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Genetics, Vol. 173, 1991-2004, August 2006, Copyright © 2006
doi:10.1534/genetics.106.057562
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Biology Department, Carleton University, Ottawa, Ontario K1S 5B6, Canada
2 Corresponding author: Biology Department, Carleton University, 1125 Colonel By Dr., Ottawa, Ontario K1S 5B6, Canada.
E-mail: mysmith{at}ccs.carleton.ca
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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10 loci that control heterokaryon incompatibility during vegetative growth of N. crassa. Previously, it was found that het-6-associated incompatibility in Oak Ridge (OR) strains involves two contiguous genes, het-6 and un-24. The OR allele of either gene causes "strong" incompatibility (cell death) when transformed into Panama (PA)-background strains. Several remarkable features of the locus include the nature of these incompatibility genes (het-6 is a member of a repetitive gene family and un-24 also encodes the large subunit of ribonucleotide reductase) and the observation that un-24 and het-6 are in severe linkage disequilibrium. Here, we identify "weak" (slow, aberrant growth) incompatibility activities by un-24PA and het-6PA when transformed separately into OR strains, whereas together they exhibit an additive, strong effect. We synthesized strains with the new allelic combinations un-24PA het-6OR and un-24OR het-6PA, which are not found in nature. These strains grow normally and have distinct nonself recognition capabilities but may have reduced fitness. Comparing the Oak Ridge and Panama het-6 regions revealed a paracentric inversion, the architecture of which provides insights into the evolution of the un-24–het-6 gene complex.
The function of vegetative nonself recognition in fungi has been debated for some time (CATEN 1972; HARTL et al. 1975; WU et al. 1998; SAUPE 2000). A favored explanation is that nonself recognition reduces the transmission of potentially deleterious genetic elements (organelles, viruses, plasmids) from one individual to another. Evidence in support of this hypothesis includes direct observation of reduced transmission of infectious agents between incompatible fungal strains (DEBETS and GRIFFITHS 1998; CORTESI et al. 2001). In Ophiostoma novo-ulmi, the causative agent of Dutch elm disease, a shift to greater diversity in vegetative incompatibility groups is associated with increased virus abundance in populations (PAOLETTI et al. 2006). In another study, the frequency of programmed cell death (PCD) associated with vegetative incompatibility was found to be negatively correlated to virus transmission in Cryphonectria parasitica (BIELLA et al. 2002). Such observations provide a potential adaptive explanation for the maintenance of heterokaryon incompatibility in fungi.
Among the incompatibility factors in N. crassa, differences at het-6 cause one of the most intense heterokaryon incompatibility reactions. Initially identified as a single locus (MYLYK 1975), the het-6 region comprises two tightly linked genes, un-24 and het-6, each having two allelic variants, Oak Ridge (OR) and Panama (PA). The two loci are in severe linkage disequilibrium such that they have been found in only two of four possible allelic combinations, un-24ORhet-6OR and un-24PAhet-6PA (MIR-RASHED et al. 2000; SMITH et al. 2000b), referred to as OR and PA haplotypes, respectively. In addition to its function in heterokaryon incompatibility, un-24 encodes the large subunit of ribonucleotide reductase, an essential enzyme that converts ribonucleotides to the deoxyribonucleotides required for DNA synthesis and repair (SMITH et al. 2000a). In contrast, het-6 appears to be a member of a repetitive gene family and to encode a protein with no known function aside from nonself recognition. OR alleles at both het-6 and un-24 were previously characterized and shown to elicit incompatibility reactions when introduced separately or together into PA recipient cells; however, failure to detect reciprocal incompatibility reactions from the PA allelic counterparts suggested that un-24 and het-6 incompatibility reactions may involve nonallelic interactions with additional factors in the het-6 region (SMITH et al. 2000b).
