Molecular mechanisms of self-incompatibility in flowering plants and fungi — different means to the same end

REVIEWS

Organisms that reproduce sexually usually display adaptations that prevent self-fertilization and promote outcrossing. These adaptations increase variation in offspring and counteract the deleterious effects of inbreeding. Many animals eliminate the possibility of selfing by having individuals of two different sexes. This strategy succeeds as a breeding system provided there is no restriction on the mixing of males and females and/or their gametes, but, when mixing is restricted (as a consequence of the organism being of limited mobility, or immobile), having only half of the individuals able to produce offspring can be a serious reproductive handicap. Many sexually reproducing organisms are therefore hermaphrodite and rely on other means to avoid selfing. Flowering plants are almost exclusively hermaphrodite, while fungi, in which sexual dimorphism is rarely seen, can be thought of as functionally hermaphrodite because the nature of their breeding systems cannot preclude self-fertilization. Adaptations to prevent selfing are astonishingly diverse in flowering plants, but by far the most widespread is self-incompatibility (52). SI is a molecular process that allows the female cells of the pistil (consisting of stigma and style) to recognize and reject self pollen. In fungi, which appear to be more conservative in their range of mechanisms to prevent selfing, self-incompatibility mediated by mating-type is again the most general mechanism. Classical

genetics

of 51 and mating

Molecular mechanisms of self-incompatibility in flower&g plants and fungi different means the same end to

Hermaphrodite

floweringplants

and fungi face the same sexual

dilemma - how to avoid self-fertilization.

Both have evolved

ingenious recognition systems that reduce or eliminate the possibility of selfing. These self-incompatibility

(SI) systems offer unique

opportunities to study recognition and signalling in non-animal

type

Most systems of SI in flowering plants are under the control of a single multiallelic S (self-incompatibility) locus. A notable exception occurs in the grasses (Poaceae) where two multiallelic loci (S and Z) control SI. S alleles act in ‘opposition’, such that, when they are matched in pollen and pistil, pollen development is inhibited. Two genetically distinct forms of SI have been identified on the basis of whether the incompatibility phenotype of the pollen is determined by its own haploid genome [gametophytic SI (GSI)] or by that of the diploid parent [sporophytic SI (SSI)]’ (Fig. 1). GSI is found typically in the Solanaceae, Scrophulariaceae, Rosaceae,Papaveraceaeand Poaceae, whereas SSIoccurs in the Brassicaceaeand Asteraceae. Gametophytic determination of SI dictates that the two S alleles present in the pistil must behave in a codominant manner, so that both allelic S products are present in the pistil, whereas sporophytic determination of SI places no such constraints and permits dominance/recessive relationships to occur between S alleles. In fungi, SI is also controlled by one or two matingtype loci, which can, like S loci, be multiallelic. However, the alleles of the mating-type loci are not oppositional; instead, they function in a complementary way, such that, when different alleles are present in mating cells, the interaction is compatible. Matingtype systems with one locus are referred to as ‘bipolar’, whereas mating-type systems controlled by two loci are referred to as ‘tetrapolar’. The terms bipolar and tetrapolar refer to the number of mating types (2 or 4, respectively) that are possible in the progeny of a given cross. In simple bipolar systems found in

ascomycetes such as Saccharomyces cerevisiae and Neurospora crassa,the single mating-type locus (MAT) is diallelic, whereas in bipolar basidiomycetes such as Polypoms palustris the single mating-type locus is multiallelic. In tetrapolar systems, two loci, usually designated A and B, determine compatibility; in the basidiomycetes Coprinus cinereus and Schizophyllum commune, these loci are multiallelic, but in the hemibasidiomycete Ustilago maydis only one locus (b) is multiallelic, the other (a) is diallelic. In both bipolar and tetrapolar systems, matings are only compatible if the alleles at the mating-type loci of the two mating cells are different (for reviews, see Refs 2 and 3). Thus, in terms of their classical genetics, the SI systems of flowering plants and fungi appear very similar. However, the fact that, in flowering plants, S alleles act in opposition (leading to rejection of self), and, in fungi, mating-type alleles act by complementation (leading to acceptance of non-self), predicts that the molecular mechanisms involved will be quite distinct. Recent molecular studies have revealed just how different these mechanisms are.

trends

0 1996 Elsevier Science Ltd

in CELL BIOLOGY

(Vol.

