Proc. Natl. Acad. Sci. USAVol. 93, pp. 7783-7788, July 1996Genetics

Retrotransposons of rice involved in mutations inducedby tissue culture

(retroelements/transposable elements/stress/insertion mutations)

HIROHIKo HIROCHIKA*t, KAZUHIKO SUGIMOTO*, YOSHIAKI OTSUKIt, HIDEHITO TSUGAWAt, AND MARI KANDA§*Department of Molecular Biology, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305, Japan; *Department of Crop Breeding, NationalAgriculture Research Center, Tsukuba, Ibaraki 305, Japan; and §Shiga Agricultural Experiment Station, Azuchi, Shiga 521-13, Japan

Communicated by Ronald L. Phillips, University of Minnesota, St. Paul, MN, March 21, 1996 (received for review July 17, 1995)

ABSTRACT Five retrotransposon families of rice (Tosi-Tos5) have been reported previously. Here we report 15 newretrotransposon families of rice (Tos6-Tos2O). In contrast toyeast and Drosophila retrotransposons, all ofthe rice retrotrans-posons examined appear inactive (or almost inactive) undernormal growth conditions. Three of the rice retrotransposons(ToslO, Tosl7, and Tosl9) are activated under tissue cultureconditions. The most active one, Tosi 7, was studied in detail. Thecopy number of Tosl7 increased with prolonged culture period.In all of the plants regenerated from tissue cultures, includingtransgenic plants, 5 to 30 transposed Tosl7 copies were detected.The transcript of Tosl7 was only detected under tissue cultureconditions, indicating that the transposition of Tosl7 is mainlyregulated at the transcriptional level. To examine the target-sitespecificity of Tosl7transposition, sequences flanking transposedTosl7 copies were analyzed. At least four out of eight target sitesexamined are coding regions. Other target sites may also be ingenes because two out of four were transcribed. The regeneratedplants with Tosl7-insertions in the phytochrome A gene and theS-receptor kinase-related gene were identified. These resultsindicate that activation of Tos17 is an important cause of tissueculture-induced mutations. Tissue culture-induced activation ofTosl7 may be a useful tool for insertional mutagenesis andfunctional analysis of genes.

Tissue culture of plants is an important means to propagategenetically identical individuals asexually and to produce trans-genic plants. However, undesired genetic and cytogenetic mod-ifications are frequently induced during tissue culture. Thesetissue culture-induced mutations have been reported in manyplant species and seem to be ubiquitous in plants (1, 2). Althoughtissue culture-induced mutations have been studied extensively asa source of plant improvement, little is known about theirmolecular causes. Larkin and Scowcroft (1) proposed that theactivation of transposable elements might be responsible fortissue culture-induced mutations. The first evidence supportingthis hypothesis was the detection of the activity of the maizetransposable element Activator (Ac) in the progeny of 3% ofregenerated maize plants (3). The activation of another trans-posable element Suppressor-Mutator (Spm) was also observed inthe progeny of 1% of regenerated maize plants (4). Our recentstudies have shown that another type of transposable elements,retrotransposons, are also activated by tissue culture of tobacco(5). Retrotransposons are both functionally and structurally dif-ferent from the well-characterized transposable elements such asAc and Spm. Therefore, the mechanism of activation by tissueculture is most likely different between retrotransposons and themaize transposable elements. Because retrotransposons undergoreplicative transposition through RNA as an intermediate (6),they induce only stable mutations. Therefore, stable tissue cul-ture-induced mutations (1, 2) may be explained by the activationof retrotransposons. Here we report the first active retrotrans-

posons of rice that are activated by tissue culture. Rice was chosenas a representative of monocotyledonous plants to examine theubiquity of retrotransposons activated by tissue culture and thepossible involvement of retrotransposons in tissue culture-induced mutations because rice is being studied as a model plantsuitable for molecular genetic analysis (7) and many studies ontissue culture-induced mutations have been carried out (8). Thepresent data indicate that retrotransposons are involved in tissueculture-induced mutations.

MATERIALS AND METHODSPlant Materials. Calli of thejaponica variety Nipponbare of

Oryza sativa were induced by culturing germinating rice seedson Murashige-Skoog solid medium (9) containing 2,4-dichlorophenoxyacetic acid at 2 jig/ml. After incubation forabout 1 month, calli were transferred into N6 medium (10) toinduce suspension cultured cells, which were cultured in thesame medium. Oc cell line (11) derived from the indica varietyC5924 of 0. sativa was cultured in Muller-Grafe's AA medium(12). Protoplast formation and transformation of rice byelectroporation were carried out as described (13).