This article reports on the isolation and characterization of functional PA alleles of un-24 and het-6 and describes a paracentric inversion polymorphism that provides a proximate reason for decreased recombination within the region and linkage disequilibrium of these two genes. The inversion breakpoints are immediately adjacent to regions in both un-24 and het-6 that appear to be under diversifying selection. We constructed strains that express the novel allelic combinations un-24PA het-6OR or un-24OR het-6PA and observed that, unlike their naturally occurring counterparts, these strains have lower fitness and compromised incompatibility function. Our observations suggest that evolution within the un-24–het-6 gene complex has had an adaptive role in heterokaryon incompatibility function in the species.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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DNA was obtained by the liquid lysis method (SAMBROOK et al. 1989). Oligonucleotides used for PCR amplification (Table 2) were synthesized by Invitrogen (Carlsbad, CA) and PCR amplifications were done with Taq DNA polymerase (Invitrogen) or an Expand High Fidelity PCR system (Roche, Laval, PQ, Canada). When required, PCR products were cloned into pCRII or TOPO pCRII (Invitrogen) and subclones were prepared in pUC118 and pCB1004, which carries hygromycin resistance (CARROL et al. 1994). Southern hybridizations with [
-32P]dCTP-labeled probes were by standard methods (SAMBROOK et al. 1989). For a -library of FGSC1131, genomic DNA was partially digested with MboI and separated on a 10–40% sucrose gradient by ultracentrifugation at 26,000 rpm for 42 hr in a Beckman swinging bucket SW27 rotor. Fragments between 10 and 20 kbp in length were ligated into Lambda FixII arms and packaged into a -phage according to the manufacturer's recommendations (Stratagene, La Jolla, CA).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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IIIR)AR18 translocated region was previously determined by partial diploid analyses (MYLYK 1975; SMITH et al. 1996). Colonies with heterozygous duplications of the region [Dp(AR18)] display inhibited growth due to het-6 self-incompatibility. Escape from self-incompatibility by Dp(AR18) colonies was associated with deletions within either the OR or the PA haplotype DNA of at least an 35-kbp region that includes both un-24 and het-6 (SMITH et al. 2000b). However, PCR products obtained from PA strains that were equivalent to active un-24OR and het-6OR PCR products did not have incompatibility activity when transformed into OR spheroplasts, suggesting that un-24PA and het-6PA do not have incompatibility activity.
Using a different approach to isolate the PA-specific incompatibility factor(s) in the 35-kbp region, a -library of strain FGSC1131 was constructed. -Clones from the het-6PA region were identified by hybridization to probes corresponding to het-6OR, het-6PA, and un-24OR and a chromosome walk was done from het-6PA both toward and away from the centromere (Figure 1). Within this walk, clone B was found to hybridize to both un-24 and het-6 and was chosen for further study. The B insert was subcloned using NotI into pCB1004 as pB and transformed into C9-2 (het-6OR) and C2(2)-1 (het-6PA) spheroplasts (Figure 2). pB caused an 83% decrease (±16% in four independent transformation experiments) in the number of C9-2 (OR haplotype) transformants recovered as compared to the pCB1004 vector control, but no decrease in the number of transformants when introduced into PA-haplotype C2(2)-1 spheroplasts. This indicated that pB has PA-specific incompatibility activity. Subclones of pB were constructed and tested in transformation assays to further define the incompatibility region, as summarized in Figure 2. The incompatibility activity associated with un-24PA is notably weaker than that of un-24OR, which yields no, or few, viable colonies when transformed into un-24PA strains (SMITH et al. 2000b). Construct pBP/C contains the entire un-24PA coding region along with 550-bp upstream and 380-bp downstream regions. When transformed into OR spheroplasts, on average 52% (± 20% in three independent trials) fewer colonies were observed as compared to the pCB1004 vector control. Of these pBP/C transformants, 63% appeared inhibited in their growth with a star-like appearance on the original transformation plates (Figure 2C). The remaining colonies had a cloud-like morphology characteristic of compatible transformants. When pBP/C transformants were transferred to 1x Vogel's medium containing no sorbose, the star-like colonies grew significantly more slowly, did not conidiate, and displayed a "spidery" morphology whereas cloud colonies grew at wild-type rates and conidiated normally. A similar spidery morphology is typical of incompatibility due to heteroallelism at mat and het-c in N. crassa (BEADLE and COONRADT 1944; SAUPE et al. 1996). Early escape events or disruption of the un-24PA gene during integration of the transforming DNA may account for the cloud colonies obtained when un-24PA is transformed into OR cells. Within 3–6 days after transfer to Vogel's medium, the self-incompatible transformants were observed to give rise to fast-growing sectors, indicative of escape (Figure 2D). Thus, incompatibility activity is evident when un-24PA is transformed into un-24OR spheroplasts by a modest reduction in transformant numbers but a high frequency of transformants that are self-incompatible and consequently undergo escape.