6) November

1996

cells and also represent model systems for studying the evolution of breeding systems at a molecular level. In this review, the authors discuss recent molecular data that predict an astonishing diversity in the cellular mechanisms of SI operating in flowering plants and fungi.

PII: SO962.8924(96)10037-4

Simon Hiscock and Hugh Dickinson are at the Dept of Plant Sciences, University of Oxford, South Parks Road, Oxford, UK OX1 3RB; and Ursula Kiies is at the lnstitut fur Mikrobiologie, ETH Zurich, Schmelzbergstr. 7, CH-8092 Zurich, Switzerland. E-mail: SimonHiscock @plant-sciences. oxford.ac.uk

421

(a) si

52 Pollen Stigma

style

Pollen tube

Ovule

(b) sis2

SlS2

sis2

s3s4

FIGURE Genetic

control

of gametophytic

sporophytic

self-incompatibility

self-incompatibility by its own

generally Papaver

rhoeas

of the stigma).

with

of the two

pollen

is rendered

S,S, individual. pollen

that

derived

products from

is usually

When

with

Pollen from

the diploid inhibited

Molecular

indicate

from

an S,S, plant

coating’*,

tissue

of the anther

tapetum),

of the stigma.

genotype,

roman

basis of sporophytic

text

is is

of the

of the parent

in the pollen

on the surface

the

of any other

located

if S-alleles are codominant, all pollen derived an S,S, plant is inhibited Italics

in the pistil,

(b) In SSI, the SI phenotype genome

are

are inhibited

an S,S, parent

pistil or that

SO% of the pollen

is are

S-allele is matched

S-alleles

its own

tubes

exceptions

tubes

a pollen

by the diploid being

and pollen

pollen

codominant

the S,S, pistil.

is determined

(S-allele pollen

Note

with

where

incompatible.

incompatible compatible

genome,

plants.

of the pollen

in the style (notable

and the grasses,

on the surface either

phenotype

haploid

inhibited

(a) and

(SSI) (b) in flowering

(a) In GSI, the self-incompatibility determined

(CSI)

1

plant

which

is

and the Note

that,

from, for example, on an S,S, stigma. indicates

phenotype.

self-incompatibility

Molecular studies of SSI have focused mainly on one species - Brassica oleracea, cultivars of which include cabbages, kales and Brussels sprouts. The S locus of B. oleracea spans in excess of 200 kb of DNA and has been demonstrated to contain at least two genes, SLG (Slocus glycoprotein) and SRK(Sreceptor kinase), both of which are required for the correct functioning of S14r5.The molecular complexity of the locus, together with the extensive allelic polymorphisms 422

exhibited by SLG and SRK, has prompted the redesignation of S alleles as S haplotype@. SLG encodes a secreted glycoprotein that localizes primarily to the cell wall of the epidermal cells (papillae) of the stigma, whereas SRK encodes a functional transmembrane receptor serine/threonine kinase that spans the plasma membrane of the papillae7 (Fig. 2). The extracellular domain of SRK,which extrudes into the cell wall, shares considerable homology with SLG; for a given haplotype, this homology may represent >90% identity6J7, suggesting that there was a very close co-evolution of these two sequences. In certain S haplotypes, intermediate ‘variants’ of SLG and SRK have been identified, for example the SLG2 gene produces an alternative transcript that encodes a membrane-anchored version of SLG2 (Ref. S), while SRK3 produces a number of alternative transcripts - one of which encodes a soluble truncated form of the extracellular domain of SRK (Ref. 9). These findings suggest that SLG may have evolved from SRKby gene duplications. Another level of complexity came from the discovery that the S genes are just part of a much larger ‘superfamily’ of S-locus-related genes and sequences found in Brassica, Arabidopsis and other species within and outside the Brassicaceae’jJO.None of these sequences is linked to the S locus, although two S-locus-related glycoprotein genes, SLRI and SLR2, exhibit expression patterns and regulation almost identical to SLGsll. Current models for the operation of SSIin Brassica (Fig. 2) predict a central role for SRK in the initiation of a signal-transduction pathway within a papillar cell that culminates in the rejection of incompatible pollen6,7. The rejection process must be highly focused within the cell because a single papillar cell is, at the same time, able to inhibit the development of an incompatible grain while permitting the development of a compatible grainlz. So far, the pollen S determinant, which is presumed to bind to the extracellular domain of SRK, is unknown. Sporophytic determination of the incompatibility phenotype of the pollen predicts that the male determinant will reside in the pollen coating, which is diploid in origin, being added to the pollen in the anther. Indeed, physiological evidence has confirmed this prediction12. Analysis of the spectrum of proteins contained within the ppllen coating of Bras&u has identified a group of small proteins (mol. mass 6-8 kDa), one of which, PCP7 (pollen coat protein 7 kDa), interacts in vitro with stigmatic S-classmolecules13J4. Amino acid sequence data from PCP7 reveal that it shares homology with a group of thionin-like plant defence proteins u. Doughty, pers. commun.). However, there is no firm evidence as yet to suggest that these in vitro interactions are involved in SI. Recently, Boyes and Nasrallahls identified an anther-expressed gene at the S locus of the S2 haplotype of B. oleracea. This gene, SLA (S locus anther) expresses two alternative transcripts, which predict proteins of 7.5 kDa and 10 kDa. The predicted amino acid sequences of these transcripts show no homology to PCPs or indeed to any other known proteins. Nevertheless, the linkage of SLA to the S locus would suggest a role in SSIprovided that these transcripts are translated. trends

