Plant Molecular Biology 11:791-794 (1988) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands
791
T h e Agrobacterium tumefaciens T - D N A gene 6 b is an onc gene P. J. J. Hooykaas, H. den Dulk-Ras and R. A. Schilperoort
Department of Plant Molecular Biology, Leiden University, Wassenaarseweg 64, 2333 A L Leiden, Netherlands Received 29 June 1988; accepted in revised form 12 September 1988
Key words." Agrobacterium tumefaciens, t u m o r induction, onc gene, T-DNA Abstract In this article it is shown that the T-DNA of Agrobacterium tumefaciens contains besides the well-known cyt and aux genes another gene with an oncogenic effect in plants. The gene in question is called 6 b and causes the formation of small tumors in plant species such as Nicotiana glauca and Kalanchoe tubiflora.
Introduction The soil bacterium Agrobacterium tumefaciens is a phytopathogen by virtue of its ability to transform normal plant cells into t u m o r cells. This eventually results in t u m o r formation in dicotyledonous plants. The molecular mechanism underlying this bacterium-plant interaction has been described in some detail (for a recent review see [11]). Transfer of a piece of oncogenic DNA, the T-DNA, originating from the large, bacterial Ti plasmid, to plant cells at the infection sites is the key event of the process. The TD N A contains onc genes, the expression of which is responsible for the tumorous character of the transformed plant cells. Mutational analysis has shown that three one genes are of prime importance, viz. the genes cyt (ipt), auxl (iaaM) and aux2 (iaaH). These genes code for enzymes involved in the biosynthesis of the cytokinin isopentenyl-AMP (cyt) and the auxin indole acetic acid (auxl, aux2). Mutations in Agrobacterium T-DNA genes other than cyt, auxl and aux2 did not lead to avirulence [4]. Therefore, it is questionable whether any of the other genes that naturally are present in the T-DNA, are involved in tumorigenesis. Only some indirect data point to a possible accessory or regulatory role of some of these
genes (e.g. T-DNA genes 5 and 6 a, 6 b) in tumorigenicity. Mutations in the area covering genes 6 a and 6 b of the wild-type octopine Ti plasmid were found to lead to an increased size o f tumors induced on kalanchoe [4] but similar mutations in the nopaline Ti plasmid had no such effect [9]. Recent data showed that gene 6 a determines a permease system for the excretion of octopine and related opines from transformed plant cells [12], which makes a role of this gene in determining t u m o r size difficult to understand. In this paper we show that T-DNA gene 6 b is an onc gene by itself, capable of inducing t u m o r formation in a limited set of plant species.
Materials and methods
Bacterial strains and plasmids Escherichia coli strain K M B L l I 6 4 thi pro lac (P, van de Putte) was used as a recipient for plasmid constructs in transformation experiments. Transformed plasmids were subsequently transferred to Agrobacterium via conjugation in triparental matings with HB101 (pRK2013). The binary vector pBinl9, which contains a K m r marker, was described by Bevan [2].
792
Ba m H
Agrobacterium strain LBA4404 [6] was used as a recipient for the b i n a r y constructs. S t r a i n LBA1834 is a cyt aux d o u b l e m u t a n t [5], while LBA4443 is a derivative o f LBA4404 c o n t a i n i n g a b i n a r y vector, which still c o n t a i n s o c t o p i n e Ti T - D N A genes 5, 6 b a n d 3 [7]. S t r a i n LBA1010 c o n t a i n s the w i l d - t y p e Ti p l a s m i d a n d is used as a c o n t r o l in virulence assays.
Conjugation, cloning, D N A isolation C l o n i n g experiments, D N A i s o l a t i o n s etc. were d o n e a c c o r d i n g to the p r o t o c o l s in the l a b o r a t o r y m a n u a l o f M a n i a t i s et al. [10]. C o n j u g a t i o n s were p e r f o r m e d o n S a r t o r i u s filters as d e s c r i b e d previously [8]. Transc o n j u g a n t s were selected by the r i f a m p i c i n resistance (20 # g / m l ) o f the Agrobacterium recipient L B A 4 4 0 4 a n d the k a n a m y c i n resistance ( 5 0 / ~ g / m l ) o f the p B i n l 9 vector o n SM m i n i m a l m e d i u m plates [8].