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Overall, the un-24PA (accession DQ525966) and un-24OR DNA sequences are 95% identical; however, they differ significantly at the 3'-end wherein the specificity region for nonself recognition is likely encoded (SMITH et al. 2000b). Deletion analysis and transformation assays were used to delimit the incompatibility function of un-24PA to an 1.6-kbp region within the 3'-coding region (e.g., pBX/C, Figure 2A). Loss of both incompatibility and catalytic functions are observed for the pBP/Nr construct, which has a small 34-nucleotide region of 3'-coding sequence deleted. Similar to deletions within un-24OR (SMITH et al. 2000b), the 1.6-kbp 3'-coding sequence with incompatibility activity does not have ribonucleotide reductase catalytic function. This provides further evidence that incompatibility function involves a mechanism that is independent of ribonucleotide reductase activity for the UN-24 protein.
Construct pBC/N was derived from pB and used to further test whether het-6PA has incompatibility activity (Figure 2A). This construct carries a full-length copy of het-6PA flanked by 3030 bp of 5' upstream and 880 bp of 3' downstream noncoding sequences. Transformation of pBC/N into OR cells resulted in an average 69% (±10% in three independent trials) decrease in the number of OR transformants compared to the pCB1004 vector control but did not cause a significant decrease in the number of transformants when PA cells were used as recipients (Figure 2B). Therefore, pBC/N was considered to carry het-6PA-associated incompatibility activity that is notably weaker than that of het-6OR, which, when introduced into PA spheroplasts, results in a nearly complete absence of transformants. Construct 6VPA3/6VPA4 spans the entire coding region of het-6PA as well as 340 bp upstream of the translation initiation site, but lacks heterokaryon incompatibility activity. In contrast, clone pBBg/A carries an additional 1097 bp in the 5' region upstream from the start codon and displays heterokaryon incompatibility activity. Thus, a minimum region required for het-6PA incompatibility activity encompasses 1100 bp upstream from the start codon and 110 bp downstream from the stop codon (Figure 2A). In contrast, the additional noncoding sequences outside of the 6VPA3/6VPA4 primer sites are not required for het-6OR activity, accounting for the lack incompatibility activity reported earlier for het-6PA (SMITH et al. 2000b). This observation also suggests that OR and PA forms of het-6 have distinct mechanisms of gene regulation since the additional 5'- and 3'-untranslated regions required for het-6PA activity do not appear to contain additional genes.
The het-6PA sequence was reported previously (SMITH et al. 2000b). The OR and PA alleles at het-6 share 78% identity at the nucleotide level and only 68% identity at the amino-acid level. Nucleotide sequence differences are spread over the entire length of the het-6 open reading frame. As is the case for het-6OR, no intron consensus sequences occur in het-6PA (BRUSCHEZ et al. 1993; EDELMAN and STABEN 1994). The gene putatively encodes a 680-amino-acid peptide with three regions of identity to at least 13 other putative N. crassa proteins of unknown function (Blast cutoff score >38, E < 1 x 10–4; Broad Institute). The three common regions of 10, 27, and 21 amino acids in length form the HET domain identified in other heterokaryon incompatibility-associated proteins from N. crassa (TOL) and Podospora anserina (HET-E) (SMITH et al. 2000b). Genes similar to het-6 are also found in Aspergillus nidulans, Magnaporthe grisea, C. parasitica, and Fusarium graminearum sequence databases, for example, but not in the Saccharomyces cerevisiae genome, suggesting that this gene family may be restricted to filamentous fungi. In addition to the HET domain, there is a region bound by amino acids 434–516 in het-6 that displays amino acid similarity (50%) to a domain of unknown function in bacterial aconitases (data not shown).