in CELL BIOLOGY

(Vol.

6) November

1996

The identification of molecules involved in signal transduction downstream of SRK will also be essential in determining the mechanism of SSI. Circumstantial evidence suggests that phosphorylation and/or dephosphorylation events are involvedr6,r7, but the molecular targets of these events are not known. Interestingly, two thioredoxin H clones have been identified recently that interact specifically with the phosphorylated kinase domain of SRK-910 from B. napus; one of these thioredoxins, THL-1, is phosphorylated as a consequence of this interaction in vitro (D. Goring, pers. commun.). Molecular

basis of gametophytic

self-incompatibility

In Nicotiana ala& (ornamental tobacco), Petunia inflata and other members of the Solanaceae with GSI, genes encoding S-associated glycoproteins have been cloned and shown to be ribonucleases (for review, seeRef. 18). Genes for similar so-called S-RNases have been cloned from species in the Rosaceae and Scrophulariaceae 1g,20 . Like SLG and SRK, genes encoding S-RNase are extremely polymorphic and can only be recognized by a number of small conserved regions-one of which is the RNase active site. S-RNases are found primarily in the cells of the stigma and in the conductive tissue of the style, where pollen tubes are arrestedzl. Conclusive proof that S-RNases are essential for the operation of GSI was demonstrated by transformation-based bioassays for loss and gain of S-RNase function2z-24. Interestingly, it has been demonstrated recently that S-RNases also mediate certain types of interspecific incompatibility in Nico&‘ana25. Together, these findings support a model for GSI based on specific degradation of pollen RNAs (messenger and/or ribosomal) by S-RNasesduring an incompatible reaction18,24. How, then, is S-specificity achieved? Since no S genes specific to pollen have yet been identified, only speculation is possible. Two candidates for the pollen S-determinant have been proposed: an S-specific S-RNase translocator located within the plasma membrane of the pollen tube, which recognizes and internalizes the allelic S-RNase, and an inhibitor of S-RNaseswhich is unable to inhibit the allelic S-RNase (see Ref. 18). Emerging data from an analysis of mutant plants that produce pollen lacking a normal self-incompatibility response tend to support the inhibitor model (J. Golz and E. Newbigin, pers. commun.). GSI is not only mediated through S-RNases:in the field poppy (Papaver rkoeas), a different mechanism operates based on a Ca2+-mediated signalling pathway in the pollen26 (Fig. 3). So far, four stigmatically expressed S genes have been cloned from Papaver rkoeas27,28.These encode small extracellular glycoproteins with no significant homology to any known proteins. Using an in vitro bioassay system, FranklinTong et al. 2g have demonstrated that these stigmatic S glycoproteins induce the arrest of growing pollen tubes in an S-allele-dependent manner. This inhibition has been correlated with a transient rise in cytosolic Ca2+within the pollen tube and with the specific phosphorylation of a number of pollen proteins, one of which (~26) has been partially characterizedao, trends

in CELL BIOLOGY

(Vol.