Southern and Northern Blot Hybridizations. Extraction ofnucleic acids was carried out as described (5). About 2 jig ofgenomic DNA and 1 ,ug of poly(A)+ RNA were electropho-resed and transferred to a nylon membrane as described (14).Filters were hybridized and washed as described (14), exceptthat hybridization and washing were carried out at 42°C and65°C, respectively. Cloned sequences of the reverse transcrip-tase domain were used as probes.

Amplification of Reverse Transcriptase Domain of Retro-transposons. The genomic DNA or the first strand cDNA wasamplified as described (14). The cDNA was generated frompoly(A)+ RNA isolated from protoplasts of cultured cellsusing random primers and a cDNA cycle kit (Invitrogen).

Amplification of Sequences Flanking Transposed Tosl7.Sequences flanking transposed Tosi 7 (target-site sequences)were amplified by inverse PCR. Target-site sequences 1-6 and7-8 were amplified using A clones and the rice total DNA,respectively, as described (13) except that the total DNA wasdigested with XhoI-SalI. Two sets of primers were used fortwo-step PCR: Tosl7LTR-1, TTGGATCTTGTATCTTGTA-TATAC and Tosl7LTR-3, CCAATGGACTGGACATCCG-ATGGG were used for the first PCR; the primers Tosl7LTR-2,GCTAATACT7ATTGTTAGGTTGCAA and Tosl7LTR-4, CT-GGACATGGGCCAACTATACAGT were used for the secondreaction. An empty target site of normal plants was amplified byusing primers TGAGTTCCCCTTGAGTCAGC and GTAGG-ACACTGGACAGTTGC.

Sequence Analysis and Database Search. Handling of pri-mary sequences and multiple sequence alignment was carriedout using the GENEWORKS 2 software (IntelliGenetics). Com-

Abbreviation: LTR, long terminal repeat.Data deposition: The sequences reported in this paper have beendeposited in the GenBank data base (accession nos. D85865-D85879).tTo whom reprint requests should be addressed.

7783

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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puter-based amino acid similarity searches of the ProteinIdentification Research (release 44), DNA Data Bank ofJapan(release 21), GenBank (release 89), and European MolecularBiology Laboratory (release 42) databases were carried outwith the BLAST program.

RESULTSCloning of Retrotransposons of Rice. We have isolated five

families (Tosl-TosS) of rice retrotransposons by two generalmethods (14). Here, we further screened rice retrotransposonsby using one of these methods based on the fact that certainamino acids are highly conserved in the reverse transcriptasesencoded by retrotransposons. Reverse transcriptase domainswere amplified by PCR from genomic DNA and cloned.Eleven new families (Tos6-Tosl6) were identified by screeningabout 200 clones by cross-hybridization and their identitieswere confirmed by sequencing [the criterion for a new familywas <90% amino acid identity (15)]. Deduced amino acidsequences are shown in Fig. 1.The activity of the new retrotransposons was examined in both

plants and cultured cells by assaying the copy number changeamong individuals and varieties and between plants and culturedcells. No copy number change among individuals of normallypropagated plants was observed with any retrotransposon probesexamined. Only minor copy number changes were observed withsome retrotransposon probes among rice varieties (data notshown). This is in contrast to yeast and Drosophila retrotrans-posons, the distribution of which in the genome can differbetween different stocks of the same strain (16). In one estab-lished cell line, an increase in the copy number of ToslO wasobserved and is described in the following section.

TOS14TOS15TOS16TOS9TOS4TOS13TOS18TOS12TOS19TOS17TOS20TOS1lTOS6TOS8TOS7TOS10

Consensus

TOS5TOS14TOS15TOS16

TOS9TOS4TOS13TOS18TOS12TOS19TOS17TOS20TOSilTOS6TOS8TOS7

TOS10

Consensus

AFLNGELEEAFLNGELDEEAFLNGELD8EAFLNGDLEEETFLHGELEEEAFLEGELEEDAFLEGELEEDAFLEGELGED

IYMDQPDGYV LEGQEGMVC- -----KLLKSIYMEQPDGFV ALGQEGKVV- -----KLLKSIYMQQPDGFV VNGQERKVC- -----KLVKSVYVSQPLGFE VTGEEAKVY- -----RLHKAIYMDQPEGFI VPGKEDYVC- -----KLKRSIYMDQPEGFI VPGKEKYVC- -----KLKRSIYMEQPEGFV VPGKENLVCR LKKSLRLKKSINMEQPEGFF VPGKENLVC- -----RLKKS

