Genetic Analysis of Wild-Isolated Neurospora crassa Strains Identified as Dominant Suppressors of Repeat-Induced Point Mutation

Corresponding author: Durgadas P. Kasbekar, Uppal Rd., Hyderabad 500 007, India., kas{at}ccmb.res.in (E-mail)

Communicating editor: J. J. LOROS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

Repeat-induced point mutation (RIP) in Neurospora results in inactivation of duplicated DNA sequences. RIP is thought to provide protection against foreign elements such as retrotransposons, only one of which has been found in N. crassa. To examine the role of RIP in nature, we have examined seven N. crassa strains, identified among 446 wild isolates scored for dominant suppression of RIP. The test system involved a small duplication that targets RIP to the easily scorable gene erg-3. We previously showed that RIP in a small duplication is suppressed if another, larger duplication is present in the cross, as expected if the large duplication competes for the RIP machinery. In two of the strains, RIP suppression was associated with a barren phenotype—a characteristic of Neurospora duplications that is thought to result in part from a gene-silencing process called meiotic silencing by unpaired DNA (MSUD). A suppressor of MSUD (Sad-1) was shown not to prevent known large duplications from impairing RIP. Single-gene duplications also can be barren but are too short to suppress RIP. RIP suppression in strains that were not barren showed inheritance that was either simple Mendelian or complex. Adding copies of the LINE-like retrotransposon Tad did not affect RIP efficiency.

A mutational process called repeat-induced point mutation (RIP) occurs during the premeiotic stage of the Neurospora sexual cycle and causes the hypermutation of any DNA sequences that are duplicated in an otherwise haploid genome (for reviews see SELKER 1990 ; IRELAN and SELKER 1996 ). It had been suggested that RIP might serve to protect the genome against the proliferation of transposable elements. This hypothesis gained experimental support with the demonstration that the only wild-isolated Neurospora strain in which an active transposable element was found was also a dominant partial suppressor of RIP in the erg-3 test system (NOUBISSI et al. 2000 ). This was the Neurospora crassa Adiopodoumé [Fungal Genetics Stock Center (FGSC) 430] strain that contains the LINE-like transposable element Tad (KINSEY and HELBER 1989 ). All other Neurospora strains examined contained only RIP-inactivated relics of Tad (KINSEY 1990 ; KINSEY et al. 1994 ). Subsequently, we reported the identification of two more RIP suppressor strains: Adiopodoumé-7 (a Tad-free strain from the same Ivory Coast location as Adiopodoumé 430) and Sugartown (from Louisiana; NOUBISSI et al. 2001 ). In double-blind experiments all three suppressors could be distinguished from other wild strains solely on the basis of their dominant RIP suppressor phenotype. In this article the term "RIP suppressor" is used as an abbreviation for any factor that suppresses the RIP of erg-3 in our test system (described below).

In addition to suppressing RIP, crosses with Sugartown as one parent also displayed a barren phenotype; that is, they produced perithecia that yielded only very few ascospores (NOUBISSI et al. 2001 ). Barrenness is a characteristic of strains that contain large duplications (RAJU and PERKINS 1978 ; PERKINS 1997 and references therein). The duplications trigger a gene-silencing process called meiotic silencing by unpaired DNA (MSUD) whereby any DNA sequence that is unpaired in meiosis is silenced itself and causes the silencing of all sequences that are homologous to it, including genes that may themselves be paired (ARAMAYO and METZENBERG 1996 ; SHIU et al. 2001 ; SHIU and METZENBERG 2002 ). Since large duplications can contain many genes, including some that may be required for meiosis or ascospore development, and one copy necessarily remains unpaired in meiosis of a heterozygous cross, gene silencing by MSUD renders the cross barren. The semidominant mutation Sad-1 imposes a defect for MSUD and thereby suppresses the barren phenotype of duplication strains (SHIU et al. 2001 ; SHIU and METZENBERG 2002 ).

The present study follows the identification by FEHMER et al. 2001 of wild-isolated strains that suppress the barren phenotype of large duplications. It also makes use of the demonstration that presence of a large duplication suppresses RIP in a smaller duplication (BHAT and KASBEKAR 2001 ; FEHMER et al. 2001 ). We have proposed that large duplications do so by titrating out limiting amounts of the RIP machinery.

The ergosterol-3 (erg-3) gene is located in distal linkage group (LG) III and it encodes the ergosterol biosynthetic enzyme C-14 reductase (ELLIS et al. 1991 ; PAPAVINASASUNDARAM and KASBEKAR 1994 ; PRAKASH and KASBEKAR 2002 ). We have constructed strains in which a tagged duplicate copy of a 1.3-kb fragment of erg-3, designated Dp 1.3^ec hph, was introduced ectopically to target RIP to erg-3 (PRAKASH et al. 1999 ). RIP-induced erg-3 mutants are viable and have altered sensitivities to isoflavonoids and to the steroidal glycoside -tomatine (SENGUPTA et al. 1995 ). Whereas the wild type is resistant to isoflavonoids and sensitive to tomatine, erg-3 mutants are resistant to tomatine and sensitive to isoflavonoids. Most important for our objective, the colonies generated from erg-3 mutant ascospores exhibit a characteristic growth morphology on Vogel's sorbose agar medium that allows them to be unambiguously identified by inspection under a dissection microscope (for a picture see NOUBISSI et al. 2000 ).

In our screens we had crossed the wild strains with strains bearing the Dp 1.3^ec hph transgene. Although both parents in these crosses were erg-3⁺, the presence of the duplicated fragment resulted in the generation of erg-3 mutant progeny. By determining the frequency of erg-3 mutants produced in these crosses, we were able to identify the wild isolates that conferred a dominant RIP defect. We have now extended our screens to another 242 wild-isolated strains and identified four more dominant RIP suppressors: Golur-1 (P0334), Fred (P0833), Coon (P0881), and Bayan Lepas (P2663; see Appendix). Crosses of Dp 1.3^ec hph strains with the standard Oak Ridge (OR) strains yield erg-3 mutant progeny at frequencies typically in the range 8–13%, whereas in crosses with the RIP suppressor strains this frequency is generally <<0.5%.

