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A gene influencing spiral growth ofNeurospora crassa hyphae
EXPERIMENTAL
MYCOLOGY
4, 338-342 (1980)
A Gene Influencing
Spiral
Growth
of Neurospora
crassa Hyphae
Ross E. BEEVER Plant Diseases Division, Department of Scientific and Industrial Research, Private Bag, Auckland, New Zealand Accepted for publication July 17, 1980 BEEVER, R. E. 1980. A gene influencing spiral growth of Neurospora crassa hyphae. Experimental Mycology 4, 338-342. Young hyphae of wild-type Neurospora crassa curve clockwise, as viewed from above, when grown on agar medium. Mutant strains carrying the gene coil-l (linkage group IVR) show this spiral growth in a more pronounced form. Two coil-l alleles, differing slightly in expression, have been isolated. Coiling is not affected by temperature, lateral light, or the orientation of the agar surface. Coiling may result from axial rotation of the growing hyphal tips. INDEX DESCRIPTORS: Neurospora crassa; spiral growth; coil-l gene; morphological mutant; hyphal growth.
The term “spiral growth” was used by Ritchie (1960) to describe the curved growth sometimes shown by vegetative hyphae of fungi growing on agar media. He found that hyphae of species ofAspergilEus, Phoma, and Curvularia showed a tendency to grow to the left, giving the resultant colony an anticlockwise spiral growth pattern when viewed from above. The phenomenon is widespread. In a recent study Madelin et al. (1978) examined 157 different isolates and found that 60, including representatives of all five fungal subdivisions, showed spiral growth. Growth was anticlockwise in 39 instances and clockwise in the remainder. Spiralling occurs only on the surface of solid media and is not influenced by the orientation of that surface. It is not shown by colonies grown in liquid culture (Ritchie, 1960; Madelin et al., 1978). Madelin et al. (1978) found it was best displayed by colonies growing on weak nutrient media; Trinci et al. (1979) found it was manifest most clearly by young colonies. Relatively weak spiral growth has been observed in wild type Neurospora crassa Shear and Dodge (see Fig. 1A in Brody, 1973; Trinci et al., 1979). This paper describes a mutant gene of N. crassa that causes hyphae to exhibit very pronounced spiral growth.
METHODS
N. crassa strains were used: wild types STA4 and Sta (St. Lawrence) from J. R. S. Fincham; leu-2 (37501) and arg-2 (33442) from the Fungal Genetics Stock Center, Humboldt State University Foundation, Arcata, California (FGSC Nos. 117 and 530, respectively); coil-l (JI31) originated from strain 5131, an au-6 strain received from R. B. Flavell; strain P4120 (putative coil-l allele) received from D. D. Perkins. Strains of coil-l (J131) were backcrossed five times to wild types before being used here. Representative coil-l strains have been deposited with the Fungal Genetics Stock Center, Humboldt State University Foundation, Arcata, California: FGSC No. 3648, coil-l (JI31), A; FGSC No. 3649, coil-l (JI31), a; FGSC No. 3650, coil-l? (P4120), A. Media and scoring methods. Cultures for morphological and physiological studies were grown on Vogel’s medium N (Vogel, 1964) supplemented with sucrose (20 g/liter) and agar (15 g/liter). Leucine (0.2 g/liter) and/or arginine (0.2 g/liter) were added for auxotrophs. The coil-l character was scored visually by point inoculating conidial suspensions onto this medium and examining the developing colony at 10-30x magnification after 16-24 h growth. Auxo338
0147-5975/80/040338-05$02.00/O Copyright AU rights
@ 1980 by Academic Press, Inc. of reproduction in my form reserved.
AND MATERIALS
Strains. The following
SPIRAL
GROWTH
MUTANT
OF Neurospora
FIGS. 1,2. Appearance of wild type, STA4 (Fig. l), and coil-l (X31) mutant (Fig. 2) after 16 h growth on agar medium. Petri dishes were inoculated with a loopful from a dense conidial suspension. The margin of the inoculated region is visible in the lower left of each photomicrograph. Bar = 1 mm.
