The EMBO Journal vol. 1 1 no.6 pp. 2159 - 2166, 1992

cot-1, a gene required for hyphal elongation in Neurospora crassa, encodes a protein kinase

Oded Yarden1'2, Michael Plamann3, Daniel J.Ebbole3'4 and Charles Yanofskyl 5 'Department of Biological Sciences, Stanford University, Stanford, CA 94305 and 3Department of Biology, Texas A&M University, College Station, TX 77843, USA 2Present address: Department of Plant Pathology and Microbiology, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel 4Present address: Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA 5Corresponding author Communicated by U.Henning

Neurospora crassa is a filamentous fungus that grows on semisolid media by forming spreading colonies. Mutations at several loci prevent this spreading growth. cot-i is a temperature sensitive mutant of N.crassa that exhibits restricted colonial growth. At temperatures above 32'C colonies are compact while at lower temperatures growth is indistinguishable from that of the wild type. Restricted colonial growth is due to a defect in hyphal tip elongation and a concomitant increase in hyphal branching. We have isolated a genomic cosmid clone containing the wild type allele of cot-i by complementation. Sequence analyses suggested that cot-i encodes a member of the cAMP-dependent protein kinase family. Strains in which we disrupted cot-i are viable but display restricted colonial growth. Duplication, by ectopic integration of a promoter-containing fragment which includes the first one-third (209 codons) of the structural gene, unexpectedly resulted in restricted colonial growth. Our results suggest that an active COTi kinase is required for one or more events essential for hyphal elongation. Key words: hyphal growth/cAMP-dependent protein kinase/fungal development

of specialized hyphae. The frequency of hyphal branching is markedly influenced by compounds such as tergitol (Springer, 1991) or L-sorbose (Tatum et al., 1949). Exposure to these substances results in the formation of dense, restricted growth characterized by extensive branching. Colonial growth on L-sorbose has been utilized extensively in screening procedures with N.crassa. Many 'colonial' genes have been identified which, when mutated, result in compact colonial growth under conditions favoring spreading growth in wild type (Perkins et al., 1982). One of these mutant genes, colonial temperature sensitive-I (cot-i), causes colonial growth at 32°C and above, but normal growth, morphology and fertility at or below 25°C (Mitchell and Mitchell, 1954; Galsworthy, 1966). When cultured at temperatures above 32°C, cot-i colonies grow slowly, their hyphae branch excessively and they do not conidiate. These cultures rapidly resume normal growth when shifted to a permissive temperature. Because of its high viability and compact growth at restrictive temperatures and normal growth and development at 25°C, cot-i strains have been used widely in procedures requiring compact colonial growth (Perkins et al., 1969, 1982). We have cloned, sequenced and characterized the wild type cot-i gene in order to understand the molecular basis of hyphal elongation and branching in filamentous fungi. cot-i was disrupted and shown not to be essential for viability, but required for normal hyphal elongation. The predicted sequence of COT I is homologous to polypeptides in the cAMP-dependent protein kinase family. The results of this study suggest that the COTI protein kinase is essential for normal hyphal elongation. Our studies provided an unexpected result; ectopic duplication of the initial segment of the cot-i clone, encoding the N-terminal 209 residues of COT1, was negative complementing.

Results Introduction In most filamentous fungi, newly formed hyphae elongate and branch to generate the mycelial mat which is typical of

the growth of these organisms. Under standard laboratory growth conditions, the biomass of the filamentous ascomycete Neurospora crassa is mainly comprised of hyphae. Hyphae first appear upon germination of macroconidia, the predominant asexual propagules of N.crassa. The hyphal germ tube is characteristically tubular in shape IOtM (Schmit and and has an average diameter of 4-10 Brody, 1976). Hyphae typically branch every 15-150 AtM. Continuous hyphal elongation and branching results in the formation of spreading radial colonies on solid media and spherical cellular aggregates during submerged aerated growth in liquid media. Appropriate environmental stimuli (i.e. nutrient deprivation, desiccation and light etc.) induce sexual or asexual development by promoting the formation ©) Oxford University Press

cot-1 morphology In order to determine the extent of the morphological changes conferred by the cot-i mutation, we conducted a microscopic analysis of a cot-i mutant grown under permissive and restrictive temperatures. When grown at 25°C, no distinct differences (either changes in rate of hyphal elongation or frequency of hyphal branching) were observed between hyphae of cot-i and wild type strains (Figure la and b). Within 2 h of transferring cot-i cultures from 25°C to a nonpermissive temperature, 37°C, there was a rapid reduction in the rate of hyphal elongation accompanied by the appearance of numerous branches (Figure ic). The temperature shift had no apparent effect on growth of wild type cultures. In cot-i cultures, cessation of hyphal elongation was evident both at the tips of pre-existing hyphae (which had grown prior to the temperature shift) and at the tips of newly formed branches. The initial branching that occurred when cultures were shifted at 37°C was not