The primary objective of this work was to analyze the genetic basis of dominant RIP suppression in the seven strains. For this, each suppressor strain was crossed with the standard OR strains and the f₁ progeny from these crosses were scored for inheritance of the suppressor phenotype. Our studies reveal that natural populations of N. crassa harbor two classes of dominant RIP suppressor strains. One class, represented by the Sugartown strain, was barren in crosses, and this is attributed to the presence of a large duplication. The second class was not barren.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

Strains:
All strains, unless otherwise indicated, were from the FGSC, University of Kansas Medical Center, Kansas City. These included the 242 wild-isolated mat a strains, the standard laboratory wild-type strains 74-OR23-1 A (FGSC 987) and OR8-1 a (FGSC 988), and the wild-isolated mat A strains Adiopodoumé (FGSC 430), Adiopodoumé-7 (P 4305), and Sugartown (P 0854). The T-430-a (Hyg^r; FGSC 8609) strain is derived from the Adiopodoumé strain by switching the mating type to mat a by transformation (ANDERSON et al. 2001 ).

The helper-1 strain a^m1 ad-3B cyh-1 (FGSC 4564; see PERKINS et al. 2001 ) was used to construct heterokaryons with OR-compatible mutant strains of either mating type. The helper-1 component of such heterokaryons is a passive partner in a cross because the mat a allele a^m1 is inactive.

The Dp 1.3^ec hph a and Dp 1.3^ec hph A strains were from our laboratory collection and have been described by PRAKASH et al. 1999 . Dp 1.3^ec hph is essentially a tagged duplication of a 1.3-kb HindIII fragment from erg-3 that is inserted as a single copy locus into an ectopic site unlinked to erg-3. The duplicated segment is marked by the bacterial hph gene for resistance to the antibiotic hygromycin B. It does not encode a functional sterol C-14 reductase but serves to target RIP to the erg-3 gene. Double duplication strains were constructed that contain the Dp 1.3^ec hph transgene together with another larger duplication (BHAT and KASBEKAR 2001 ; FEHMER et al. 2001 ). The large duplications are Dp(AR17) (in A40), Dp(OY329) (in C25-3 and C25-8), and Dp(IBj5) (in E30 and E42). The large duplications were obtained as segregants from crosses between translocation and normal sequence strains containing the Dp 1.3^ec hph transgene. The double duplication strains were barren in crosses with the wild type (BHAT and KASBEKAR 2001 ; FEHMER et al. 2001 ). The strain designated 39 was obtained in this work as a segregant from an Adiopodoumé x Dp 1.3^ec hph a. It has an abnormal growth morphology and contains active Tad elements.

The MSUD suppressor strains 96-01 (Sad-1 A) and 96-02 (Sad-1 a) were kindly provided by Robert L. Metzenberg (Stanford University). We generated Sad-1; Dp 1.3^ec hph A and Sad-1; Dp 1.3^ec hph a strains as segregants from the crosses Dp 1.3^ec hph a x Sad-1 A and Dp 1.3^ec hph A x Sad-1 a. The presence of Sad-1 in these strains was confirmed by verifying their ability to suppress the barren phenotype in crosses with Dp(IBj5) strains, and the presence of Dp 1.3^ec hph was confirmed by verifying that RIP-induced erg-3 mutant progeny were generated in crosses with 74-OR23-1 A or OR8-1 a. Sixty segregants were examined from each of the crosses Dp 1.3^ec hph a x Sad-1 A and Dp 1.3^ec hph A x Sad-1 a, and segregants 19 and 21, respectively, had a hygromycin-sensitive phenotype. Since both Dp 1.3^ec hph and Sad-1 are marked by the hph gene, these results suggested that Dp 1.3^ec hph and Sad-1 were unlinked. Since Sad-1 is linked to mat (SHIU and METZENBERG 2002 ), these results cast doubt on earlier results suggesting linkage between Dp 1.3^ec hph and mat (PRAKASH et al. 1999 ). More extensive linkage experiments have now shown that the Dp 1.3^ec hph transgene is linked to al-3 on LG VR (3/80 crossovers) and that it segregates independently of markers for all other linkage groups (data not shown).

Metzenberg also provided the strains FGSC 8752, FGSC 8754, FGSC 8756, FGSC 8758, and FGSC 8760. These are pan-2 a strains that contain, respectively, functional copies of the "meiosis-essential" genes act⁺ (actin), hH3hH4-1⁺ (histones H3 and H4-1), Bml^R (ß-tubulin), pma-1⁺ (plasma membrane ATPase), and mei-3⁺ (the Neurospora RecA/Rad51 ortholog meiotic-3) inserted into the his-3 locus. In heterozygous crosses, the induction of MSUD silences these genes and thereby causes the cross to become barren (SHIU et al. 2001 ). The strain FGSC 8750 that contained an insertion of the his-3 vector alone was used as the control. These strains were used to test whether gene-sized duplications that are capable of inducing barrenness can function as dominant RIP suppressors.

For linkage analysis of the barren phenotype of the Sugartown strain we used the single mutant strains ad-8 a (FGSC 453), al-1 a (FGSC 2085), al-2 a (FGSC 3448), al-3 a (FGSC 4073), arg-5 a (FGSC 4035), col-18 a (FGSC 8284), cot-1 a (FGSC 4066), cum a (FGSC 3878), cys-10 a (FGSC 4054), dow a (FGSC 4052), lys-1 a (FGSC 4070), met-9 a (FGSC 3280), pi a (FGSC 4027), pyr-1 a (FGSC 8250), slo-2 a (FGSC 1533), spco-4 a (FGSC 1372), thi-3 a (FGSC 4084), and ylo-1 a (FGSC 4100). The mutations are described by PERKINS et al. 2001 .

