunder different aeration conditions. Conidia from either Wt or cat-360

mutant cells were inoculated in liquid medium. Volumes of 20, 15, 10,or 5 ml were then poured into 25-ml Erlenmeyer flasks and incubatedfor 15 h at 30°C and 250 rpm. Cultures were transferred to petri dishesfor documentation of hyphal adhesion, and thereafter, mycelium wasrecovered for determination of protein carbonyl content. Panels: A,adhesion of Wt and cat-360 mutant hyphae (lanes: 1, 5 ml [4:1 ratio]; 2,10 ml [3:2 ratio]); B, protein carbonyl content from Wt or cat-360

mutant mycelium growing at an air-to-liquid ratio of 1:4 (columns 1),2:3 (columns 2), 3:2 (columns 3), or 4:1 (columns 4). The results shownare averages of two independent experiments.



.org/D

ownloaded from

http://ec.asm.org/

induction during conidiation is independent of light and WCgenes (16, 24). 1O2 is an inducer of carotene synthesis inPhaffia rhodozyma (33). 1O2 is produced by blue light throughphotosensitization reactions. However, in the dark, H2O2 is amain source for 1O2 generation (reviewed in reference 27). N.crassa carotene mutant strains are sensitive to light and 1O2

(37). 1O2 is generated during N. crassa conidial germination(26) and conidiation and under different stress conditions(heat shock or paraquat treatment) (30). Wt colonies hardlymade carotenes when grown in the dark and synthesized themmainly when in the presence of light. Instead, cat-360 mutantcolonies grown in the dark had increased carotene content.These results are consistent with 1O2 generation and 1O2 in-duction of carotene synthesis in mutant strains growing in thedark. Carotene synthesis in the Wt and cat-360 mutant strains

was enhanced further by light and/or by 1O2 generated byphotosensitization.

Besides carotene synthesis, cat-360 mutant hyphae tended toadhere to each other when grown in liquid cultures. Hyphaladhesion is related to a carbohydrate that is secreted andpolymerized at the cell wall, functioning as cement betweenhyphae (W. Hansberg, unpublished observations). When a my-

FIG. 7. Enhancement of aerial hyphae and conidium formation ina cat-360 mutant strain. Wt and cat-360 mutant cultures (100 ml) werefiltered, and the resulting mycelial mats were exposed to air for 24 h.Side (A) and top (B) views of the mycelial mats showing aerial myce-lium (A) and conidia (B) from Wt and cat-360 mutant strains and aerialmycelium dry weight and numbers of conidia from Wt and cat-360

mutant strains (C) are depicted. The results shown in panel C areaverages of four independent experiments.

FIG. 8. Conidiation of Wt and cat-360 mutant colonies. Colonieswere grown on VSM for 7 days at 30°C. Panels: A, 7-day-old Wt andcat-360 colonies; B, number of conidia per colony (averages of threegroups of 20 colonies picked at random are shown); C, protein car-bonyl contents of Wt (dark bar) and cat-360 mutant (gray bar) coloniesgrown in the dark on cellophane-covered VSM plates after 3 (columns1) or 7 (columns 2) days. The results shown are averages of fourindependent experiments.



.org/D

ownloaded from

http://ec.asm.org/

celial mat is exposed to air, a layer of adherent hyphae isformed within minutes. This layer of adherent hyphae is oftenmistakenly interpreted as desiccation. A layer of adherent hy-phae also forms in standing liquid cultures and is not formed inmycelial mats in the absence of air or in the presence of anti-oxidants. Aerial hyphae develop only from the layer of adher-ent hyphae and represent the first step of the conidiationprocess (38). Only in this layer of the mycelial mat are proteinsand specific enzymes oxidized and degraded and are some ofthem resynthesized (41). Hyphal adhesion correlated with pro-tein oxidation in liquid cultures. cat-360 mutant hyphae grew asaggregates that increased in size depending on aeration; Wthyphae also tend to form aggregate at a high aeration rate. Hy-phal adhesion is a cellular response to oxidative stress. It prob-ably has the effect of reducing the local concentration of O2 byactive respiration. Deposited carbohydrates at the cell wallcould also reduce the entrance of O2 into the adherent hyphae.

