Synergistic Function of rolb, rolc, ORF13 and ORF14 of TL-DNA of Agrobacterium rhizogenes in Hairy Root Induction in Nicotiana tabacum

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1 Plant Cell Physiol. 40(2): (1999) JSPP 1999 Short Communication Synergistic Function of rolb, rolc, ORF13 and ORF14 of TL-DNA of Agrobacterium rhizogenes in Hairy Root Induction in Nicotiana tabacum Seishiro Aoki' and Kunihiko Syono 2 Department of Life Science, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo, Japan Comparison of the frequency of rooting in the tobacco leaf segments inoculated with Agrobacterium tumefaciens harboring various combinations of rolb, rolc, ORF13 and ORF14 of TL-DNA of Ri plasmid (prihri) revealed that the genes differ in their function to stimulate adventitious root induction. A single gene rolb induced roots, while rolc, ORF13 and ORF14 independently promoted the root induction by the rolb gene. The effects of these genes on the ro/fl-mediated rooting were in the order of ORF13> rolc^ ORF14. Key words: Adventitious root induction Agrobacterium rhizogenes Hairy root Nicotiana tabacum plast gene family rol genes. Hairy root disease is the consequence of the introduction of the segments of DNA (T-DNA) of the Ri plasmid of Agrobacterium rhizogenes into the plant genome (Chilton et al. 1982, White et al. 1982, Willmizer et al. 1982). The agropine-type Ri plasmid has two fragments of T-DNA, TL-DNA and TR-DNA (Huffman et al. 1984, Jouanin 1984). TL-DNA may be essential for the induction of hairy roots since cucumopine-type and mannopine-type Ri plasmids have a single T-DNA that is homologous to the TL-DNA of the agropine-type Ri plasmid (Lahners et al. 1984, Cardarelli et al. 1985). Cardarelli et al. (1987) suggested that TR-DNA genes (auxin biosynthetic genes) assist in the induction of hairy roots. Previous studies on transgenic plants that carried the genes in TL-DNA have shown that rol genes (rola, rolb, rolc and rold) play key roles in the induction of hairy roots (White et al. 1985, Spena et al. 1987, Schumulling et al. 1988). Regarding other open reading frames (ORFs) in prc1314 prb1314 prbc14 prbc13 prb prbc prb13 prb14 PR1314 TL-DNA, Capone et al. (1989) showed that the segment containing both ORFs 13 and 14, in addition to the rola, rolb and rolc region, is necessary for adventitious root induction on carrot root disks. Co-inoculation of the rola, rolb and rolc region with either ORF13 or ORF14 resulted in limited root induction on carot disks (Capone et al. 1989). In other plants, the function of the ORF13 and ORF14 in root induction remains unknown. Among these TL-DNA genes, the rolb, rolc, ORF13 and ORF14 constitute a divergent gene family known as the 'plast gene family' and are believed to have evolved from a common ancestor (Levesque et al. 1988). Levesque et al. (1988) proposed that these genes have similar functions. To obtain basic information about the function of these four genes, we have analyzed the synergistic function of these genes in producing adventitious roots in Nicotiana tabarola rolb Sm rolc ORF13 ORF14 rold E A 1 kb Abbreviation: ORF(s), open reading frame(s). 1 To whom correspondence should be addressed. Present address: Laboratory of Phylogenetic Botany, Department of Biology, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba, Japan. 2 Present address: Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, Mejirodai, Bunkyo-ku, Tokyo, Japan. Fig. 1 Schematic drawing of the constructs. The top lines represent TL-DNA region from prihri and relative locations of each ORF (Jouanin 1984, Slightom et al. 1986). Below the map are shown the relevant restriction sites that were used in this paper. The different constructs are drawn by solid bars below the restriction endonuclease map. Fragments were subcloned in the binary vector pmm454-/f m. Abbreviations: Sm, Smal; X, Xhol; C, CM; Sa, So/I; A, Accl; E, EcoKl; K, Kpnl; P, Pvul. 252

Root induction by rot genes cum. A DNA fragment harboring rolb, rolc, ORF13 and ORF14 was subcloned from plasmid pljl, which contained the TL-DNA region of prihri of A.