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Fig. 1. (a) Wild type N.crassa grown in Vogel's minimal salts medium for 18 h at 25°C (shaking liquid culture). (b) cot-i grown in Vogel's minimal salts medium for 18 h at 25°C (the permissive temperature for this temperature sensitive mutant). (c) cot-i grown for 16 h at 25°C, followed by 2 h of growth at 37°C (the non-permissive temperature). Note the uniform initiation of multiple branches along the hyphal filaments, resulting in a 'barbed-wire' morphology. (d) A 30 h culture of cot-i grown in liquid on a microscope slide at 37°C. In time, hyphal tips originating from the newly formed branches (shown in picture c) will cease to elongate. In some cases, secondary and tertiary branching occurs prior to arrest of hyphal tip growth. (e) A pMP30 transformant grown for 20 h at 20°C in the presence of hygromycin. cot-i-like structures and hyphae resembling those of wild type are present. Bars indicate 100 yM.

terminal, i.e. secondary and tertiary branches appeared close to primary branches (Figure Id). Bulb-like structures at branch junctions and deterioration of hyphal tips were common morphological abnormalities in cot-i cultures grown for long periods at a restrictive temperature. Cloning of the N.crassa cot-1 gene The wild type cot-i gene was cloned by complementation of the N. crassa cot-i mutant. Spheroplasts of a cot-i strain (prepared from conidia produced at 25°C) were transformed with 10 pools of a N. crassa genomic cosmid library (Orbach/Sachs, Fungal Genetics Stock Center), plated onto sucrose medium containing hygromycin and incubated overnight at 37°C. Four of the cosmid pools yielded hygromycin resistant, cot-i + transformants, i.e. colonies that displayed rapid, spreading growth. One of these cosmid pools was used to obtain sub-pools in Escherichia coli, which were used in a second round of cot-i complementation analyses. Following a third round, with cosmids isolated from individual E. coli transformants, two cosmids containing cot-i complementing DNA were identified (pCT1A and pCT1B). These cosmids were determined to be identical by restriction enzyme analysis. cot-i was co-transformed with aliquots of one of these clones, pCTlA, which had been digested separately with a number of endonucleases and pMP6, a plasmid containing a cpc-i -hph gene fusion which confers hygromycin resistance on N. crassa (Plamann,M. and Yanofsky,C., unpublished results). BamHI, EcoRI, XhoI and SnaI digested pCTIA complemented the cot-i mutation, suggesting that these enconucleases do not cleave within the complementing genetic segment. A 4.2 kb EcoRI-SmaI fragment of pCT 1A which complements cot-i was subcloned (pOYl8; Figure 2). 2160

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Fig. 2. Molecular organization of the N. crassa cot-I gene. Sm (= SmaI) and E (= EcoRI) mark the boundaries of the genomic clone containing cot-i (pOY18). The predicted initiation codon, stop codon and polyadenylation signal sequence are marked, as are the boundaries of coding and non-coding sequences. pOY311 and pOY411 are cot-i cDNA clones used to verify the intron boundaries of the cot-i gene. Intervening sequences absent from the cDNA clones are marked.

Sequence of cot- 1 A 3.3 kb segment of pOYl 8 containing the cot-i gene was completely sequenced on both strands. The nucleotide sequence of cot-i and the predicted amino acid sequence of the COTI polypeptide are presented in Figure 3. The predicted cot-i open reading frame showed a distinct bias for codons preferred by N. crassa. The sequence was also analyzed for the presence of additional hallmarks of N. crassa genes. The context of the presumed cot-i translation initiation codon (ACCAAGATGG) is similar to the N. crassa consensus (A/GTCAA/CAATGG) compiled for 26 genes (Paluh et al., 1988). Three tentative intervening sequences were identified on the basis of consensus 5' and 3' splice junction sequences (Orbach et al., 1986; Hager and Yanofsky, 1990). The existence of these introns was verified

N.crassa cot-1 is required for hyphal elongation 1

CC CGGGAGTGCC CGCT CC CAGC CAGTAACAAATTCC CGGACCAGAGTC TC GATGCT CT CGAGGT CCAATG CC GTTGCGTC GAGC TT CAACACTC GGGGGA

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CTGGTGGTTTACCTATAGTGTGCTTGTGAGGCACGAGAAGATGGCAGCAGTGCGGAACAGACAGCAACGAGTTGACGTCTGACGATGAGGTTGGAGTCAG