Previously, we have adopted the reverse transcription-PCRmethod to screen retrotransposons of tobacco activated by tissueculture (5). This method was applied for isolation of rice retro-transposons activated by tissue culture. cDNA was made frompoly(A)+ RNA prepared from protoplasts of cultured cells andsubjected to PCR amplificatiorl. By analyzing 20 clones, four newretrotransposon families (Tosl7-Tos20) (Fig. 1) in addition toTosS were identified. These new retrotransposons and ToslOwerecharacterized further.

Activation of Transposition by Tissue Culture. An increase inthe copy number of ToslO and Tosl7 was observed in the Oc cellline (11) derived from the indica variety (Fig. 2), indicating thatthese retrotransposons are activated in this cell line. The copynumber of Tosl 7 increased in cells ofthejaponica variety culturedfor relatively short periods of time [Fig. 2, lane 2 (3 months), lane3 (12 months), and lane 4 (24 months)]. The copy numberincreased with the time of incubation (Fig. 2, lanes 2-4). A slightincrease in the copyX number of Tosl9 was observed in the24-month-old culture, indicating that TosJ9 is weakly activated incultured cells. An increase in the copy number of Tosl9 was moreclearly shown by the analysis of regenerated plants (data notshown). Because Oc cells have been cultured for more than 10years, the observed difference between Oc cells and cultured cellsmay be due to the duration of culture. Alternatively, the differ-ence may be due to the variety or subspecies (indica/japonica)difference. No change in the copy number of Tosl8 and Tos20wasobserved in either cultured cells or Oc cells (data not shown). Themost active Tosl7 retrotransposon was further characterized.The copy number change in regenerated plants was exam-

ined (Fig. 3B). In all of the regenerated plants, about 5 to 30transposed Tosl7 copies were detected. Different hybridiza-tion patterns expected as a result of random transposition were

LYGRKQAPKQLYGLKQAPKQLYGLRQAPKQLYGLRQAPRALYGLKQSPRQLYSLKQSPRQIYRLKQSPRQLYGLKQSPRQ

AFLHGDLKEB VYMKPPPGVD APANYVF--- -----RLNRA LYGLKQAPRAAFLHGDLHEE VYMHPPPGVE APPGHVF--- -----GLRRA LYGLKQAPRANFLNENLDED VYMTQPKGFV DPESAKRIG- -----QLPKS IYGLTKASHSAILNGNLDED VYMTEPKGFV DPQLAKKIC- -----KLQKS IYGLKQASRSAFLNGDLYED VYMAQPEGFV TKGKEHMGC- -----HLNKS IYGLKQASRQAFLEGVLEEE VYMRQPPGYE NSSKPDFIC- -----KLDKA LYGLKQAPRAAFLHGVLEEE VYMEQPPGYE KKSMPNYVC- -----KLDKA LYGLKQAPRAAFLHGDLHEE VYMDIPLGFG NSQTVEKVC- ----- KLKKS LYGLKQSPRA

AFLHG.L.EE VYM.QP.GF. ..G.E..VC- -----KL.KS LYGLKQAPRQ

WHEKFDKTLT STGFAVNEAD KCVYYRHGGG EGVILCLWHEKFDTTLT SAGFVVNEAD KCVYYRYGGG EGVILCLWHEKFDRTLT SAGFIVNEAD KCVYYRFGGG EGVILCLWHEKFNTTPT SVGFVVNEAE KCVYYRYGGG EGVILCLWYAKLDATLI KMGFEGSTSD PAVYKRNSEH STLIVGVWYKRFDSFML SEGFKRSEFD SCVYIKFVNG SPIYLLLWNKRFDSFML SHSFKRSKYD SCVYIKHVNG SPIYLLLCYKRFDSFML SQKFRRSNYD SCVYLKVVDG STIYLLLWYKRFDSFML SQKFRRSNYD SCVYLKVVDG SAIYLLLWFERFSSVVC AAGFSPSDHD PALINHVSDH GRTLMLLWFARFSSVVL AAGFSPSDHD PALFIHTSSR'GRTLLLLRNIRFDEVVR HWGFVKNEEE PCVYKKNSGN APVFLILWNIRFDEVVK ALGFVKNERE PCVYKKISGS ALVFLILWYLKFDQIIR QFGFKENKKD NCIYANFKES KFIFLILWYARLSGKLQ QLGLSHLKAD TSLFYFNKGN VTMFILIWYSRLSTKLS ELGFVPSKAD TSLFFYKKGQ VSIFLLI