Virulence assays These were d o n e using Kalanchoe tubiflora a n d Nicotiana glauca p l a n t s as d e s c r i b e d p r e v i o u s l y [8].
Results Several years ago the Ti p l a s m i d was d i s a r m e d in several l a b o r a t o r i e s i n c l u d i n g o u r own via the inactiv a t i o n o f the T-cyt gene as well as o n e o r b o t h o f the T-aux genes. Agrobacterium strains c a r r y i n g such p l a s m i d s were said to be d i s a r m e d , b e c a u s e they were no l o n g e r c a p a b l e o f i n d u c i n g t u m o r s on p l a n t species such as t o b a c c o a n d t o m a t o . However, w h e n a n a l y z i n g such strains o n a wider range o f p l a n t species we a n d others [14] f o u n d u n e x p e c t e d l y t h a t s o m e were able to f o r m small t u m o r s o n Kalanchoe tubiflora a n d Nicotiana glauca. A s a n e x a m p l e strain LBA1834 c a n be m e n t i o n e d . This strain contains a derivative o f the o c t o p i n e Ti p l a s m i d with ina c t i v a t e d genes aux2, cyt a n d 6 a [5]. Therefore, in this case any o f the T - D N A genes auxl, 5, 7, 6 b o r 3 m i g h t be r e s p o n s i b l e for o n c o g e n i c i t y on the two p l a n t species m e n t i o n e d . A n o t h e r example c o n c e r n s strain LBA4443, which is a derivative o f the h e l p e r vi-
BP
P
I I D
117 a
S
B
t t ~-\\\\~ I p
I.
11
6a 6b Fig. 1. On the map of the Bam HI fragment 17a (coordinates 9062-13774) of the octopine Ti plasmid [2, 3], the cleavage sites for the restriction enzymes Bam HI (B), Pst I (P) - coordinates 9211 and 10069 - and Sma I (S) - coordinate 11207 - are indicated according to Barker et aL [1]. The symbols p and t denote promoter and terminator, respectively.
rulence strain LBA4404 with a b i n a r y vector e m b r a c ing the T - D N A genes 5, 6 b a n d 3 [7]. In o r d e r to find o u t directly w h e t h e r gene 6 b h a d a n y t h i n g to do with this p h e n o m e n o n , we c l o n e d the Bam H I fragm e n t 17 a, which c o n t a i n s genes 6 a a n d 6 b (Fig. 1) f r o m the w i l d - t y p e o c t o p i n e Ti p l a s m i d in b o t h o r i e n t a t i o n s into the b i n a r y vector pBinl9. A f t e r transfer to the helper virulence strain LBA4404 we assayed the resulting strains LBA4463 a n d LBA4464 for virulence a n d f o u n d t h a t b o t h were able to induce small t u m o r s on K. tubiflora a n d N. glauca indeed (Fig. 2). This shows t h a t either gene 6 a o r 6 b or b o t h are onc genes by themselves. In o r d e r to determ i n e w h e t h e r gene 6 b was solely responsible for t u m o r i g e n i c i t y , we deleted a Pst I segment - c o o r d i n a t e s 9211-10069 [1] f r o m f r a g m e n t Bam H I 17 a (Fig. 1) in vector pACYC184, recloned the deleted Bam H I 17 a fragment, which carries gene 6 b b u t n o t 6 a, into p B i n l 9 in b o t h o r i e n t a t i o n s a n d transferred the resulting p l a s m i d s to LBA4404. T h e resulting strains called LBA4465 a n d LBA4466 were i n d e e d able to i n d u c e small t u m o r s on N. glauca a n d K. tubiflora whereas the virulence helper strain (with o r w i t h o u t the p B i n l 9 vector) did n o t i n d u c e any p r o l i f e r a t i o n at all. This latter result proves t h a t in fact gene 6 b is an onc gene with a t u m o r i g e n i c effect o n l y o n p a r t i c u l a r p l a n t species such as N. glauca a n d K. tubiflora.