2 Root induction by rot genes cum. A DNA fragment harboring rolb, rolc, ORF13 and ORF14 was subcloned from plasmid pljl, which contained the TL-DNA region of prihri of A. rhizogenes strain HRI (Jouanin 1984). A plasmid was constructed by subcloning the Smal-Pvul fragment of 7,099 bp of pljl in the T-DNA region of binary vector pmm454-/fm (Fig. 1). in A. tumefaciens strain EHA101 was tested for its ability to induce the formation of roots on leaves of N. tabacum cv. Xanthi. Tobacco leaves that were cut transversely into segments of about 2 cm in length were placed in the solid MS medium; care was taken to insert each segment into the medium with the apical end downwards (Murashige and Skoog 1962). One to three segments were cut near the basal side of each leaf, and a total of 96 leaf segments in 16 flasks were used for each construct. A small amount of A. tumefaciens was applied from a fresh plate to the protruding cut surface of each leaf segment. Three to five days after the inoculation, segments were transferred to solid MS medium supplement- 253 ed with carbenicillin (500 mg liter ') and scored daily for root formation for 30 d. Almost all the leaf segments after introduction of developed vigorous roots (Fig.2A). Inoculated leaf segments always produced roots from the cut midribs at the wound edges. The roots of segments inoculated with A. tumefaciens harboring exhibited the thick, curly and reduced geotropic growth pattern that is characteristic of hairy roots (Tepfer 1984). The frequency of leaf segments differentiating roots was shown by the percentage of the number of segments forming roots against the total number of leaf segments (Fig. 3A). Six to 8 d after inoculation with A. tumefaciens that harbored, roots began to emerge from some leaf segments, and three to five days later, half of the leaf segments had produced roots. By 20 d, regeneration of roots was apparent on more than 80% of the inoculated segments. By contrast, only a few segments infected with A. tumefaciens that harbored the control vector (pmm454-a"m) produced roots, and the induced roots lacked the characteric features of hairy roots Fig. 2 Comparison of adventitious roots elicited on leaf segments of N. tabacum by introduction of each plasmid. (A) rolb, rolc, ORF13 and ORF14 genes in pmm454-xm (). (B) pmm454-atm (control vector). (C) pria4b. (D) rolb gene in pmm454-/tm (prb). Bars=l cm.

3 254 Root induction by rol genes 100 5? 100 pria4b V phb1314 prbc13 O prbc14 D prc1314 A Control Days after infection Days after infection 30 Fig. 3 Root formation in tobacco leaf segments after infection with A. tumefaciens harboring various constructs of rol genes. The number of segments with roots is shown as the persentage to the total number of segments. Ninety-six leaf segments in 16 flasks were used for each construct. Bars indicate standard errors (n=16). (A) Leaf disks were inoculated with A. tumefaciens harboring construct ( ), prc1314 ( ), prb1314 (v), prbc14 (O), prbc13 ( ), or pria4b (A), or with a control vector (A). (B) Leaf disks were inoculated with A. tumefaciens harboring construct prb ( ), ( ), prbc ( ), prb13 (*),OT prb14 (o), or with a control vector (A). (Fig. 2B). A. tumefaciens strain R1000, which had been cured of Ti plasmid and had been transformed with pria4b, a wild-type Ri plasmid (Dr. H. Kamada, personal communication), was used as a positive control in the experiments for root induction. More intense traits of hairy root syndrome were observed with pria4b than with (Fig. 2C). pria4b has many genes other than the rolb, rolc, ORF13 and ORF14 in TL and TR-DNA regions (Huffman et al. 1984, Jouanin 1984, White et al. 1985, Slightom et al. 1986). Several genes, such as rol A of TL-DNA, may also be necessary for the full establishment of hairy root symptom (Spena et al. 1987). Further analysis of independent and synergistic functions of these four genes was made by introducing these genes at various combinations into tobacco leaves. According to loss-of-function strategy, four constructs, prc1314, prb1314, prbc14 and prbc13, with disruption in each one of the four ORFs of were prepared (Fig. 1). prc1314 was constructed by subcloning the Xhol-Pvul fragment of 5,959 bp of pljl in pmm454-/s: m. Similarly prb1314 contained the Smal- Clal fragment of 2,735 bp and the Accl-Pvul fragment of 4,306 bp. prbc14 was obtained by subcloning the Smal- EcoRl fragment of 3,582 bp and the Kpnl-Pvul fragment of 2,829 bp. prbc13 was a subclone of the Smal-EcoRl fragment of 5,495 bp. Abundant roots were induced by introduction of prb1314 and of prbc13, as well as of (Fig. 3A). Some depression of rooting capability was observed after introduction of prbc14, which lacked ORF13. prc1314 induced a similar number of roots on tobacco leaf segments as the control vector. Like prc1314, a construct pr1314 that harbored the Clal- Pvul fragment of 4,364 bp of pljl did not provoke vigorous root induction (Aoki and Syono 1999). These results showed the requirement of the rolb locus for hairy root induction on tobacco leaves. The importance of the rolb gene was verified by the introduction of a construct prb into leaves (Fig. 3B). prb was obtained by subcloning the Smal-Safl fragment of 2,792 bp of pljl that contained the rolb gene alone. Spena et al. (1987) and Capone et al. (1989) have also suggested that the rolb gene has a crucial function for hairy root formation. The properties of hairy roots induced by prb were less prominent than Table 1 Induction of roots by various constructs Plasmids pria4b PRC1314 prb1314 prbc14 PRBC13 prb prbc prb13 prb14 Control vector" Rooting " Binary vector pmm454-/f m was used as a conrol. 6 The rooting frequency shown in Figure 3 is expressed here on 6-grade scale from none ( ) for the control vector, which did not include the external egent, to the highest frequency ( ) which was produced in the positive control (pria4b). ±

