Isolation of Two Different Phenotypes of Mycorrhizal Mutants in the Model Legume Plant Lotus japonicus after EMS-Treatment

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1 Plant CellPhysiol. 41(6): (2000) JSPP 2000 Isolation of Two Different Phenotypes of Mycorrhizal Mutants in the Model Legume Plant Lotus japonicus after EMS-Treatment Keishi Senoo lj 4, M. Zakaria Solaiman \ Masayoshi Kawaguchi 2, Haruko Imaizumi-Anraku 3, Shoichiro Akao 3, Akiyoshi Tanaka ' and Hitoshi Obata ' 1 Faculty of Bioresources, Mie University, Tsu, Mie, Japan 2 Graduate School of Arts and Science, University of Tokyo, Komaba, Meguro-ku, Tokyo, Japan 3 National Institute of Agrobiological Resources, Kannondai, Tsukuba, Ibaraki, Japan Lotus japonicus has been proposed as a model plant for the molecular genetic study of plant-microbe interaction including Mesorhizobium loti and arbuscular mycorrhizal (AM) fungi. Non-mycorrhizal mutants of Lotus japonicus were screened from a collection of 12 mutants showing non-nodulating (Nod), ineffectively nodulating (") and hypernodulating (Nod ++ ) phenotypes with monogenic recessive inheritance induced by EMS (ethylmethane sulfonate) mutagenesis. Three mycorrhizal mutant lines showing highly reduced arbuscular mycorrhizal colonization were obtained. All of them were derived from Nod" phenotypes. In Ljsym72, the root colonization by Glomus sp. R-10 is characterized by poor development of the external mycelium, formation of extremely branched appressoria, and the blocking of hyphal penetration at the root epidermis. Neither arbuscules nor vesicles were formed in Ljsym72 roots. Fungal recognition on the root surface was strongly affected by the mutation in the LjSym72 gene. Unique characteristics in mutant lines Ljsym71-1 and Ljsym71-2 were the overproduction of deformed appressoria and arrested hyphal penetration of the exodermis. Small amounts of internal colonization including degenerated arbuscule formation occurred infrequently in these types of mutants. Not only fungal development on the root surface but also that in the root exodermis and cortex was affected by the mutation in LjSym71 gene. These mutants represent a key advance in molecular research on the AM symbiosis. Key words: Arbuscular mycorrhiza Glomus sp. R-10 Lotus japonicus Mycorrhizal mutants Non-nodulating mutants. Arbuscular mycorrhizal (AM) fungi belong to the Zygomycetes and are capable of making a non-specific relationship with roots of more than 80% of terrestrial plant species. Colonization of roots by AM fungi involves Abbreviations: AM, arbuscular mycorrhiza; EMS, ethylmethane sulfonate; LSD, least significant difference. 4 Corresponding author: , senoo@bio.mie-u.ac.jp; Fax, several specific developmental steps. These steps are: (i) spore germination and hyphal elongation; (ii) appressoria formation; (iii) penetration of hyphae into root cortical tissue; and (iv) formation of arbuscules in the cortex layer. The arbuscule is the site of mutual nutrient exchange between the fungi and host plant. The fungi obtain photosynthates from the host plant in exchange for phosphate absorbed from soil through the external hyphae. The AM symbiosis is of considerable importance in agriculture, enhancing plant growth and making the plant more tolerant to various stresses including low.soil fertility, drought stress, and pathological stress. The highly compatible plant-microbe relationship and the beneficial effect on plant growth shown by the AM symbiosis have attracted the interest of plant scientists in various fields. Although extensive research effort has been made, current knowledge on the molecular and genetic aspects of the development of the AM symbiosis is very limited (Smith 1995, Harrison 1997). Several mycorrhizal mutants have been screened as tools to study the mechanism of the AM symbiosis (Gianinazzi-Pearson 1996). These mutants were mostly obtained from legumes, including pea, faba bean (Due et al. 1989, Gianinazzi-Pearson et al. 1991), alfalfa (Bradbury et al. 1991, 1993a, b), Phaseolus vulgaris (Shirtliffe and Vessey 1996), and Medicago truncatula (Sagan et al. 