Evidence for balancing selection operating at the het-c heterokaryon incompatibility locus in a group of filamentous fungi

In filamentous fungi, het loci (for heterokaryon incompatibility) are believed to regulate self/nonself-recognition during vegetative growth. As filamentous fungi grow, hyphal fusion occurs within an individual colony to form a network. Hyphal fusion can occur also between different individuals to form a heterokaryon, in which genetically distinct nuclei occupy a common cytoplasm. However, heterokaryotic cells are viable only if the individuals involved have identical alleles at all het loci. One het locus, het-c, has been characterized at the molecular level in Neurospora crassa and encodes a glycine-rich protein. In an effort to understand the role of this locus in filamentous fungi, we chose to study its evolution by analyzing het-c sequence variability in species within Neurospora and related genera. We determined that the het-c locus was polymorphic in a field population of N. crassa with close to equal frequency of each of the three allelic types. Different species and even genera within the Sordariaceae shared het-c polymorphisms, indicating that these polymorphisms originated in an ancestral species. Finally, an analysis of the het-c specificity region shows a high occurrence of nonsynonymous substitution. The persistence of allelic lineages, the nearly equal allelic distribution within populations, and the high frequency of nonsynonymous substitutions in the het-c specificity region suggest that balancing selection has operated to maintain allelic diversity at het-c. Het-c shares this particular evolutionary characteristic of departing from neutrality with other self/nonself-recognition systems such as major histocompatibility complex loci in mammals and the S (self-incompatibility) locus in angiosperms.

The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog

The het-s locus of Podospora anserina is a heterokaryon incompatibility locus. The coexpression of the antagonistic het-s and het-S alleles triggers a lethal reaction that prevents the formation of viable heterokaryons. Strains that contain the het-s allele can display two different phenotypes, [Het-s] or [Het-s*], according to their reactivity in incompatibility. The detection in these phenotypically distinct strains of a protein expressed from the het-s gene indicates that the difference in reactivity depends on a posttranslational difference between two forms of the polypeptide encoded by the het-s gene. This posttranslational modification does not affect the electrophoretic mobility of the protein in SDS/PAGE. Several results suggest a similarity of behavior between the protein encoded by the het-s gene and prions. The [Het-s] character can propagate in [Het-s*] strains as an infectious agent, producing a [Het-s*] → [Het-s] transition, independently of protein synthesis. Expression of the [Het-s] character requires a functional het-s gene. The protein present in [Het-s] strains is more resistant to proteinase K than that present in [Het-s*] mycelium. Furthermore, overexpression of the het-s gene increases the frequency of the transition from [Het-s*] to [Het-s]. We propose that this transition is the consequence of a self-propagating conformational modification of the protein mediated by the formation of complexes between the two different forms of the polypeptide.

The pollen determinant of self-incompatibility in Brassica campestris

Many flowering plants possess self-incompatibility (SI) systems that prevent inbreeding. In Brassica, SI is controlled by a single polymorphic locus, the S locus. Two highly polymorphic S locus genes, SLG (S locus glycoprotein) and SRK (S receptor kinase), have been identified, both of which are expressed predominantly in the stigmatic papillar cell. We have shown recently that SRK is the determinant of the S haplotype specificity of the stigma. SRK is thought to serve as a receptor for a pollen ligand, which presumably is encoded by another polymorphic gene at the S locus. We previously have identified an S locus gene, SP11 (S locus protein 11), of the S9 haplotype of Brassica campestris and proposed that it potentially encodes the pollen ligand. SP11 is a novel member of the PCP (pollen coat protein) family of proteins, some members of which have been shown to interact with SLG. In this work, we identified the SP11 gene from three additional S haplotypes and further characterized the gene. We found that (i) SP11 showed an S haplotype-specific sequence polymorphism; (ii) SP11 was located in the immediate flanking region of the SRK gene of the four S haplotypes examined; (iii) SP11 was expressed in the tapetum of the anther, a site consistent with sporophytic control of Brassica SI; and (iv) recombinant SP11 of the S9 haplotype applied to papillar cells of S9 stigmas, but not of S8 stigmas, elicited SI response, resulting in inhibition of hydration of cross-pollen. All these results taken together strongly suggest that SP11 is the pollen S determinant in SI.

