Proc. Natd. Acad. Sci. USAVol. 91, pp. 1992-1997, March 1994

Review

Self-incompatibility: How plants avoid illegitimate offspringMatton,

NorbertClarke*,

Plant Cell Biology Research Cont"u, School of Botany, Melbourne University, ParkWle, VIctoria 3052, Australia

ABSTRACT In some families of flow-ering plants, a single self-incompatibility(S) locus prevents the fertilization of flow-ers by pollen from the same plant. Self-incompatibility of this type involves theinteraction of molecules produced by the Slocus in pollen with those present in thefemale tissues (pistil). Until recently, thepistil products of the S locus were knownin only two families, the Brassicaceae(which indudes the cabbages and mus-tards) and Solanaceae (potatoes and toma-toes). A paper in this issue of the Proceed-ings describes the molecules actedwith self-incompatibility in a third family,the Papaveraceae (poppies). We reviewcurrent research on self-incompatibility inthese three families and discuss the impli-cations of the latest findin in poppy onthe likely evolution of self-incompatibilityin flowering plants. We also compare re-searchinto self-icom bilit with recentprogress in understanding the mechanisby which plants overcome infection bycertain pathogens.

Why Are BiolWgists So Interested inSelf-Incompatibility?

Self-incompatibility, the inability of ap-parently healthy plants to produce seedwhen self-pollinated, was described byCharles Darwin as "one of the mostsurprising facts which I have ever ob-served" (1). A similar sense of wonderhas led generations of plant biologistssince Darwin to study self-incompatibil-ity and more recently to characterizesome of the molecules involved in this"fsurprising fact." In this issue of theProceedings (2), Foote and others de-scribe one of the molecules involved inself-incompatibility in field poppy (Pa-paver rhoeas), and in this briefreview weplace this new data in the context of ourknowledge of other systems of self-incompatibility. It has been postulatedthat self-incompatibility arose a numberoftimes during the evolution offloweringplants [angiosperms (3)], and the datapresented by Foote et al. provides thefirst molecular insights into self-incom-patibility in a family that diverged early inthe evolution of the angiosperms, thePapaveraceae.The strategies that plants use to recog-

nize and reject "self' pollen while ac-

cepting "nonself' pollen are necessarilydifferent from the strategies for "self'and "nonself 'discrimination in animals.Plants lack both circulating lymphocytesand immunoglobulins and have no knownequivalents to the tissue transplantationantigens [although some proteoglycans,the arabinogalactanproteins, may beanalogous (4)]. Nonetheless, plant cellsdo recognize and respond to each other.The way in which this recognition ismediated is ofprofound interest, not onlyto an understanding of plant fertility andreproduction but also to an understand-ing of the way in which plants respond topathogenic or symbiotic microorganismsin the environment.

Systems in Which Self-IncompatibuityIs Studied

Self-incompatibility is a relatively simpleand genetically defined example of cell-cell recognition in plants. Self-incompat-ible plants are able to distinguish betweenself pollen and nonself pollen within thefemale reproductive tissue (the pistil) andarrest the further growth of self pollen(see ref. 5 and the references therein). Byrecognizing and rejecting self pollen be-fore fertilization, self-incompatibleplants promote outbreeding and maintaingenetic variability, a factor consideredimportant in the evolutionary success offlowering plants (6). The molecular ge-netics of two types of self-incompatibil-ity, gametophytic and sporophytic self-incompatibility, have been studied inten-sively (5, 7).Gametophytic self-incompatibility is

well-characterized in plants from the fam-ily Solanaceae-such as the ornamentaltobacco (Nicotiana alata), petunia (Petu-nia inflata and Petunia hybrida), potato(Solanum tuberosum and Solanum cha-coense), and wild tomato (Lycopersiconperuvianum). Some molecular informationis also available for gametophytically self-incompatible species from the Papaver-aceae, Rosaceae, and Scrophulariaceae(Fig. 1, see below). In each case, self-incompatibility is controlled by a singlegenetic locus (S locus) with many alleles.Rejection of pollen occurs when the singleS allele present in the haploid pollen grainmatches either of the S alleles present inthe diploid tissues of the pistil. Andersonand others (10) showed that the S loci ofN.