1995). Mycorrhizal mutants were found by extensive screening of mutants for nodulation or nitrogen fixation. One exception was the identification of a mycorrhizal mutant in tomato with highly reduced mycorrhizal colonization (Barker et al. 1998). The fact that some nodulation mutants among various legumes are unable to form normal AM symbioses indicates the root nodule symbiosis and the AM symbiosis share common functions. Lotus japonicus has been proposed as a model legume plant for the molecular genetic study of legume-rhizobium interactions (Handberg and Stougaard 1992) and the AM symbiosis (Wegel et al. 1998). The basis of the molecular genetic analysis of L. japonicus has been established, including a gene tagging system using the Ac transposable element (Thykjaer et al. 1995), in planta transformation (Oger et al. 1996), the construction of a linkage map (Jiang and Gresshoff 1997), and the isolation of symbiotic mutants (Imaizumi-Anraku et al. 1997, Szczyglowski et al. 726

2 Mycorrhizal mutants in Lotus japonicus , Schauser et al. 1998, Wegel et al. 1998). We are currently building a collection of L. japonicus mutants generated by the chemical mutagen ethylmethane sulfonate (EMS). These mutants have phenotypes that are non-nodulating (Nod~), ineffectively nodulating ("), or hypernodulating (Nod ++ ). In this study, five stable Nod mutants, six " mutants, and one Nod ++ mutant were screened for mycorrhizal mutations. We report three mycorrhizal mutant lines which are affected in the initial stage of mycorrhizal colonization. Materials and Methods Table 1 Lotus japonicus symbiotic mutants used in this study Locus Ljsym70 Ljsym71-1 Ljsym71-2 Ljsym72 Ljsym73 Ljsym74-1 (albl) Ljsym74-2 Ljsym75 Ljsym76 (fenl) Ljsym78 Ljsym79 Ljsym81 Nodulation phenotypes Nod - Nod Nod Nod Nod (low nodulating) ^ + Nod + (hypernodulating) Generation F4 F4 M5 F4 Plant material Screening for phenotypes defective in stages of mycorrhizal colonization was carried out using a population of Lotus japonicus B-129 'Gifu' mutants that had been generated by EMS-treatment (Imaizumi-Anraku et al. 1997). Characteristics of these mutant lines (Kawaguchi et al. in preparation) are shown in Table 1. The mutants Ljsym70, Ljsym71-1, Ljsym71-2, Ljsym72 and Ljsym73 are non-nodulating (Nod~) mutants. Ljsym73 shows low nodulating phenotype and is included in Nod mutant in this study. In these mutants, the initial stage of nodulation prior to nodule primordia formation is blocked. Ljsym74-1 (albl), Ljsym74-2, Ljsym75, Ljsym76 (fenl), Ljsym79 and Ljsym81 are ineffectively nodulating () mutants. In albl, the stage of nodule organogenesis is affected by the mutation, which produces aberrant localization of bacteria inside the nodule (Imaizumi-Anraku et al. 1997). In the nodules of fenl, differentiated bacteroids fail to become enlarged by the cell expansion and show low activity of nitrogen fixation (Imaizumi-Anraku et al. 1997). Ljsym78 is a hypernodulating mutant. All of the mutated loci were confirmed to be monogenic and recessive. Potting medium and inoculum Washed sand was mixed in equal volumes with Akadama soil, which is subsoil of a volcanic ash soil (Kodaira Engei Shizai Co., Ltd., Japan). The soil-sand mix was sterilized by autoclaving at 121 C for 1 h, and amended with NH4NO3, KH 2 PO 4 and KC1 at the concentrations of 0.53 g, g, and g liter ' mix, respectively. AM fungal inoculum containing spores of Glomus sp. R-10 (Dr. Kinkon, Idemitsu Kosan Co., Ltd., Japan) was mixed well with the potting mixture (1 : 10, v/v). Glomus sp. R-10 inoculum gave the highest colonization rate among several Glomus species in tests with various crops including legumes (Narutaki et al., personal communication). Screening procedure Seeds from wild-type 'Gifu' and Nod, or Nod ++ mutants were scratched with sand paper, surface sterilized with 2% sodium hypochlorite containing 0.02% Tween 20 for 10 min, rinsed in sterile distilled water, and then germinated on sterilized moist filter paper in petri dishes at 25 C under dark conditions. After germination, the seedlings were transplanted individually into nursery trays