201

GGGTTTTCAGGTTGACCAGGGCACCCCATAGTTAGGTGGTCCTGTTTCGGCCTGGAGCTCACCACTGGACCTGGGCATGTCATCCCAAACATGGAACCCC

301

TCTTTCTACGACCTCCTTATTCCTGGGCATCGTGTACTAGATTCACATCGAGGCCAGCTTCCTGTGGCAACAGAAAACGGAAACGTAAAGTCCATTGTTG

401

TCTGTTGTTATTCAAGGTGTCCTTTGAATGCGCCCGTGGCTGTCTCATATCATCACAGTCTATGTCTTGGTCACTTATAGATGGGATCTGTTAGGTTCCA

501

TGTCACCTCTATACGTCTAGGTCTTTCCTTTTATACTGATACCGCACGGCACCCTGCGCCCACCTGCCTTTGGCCTTTGCGTTGCTGATTCGTGCGCCTA

601

GGTTCTCGAATTACAGAGGCCCTGAATCGCCTCCAGCCAGTGCTCTCCTGTCAGACTCCTTCCTGTAGGTAGGCTGGAGAGGCTCACGTCGGTCACGCTT

701

CCACTTTGCTTCCACTTCCACACTGCGCCTATCGACTGCACGTTCCAGCGGGACACTTCCACTTCGGACGGTGCCACTCAACCACCTGCCCCCCCAGCCT

801

GGCCGCCCTCGATTCCCATCCCTACCATCTACAGTAGCCAACCGTCGTTCGATCCTCGTTGGACCCTGGTCCCAAGAAAAAGCAACCTCCTTTCCTCCCG

901

TCTCCCCATACCTATCCCGTCTTGTACACAAACCCCTTTCTGTGCGCATAGATCCCATCGATCGGTCTGGGAGCTCGACTCTGATCGTTTCTCCATCTGC

1001

CGTCCCTCGGAATCTCGTCTGAGCTCGCTTGCGCCCTTCCCGCTACATACACCCGGAATCCACTGTTTACCGTTTAGAGACAAGGTACCAAGATGGACAA

1101

CACCAACCGCCCCCATCTCAACCTGGGCACCAACGATACCCGCATGGCTCCAAACGATCGTACCTATCCCACCACCCCGTCCACCTTCCCCCAACCCGTC

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CAACCAGAAGAAGTGCTCACAGCTGGCCTCTGACTTCTTTAAGGACAGCGTCAAGCGCGCCAGGGAGCGC T

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TCGATCTGGTCAACCGCTGGCCGGAAGGAGGGCCAGTATTTGCGCTTCCTGAGAACCAAGGACAAGCCCGAGAACTACCAGACCATCAAGATCATCGGCA S

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TCGTTTGATTGCGATTGGTGGTCTTTGGGTACCATCATGTTCGAGTGCTTGGTCGGCTGGCCTCCTTTCTGCGCCGAGGATAGCCACGACACTTACCGCA S

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ACTACTACACACAACTGCTCCAGGGCAAATCCAACAAGCCGCGCGACAACCGCAACTCGGTTGCTATTGACCAAATCAACCTGACGGTCAGCAACCGTGC Q

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AaAGATATTAAGCCAGACAACATCCTTCTTGATAGGGGTGGTCATGTCAAGCTTACAGATTTCGGTCTCTCTACCGGCTTCCACAAGCTCCATGATAACA Y

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ATGCTCATGGAGTTCTTGCCCGGTGGTGACTTGATGACCATGCTGATCAAATACGAAATCTTCTCGGAGGATATTACCCGTTTCTACATTGCCGAAATCG M

2201

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AGATCGTGAACTGGAGGCACTCGCTTTACTTCCCGGATGACATCACCCTTGGTGTAGATGCCGAGAACCTTATCAGAAGGaILZCATGCTTTTGACTTCC I

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CCCAAAATCACGCGCTCACCATATTGACTTGTCATAGCCTCATCTGCPACACTGAGAACCGTCTCGGCCGTGGTGGTGCTCACGAAATCAAGAGCCACGC

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CTTCTTCCGCGGCGTTGAGTTCGACAGCTTGCGTCGCATCCGTGCGCCCTTCGAGCCCCGTCTTACATCCGCCATCGATACCACATACTTCCCTACGGAC

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GGGGGAGGAAGGGGGCGTAATAGTGATG GC GAAAGGGAGAAGAGGTTGTGCTGCGGGGCGGCAGTATCTGGGTACCCCTTATAC TT GGCTTT GACGTC TT

3301

TGCTAGCTCGCGGTGCTGTTGAAATTTCTGATGGCAGAGACACTTTGCTGATAGTACGCGAATTTTCCGTTCCGCTC

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Fig. 3. Nucleotide sequence of the N.crassa cot-l gene and flanking regions and the predicted amino acid sequence of COTI. Conserved intron boundaries and presumed lariat sequences are underlined, as is the putative polyadenylation signal. 2161

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by isolation and characterization of two cot-i cDNA clones (obtained from a N. crassa cDNA library provided by Exley and Garrett). Sequence analysis of these cDNA clones (pOY3 11, pOY41 1; Figure 2) confirmed the location and length of the intervening sequences.

Northern analyses showed that the cot-i transcript is 2.4 kb in length (Figure 4A). The site of transcription initiation was not determined. cot-i transcripts were detected in RNA prepared from cot-i and wild type strains, regardless of growth temperature (25°C or 37°C), suggesting that the colonial phenotype of the mutant was not due to the absence of the cot-i transcript (Figure 4A). While almost undetectable in conidia, high levels of cot-i transcript were observed in mycelial samples from early phases of germination and throughout growth (Figure 4B). During conidiation, a stage in which hyphal elongation is significantly reduced, cot-i transcript levels were markedly reduced. This finding is consistent with the observed reduction in transcript levels of many other biosynthetic genes during condiation (Sachs and Yanofsky, 1991).