3
340
ROSS E. BEEVER
trophic characters were scored by inoculating onto appropriately supplemented Vogel’s medium N containing agar but with the carbohydrate source changed to sucrose (2 g/liter) and sorbose (9 g/liter) to induce colonial growth; this allowed a T 3.5h ( 5.0 h number of strains to be scored simultaneously on one petri dish. Linear growth was measured by the race tube method (Ryan et al., 1943). Crosses were made on the medium described by Westergaard and Mitchell (1947) with appropriate supplements. Cultures were incubated in the dark at 25°C. Photomicrographs were taken on a Wild M7 stereomicroscope. The time-lapse seFIG. 3. Time-lapse sequence of coil-l (J131) mutant quence was traced from selected photogrowing on agar medium overlaid with cellophane to graphic prints of a colony grown on medium overlaid with sterile “cellophane” (4OOP, prevent hyphae penetrating the medium. British Cellophane Ltd.) to prevent hyphae penetrating the medium. clockwise coils. Coiling is only shown by hyphae that grow on the agar surface and RESULTS not by hyphae that penetrate the agar, nor The property of pronounced spiral by aerial branches. It is best shown by growth (coiling) was first observed in young colonies and is not well shown by the crosses involving the acetate-nonutilizing vigorous leading hyphae that are produced strain J131 (acud), which is defective in as the colonies mature. Linear growth of the structural gene for PEP carboxykinase coil-l (J131) strain FGSC 3648 was indistin(Beever and Fincham, 1973). The property of guishable from that of wild-type STA4 both coiling segregated as a single gene desig- in the length of its lag period and in its final nated here as coil-l, distinct from, and un- linear growth rate. As with spiral growth linked to, acud. Subsequent crosses lo- shown by wild-type N. crassa, and other cated it in linkage group IV between arg-2 fungal species, coiling was not influenced and leu-2 (1.8 and 12.2% recombination, by incubating the plates in an inverted porespectively). (Analysis based on scoring sition or on edge. Nor was it influenced by 164 progeny from the cross coil-l (JI31), exposing the colonies to daylight from one leu-2; a x arg-2; A. Germination was 73% side, and it was still manifest by cultures and three double crossovers were found.) incubated at 35 and 11°C despite differences Figure 1 shows the typical clockwise spiral in growth rate at these temperatures. It thus growth pattern of wild-type strains, where appears that the coiling of coil-l mutants is the radius of hyphal curvature in young basically the same phenomenon as the spicolonies is usually about 1.0 mm. Figure 2 ral growth shown by wild types and that it shows the coiling pattern shown by coil-l differs only in the degree of expression. (J131) strains. The radius of hyphal curvaA second mutant strain of independent ture in this instance is about 0.2 mm and origin, P4120, also showing the property of complete circles are frequently formed. The coiling, was obtained from D. D. Perkins, time-lapse sequence (Fig. 3) shows that the who noticed it segregating in crosses carcoils are produced by hyphae curving to the ried out for other purposes and showed that right as they grow, thus giving rise to it segregated as a single gene. Strains car-
“&.J--2’5hh
SPIRAL
GROWTH
MUTANT
rying P4120 resemble coil-l (JI31), but the coils tend to be larger with a radius usually about 0.5 mm, and in addition the branch hyphae often have a sinuous appearance (Fig. 4). The coiling property and the sinuous appearance did not segregate when strain P4120 was crossed with wild type (STa; germination 90%, 45 progeny scored). When strain P4120 was crossed with coil-l (JI31), progeny could be scored as resembling one or other parent (germination 80%, 40 progeny scored), and no wild-type recombinants were recovered, indicating that P4120 is probably allelic with coil-l. DISCUSSION
Madelin et ek2. (1978) hypothesized that spiral growth of hyphae on agar results from the axial rotation of the extension
OF
Neurospora
341
zone of the hyphal tip. They suggeste in fungi that show spiral growth the sion zone rotates as the hyp ward; because the older suba of the hyphae are anchored to this rotation causes the somewhat plastic tip to be displaced either to the right or depending on the direction of rotatio though Madelin et al. (1978) were un directly detect axial rotation in the species they studied, Trim5 et ak. (1979) vided experimental evidence link rotation and spiral growth. They that elongating Stage I sporangio Phycomyces b~~kesl~e~nu~ conidiophores of Aspergillus g~g~~r~~s Wehmer both form coils when ahowed to grow up against the flat surface of a petri dish lid. It is known that these s~e~i~~i~e structures both show axial rotation durin
FIG. 4. Appearance of strain P4120 (putative coil-l allele) after 23 h growth on agar medium. The petri dish was inoculated with a loopful from a dense conidial suspension. The margin of the inoculated region is visible in the lower right. Bar = 1 mm.