un-24 and het-6 show evidence of diversifying selection:
The isolation of active PA alleles at un-24 and het-6 suggests that allelic interactions are responsible for incompatibility function, rather than nonallelic interactions as previously hypothesized (SMITH et al. 2000b). Therefore, the allelic specificity domains of un-24 and het-6 may be recognized by comparing the respective OR and PA forms since nonself recognition loci are often subject to diversifying selection (APANIUS et al. 1997; MCDOWELL et al. 1998; ROALSON and MCCUBBIN 2003). Under diversifying selection, substitutions leading to amino-acid changes are favored to presumably allow for broader specificity of nonself recognition during protein interactions (HUGHES 1999) as exemplified with the MHC in humans (AGUILAR et al. 2004), the S locus in plants (ROALSON and MCCUBBIN 2003), and the het-c locus in N. crassa (WU et al. 1998). This is in contrast to most genes undergoing neutral or directional selection, where the rate of synonymous substitutions typically exceeds that of nonsynonymous substitutions.
We tested for indicators of diversifying selection at un-24 and at het-6 by determining the number of synonymous and nonsynonymous nucleotide substitutions in the OR and PA alleles for each gene. For this analysis, DNA sequences corresponding to the entire coding region of OR and PA alleles of both genes were used (GenBank accession nos. AF206700, AF208542, AF171697, and DQ525966). Intron sequences were removed from un-24 sequences and pairwise alignments in all combinations were done using ClustalW (http://clustalw.genome.ad.jp/), adjusted by hand and analyzed for the distribution of synonymous and nonsynonymous substitutions.
het-6 has a significantly greater frequency of nonsynonymous than synonymous substitutions throughout most of the coding region (overall dN/dS = 1.47; Figure 3A) on the basis of both parametric (two-tailed test, P < 0.001) and nonparametric (Kruskal–Wallis test, 
2 = 8.16, d.f. = 1, P = 0.004) statistics. Small islands with more synonymous than nonsynonymous substitutions are evident between codons 337 and 378, 389 and 403, and 638 and 670, and notably in the regions that correspond to part of the conserved HET domain (codons 53–95) and within the aconitase-like domain (codons 455–475). In contrast, two distinct patterns of nucleotide substitutions are apparent within un-24 (Figure 3B). un-24PA and un-24OR are very similar across the 5' region of the open reading frame with only 7 of the 38 nucleotide substitutions in the first 871 codons resulting in significantly more synonymous than nonsynonymous substitutions across this region (dN/dS = 0.22; two-tailed test, P < 0.001; Kruskal–Wallis test, 2 = 17.25, d.f. = 1, P < 0.001). This N-terminal region of un-24 is essential for ribonucleotide reductase function and is highly conserved in amino-acid sequence across taxa (SMITH et al. 2000a). However, in the putative allelic-specificity domain located at the 3'-end of un-24 from codons 872 to 925, there is a strong bias toward nonsynonymous substitutions (dN/dS = 2.32; two-tailed test, P < 0.016; Kruskal–Wallis test, 2 = 3.71, d.f. = 1, P = 0.05). Thus, both un-24 and het-6 display high levels of nonsynonymous substitutions in regions that are likely associated with heterokaryon incompatibility function, consistent with the view that diversifying selection is operating within both genes.
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In addition to mating capacity, we evaluated heterokaryon incompatibility characteristics of escape strains. If heterokaryon incompatibility has an adaptive function in maintaining individuality and in preventing the transmission of infectious elements, strong incompatibility reactions would presumably have a selective advantage. To assess the strength of heterokaryon incompatibility reactions, heterokaryon incompatibility tests were done with all possible allelic combinations at un-24 and het-6 (Table 3). For these experiments, functionally un-24PA het-6OR and un-24OR het-6PA strains were derived through escape as described above. The resulting heterokaryons were assessed for growth rate, colony morphology, and conidiation. As shown in Table 3, strains with the novel allelic combinations, un-24PA het-6OR and un-24OR het-6PA, displayed weak incompatibility reactions with strains that had differences at one or both het genes. These novel haplotypes are apparently compromised for nonself recognition and, in this context, individuals with these genetic combinations at un-24 and het-6 may be selected against in nature.