ow

COT1 amino acid sequence homology to cAMP-dependent protein kinases cot-i is predicted to encode a 622 residue polypeptide with a calculated mass of 70 kDa and a relatively high isoelectric point, 8.6. A database search revealed that COTI was homologous to protein kinases. The greatest extent of amino acid sequence identity was observed between COT1, the catalytic subunit of the mouse cAMP-dependent protein kinase and the Saccharomyces cerevisiae DBF2 (Johnston et al., 1990) gene product (34% and 32%, respectively; Figure 5). Similar sequence identities (data not shown)

Fig. 4. (A) Northern analysis

of cot-i transcript production in cot-i and wild type strains of N.crassa. Duplicate shake cultures of each strain were grown for 16 h at 25°C. Flasks were either maintained for an additional 4 h at 25°C or transferred to 37°C for various time periods (lanes 2 and 4) prior to RNA extraction. Ten jig of RNA were loaded in each lane. pOY311 was used as a template for preparing the RNA probe. (B) Northern analysis of cot-i transcript presence during development of N.crassa. Ten lAg of RNA, isolated from germinating or conidiating cultures taken at times indicated, were loaded in each lane and probed with a labeled pOY311 RNA probe.

50 60 45 5 55 10 1 15 40 20 25 35 30 DBF2 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -IM A GN M S N COT1 M D N T N R P H L N L G T N D T R M A P N D R T Y P T T P S T F P Q P V F P G Q Q A G G S Q Q Y N Q A Y A Q S G |Y Y Q

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190 195 185 V I S R RQ R T K Q V L E Y L Q H P A V I G A D T L F R QmE M - - - - - - - - - - - Q E V K

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295 285 290 300 L TE R D I LIT T T R LL L V KL Y RAERD I L|A E S D P MPKA T L T R MLK E L L K I E L NEK IL A V N 301 305 3 355 350 330 345 335 340 360 X 3 DBF2 Y A rL.L LQSIL YLAI EF|VP GGD|F RTLIL IIN T R C L K S G H A RF VIV N L D YMTHRL D COT1 T T|E G D I TR F YAEIVLAI L M M K Y E IF VIHIKALFJHRDl MPKA F SIf L L L F S HR R I G RIF S EIP H AR E N S N| Y MV D 365 370 375 380 415 410 385 405 390 400 395 361 420 DBF2 KPIE FIA K I LTD G1A ArT I S N E R I E SM K I RLE K I K D L E F P A F T E K SlE D - - - - COTI IK PIDINIIILLIDIR GIG VILTDF G S TUJFH K L H D N N Y Y T Q LLQ G K S N K P R D N R N S V AWD Q I N L T V MPKA K Y I E L I QQ FA K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 421 425 430 435 475 440 470 465 445 460 480 450 455 LfwE K E I N Y Nr M M T V WS M L| ES| V1 L EGKlK D8F2 - R -K M Y TIPFE C E P S D D L L S R TWH COT1 GWIP P Fl L VG GITI WW RS IF TPDYIIA AyS T - - - AQINDw MPKA SN - - - - - -kV K ScL GTP LA P E I|I L S K N K A VDV L I M -FW YIP P FI 481 485 490 495 535 500 530 525 540 505 520 515 510 DBF2 S G S S T N E D N L R R K Q TLRR R QS D G R A A F S D R T W D IT R L I A D P I N ML R S F E H V R M S COT1 CrE D S H DYRf I VN R H SLY F D D I TmG V D A EW LI R S| C - N T E R L G RIG G A - H E IIS HnA MPKA FJD Q P I Q I JELI S G K V R F P S H F S S DIDK D L L RIN LIL Q V T K R F G L K D GV N D I K N HKW FA 541 545 550 595 555 590 585 560 580 600 565 575 570 DBF2 YMA D I NrnS T SI M I TTmQI FD D F T S E A D M A K Y A D V F K R Q D K L T M V D D S A G DMET Q T D N A T L L K A Q Q A ArG A SA P A Q Q E COTi1 F TFjDR D G V ELFJ DFI DrEI R I R E R T A I P T T MPKA T T D W I A I - YIJK V E I - - - - - - - - - - - -LJVS I NE KCGK I K F K G P Gl S NWD D gE EE E 601 605 610 615 620 640 635 625 630 DBF2 S V G F T F R H R N G K Q G S S G I LF N G L E H S D P F S T F Y P IS L P F I G Y T F K R F D N N F R - - - - - - - - - - - - - - COT1 MPKA F-- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - FT DBF2

COT1

Q V I I

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Fig. 5. Multiple sequence alignment of COTI, S.cerevisiae DBF2 and the catalytic domain of the mouse cAMP-dependent protein kinase (MPKA) predicted polypeptides. Yeast and mouse amino acid residues identical in the N.crassa polypeptide are boxed. Hyphens mark gaps. 2162