342
ROSS E. BEEVER
unconstrained growth. However, the results were not in complete agreement with the hypothesis of Madelin et al. (1978) because, although the direction of coiling was in the predicted direction for P. blakesleeanus, it was not for A. giganteus. The hypothesis of Madelin et al. (1978) forms a useful basis on which to consider the behavior of coil-1 mutants of N. crassa. The mechanism of axial rotation itself is not well understood but is thought to be related in some way to the presence of spiral structures in the cell wall. As the cell wall expands under the influence of turgor pressure these structures “stretch” and rotate the cell (Preston, 1948; Frei and Preston, 1961; Trinci et al., 1979). The coil-l mutants could differ from wild type either in the orientation of their spiral wall structures, or in the way in which the extension zone and the turgor pressure of the cell interact. Whatever the explanation, the existence of coil-l mutants provides a new tool to explore the problem of spiral growth and axial rotation in more detail. ACKNOWLEDGMENTS This study was started at the Department of Genetits, University of Leeds, England, while I was a recipient of a Sir Walter Mulholland Fellowship provided by the New Zealand Meat Producers Board. Professor D. D. Perkins generously supplied strains
and gave valuable advice. Virginia Ramsay and Joy Leonard provided technical assistance. REFERENCES AND FINCHAM, J.R.S. 1973. BEEVER, R.E., Acetate-nonutilizing mutants of Neurospora crassa: acu-6, the structural gene for PEP carboxykinase and inter-allelic complementation at the acu-6 locus. Mol.
Gen.
Genet.
126: 217-226.
BRODY, S. 1973. Metabolism, cell walls, and morphogenesis. In Develomental Regulation (S. J. Coward, Ed.), pp. 107- 154. Academic Press, New York. FREI, E., AND PRESTON, R. D. 1961. Cell wall organization and wall growth in the filamentous green algae Cladophora and Chaetomorpha. II. Spiral structure and spiral growth. Proc. Roy. Sot. London B 155: 55-77.
MADELIN, M. F., TOOMER, D. K., AND RYAN, J. 1978. Spiral growth of fungus colonies. J. Gen. Microbiol. 106: 73-80. PRESTON, R. D. 1948. Spiral growth and spiral structure. I. Spiral growth in sporangiophores of Phycomyces. Biochim. Biophys. Acta 2: 155-166. RITCHIE, D. 1960. Spiral growth of fungus colonies. Growth 24: 391-400. RYAN, F. J., BEADLE, G. W., AND TATUM, E. L. 1943. The tube method of measuring the growth rate of Neurospora. Amer. J. Bot. 30: 784-799. TRINCI, A. P. J., SAUNDERS, P. T., GOSRANI, R., AND CAMPBELL, K. A. S. 1979. Spiral growth of mycelial and reproductive hyphae. Trans. Brit. Mycol. Sot. 73: 283-293. VOGEL, H. J. 1964. Distribution of lysine pathways among fungi: evolutionary implications. Amer. Natur.
98: 435-446.
WESTERGAARD, M., AND MITCHELL, H. K. 1947. Neurospora. V. A synthetic medium favoring sexual reproduction. Amer. .I. Bot. 34: 573-577.