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B, encompassing both un-24 and het-6 PA alleles, allowed us to address this hypothesis through structural comparisons of OR and PA haplotypes. The cosmid G8:G1 (which spans the un-24OR–het-6OR region) and DNA sequence from contig B2A19 (MIPS, http://mips.gsf.de/proj/neurospora/) were used as reference points for the OR haplotype. -Clones from the FGSC1131 library were used to study the structure of the PA haplotype. pB and pA3 (derived from FGSC1131 via FGSC2190; SMITH et al. 1996; MIR-RASHED et al. 2000) were sequenced in the un-24PA–het-6PA region and used for RFLP analyses.
Southern hybridizations were done to compare the marker distribution and general structure of clones carrying PA- or OR-derived sequences (Figure 1). Hybridization membranes containing DNA from clones pA3, B, TLP131, and 11-1 (PA haplotype) and G8:G1 (OR haplotype) were probed sequentially with radioactively labeled PCR products of primer pairs 6J-P11/6J-P6 (un-24OR) and cys3-P1/cys3-P2 (cys-3OR). Hybridization of the un-24OR probe to G8:G1, pA3, and B was observed and RFLP patterns were consistent with previously published restriction maps (MIR-RASHED et al. 2000; SMITH et al. 2000b). On the basis of hybridization to the cys-3OR probe, neither pA3 nor B harbors this gene and this was verified by the absence of a PCR amplicon from either clone with cys-3-specific primers. However, cys-3 amplicons were obtained from G8:G1 (OR) and from the TLP131 (PA) clone that maps centromere distal to un-24PA (Figure 1). Given that the intergenic distance between un-24 and het-6 is 5.2 kbp in the PA haplotype compared to 19 kbp in the OR haplotype, these results suggested the presence of an inversion in the region. This inversion, designated here as In(IIL)1131 het-6, for inversion associated with het-6, was verified by determining the DNA sequences of the termini of pA3. In pA3 (PA haplotype), un-24 and het-6 are transcribed from the same strand of DNA whereas in the OR haplotype (MIPS database) the genes are transcribed from opposite strands.
Characterization of inversion breakpoints brk-1 and brk-2:
The location of the In(IIL)1131 het-6 [abbreviated as In(het-6) hereafter] breakpoints were determined by comparisons of the OR and PA haplotypes. On the basis of the map data (above), we surmised that brk-1, the centromere-proximal breakpoint, was located close to Gene7a or Gene7b with respect to the OR map (Figure 4A). The location of the distal breakpoint, brk-2, had to account for the shorter distance between un-24 and het-6 in PA relative to OR haplotypes and was inferred to be just downstream of un-24OR, likely between un-24OR and Gene2 relative to the OR map. We sought confirmation of these hypothetical maps by designing PCR primers based on the contig B2A19 sequence (MIPS) that would differentially amplify across the OR and PA configurations of brk-1 and brk-2 (Table 2 and Figure 4A). Genomic DNAs from RLM58-18 (PA) yielded amplicons with primer pairs Gene7b-P1/6J-P11 (brk-1PA) and Gene2-P3/Gene7a-P5 (brk-2PA), while 74-OR23-1V (OR) DNA produced amplicons with primers Gene7b-P1/cys3-P4 (brk-1OR) and Gene2-P3/6J-inv2 (brk-2OR). On the basis of these inversion breakpoint positions, In(het-6) spans an 18.6-kbp region from 4.2 kbp upstream of het-6 to 100 bp upstream of Gene 2 (based on the OR haplotype).
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G12 (Figure 1) using primers cys3-P4 and Gene2-P1, subcloned and sequenced. brk-1PA and brk-2PA sequences were compared to the OR genomic sequence available at MIPS. Sequence comparisons around the two inversion breakpoints are summarized in Figure 4 using sliding-window analyses. For the analyses, OR and PA sequences were aligned and sequence identity was calculated as the number of identical nucleotides in a window of 60 bp. Successive identity values were calculated as the window was incrementally shifted by 30 bp until the breakpoint region was covered. The inversion breakpoints are characterized by regions of sequence dissimilarity between OR and PA haplotypes of 2 kbp (brk-1) and 0.6 kbp (brk-2), flanked by regions of significant sequence identity. The sequence identity patterns to the right of brk-1 in Figure 4B suggested that an additional small inversion polymorphism just off the 5'-end of het-6 also differentiates the OR and PA haplotypes (Figure 4B).