The strains In(H4250) aur R A (FGSC 3156), R A (FGSC 4022), and R a (FGSC 4023) contain the dominant Round spore mutation that confers a female-sterile phenotype. These strains were used in experiments that revealed that a high proportion of the progeny of Adiopodoumé and T-430-a (Hyg^r) are female sterile.

The rid A (N1977) and rid a (N2148) strains were kindly provided by Eric U. Selker and Michael Freitag (University of Oregon) and are described by FREITAG et al. 2002 . The rid A strain was found to have a hygromycin-sensitive phenotype whereas the rid a strain was hygromycin resistant. We obtained rid; Dp 1.3^ec hph A and rid; Dp 1.3^ec hph a segregants (designated 10 and 2, respectively) from the crosses Dp 1.3^ec hph a x rid A and Dp 1.3^ec hph A x rid a. The genotypes of these segregants was confirmed by verifying that RIP-induced erg-3 mutant progeny failed to be produced (frequency <0.5%) in crosses with rid strains of the opposite mating type but were produced (frequency >>0.5%) in crosses with 74-OR23-1 A or OR8-1 a. These strains were used in experiments to test linkage between rid and the Adiopodoumé RIP suppressor.

Growth and cross conditions:
Crossing and maintenance of the Neurospora strains was essentially as described by DAVIS and DE SERRES 1970 . Antibiotic resistance was scored by streaking conidia onto 1.5% agar plates containing Vogel's N medium plus "sorbose" (0.05% fructose, 0.05% glucose, and 2% sorbose) and supplemented with the antibiotic. The antibiotics tested were -tomatine (Sigma, St. Louis) at 90 µg/ml made from a 25-mg/ml stock solution in dimethylformamide and hygromycin B (Sigma) 200 µg/ml made from a 100-mg/ml aqueous stock solution. After an overnight incubation at 30° on tomatine-supplemented medium, growth can be observed of only the erg-3 mutant strains (SENGUPTA et al. 1995 ). Only strains expressing the hph gene could grow on hygromycin medium.

Crosses were performed by confrontation between mycelia inoculated as plugs on synthetic crossing medium in petri dishes. Generally ascospores began to be shot within 14–16 days. Ascospores were harvested by washing the lids with 1 ml water. Unless indicated otherwise, a first harvest was made 31 days after the crosses were set up. Then the petri dish lids were replaced, and a second harvest was made after an additional 14 days.

Determination of RIP efficiencies:
RIP was assayed in crosses with strains bearing the Dp 1.3^ec hph transgene. Progeny ascospores were germinated on Vogel's N-sorbose agar plates and the fraction of colonies with the erg-3 mutant morphology was counted under a dissection microscope. Reliability of identifying the erg-3 mutant phenotype in this way was established by confirming the ability of the conidia to germinate and grow on tomatine medium (SENGUPTA et al. 1995 ). This assay enabled us to score even very rare RIP events (e.g., <0.04%) in large numbers of crosses. In this article we use the frequency of erg-3 mutant progeny as a measure of RIP efficiency. In general, this frequency is one-half of the RIP frequency, the proportion of asci that have undergone RIP, since in crosses heterozygous for Dp 1.3^ec hph only one-half of the progeny ascospores inherit an erg-3 gene that was exposed to RIP.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

Barren RIP suppressors:
Seven RIP suppressor strains have now been identified, including Golur-1 (P0334), Fred (P0833), Coon (P0881), and Bayan Lepas (P2663; see Appendix), and Adiopodoumé (FGSC 430), Adiopodoumé-7 (P4305), and Sugartown (P0854) (from previous surveys). Of these suppressor strains, Sugartown and Golur-1 were barren in crosses, suggesting that suppression might be caused by a segmental duplication. In addition, the screens also identified three other barren strains: Georgetown-6 (P2622), Batu Ferringi-1 (P2681), and Brabadougou (P4296).

The Sugartown strain: A cross was performed between Sugartown and OR8-1 a, and 81 mat A f₁ progeny were crossed with Dp 1.3^ec hph a. Of these crosses, 40 were barren, 37 were fertile, and 4 had an intermediate productivity. This showed that the barren phenotype segregated 1:1 and was unlinked to mat. Among the barren crosses, 13 produced <100 ascospores so it was not possible to obtain a meaningful erg-3 mutation frequency for them (however, none of the f₂ progeny examined from these 13 crosses was mutant in erg-3). In the 27 barren crosses that could be tested, the mutation frequencies were generally <0.5%, whereas in all the fertile crosses they were in the 2–24% range. Of the four crosses with intermediate productivity, one gave a mutation frequency <0.5% and the other three were in the 0.5–1% range. These results showed a tight linkage between RIP suppression and the barren phenotype. Since barrenness is a characteristic of strains bearing large duplications and large duplications suppress RIP in a smaller duplication, the linkage between the two phenotypes suggested that both were caused by the presence of one or more large duplications in the Sugartown strain.