1992

alata and L. peruvianum encode extracel-lular glycoproteins that are abundantwithin the pistil, and the discovery thatthese glycoproteins are ribonucleases re-lated to extracellular ribonucleases ofsome fungi was a major surprise (11). Untilrecently, evidence for the involvement ofthese glycoproteins (now called S-RNases)in self-incompatibility was indirect and re-lied on a number of correlations: for ex-ample, the genes that encode S-RNasescosegregate with alleles ofthe S locus, andthe timing of expression of S-RNases iscoincident with the onset of self-incompat-ibility in the pistil (5). There is now directevidence that S-RNases determine the self-incompatibility phenotype of the pistil andthat the ribonuclease activity of these gly-coproteins is required for rejection of in-compatible pollen (refs. 12 and 13; J. Royo,Y. Kowyama, and A.E.C., unpublishedwork). These findings strengthen the viewthat if S-RNases enter incompatible pollentubes, then they would act ascytotoxins bydegrading RNA, including ribosomal (r)RNA (14). As rRNA genes do not appearto be expressed in mature pollen (15), de-grading the fixed amount ofrRNA synthe-sized during early pollen developmentwould be an effective way of arrestingpollen-tube growth.

Sporophytic self-incompatibility,, theother extensively studied system of self-incompatibility, is found in members ofthe Brassicaceae, and molecular informa-tion is available for three species, Bras-sica oleracea, Brassica napus, and Bras-sica campestris (7). Sporophytic self-incompatibility in the Brassicaceae, likegametophytic self-incompatibility in theSolanaceae, is controlled by a single,multiallelic S locus. However, rejectionin the sporophytic system is controlledby the interaction of the self-incompati-bility genotype of the pistil with the gen-otype of the pollen parent and not withthe haploid genotype of the pollen, as isthe case in the gametophytic system.Thus, each pollen grain in plants withsporophytic self-incompatibility presentsthe products of two S alleles, and rejec-tion occurs when either one of thesealleles matches either of the S alleles

Abbreviations: SLG, S-locus glycoprotein;SRK, S-locus receptor kinase.*To whom reprint requests should be ad-dressed.

Proc. Natl. Acad. Sci. USA 91 (1994) 1993

expressed in the pistil; complex domi-nant or codominant interactions oftenoccur between S alleles, affecting theoutcome of particular crosses.At least two multiallelic genes are

found within the Brassica S locus, and ithas been suggested that the complementof allelic genes at the S locus be de-scribed as a "haplotype", a term alsoapplied to a complex of highly allelicgenes within the major histocompatibil-ity locus of mammals (7, 16). One of thegenes within the S haplotype of theBrassicaceae encodes an extracellularglycoprotein called the S-locus glyco-protein (SLG) and the other, called theS-locus receptor kinase (SRK), encodesa membrane-associated protein able tophosphorylate serine/threonine resi-dues (17, 18). The two genes are withina few hundred kilobases of each other inthe genome (16), and a number of inde-pendent lines of evidence have impli-cated both in sporophytic self-incom-patibility (19-21). The genes for SLGsand SRKs are expressed within the re-productive structures of the flower (7),and SLG is particularly abundant instigmatic papillae, the cells of the pistilthat receive the pollen. The predictedsequence of the SRK contains at the Nterminus a potentially glycosylated, ex-tracellular domain with extensive simi-larity to SLG (17): indeed, on the basisof sequence comparisons of SLG/SRKgene pairs from the same S haplotype, itappears that the SLGs may be derivedby duplication from the SRKs (20). TheC terminus of the SRK encodes a do-main with similarity to serine/threoninekinases, and it is thought that this do-main is located within the cytoplasm andjoined to the extracellular SLG-like do-main by a membrane-spanning domain(17).

