D.J. Jacobson(1), J. Ohrnberger(2), and R.A. Akins(2) - (1)Department of Botany and Plant Pathology, Michigan State University, East Lansing, MI 48824-1312 (2)Department of Biochemistry, School of Medicine, Wayne State University, Detroit, MI 48201
Recent interest and accelerated research into the genetics of heterokaryon incompatibility (HI) in Neurospora crassa has led to increased use of the original Wilson-Garnjobst HI tester strains available from FGSC (1994 Catalog of Strains, Part VII.D.1.). We have found inconsistencies and abnormalities in both growth of heterokaryons and segregation of markers in crosses using these strains. First noticed was a lack of vigor and incomplete complementation of markers in forced heterokaryons when compared to compatible heterokaryons with known Oak Ridge (OR) background. Secondly, skewed allele ratios were recorded in crosses between the Wilson-Garnjobst strains and strains with OR background. Perkins and Bjorkman raised a cautionary note about these strains (1978 Neurospora Newsl. 25:24-25), however, they concentrated primarily on the scot mutant present in these and other strains originating from the Rockefeller-Lindegren (RL) background. We have attempted to further characterize the erratic behavior of Wilson-Garnjobst strains and determine if the scot mutant or other modifiers of HI are responsible.
To date, all Wilson-Garnjobst strains tested by us have show the scot phenotype at 39°C, confirming results of Perkins and Bjorkman (Figure 1). FGSC 1527 is the only strain listed in Part VII.D.1. that was not expected to contain scot because it is of OR rather than RL background; this proved to be true (data not shown).
Linear growth rate in race tubes was used to quantify and compare heterokaryon formation among the strains tested. Loopfuls of conidia from two strains were co- inoculated at one end of a 25 ml disposable pipet containing 15 ml Vogel s minimal medium N. (A similar method is described by White and Woodward 1995 Fungal Genetics Newl. 42) The experiment was limited to the Wilson-Garnjobst strains that differ at het-c (Table 1); the am1 ad-3B cyh-1R helper strain (FGSC 4564) was included as an OR background standard. Strains of like mating type with complementary markers were co-inoculated in all possible combinations. No attempt was made to control nuclear ratio. Single strains inoculated on race tubes were used as controls. All tubes were incubated at 34°C and each combination of strains was tested three times. The starting point was marked as the leading edge of the colony after overnight growth; measurements were recorded at 24 hr intervals (Figure 2). Although scot is reported to have a slightly abnormal phenotype at 34°C, it is erratic and difficult to see on Vogel s minimal medium N (Perkins and Bjorkman 1978 Neurospora Newsl. 25:24- 25). We saw no evidence of the scot phenotype in these experiments. Heterokaryon formation was confirmed by testing hyphal tips for prototrophic growth. The leading margin of the colony from the race tube was transferred to a fresh plate of minimal medium. These cultures were incubated for 1 day at 34°C and 10 single hyphal tips from the colony were subcultured on to separate 10 x 75 mm tubes of minimal medium. Prototrophic growth in any of the 10 tubes, scored at 2 days, was considered a positive heterokaryon. The results of hyphal tip subculturing is indicated on Figure 2 with either a (+) or (-) associated with the line of each combination tested.
The combination of strains with known OR background, FGSC 1527 and FGSC 4564, consistently gave the fastest growth; we consider this characteristic of a strong positive heterokaryon. All other combinations, whether alike or different at het-c, showed both slower or stalled growth and inconsistency among replications (Figure 2). Only FGSC 1527 + FGSC 1438, het-C + het-C, grew the entire length of the race tube (25 cm). However, this happened only twice in the three replications, with one replication growing less than 1 cm in 9 days. Moreover, FGSC 1438 + FGSC 4564, the other OR strain, never grew more than 5 cm. This was generally true of the other het-Cde strains with FGSC 4564 (data not shown).