The barren phenotype was tested for linkage with several markers (see MATERIALS AND METHODS). It was unlinked to the following markers (numbers in parentheses indicate nonparental/total progeny): mat on IL (50/101), pi on IIL (43/85), arg-5 on IIR (18/40), cum on IIIL (39/71), dow on IIIR (21/40), cys-10 on IVL (43/82), cot-1 on IVR (47/86), pyr-1 on IVR (19/34), lys-1 on VL (41/85), al-3 on VR (45/84), ade-8 on VIL (42/87), ylo-1 on VIL (15/41), and col-18 on VIR (34/68). The table provided by PERKINS 1994 was used to decide whether these observed ratios indicated deviations from 1:1 segregation of parental:nonparental. Significant deviation from the 1:1 ratio was expected if there was no linkage in tests with the markers al-1 on IR (23/68), al-2 on IR (20/67), spco-4 on VIIL (14/49), and met-9 on VIIR (24/63). The tests for linkage with al-1 and al-2 were repeated and the results (11/38 and 12/38, respectively) once again showed significant deviation from the 1:1 ratio. Linkage of the barren factor was seen also with an additional LG VII marker, slo-2 (11/38).

The hypothesis that the barrenness and the RIP suppression phenotypes of the Sugartown strain might be due to one or more large duplications received indirect support with the recovery of a morphological mutant among the progeny from a cross of the Sugartown strain with the auxotrophic strain thi-3 a. The mutant was prototrophic and it was barren in crosses with OR8-1 a. The mutation segregated in 10/36 progeny of this barren cross. RIP in the presumptive Sugartown duplication might have been responsible for generating this mutation. Alternatively, crosses with some duplications can also increase mutations in loci that are not covered by the duplication or even in unlinked loci, although it is not known whether such mutations have the molecular hallmarks of RIP (PERKINS et al. 1997 ).

The Golur-1 strain: Although the barren phenotype of Golur-1 is stable, it appeared to become unstable in the f₁ progeny from Golur-1 x 74-OR23-1 A and Golur-1 x Dp 1.3^ec hph A. Of the exceptional f₁ progeny produced, 60 were tested from each cross. The crosses with these f₁ progeny appeared to be potentially barren or fertile until 24 days, but by the 31st day all the crosses had comparable fertility and showed no evidence of suppression of RIP (data not shown). These results suggested that the presumptive duplication in Golur-1 is subject to a genotype-dependent instability. This instability foiled our attempts to test for linkage between the RIP suppressor and barren phenotypes of the Golur-1 strain.

Failure of the Sad-1 suppressor of MSUD-induced barrenness to impair suppression of RIP by duplications in the erg-3 test system:
The semidominant Sad-1 mutation imposes a defect for MSUD (SHIU et al. 2001 ; SHIU and METZENBERG 2002 ). The barren phenotype of the Sugartown, Golur-1, and Georgetown-6 strains, but not that of the Batu Ferringi-1 and Brabadougou strains, was suppressed in crosses with Sad-1 strains (data not shown). These findings agreed with our model that the barren phenotype of the Sugartown and Golur-1 strains stems from MSUD-induced silencing of duplication-borne genes (see above).

We investigated whether large duplications retain their RIP suppression ability when their barren phenotype is suppressed by Sad-1. For this we used strains carrying Dp 1.3^ec hph together with one of the large duplications Dp(AR17), Dp(OY329), or Dp(IBj5). The double duplication strains were crossed with Sad-1 strains of the appropriate mating type. Control crosses were done between Dp 1.3^ec hph and Sad-1 strains. As expected, the barren phenotype of all three large duplications was suppressed by Sad-1 but as can be seen in the results summarized in Table 1A, the erg-3 mutation frequencies continued to be very low. These results indicated that the defect for MSUD did not affect the ability of large duplications to suppress RIP in a smaller duplication.

Of 35 progeny examined from the A40 x Sad-1 cross one had the downy (dow) phenotype. The Dp(AR17) duplication covers the dow locus; therefore, the dow mutant must have been generated by RIP. The dow mutation frequency (1/35 = 2.8%) was in agreement with results from previous crosses with Dp(AR17) (PERKINS et al. 1997 ; BHAT and KASBEKAR 2001 ). These results demonstrated that the Sad-1 mutation does not impair RIP in either Dp 1.3^ec hph or Dp(AR17).

Failure of barren gene-sized duplications to modify RIP in the erg-3 test system:
Even relatively small duplications can render a cross barren, for example, Dp (IVR>I)B362i, which covers only one known gene, met-1 (see PERKINS 1997 ). Sad-1-suppressible barrenness has also been demonstrated in crosses heterozygous for ectopic insertions of genes that code for functions required during meiosis (SHIU et al. 2001 ). These include ß-tubulin (Bml^R), actin (act⁺), histones H3 and H4-1 (hH3hH4-1⁺), plasma membrane ATPase (pma-1⁺), and the Neurospora RecA/Rad51 ortholog meiotic-3 (mei-3⁺). The titration model, however, predicts that such gene-sized duplications might not be large enough to titrate out the RIP machinery and therefore would be incapable of functioning as dominant RIP suppressors. To test this we performed crosses between the strains bearing duplications for the "meiosis-essential" genes and Sad-1 Dp 1.3^ec hph A and scored the frequency of erg-3 mutant progeny. The results summarized in Table 1B show that the gene-sized duplications did not suppress RIP in erg-3. This is consistent with the idea that gene-sized duplications are too small to act as dominant RIP suppressors.

A barren strain that is not a RIP suppressor:
Although the Georgetown-6 (P2622) strain showed a Sad-1-suppressible barrenness, it did not display a RIP suppressor phenotype (see Appendix). The frequency of erg-3 mutant progeny from the cross of Georgetown-6 with Sad-1 Dp 1.3^ec hph A was 1.8% (n = 709). It is conceivable that this strain contains a gene-sized duplication of a "meiosis-essential" gene that is capable of inducing barrenness but is not large enough to titrate the RIP machinery.

Nonbarren RIP suppressor strains:
The Adiopodoumé, Adiopodoumé-7, Bayan Lepas, Coon, and Fred RIP suppressor strains did not have an associated barren phenotype. It was conceivable that these strains contained a large duplication as well as a "Sad-1-like" mutation. To test such possibilities, each of these strains was crossed with 74-OR23-1 A or OR8-1 a and the f₁ progeny were examined for segregation of the suppressor and any cryptic barren phenotype.