WFDRFRRAVC GMGYSQCNGD HMVFYKHRGA HITILAV

W. .RFD .. GF D .CVY....G. L.L

44444444444450444242444444444444

50

818181 FIG. 1. Alignment of sequences81 corresponding to the reverse tran-81 scriptase domain of retrotransposon81 families of rice. Deduced amino acid81 sequences of clones of sequences am-87 plified by PCR (Tos4-Tosl6) or re-81 verse transcription-PCR (Tosl7-79 Tos20) are organized according to79 their phylogenetic relationships. QM-

DVKT and YVDDM sequences at the81 amino and carboxyl ends, respectively,81 corresponding to the oligonucleotide81 primers used are not included, because81 they may not represent the true81 genomic or cDNA sequences. The for-81 merly isolated Tos4 and TosS se-

87 quences (14) are included for compar-ison.

7784 Genetics: Hirochika et aL

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kb

23.1-

9.4

6.6-

ToslO1 23456

Tosl71 23456

Tosl91 23456

4.4 -

qv .. .%

FIG. 2. An increase in the copy number of ToslO, Tosl7, and Tosl9during tissue culture. DNAs of leaves or cultured cells were digestedwith XbaI and analyzed by Southern blot hybridization using 32p-labeled ToslO, Tos17, and Tosl9 probes. Lanes 1 and 5, leaves ofNipponbare and C5924 varieties, respectively; lanes 2-4, cultured cellsderived from Nipponbare cultured for 3, 12, and 24 months, respec-tively; lane 6, the Oc cell line derived from C5924.

observed in plants regenerated from 3- and 9-month-oldcultures. On the other hand, a nearly identical pattern wasobserved in plants regenerated from the 16-month-old culture,suggesting that a specific cell type was selected during a longculture period. As a control, 12 individuals of normally prop-agated plants were analyzed (Fig. 3A). Although the copynumber difference was observed between two varieties (twoand one copies in Nipponbare and Koshihikari, respectively),there was no difference among individuals of each variety. Theactivation of Tosi 7 during the production of transgenic ricewas also examined, because tissue culture is used for theproduction of transgenic rice. Transgenic rice plants carryinga hygromycin-resistance gene were produced by an electropora-tion method using protoplasts. In all of the transgenic plantsexamined, an increase in the copy number of Tosl7was observed(Fig. 3C). Even in clones derived from a single transformed cell,

the pattern is different, indicating that the transposition of Tosl 7occurred also during the propagation of clones.

Structural Analysis of Tosl7. The genomic DNA library ofcultured cells was screened for Tosl7 using the cloned sequenceof Tosl7 as a probe. Eight clones carrying the entire Tosl7sequences were isolated. Restriction maps of these Tosl7 se-quences were shown to be identical. One clone, Tosl7-1, wasanalyzed further. This clone carries the Tosl7 sequence of 4.3 kbin which two identical long terminal repeats (LTRs) of 138 bpwere found (Fig. 4). The LTRs of all of the plant retrotransposonsexamined begin with TG and end with CA (17). Exceptionally,the LTRs of the Tosl 7-1 clone end with GA. The GA sequenceis an attribute of Tosl 7, since the same sequence was found infive other clones. The Tosl 7-1 sequence was flanked by directrepeats of five base pairs. This was shown to be the conse-quence of a duplication of the target sequence during theinsertion into the genome by sequencing an empty target site,which was amplified by PCR. Two other structural featurescommon to retrotransposons (6) were identified: (i) a primerbinding site (PBS) that is homologous to the 3' end of atransfer RNA (tRNA) and (ii) a polypurine tract (PPT) (Fig.4). The above structural features, except the terminal se-quence, indicate that Tos17-1 is a typical retrotransposon.