Aerial hyphae develop from adherent hyphae and conidiaare formed at their tips, representing the second and thirdsteps of the conidiation process. The most conspicuous fea-tures of cat-3-null strains are the increased amount of aerial

hyphal mass and the number of conidia produced; both weresix times the amount determined in the Wt strain. The in-creased amount of aerial hyphae was due to a combination ofhigh density and a prolonged growth period, producing anaerial hyphal mass of increased height and density. The in-creased amount of aerial hyphae produced an increasedamount of conidia. Conidiation was retarded in both the air-exposed mycelial mat and the standing liquid culture systems.The aerial hyphal morphology analyzed under a light micro-scope was similar in both strains.

Interestingly, the N. crassa Cu, Zn SOD-null mutant strainsod-1 (5) has a phenotype similar to that of the cat-3 mutantstrain with respect to carotene production, adhesion of hyphae,and increased amount of aerial mycelium and conidium for-mation (Hansberg, unpublished). However, and as expected,the sod-1 mutant is more resistant to H2O2 and more sensitiveto paraquat treatment than is the cat-3 mutant.

How oxidative stress triggers conidiation remains to be de-termined. During a hyperoxidant state, the amount and ratioof NAD(P)H/NAD(P) and glutathione/glutathione disulfidechanges dramatically (40, 42) and this will change many met-

FIG. 9. Aerial hyphal height in mycelial mats and in standing liquid cultures. (A) Mycelial mats were covered with darkly stained filter paperat the start of development. Aerial hyphae grew through the filter paper. Photographs taken after 24 h show increased density of aerial hyphaein the cat-360 mutant strain. Aerial mycelium heights in mycelial mats (B) and in standing liquid cultures (C) are also shown. Wt, open circles;cat-360, closed circles. The arrow labeled “c” indicates the time when conidiation was observed under a light microscope. (D) Amounts of aerialhyphae that stuck to 1-cm2 microscope slides placed on mycelial mats (columns 1) and on standing liquid cultures (columns 2). The results in panelsB to D are averages of two independent experiments.



.org/D

ownloaded from

http://ec.asm.org/

abolic fluxes. Besides, the redox state in cells is sensed througha cascade of protein kinases. Cytoplasmic catalase in Saccha-romyces cerevisiae is induced by the HOG1 pathway (34). Un-der these conditions, SOD is also regulated although throughthe protein kinase A-cyclic AMP (cAMP) and Skn7 pathways(11). In Schizosaccharomyces pombe (7) and Aspergillus nidu-lans (22), osmotic and oxidative stress activates the HOG1homologues Spc1 and SakA, respectively. In N. crassa, the S.cerevisiae mitogen-activated protein kinase kinase kinase Ste11homologue NRC-1 and also a Tre/Ser protein kinase, NCR-2,have been found to be required for vegetative growth and torepress the onset of conidiation (23). The HOG1 homologueos-2 has been cloned, and its deletion has been shown to beosmosensitive and resistant to some fungicides, but there is noindication of an effect on conidiation (48). It would be inter-esting to find out if a catalase is regulated by one of theseprotein kinases in N. crassa.

Deletion of N. crassa gna-1, which encodes a G�i protein,reduces the cAMP level, causing a decreased apical extensionrate, carotene accumulation, increased tolerance to heat shockand H2O2 treatment, short aerial hyphae, and hyperconidia-tion, a phenotype similar to the adenylate cyclase-deficient cr-1mutant strain. Mutant strains with G�i permanently activatedshow an increased cAMP level, have an apical extension rateclose to that of the Wt, a low carotene content, decreased tol-erance to heat shock and H2O2 treatment, and increased pro-liferation of aerial hyphae but a low level of conidium forma-tion (47).

cAMP levels are probably inversely related to stress intensityin N. crassa. In the cat-3 mutant, the stress is increased, and inthe gna-1 mutant, the signal is increased. The stress and thesignal both will induce antioxidant mechanisms (carotene syn-thesis). Thus, we expect low levels of cAMP in cat-3 mutantstrains and induction of CAT-3 in gna-1 mutant strains.

During the conidiation process, oxidative stress is not con-tinuous but is only generated at the start of each morphoge-netic transition. Thus, in a cat-3RIP mutant strain, both aerialhyphal growth and conidiation are stimulated. In a gna-1 mu-tant strain, the signal is always on at its maximum (no cAMP)and the response is correspondingly maximal. Thus, the strainconidiates profusely, with no delay and without much growthof aerial hyphae.