Discussion In this p a p e r we have shown t h a t the T - D N A o f the Ti p l a s m i d contains, besides the three well-
793
Fig. 2. Tumor formation on Nicotiana glauca by LBA4404 (left: no tumors formed), LBA1010 (middle: large tumors formed) and LBA4463, LBA4464 (right: small tumors induced). LBA4404 only contains the virulence helper plasmid; LBA1010contains the wild-type Ti plasmid; LBA4463, LBA4464 are derived from LBA4404 and have the pBinl9 vector with the Barn HI 17a fragment in two orientations.
c h a r a c t e r i z e d onc genes cyt, a u x l a n d aux2, at least o n e m o r e gene called 6 b with a n o n c o g e n i c effect o n c e r t a i n p l a n t species. In the literature certain m o r p h o g e n i c effects have b e e n a s c r i b e d to gene 6 b. M u t a t i o n s in the a r e a covering genes 6 a a n d 6 b o f the o c t o p i n e Ti p l a s m i d were r e p o r t e d to result in the f o r m a t i o n o f larger t u m o r s o n k a l a n c h o e [4], a l t h o u g h a similar effect was n o t seen in the n o p a l i n e Ti system [9]. A n y i n h i b i t o r y role o f gene 6 b o n t u m o r f o r m a t i o n o n k a l a n c h o e is difficult to reconcile with its o n c o g e n i c effect p e r se on the s a m e p l a n t species as s h o w n in this article. D o u b l e m u t a n t s with a m u t a t i o n in gene cyt as well as in 6 b i n d u c e t u m o r s o n K. tubiflora f r o m which m o r e r o o t s proliferate t h a n f r o m t u m o r s f o r m e d v i a cyt m u t a n t s [14]. R e a m et al. [14] have s h o w n also t h a t d o u b l e m u t a n t s with m u t a t i o n s in a u x a n d the 6 b gene cause t u m o r s t h a t are less p r o n e to s h o o t s f o r m a t i o n as c o m p a r e d to a u x m u t a n t s . These l a t t e r d a t a reveal t h a t gene 6 b has a n i n h i b i t o r y effect o n r o o t f o r m a t i o n a n d s t i m u l a t e s s h o o t i n g . Such effects c o u l d be e x p l a i n e d
if the p r o d u c t s d e t e r m i n e d by gene 6 b led to breakdown or inactivation of auxin or modify a cytokinin into a m o r e p o t e n t form. Alternatively, the gene m i g h t m a k e t r a n s f o r m e d p l a n t cells m o r e sensitive to c y t o k i n i n o r less sensitive to auxin. T h e fact t h a t gene 6 b acts as a n onc gene by itself shows t h a t if the p r o d u c t s o f gene 6 b are involved in the m o d i f i c a t i o n o f a class o f p h y t o h o r m o n e s , the target m o l e c u l e s are n o t (only) the p l a n t g r o w t h regulators f o r m e d via the T - D N A genes, b u t rather c o m p r i s e t h o s e f o r m e d by the p l a n t cells naturally. G e n e 6 b is n o t o n l y present in the o c t o p i n e Ti p l a s m i d T - D N A , b u t also in t h a t o f the n o p a l i n e Ti p l a s m i d [9, 15]. Moreover, h o m o l o g o u s sequences have also been detected in the T - D N A s o f the limited h o s t range t y p e Ti p l a s m i d pTiAG162 [16]. T h e wides p r e a d o c c u r r e n c e o f these 6b-like sequences suggests t h a t the gene plays a role in the i n t e r a c t i o n o f m a n y different a g r o b a c t e r i a a n d p l a n t cells. A l t h o u g h n o t essential for t u m o r f o r m a t i o n o n the p l a n t species s t u d i e d thus far, it m i g h t be t h a t the
794 gene has a much more prominent role in tumorigenicity on some plant species. Even on plant species where no direct oncogenic
7.