Few combinations other than FGSC 1527 + FGSC 4564 yielded heterokaryotic hyphal tips, including those with like het-c alleles. Correlated with this was apparent incomplete complementation of the al-2 mutant seen as either white or mixed white and orange conidia along the length of the race tube. We interpret these two results as either failure to establish a true heterokaryon or maintain a stable heterokaryon during continued growth in the race tube. Of interest is the one het-c + het-C pairing that appeared to form a heterokaryon. This was probably due to escape from incompatibility.
Additional growth rate tests were performed with other Wilson-Garnjobst strains at 30°C. One set was inoculated with equal number of conidia in an attempt to control for nuclear ratios. All these additional tests gave similar results as described above (data not shown).
The growth curves in our Figure 2 resemble the seven types of heterokaryotic growth reported by Holloway (1955 Genetics 40:117-129, see his Figure 1). In fact, Holloway used some strains from the Garnjobst collection in his study. In addition, personal communication from James F. Wilson to David D. Perkins (an unpublished letter) states, "The inos testers do show a single gene difference in the ability to form a heterocaryon which will grow the length of a growth tube, but it is not C, D, or E, does not involve cytoplasmic incompatibility and does not affect heterocaryon test on slants." Crosses were performed to determine if het-like genes (such as Holloway s W, X, Y, and Z) were responsible for the growth curves observed, especially the reduced vigor of OR and Wilson-Garnjobst het-Cde combinations.
Four Wilson-Garnjobst strains were crossed with multiply marked centromere (multicent) linkage testers (Table 1), which are OR background and fully heterokaryon compatible with FGSC 4564. Progeny were scored for all markers and compatibility with the OR parental background (Table 2). Heterokaryon formation was tested by pairing all progeny containing a forcing marker with FGSC 4564 in 10 x 75 mm tubes of minimal medium. The difference between strong positive compatibility and weak or no prototrophic growth was clear at 2 days. This trait segregated in a 1:1 ratio in the three crosses homozygous for het-C. Alleles of at least one other marker in each cross showed deviation from the expected 1:1 ratio, affecting linkage calculations from these data. Skewed allele ratios were also seen when strains derived from the Wilson-Garnjobst strains were crossed with other strains of OR background (compatible with FGSC 4564) and in one cross between Wilson-Garnjobst inl and pan-1 strains (data not shown). No marker was consistently skewed in any of these crosses when repeated. However, het-c and mt (the only scorable markers) were not skewed in crosses between pan-1 strains (data not shown).
Of particular interest is linkage between compatibility with OR background and other markers, as this should indicate approximate location of gene(s) affecting compatibility. The clearest instance of linkage to OR compatibility is inl in cross 1027. However, this is complicated by the skewed allele ratio of acr-2. Both inl and at (linkage group V) show linkage to acr-2 (III) and as such OR compatibility also appears linked to acr-2. In contrast, cross 1028 shows an apparent linkage between OR compatibility and each ylo-1 (VI) and wc-1 (VII). Again, a skewed allele ratio of wc-1 confounds this analysis. Unlike cross 1027, there is no apparent linkage between OR compatibility and linkage group III or V markers.
An unexplainable excess of recombinant progeny is seen between OR compatibility and each pan-1, psi, and wc-1 in crosses 1030 and 1034. If the locus modifying compatibility with OR is independent of het-c, as indicated by the lack of linkage to arg-5, the expectation for the cross heterozygous at het-c would be a 1:3 ratio of OR compatible to OR incompatible. The data reveal an opposite ratio of 3:1 compatible:incompatible. This does not correlate with the observed skewed ratio of mating type. At present, we do not have a good explanation for the distorted segregation patterns seen in these crosses.