The Adiopodoumé (FGSC 430) strain: The T-430-a (Hyg^r) strain is a derivative of the Adiopodoumé 430 strain in which the mating type has been switched to mat a by transformation (ANDERSON et al. 2001 ). A total of 116 f₁ progeny from T-430-a (Hyg^r) x 74-OR23-1 A were crossed with Dp 1.3^ec hph strains of the appropriate mating type. None of the crosses were barren, indicating that T-430-a (Hyg^r) does not contain any conventional duplication. We determined the frequency of RIPinduced erg-3 mutant progeny from these crosses. In the crosses with the 59 mat a progeny, 52 (88.1%) gave erg-3 mutation frequencies <0.5%, and in the crosses with the 57 mat A progeny, 50 (87.7%) gave erg-3 mutation frequencies >1%. These results suggested that the suppressor was linked to the mat locus on LG IL and that the 7 mat a progeny with the nonsuppressor phenotype (i.e., erg-3 mutation frequencies >0.5%) and the 7 mat A progeny with the suppressor phenotype (i.e., erg-3 mutation frequencies <1%) represent the crossover types. From this we can infer that the suppressor is 14/116 (12.1%) units away from the mat locus.

Linkage between the RIP suppressor and the mat locus was also evident in the f₁ progeny from Adiopodoumé x OR8-1 a. Of the 54 mat A progeny tested, 32 (59.3%) gave a low erg-3 mutation frequency (<0.5%), 7 (12.9%) gave an intermediate frequency (0.5–1%), and 15 (27.8%) gave a high frequency (>1%). And of the 30 mat a progeny tested, the mutation frequencies were high for 25 (83.3%), intermediate for 2 (6.7%), and low for 3 (10%). Thus, most of the mat A progeny had the suppressor phenotype whereas most of the mat a progeny had the nonsuppressor phenotype. The proportion of putative crossovers in this experiment was 27/84 (32.1%).

A newly identified locus on LG IL, designated rid (RIP defective), codes for a putative DNA methyltransferase and homozygous rid mutant crosses are defective for RIP (FREITAG et al. 2002 ). We tested the Adiopodoumé RIP suppressor for linkage with rid. A total of 100 f₁ progeny from the cross T-430-a (Hyg^r) x rid A were crossed with rid; Dp 1.3^ec hph strains of the opposite mating types and the f₂ ascospores from these crosses were examined for erg-3 mutants. Only two crosses produced any erg-3 mutant progeny in the f₂. These two f₁ strains presumably represent crossover segregants that are wild type at both rid and the dominant RIP suppressor locus. Control crosses were done in which the f₁ progeny from OR8-1 a x rid A were crossed with the rid; Dp 1.3^ec hph strains and the rid and rid⁺ markers showed 1:1 segregation in the f₁ progeny (data not shown). Taken together, these results allow us to conclude that the Adiopodoumé RIP suppressor is distinct from, but linked to, rid (4/100).

Infertility factors in the Adiopodoumé strain: The Adiopodoumé x T-430-a (Hyg^r) cross is infertile (ANDERSON et al. 2001 ). For all practical purposes this is a self-cross; therefore, it follows that the Adiopodoumé strain contains one (or more) recessive fertility defect(s). The 116 f₁ progeny from T-430-a (Hyg^r) x 74-OR23-1 A were backcrossed to Adiopodoumé or T-430-a (Hyg^r). Of the 59 mat a segregants, 56 (95%) were infertile in the backcrosses with Adiopodoumé and 3 (5%) were fertile, and of the 56 mat A segregants, 33 (59%) were fertile in the backcross with T-430-a (Hyg^r) and 23 (41%) were infertile. We interpret these results to suggest that the T-430-a (Hyg^r) strain (and therefore also the Adiopodoumé strain) contains two recessive infertility mutations: one with 95% linkage to mat and another unlinked to mat. Only 1 of the 3 mat a crossover types for the infertility factor was also a crossover type for the RIP suppressor. Since the linkage between the LG I infertility factor and mat was greater than that between the RIP suppressor and mat, the infertility locus and the dominant RIP suppressor are likely to be on opposite sides of the mat locus.

Crosses parented by the Adiopodoumé strain produce many female-sterile progeny: A high proportion of progeny from the crosses parented by the Adiopodoumé and T-430-a (Hyg^r) strains were found to have a female-sterile phenotype and could participate in crosses only as males. This was discovered in crosses of the progeny with a strain bearing the Round spore (R) mutation, which confers a female-sterile phenotype. Since the erg-3 mutation also confers a female-sterile phenotype, in subsequent studies the female sterility of such progeny was tested in crosses with erg-3 strains. Of 40 progeny examined from the cross Adiopodoumé x OR8-1 a, 14 (35%) were female sterile and of 113 examined from T-430-a (Hyg^r) x 74-OR23-1 A, 20 (17.7%) were female sterile. Since all four parental strains are fertile as both male and female, these results suggest that one or more mutations that cause female sterility occur at a high frequency in crosses parented by the Adiopodoumé strains. In control crosses none of the 50 progeny tested from 74-OR23-1 A x OR8-1 a were female sterile. Crosses of the Adiopodoumé and T-430-a (Hyg^r) strains with non-Oak Ridge partners also yielded high frequencies of female-sterile progeny. The Adiopodoumé strain was crossed with the mat a strains Venkatavarum (FGSC 4722), Madurai (FGSC 4718), Iowa (P0529), and Bichpuri-1 (P0748) and the frequencies of female-sterile progeny were, respectively, 37.5% (40), 35% (20), 40% (40), and 55% (20), (numbers in parentheses indicate the number of progeny examined). T-430-a (Hyg^r) was crossed with the mat A strains Esterillo Este Rd-3 (P4000) and Rondon (P4214) and the frequencies of female-sterile progeny from these crosses were, respectively, 12.5% (40) and 12.5% (40).