Evolution of Self-Incompatibility

The new data from field poppy (P.rhoeas; Papaveraceae) published in thisissue of the Proceedings (2) can be com-pared to data from other systems andallows us to reexamine the question ofthe evolution of self-incompatibility.Foote and others describe a small glyco-protein associated with self-incompatibil-ity in P. rhoeas that is unrelated in se-quence to either the S-RNases of solan-aceous plants or the SLGs and SRKsfrom Brassica spp. (see below). This re-sult is consistent with earlier findings thatshowed no correlation between self-incompatibility and ribonuclease activityin P. rhoeas (22), even though the genet-ics of self-incompatibility in this speciesare similar to that of solanaceous plants,such as N. alata (23). The new moleculardata indicates that the self-incompatibil-ity systems found in the Brassicaceae,Papaveraceae, and Solanaceae are unre-

lated and supports the conclusion ofBateman (3) that self-incompatibilityarose independently many times withinthe angiosperms.Most flowering plants (angiosperms)

are self-compatible, which is generallyconsidered to be the primitive condition(3). Self-incompatibility, the presumedderived condition, is found scattered inmost major lineages. Fig. 1 shows thebroad taxonomic relationships withinangiosperms, together with the locationof some families in which self-incompat-ibility is controlled by a single locus(monofactorial). Only one type of self-incompatibility (gametophytic or sporo-phytic) is found within any one family(24): for example, gametophytic self-incompatibility is found within the So-lanaceae, Scrophulariaceae, Rosaceae,and Papaveraceae, whereas sporophyticself-incompatibility is found within theBrassicaceae, Asteraceae, and Convol-vulaceae. The Papaveraceae are in-cluded in the ranunculids, a group that isbasal in nonmagnolid dicotyledons andquite widely separated from the Aster-idae (see Fig. 1). However, within theAsteridae, the closest family to the So-lanaceae is the Convolvulaceae (8, 25),and yet these two families have game-tophytic and sporophytic self-incompat-ibility systems, respectively. It thus ap-pears that self-incompatibility arose

quite late in the evolution of the fami-lies, as closely related families do notshare the same system of self-incompat-ibility.

Despite their apparently independentorigins, gametophytic self-incompatibil-ity systems in the Scrophulariaceae andRosaceae appear to involve ribonucle-ases that are biochemically similar tothose described for solanaceous plants(Y. Xue, H. Dickinson, and E. Coen,personal communication; ref. 26); theSolanaceae and Scrophulariaceae areclosely related within Asteridae, whereasthe Rosaceae are more distantly relatedand lie within the Rosidae. This resultsuggests that at least one and possiblytwo families other than the Solanaceaehave acquired gametophytic self-incom-patibility mechanisms that involve ribo-nucleases, although it is not knownwhether other aspects of the signal-transduction pathway in these familiesare related. Secreted ribonucleases havebeen described in the flowers, leaves,and seeds of many unrelated plant spe-cies (27, 28) and could possibly have beenrecruited to a cell-recognition role onmore than one occasion.One interesting possibility is that the

molecules involved in self-incompatibil-ity in the Convolvulaceae and Solan-aceae are related, despite these familieshaving different self-incompatibility sys-

Self-Incompatibility

type

SolanaceaeConvolvulaceaeScrophulariaceae

Asteraceae

Rosaceae

Fabaceae

GSG

RNases

S

+G

G

Brassicaceae S

Onagraceae G

Papaveraceae G

FIG. 1. Simplified version of the family tree of major angiosperm clades as given by Chaseet al. (8). For clarity, the Hamamelidae have been omitted from the nonmagnolid dicots. Thenames of the other orders and of some of the families in which simple (monofactorial)self-incompatibility occurs are shown. The position offamily names approximates their locationin the order as given by Chase et al. (24), and the distance between names approximates theirtaxonomic relationship. The type of self-incompatibility in each family is indicated: G,gametophytic self-incompatibility; S, sporophytic self-incompatibility. A second column indi-cates whether ribonucleases (RNases) have been implicated (+) or are known not to be involved(-) in self-incompatibility in each family. The S-locus products of the Convolvulaceae,Asteraceae, Fabaceae, and Onagraceae are not known. The classification of the Brassicaceaein the Rosidae is debatable (9).

Review: Matton et al.