6) November

1996

Key

I

Pollen ligand

SRK

SLG

(THL-I

E f

FIGURE

2

Hypothetical Inhibition before

model

for the mechanism

of the pollen germination

transmembrane

of stigmatic

glycoprotein

(SLC),

>90%

S-haplotype, identity.

extracellular interaction

kinase

An S-specific

with

culminates

ligand

SLC and induce

in localized domain

and a secreted

domain

through

inhibition

to bind with

of SRK with

of pollen

the

a primary

dimerization

pathway

For a

of SRK share

of SRK molecules.

on serine and threonine pathway within a papilla

of the signal-transduction

of the kinase

by a

of papillae.

is thought

olerucea.

usually

in the plasma

cells (papillae),

in the cell walls

of SRK, perhaps

SRKs then autophosphorylate initiate a signal-transduction stage

to be mediated

SLC and the receptor

domain

surface

(SRK), located

epithelial

located

of SSI in Brassica

on the stigma

and is thought receptor

membrane given

occurs

residues that

development. may

a thioredoxin

involve

to

An early interaction

H protein

(THL-1)

(see text).

Synthesis of specific ‘response’ mRNAs has also been detected in incompatible pollen26. So far, no S-linked receptor protein has been identified in the pollen of P. rkoeas, but a recently characterized membraneassociated glycoprotein, SBP (S-binding protein), from pollen has been shown to bind to stigmatic S-glycoproteins, albeit in a non-S-specific manner31. It is thought that this protein may ‘present’ the S-glycoprotein to the pollen S-receptor in a manner similar to the accessory receptors of certain mammalian growth-factor receptors31. The two-locus system of GSI found in the grasses predicts yet another molecular variation in the way in which GSI is achieved. This promise was fulfilled by the cloning of the first S alleles from a grass, Pkalaris coerulescens,by Li et a1.32,who showed that these genes encode active thioredoxin H proteins, which are expressed predominantly in the pollen. 423

Stigma

Key

&

SW

i

cell

S receptor

S-glycoprotein

FIGURE

3

Hypothetical model for the mechanism of gametophytic self-incompatibility (CA) in Papaver rhoeas. Inhibition of the growing pollen tube is on the surface of the stigma and is mediated by a Ca’+-based signal-transduction pathway within the pollen tube. The stigmatic S-glycoprotein is thought to be divalent (C. Franklin, pers. commun.) one domain binding non-specifically to an accessory membrane-bound receptor protein (SBP) that ‘presents’ the S-glycoprotein to a hypothetical S-receptor protein that binds with the S-specific domain of the S-glycoprotein. This interaction induces a rise in the level of cytosolic free Ca2+, which could activate calmodulin-dependent kinase(s) and result in phosphorylation of specific proteins such as p26 (see text), leading to gene transcription and pollen tube arrest.

This finding is intriguing because thioredoxin H molecules have also been implicated in the stigmatic SSI response of Brassica (see previous section). The role of these thioredoxins in GSI in P. coerulescensand SSIin Brassica remains to be determined. Molecular in fungi

mechanisms

controlling

mating-type

All of the mating-type (MAT) loci characterized so far from bipolar ascomycetes encode transcription factors that regulate, directly or indirectly, the expression of certain mating-type-specific, haploid-specific and diploid-specific genes - including those that encode pheromones and pheromone receptors2. The sequences of the transcription factors encoded by the two ‘allelic’ forms of MAT loci are usually so different that they are now often referred to as idiomorphs3,33. So far, three classesof transcription factor have been characterized at the MAT loci of ascomycetes homeodomain, high-mobility group (HMG) domain and al-domain - different combinations of these 424