containing the sand-soil-inoculum mixture. Wild-type seedlings were grown without inoculum to check for contamination with pathogens in the sterilized mix. Plants were grown for one month in a growth chamber with a 20 h day at 25 C, a photosynthetic photon flux density of 45^mol m" 2 s" 1, and a 4h night at 22 C. After 30d, the plants were transplanted into 300-ml pots and allowed to grow for 2 more months. The growth chamber conditions were the same as described above, except the light intensity was 70//molm 2 s"'. Plants were sampled after 90 d of growth for assessment of mycorrhizal colonization. Evaluation of mycorrhizal colonization Root samples were cleared in 10% KOH and stained with trypan blue using a modification of the method of Philips and Hayman (1970), in which lactoglycerol was used instead of lactophenol. Colonization in the inoculated control 'Gifu' plants and the mutagenized plants was determined as the percentage of root length colonized, under a dissecting microscope and the grid intersect method (Tennant 1975). If the AM colonization of mutagenized plants appeared reduced, more detailed assessment of colonization was carried out by the method of McGonigle et al. (1990). Briefly, cleared and stained whole root segments were mounted on slides. Intersects between the roots and an ocular crosshair were scored for the presence of different mycorrhizal structures at x 100 magnification. The evaluation of mycorrhizal colonization was carried out in three replicates. Results are expressed as the percentage of intersects having appressoria, arbuscules, vesicles and hyphal colonization. Results Root colonization of wild-type and mycorrhizal mutant lines in Lotus The percentage of root length colonized by Glomus sp. R-10 in twelve Lotus mutant lines and the wild-type are shown in Fig. 1. The colonization was highly reduced in mutants Ljsym72 (Nod"), Ljsym71-1, and 77-2 (Nod"). The percentage of root length colonized by appressoria, arbuscules, vesicles and hyphae was individually estimated at 90 d after transplanting of seedlings (Table 2). In the Ljsym72 mutant, the percentage of colonization by appressoria was not significantly (P>0.05) different from that of the wild-type 'Gifu' according to the Fisher's LSD (least significant difference) test, but the appressoria were strikingly abnormal in shape and hyphal penetration into the root cortex was blocked. Neither arbuscule nor vesicle formation was observed. Most of the hyphal colonization observed was on the root surface (Table 2). Mutants Ljsym71-1 and 71-2 showed higher

3 728 Mycorrhizal mutants in Lotus japonicus Root length colonized (%) Fig. 1 Percentage of root length colonized by Glomus sp. R-10 in wild-type and different nodulation or nitrogen fixation mutants of Lotus japonicus. Three plants for each mutants and wild-type were used. The determination was conducted five times for each root, and the reported data are the means from these determinations. Bar ( ) indicates LSD (least significant difference) at 5% level. percentage of root length colonization by the appressoria, however, the rate of arbuscule and vesicle formation was remarkably reduced compared with the wild-type. The hyphal colonization was also reduced in these two lines. The level of root colonization in other mutants was not remarkably different from that in the wild-type (Fig. 1). Characteristics of normal mycorrhizal colonization in Lotus Light microscopic observations following clearing and staining of the roots with trypan blue indicated that Table 2 Percentage of appressorial, arbuscular, vesicular and hyphal colonization in roots of Lotus japonicus' a Mycorrhizal mutants Gifu (wild-type) Ljsym71-1 Ljsym71-2 Ljsym72 LSDo.os 7 ApC vc HC ** 10.5* * 2.7* 0* * 0.2* 0* * 27.5* 18.5* 8.3 a Three plants for each mutants and wild-type were used. The determination was conducted five times for each root, and the reported values are the means from these determinations. b ApC, appressorial colonization. c AC, arbuscular colonization. d VC, vesicular conization. e HC, hyphal colonization. f LSD o.o5, least significant difference