Proc. Natl. Acad. Sci. USA 91 (1994)

tems. This idea is supported by the close-ness of the two families and by the ob-servation that allelism of the S locusappears to predate speciation of the So-lanaceae (29). Thus, the alleles present atthe solanaceous S locus could havearisen in a species that was ancestral toboth the Convolvulaceae and Solan-aceae. The possibility that gametophyticand sporophytic self-incompatibility sys-tems may, in fact, be more closely relatedthan is at first apparent has been dis-cussed previously (for example, see ref.30): thus, a shift in the timing of expres-sion of the S locus in pollen from beforemeiosis to after meiosis could convert asporophytic incompatibility system to agametophytic one. The limited molecularinformation available for the Convolvu-laceae indicates that the molecules in-volved in sporophytic self-incompatibil-ity in this family differ from those in theBrassicaceae (31). At this stage, any as-sociation between ribonucleases and self-incompatibility in the Convolvulaceae isuntested.

Models of Self-Incompatibility

Most of the studies on self-incompatibil-ity focus on the identification and char-

acterization of the molecules associatedwith self-incompatibility present in thepistil. The complete process of signalperception and transduction is, however,not understood for any one system be-cause of the lack of data on the pollenpart of the S locus. Nevertheless, on thebasis ofthe limited information available,the postulated pathways for cell recogni-tion in self-incompatibility in the Papa-veraceae, Brassicaceae, and Solanaceaeshow striking differences.The availability of an in vitro assay for

self-incompatibility in P. rhoeas putFranklin-Tong and coworkers (2, 32, 33)in a position to identify the S-locus prod-uct of the pistil directly and then to studythe signal-transduction mechanism. Theyshowed that pollen tubes grown in cul-ture respond to the presence of extractsfrom pistils of the same S genotype witha transient increase of calcium and, sub-sequently, altered gene expression andprotein phosphorylation. Using their pol-len-tube bioassay to monitor purification,they isolated a small glycoprotein frompistil extracts and cloned the correspond-ing cDNA. This cDNA hybridized to asingle gene in the poppy genome thatcosegregated with the S locus. Remark-

ably, the recombinant protein isolatedfrom a strain ofEscherichia coli express-ing this S-locus gene elicited the sameeffects on pollen-tube growth in cultureas did the glycoprotein isolated from pis-tils, indicating that the carbohydratecomponent of the glycoprotein is notrequired for biological activity. Based onthe involvement of calcium, they specu-late that the S glycoprotein binds to areceptor in the pollen tube and induces acellular response via the inositol phos-phate pathway (34), but there are noexperimental data at this stage for theexistence ofa receptor (which may be theproduct of the S locus in pollen) or theinvolvement ofthis pathway. P. rhoeas isperhaps the best system so far for study-ing the incompatibility responses of pol-len. Fig. 2 shows the essential features ofthe model and uncertainty in correlatingthe in vitro data and the in vivo situation.For Brassica spp., Nasrallah and col-

leagues (7) have identified two gene prod-ucts of the S locus-namely, SLG andSRK (see above). These genes are ex-pressed both in the pistil and in the pollengrain and anther, although the level ofexpression found in these latter two tis-sues is much lower than that found in the

pollen grain / tube

pollen growth arrest

gene activation

calcium flux4

rcepto

4

FIG. 2. Schematic representation of the signal-transduction pathway governing self-incompatibility in field poppy (P. rhoeas). Solid arrowsin the figure indicate known steps in the pathway, and dashed arrows indicate hypothetical steps. The accompanying table highlights some ofthe stages of the signal-recognition process listed according to the degree of certainty.

level of confidence S-locus products perception and response

recognition

known secreted arrest of pollen

glycoprotein growth

produced by pistils

implicated secreted * pollen gene

glycoprotein activation

functions as a * calcium flux in

ligand for a pollen tube

receptor on pollen * activation of

tubes calcium depen-dent enzymes

unknown Is product of the S- * basis of allelic correlation

locus in pollen a specificity between in vitro

receptor? * pollen-stigma and in vivo

signalling effects

leading

* to callose depo-

sition in the

stigma

1994 Review: Matton et al.

Proc. Natl. Acad. Sci. USA 91 (1994) 1995

pistil. A specific interaction between thepollen and pistil S products may be me-diated by another compound present inthe pollen coat (Fig. 3), and an interactionbetween a coat-derived peptide fromBrassica pollen and the pistil SLG hasbeen reported (35). Nasrallah and Nas-rallah (7) have postulated that protein-phosphorylation events in the pistil leadto inhibition of pollen germination andultimately deposition of callose (a poly-saccharide essentially composed of (,