transcription factors being used by different species for mating-type determination2,3. In the yeast S. cerevisiae,the MATa idiomorph encodes a homeodomain transcription factor (~2) and an additional transcription factor (al), while the MATa idiomorph encodes an unrelated homeodomain transcription factor (al) and a non-functional protein (a.??).All haploid cells contain genes for the a and a pheromones and the a and a pheromone receptors, but only the appropriate pair of genes is expressed in each cell type: the genes for the a pheromone and the a pheromone receptor are expressed constitutively in MATa cells, whereas, in MATcl cells, their expression is repressed by the a2 transcription factor. By contrast, the genes for the a pheromone and the a pheromone receptor are only active in MATa cells because the al transcription factor is required for their transcriptional activation. Complementary interaction between each pheromone and its receptor (Fig. 4a) leads to cell fusion which is followed by nuclear fusion and dimerization of the al and a2 transcription factors (Fig. Sa) to form an ‘active complex’ that represses a-specific and haploid-specific genes - a prerequisite for the initiation of sexual (diploid) differentiation2,34,35. The mating-type system in S. cerevisiae remains the best characterized of all fungal matingtype systems and serves as a paradigm for the study of mating-type systems in all other fungi. In the fission yeast Schizosaccharomycespombe, the two MAT idiomorphs, ‘plus’ (matl-P) and ‘minus’ (matl-M) also contain two genes. One idiomorphic pair, rnatl-Pc (unclassified) and matl-Mc (encoding an HMG domain transcription factor), is responsible for establishing a pheromone communication system that mediates conjugation in a similar way to S. cerevisiae.However, unlike S. cerevisiae, entry into meiosis requires a pheromone-induced signal to activate the additional MAT genes mat1 -Pm (encoding a homeodomain transcription factor related to 132)and matl-Mm (unclassified) that together induce expression of the mei gene, which activates meiosis36,37.In common with the yeasts, mating type in filamentous ascomycetes, such as N. crassaand Podosporaanserina, is conferred by alternative idiomorphic transcription factors, although it has yet to be demonstrated that mating-type-specific pheromones and their receptors are among the genes that they regulate3. In basidiomycetes, the majority of molecular studies have focused on species with tetrapolar mating-type systems. The two mating-type loci contain genes that encode, respectively, pheromones and pheromone receptors, and transcription factors38-41. In Ustilago may&s, the two alleles of the a locus, al and a.2, each contain a pair of genes encoding a pheromone and a pheromone receptor; these alleles are so dissimilar in sequence that they can (like MAT ‘alleles’) be described as idiomorphs 42.Complementary interaction between a pheromone and a pheromone receptor derived from opposite idiomorphs leads to fusion of unicellular ‘yeast-like’ cells and the establishment of a dikaryotic mycelium43 (Fig. 4a). Interestingly, it has been shown recently that this interaction activates the genes for transcription factors at the b locus, which in turn are responsible for the fine-regulation of the a genes trends

in CELL BIOLOGY

(Vol.

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1996

-this dialogue between the two different mating-type genes being mediated by the novel transcription factor Prf144,45. In the higher basidiomycetes Schizophyllum commune and Coprinus cinereus, it is the B locus that encodes pheromone and pheromone receptor genes, but, unlike the a locus of U. maydis, the multiallelic B locus contains several copies of these genes; in common with other mating-type loci, the ‘allelic’ forms of the B loci are extremely polymorphic41,46 (S. O’Shea and L. Casselton, pers. commun.). In S. commune, each B allele consists of two similar, but distinct, units of three genes encoding pheromones and one gene encoding a pheromone receptor (Fig. 4b). Functional analysis in vivo suggests that only pheromones and pheromone receptors derived from the same allelic unit, but different alleles, can interact4r (Fig. 4b). Functional redundancy occurs because only one of the three pheromones from a given unit is required to activate the receptor (of that unit) from the different allelic forms41,46,and the receptors can interact with one or more pheromones of the same unit derived from different alleles (Fig. 4b). It is believed that there is no recognition between products of the same allele, nor between the products of the two independent units of different B alleles41,46(C. A. Raper, pers. commun.). At present, it is not clear where or when the pheromones and their receptors interact during compatible matings because the filamentous hyphae of S. commune and C. cinereus can fuse freely throughout their life cycles, even within the same mycelium38. In U. maydis, the interaction between pheromones and their respective receptors is clearly extracellulati3 and so the receptors must be situated in the plasma membrane (Fig. 4a). In S. commune and C. cinereus, the most likely times for a similar extracellular interaction to take place are either during the apparent attraction of hyphae towards asexual spores (oidia)47 or during fusion of ‘clamp’ connections in the dikaryotic mycelium38. The genes encoding transcription factors at the A loci of C. cinereusand S.commune and the b locus of V. maydis encode two different types of homeodomain transcription factors (Fig. 5b), and, within a particular species, the different alleles of these multiallelic loci have retained a degree of sequence similarity38,3g. In the simplest locus, b, of U. maydis, there are two divergently transcribed genes, bE and bW, and this pattern of pairs of divergently transcribed genes is repeated trends

in CELL BIOLOGY

(Vol.

6) November

1996

(4

Key:

I.