Transcriptional Activation ofRetrotransposons. The presenceof RNA, which is a putative intermediate for transposition, wasexamined (Fig. 5). In the 6-month-old cultured cells, two RNAspecies (8.0 and 4.5 kb) of Tosl 7were detected (lane 2), whereasonly one species of 8.0kbwas detected in leaf tissues (lane 1). The8.0-kb RNA is likely to be the read-through transcript from acellular gene because its transcription starting point was notmapped within the LTR by primer extension (Fig. 6B, leafsample). This RNA was not detected in the indica rice variety(Fig. 5, lane 4), suggesting the different distribution of Tosl7between varieties or subspecies. Southern blot hybridizationanalysis supports this interpretation (see Fig. 2, lanes 1 and 5).The 4.5-kb RNA must be a full-length transcript of Tosl 7becauseit is the only transcript starting within the LTR (nt 120) in thecultured cells (Figs. 5 and 6B, culture sample). The TATA boxwas found at the expected site. These results, together with theresults of the analysis of transposition, indicate that the transpo-sition of Tosl7 is regulated mainly at the transcriptional level. The16-bp repeats identified upstream of the TATA box may beinvolved in the transcriptional activation. The 4.5-kb RNAwas alsodetected in Oc cells, suggesting that Tosl 7 is still active in Oc cells.

ANipponbare Koshihikari

kb 1 2 3 4 5 6 7 8 91011 12

23.1-

9.4-

6.6-# w*" I,W.* ,.,: ~~b il.

B "ICopy 3 months

kb 10 2 1

23.1-

9.4-

6.6-4.4 -

Cco

9 months 16 months

.*5

4.4-

* * F i' j ::>!4: :- :: iF

....._ . , .. .. .... ...:::i:.i: .::.

jiiiii, :iti *' f ''" '" '''" f§ * 4

_ 4 ''' ; Z- Za 'X :.: ,, :.

:::. ., :3w :i.F,: w: X w : :Z: t.* * i*:_:*::j ............ w:-w.i*..t,': :ei ::: :. ,. - ::.: : .**:w*:*.^.... ... '' .. r5

.X.tF: :F. F g., - - w I w ww: .,ji. | i.:, i w: w ,.' | a. i:.

* w w w 7i*::m: : ::. .. - Z:,:.:2.3-

2.0-

2.3-

2.0-

FIG. 3. An increase in the copy number of Tosl7 in plants regenerated from culture and transgenic plants. DNAs were prepared from leavesand analyzed by Southern blot hybridization after digestion with HindIII (A) or XbaI (B and C). (A) Lanes 1-8 and 9-12: normally propagatedplants of Nipponbare and Koshihikari varieties, respectively. (B) Plants regenerated from tissue cultures of Nipponbare. Plants were regeneratedfrom 3-, 9-, and 16-month-old cultures. Left three lanes: cloned Tosl7-1 digested with XbaI as a copy number control. (C) Lane 1, the controlNipponbare plant; lanes 2-14, transgenic plants (lanes 7-14, clones derived from a single transformed cell).

Clones

1 2 3 4 5 6 7 8 9 101121314

Genetics: Hirochika et al.

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Proc. Natl. Acad. Sci. USA 93 (1996)7786 Genetics: Hirochika et al.

A 5'-LTR PBS

BstXl

0

B

1 2 3

5'-LTR(138bp) PBSGTCTITGTTA-- --GTGGTATCAGAGCAATGG----TS *******

tRNAimetACCAUAGUCUCGGUCCA

3'-LTR (138 bP)----_GAAGGGGGGt~GTTA_ - -_GN

PPT

Two RNA species (9.5 and 6.2 kb) of ToslO were detected overan intense smear background in Oc cells, whereas no RNA wasdetected in leaf tissues (Fig. 5). These results suggest that thetransposition of ToslO is also regulated at the transcriptional level.

Target-Sites of Tosl7 Transposition. The target-site specificityof Tosl 7 transposition induced during tissue culture was analyzedto examine the possibility that Tosl7 may be involved in tissueculture-induced mutations. Six (TS-1 - TS-6) and two (TS-7 andTS-8) sequences flanking transposed Tosl 7 were amplified byinverse PCR from cultured cells and regenerated plants, respec-tively, and their sequences were analyzed. Four of them (TS-1 andTS-6-TS-8) showed significant amino acid similarity to knowngenes, the thiC ofEscherichia coli involved in thiamin biosynthesis(18), NADP+-dependent malic enzyme genes of several plantspecies (19, 20), the phytochrome A gene (phyA) of rice (21), andthe S-receptor kinase-like gene of Brassica oleracea (22), respec-tively (Fig. 7). Partial cDNA sequences of plants showing signif-icant amino acid similarity to the thiC and TS-1 were alsoidentified (GenBank accession nos. T42978 and L33637, respec-tively). Although a partial cDNA sequence of a malic enzymegene of rice (GenBank accession no. D21287) has been deter-mined, this sequence is distinct from TS-6. Sequences oftwo typesof NADP+-dependent malic enzyme, cytoplasmic (20) and chlo-roplastic (19), have been reported and share 84% amino acidsimilarity to each other. The two rice sequences probably code fordifferent types of malic enzyme. The nucleotide sequence of TS-7is identical to the ricephyA gene, indicating that Tosl 7 transposedinto the phyA gene.

