Fusarium oxysporum as a Multihost Model for the Genetic Dissection of Fungal Virulence in Plants and Mammals

Fungal pathogens cause disease in plant and animal hosts. Theextent to which infection mechanisms are conserved between bothclasses of hosts is unknown. We present a dual plant-animalinfection system based on a single strain of Fusarium oxysporum,the causal agent of vascular wilt disease in plants and an emergingopportunistic human pathogen. Injection of microconidia of awell-characterized tomato pathogenic isolate (isolate 4287)into the lateral tail vein of immunodepressed mice resultedin disseminated infection of multiple organs and death of theanimals. Knockout mutants in genes encoding a mitogen-activatedprotein kinase, a pH response transcription factor, or a classV chitin synthase previously shown to be implicated in virulenceon tomato plants were tested in the mouse model. The resultsindicate that some of these virulence factors play functionallydistinct roles during the infection of tomato plants and mice.Thus, a single F. oxysporum strain can be used to study fungalvirulence mechanisms in plant and mammalian pathogenesis.

INTRODUCTION

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Introduction

Fungi are an extremely versatile class of organisms comprisedmostly of saprophytes thriving on dead organic material. A relativelysmall number of fungal species have developed a parasitic lifestyle,associated with the ability to recognize and penetrate a specifichost, exploit its nutrient reserves, overcome its innate defenseresponses, and cause disease. The list of organisms attackedby fungi encompasses evolutionary distinct groups from lowerto higher eukaryotes, most prominently plants, insects, andmammals, including humans. To cause disease, fungal pathogensrely on an arsenal of pathogenicity and virulence factors, whosespatially and temporally correct deployment determines the basicpathogenic potential and the extent of infection, respectively.The advent of protocols for targeted gene knockout or randominsertional mutagenesis has led to the identification of anincreasing number of genes encoding pathogenicity or virulencefactors from plant and animal pathogens (12, 34). In spite ofthese advances, many crucial aspects underlying fungal pathogenesisremain poorly understood.

One of these questions concerns host specificity. Certain fungicause disease on a single host species, whereas others haveextremely broad host ranges. The molecular mechanisms that determinefungal host range specificity are not fully understood. Thus,although a number of virulence determinants are clearly hostspecific (36), there is also evidence for the existence of universalvirulence mechanisms shared by fungal pathogens with highlydiverse host ranges (17, 20). Interestingly, common patternsof host defense also are found in evolutionary diverse groupssuch as plants, insects and mammals (23). Thus, evolutionaryancient mechanisms of fungal virulence and host defense mightcoexist with highly specific virulence and resistance traitsthat have arisen during later stages of pathogen-host coevolution.

The availability of the complete genome sequences of fungalplant and human pathogens and their model hosts, as well asthe development of high-throughput protocols for gene functionanalysis, hold a huge potential for advancing our understandingon fungal pathogenesis and host defense (33). However, pathogenhost range imposes an important limitation on the comparativeanalysis of virulence mechanisms in evolutionary distant hostsbecause it requires gene identification and knockout in differentpathogenic organisms. This constraint has been overcome in bacterialsystems by developing multihost pathogenesis models, as exemplifiedby the Pseudomonas aeruginosa strain PA14 (29). Applicationof these models to phylogenetically diverse host species suchas Arabidopsis spp., Caenorhabditis elegans, or mice has ledto the identification of both common and contrasting virulencemechanisms in bacterial plant and animal pathogenesis (28, 32).In fungi, no such model system allowing simultaneous testingof virulence factors on plant and mammalian hosts is currentlyavailable.

The soil-borne fungus Fusarium oxysporum is the causal agentof vascular wilt, a disease that affects a large variety ofeconomically important crops worldwide (1). Besides its well-studiedactivity as a plant pathogen, F. oxysporum is known as a seriousemerging pathogen of humans due to the increasing number ofsevere cases reported and to its broad resistance to the availableantifungal drugs (2, 24). Fusarium now represents the secondmost frequent mold causing invasive fungal infections in immunocompromisedpatients, frequently with lethal outcomes (22, 27, 35). F. oxysporum,together with F. solani and F. verticillioides, are responsiblefor practically all of the cases of invasive fusariosis in humans(14). Given the dual ability to cause disease both on plantsand on humans, we reasoned that F. oxysporum could serve asa universal model for studying fungal virulence mechanisms.In the present study, we tested the ability of F. oxysporumf.sp. lycopersici strain 4287, a well-characterized model pathogenof tomato plants, to cause disease in a mammalian pathogenesismodel. We found that strain 4287 is able to produce systemicinfections in immunodepressed mice, resulting in a high deathrate. By applying the mouse model to a number of knockout mutantspreviously shown to exhibit altered virulence on tomato plants,we show that specific virulence factors in a single fungal strainplay distinct functional roles in plant and animal pathogenesis.