Pheromone Pheromone

receptor

YY

@I Pheromone genes

Pheromone receptor gene

Pheromone genes

Pheromone receptor gene

Pheromone genes

Pheromone receptor gene

BI

82 Pheromone genes FIGURE

Pheromone receptor gene

4

(a) Schematic diagram to illustrate the role of pheromones and pheromone receptors during recognition between single mating cells of yeasts and the smut fungus Usfilago muydis. In the yeasts Sacchoromyces cerevisiae and Schizosaccboromyces pombe, all haploid cells contain the genes encoding the two different pheromones and their receptors, but the mating-type genes ensure that only one pheromone and one pheromone receptor are produced by each cell. In U. maydis, the genetic situation is different in that the genes that encode the pheromones and pheromone receptors are themselves mating-type genes. Each haploid cell can therefore only produce one pheromone and one pheromone receptor. Pheromone receptors belong to the class of G-protein-coupled seven-bypass transmembrane receptors; binding to the correct pheromone initiates a signal-transduction pathway that leads to cell fusion and sexual development4 OJ’. (b) Schematic representation of the products of the genes for pheromones and the genes for pheromone receptors from two alleles of the B mating-type locus of the basidiomycete fungus Schizophyllum commune. Each B allele encodes two units of one receptor and three genes for pheromones. Arrows denote the possible positive interactions between the products of these genes. Note that interactions may only take place between pheromones and receptors derived from the same allelic units from different alleles41(C. Raper, pers. commun.). The exact cytological location of the interaction between the pheromones and their receptors remains to be determined.

425

and amplified in the more complex A loci of C. cinereus and S. commune38~3g~48. The products of these gene pairs have been designated HDl and HD2 proteins, and their homeodomains share significant homology with those of the S. cerevisiaea2 and al transcription factors, respectively38. Heterodimerization of HDl and HD2 proteins from different A alleles or different b alleles determines intracellular recognition and triggers subsequent sexual development3g (Fig. 5~). Opposition

Compatible interaction active

Activation

or repression

leading to sexual

(4

Incompatible-no

Keyq

and

n

DNA-binding

Specificity

complex

of genes

development

active

complex

,‘:’ 0

domain

domain

and

w

found

Transactivation

Double-stranded

domain

DNA, promoter

region

in red

FIGURE Schematic

representation

of the possible

classes of homeodomain intracellular cerevisiae

recognition

transcribed

different (HDl there sequence

and HD2) may

which

dimerization,

have

contribute

specificity

as indicated hollow

triangular ‘key-key’ number of possible number interactions. and green

426

by the different

genes

positive

interactions

to target

DNA-binding ‘+’ indicates

DNA

result

HDl

a compatible all differing

and HD2

sequences,

so-called

interaction38,3g. neutral ‘lock

to regulate

indicate that the proteins are from different represent the DNA-binding homeodomains,

proteins.

acids

effect

at the

on protein

and key’ fits denote

positive

neutral

and the

interactions,

Given the large a population, the

will far outnumber

in dimerization

in

model specificity

Amino

or negative

denote

interactions

sequences

denote domains

and 5. commune,

at the A locus,

(d) between

shapes:

is a pair of

(c) and (d): Hypothetical

N-terminal

interactions

there

and green

In C. cinereus

present

control

is only one functional

in (d) denotes a negative interaction. of HDl and HD2 proteins within

of compatible

that binds

respectively.

have a positive,

‘lock-lock’

there

of the two

which

in Saccharomyces

in U. may&,

reaction.

interactions variable

may

mating

different

redundancy38,3g,48.

highly

interaction combinations

Positive

the two

to the protein-protein

domain

interactions,

complex

pairs of these

(c) and incompatible proteins

after

b allele. Yellow

at every

in red and blue,

functional

and HD2),

whereas

encoding

the products

(HDI

(b). In 5. cerevisiae,

and green),

an incompatible

be up to four

and showing

and HD2

domains,

are shown

between genes

development

HD1 and HD2 genes

and ‘-’ indicates

for compatible HDI

fungi

in yellow

alleles, and the regions

interaction

factor

and sexual

(a) and basidiomycete

gene of each class (shown divergently

interactions

transcription

5

and formation gene

the negative of an active

transcription.