The spreading colonial phenotype of scot is probably not responsible for the inconsistent growth or incomplete complementation seen, even though OR compatibility is apparently linked to linkage group V in cross 1027. First, there is no such linkage in the other three crosses. Second, although scot is erratic at 34°C, similar results were obtained from a subsample of pairings repeated at 30°C and 25°C, both permissible temperatures (data not shown). It is unclear if scot may influence growth rate when heterozygous in heterokaryons, for example the Wilson-Garnjobst + OR combinations, or if the gene has a more general pleiotropic effect in modifying heterokaryotic growth. The linkage data leave this possibility open, however, other loci on linkage group V or other linkage groups may also be responsible for modifying compatibility. Since scot expression may affect growth rate directly, it is also possible that modifiers of the scot gene cause growth variability in heterokaryons. In any case, inconsistencies among the pan-1; al-2 strains (crosses 1028, 1030, 1034) and in other repeated crosses does not allow the interpretation of these data as indicating the location of any loci responsible for skewing or incompatibility with OR background.
The skewed ratios and apparent linkage of markers on separate chromosomes may suggest a chromosomal difference between the Wilson-Garnjobst and multicent OR parents. Although a relatively high number of white inviable ascospores were noted in the crosses listed in Table 2, there was no regular patterns of black to white ascospores among shot asci, which would indicate chromosome aberrations (data not shown). Although we could not completely reject chromosomal differences, presence of other inviability factors or segregation distorters seems a more likely explanation.
To further disseminate this information and warn workers of possible complications using the Wilson-Garnjobst strains, we have requested that the following statement be added to Part VII.D.1. of the FGSC Catalog: These strains contain scot and probably other genes in their background that affect both growth of heterokaryons and segregation of markers in crosses. Heterokaryon tests should be perfomed at permissible temperature (25°C) and caution should be exercised when using these strains for genetic studies.
These results also raise the issue of standardizing definitions of compatibility and incompatibility. This is becoming more important as workers proceed with the molecular dissection of HI. There is a need to further identify these factors which may modify the reactions of het genes at the molecular, cellular, and colony level. In an effort to facilitate standardization in future studies of HI in N. crassa we suggest that 1) OR background (het-Cde and OR alleles at het-6 through het-10) be considered the standard against which HI is measured (FGSC 4564 is especially useful in this context, see Perkins 1984 Neurospora Newsl. 31:41-42); 2) genes of interest (het genes, modifiers, suppressors, etc.) be crossed into OR background to measure their effect; and 3) characterizations of compatibility or incompatibility be quantified, when necessary, by growth rates in race tubes.
Acknowledgments: This work was supported in part by MSU All-University Research Initiation Grant to DJJ and NIH Award GM43309 to RAA.
Figure 1. Phenotype of scot at permissive (30°C) and restrictive
(39°C ) temperatures. Plates show 3 days growth on Vogel s minimal medium N
supplemented with pantothenic acid where required. Scot- strain is
FGSC 2659 -- Wilson-Garnjobst het-cDe; pan-1; al-2 a. Scot+ is FGSC
6714 -- Oak Ridge background, T(VL )MB67.
Figure 2. Growth rates of forced heterokaryons in race tubes at 34 C. The het-c alleles of
each pair is indicated at the top of each graph. Each line represents a pair of strains;
each pair was tested three times. Pairings tested for heterokaryotic hyphal tips have a symbol
in parentheses at the end of the line, with (+) indicating prototrophic hyphal tips recovered
and (-) indicating only auxotrophic hyphal tips recovered (see text for details).
Table 1. Strains used for heterokaryon growth rate tests illustrated in Fig. 1 and crosses listed in Table 2.