Adiopodoumé-7: Sixty mat A f₁ progeny from a cross between Adiopodoumé-7 (P4305) and OR8-1 a were crossed with Dp 1.3^ec hph a. Only six crosses gave a very low frequency of erg-3 mutants in progeny ascospores harvested at 31 days and of these only three continued to exhibit this low frequency even in ascospores harvested at 45 days (data not shown). Additionally, 37 mat a f₁ progeny were crossed with Dp 1.3^ec hph A, but none of them appeared to have inherited the dominant RIP suppressor phenotype (data not shown). Thus the dominant RIP suppressor phenotype of Adiopodoumé-7 was inherited by only 1/10–1/20 of the mat A progeny and by an even lower fraction (<1/37) of the mat a progeny. These results are consistent with models in which RIP suppression by Adiopodoumé-7 requires the inheritance of four or five unlinked loci of which one might be located on LG IL. At any rate, the RIP suppressor of Adiopodoumé-7 does not appear to segregate as a single locus linked to mat and therefore the genetic basis of suppression appears to be different from that in the Adiopodoumé (FGSC 430) strain. It is noteworthy that two strains isolated from the same geographical region contain nonidentical RIP suppressors.

Bayan Lepas: We examined 59 f₁ progeny from Bayan Lepas x 74-OR23-1 A. Of these, 14 gave low erg-3 mutation frequencies (<0.5%), 10 were intermediate (0.5–1.0%), and 35 were high (>1.0%). If the low and intermediate mutation frequencies indicate the presence of the suppressor, then the segregation of suppressor and wild-type phenotypes is 24:35, which is not significantly different from 1:1 (PERKINS 1994 ). However, when 70 f₁ progeny from Bayan Lepas x Dp 1.3^ec hph A were examined, only 9 (1/8) appeared to have inherited the RIP suppressor phenotype. This result suggests that the suppressor phenotype might require the inheritance of as many as three unlinked loci from the Bayan Lepas parent. Thus dominant RIP suppression in Bayan Lepas appears to have a complex genetic basis.

Coon and Fred: The Coon and Fred RIP suppressor strains were analyzed in a similar manner. Again only a minority of the f₁ progeny were found to inherit their RIP suppressor phenotype. Among the f₁ progeny parented by the Coon strain, 6 gave low erg-3 mutation frequencies (<0.5%), 4 were intermediate (0.5–1.0%), and 54 were high (>1.0%), which indicated that the suppressor was inherited only by 10/64 progeny. The corresponding frequencies for the f₁ progeny parented by the Fred strain were 5 low, 7 intermediate, and 43 high, which indicated that the suppressor phenotype was inherited by only 12/55 progeny. These deviations from 1:1 segregation suggest that the RIP suppressor phenotype of these strains might require the inheritance of more than one locus.

Failure of the Tad retrotransposon to alter the efficiency of RIP:
A Dp 1.3^ec hph; col-18 A strain that does not contain any Tad sequences was used as the naive strain into which Tad sequences were introduced by infection from a donor strain. Another strain, no. 39 (see MATERIALS AND METHODS), which was obtained as a segregant from Adiopodoumé x Dp 1.3^ec hph a, was found to contain active Tad elements and used as the donor strain. Strain 39 also has an abnormal growth phenotype (different from the col-18 phenotype). Heterokaryons were formed between these two morphologically abnormal strains and isolated on the basis of their wild-type morphology. The heterokaryons were passaged through 30 vegetative transfers during which time the naive nuclear component became infected with Tad. The newly infected nuclei were isolated in homokaryotic conidia, now designated as "infected derivatives," and these were maintained as heterokaryons with the helper-1 strain. Fig 1 presents the results of the Southern analysis done to confirm the acquisition of Tad sequences by the infected derivative strains. When the infected derivatives were crossed with the wild type, there was no difference in the erg-3 mutation frequencies relative to control crosses with the naive strain (Table 2). These results lead us to conclude that the RIP efficiency of a naive strain is not affected following its infection by Tad.

View larger version (70K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Acquisition of Tad sequences by "infected derivatives" of the naive strain. Genomic DNA of the Adiopodoumé strain (lane 1), the Dp 1.3ec hph; col-18 A naive strain (lane 9), and seven infected derivative strains (lanes 2–8) was digested with EcoRI and subjected to Southern analysis. The blot was probed for Tad (A), hph (B), and erg-3 (C). Note that Tad sequences are present in the Adiopodoumé strain and in all the infected derivatives but are absent from the naive strain. The Dp 1.3ec hph transgene is present in the infected derivatives and the naive strain but not in the Adiopodoumé strain. The relatively constant hybridization intensities in B and C indicate that the proportion of infected derivative nuclei in the heterokaryons with helper-1 are roughly equal (see text for details).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

Of the 446 N. crassa wild isolates that we screened, 7 dominant RIP suppressor strains were identified. Each of these strains was crossed with the standard OR background and the inheritance of the suppressor phenotype was examined in the f₁ progeny. The RIP suppressors from the Adiopodoumé and Sugartown strains showed 1:1 segregation in the f₁ progeny, whereas the suppressors of the Adiopodoumé-7, Bayan Lepas, Coon, and Fred strains appeared to show a more complex inheritance pattern. We could not establish the segregation pattern of the Golur-1 suppressor. Additionally, our studies reveal that the dominant RIP suppressors fall into two classes. One class, represented by the Sugartown strain and possibly by the Golur-1 strain, appears to use large duplications to suppress RIP. To our knowledge, this is the first report identifying duplications in natural populations of Neurospora. The second class, which included the Adiopodoumé, Adiopodoumé-7, Bayan Lepas, Coon, and Fred strains, did not appear to involve duplications. However, we cannot rigorously exclude the possibility that a subset of these strains might in fact contain duplications that do not cover any "meiosis-essential" genes whose silencing by MSUD would evoke barrenness.