MATERIALS AND METHODS

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Materials and Methods

Fungal isolates and culture conditions. F. oxysporum f.sp. lycopersici strain 4287 (race 2) was originallyobtained from J. Tello, Universidad de Almería, Almería,Spain, and stored at -80°C with glycerol as a microconidialsuspension (11). The pathotype of the isolate on tomato wasroutinely confirmed by plant infection assays. The generationand characterization of the following mutant strains, all derivedfrom wild-type strain 4287, were described previously: mitogen-activatedprotein kinase (MAPK) mutant ${Delta}$ fmk1 (9), pacC loss-of-functionmutant pacC^+/-12 and dominant activating mutant pacC^c9 (5),and class V chitin synthase mutant D1 and complemented strainC2 (21).

For preparation of inocula, cultures were grown in potato dextrosebroth (Difco, Detroit, Mich.) at 28°C and 150 rpm for 4days, and microconidia were obtained by filtration as describedpreviously (11). The microconidial suspension was centrifugedand the pellet was resuspended in sterile saline. The concentrationof conidia was adjusted to 10⁸ CFU/ml with a hemocytometer andchecked by plating serial dilutions on potato dextrose agar(PDA; Pronadisa, Madrid, Spain) plates that were incubated at28°C for 3 days. To prepare heat-killed conidia for testingtheir activity in the murine model, conidial suspensions wereincubated in a thermostatic bath at 60°C for 60 min. Completeheat-killing of conidia was confirmed by plating serial dilutionson PDA plates.

Animal infection. OF-1 male mice (Charles River, Criffa S.A., Barcelona, Spain)weighing ca. 30 g were used. Five mice were housed per cagein standard conditions with free access to food and water. Conditionswere approved by the Animal Welfare Committee of the Facultyof Medicine of Universitat Rovira i Virgili. Mice were immunosuppressedwith a single intraperitoneal 200-mg/kg dose of cyclophosphamide(Laboratorios Funk S.A., Barcelona, Spain) and with an intravenous150-mg/kg dose of 5-fluorouracil (Fluoro-uracil; Roche S.A.,Madrid, Spain) on day 0.

Groups of 10 animals were infected by injecting 0.2 ml of aninoculum of 10⁸ conidia/ml into a lateral vein of the tail onday 0. Survival was recorded each day for 13 days. Infectionexperiments with each individual strain were performed between3 and 6 times. Survival was estimated by the Kaplan-Meier methodand compared among groups by using the log-rank test.

Tissue burden and histopathology. Randomly chosen surviving mice were sacrificed 13 days afterinoculation with an overdose of halotane (Fluothane; ZenecaFarma, S.A., Pontevedra, Spain). Livers, spleens, kidneys, lungs,and brains were aseptically removed, and one-half of each organwas weighed and homogenized in 1 ml of sterile saline. Tenfoldserial dilutions of this homogenate were made with sterile salineand spread onto PDA. Plates were incubated at 28°C, colonieswere counted after 3 days, and the numbers of CFU per gram oforgan were calculated. Fungal colony counts were converted tolog₁₀ values and compared by using the analysis of variancetest. Calculations were performed by using SPSS for Windows,version 10.0.

The remaining halves of the organs were fixed for 10 days in10% neutral buffered formaldehyde, embedded in paraffin wax,and automatically processed. Sections of the embedded tissues(3 µm in thickness) were stained with hematoxylin-eosin,periodic acid-Schiff, and methenamine silver (Grocott) for lightmicroscopy observations.

RESULTS

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Results

F. oxysporum f.sp. lycopersici causes systemic infection and death in immunodepressed mice. F. oxysporum strains have been reported either as plant or humanpathogens. We tested the hypothesis that a single strain ofF. oxysporum can produce disease both on plant and mammalianhosts. As a candidate we chose strain 4287, a tomato pathogenicisolate belonging to F. oxysporum f.sp. lycopersici race 2,because the genetic interaction between this pathogen race andits host plant has been well characterized (1, 31). Isolate4287 has been previously used in numerous molecular studies,resulting in the availability of genomic and cDNA librariesand optimized plant infection assays (10). Studies in our laboratorywith targeted gene knockout mutants (5, 9, 21) have led to theidentification of several genes involved in the virulence ofF. oxysporum on tomato plants (Fig. 1).