Double and triple catalase mutant strains of A. nidulans hadno apparent effect on conidiation, although colonies from catBmutant strains and conidia from catA mutant stains are sensi-tive to H2O2 (20, 21, 31). This could indicate different fungalstrategies by which to cope with oxidative stress and to startcell differentiation. A. nidulans has been used mainly for ge-netic studies, and growth conditions and strains have beenselected for rapid and abundant production of conidia. A.nidulans laboratory strains grow in pellets, which could be aprotection mechanism against oxidative stress. Thus, we willtest the phenotype of CatB-null strains, the CAT-3 homologue,under growth conditions similar to those used for N. crassa.

ACKNOWLEDGMENTS

This research was supported in part by grant IN214199 from DGAPA,Universidad Nacional Autonoma de Mexico, and grant 33148-N fromthe Consejo Nacional de Ciencia y Tecnología, Mexico.

We are grateful to Dan Ebbole (Texas A&M University, CollegeStation, Texas) for help with the RIP protocol. We thank Jesus Aguirre(IFCE-UNAM) for critically reviewing the manuscript and LeonardoPeraza for the photographs in Fig. 9.

REFERENCES

1. Ahn, B., S. G. Rhee, and E. R. Stadtman. 1987. Use of fluorescein hydrazideand fluorescein thiosemicarbazide reagents for the fluorometric determina-tion of protein carbonyl groups and for the detection of oxidized protein onpolyacrylamide gels. Anal. Biochem. 161:245–257.

2. Bagyan, I., L. Casillas-Martinez, and P. Setlow. 1998. The katX gene, whichcodes for the catalase in spores of Bacillus subtilis, is a forespore-specificgene controlled by �F, and KatX is essential for hydrogen peroxide resis-tance of the germinating spore. J. Bacteriol. 180:2057–2062.

3. Calera, J. A., S. Paris, M. Monod, A. J. Hamilton, J. P. Debeaupuis, M.Diaquin, R. Lopez-Medrano, F. Leal, and J. P. Latge. 1997. Cloning anddisruption of the antigenic catalase gene of Aspergillus fumigatus. Infect.Immun. 65:4718–4724.

4. Cambareri, E. B., B. C. Jensen, E. Schabtach, and E. U. Selker. 1989.Repeat-induced G-C to A-T mutations in Neurospora. Science 244:1571–1575.

5. Chary, P., D. Dillon, A. L. Schroeder, and D. O. Natvig. 1994. Superoxidedismutase (sod-1) null mutants of Neurospora crassa: oxidative stress sensi-tivity, spontaneous mutation rate and response to mutagens. Genetics 137:723–730.

6. Davis, R. H., and J. F. De Serres. 1970. Genetic and microbiological researchtechniques for Neurospora crassa. Methods Enzymol. 17A:79–143.

7. Degols, G., K. Shiozaki, and P. Russell. 1996. Activation and regulation ofthe Spc1 stress-activated protein kinase in Schizosaccharomyces pombe. Mol.Cell. Biol. 16:2870–2877.

8. Díaz, A., P. Rangel, Y. Montes de Oca, F. Lledías, and W. Hansberg. 2001.Molecular and kinetic study of catalase-1, a durable large catalase of Neu-rospora crassa. Free Radic. Biol. Med. 31:1323–1333.

9. Ebbole, D. 1990. Vectors for construction of translational fusions of �-ga-lactosidase. Fungal Genet. Newsl. 37:15–16.

10. Engelmann, S., C. Linder, and M. Hecker. 1995. Cloning, nucleotide se-quence, and regulation of katE encoding a �B-dependent catalase in Bacillussubtilis. J. Bacteriol. 177:5598–5605.

10a.Galagan, J. E., S. E. Calvo, K. A. Borkovich, et al. 2003. The genomesequence of the filamentous fungus Neurospora crassa. Nature 422:859–868.

11. Garay-Arroyo, A., F. Lledías, W. Hansberg, and A. A. Covarrubias. 2003.Nature 422: 859–868 The cytoplasmic Cu, Zn superoxide dismutase of Sac-charomyces cerevisiae is required for resistance to hyperosmosis. FEBS Lett.539:68–72.