Duplications can affect Neurospora crosses in two ways. During the premeiotic stage they serve as substrates for RIP and in this way they can lead to the generation of novel mutations among the progeny. Witness the morphological and dow mutants produced, respectively, in the crosses with the Sugartown and Dp(AR17) duplications (also see PERKINS et al. 1997 ). By acting as substrates for RIP, large duplications might also titrate out the RIP machinery and thereby act as suppressors of RIP in a smaller duplication. Subsequently during meiosis, duplications can cause the silencing of unpaired genes by MSUD and thereby render the cross barren. RIP and MSUD are independent processes. It was shown previously that the barren phenotype of a duplication strain was not alleviated in crosses with the Adiopodoumé-dominant RIP suppressor strain (NOUBISSI et al. 2000 ). Now we have shown that suppression of the barren phenotype by Sad-1 does not reduce the RIP suppressive effect of large duplications. The picture emerging from these studies is that the size of the duplication ("quantity") determines whether it will act as a RIP suppressor, and its coverage of meiosis-essential genes ("quality") determines whether or not it causes barrenness.

It is not known whether rearrangements other than duplications can trigger MSUD when they are heterozygous. Pairing would be disrupted in the vicinity of breakpoints or with short inversions. Such rearrangements should look like dominant-barren mutations that are suppressible by Sad-1 but do not suppress RIP. The Georgetown-6 strain is a candidate for such a rearrangement. The inability of Sad-1 to suppress the barren phenotype of the Batu Ferringi-1 and Brabadougou strains suggested that these two strains might not contain duplications. Although Brabadougou was not tested for RIP suppression, the Batu Ferringi-1 strain happened to be tested en passant in a double-blind experiment (see Appendix) and found not to behave as a dominant RIP suppressor. This result also argues against the presence of a duplication in this strain.

Infection of a naive strain by Tad did not affect RIP. But we were somewhat surprised to find that Tad sequences made no contribution at all to the RIP suppression phenotype of the Adiopodoumé strain. This strain contains 40 copies of Tad dispersed throughout the genome. A complete Tad element is 7 kbp in size; therefore, the Adiopodoumé strain could harbor as much as 280 kb of duplicated DNA, which is comparable in size with some large duplications (SMITH et al. 1996 ). But its RIP suppressor phenotype appeared to show quite straightforward linkage to the rid and mat loci on LG IL rather than to multiple dispersed loci that would be expected to represent the Tad elements. Although some copies of Tad might have already suffered enough sequence alterations to preclude their involvement in further rounds of RIP, Southern analysis of suppressor and nonsuppressor f₁ progeny did not reveal any consistent difference in Tad sequences (data not shown). Thus our results suggest that duplicated DNA might be effective in titrating out the RIP machinery only if it is present in one contiguous stretch, as in a segmental aneuploid strain, and not if it is composed of relatively shorter segments dispersed in the genome.

Of the 116 progeny tested from T-430-a (Hyg^r) x 74-OR23-1 A, 12.1% represented products of crossovers between the RIP suppressor and mat loci, whereas the crossover frequency was considerably higher (32.1%) among the 84 progeny tested from Adiopodoumé x OR8-1 a. One explanation for this difference could be that the T-430-a (Hyg^r) strain, which represents a transformed nucleus that was essentially "cloned" out of the Adiopodoumé strain, is homokaryotic for an inversion for which the parental Adiopodoumé strain is heterokaryotic. Homokaryosis for an inversion could have reduced the crossover frequency. Chromosome rearrangements have been noted to be frequent in the Adiopodoumé strain (by KINSEY and HELBER 1989 , as a personal communication from David Perkins). Alternatively, the putative inversion might have been generated during the transformation done to switch the mating type. PERKINS et al. 1993 have reported that chromosome rearrangements can accompany transformation at surprisingly high frequencies. However, we do not have any explanation for our observation that the frequencies of female-sterile progeny were consistently lower (12.5–17.5%) in crosses parented by the T-430-a (Hyg^r) strain than in crosses parented by Adiopodoumé (35–55%).

We have shown here that the Adiopodoumé suppressor was distinct from, but closely linked to, rid. Another LG I candidate gene that should probably be tested for allelism with the Adiopodoumé suppressor is eth-1 (ethionine resistant-1), the structural gene for S-adenosylmethionine synthetase (see PERKINS et al. 2001 ). SELKER 1990 had proposed an attractive model in which limiting cellular levels of S-adenosylmethionine could increase the efficiency of RIP. The model was considered in further detail by MAUTINO and ROSA 1998 . In this model a putative DNA-(5-cytosine) methyltransferase (possibly the enzyme encoded by rid) interacts with the C6 of cytosine to form an unstable intermediate 5,6-dihydrocytosine complex with the enzyme. The intermediate either can receive the methyl group from S-adenosylmethionine to generate 5-methylcytosine or, alternatively, in S-adenosylmethionine-limiting conditions, can tautomerize to an imino group, which can be easily hydrolyzed to generate uracil. Conceivably, if an overactive S-adenosylmethionine synthetase of the Adiopodoumé strain caused overaccumulation of S-adenosylmethionine, the efficiency of RIP might become depressed. In parallel with the testing of candidate genes we are continuing our efforts to obtain a more accurate localization of the suppressor locus on IL as a prelude to its molecular characterization.

Note added in proof:
The Adiopodoumé suppressor was found not to be allelic with eth-1 (6/80) (Ranjan TAMULI, unpublished results).