Structural Analysis and Molecular Model of a Self-Incompatibility RNase from Wild Tomato1

Self-incompatibility RNases (S-RNases) are an allelic series of style glycoproteins associated with rejection of self-pollen in solanaceous plants. The nucleotide sequences of S-RNase alleles from several genera have been determined, but the structure of the gene products has only been described for those from Nicotiana alata. We report on the N-glycan structures and the disulfide bonding of the S3-RNase from wild tomato (Lycopersicon peruvianum) and use this and other information to construct a model of this molecule. The S3-RNase has a single N-glycosylation site (Asn-28) to which one of three N-glycans is attached. S3-RNase has seven Cys residues; six are involved in disulfide linkages (Cys-16-Cys-21, Cys-46-Cys-91, and Cys-166-Cys-177), and one has a free thiol group (Cys-150). The disulfide-bonding pattern is consistent with that observed in RNase Rh, a related RNase for which radiographic-crystallographic information is available. A molecular model of the S3-RNase shows that four of the most variable regions of the S-RNases are clustered on one surface of the molecule. This is discussed in the context of recent experiments that set out to determine the regions of the S-RNase important for recognition during the self-incompatibility response.

Geometric incompatibility in a fault system.

Interdependence between geometry of a fault system, its kinematics, and seismicity is investigated. Quantitative measure is introduced for inconsistency between a fixed configuration of faults and the slip rates on each fault. This measure, named geometric incompatibility (G), depicts summarily the instability near the fault junctions: their divergence or convergence ("unlocking" or "locking up") and accumulation of stress and deformations. Accordingly, the changes in G are connected with dynamics of seismicity. Apart from geometric incompatibility, we consider deviation K from well-known Saint Venant condition of kinematic compatibility. This deviation depicts summarily unaccounted stress and strain accumulation in the region and/or internal inconsistencies in a reconstruction of block- and fault system (its geometry and movements). The estimates of G and K provide a useful tool for bringing together the data on different types of movement in a fault system. An analog of Stokes formula is found that allows determination of the total values of G and K in a region from the data on its boundary. The phenomenon of geometric incompatibility implies that nucleation of strong earthquakes is to large extent controlled by processes near fault junctions. The junctions that have been locked up may act as transient asperities, and unlocked junctions may act as transient weakest links. Tentative estimates of K and G are made for each end of the Big Bend of the San Andreas fault system in Southern California. Recent strong earthquakes Landers (1992, M = 7.3) and Northridge (1994, M = 6.7) both reduced K but had opposite impact on G: Landers unlocked the area, whereas Northridge locked it up again.

Wolbachia, normally a symbiont of Drosophila, can be virulent, causing degeneration and early death

Wolbachia, a maternally transmitted microorganism of the Rickettsial family, is known to cause cytoplasmic incompatibility, parthenogenesis, or feminization in various insect species. The bacterium–host relationship is usually symbiotic: incompatibility between infected males and uninfected females can enhance reproductive isolation and evolution, whereas the other mechanisms enhance progeny production. We have discovered a variant Wolbachia carried by Drosophila melanogaster in which this cozy relationship is abrogated. Although quiescent during the fly’s development, it begins massive proliferation in the adult, causing widespread degeneration of tissues, including brain, retina, and muscle, culminating in early death. Tetracycline treatment of carrier flies eliminates both the bacteria and the degeneration, restoring normal life-span. The 16s rDNA sequence is over 98% identical to Wolbachia known from other insects. Examination of laboratory strains of D. melanogaster commonly used in genetic experiments reveals that a large proportion actually carry Wolbachia in a nonvirulent form, which might affect their longevity and behavior.

The role of Wolbachia bacteria in reproductive incompatibilities and hybrid zones of Diabrotica beetles and Gryllus crickets