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FIG. 1. Invasive growth of F. oxysporum strains on tomato fruits. Fruits were inoculated by injecting 5 x 10⁵ microconidia of the following strains: wild-type strain 4287, MAPK mutant ${Delta}$ fmk1, pacC loss-of-function mutant pacC^+/-, dominant-activating mutant pacC^c, chitin synthase mutant ${Delta}$ chsV complemented with the wild-type chsV allele, and chitin synthase mutant ${Delta}$ chsV. The photograph was taken after 5 days of incubation at 100% relative humidity.

In preliminary experiments, we found that strain 4287 grew andsporulated well in submerged culture on porcine blood plasmaat 37°C (data not shown). We next tested the capacity ofthe strain to cause infections in mice. The murine model waschosen because of its high relevance to infectious diseasesin humans (3). Inoculation of immunocompetent mice with microconidiaof strain 4287 did not produce any detectable symptoms (datanot shown). In contrast, inoculation of immunodepressed micewith 2 x 10⁷ microconidia reproducibly resulted in the deathof ca. 70% of the animals (Fig. 2A and Table 1). Importantly,injection of heat-killed conidia caused no mortality, suggestingthat death was not simply a consequence of physical presenceof the microconidia but required living fungal propagules. Tostudy the ability of F. oxysporum strain 4287 to produce disseminatedinfections in different organs, fungal tissue burden was determinedin the livers, spleens, kidneys, and lungs of surviving micesacrificed 13 days after challenge. The presence of fungal propaguleswas detected in multiple organs, with the highest concentrationfound in the spleen, followed by kidney, liver, and lung (Fig.2B).

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FIG. 2. F. oxysporum strain 4287 causes systemic infection and death in immunodepressed mice. (A) Groups of 10 immunodepressed mice were infected with 2 x 10⁷ living (•) or heat-killed ( ${triangledown}$ ) microconidia by lateral tail vein injection. The percent survival was plotted for 13 days. The data shown are from one representative experiment. (B) Four surviving mice were sacrificed on day 13 postinfection, and homogenates from the indicated organs were quantitatively cultured on PDA medium.

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TABLE 1. MST of mice infected with the F. oxysporum wild-type strain 4287 and different mutants derived therefrom^a

Role of fungal virulence factors in plant and animal infection. The ability of F. oxysporum 4287 to survive and reproduce inimmunodepressed mice, colonize multiple organs, and kill thehost suggested that this strain contains the basic pathogenicitydeterminants required to cause disease on mammalian hosts. Tofurther test the usefulness of the F. oxysporum model for comparingmechanisms of plant and animal pathogenesis, we examined a numberof previously obtained knockout mutants in genes involved invirulence on tomato. Among these mutants, ${Delta}$ fmk1 strains thatlack an MAPK structurally related to the yeast MAPKs Fus3 andKss1 exhibit an extreme nonpathogenic phenotype on plants (9). ${Delta}$ fmk1 mutants not only fail to produce vascular wilt symptomswhen inoculated onto tomato roots but also are unable to growinvasively on living tomato fruit tissue (Fig. 1). We foundthat the ${Delta}$ fmk1 mutant was at least as virulent as the wild-typestrain in the murine model (Fig. 3 and Table 1), indicatingthat Fmk1 is dispensable for virulence on immunodepressed mice.

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FIG. 3. Virulence of gene knockout mutants of F. oxysporum on immunodepressed mice. Groups of 10 immunodepressed mice were infected with 2 x 10⁷ microconidia of the wild-type strain 4287 (•), MAPK mutant ${Delta}$ fmk1 ( ${circ}$ ), loss-of-function mutant pacC^+/- ( ${blacktriangledown}$ ), or dominant-activating mutant pacC^c ( ${triangledown}$ ). The percent survival was plotted for 13 days. The data shown are from one representative experiment.