'-

N.crassa cot-1 is required for hyphal elongation

existed between COT 1 and the deduced product of the DBF2 homologue DBF20 (Toyn et al., 1991). The sites in the catalytic core which are believed to be essential for kinase activity (Hanks et al., 1988; Taylor, 1989) are conserved in residues 196-570 of COTI (Figure 5). The conserved functional sites include the glycine-rich loop containing the MgATP binding site (Gly243 -Lys265), the catalytic loop (Arg358-Asn364) and carboxyl groups that participate in substrate recognition (Asp377-Gly379). A 47 residue segment (residues 383 -429) is located between conserved presumed catalytic subdomains VII and VIII (Hanks et al., 1988) of the COTI polypeptide. This segment is similar in length (though not in sequence) to comparable segments in S. cerevisiae DBF2, DBF20 and CDC7 (Patterson et al., 1986), but is absent from other cAMP-dependent protein kinases characterized to date. The overall similarity between COT1 and CDC7 is not high (25% homology). Chromosomal localization of cot- 1 cot-i has been mapped to the right arm of linkage group IV (Mitchell and Mitchell, 1954). To establish that the gene we cloned was cot-l, we mapped its location by RFLP procedures. We probed a PstI digest of genomic DNA isolated from 18 random progeny (Metzenberg et al., 1985) with hexamer-labeled fragments of the EcoRI -SmaI insert in pOY18. The polymorphism pattern (data not shown) placed the cloned DNA on the right arm of linkage group IV, in full agreement with the segregation of cot-i progeny from the cross (one of the parents used in the cross was cot-i ). Disruption of cot-1 In N. crassa, as in other filamentous fungi, stable transformants mainly result from ectopic integration of transforming DNA, but homologous integration of trans-

A

forming DNA is observed (for review see Rambosek and Leach, 1987; Fincham, 1989). Since N.crassa is a coenocytic haploid organism, it is possible to disrupt an essential gene and have the mutated nucleus survive in a heterokaryon. If the disrupted gene is essential it should not be possible to obtain homokaryons containing only the transformed nuclei. We constructed two plasmids for disruption of cot-i, pMP30 and pMP41 (Figure 6A). pMP30 has the DNA segment from the NruI site to the distal SphI site removed and replaced by the trpC-hph construct (Materials and methods). In pMP41 the DNA segment extending from the KpnI site, just proximal to the ATG initiation codon to the distal SphI site, was removed and replaced by trpC-hph DNA. Wild type N. crassa was transformed with a SnaI -EcoRV DNA fragment from pMP30 (containing the intact DNA segment encoding the N-terminal region of COT1). Unexpectedly, -95% of the stable Hygr pMP30 transformants showed varying degrees of colonial growth. Some transformants had a severe colonial phenotype that was evident on the primary selection plates. This result was unexpected since primary transformants should be heterokaryons. Southern analysis of a number of these transformants revealed that they had one or more copies of the truncated cot-i gene integrated at ectopic sites and they contained intact cot-i DNA at its normal chromosomal location. These findings suggest that the truncated cot-i gene acted in trans to confer a colonial phenotype. It is possible that production of a truncated polypeptide containing the first 209 amino acids of COT 1, encoded by pMP30 DNA, inhibits the function of wild type COT 1. Alternatively; the cot-i DNA segments of pMP30 which flank the trpC-hph gene contains sequences that can titrate limiting factors that are required for cot-i expression. A photograph of a representative transformant containing

3 C D T.. 7'

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Fig. 6. Southern blot analysis of cot-I::trpC-hph transformants. (A) Restriction maps of pOY18 and two plasmids, pMP30 and pMP41, used in cot-I disruption experiments. Relevant restriction sites are indicated: E = EcoRI, EV = EcoRV, K = KpnI, N = Nrul, Sm = SnaI, S = SphI and X = Xhol. The sizes of the diagnostic fragments identified in the Southern blot (B) are indicated. (B) Total DNA was isolated from a wild type strain (lane A), two heterokaryons containing both wild type nuclei and nuclei containing cot-i disrupted by homologous integration of the XhoI-EcoRV fragment of pMP41 (lanes B and C) and a homokaryon containing one or more copies of the Smal-EcoRV fragment of pMP30 ectopically integrated into the N.crassa genome. All DNA samples were digested with SnaI and EcoRV prior to electrophoresis. The probe used was the entire pMP41 plasmid. Note the presence of both the 2.0 kb band, characteristic of the wild type cot-i gene and the 4.1 kb and 1.1 kb bands, diagnostic of the cot-i::trpC-hph homologous replacement allele, in DNA from the heterokaryons shown in lanes B and C.