A BLAST analysis of brk-1OR and brk-1PA revealed sequence identity to regions located elsewhere within the N. crassa genome. Notably, within brk-1PA, but not within brk-1OR, a 397-bp stretch of sequence shows 75% identity to at least 14 different regions within the N. crassa genome. This sequence also contains 53-bp inverted repeats that share 88% identity. Among these 14 regions, contigs 3.213 (mapping to LGVI), 3.622 (mapping to LGII), and 3.400 (LGVII) also carry similar inverted repeats (Figure 5). The remaining 11 sequences lack the inverted repeats but otherwise share significant sequence identity to the 397-bp internal sequence. In summary, this 397-bp sequence has several features that qualify it as a bona fide repetitive sequence (RS): inverted terminal repeats, short imperfect direct repeats (CCTAACC), and its presence in multiple (at least 14) copies in the genome of N. crassa. A BLAST survey indicated that neither the inverted repeats nor the internal repeated sequence matched any known mobile genetic elements or repeated sequences in the databases and that the region had no similarity to known transposases, which suggests that this is a novel RS element. Several RS elements and transposons have been implicated in chromosomal rearrangements in maize, Drosophila, and Anopheles species (MATHIOPOULOS et al. 1998; CÁCERES et al. 1999; ZHANG and PETERSON 1999). In a majority of cases, inversion breakpoints were associated with mobile genetic elements, suggesting that recombination between the two homologous mobile genetic element sequences was responsible for the genetic rearrangement. The close spatial association of a repetitive genetic element, brk-1, and incompatibility genes under diversifying selection suggests a role for RS elements in the evolution of nonself recognition in N. crassa.
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To explore this, 20 N. crassa strains (10 un-24OR het-6OR and 10 un-24PA het-6PA) were selected from different geographic origins to represent strains that have likely been separated by many generations (Figure 6). The het-6 and un-24 genotype of each strain was verified using a PCR–RFLP-based assay as described previously (MIR-RASHED et al. 2000). Inversion forms were determined using primers designed for the isolation of brk-1 and/or brk-2 and, in all strains tested, there was a perfect correlation between genotypes at het-6 and un-24 and inversion type (P < 0.001, Figure 6). This strongly supports the idea that the In(het-6) is responsible for linkage disequilibrium of the two incompatibility genes, het-6 and un-24.
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To test whether there is a correlation between genotype at het-6 and genotype at each of the other markers, pairwise allelic associations were calculated using Fisher's exact test (Figure 6; http://www.physics.csbsju.edu/stats/exact.html; JERROLD 1984). Polymorphisms at het-6, un-24, and In(het-6) are perfectly correlated with no exceptions, as described previously. Other markers within the inversion breakpoints were significantly correlated to the allelic form of het-6 except for marker Gene4-2 (P = 0.152), which is situated toward the center of the inverted region. Markers outside the inverted region were not correlated to the het-6 genotype (P = 0.328 for Gene2, P = 0.328 for Gene9). Therefore, polymorphisms are more frequently shared between PA- and OR-haplotype strains toward the center of, and outside of, the inverted region. The presence of the shared polymorphisms at markers within In(het-6) is consistent with the view that gene conversion events and double crossovers are possible in the center of inversions, where chromosomal pairing is more efficient than at inversion breakpoints (CHOVNIK 1973). Apparently, as one approaches the breakpoints, and homologous pairing is not possible due to divergent sequences at each breakpoint, the correlation between markers increases to unity. Therefore, the proximity of the un-24 and het-6 incompatibility genes to the In(het-6) breakpoints, rather than the inversion per se, is responsible for linkage disequilibrium of un-24 and het-6 in N. crassa.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Sequence differences in alleles of un-24 and het-6 are found mainly adjacent to the In(het-6) breakpoints and are characterized by high dN/dS ratios in un-24 and het-6, suggesting that diversifying selection is operating within the gene complex. The signature of diversifying selection is particularly apparent in the 3' coding sequence of un-24, where the encoded protein sequences of OR and PA alleles are only 70% similar. Moving toward the 5'-end of the gene, an abrupt shift to high sequence similarity between the OR and PA alleles corresponds to the ribonucleotide reductase catalytic domain that is conserved across taxa (SMITH et al. 2000a). Thus, the un-24 locus represents a battlefield between opposing selection pressures: diversifying selection acting in the incompatibility domain and purifying selection acting in the rest of the gene. Similarly, distinct selection pressures on various domains of nonself recognition loci were described in R-genes of plants and in MHC loci in vertebrates. In plants, protein domains involved in direct or indirect interactions with pathogen protein moieties [e.g., the ligand-binding leucine-rich repeat (LRR)] are hypervariable and carry the mark of diversifying selection, whereas structural domains and domains involved in host signaling are highly conserved (NOËL et al. 1999; BERGELSON et al. 2001). Given the conserved nature of the large subunit of ribonucleotide reductase across diverse taxa, it would appear that nonself recognition function by un-24 is a derived character in Neurospora.