12. Garre, V., U. Muller, and P. Tudzynski. 1998. Cloning, characterization, andtargeted disruption of cpcat1, coding for an in planta secreted catalase ofClaviceps purpurea. Mol. Plant-Microbe Interact. 11:772–783.

13. Hansberg, W., and J. Aguirre. 1990. Hyperoxidant states cause microbial celldifferentiation by cell insulation from dioxygen. J. Theor. Biol. 142:201–221.

14. Hansberg, W., H. de Groot, and H. Sies. 1993. Reactive oxygen speciesassociated with cell differentiation in Neurospora crassa. Free Radic. Biol.Med. 14:287–293.

15. Hansberg, W. 1996. A hyperoxidant state at the start of each developmentalstage during Neurospora crassa conidiation. Cienc. Cult. 48:68–74.

16. Harding, R. W., and R. V. Turner. 1981. Photoregulation of carotenoidbiosynthetic pathway in albino and white collar mutants of Neurosporacrassa. Plant Physiol. 68:745–749.

17. Hengge-Aronis, R. 1993. Survival of hunger and stress: the role of rpoS inearly stationary phase gene regulation in E. coli. Cell 72:165–168.

18. Hoffman, B., H. J. Hecht, and L. Flohe. 2002. Peroxiredoxins. Biol. Chem.383:347–364.

19. Johnson, C. H., M. G. Klotz, J. L. York, V. Kruft, and J. E. McEwen. 2002.Redundancy, phylogeny and differential expression of Histoplasma capsula-tum catalases. Microbiology 148:1129–1142.

20. Kawasaki, L., D. Wysong, R. Diamond, and J. Aguirre. 1997. Two divergentcatalase genes are differentially regulated during Aspergillus nidulans devel-opment and oxidative stress. J. Bacteriol. 179:3284–3292.

21. Kawasaki, L., and J. Aguirre. 2001. Multiple catalase genes are differentiallyregulated in Aspergillus nidulans. J. Bacteriol. 183:1434–1440.

22. Kawasaki, L., O. Sanchez, K. Shiozaki, and J. Aguirre. 2002. SakA MAPkinase is involved in stress signal transduction, sexual development and sporeviability in Aspergillus nidulans. Mol. Microbiol. 45:1153–1163.

23. Kothe, G. O., and S. J. Free. 1998. The isolation and characterization of nrc-1and nrc-2, two genes encoding protein kinases that control growth anddevelopment in Neurospora crassa. Genetics 149:117–130.

24. Linden, H., and G. Macino. 1997. White collar 2, a partner in blue-lightsignal transduction controlling expression of light-regulated genes in Neuro-spora crassa. EMBO J. 16:98–109.

25. Lledías, F., P. Rangel, and W. Hansberg. 1998. Oxidation of catalase bysinglet oxygen. J. Biol. Chem. 273:10630–10637.

26. Lledías, F., P. Rangel, and W. Hansberg. 1999. Singlet oxygen is part of a



.org/D

ownloaded from

http://ec.asm.org/

hyperoxidant state generated during spore germination. Free Radic. Biol.Med. 26:1396–1404.

27. Lledías, F., and W. Hansberg. 2000. Catalase modification as a marker forsinglet oxygen. Methods Enzymol. 319:110–119.

28. Loewen, P. C., and J. Switala. 1988. Purification and characterization ofspore-specific catalase-2 from Bacillus subtilis. Biochem. Cell Biol. 66:707–714.

29. Margolin, B. S., M. Freitag, and E. U. Selker. 1997. Improved plasmids forgene targeting at the his-3 locus of Neurospora crassa by electroporation.Fungal Genet. Newsl. 44:34–36.

30. Michan, S., F. Lledías, J. D. Baldwin, D. O. Natvig, and W. Hansberg. 2002.Regulation and oxidation of two large monofunctional catalases. Free Radic.Biol. Med. 33:521–532.

31. Navarro, R. E., M. A. Stringer, W. Hansberg, W. E. Timberlake, and J.Aguirre. 1996. catA, a new Aspergillus nidulans gene encoding a develop-mentally regulated catalase. Curr. Genet. 29:352–359.

31a. Peraza, L., and W. Hansberg. 2002. Neurospora crassa catalases, singletoxygen and cell differentiation. Biol. Chem. 383:569–575.