Northern blot analysis showed that the target-site sequences,except TS-8, are transcribed in both leaf and callus (data notshown). The transcription of TS-8 may be restricted to specifictissues as in the case of the S-receptor kinase gene (23). Theseresults, together with the sequence data, indicate that TS-1 and

4 4.3kb

FIG. 4. Structural features of Tosl 7-1. (A) Restric-tion map of Tosl7-1. (B) Nucleotide sequences offlanking the LTRs of TosJ7-1. LTRs are boxed, andthe primer binding site (PBS) and polypurine tract(PPT) are indicated by thick and thin underlining,respectively. The site of insertion target sequence (TS)and the resulting sequence duplication are denoted by

GTCSC arrows. The complementation between the PBS andTS the 3' end of the initiator methionine tRNA is shown

by asterisks.

TS-6-TS-8 are parts of functional genes. Three of the othertarget-sites are present in the rice genome as single copies and twoof them are transcribed. These data strongly suggest that some ofthese sequences are also parts of genes, although these sequencesdo not share significant similarity to known genes.

DISCUSSIONCharacteristics of Rice Retrotransposons. ToslO, Tos17,

and Tosl9 described in this paper are, to our knowledge, thefirst active transposable elements of rice, and, furthermore,Tosl 7 is the first retrotransposon of monocotyledonous plantswhose transcriptional and transpositional activities have beendemonstrated. ToslO, Tos1 7, and Tosl9 are inactive (or almostinactive) under normal growth conditions and become activeunder tissue culture condition. Retrotransposons activated bytissue culture (Ttol and Tto2) were first found in tobacco, adicotyledonous plant (5). The presence of the retrotrans-posons activated by tissue culture in a distantly related mono-cotyledonous plant, rice, suggests that retrotransposons acti-vated by tissue culture are widely distributed. Although theubiquity of retrotransposon sequences in plants has beendemonstrated (15, 24, 25), their activity remains unknown.

Tntl (26), Ttol (5), and Tosl7 are the only plant retrotrans-posons whose transcriptional and transpositional activities havebeen demonstrated. Each of these retrotransposons is regulateddifferently. The transcription of Ttol was activated by tissueculture and further enhanced by protoplast formation. Recently,we have found that virus infection also activates the transcriptionof Ttol (H.H., H. Otsuki, and Y. Ohashi, unpublished data). Thetranscription of Tosl7 was activated by tissue culture, but notenhanced by protoplast formation (H.H., unpublished data). Thetobacco retrotransposon Tntl was found as insertion elements inthe nitrate reductase gene of nitrate reductase-deficient mutants,

ToslO1 2 3 4 5

..

kb kb

Tosl71 2 3 4 S kb

_-109.5 8.0-

5.9

4.5- ~-4.5-3.8

2.3 ~~........

FIG. 5. Activation of transcription of ToslO andTosJ7 in cultured cells. Poly(A)+ RNA prepared fromleaves (lanes 1 and 4) and cultured cells (lanes 2, 3 and5) were analyzed by Northern blot hybridization. Lanes1 and 4, leaves of Nipponbare and C5924, respectively;lanes 2 and 3, cultured cells derived from Nipponbarecultured for 6 and 24 months, respectively; lane 5, the Occell line derived from C5924.

XbaI

Sall BamHI

PPT 3'-LTR

XhoI TBstXI

kb

5.6 -

4.2 -

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Proc. Natl. Acad. Sci. USA 93 (1996) 7787

A10 20 J311 40

5- TGTTAAATATATATACAAGCTAATGTACTGTATAGTTGGC5016-bp 60 16-bp 70 80

CCATGTCCAGCCCATCG ^TGT CCAGTCCATTGgTCTTG90TATA 100 110 120)k

TATCTTGA TTCTCTATTGCTAATACTATTGTTAG130 1 0 150 160

Gl4C-AAGTTAGTTAG TGGTATCAGAGCAATGGTCTGA170 180 190 200

ACCCTAGCTGCCAATTCCCCATCGCCGGCCGGCGCGGCGG210 220 230

CGTGCCGCCGGCGTTTCACTGCTAAGCAACCTCCTCCA 3'

B

culturele

T!E l ,............