1-3 glucan) on both pollen and the sur-face of the pistil. However, in this sys-tem, callose deposition may not be caus-ally related to self-incompatibility (36).The main events and points of uncer-tainty in the pathway of cell recognitionin sporophytic self-incompatibility ofBrassica spp. are summarized in Fig. 3.As mentioned above, the products of

the S locus expressed in the pistil ofsolanaceous plants are active ribonucle-ases (S-RNases), and there is one reportthat low levels of S-RNases are producedduring pollen development (37). The ba-sis of the allelic interaction in this systemis not understood, although our currentthinking on the mode of action ofS-RNases is that they are specificallytaken up by incompatible pollen tubes.This specific uptake may involve do-mains on the surface of the protein thatare very different in sequence between

different S-RNase alleles ("hypervari-able domains"; ref. 38). An alternativehypothesis is based on nonspecific up-take of S-RNases into the pollen tubefollowed by specific inactivation or othermodification (39). In both cases, theRNase is thought to act as a cytotoxinand degrade the RNA essential for pro-tein translation; arrest of pollen-tubegrowth would follow (Fig. 4).

Self-Incompatibility and DiseaseResistance

All cell-cell interactions have certain fea-tures in common, including some kind ofdiscrimination and differential response.Many authors have compared self-incompatibility and host-pathogen inter-actions, another process in plants thatrequires specific cell-cell signaling (forexamples, see refs. 40 and 41).

Plants lack an immune system and relyon several different strategies to resist orovercome infection. In many cases, re-sistance to a particular pathogen is con-trolled by a single, multiallelic locus (Rlocus). There can be many R loci in asingle species, and these encode resis-tance to a variety of pathogens; there arealso examples of multiple-resistance locifor a single pathogen. Some types ofresistance loci direct a strategy to combatinfection that results in the death of cells

around the site of infection (a "hypersen-sitive response"). This response pre-vents further colonization of the plant bythe pathogen. The genetic basis underly-ing the hypersensitive response ofa plantto a specific race of a pathogen wasoriginally provided by Flor (42), whodescribed it as a gene-for-gene interac-tion (for reviews, see refs. 40, 41, and 43).Flor hypothesized that certain types ofinfection are controlled by two dominantgenes, one gene for resistance in the hostand another gene for avirulence in thepathogen. An incompatible (hypersensi-tive) response is triggered when the com-plementary products of the plant's resis-tance gene and the pathogen's avirulencegene interact. A single avirulence/resistance gene combination is sufficientto activate the hypersensitive response,regardless of how many other virulenceand susceptibility combinations exist. Anumber of such avirulence/resistancegene pairs have been described for dif-ferent plant-pathogen combinations.There are several parallels between

self-incompatibility and gene-for-gene in-teractions. (i) Both are genetically simpleand based on a specific interaction be-tween the products of complementarygenes expressed in separate cells. (ii) Inboth systems, there are only two possibleoutcomes of an interaction, either a com-patible reaction (leading to pollen growth

(SLG.U*.e specific

M or L

I- -- -- phosphorylation

,.-- cell wall changes

.' callose deposition

pistil

FIG. 3. Schematic representation of the signal-transduction pathway governing self-incompatibility in Brassica. Details of the figure aredescribed in the legend to Fig. 2.

pollen grain

pollengrowtharrest4

level of confidence S-locus products perception and response

recognition

known secreted * inhibition of

glycoproteins pollen ger-

(SLGs), and mination

membrane bound

receptor kinases

(SRKs)

implicated allelic specificity SRKs interact with *Protein phos-results from SLGs and a pollen phorylation

sequence product (either a * callose depo-differences in the ligand (L) or sition in stigma

SLGs and SRKs modifier (M)) and pollen

unknown S-locus other interacting substrate for SRK

product(s) moleculesin pollen

Review: Matton et al.