We next examined two different classes of mutants in pacC, encodinga zinc finger transcription factor that mediates ambient pHresponse in fungi (26) and negatively regulates virulence ofF. oxysporum on tomato (5). pacC^+/- loss-of-function mutantsmimic growth at acidic ambient pH and exhibit increased virulencein a root inoculation assay. In contrast, merodiploid pacC^cstrains expressing a dominant-activating pacC allele mimic growthat alkaline pH and show significantly reduced virulence in theroot inoculation assay (5), although invasive growth on tomatofruits is not affected (Fig. 1). Inoculation of immunosuppressedmice with a pacC^+/- loss-of-function mutant resulted in a significantlyhigher survival rate and mean survival time (MST) than in miceinoculated with the wild-type strain (Fig. 3; Table 1). In contrast,survival and MST of mice infected with a dominant-activatingpacC^c mutant were not statistically different from those infectedwith the wild-type strain. Thus, PacC is required for full virulenceof F. oxysporum on mice but not on tomato plants.

A third class of knockout mutants tested were those lackinga functional copy of the chsV gene (21), encoding a class Vchitin synthase required for virulence on tomato (Fig. 1). ${Delta}$ chsVmutants have defects in cell wall integrity and show morphologicalalterations such as abnormally enlarged, lemon-like microconidia.When mice were inoculated with 2 x 10⁷ microconidia of the ${Delta}$ chsVmutant, most of the animals died within 24 h (Fig. 4 and Table1). We termed this response "fast killing" as opposed to "slowkilling" caused by the wild-type strain 4287. A ${Delta}$ chsV mutantthat had been transformed with the wild-type chsV allele revertedto the slow-killing phenotype, indicating that fast killingwas determined by lack of a functional ${Delta}$ chsV allele. Importantly,fast killing was provoked both by living and heat-killed conidiaof the chsV mutant (Fig. 4) and occurred both in immunocompetentand in immunosuppressed mice (results not shown). Taken together,these data indicate that the slow- and the fast-killing responseshave distinct underlying mechanisms.

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FIG. 4. F. oxysporum mutants lacking the chitin synthase ChsV cause "fast killing" on mice. (A) Groups of 10 immunodepressed mice were infected with 2 x 10⁷ microconidia of the wild-type strain 4287 (•), chitin synthase mutant ${Delta}$ chsV ( ${blacksquare}$ ), heat-killed conidia of the ${Delta}$ chsV mutant ( ${triangleup}$ ), or a ${Delta}$ chsV mutant complemented with a wild-type chsV allele ( ${diamond}$ ). The percent survival was plotted for 13 days. The data shown are from one representative experiment.

Postmortem studies in mice that had died 24 h after injectionwith conidia of the ${Delta}$ chsV mutant revealed an important amountof serum in the upper respiratory tract, suggesting respiratoryinsufficiency as a possible cause of death. In support of thishypothesis, histolopathological analysis of lung tissue of theseanimals showed an enlargement of the interstitial walls at thealveolar level. We noted the presence of numerous large (30by 25 µm), lemon-shaped or irregularly swollen conidia(Fig. 5C to E) which obstructed the blood flow in the interstitialcapillaries, leading to congestion, edema, and haemorrhagicfoci (Fig. 5E). Some of the conidia had germinated and, ocasionally,branching of the germ tubes was observed. Lung sections of miceinfected with heat-killed conidia of the chsV strain revealeda highly similar pattern except that these conidia failed togerminate (data not shown). In contrast, the lungs of animalsinfected either with the wild-type strain or with the complementedstrain were considerably less damaged (Fig. 5A) than those ofthe group infected with the ${Delta}$ chsV mutant. The presence of germinatedand ungerminated microconidia of normal shape and size (10 by3 µm) was detected in multiple organs (Fig. 5A and B).Taken together, these findings indicate that the fast-killingresponse might be caused by respiratory insufficiency due toearly lung damage provoked by the conidia of the chsV strain.

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FIG. 5. Histological analysis of mice infected with F. oxysporum strains. Sections of embedded tissues from different organs (A and C to E, lung; B, heart) of mice inoculated with microconidia of F. oxysporum wild-type strain (A and B) or chitin synthase mutant ${Delta}$ chsV (C to E) and sacrificed 24 h after inoculation were stained with hematoxylin-eosin, periodic acid-Schiff, and methenamine silver and then examined by light microscopy. (A) Characteristic ellipsoidal microconidia of wild-type strain 4287 in the interstitial space; (B) germinated microconidium of wild-type strain 4287; (C) lemon-shaped microconidium of the ${Delta}$ chsV mutant; (D) swollen ungerminated and germinated microconidia of the ${Delta}$ chsV mutant; (E) germinated conidia of the ${Delta}$ chsV mutant growing in the interstitial spaces and provoking an edema. Bar, 20 µm.