at 5% level. g Means followed by an asterisk within a column are significantly different (P<0.05) from values obtained in wild-type 'Gifu' according to the Fisher's LSD test. mycorrhizal colonization of the wild-type L. japonicus by Glomus sp. R-10 was typical (Fig. 2A, B). All steps in the colonization processes were observed, including appressoria on the root surface, internal hyphae, arbuscules, and vesicles. Glomus sp. R-10 often produced vesicles that completely filled up the root at mature stage (Fig.2C). Uninoculated control plants did not show any colonization with AM fungi or with pathogens. Microscopic observation on phenotypes of mycorrhizal mutants The hyphal colonization in the root of Ljsym72 was highly reduced as shown in Table 2, and most hyphae on the root surface grew in the form of runner hyphae with remarkable branching (Fig.2D). Appressoria were visible on the root surface but they were extremely abnormal in shape, showing extraordinary branching and swelling (Fig. 2E, F). The number of appressoria was not statistically different from that of the wild-type. Following appressoria formation, hyphae tried to enter into the root exodermis, but their growth was halted at the root epidermis and the hyphae showed abnormal branching and swelling (Fig. 2D, E, F). Absolutely no arbuscules and no vesicles were present in any root of mutant Ljsym72 examined. In Ljsym71-1 and Ljsym71-2 mutants, the hyphal colonization of the root was significantly reduced compared with the wild-type (Table 2). The development of deformed appressoria associated with aborted internal hyphae were often observed. Several types of appressoria irregular in shape were observed on the root surface or along the adjacent walls of epidermal cells. The appressoria showed complex branching and a swollen appearance (Fig. 3A-C, E-G), which were features similar to those of irregular appressoria observed in mycorrhizal mutants of pea (Due et al. 1989), alfalfa (Bradbury et al. 1991) and tomato (Barker et al. 1998). Also, the percentage of appressoria formation on the root surface was significantly higher than those on wild-type root. The elongation of internal hyphae from the appressoria were observed, but they were aborted soon after penetrating one or two cell layers, and sometimes appeared swollen (Fig. 3B). Most often, the mycorrhizal colonization was arrested at this stage. Colonization by internal hyphae at the root cortex was observed at a low frequency, but rather complex branching or swelling of hyphae were visible (Fig. 3C, G). Only a limited number of hyphae formed arbuscules, but the morphology of these arbuscules occasionally appeared to be degenerated (Fig. 3D, H). Phenotype of mutants Ljsym71-1 and Ljsym71-2 was similar except that the amount of internal hyphae seemed to be more reduced in Ljsym71-2. Discussion Among twelve nodulation or nitrogen-fixation mutants, Ljsym72, Ljsym71-1, and Ljsym71-2 exhibit resist-

4 Mycorrhizal mutants in Lotus japonicus 729 ance to mycorrhizal colonization. In the mutant Ljsym72, typical features were limited growth of external hyphae, formation of branched runner hyphae, formation of appressoria extremely abnormal in shape, aborted penetration of hyphae into root, and abso- lutely no formation of arbuscules and vesicles. These observation indicate that the colonization was blocked at the root surface in this mutant, just like the myc~ mutants in other legumes (Due et al. 1989, Shirtliffe and Vessey 1996, Sagan et al. 1995). The appressoria on the root surface of Fig. 2 Light microscopy of cleared and trypan blue stained roots of wild-type 'Gifu' (Fig. A-C) and mutant Ljsym72 (Fig. D-F) of Lotus japonicus, colonized by Glomus sp. R-10. (A) Typical colonization showing appressorium (ap), internal hyphae (ih) and arbuscules (ar). (B) External hyphae (eh), internal hyphae and vesicles (v). (C) Roots were completely filled up with vesicles. (D) External and runner hyphae (rh) anchored on the root surface. (E) Abnormal appressoria on the root surface. (F) Complex appressoria and hyphal swelling on the root surface. Bar represents 50 /um.