MYCOLOGY
4, 338-342 (1980)
A Gene Influencing
Spiral
Growth
of Neurospora
crassa Hyphae
Ross E. BEEVER Plant Diseases Division, Department of Scientific and Industrial Research, Private Bag, Auckland, New Zealand Accepted for publication July 17, 1980 BEEVER, R. E. 1980. A gene influencing spiral growth of Neurospora crassa hyphae. Experimental Mycology 4, 338-342. Young hyphae of wild-type Neurospora crassa curve clockwise, as viewed from above, when grown on agar medium. Mutant strains carrying the gene coil-l (linkage group IVR) show this spiral growth in a more pronounced form. Two coil-l alleles, differing slightly in expression, have been isolated. Coiling is not affected by temperature, lateral light, or the orientation of the agar surface. Coiling may result from axial rotation of the growing hyphal tips. INDEX DESCRIPTORS: Neurospora crassa; spiral growth; coil-l gene; morphological mutant; hyphal growth.
The term “spiral growth” was used by Ritchie (1960) to describe the curved growth sometimes shown by vegetative hyphae of fungi growing on agar media. He found that hyphae of species ofAspergilEus, Phoma, and Curvularia showed a tendency to grow to the left, giving the resultant colony an anticlockwise spiral growth pattern when viewed from above. The phenomenon is widespread. In a recent study Madelin et al. (1978) examined 157 different isolates and found that 60, including representatives of all five fungal subdivisions, showed spiral growth. Growth was anticlockwise in 39 instances and clockwise in the remainder. Spiralling occurs only on the surface of solid media and is not influenced by the orientation of that surface. It is not shown by colonies grown in liquid culture (Ritchie, 1960; Madelin et al., 1978). Madelin et al. (1978) found it was best displayed by colonies growing on weak nutrient media; Trinci et al. (1979) found it was manifest most clearly by young colonies. Relatively weak spiral growth has been observed in wild type Neurospora crassa Shear and Dodge (see Fig. 1A in Brody, 1973; Trinci et al., 1979). This paper describes a mutant gene of N. crassa that causes hyphae to exhibit very pronounced spiral growth.
METHODS
N. crassa strains were used: wild types STA4 and Sta (St. Lawrence) from J. R. S. Fincham; leu-2 (37501) and arg-2 (33442) from the Fungal Genetics Stock Center, Humboldt State University Foundation, Arcata, California (FGSC Nos. 117 and 530, respectively); coil-l (JI31) originated from strain 5131, an au-6 strain received from R. B. Flavell; strain P4120 (putative coil-l allele) received from D. D. Perkins. Strains of coil-l (J131) were backcrossed five times to wild types before being used here. Representative coil-l strains have been deposited with the Fungal Genetics Stock Center, Humboldt State University Foundation, Arcata, California: FGSC No. 3648, coil-l (JI31), A; FGSC No. 3649, coil-l (JI31), a; FGSC No. 3650, coil-l? (P4120), A. Media and scoring methods. Cultures for morphological and physiological studies were grown on Vogel’s medium N (Vogel, 1964) supplemented with sucrose (20 g/liter) and agar (15 g/liter). Leucine (0.2 g/liter) and/or arginine (0.2 g/liter) were added for auxotrophs. The coil-l character was scored visually by point inoculating conidial suspensions onto this medium and examining the developing colony at 10-30x magnification after 16-24 h growth. Auxo338
0147-5975/80/040338-05$02.00/O Copyright AU rights
@ 1980 by Academic Press, Inc. of reproduction in my form reserved.
AND MATERIALS
Strains. The following
SPIRAL
GROWTH
MUTANT
OF Neurospora
FIGS. 1,2. Appearance of wild type, STA4 (Fig. l), and coil-l (X31) mutant (Fig. 2) after 16 h growth on agar medium. Petri dishes were inoculated with a loopful from a dense conidial suspension. The margin of the inoculated region is visible in the lower left of each photomicrograph. Bar = 1 mm.