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ectopically integrated pMP30 DNA is shown in Figure le. This transformant is a homokaryon and has a phenotype intermediate between wild type and the pMP41 cot-i disruptant (see below). Southern analyses with the transformant revealed the two 2.0 kb SmaI-EcoRV and EcoRV-EcoRV fragments characteristic of the wild type cot-i allele and a larger SmaI-EcoRV fragment containing ectopically integrated pMP30 sequences (Figure 6B, lane D). To disrupt the initial segment of the cot-i coding region and avoid the complication introduced by expression of the initial portion of the cot-i gene, we constructed pMP41 and performed a homologous replacement analysis. In pMP41 the cot-i structural gene segment upstream of the distal SphI site is removed (Figure 6A). Transformation of N.crassa with linear pMP41 sequences resulted in the production of many transformants, most of which displayed wild type phenotype. Among -50 stable Hygr transformants that were isolated, two displayed a phenotype that was intermediate between cot-i strains at the restrictive temperature and wild type. When conidia from these two transformants were plated we observed large colonies with wild type characteristics and very small colonies that resembled cot-i strains growing at the restrictive temperature. The small colonies presumably were homokaryons in which there had been homologous replacement of cot-i by cot-i::trpC-hph. It was difficult to obtain sufficient mycelium for DNA isolation from the slowly growing putative disrupted homokaryons. Accordingly, DNA was isolated from the two heterokaryons and from wild type, the DNA was cut with SmaI and EcoRV, and analyzed by Southern analysis using the entire pMP41 plasmid as a probe. As shown in Figure 6B lane A, a single 2.0 kb band was observed with wild type DNA since the SmaI-EcoRV and EcoRV-EcoRV fragments are both 2.0 kb in size (Figure 6A). With the two heterokaryotic strains we observed the 2.0 kb fragments characteristic of wild type nuclei as well as the 4.1 kb and 1.1 kb fragments expected from nuclei containing the homologous cot-i replacement (Figure 6). When the XhoI -EcoRV fragment of pMP41 integrates ectopically the 4.1 kb SmaI-EcoRV and 1.1 kb SmnaI-SmaI fragments are not observed. Southern analyses employing additional restriction endonuclease digestions of the DNA of heterokaryotic strains verified further that homologous replacement had occurred in these two transformants (data not shown). We could obtain homokaryons from primary transformants containing the homologously integrated pMP41 DNA either by plating conidia from the primary transformants or by crossing the transformants with a wild type strain. cot-i::trpC-hph homokaryons grew as very small dense colonies in a temperature independent manner. These results establish that cot-i function is not essential for viability or for germination of asexually or sexually derived spores.

Discussion cot-i of N. crassa encodes a polypeptide that is homologous to members of the cAMP-dependent protein kinase family. Gene disruption experiments were used to show that cot-i is dispensible for viability but essential for normal hyphal growth. This finding implies that the restricted colonial growth of the cot-i mutant at the non-permissive temperature

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is due to loss of COTI function. The presence of cot-i transcript in extracts prepared from the cot-i mutant grown at the non-permissive temperature suggests that the cot-i phenotype is a consequence of a post-transcriptional defect, most likely temperature sensitivity of the COT1 polypeptide. Normal growth of filamentous fungi proceeds by apical extension of hyphal tips. It is conceivable that once the biosynthetic capacity or volume of the cytoplasm in a hyphal tip has reached a level exceeding the ability of the tip to respond simply by elongation, a signal is generated promoting hyphal branching and the concomitant formation of a new growing tip. Therefore in cot-i strains growing at the restrictive temperature, cessation of hyphal tip elongation would result in excessive induction of hyphal branching. The formation of new hyphal tips presumably accommodates the increased volume or metabolic activity. However, since the new tips of cot-i strains cannot elongate normally, tip inhibition causes an abnormal increase in hyphal branching (Figure lb and c). COT1 may function as a regulator that couples cell volume or biosynthetic capacity with hyphal tip elongation. Following a shift to the non-permissive temperature, branching of cot-i hyphae proceeds uniformly throughout the mycelium (Figure lc). This rapid morphological change associated with a halt of hyphal elongation raises the possibility that elongation and branching are interrelated processes. Nonetheless, extensive branching does not always accompany the inhibition of hyphal tip growth; deterioration of hyphal tips has been shown to occur in a N.crassa strain in which the chitin synthase 1 gene has been inactivated, yet in this chs-iRIP strain no increase in the rate of branching was detected (Yarden and Yanofsky, 1991). The predicted COTl polypeptide contains a segment with appreciable sequence identity to the catalytic domain of cAMP-dependent protein kinases. The three-dimensional structures of the catalytic domain of the mouse cAMPdependent protein kinase (Knighton et al., 1991a) and its complex with a substrate analog (Knighton et al., 1991b) have confirmed the importance of this domain which is highly conserved in the various protein kinases and in COT 1. Unlike many cAMP-dependent kinases, COTI contains a relatively long amino-terminal segment as well as an additional sequence of 47 residues within its presumed catalytic domain. Knighton et al. (199la) suggest that these non-conserved regions may determine substrate specificity. This suggestion is supported by the observation that the similar segment of the S.cerevisiae CDC7 polypeptide is necessary for in vivo function but not for protein kinase activity in vitro (Bahman et al., 1988). The observation that ectopic integration of pMP30 DNA confers a colonial phenotype was unexpected. We presume that the additional copy of the cot-i promoter and first 209 codons of the structural gene interferes with the normal function of COTI specified by the intact resident copy of cot-i. This interference could be attributed to competition for factors involved in transcription of cot-i or more likely, the truncated COT1 polypeptide produced in pMP30 transformants interferes with COT1 function. The latter hypothesis is supported by the observation that ectopic transformants obtained with pMP41 (a construct containing the cot-i promoter but lacking the first 209 codons of COT1) display a wild type phenotype. It is possible that the putative truncated COT 1 product of pMP30 DNA contains a