5 730 Mycorrhizal mutants in Lotus japonicus Fig. 3 Light microscopy of cleared and trypan blue stained roots of mutants Ljsym71-1 (Fig. A-D) and Ljsym71-2 (Fig. E-H) of Lotus japonicus, colonized by Glomus R-10. (A) Several appressoria on the root surface, and external and runner hyphae anchored on the root surface. (B) Penetration by appressoria and hyphal development restricted in the epidermis. (C) Aborted internal hyphal growth. (D) Degenerated arbuscules and infrequent vesicle formation. (E) External hyphae and abnormal appressoria (ap) on the root surface. (F) Abnormal complex appressoria (ap). (G) Abnormal appressoria (ap) and swollen internal hyphae. (H) Colonization aborted and arbuscules degenerated. Bar represents 50 fim.

6 Mycorrhizal mutants in Lotus japonicus 731 mutant Ljsym72 were similar to that of the wild-type in number, but they had a highly branched shape. This phenotype is different from that with increased production of appressoria on myc~ phenotypes of Medicago sativa (Bradbury et al. 1991) and the phenotype with fewer appressoria of pea (Due et al. 1989). The formation of highly branched runner hyphae and appressoria indicates morphogenetic changes in fungal branching pattern, which are similar to those that occur when contact between the fungus and the plant is prevented by a membrane filter (Giovannetti et al. 1993, 1994, for reviews see Smith and Read 1997). Therefore, the mutual recognition between host plant and mycorrhiza at the hyphae-root interface might be affected by the mutation locus of Ljsym72. In addition, the existence of arrested internal hyphae at the root epidermis suggests that the Ljsym72 locus also affects fungal development at the root epidermis. Neither arbuscules nor vesicles was observed in this mutant, but it is unclear whether arbuscule formation itself was blocked by the mutation or complete abortion of internal hyphae at root exodermis resulted in the failure of arbuscule and vesicle formation. The phenotype of this mutant is different from myc 1 in the presence of extraordinarily deformed appressoria and runner hyphae on the root compared with those reported in pea (Due et al. 1989), alfalfa (Bradbury et al. 1991), Medicago (Sagan et al. 1995) and Phaseolus (Shirtliffe and Vessey 1996), and in the complete lack of formation of arbuscules and vesicles. This phenotype is also different from the Coi mutant of L. japonicus (Wegel et al. 1998) in that the colonization is blocked at the root surface and epidermis. According to the unique phenotype observed, we designate the mutant Ljsym72 as mcbep (mycorrhizal colonization blocked at epidermis). In Ljsym71-1 and Ljsym71-2 mutants, typical features were the reduction of external hyphae and the overproduction of deformed appressoria associated with no hyphal penetration of the exodermis. Overproduced and deformed appresorria may be the result of repeated failed attempts at appresorria formation on the root surface by AM fungi as observed in alfalfa (Bradbury et al. 1991). Very rarely, internal hyphae grew up to the cortical cell layer and formed arbuscules, but they are often abnormal in morphology. These phenotypes of Ljsym71 were considered to be similar to the myc" 1 mutant of alfalfa (Bradbury et al. 1991), but the formation of degenerated arbuscules is a characteristic unique to this Lotus mutant. According to the phenotype, the mutation at the Ljsym71 locus firstly affects the recognition of fungal hyphae on root surface, and it also affects fungal penetration of the exodermis, and thereby the development of internal hyphae in the root cortex and arbuscule formation. These observations are in contrast to the interpretation of Wegel et al. (1998) that different genetic requirements