3
340
ROSS E. BEEVER
trophic characters were scored by inoculating onto appropriately supplemented Vogel’s medium N containing agar but with the carbohydrate source changed to sucrose (2 g/liter) and sorbose (9 g/liter) to induce colonial growth; this allowed a T 3.5h ( 5.0 h number of strains to be scored simultaneously on one petri dish. Linear growth was measured by the race tube method (Ryan et al., 1943). Crosses were made on the medium described by Westergaard and Mitchell (1947) with appropriate supplements. Cultures were incubated in the dark at 25°C. Photomicrographs were taken on a Wild M7 stereomicroscope. The time-lapse seFIG. 3. Time-lapse sequence of coil-l (J131) mutant quence was traced from selected photogrowing on agar medium overlaid with cellophane to graphic prints of a colony grown on medium overlaid with sterile “cellophane” (4OOP, prevent hyphae penetrating the medium. British Cellophane Ltd.) to prevent hyphae penetrating the medium. clockwise coils. Coiling is only shown by hyphae that grow on the agar surface and RESULTS not by hyphae that penetrate the agar, nor The property of pronounced spiral by aerial branches. It is best shown by growth (coiling) was first observed in young colonies and is not well shown by the crosses involving the acetate-nonutilizing vigorous leading hyphae that are produced strain J131 (acud), which is defective in as the colonies mature. Linear growth of the structural gene for PEP carboxykinase coil-l (J131) strain FGSC 3648 was indistin(Beever and Fincham, 1973). The property of guishable from that of wild-type STA4 both coiling segregated as a single gene desig- in the length of its lag period and in its final nated here as coil-l, distinct from, and un- linear growth rate. As with spiral growth linked to, acud. Subsequent crosses lo- shown by wild-type N. crassa, and other cated it in linkage group IV between arg-2 fungal species, coiling was not influenced and leu-2 (1.8 and 12.2% recombination, by incubating the plates in an inverted porespectively). (Analysis based on scoring sition or on edge. Nor was it influenced by 164 progeny from the cross coil-l (JI31), exposing the colonies to daylight from one leu-2; a x arg-2; A. Germination was 73% side, and it was still manifest by cultures and three double crossovers were found.) incubated at 35 and 11°C despite differences Figure 1 shows the typical clockwise spiral in growth rate at these temperatures. It thus growth pattern of wild-type strains, where appears that the coiling of coil-l mutants is the radius of hyphal curvature in young basically the same phenomenon as the spicolonies is usually about 1.0 mm. Figure 2 ral growth shown by wild types and that it shows the coiling pattern shown by coil-l differs only in the degree of expression. (J131) strains. The radius of hyphal curvaA second mutant strain of independent ture in this instance is about 0.2 mm and origin, P4120, also showing the property of complete circles are frequently formed. The coiling, was obtained from D. D. Perkins, time-lapse sequence (Fig. 3) shows that the who noticed it segregating in crosses carcoils are produced by hyphae curving to the ried out for other purposes and showed that right as they grow, thus giving rise to it segregated as a single gene. Strains car-
“&.J--2’5hh
SPIRAL
GROWTH
MUTANT
rying P4120 resemble coil-l (JI31), but the coils tend to be larger with a radius usually about 0.5 mm, and in addition the branch hyphae often have a sinuous appearance (Fig. 4). The coiling property and the sinuous appearance did not segregate when strain P4120 was crossed with wild type (STa; germination 90%, 45 progeny scored). When strain P4120 was crossed with coil-l (JI31), progeny could be scored as resembling one or other parent (germination 80%, 40 progeny scored), and no wild-type recombinants were recovered, indicating that P4120 is probably allelic with coil-l. DISCUSSION
Madelin et ek2. (1978) hypothesized that spiral growth of hyphae on agar results from the axial rotation of the extension
OF
Neurospora
341
zone of the hyphal tip. They suggeste in fungi that show spiral growth the sion zone rotates as the hyp ward; because the older suba of the hyphae are anchored to this rotation causes the somewhat plastic tip to be displaced either to the right or depending on the direction of rotatio though Madelin et al. (1978) were un directly detect axial rotation in the species they studied, Trim5 et ak. (1979) vided experimental evidence link rotation and spiral growth. They that elongating Stage I sporangio Phycomyces b~~kesl~e~nu~ conidiophores of Aspergillus g~g~~r~~s Wehmer both form coils when ahowed to grow up against the flat surface of a petri dish lid. It is known that these s~e~i~~i~e structures both show axial rotation durin
FIG. 4. Appearance of strain P4120 (putative coil-l allele) after 23 h growth on agar medium. The petri dish was inoculated with a loopful from a dense conidial suspension. The margin of the inoculated region is visible in the lower right. Bar = 1 mm.