Root induction by rol genes 255 those induced by (Fig.2D). Analysis of the relationship between the characteristics of hairy roots and the expression of TL-DNA genes is in progress.

4 Root induction by rol genes 255 those induced by (Fig.2D). Analysis of the relationship between the characteristics of hairy roots and the expression of TL-DNA genes is in progress. Root induction occured less frequently by prb than by (Fig. 3B). The intensity of root induction by the constructs is summarized in Table 1. To summarize the root induction by the constructs, we expressed the rooting frequency shown in Figure 3 on a 6-grade scale from none ( ) for the control vector (pmm454-a m ), which did not include the external agent for root induction in its T-DNA region, to the highest frequency ( ) which was produced by the wild-type Ri-plasmid (pria4b). In order to test whether rolc, 0RF13 or 0RF14 is necessary to promote the root-inducing function of the rolb gene, we further dissected this region of TL-DNA. The resulting constructs, prbc, prb13 and prb14 were introduced to tobacco leaves. prbc was a subclone of the Smal-EcoRl fragment of 3,582 bp of pljl. prb13 was constructed by subcloning the Smal-Clal fragment of 2,735 bp and the Accl fragment of 3,166 bp. prb14 contains the Smal-Sall fragment of 2,792 bp and Kpnl-Pvul fragment of 2,829 bp. The construct prb13 elicited the strongest biological response (Fig. 3B). Introduction of prb13 resulted in more intense and earlier root formation than that of prb. prb13 elicited root induction as strongly as prbc13, prb1314 and. prbc and prb14 also induced more adventitious roots than did prb. The rolb and rolc genes have been reported to have a synergistic function in the induction of roots (Spena et al. 1987, Schmulling et al. 1988). In the present study, the promotion of root formation by prbc and prb14 was weaker than that by prb13. Further experiments revealed that the promotion by prb14 was somewhat weaker than that by prbc (data not shown). Detailed analysis is required to understand the function of 0RF14 in hairy root formation. These findings indicate that the rolc, 0RF13 and ORF14 collaborate with the rolb gene in the process of hairy root induction in TV. tabacum. Genes in TL-DNA of A. rhizogenes act in plant cells as effectively as eukaryotic genes do. A sequence similar to the TL-DNA region has been found in the genome of uninfected plants of some species of the genus Nicotiana (Furner et al. 1986). Sequencing analysis by Furner et al. and us revealed four novel genes (Ngrol genes) in the iv. glauca genome that are similar to the rolb, rolc, 0RF13 and 0RF14 (Furner et al. 1986, Aoki et al. 1994). These Ngrol genes may have been introduced from A. rhizogenes like the ancestor to an progenitor of N. glauca (Furner et al. 1986). We have shown that three of these genes (NgrolC, NgORF13 and NgORF14) can function in tobacco plants (Aoki and Syono 1999, unpublished data). Further studies on these TL-DNA genes of A. rhizogenes should help to clarify the physiological function of the genes of bacteria and plants. We are indebted to Dr. H. Kamada (University of Tsukuba, Japan) for the gift of pria4b in A. tumefaciens strain R1000 and Dr. L. Jouanin and Dr. D. Tepfer (INRA, France) for their gift of the cosmid clone, pljl. We are also grateful to Dr. M. Sekine (Nara Institute of Science and Technology, Japan) for providing the plasmid pmm454-# m. We wish to thank Drs. Hiroyuki Adachi, Masaki Edamatsu and Motomi Ito for their valuable suggestions and technical advice. This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas and a Grant-in-Aid for Scientific Research to K.S. from the Ministry of Education, Science, Sports and Culture of Japan. References Aoki, S., Kawaoka, A., Sekine, M., Ichikawa, T., Fujita, T., Shinmyo, A. and Syono, K. (1994) The sequence of cellular T-DNA in the genome of Nicotiana glauca that is homologous to ORFs 13 and 14 of the Ri plasmid and the analysis of the expression of the cellular genes in genetic tumors of hybrids between N. glauca and N. langsdorffii. Mol. Gen. Genet. 