Yellow

alleles, and red and blue boxes HDI and HD2, respectively.

versus

complementation

The molecular events that lead to SI in flowering plants and fungi are clearly very different - in fungi, pheromone-pheromone-receptor interactions and transcription factors play a key role, whereas, in flowering plants, RNases, receptor kinases, thioredoxins and Ca2+-mediated signal-transduction pathways are involved. Full elucidation of the molecular pathways to SI in flowering plants has been confounded by the failure, so far, to identify S-determinants in pollen (P. coerulescensexcepted). However, the speculative models for SSI and GSI described here support the classical ‘oppositional’ model for active rejection of pollen in response to an extracellular interaction between gene products derived from identical alleles. In fungi, the products of mating-type loci are better characterized, allowing more accurate predictions to be made about the mechanisms through which they mediate SI. To date, compatibility has been shown to involve extracellular and intracellular interactions between different ‘allelic’ products leading to ‘complementary’ acceptance of non-self. This crucial difference between the two overall mechanisms of SI is a consequence of the different life cycles of flowering plants and fungi: in flowering plants, self-recognition occurs when a haploid pollen grain makes contact with or invades a diploid pistil, whereas, in fungi, recognition occurs between two single cells or hyphal filaments derived from haploid spores. These spores are analogous to the gametes of flowering plants, so, if cells produced by spores derived from the same meiosis mate, this is equivalent to selfing. The oppositional systems of flowering plants totally preclude the possibility of selfing, but the complementary mating-type systems of fungi vary greatly in their ability to prevent selfing. For example: diallelic bipolar mating-type systems only reduce the chance of selfing by 50%, because just two matingtypes are possible. Multiallelic tetrapolar mating-type systems (in which four mating-types are possible) are much more effective, and reduce the chance of selfing to 25%, but, with large numbers of alleles and efficient spore dispersal, the actual risk of selfing within a natural population will be negligible38. Thus, despite the constraints imposed on the breeding system by haploid-haploid complementary recognition, the efficiency of SI in the multiallelic tetrapolar mating-type systems of basidiomycetes approaches and probably equals that of the haploid-diploid oppositional systems of flowering plants. Evolution

of self-incompatibility

systems

The disparate nature of the molecules that mediate SI in flowering plants suggests that, during the course trends

in CELL BIOLOGY

(Vol.

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of their evolution, different molecules have been recruited independently a number of times and modified for a role in SI. A similar polyphyletic origin for SI in fungi appears less obvious because the general mechanisms are very similar and appear conserved between ancient groups. However, the clearly unrelated nature of the different families of mating-type transcription factors suggests that this type of molecule has been recruited into SI more than once during the evolution of fungi. In principle, any protein with appropriate function could be recruited into SI provided that it somehow became linked to the sexual cycle. This appears to be what has happened in the protozoan ciliate Euplotes raikovi where the pheromone-mediated SI system is proposed to have evolved through diversification of an extant autocrine pheromone system involved in the promotion of asexual reproduction4g. It is probable that, in fungi, the pheromones and pheromone receptors were recruited into a mating-type role from a similar cellular functio#, and the situation in ascomycetes may represent the half-way stage towards full mating-type status seen in basidiomycetes. There are many theories for the origin of SI in flowering plants, but two are particularly appealing: modification of a previously existing pathogendefence system50 and elaboration of an ancient interspecific incompatibility systems’. The superficial similarities between SSIin Brassicaand host-pathogen interactions are very striking5y, and the recent cloning of resistance genes from plants that encode a receptor kinase and a cytosolic kinase (reviewed in Ref. 52) provides strong evidence that both SSI and certain types of disease resistance are mediated by phosphorylation. Evidence that S-RNasesare required for certain types of interspecific incompatibility in Nicotianazs gives renewed support to Pandey’s ‘twinspecificities’ theorysl for the origin of SI. In this theory, SI (‘secondary specificity’) is proposed to have arisen through gene duplication of an original ‘primary specificity’ that restricted crossing in early wind-pollinated gymnosperms. Secondary specificity evolved in response to insect pollination and the increased likelihood of self-pollination. Despite some evidence to the contrary (reviewed in Ref. 53), a large body of data suggests that S loci are involved in interspecific incompatibility phenomenaz5J1~54, but molecular data do not support Pandey’s monophyletic origin of SI. It is conceivable, however, that a number of different primary specificities existed in the ancestors of flowering plants and these were the original molecular ‘recruits’ (perhaps from pathogendefence systems) that became adapted for a role in SI after having first acquired the necessary pollenspecific and pistil-specific elements through their role in interspecific incompatibility. Support for ancient origins of SI in flowering plants has come from phylogenetic analyses of S-allele sequences, which predict that GSI, as controlled by S-RNases,is approximately 70 million years oldzOand that SSI, as mediated by SRK, is at least 50 million years old (and possibly 120 million years old)s5. These studies also reveal that new S-alleles evolve very slowly and that allelic polymorphisms frequently trends