N.crassa cot-l is required for hyphal elongation

regulatory domain that inactivates COTI kinase. However, the predicted amino acid sequence of the N-terminal segment of COT1 does not resemble any known kinase regulatory domain. Another possibility is that COTI activity is dependent on dimerization or complex formation with other proteins, in which case the putative truncated COTI polypeptide might act by titrating binding surfaces essential for COTl activity. Since cot-i disruption halts hyphal elongation predominantly, it is possible that COTI and the COT1 substrate are localized at hyphal tips, perhaps as components of a complex responsible for cell wall biogenesis (Grove and Bracker, 1970). Many mutants of N. crassa have been identified, so called gulliver mutants, which contain suppressors of the cot-i mutation (Perkins et al., 1982). Analysis of these mutants may elucidate the functions and regulation of cot-l. Further studies of colonial mutants should increase our understanding of the factors and events responsible for hyphal elongation and branching. We have learned recently that M.Robb and J.P.Vierula have also cloned the N.crassa cot-i gene and are studying its function (J.P.Vierula, personal communication).

Materials and methods Strains and media Wild type N.crassa strains 74-OR23-1A (FGSC 987), 74-ORS-6a (FGSC 4200) and cot-i (FGSC 4065) were used throughout. Procedures used in growth studies, crosses etc. have been described in Davis and de Serres (1970). Where appropriate, the medium was supplemented with hygromycin B (Calbiochem) at 150 ytg/ml. Induction of conidiation was performed as described by Berlin and Yanofsky (1985). DNA mediated transformation was carried out as described by Vollmer and Yanofsky (1986). Isolation and analysis of nucleic acids from N.crassa Genomic DNA was isolated as follows: two day-old mycelial cultures grown in 25 ml of Vogel's N medium (Vogel, 1956) were collected by filtration on Whatman No. 2 filter paper on a Buchner funnel. Samples were quickly frozen in liquid nitrogen and lyophilized. The dry samples were powdered by grinding and were suspended in an equal volume of lysis buffer (50 mM Tris-HCI pH 8.0, 50 mM EDTA, 2% SDS, 1% ,B-mercaptoethanol) containing 25 tg/ml RNase A. Following a 30 min incubation at 37°C, 100 ttg/ml proteinase K was added to the solution and incubation was continued for 1 h at 65°C. Two phenol:chloroform (1:1) extractions were performed; these were followed by a single chloroform extraction, an ethanol precipitation and a 75% ethanol wash. The DNA pellet was dried and dissolved in TE buffer (10 mM Tris-HCI pH 7.4, 1 mM EDTA pH 8.0). Total RNA was isolated as follows: mycelia were harvested as described above. Following a quick freeze in liquid nitrogen, 25 mg were transferred to a 2 ml screw cap tube containing 480 jsl of extraction buffer (100 mM Tris-HCI pH 7.5, 100 mM LiCl, 10 mM EDTA, 20 mM dithiothreitol), 420 Il phenol, 420 il chloroform, 84 Al 10% SDS, 2 g zirconium beads (Biospec Products Inc.). The samples were shaken twice for 30 s in a mini bead-beater (Biospec Products Inc.). Following a 15 min centrifugation in a microftige, the aqueous phase was transferred to a new tube and re-extracted with phenol:chloroform (1:1). After an additional chloroform extraction the RNA was precipitated, washed, dried and dissolved in 10 mM Na-HEPES pH 7.5 containing 1 mM EDTA. Pools from the Orbach/Sachs cosmid library (Fungal Genetics Stock Center) were used to complement the cot-i mutant. cDNA from the Exley and Garrett library was screened as described by Benton and Davis (1977). Southern analyses were carried out as described by Sambrook et al. (1989) as were all other DNA modification and cloning procedures. Bluescript SK- (Stratagene) and pUCl 19 (Vieira and Messing, 1987) were used for cloning and preparation of various constructs. Northern analyses were performed as described (Sambrook et al., 1989). cot-i RNA probes were generated from pOY311 (Figure 2) using phage T7 RNA polymerase as described by Sambrook et al. (1989) with [c-32P]CTP as the label. pDH25 was the source of the hph gene, encoding hygromycin phosphotransferase, which confers hygromycin resistance (Cullen et al., 1987). This gene was driven by the Aspergillus nidulans trpC promoter region and was used as a dominant selectable marker in the isolation of N.crassa transformants.

Mapping of cot-i was carried out by restriction fragment length polymorphism (RFLP) analysis using DNA from 18 random progeny according to the procedure of Metzenberg et al. (1985). DNA sequencing DNA sequencing was performed by the dideoxy chain termination method of Sanger et al. (1977) and [ci-35S]dATP. Clones used for sequencing were generated by insertion of restriction fragments obtained from pOY18 into the Bluescript vector. Oligonucleotide primers for sequencing portions of the gene were synthesized on an Applied Biosystems DNA synthesizer. Microscopy and computer methods Samples were viewed with a Nikon Microphot FX epifluorescence microscope. Programs of The University of Wisconsin Genetics Group were used for analysis of nucleic acid sequences (Devereux et al., 1984). Multiple sequence alignments for comparison of predicted amino acid sequences corresponding to different cAMP-dependent protein kinase genes were performed using the Tulla program (Subbiah and Harrison, 1989).