het Mating
genotype type Markers FGSC Number
het-Cde A inl 1453
het-Cde A pan-1;al-2 2658
het-Cde a inl 1438
het-Cde a pan-1;al-2 2657
het-Cde a arg-12 1527
het-Cde am1 ad-3B cyh-1R 4564
het-Cde a multicent-4
(arg-5;acr-2;psi; 6829
at;ylo-1;wc-1)
het-Cde A multicent-4
(arg-5;acr-2;psi; 6828
at;ylo-1;wc-1)
het-cde A inl 1422
het-cde A pan-1; al-2 2662
het-cde a inl 1436
het-cde a pan-1; al-2 2660
Note: All strains are Wilson-Garnjobst heterokaryon
incompatibility testers except FGSC 1527, 4564, 6828, and
6829. They are, respectively, an arg-12 mutant, a mating-
type mutant with inactive heterokaryon incompatibility
function of the mating type gene, and the two multiply
marked centromere testers; these strains are all Oak Ridge
background.Table 2. Allele ratios of markers and apparent linkage among markers in crosses of Wilson-Garnjobst testers and multiply marked centromere (multicent) testers.
Cross 1027: het-Cde; inl A 1453 X multicent-4 a 6829 Allele Ratios of Markers
mt arg-5 acr-2 psi at ylo-1 wc-1 inl OR(a) A:a +:- +:- +:- +:- +:- +:- +:- +:- 54:50 48:56 74:30* 48:56 51:53 58:46 60:44 51:53 46:45Linkage Among Markers
inl & at inl & acr-2 at & acr-2 inl & OR acr-2 & OR Parentals 65* 75* 65* 80* 62* Recombinants 38 29 39 11 29Cross 1028: het-Cde; pan-1; al-2 A 2658 multicent-4 a 6829 Allele Ratios of Markers
mt arg-5 acr-2 psi at ylo-1 wc-1 pan-1 al-2 OR A:a +:- +:- +:- +:- +:- +:- +:- +:- +:- 26:36 26:36 32:30 38:24 28:34 18:16 27:7* 33:29 34:28 28:30Linkage Among Markers
mt & al-2 psi & pan-1 ylo-1 & OR wc-1 & OR Parentals 40* 51* 22* 22* Recombinants 22 11 10 10Cross 1030: het-Cde; pan-1; al-2 a 2657 multicent-4 A 6828 Allele Ratios of Markers
mt arg-5 acr-2 psi at ylo-1 wc-1 pan-1 al-2 OR A:a +:- +:- +:- +:- +:- +:- +:- +:- +:- 66:55 65:56 52:69 63:58 67:54 44:22* 33:31 64:57 64:57 53:68Linkage Among Markers
mt & al-2 psi & pan-1 pan-1 & OR psi & OR wc-1 & OR Parentals 77* 101* 46* 37* 23* Recombinants 44 20 76(b) 84(b) 42(b)Cross 1034: het-cde; pan-1; al-2 a 2660 multicent-4 A 6828 Allele Ratios of Markers
mt arg-5 acr-2 psi at ylo-1 wc-1 pan-1 al-2 OR A:a +:- +:- +:- +:- +:- +:- +:- +:- +:- 63:38* 54:47 50:51 49:52 57:44 25:27 28:24 55:46 52:49 71:30*Linkage Among Markers
mt&al-2 psi&pan-1 pan-1&OR psi&OR mt&OR al-2&OR arg-5&OR Parentals 60* 82* 39* 36* 61 54 49 Recombinants 41 19 62(b) 55(b) 40 47 52
Notes: Strain numbers are FGSC numbers. Number of progeny tested are listed for each ratio; number of progeny tested for ylo-1 and wc-1 were limited to albino+ progeny where al-2 was present in one parent. An (*) for any ratio indicates deviation from a 1:1 ratio at a 5% significance level. Other unlisted linkage tests betwen marekers are all not significantly different from 1:1. Marker locations are: mt, linkage group I; arg-5, II; acr-2, III; psi, IV; at, V; ylo-1, VI; wc-1, VII; inl, VR; pan-1, IVR; al-2, IR.
(a)OR signifies Oak Ridge het background (multicent parent), with (+) indicating
heterokaryon compatibility and (-) indicating incompatibility with FGSC 4564 (am1
ad-3B cyh-1R). Number of progeny tested were limited to those containing an
appropriate forcing marker.
(b) Ratios with unexplained excess of Recombinant progeny.