DISCUSSION

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Discussion

Within the fungal kingdom, two highly successful groups of pathogenshave evolved the ability to infect either plant or animal hosts.Whether these two classes of pathogens use similar mechanismsto cause disease in phylogenetically divergent hosts is currentlyan open question. Bacterial multihost pathogen models have beenhighly successful in revealing both conserved and contrastingvirulence mechanisms in plant and animal pathogenesis (28, 32).Therefore, the availability of such multihost models for fungalpathogenesis might prove similarly useful. In the present studywe have developed a dual plant-animal pathogen system basedon a single strain of the vascular wilt fungus F. oxysporum,which allows the simultaneous testing of fungal virulence factorsin plant and mammal host models.

In search for a fungal multihost pathogen model, F. oxysporumwas an obvious candidate because of its well-documented abilityto infect both plants and immunocompromised humans. Due to theeconomical and historical importance as a causal agent of vascularwilt, a much larger body of information is currently availableon the phytopathogenic aspects of F. oxysporum than on its facetas an emerging human pathogen. Plant pathogenic strains of F.oxysporum exhibit a complex pattern of host specificity, promptingtheir classification into formae speciales and physiologicalraces (1). Whereas race-cultivar specificity within a givenforma specialis was recently shown to be controlled by a classicalgene-for-gene system (16, 31), the genetic basis underlyingplant host specificity in F. oxysporum remains unclear. Thefinding that isolates from a particular forma specialis whichinfect the same host plant have independent evolutionary originssuggests that the ability to infect a given plant species mayhave arisen convergently (25).

We decided to examine the ability of a genetically well-characterizedplant pathogenic strain of F. oxysporum to cause disease ina standard animal infection model. An implicit concern withthis approach was whether the behavior of a plant pathogenicstrain would reflect that of a "true" human pathogen in themurine model. Our results suggest that strain 4287 is not ahighly virulent animal pathogen, as shown by the inability tocause disease in immunocompetent mice. However, they also clearlydemonstrate that this strain has the basic capacity to infectand to kill immunosuppressed animals in a highly reproduciblemanner. These findings are consistent with the fact that systemicinfections in humans caused by F. oxysporum are predominantlyreported in immunocompromised individuals (22, 27, 35). Thepresence of germinating microconidia in different organs suggeststhat strain 4287 can grow actively on mammalian tissue and mayalso undergo cycles of conidiation in the host, as reportedpreviously for pathogenic Fusaria (19). This notion is furthersupported by the fact that significant amounts of fungal propaguleswere detected in multiple organs 13 days after inoculation.Even though quantitative CFU data obtained from filamentousfungal pathogens have to be interpreted with caution since theymay vary depending on the spore/mycelium ratio, these resultsclearly indicate that strain 4287 has a high persistency withinthe mammalian host.

Once we confirmed the basic ability of F. oxysporum strain 4287to cause disease in a mammalian model, we further tested itsusefulness by examining knockout mutants in three functionallydistinct classes of genes that were previously found to be involvedin virulence on tomato plants. One of them, fmk1, encodes anMAPK that functions in a highly conserved signaling module thatis essential for virulence in all fungal plant pathogens examinedso far (18). Interestingly, we found that Fmk1 is dispensablefor the virulence of F. oxysporum in the disseminated mousemodel. This finding is intriguing, because it was suggestedthat this highly conserved signaling cascade might play functionallysimilar roles in fungal plant and animal pathogenesis (18).However, a recent study showed that the orthologous MAPK cascadeof the human pathogen C. neoformans is also dispensable forvirulence in a disseminated mouse model (7). Moreover, the samepathway in C. albicans was previously reported to contributeonly partly to virulence (6). Although the role of this pathwayhas not been tested thus far in topical fungal infection modelssuch as the skin or the oral cavity, these results, taken together,indicate that the Fus3/Kss1 MAPK cascade plays functionallydistinct roles in plant and animal pathogenesis. Our data furthersuggest that such divergent functions not only apply to differentpathogen species but also reside within a single fungal strain.

Similarly contrasting results were obtained with mutants inthe zinc finger transcription factor PacC. In this case, wefound that PacC is required for full virulence on mice, althoughit was previously shown to be dispensable or even negativelyaffected virulence on tomato plants (5). Interestingly, thepacC homologue of C. albicans, RIM101, is also required forvirulence in a hematogenously disseminated murine model (8).A likely explanation for the contrasting role of PacC in animaland plant pathogenesis is the diverging pH of the host niche:slightly alkaline during systemic animal infection versus acidicin plant tissue. Depending on the ambient pH, PacC may functioneither as a positive or a negative virulence determinant byactivating or preventing expression of distinct groups of pathogenicitygenes.