exist for the infection of different cell types by the symbiotic fungi. According to the uniqueness of the phenotype reported here, we designate the mutants Ljsym71-1 and 71-2 as mcbex (mycorrhizal colonization blocked at exodermis). The unique phenotypes of mycorrhizal colonization observed in the mutants Ljsym71-1, 71-2 and 72 may possibly be limited to the mycorrhizal strain used in this study {Glomus sp. R-10) although different mycorrhizal strains were found to show similar colonization phenotypes on the myc mutants of pea and fava bean (Due et al. 1989). In our study, two types of phenotypes of mycorrhizal mutants were found among Nod~ mutants. In these mutants, the early steps of nodule formation prior to nodule primordia formation were blocked (Kawaguchi et al., in preparation). This result suggests that several parts of the genetic systems controlling the initial stage of nodule formation are shared with those that control colonization of roots by AM fungi. The non-nodulating mutants Ljsym70 and Ljsym73 exhibited normal mycorrhizal colonization, suggesting that a certain step of the genetic system might be blocked in these mutants. This certain step is specific to the initiation of nodule formation, such as a step for the recognition of Nod factors and the upstream region of related signal transduction pathway. All of six ~ mutants tested in this study had normal mycorrhiza, suggesting the genes controlling nodule organogenesis and nodule maturation are not always shared with the process of mycorrhizal colonization. Our goal is to determine the mechanism of the AM symbiosis at the molecular level. The discovery of genetically defined myc~ mutants that are isogenic except for the mutated genes opens possibilities for identifying some of the genes involved in the key stages leading to the establishment of mycorrhiza. In mutant lines used in this study, all of the mutated loci had already been confirmed to be monogenic. Molecular cloning of the genes affected in the Lotus mycorrhizal symbiosis can be achieved through map-based cloning. In order to draw a detailed picture of the mycorrhizal symbiosis, the collection of more mutants with a variety of unique phenotypes will be necessary. We are currently screening and F4 progenies of Lotus Nod and " mutants to get additional unique mycorrhizal phenotypes as the potent materials of map-based cloning. Several symbiotic defective mutants of L. japonicus showing non-mycorrhizal, non-nodulating and ineffective nodulating phenotypes have already been reported (Wegel et al. 1998, Szczyglmski et al. 1998). It will be of great importance to perform allelic tests in future among those mutants including ours, in order to dissect genetically the processes of both mycorrhization and nodulation.

7 732 Mycorrhizal mutants in Lotus japonicus The authors thank Dr. Motoshi Suzuki and Mr. Akihiko Narutaki of Idemitsu Kosan Co, Ltd., Tokyo, Japan for supplying spores of Glomus sp. R-10. We also thank Dr. Masanori Saito of National Grassland Research Institute, and Dr. Keitaro Tawaraya of Yamagata University for invaluable suggestion and Dr. Terence P. McGonigle for his critical reading of this manuscript. M.Z.S. is grateful to Japan Society for the Promotion of Science for providing postdoctoral fellowships. Some parts of this study was supported by Grant-in-Aid for Scientific Research on Priority Areas (grant no ). References Barker, S.J., Stummer, B., Gao, L., Dispain, I., O'Connor, P.J. and Smith, S.E. (1998) A mutant in Lycopersicon esculentum Mill, with highly reduced VA mycorrhizal colonization: isolation and preliminary characterization. Plant J. 15: Bradbury, S.M., Peterson, R.L. and Bowley, S.R. 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