243: Aoki, S. and Syono, K. (1999) Function of Ngrol genes in the evolution of Nicotiana glauca: conservation of the function of NgORF13 and NgORF14 after ancient infection by an Agrobacterium rhizogeneslike ancestor. Plant Cell Physiol. 40: Capone, I., Spano, L., Cardarelli, M., Bellincampi, D., Petit, A. and Costantino, P. (1989) Induction and growth properties of carrot roots with different complements of Agrobacterium rhizogenes T-DNA. Plant Mol. Biol. 13: Cardarelli, M., Spano, L., De Palios, A., Mauro, M.L., Vitali, G. and Costantino, P. (1985) Identification of the genetic locus responsible for non-polar root induction by Agrobacterium rhizogenes Plant Mol. Biol. 5: Cardarelli, M., Spano, L., Mariotti, D., Mauro, M.L., Sluys, M.A.V. and Costantino, P. (1987) The role of auxin in hairy root induction. Mol. Gen. Genet. 208: Chilton, M.D., Tepfer, D.A., Petit, A., David, C, Casse-Delbart, F. and Tempe, J. (1982) Agrobacterium rhizogenes inserts T-DNA into the genome of host plant root cells. Nature 295: Furner, I.J., Huffman, G.A., Amasino, R.M., Garfinkel, D.J., Gordon, M.P. and Nester, E.W. (1986) An Agrobacterium transformation in the evolution of the genus Nicotiana. Nature 319: Huffman, G.A., White, F.F., Gordon, M.P. and Nester, E.W. (1984) Hairy-root-induction plasmid: physical map and homology to tumorinducing plasmids. J. Bacteriol. 157: Jouanin, L. (1984) Restriction map of an agropine-type Ri plasmid and its homologies to Ti plasmids. Plasmid 12: Lahners, K., Byrne, M.C. and Chilton, M.D. (1984) T-DNA fragments of hairy root plasmid pri8196 distantly related to octopine and nopaline Ti plasmid T-DNA. Plasmid 11: Levesque, H., Delepelaire, P., Rouse, P., Slightom, J. and Tepfer, D. (1988) Common evolutionary origin of the central portions of the Ri TL-DNA of Agrobacterium rhizogenes and the Ti-T-DNAs of Agrobacterium tumefaciens. Plant Mol. Biol. 11: Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15: Schmulling, T., Schell, J. and Spena, A. (1988) Single genes of Agrobacterium rhizogenes influence plant development. EMBO J. 7: Slightom, J.L., Durand-Tardif, M., Jouanin, L. and Tepfer, D. (1986) Nucleotide sequence analysis of TL-DNA of Agrobacterium rhizogenes agropine-type plasmid: identification of open reading frames. J. Biol. Chem. 261: Spena, A., Schmulling, T., Koncz, C. and Schell, J. 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5 256 Root induction by rol genes type and phenotype. Cell 37: E.W. (1985) Molecular and genetic analysis of the transferred DNA White, F.F., Ghidossi, G., Gordon, M.P. and Nester, E.W. (1982) Tu- regions of the root-inducing plasmid of Agrobacterium rhizogenes. J. mor induction by Agrobacterium rhizogenes involves the transfer of Bacteriol. 164: plasmid DNA to the plant genome. Proc. Natl. Acad. Sci. USA 79: Willmizer, L., Sanchez-Serrana, J., Buschfeld, E. and Schell, J. (1982) DNA from Agrobacterium rhizogenes involves the transfer of plasmid White, F.F., Taylor, B.H., Huffman, G.A., Gordon, M.P. and Nester, DNA to plant genome. Mol. Gen. Genet. 186: (Received August 24, 1998; Accepted December 6, 1998)

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