A third class of knockout mutants tested were those affectedin the class V chitin synthase ChsV. Chitin synthases catalyzethe biosynthesis of chitin, a ß-1,4-linked polysaccharidemade of N-acetylglucosamine and an essential component of thefungal cell wall (4). F. oxysporum mutants that lack ChsV areunable to infect tomato and hypersensitive to antifungal plantdefense compounds such as ${alpha}$ -tomatine or H₂O₂ (21). Surprisingly,most of the mice injected with microconidia of the ${Delta}$ chsV straindied within 24 h, a phenomenon that we termed "fast killing."Postmortem studies and histopathological analysis suggestedthat in these animals death was caused by respiratory insufficiencyas a consequence of severe lung damage. In contrast, death inmice infected by filamentous fungi is usually not provoked byinitial lung damage but by more generalized lesions affectingnumerous organs (13). We found evidence suggesting that thisunusual fast-killing effect could be caused by physical obstructionof interstitial capillaries by the abnormally enlarged ${Delta}$ chsVmutant conidia. The presence of morphological aberrations infungal class V chitin synthase mutants such as balloon-likeswellings and abnormally shaped, lemon-like microconidia, hasbeen reported previously and is probably caused by defects incell wall integrity, because it can be reversed by additionof an osmoprotectant to the growth medium (15, 21). Two linesof evidence strongly suggest that fast killing is mechanisticallyunrelated to slow killing caused by the wild-type F. oxysporumstrain. First, unlike slow killing, fast killing was inducedboth by living and heat-killed conidia of the ${Delta}$ chsV mutant, suggestingthat the sole physical presence of the conidia is sufficientto provoke death. Second, in contrast to slow killing, fastkilling occurred both in immunocompetent and in immunosuppressedmice, indicating that this mechanism acts independently of theimmunological status of the animals.

F. oxysporum strain 4287 is to our knowledge the first fungalmodel strain reported to reproducibly infect both plant andmammalian hosts. The availability of such a multihost modelstrain represents a key step toward improving our understandingof how fungal pathogenesis on evolutionary distant hosts hasevolved. The results obtained with the F. oxysporum knockoutmutants suggest that certain virulence factors essential inplant pathogenesis are dispensable in animal pathogenesis andvice versa. In both cases where such divergent roles were detectedbetween the tomato and the mouse model, the virulence patternof the F. oxysporum mutants in mice mimics that previously reportedfor two well-established animal model pathogens, C. albicansand C. neoformans. This finding is important because it addressesthe underlying concern whether the virulence pattern of an opportunisticpathogen such as F. oxysporum will reflect that of a "true"human pathogen. Our results with the fmk1 and the pacC mutantssuggest that F. oxysporum strain 4287 indeed behaves like a"true" animal pathogen in the immunosuppressed mouse model.Thus, F. oxysporum strain 4287 could be a useful model for theidentification of conserved and divergent fungal virulence determinantsin plant and animal pathogenesis, similar to the P. aeruginosastrain PA14 (30). Importantly, 4287 is also amenable to randominsertional mutagenesis (21), and rapid and reliable in vivovirulence assays on tomato fruits are already available forthis strain (9). The F. oxysporum model may therefore provehighly useful in providing new insights into the molecular basisof host specificity in fungal pathogenesis.

ACKNOWLEDGMENTS

The research was supported by grants PI-020114 from the SpanishMinisterio de Sanidad y Consumo and BIO2001-2601 from Ministeriode Ciencia y Tecnología. M.O. has a Ph.D. fellowshipfrom Universitat Rovira i Virgili. M.P.M. and Z.C. have Ph.D.fellowships from the Ministerio de Ciencia y Tecnologíaand the Ministerio de Educación, Cultura y Deporte, respectively.A.D.P. is recipient of a Ramón y Cajal grant from Ministeriode Ciencia y Tecnología.

FOOTNOTES

* Corresponding author. Mailing address: Departamento de Genética, Universidad de Córdoba, Campus de Rabanales C5, 14071 Córdoba, Spain. Phone: (34) 957218981. Fax: (34) 957212072. E-mail: ge2dipia{at}uco.es.

Editor: T. R. Kozel

REFERENCES

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