The un-24–het-6 region also appears to be under balancing selection, which helps to maintain variability at nonself recognition loci over long time periods (RICHMAN 2000). Two characteristics of balancing selection, which are not necessarily observed with diversifying selection, are balanced frequencies of alleles within populations and the preservation of specific polymorphisms across different species. Previous studies showed that the PA and OR haplotypes occur at about equal frequencies in N. crassa populations (MIR-RASHED et al. 2000; SMITH et al. 2000b). In addition, evidence for linkage disequilibrium of the PA and OR haplotypes was also found in N. intermedia and N. tetrasperma (MIR-RASHED 1998; POWELL 2002). These observations provide support for the trans-species maintenance of OR and PA alleles at un-24 and het-6 and for balancing selection acting on the gene complex. Significantly, the maintenance of linkage disequilibrium of the PA and OR haplotypes would suggest that In(het-6) also has a trans-species distribution.
That In(het-6) is old is suggested by two observations in addition to its inferred trans-species distribution. First is the extensive sequence divergence between PA and OR haplotypes immediately adjacent to In(het-6) breakpoints. While this may suggest an ancient haplotype split, strong diversifying selection could also result in rapid sequence divergence at the breakpoints. The second observation is that disequilibrium is limited to markers close to the inversion breakpoints. Since regional decay of linkage disequilibrium at neutral markers is expected to occur through time (SABETI et al. 2002), the presence of shared polymorphisms in and around the inversion suggests that In(het-6) is old. A comprehensive taxonomic analysis of the distribution of In(het-6) would provide additional insight into the evolution of the incompatibility gene complex. In this context, it would be interesting to ask whether the association of un-24 with this inversion breakpoint marks the acquisition of heterokaryon incompatibility function or whether the two events are independent, with, perhaps, the inversion event locking a beneficial allelic combination of un-24 and het-6 into a supergene configuration in N. crassa. As for the former of these two possibilities, genetic rearrangements such as gene duplications, inversions, and somatic recombination are responsible for generating diversity at nonself recognition loci in other organisms. In tomato, for example, unequal crossing over between Cf-2 and Cf-5 R-genes can lead to deletions or duplications of open reading frames that affect plant disease resistance. In addition, intragenic recombination between repeated LRR domains within R-genes generates variation that directly affects R-gene function (HAMMOND-KOSACK and JONES 1997). The MLA locus involved in powdery mildew resistance in barley is another example of a gene cluster that has undergone multiple rearrangements (WEI et al. 2002). A final extreme example is evident in somatic recombination within V, D, and J gene segments of B-lymphocytes (class switch recombination) to generate antibody diversity and thus increase nonself recognition potential in mammals (LI et al. 2004). Additional rearrangements around un-24–het-6, aside from the inversions described herein, are suggested by a general lack of synteny around the large subunit of ribonucleotide reductase among N. crassa and the other Sordariomycetes, F. graminearum, Chaetomium globosum, and M. grisea (S. QADRI and M. L. SMITH, unpublished results).