A rickettsial bacterium in the genus Wolbachia is the cause of a unidirectional reproductive incompatibility observed between two major beetle pests of maize, the western corn rootworm, Diabrotica virgifera virgifera, and the Mexican corn rootworm, D. v. zeae. These subspecies are allopatric except for two known regions of sympatry in Texas and Mexico. We demonstrate that populations of D. v. virgifera, with the exception of two populations in southern Arizona, are infected with a strain of Wolbachia. Populations of D. v. zeae are not infected. Treatment of D. v. virgifera with tetracycline eliminated the Wolbachia and removed the reproductive incompatibility. Similar patterns of reproductive incompatibility exist among taxa of the cricket genus Gryllus. Gryllus assimilis, G. integer, G. ovisopis, G. pennsylvanicus, and G. rubens are infected with Wolbachia whereas G. firmus is usually not. Populations of G. rubens and G. ovisopis carry the same Wolbachia strain, which is distinct from that of G. integer. G. pennsylvanicus is infected with two Wolbachia strains, that found in G. rubens and one unique to G. pennsylvanicus. Moreover, a proportion of G. pennsylvanicus individuals harbors both strains. Wolbachia may have influenced speciation in some members of the genus Gryllus by affecting the degree of hybridization between species. Given that Wolbachia infections are relatively common in insects, it is likely that other insect hybrid zones may be influenced by infections with Wolbachia.

Striking sequence similarity in inter- and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris: Implications for the evolution and recognition mechanism

Self-incompatibility in Brassica is controlled by a single multi-allelic locus (S locus), which contains at least two highly polymorphic genes expressed in the stigma: an S glycoprotein gene (SLG) and an S receptor kinase gene (SRK). The putative ligand-binding domain of SRK exhibits high homology to the secretory protein SLG, and it is believed that SLG and SRK form an active receptor kinase complex with a self-pollen ligand, which leads to the rejection of self-pollen. Here, we report 31 novel SLG sequences of Brassica oleracea and Brassica campestris. Sequence comparisons of a large number of SLG alleles and SLG-related genes revealed the following points. (i) The striking sequence similarity observed in an inter-specific comparison (95.6% identity between SLG14 of B. oleracea and SLG25 of B. campestris in deduced amino acid sequence) suggests that SLG diversification predates speciation. (ii) A perfect match of the sequences in hypervariable regions, which are thought to determine S specificity in an intra-specific comparison (SLG8 and SLG46 of B. campestris) and the observation that the hypervariable regions of SLG and SRK of the same S haplotype were not necessarily highly similar suggests that SLG and SRK bind different sites of the pollen ligand and that they together determine S specificity. (iii) Comparison of the hypervariable regions of SLG alleles suggests that intragenic recombination, together with point mutations, has contributed to the generation of the high level of sequence variation in SLG alleles. Models for the evolution of SLG/SRK are presented.

Restriction-modification gene complexes as selfish gene entities: Roles of a regulatory system in their establishment, maintenance, and apoptotic mutual exclusion

We have reported some type II restriction-modification (RM) gene complexes on plasmids resist displacement by an incompatible plasmid through postsegregational host killing. Such selfish behavior may have contributed to the spread and maintenance of RM systems. Here we analyze the role of regulatory genes (C), often found linked to RM gene complexes, in their interaction with the host and the other RM gene complexes. We identified the C gene of EcoRV as a positive regulator of restriction. A C mutation eliminated postsegregational killing by EcoRV. The C system has been proposed to allow establishment of RM systems in new hosts by delaying the appearance of restriction activity. Consistent with this proposal, bacteria preexpressing ecoRVC were transformed at a reduced efficiency by plasmids carrying the EcoRV RM gene complex. Cells carrying the BamHI RM gene complex were transformed at a reduced efficiency by a plasmid carrying a PvuII RM gene complex, which shares the same C specificity. The reduction most likely was caused by chromosome cleavage at unmodified PvuII sites by prematurely expressed PvuII restriction enzyme. Therefore, association of the C genes of the same specificity with RM gene complexes of different sequence specificities can confer on a resident RM gene complex the capacity to abort establishment of a second, incoming RM gene complex. This phenomenon, termed “apoptotic mutual exclusion,” is reminiscent of suicidal defense against virus infection programmed by other selfish elements. pvuIIC and bamHIC genes define one incompatibility group of exclusion whereas ecoRVC gene defines another.