TA

FIG. 6. Determination of the 5' end of Tosl7 RNA by primerextension. (A) Nucleotide sequence of the LTR and the downstreamregion. The 16-bp direct repeats and the TATA box sequence areboxed. The 5' end of Tosl 7 RNA determined by primer extension isindicated by an arrowhead. The position of the oligonucleotide usedfor primer extension analysis is indicated by an arrow. (B) Primerextension analysis. Five ,ug of poly(A)+ RNA extracted from leaf tissueand cells cultured for 6 months, both derived from Nipponbare, werehybridized with the 5' end-labeled oligonucleotide. The hybrids wereextended with reverse transcriptase and the cDNA products wereelectrophoresed on a sequencing gel alongside a sequencing reactionusing the same primer as described (5). One band (indicated by anarrow) was detected only in the sample of cultured cells.

which were induced during protoplast culture (26). Because thetranscription of Tntl was detected in protoplasts but not in

cultured cells (5, 27), Tntl must have been activated by protoplastformation. Recent studies have shown that elicitor(s) present in theprotoplasting enzyme, rather than protoplast formation itself,activates Tntl (28). Although it became evident that plant retro-transposons are strictly regulated by different stresses, the biolog-ical significance of these regulations remains unclear. The activa-tion of retrotransposons by stress might be interpreted within thecontext of the genomic stress concept proposed by McClintock(29).

Retrotransposons as an Agent To Induce Tissue Culture-Induced Mutations. Ttol of tobacco transposed into new sites inthe host genome as a consequence of activation by tissue culture(5). This does not necessarily mean that Ttol can induce muta-tions during tissue culture because Ttol may have a tendency totranspose to nongene regions. Even if Ttol transposed into genes,mutant phenotypes are not always expected due to the fact thattobacco (Nicotiana tabacum) is an amphidiploid species producedby the combination of two diploid genomes. Yeast retrotrans-posons Tyl, Ty2, and Ty3 have a tendency to transpose near thetRNA genes (30). Because the transposition into genes is dele-terious to the host, yeast may have evolved this target selectionsystem for the coexistence of the host and retrotransposons. 1nplants, the system to strictly regulate the transposition may haveevolved for their coexistence (see above).

Here, we have shown that Tosl7 can induce mutations duringtissue culture at high frequency. Although activation of maizetransposable elements by tissue culture have been reported (3, 4),no direct evidence showing that mutations are induced by thesetransposable elements during tissue culture has been reported.Thus, to our knowledge, this paper is the first report demonstrat-ing a transposable element of plants can indeed induce mutationsduring tissue culture.At least four out of eight target sites of Tosl7-transposition

induced during tissue culture are coding regions of genes (Fig. 7).Other target sites may also be in genes, since two out of the fourwere transcribed. More than five transposed copies of Tosl7werefound in all of the regenerated plants examined (Fig. 3). This