Proc. Natl. Acad. Sci. USA 91 (1994)

fpollen tube(

pollen cgrowtharrest *

callosedeposition

A

RNA degradation

4

translocatorRa,

FIG. 4. Schematic representation of the signal-transduction pathway governing self-incompatibility in solanaceous plants. Details of thefigure are described in the legend to Fig. 2.

or a systemic infection) or an incompat-ible reaction (leading to the arrest ofpollen growth or a hypersensitive re-

sponse). Additionally, there are somesuperficial similarities in the biochemis-try and physiology of incompatible re-sponses in the two systems (40).Most models of gene-for-gene interac-

tions postulate that the products of theavirulence gene of the pathogen and theresistance gene ofthe plant host are anal-ogous to a ligand and a membrane-boundreceptor (44, 45). Other models recognizethe potential for intracellular processingand predict that resistance genes mayplay a critical role in preferential trans-port and/or processing of the avirulencegene product (43). Considerable progresshas been made in characterizing aviru-lence genes from various bacterial patho-gens (46), but the isolation of the firstplant resistance gene has proved moreelusive. Recently, the gene responsiblefor resistance to bacterial speck (Pseu-domonas syringae) was cloned from thePto locus of tomato using a map-basedapproach (47). Comparison of the se-quence ofthe Pto gene with sequences inDNA data bases uncovered similaritieswith catalytic domains of a number ofserine/threonine kinases, includingSRKs from Brassica spp. However, un-

like SRKs, the putative Pto protein con-tains no obvious membrane-spanning orextracellular domains and may be local-ized in the cytoplasm. Thus, both sporo-phytic self-incompatibility in Brassicaand resistance to this bacterial pathogenprobably involve phosphorylation, al-though the nature of the substrates andthe subsequent steps in the signal-transduction pathways are not known ineither case.The pto gene is only one of many R

genes used by plants to recognize aviru-lence specificities of different pathogens.Although the initial events in these inter-actions may be quite different, it isthought that all the pathways eventuallyconverge into one or a few pathways thatmediate the hypersensitive response (43).The pathways that mediate the cellularresponses found during incompatible pol-linations may prove to share some com-ponents with those involved in diseaseresistance.

Summary

The most important contribution of thepaper by Foote et al. (2) is to describe atthe molecular level a system of self-incompatibility in a representative of afamily of angiosperms for which no mo-

lecular information was previously avail-able. It allows us to extend our view ofthe evolution of self-incompatibility andconfirms the conclusion, based on otherdata, that self-incompatibility arose inde-pendently on several occasions duringevolutionary history. The molecules as-sociated with self-incompatibility fromdifferent plant families appear quite dis-similar, but the possibility that the sub-sequent pathways converge and sharesome features with pathways that medi-ate disease resistance remains open. Amajor quest in self-incompatibility re-search in many laboratories is the iden-tification of S-locus product in pollen.For reasons that are not entirely clear,the experimental approaches that led tothe cloning of the stylar products of the Slocus, the SLGs from Brassica spp.,S-RNases from solanaceous plants, andthe small glycoproteins from P. rhoeas,have not proved useful in identifying thepollen products. Possibly, map-based ap-proaches, similar to that that led to thecloning of the pto gene from tomato (47),will be required. However, althoughidentifying the product of the S locus inpollen will provide another valuablepiece of the puzzle, it will not reveal thewhole story. The questions of signaltransduction and the steps that lead to the

level of confidence S-locus products perception and response

recognition

known Secreted, active arrest of pollen

RNases produced growth associated

by pistil with changes in

cytoplasm and

pollen-tube wall

callose content

implicated allelic specificity is * uptake of RNase RNA degradation

due to hyper- into pollen tube leading to

variable regions degradation of disruption of

on S-RNase pollen RNA protein synthesis* function of S- and pollen-tube

RNase in pollen elongation

grain wall

unknown nature of the S- connection

locus product in between RNase

pollen activity, pollen-

tube growth arrest

and callose

deposition

1996 Review: Matton et al.

Proc. Natl. Acad. Sci. USA 91 (1994) 1997

arrest of pollen growth will be majorareas of research for the future.

The authors greatly appreciate the contin-ued contribution of Dr. Marilyn Anderson andthe considerable assistance that Dr. AndrewDrinnan and Dr. Steve Read made to variousparts of this work. D.P.M. is the recipient of aFellowship from the Natural Science and En-gineering Research Council of Canada, andN.N. is supported by a Studienstifung desDeutschen Volkes Fellowship.

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Review: Matton et al.