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predate speciations within a genus or familyz0~55. Similar analyses of mating-type alleles in fungi are rare, but recent sequence and recombination analysis of A mating-type alleles in Coprinuss6 has shown that allelic polymorphisms are also generally ancient and that new alleles evolve very slowly. Future

prospects

It is clear that, at a molecular level, more is known about the mechanisms of SI in fungi than in flowering plants. A future landmark in the study of SI will be the identification of the pollen S-components that interact with their stigmatic counterparts: S receptor kinase, S-RNaseand S-glycoprotein. In basidiomycete fungi, it will be extremely interesting to discover how an apparently extracellular interaction between pheromones and pheromone receptors can control intracellular recognition events after cell fusion. References 1 2 3

4 5 6

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D. (1977) incompatibility in Angiosperms, Springer KOES, U. and CASSELTON, L. A. (1992) Mycol. Res. 96,993-l 006 GLASS, N. L. and NELSON, M. A. (1994) in The Mycota I &Vessels, J. C. H. and Meinhardt, F., eds), pp. 295-306, Springer NASRALLAH, M. E., KANDASAMY, M. K. and NASRALLAH, J. B. (1992) P/ant/.2,497-506 NASRALLAH, J. B., RUNDLE, S. J, and NASRALLAH, M. E. (1994) Plant 1. 5, 373-384 NASRALLAH, J. B. and NASRALLAH, M. E. (1993) PlantCell 5, 1325-I 335

7 STEIN, J. C., DIXIT, R., NASRALLAH, M. E. and NASRALLAH, J. B. (1996) PlantCell 8,429-445 8 TANTIKANJANA, T., NASRALLAH, M. E., STEIN, J. C., CHEN, C-H. and NASRALLAH, J. B. (1993) Plant Cell 5, 657-666 9 GIRANTON, J-L., ARIZA, M. J., DUMAS, C., COCK, J. M. and CAUDE, T. (1995) P/ant/. 8, 827-834 10 DWYER, K. C. et al. (1994) Plant Cell 6, 1829-l 843 11 TANTIKANJANA, T., NASRALLAH, M. E. and NASRALIAH, J. B. (1996) Sex. Plant Reprod. 9, 107-l 16 12 DICKINSON, H. D. and ELLEMAN, C. J. (1994) in Pollen Pistil Mefactions and Pollen Tube Growth (Kao, T-H. and Stephenson, A. C., eds), pp. 45-61, Kluwer 13 DOUGHTY, J., HEDDERSON, F., McCUBBIN: A. and DICKINSON, H. C. (1993) Proc. Nat/. Acad. Sci. U. 5. A. 90, 467-471 14 HISCOCK, S. J., DOUGHTY, J., WILLIS, A. C. and DICKINSON, H. G. (1995) Planto196, 367-374 15 BOYES, 0. C. and NASRALLAH, J. B. (1995) PlantCell 7, 1283-l 294 16 SCUlT, C. P., FORDHAM-SKELTON, A. P. and CROY, R. D. D. (1993) Sex. Plant Reprod. 6,282-285 17 RUNDLE, S. J., NASRALLAH, M. E. and NASRALLAH, J. B. (1993) Plant Physiol. 103, 1165-1171 18 DODDS, P. N., CLARKE, A. E. and NEWBIGIN, E. (1996) Cell 85, 141-144 19 SASSA, H., NISHIO, T., KOWYAMA, Y., HIRANO, H., KOBA, T. and IKEHASHI, H. (1996) Mol. Gen. Cenet. 250, 547-557 20 XU, Y., CARPENTER, R., DICKINSON, H. G. and COEN, E. S. (1996) Plant Cell 8, 805-814 21 CORNISH, E. C., PETTIT, J. M., BONIG, I. and CLARKE, A. E. (1987) Nature 326, 99-l 02 22

LEE, H-S., HUANG,

S. and KAO, T-H. (1994)

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23 24 25 26 Acknowledgements We thank Lorna Casselton, James Doughty, Chris Franklin, John Golz, Daphne Goring, Regine Kahmann, Erika Kothe, Georgiana May, Bruce McClure, Ed Newbigin, Suzanne O’Shea, and Carlene Raper for helpful discussions and for allowing us to refer to data prior to publication data. We also thank Suzanne O’Shea and James Doughty for critical reading of the manuscript and Liz Paul for preparing the figures. S. J. H. is funded by the Biotechnology and Biological Sciences Research Council.

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trends

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