A Identities = 78%, Similarities = 88%

TS-1: VPIGXVPIYQALEKVNGIAXNLSWEVFRDTLIEQAEQGVDYFTIHAGVLLRYIPLTAKRM

THIC: 289 VPIGTVPIYQALEKVNGIAEDLTWEAFRDTLLEQAEQGVDYFTIHAGVLLRYVPMTAKRL 348

TS-1: TGIVSRGGSIHAKWCLTYHKENFAYEHWDEILDICNQYDVALSIGDG

THIC: 349 TGIVSRGGSIMAKWCLSHHQENFLYQHFREICEICAAYDVSLSLGDG 395

B Identities = 73%, Similarities = 83%

TS-6: QGSAVFASGSPXDXXLYEGKTYVPGQSNNAYIFPGFGLGVVI

ME : 509 QGRSIFASGSPFAPVEYEGKTFVPGQSNNAYIFPGLGLGLVI 550

C Identities = 100%

TS-7: LYGGKVWRLQNAPTESQIRDIAFWLSDVHRDSTGLSTD

PHYA: 454 LYGGKVWRLQNAPTESQIRDIAFWLSDVHRDSTGLSTD 491

D Identities = 44%, Similarities = 66%

TS-8: LKPKICDFGMSKALKADADRDCTGVVVGS

SRK: 107 LNPKISDFGLARVFEANEDAAETRRVVGT 135

FIG. 7. Genes disrupted by inser-tion of Tosi7. Amino acid sequencesdeduced from target-site sequences(TS-1, -6, -7, -8) are aligned with se-quences of THIC protein of E. coli(18) (A), malic enzyme (ME) of maize(19) (B), PHYA protein of rice (21)(C), and S-receptor kinase-relatedprotein of Brassica oleracea (22) (D),respectively. The 3'-ends of TosJ7were found downstream of TS-1, up-stream of TS-6, and upstream of TS-8,whereas the 5 '-end of Tosl 7was founddownstream ofTS-7. Although the fur-ther downstream sequence of TS-8 wasdetermined, no further homology wasdetected, probably due to the presenceof an intron sequence (22). Two dotsand one dot represent amino acid iden-tity and similarity, respectively.

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Proc. Natl. Acad. Sci. USA 93 (1996)

suggests that at least two genes are mutated by insertion of Tosl7in each regenerated plant. Because another type of mutations(point mutation) has been reported in maize (31, 32), furtherstudies are needed to understand to what extent each type ofmutations is involved in tissue culture-induced mutations.The copy number of Tosl7 ranges from one to four among six

closely related varieties examined (data not shown). This suggeststhat Tosl7 has weak activity under natural conditions. Becausecultured cells are quite similar to wound-healing tissues, Tosl7mayhave been activated by wounding. Recent studies showed thatretrotransposons are involved in a significant amount of sponta-neous mutations in maize (33). Tosl7 may also be involved in thespontaneous mutations in rice. It must be interesting to examinewhat factors activate retrotransposons under natural conditions.

Possible Use of Retrotransposons for Gene-Tagging andFunctional Analysis of Genes. Transposable elements areuseful molecular genetic tools for gene isolation. For example,the maize AciDs elements are used for cloning genes bygene-tagging in homologous and heterologous plants. How-ever, several problems and limitations associated with its modeof transposition have been noted. For example, a high pro-portion of the mutations are not tagged withAc/Ds (34). Thisis thought to be due to mutations induced by impreciseexcision. The AciDs elements transpose to linked sites (35).For successful tagging, many plants carrying the AciDs ele-ments located on different chromosomes are required. Segre-gation analysis of transposed Tosl7 copies using the progeny ofregenerated plants suggested that Tosl7 transposed throughoutthe chromosomes (data not shown). All these points suggest thatretrotransposons could be a useful alternative for gene-tagging.Tosl7 seems the best candidate because it can induce mutationsduring tissue culture at a high frequency and its original copynumber is quite low (one to four). The latter character is impor-tant to identify the retrotransposon responsible for the specificmutation. This tagging is very useful, especially for rice, becausetissue culture-induced mutations have been used as a source ofimprovement of rice and many mutants are available (8).The second possible use of retrotransposons is for the func-

tional analysis of genes. The large scale sequencing of randomcDNA of rice has been progressing (36), and many genes havebeen identified and their functions elucidated based on theirsequence similarity to genes with known functions. However,more than 70% of the sequences are of unknown function. Tounderstand their functions, retrotransposons described here, es-pecially Tosl 7, can be used. The regenerated rice plants, carryingS to 30 transposed Tosl7 copies, can be used as mutagenizedmaterials for a site-selected mutagenesis system (37,38). Mutantscarrying a Tosl7-insertion in the gene of interest can be identifiedby PCR using one primer for the ends of Tosl7 and another forthe gene of interest. Identified mutants can then be studied toascertain the function of the gene of interest. This strategy shouldbe applicable to many plant species because a general method toisolate active retrotransposons is available (5) and active trans-position of a tobacco retrotransposon Ttol in heterologousplants, such as Arabidopsis (H.H. and T. Kakutani, unpublisheddata) and rice (39), has been demonstrated.Another approach for the functional analysis of genes is to

directly analyze the target-site sequences of Tosl7-transposition.As shown in this paper, the mutant plants with Tosl7-insertionsin interesting genes, such as thephyA and the SRK-like gene, wereidentified. The characterization of these mutants will contributeto understanding of the function of these genes.

We are grateful to Drs. I. Havukkala and T. Bureau for criticalreading of the manuscript. This work was supported in part by a projectgrant from the Ministry of Agriculture, Forestry, and Fisheries ofJapan, a grant-in-aid for Scientific Research on Priority Areas from the

Ministry of Education, Science and Culture of Japan, and an enhance-ment of Center-of-Excellence, the special coordination funds forpromoting science and technology of Japan.

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