The incompatible interaction between Phytophthora parasitica var. nicotianae race 0 and tobacco is suppressed in transgenic plants expressing antisense lipoxygenase sequences

Nicotiana tabacum 46-8 cultivar displays an incompatible interaction with race 0 of Phytophthora parasitica var. nicotianae (Ppn), a fungal pathogen of most tobacco cultivars. At the plant level, incompatibility is characterized by the induction of lipoxygenase (LOX, EC = 1.13.11.12) activity and localized hypersensitive cell death before defense gene activation. To evaluate the involvement of LOX in the onset of plant defense, tobacco 46-8 plants were genetically engineered using full-length or partial-length antisense (AS) tobacco LOX cDNA constructs. AS expression strongly reduced elicitor- and pathogen-induced LOX activity. Eight independent AS-LOX lines were selected and assayed for their response to Ppn. After root or stem inoculation with race 0, all AS-LOX lines but one displayed a compatible phenotype whereas control transformed plants, not containing the AS-LOX cassette, showed the typical incompatible reaction. The presence of the fungus in transgenic lines was demonstrated by PCR amplification of a Ppn-specific genomic sequence. A linear relationship was found between the extent of LOX suppression and the size of the lesion caused by the fungus. The AS-LOX plants also showed enhanced susceptibility toward the compatible fungus Rhizoctonia solani. The results demonstrate the strong involvement of LOX in the establishment of incompatibility in plant–microorganism interactions, consistent with its role in the defense of host plants.

Regulation of S-Like Ribonuclease Levels in Arabidopsis. Antisense Inhibition of RNS1 or RNS2 Elevates Anthocyanin Accumulation1

The S-like ribonucleases (RNases) RNS1 and RNS2 of Arabidopsis are members of the widespread T2 ribonuclease family, whose members also include the S-RNases, involved in gametophytic self-incompatibility in plants. Both RNS1 and RNS2 mRNAs have been shown previously to be induced by inorganic phosphate (Pi) starvation. In our study we examined this regulation at the protein level and determined the effects of diminishing RNS1 and RNS2 expression using antisense techniques. The Pi-starvation control of RNS1 and RNS2 was confirmed using antibodies specific for each protein. These specific antibodies also demonstrated that RNS1 is secreted, whereas RNS2 is intracellular. By introducing antisense constructs, mRNA accumulation was inhibited by up to 90% for RNS1 and up to 65% for RNS2. These plants contained abnormally high levels of anthocyanins, the production of which is often associated with several forms of stress, including Pi starvation. This effect demonstrates that diminishing the amounts of either RNS1 or RNS2 leads to effects that cannot be compensated for by the actions of other RNases, even though Arabidopsis contains a large number of different RNase activities. These results, together with the differential localization of the proteins, imply that RNS1 and RNS2 have distinct functions in the plant.

Assortative fertilization in Drosophila

The concept of gametic isolation has its origins in the 1937 edition of T. Dobzhansky’s Genetics and the Origin of Species. Involving either positive assortative fertilization (as opposed to self-incompatibility) or negative assortative fertilization, it occurs after mating but prior to fertilization. Gametic isolation is generally subsumed under either prezygotic or postmating isolation and thus has not been the subject of extensive investigation. Examples of assortative fertilization in Drosophila are reviewed and compared with those of other organisms. Potential mechanisms leading to assortative fertilization are discussed, as are their evolutionary implications.

How flowering plants discriminate between self and non-self pollen to prevent inbreeding.

Flowering plants have evolved various genetic mechanisms to circumvent the tendency for self-fertilization created by the close proximity of male and female reproductive organs in a bisexual flower. One such mechanism is gametophytic self-incompatibility, which allows the female reproductive organ, the pistil, to distinguish between self pollen and non-self pollen; self pollen is rejected, whereas non-self pollen is accepted for fertilization. The Solanaceae family has been used as a model to study the molecular and biochemical basis of self/non-self-recognition and self-rejection. Discrimination of self and non-self pollen by the pistil is controlled by a single polymorphic locus, the S locus. The protein products of S alleles in the pistil, S proteins, were initially identified based on their cosegregation with S alleles. S proteins have recently been shown to indeed control the ability of the pistil to recognize and reject self pollen. S proteins are also RNases, and the RNase activity has been shown to be essential for rejection of self pollen, suggesting that the biochemical mechanism of self-rejection involves the cytotoxic action of the RNase activity. S proteins contain various numbers of N-linked glycans, but the carbohydrate moiety has been shown not to be required for the function of S proteins, suggesting that the S allele specificity determinant of S proteins lies in the amino acid sequence. The male component in self-incompatibility interactions, the pollen S gene, has not yet been identified. The possible nature of the pollen S gene product and the possible mechanism by which allele-specific rejection of pollen is accomplished are discussed.