	FOOTNOTES

This article is dedicated with affection to Professor Ramesh Maheshwari, Indian Institute of Science.
¹ These authors contributed equally to this work.

	ACKNOWLEDGMENTS

We are indebted to David D. Perkins and the two anonymous referees for several useful suggestions and for even rewriting the abstract. We are grateful to Kevin McCluskey and the Fungal Genetics Stock Center (FGSC) for readily providing us with most of the Neurospora strains and also for reviewing the manuscript. We thank Ranjan Tamuli and T. Bhavani Prasanna for technical assistance, Robert L. Metzenberg, Eric U. Selker, and Michael Freitag for generously supplying strains, and David J. Jacobson for mooting the possibility of Tad's effects on RIP. Eric Selker and Michael Freitag also suggested the testing of candidate genes for allelism with the Adiopodoumé suppressor. F.K.N. was supported by a fellowship from the Third World Organization of Women in Science. A.B. and M.V. were supported, respectively, by a Senior Research Fellowship and a Junior Research Fellowship from the University Grants Commission-Council of Scientific and Industrial Research (India). The FGSC is supported by a National Science Foundation grant BIR-9222772.

Manuscript received October 23, 2002; Accepted for publication March 25, 2003.

	APPENDIX

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

Screening of additional wild N. crassa strains for dominant suppression of RIP:
A total of 242 wild-isolated mat a strains were each crossed with Dp 1.3^ec hph A and the frequency of RIP-induced erg-3 mutants was determined in the f₁ progeny of these crosses. For crosses that gave a low frequency (<1%) of erg-3 mutants in ascospores harvested at 31 days, the mutation frequency was determined again in a second harvest taken at 45 days. Crosses with the three strains Fred (P0833), Coon (P0881), and Bayan Lepas (P2663) gave low erg-3 mutation frequencies in both harvests (Table A11).

Crosses with four other strains, Golur-1 (P0334), Georgetown-6 (P2622), Batu Ferringi-1 (P2681), and Brabadougou (P4296), were barren. This is relevant because segmental duplications make crosses barren, and long segmental duplications are also known to suppress RIP in our test system. Of the barren crosses only the one with Golur-1 produced sufficient numbers of ascospores after 45 days to allow an estimation of the erg-3 mutation frequency. This frequency was low. The other three barren crosses together produced <100 progeny. To confirm that the low frequency of erg-3 mutants in the crosses with the Coon, Fred, Bayan Lepas, and Golur-1 strains was reproducible and not merely a sampling artifact, we repeated these crosses and again the frequency of erg-3 mutant progeny was very low (data not shown). Once again the cross with the Golur-1 strain was barren and took 45 days to produce sufficient numbers of ascospores. Thus the Coon, Fred, and Bayan Lepas strains resembled the previously tested Adiopodoumé 430 strain in that the RIP suppression by these strains was not associated with a barren phenotype, whereas the Golur-1 strain was like the Sugartown strain in that its RIP suppression phenotype was associated with barrenness. It was not possible to determine from these results whether the barrenness of the Georgetown-6, Batu Ferringi-1, and Brabadougou strains was associated with a dominant RIP suppression phenotype.

Double-blind tests of the RIP suppressor phenotype:
We tested whether the dominant RIP suppression phenotype could be used as the defining character to identify the Coon, Fred, and Bayan Lepas strains in double-blind experiments. The T-430-a (Hyg^r) (FGSC 8609) strain is derived from the Adiopodoumé strain by switching of the mating type to mat a by transformation (ANDERSON et al. 2001 ). We also tested this strain to verify that it retained the ability of the Adiopodoumé strain to dominantly suppress RIP in Dp 1.3^ec hph. In each of these experiments 10 wild strains were coded and crossed with Dp 1.3^ec hph A and the frequency of RIP-induced erg-3 mutants was determined in ascospores harvested after 31 days. The challenge was to identify which of the coded cultures represented the suppressors. The experimental protocol was essentially the same as the one used by NOUBISSI et al. 2000 . The results of these experiments are summarized in Table A22 and they showed that in all cases the RIP suppressor strains could be correctly distinguished from the other wild isolates. In the double-blind test of the Bayan Lepas strain, we happened to also perform a cross with the Batu Ferringi-1 strain. This cross was barren as noted previously (see above), but nevertheless we could obtain 246 ascospores and thus determine the erg-3 mutation frequency. Despite its barren phenotype, the Batu Ferringi-1 strain did not display a RIP suppressive effect.

In the experiment to test the Coon strain, the cultures coded B, D, and E were the Coon strain. Those coded A, C, F, G, H, I, and J were the non-Coon strains, respectively, Makaba-1 (P3812), Aarey-1 (P0677), Sungai Ara (P2672), Bichpuri-1 (P0742), Homestead-3 (P1470), Fred-2 (P0828), and Klong Rangsit No. 7 (P4217). In the test of the Fred strain the cultures coded C, G, and H were the Fred strain and those coded A, B, D, E, I, and J were, respectively, Aarey-1 (P0678), Georgetown-2 (P2592), Colonia Paraiso (P1291), Welsh-1d (P0507), Georgetown-4 (P2608), and Groveland-1 (P0438). The strain coded F, Florida City (P1448), was found to behave as mat A. In the test of Bayan Lepas, the cultures coded D, H, and I were Bayan Lepas and those coded A, B, C, E, and G were Batu Ferringi-1 (P2681), Franklin (P4493), Kabah (P4126), Kabah (P4125), and Jaco-2 (P4017). The strains coded F and J, respectively, Maripasoula (P4088) and Madurai (P4360), were found to behave as mat A. In the test for T-430-a (Hyg^r) (FGSC 8609), the cultures A, E, F, G, and J were T-430-a (Hyg^r) whereas B, C, D, H, and I were OR8-1 a.

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