An argument in favor of the second possibility—that the inversion locked together a beneficial combination of preexisting alleles—takes into consideration that a striking characteristic of the un-24–het-6 gene complex is its symmetric incompatibility function, as opposed to the asymmetry of activities by either gene alone. The molecular basis for this asymmetry is not known, but it seems to be a common feature of heterokaryon incompatibility genes (WILLIAMS and WILSON 1966; BIELLA et al. 2002), and a transition from asymmetric to symmetric incompatibility may have driven the establishment of the un-24–het-6 incompatibility gene complex. Recent studies support the idea that heterokaryon incompatibility is a gatekeeper of fungal individuality. Fungal heterokaryon incompatibility genes were shown to cause cell death in a structured, organized manner analogous to PCD (BIELLA et al. 2002; DEMENTHON et al. 2003; GLASS and KANEKO 2003). Furthermore, incompatibility is negatively correlated to the probability of transmission of viruses in the chestnut blight fungus, C. parasitica (BIELLA et al. 2002). In this sense, cell death caused by heterokaryon incompatibility is analogous to the hypersensitive response in plants that limits pathogen infection and to the apoptotic response induced by killer T lymphocytes in response to viral infection (KAGI et al. 1996; GREENBERG and YAO 2004). Here, however, heterokaryon incompatibility represents a preventive strategy in the battle against viral infection, rather than a direct response to pathogen attack. One would predict that strong incompatibility reactions would be more effective in limiting the transfer of infectious elements between incompatible individuals of Neurospora, as was observed for C. parasitica (BIELLA et al. 2002). Assuming that there is a selective advantage to incompatibility, then a gene complex that provides a strong, symmetric incompatibility reaction could provide the selective force to maintain In(het-6) as a balanced polymorphism.
The adaptive significance of the un-24–het-6 gene complex requires further investigation. In particular, the observation that escape strains that were functionally un-24PA het-6OR have low fitness, as measured by the frequency of viable ascospore progeny produced, suggests that specific interactions may have developed within the respective OR and PA gene complexes. These interactions apparently do not result in incompatibility of particular combinations of un-24 and het-6, as is observed with nonallelic heterokaryon incompatibility loci in P. anserina, for example (SAUPE 2000), since, in this study, un-24PA het-6OR escape strains do not exhibit slow growth or reduced conidiation, two characteristics of self-incompatibility (NEWMEYER and GALEAZZI 1977; SMITH et al. 1996). The low fecundity of un-24PA het-6OR escapes is also distinct from the barren phenotype observed when Neurospora strains with segmental duplications are mated to wild-type strains. In contrast to the barren condition, all un-24PA het-6OR escapes that we tested produced abundant perithecia and ascospores after mating. However, unlike functionally un-24OR het-6OR escape strains, in which the un-24PA copy is silenced, the un-24PA het-6OR escapes, which have the un-24OR copy silenced, produce a significantly lower frequency of spores that are viable. Furthermore, the inability of escape strains with either un-24PA het-6OR or un-24OR het-6PA phenotypes to undergo strong incompatibility reactions with any of the other haplotypes suggests that these combinations are also impaired in nonself recognition function. Such strains could provide a hub for the propagation of infectious elements in the population at large and may have been selected against.
Finally, the repetitive element noted in the vicinity of brk-1 has interesting implications for the evolution of incompatibility function. Similar elements are often associated with genome instability and rearrangement (KAZAZIAN and GOODIER 2002). For example, a variety of transposon types are associated with inversion breakpoints in Drosophila (Galileo, CÁCERES et al. 1999; Hobo, LYTTLE and HAYMER 1993), mosquito (Odysseus, MATHIOPOULOS et al. 1998), and maize (Ac, ZHANG and PETERSON 1999). Inversions can occur by homologous recombination between two similar mobile elements situated on the same chromosome or may be the secondary consequence of a double-stranded break associated with a transposition event. It is therefore possible that the putative repetitive element associated with In(het-6) may have had a direct involvement in the inversion event. The incidence of a putative repetitive element between brk-1 and het-6, which itself is found in multiple copies in Neurospora and other genomes, is particularly interesting since it suggests that there may be a direct role for mobile elements in the origin of nonself recognition in fungi as is now hypothesized for mosquitoes (SINKINS et al. 2005).
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