Acknowledgements We thank G.Exley and R.Garrett for providing their cDNA library and M.Sachs and M.Orbach for their genomic library. The authors thank Lane Winkelmann for technical assistance in the disruption of cot-i. We thank David Perkins for helpful discussions and Ajith Kamath, Frank Lauter and Carl Yamashiro for critical reading of this manuscript. O.Y. has been supported by EMBO (European Molecular Biology Organization) and BARD (U.S.-Israel Binational Agricultural Research and Development Fund) postdoctoral fellowships for various periods of this research. D.E. was supported by a post-doctoral fellowship from the National Institutes of Health. C.Y. is a Career Investigator of the American Heart Association. These studies were supported by a grant to C.Y. from the United States Public Health Service (GM41296). The nucleotide sequence data reported in this paper have been deposited in the EMBL, Genbank and DDBJ nucleotide sequence databases under the accession number M83093.

References Bahman,M. Buck,V., White,A. and Rosamond,J. (1988) Biochem. Biophys. Acta, 951, 335-343. Benton,W. and Davis,R. (1977) Science, 1%, 180-182. Berlin,V. and Yanofsky,C. (1985) Mol. Cell. Biol., 5, 839-848. Cullen,D., Leong,S.A., Wilson,L.J. and Henner,D.J. (1987) Gene, 57, 21-26. Davis,R.H. and de Serres,F.J. (1970) Methods Enzymol., 17A, 79-143. Devereux,J., Haeberli,P. and Smithies,O. (1984) Nucleic Acid Res., 12, 387-395. Fincham,J.R.S. (1989) Microbiol. Rev., 53, 148-170. Galsworthy,S.B. (1966) Ph.D. thesis, University of Wisconsin, Madison. Diss. Abstr., 26, 6348. Grove,S.N. and Bracker,C.E. (1970) J. Bacteriol., 104, 989-1009. Hager,K.H. and Yanofsky,C. (1990) Gene, 96, 153-159. Hanks,S.K., Quinn,A.M. and Hunter,T. (1988) Science, 241, 42-52. Johnston,L.H., Eberly,S.L., Chapman,J.W., Araki,H. and Sugino,A. (1990) Mol. Cell. Biol., 10, 1358-1366. Knighton,D.R. Zheng,J., Ten Eyck,L.F., Ashford,V.A., Xuong,N., Taylor,S.S. and Sowadski,J.M. (1991a) Science, 253, 407-414. Knighton,D.R., Zheng,J., Ten Eyck,L.F., Xuong,N., Taylor,S.S. and Sowadski,J.M. (1991b) Science, 253, 414-420. Metzenberg,R.L., Stevens,J.N., Selker,E.U. and Morzycka-Wroblewska,E. (1985) Proc. Natl. Acad. Sci. USA, 82, 2067-2071. Mitchell,M.B. and Mitchell,H.K. (1954) Proc. Natl. Acad. Sci. USA, 40, 115-119. Orbach,M.J., Porro,E.B. and Yanofsky,C. (1986) Mol. Cell. Biol., 6, 2452-2461. Paluh,J.L., Orbach,M.J., Legerton,T.L. and Yanofsky,C. (1988) Proc. Natl. Acad. Sci. USA, 85, 3728-3732. Patterson,M., Sclafani,R.A., Fangman,W.L. and Rosamond,J. (1986) Mol. Cell. Biol., 6, 1590-1598. Perkins,D.D., Newmeyer,D., Taylor,C.W. and Bennett,D.C. (1969) Genetica, 40, 247-278. Perkins,D.D., Radford,A., Newmeyer,D. and Bjorkman,M. (1982) Microbiol. Rev., 46, 426-570. Rambosek,J. and Leach,J. (1987) CRC Crit. Rev. Biotechnol., 6, 357-393. Sachs,M. and Yanofsky,C. (1991) Dev. Biol., 148, 117-128.

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Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467. Schmit,J.C. and Brody,S. (1976) Bacteriol. Rev., 40, 1-41. Springer,M.L. (1991) Fungal Genetic Newsl., 38, 92. Subbiah,S. and Harrison,S.C. (1989) J. Mol. Biol., 209, 539-548. Tatum,E.L., Barratt,R.W. and Cutter,V.M.J. (1949) Science, 109, 509-511. Taylor,S.S. (1989) J. Biol. Chem., 264, 8443-8446. Toyn,J.H., Araki,H., Sugino,A. and Johnston,L.H. (1991) Gene, 104, 63-70. Vieira,J. and Messing,J. (1987) Methods Enzymol., 153, 3 -11. Vogel,H.J. (1956) Microb. Genet. Bull., 13, 42-43. Vollmer,S.J. and Yanofsky,C. (1986) Proc. Natl. Acad. Sci. USA, 83, 4869-4873. Yarden,O. and Yanofsky,C. (1991) Genes Dev., 5, 2420-2430. Received on January 9, 1992

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