342
ROSS E. BEEVER
unconstrained growth. However, the results were not in complete agreement with the hypothesis of Madelin et al. (1978) because, although the direction of coiling was in the predicted direction for P. blakesleeanus, it was not for A. giganteus. The hypothesis of Madelin et al. (1978) forms a useful basis on which to consider the behavior of coil-1 mutants of N. crassa. The mechanism of axial rotation itself is not well understood but is thought to be related in some way to the presence of spiral structures in the cell wall. As the cell wall expands under the influence of turgor pressure these structures “stretch” and rotate the cell (Preston, 1948; Frei and Preston, 1961; Trinci et al., 1979). The coil-l mutants could differ from wild type either in the orientation of their spiral wall structures, or in the way in which the extension zone and the turgor pressure of the cell interact. Whatever the explanation, the existence of coil-l mutants provides a new tool to explore the problem of spiral growth and axial rotation in more detail. ACKNOWLEDGMENTS This study was started at the Department of Genetits, University of Leeds, England, while I was a recipient of a Sir Walter Mulholland Fellowship provided by the New Zealand Meat Producers Board. Professor D. D. Perkins generously supplied strains
and gave valuable advice. Virginia Ramsay and Joy Leonard provided technical assistance. REFERENCES AND FINCHAM, J.R.S. 1973. BEEVER, R.E., Acetate-nonutilizing mutants of Neurospora crassa: acu-6, the structural gene for PEP carboxykinase and inter-allelic complementation at the acu-6 locus. Mol.
Gen.
Genet.
126: 217-226.
BRODY, S. 1973. Metabolism, cell walls, and morphogenesis. In Develomental Regulation (S. J. Coward, Ed.), pp. 107- 154. Academic Press, New York. FREI, E., AND PRESTON, R. D. 1961. Cell wall organization and wall growth in the filamentous green algae Cladophora and Chaetomorpha. II. Spiral structure and spiral growth. Proc. Roy. Sot. London B 155: 55-77.
MADELIN, M. F., TOOMER, D. K., AND RYAN, J. 1978. Spiral growth of fungus colonies. J. Gen. Microbiol. 106: 73-80. PRESTON, R. D. 1948. Spiral growth and spiral structure. I. Spiral growth in sporangiophores of Phycomyces. Biochim. Biophys. Acta 2: 155-166. RITCHIE, D. 1960. Spiral growth of fungus colonies. Growth 24: 391-400. RYAN, F. J., BEADLE, G. W., AND TATUM, E. L. 1943. The tube method of measuring the growth rate of Neurospora. Amer. J. Bot. 30: 784-799. TRINCI, A. P. J., SAUNDERS, P. T., GOSRANI, R., AND CAMPBELL, K. A. S. 1979. Spiral growth of mycelial and reproductive hyphae. Trans. Brit. Mycol. Sot. 73: 283-293. VOGEL, H. J. 1964. Distribution of lysine pathways among fungi: evolutionary implications. Amer. Natur.
98: 435-446.
WESTERGAARD, M., AND MITCHELL, H. K. 1947. Neurospora. V. A synthetic medium favoring sexual reproduction. Amer. .I. Bot. 34: 573-577.