32. Seaver, L. C., and J. A. Imlay. 2001. Alkyl hydroperoxide reductase is theprimary scavenger of endogenous hydrogen peroxide in Escherichia coli. J.Bacteriol. 183:7173–7181.

33. Schroeder, W. A., and E. A. Johnson. 1995. Singlet oxygen and peroxylradicals regulate carotenoid biosynthesis in Phaffia rhodozyma. J. Biol.Chem. 31:18374–18379.

34. Schuller, C., J. L. Brewster, M. R. Alexander, M. C. Gustin, and H. Ruis.1994. The HOG pathway controls osmotic regulation of transcription via thestress response element (STRE) of the Saccharomyces cerevisiae CTT1gene. EMBO J. 13:4382–4389.

35. Springer, M. L., and C. Yanofsky. 1989. A morphological and genetic anal-ysis of conidiophore development in Neurospora crassa. Genes Dev. 3:559–571.

36. Sundquist, A. R., K. Briviba, and H. Sies. 1994. Singlet oxygen quenching bycarotenoids. Methods Enzymol. 234:384–388.

37. Thomas, S. A., M. L. Sargent, and R. W. Tuveson. 1981. Inactivation ofnormal and mutant Neurospora crassa conidia by visible light and near-UV:

role of 1O2, carotenoid composition and sensitizer location. Photochem.Photobiol. 33:349–354.

38. Toledo, I., J. Aguirre, and W. Hansberg. 1986. Aerial growth in Neurosporacrassa: characterization of an experimental model system. Exp. Mycol. 10:114–125.

39. Toledo, I., and W. Hansberg. 1990. Protein oxidation related to morphogen-esis in Neurospora crassa. Exp. Mycol. 14:184–189.

40. Toledo, I., A. A. Noronha-Dutra, and W. Hansberg. 1991. Loss of NAD(P)-reducing power and glutathione disulfide excretion at the start of inductionof aerial growth in Neurospora crassa. J. Bacteriol. 173:3243–3249.

41. Toledo, I., J. Aguirre, and W. Hansberg. 1994. Enzyme inactivation relatedto a hyperoxidant state during conidiation of Neurospora crassa. Microbiol-ogy 140:2391–2397.

42. Toledo, I., P. Rangel, and W. Hansberg. 1995. Redox imbalance at the startof each morphogenetic step of Neurospora crassa conidiation. Arch. Bio-chem. Biophys. 319:519–524.

43. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer ofproteins from polyacrylamide gels to nitrocellulose sheets: procedure andsome applications. Proc. Natl. Acad. Sci. USA 76:4350–4354.

44. Vattanaviboon, P., and S. Mongkolsuk. 2000. Expression analysis and char-acterization of the mutant of a growth- and starvation-regulated monofunc-tional catalase gene from Xanthomonas campestris pv. Phaseoli. Gene 241:259–265.

45. Vollmer, S. J., and C. Yanofsky. 1986. Efficient cloning of genes of Neuro-spora crassa. Proc. Natl. Acad. Sci. USA 83:4869–4873.

46. Willekens, H., C. Langebartels, C. Tire, M. van Montagu, D. Inze, and vanW. Camp. 1994. Differential expression of catalase genes in Nicotiana plum-baginifolia (L.). Proc. Natl. Acad. Sci. USA 91:10450–10454.

47. Yang, Q., and K. Borkovich. 1998. Mutational activation of G�i causesuncontrolled proliferation of aerial hyphae and increased sensitivity to heatand oxidative stress in Neurospora crassa. Genetics 151:107–117.

48. Zhang, Y., R. Lamm, C. Pillonel, S. Lam, and J.-R. Xu. 2002. Osmoregula-tion and fungicide resistance: the Neurospora crassa os-2 gene encodes aHOG1 mitogen-activated protein kinase homologue. Appl. Environ. Micro-biol. 68:532–538.



.org/D

ownloaded from

http://ec.asm.org/

Asexual Development Is Increased in Neurospora crassa cat ...ec.asm.org/content/2/4/798.full.pdf ·...

Documents

Transcript of Asexual Development Is Increased in Neurospora crassa cat ...ec.asm.org/content/2/4/798.full.pdf ·...