e f f e c t o f 6 b is s e e n s u c h a s t o b a c c o , its p r e s e n c e leads
probably
to
growth
aberrations.
plants containing a truncated
Tobacco
T-DNA comprising
8.
only genes 6b and 3 (octopine synthase), form a poor root system and after flowering show premat u r e l e a f a b s c i s s i o n b e s i d e s o t h e r a b n o r m a l i t i e s [13]. F r o m all t h e s e r e s u l t s it is clear, t h e r e f o r e , t h a t t h e
9.
presence of gene 6b must be avoided in plant vectors which are to be applied for the improvement
of
c r o p s . F u r t h e r s t u d i e s will h o p e f u l l y r e v e a l t h e p r e -
10.
cise f u n c t i o n d e t e r m i n e d b y g e n e 6 b. 11.
References 12. 1. Barker RF, Idler KB, Thompson DV, Kemp JD: Nucleotide sequence of the T-DNA region from Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol Biol 2: 335-350 (1983). 2. Bevan M: Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12:8711-8721 (1984). 3. De Vos G, De Beuckeleer M, Van Montagu M, Schell J: Restriction endonuclease mapping of the octopine tumorinducing plasmid pTiAch5 of Agrobacterium tumefaciens. Plasmid 6 : 2 4 9 - 2 5 3 (1981). 4. Garfinkel DJ, Simpson RB, Ream LW, White FF, Gordon MP, Nester EW: Genetic analysis of crown gall; fine structure map of the T-DNA by site-directed mutagenesis. Cell 27: 143-153 (1981). 5. Hille J, Wullems G, Schilperoort R: Non-oncogenic T-region mutants of Agrobacterium tumefaciens do transfer T-DNA into plant cells. Plant Mol Biol 2:155-163 (1983). 6. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA: A binary plant vector strategy based on separation of vir and
13.
14.
15.
16.
T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179-180 (1983). Hoekema A, Van Haaren M J J, Fellinger A J, Hooykaas PJJ, Schilperoort RA" Non-oncogenic plant vectors for use in the Agrobacterium binary system. Plant Mol Biol 5 : 8 5 - 8 9 (1985). Hooykaas PJJ, Hofker M, den Dulk-Ras H, Schilperoort RA: A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants. Plasmid 11:195-205 (1984). Joos H, Caplan A, Sormann M, Van Montagu M, Schell J: Genetic analysis of T-DNA transcripts in nopaline crown galls. Cell 32:1057-1067 (1983). Maniatis T, Fritsch EF, Sambrook J: Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). Melchers LS, Hooykaas PJJ: Virulence of Agrobacterium. In: Miflin BJ (ed) Oxford Surveys of Plant Molecular and Cell Biology, Vol 4 pp 167-220. Oxford University Press, London (1987). Messens E, Lenaerts A, Van Montagu M, Hedges RW: Genetic basis for opine secretion from crown gall tumour cells. Mol Gen Genet 199:344-348 (1985). Peerbolte R, Leenhouts K, Hooykaas-Van Slogteren GMS, Hoge JHC, Wullems GJ, Schilperoort RA: Clones from a shooty tobacco crown gall tumor I: deletions, rearrangements and amplifications resulting in irregular T-DNA structures and organizations. Plant Mol Biol 7 : 2 6 5 - 2 8 4 (1986). Ream LW, Gordon MP, Nester EW: Multiple mutations in the T region of the Agrobacteriurn tumefaciens tumor-inducing plasmid. Proc Natl Acad Sci USA 80:1660-1664 (1983). Vanderleyden J, Desair J, De Meirsman C, Michiels K, Van Gool A, Jen G, Chilton M-D: Nucleotide sequence of the TDNA region encoding transcripts 6a and 6b of the pTiT37 nopaline Ti plasmid. Plant Mol Biol 7 : 3 3 - 4 1 (1986). Yanofsky M, Montoya A, Knauf V, Lowe B, Gordon M, Nester E: Limited-host-range plasmid of Agrobacterium tumefaciens: molecular and genetic analysis of transferred DNA. J Bact 163:341-348 (1985).
791
T h e Agrobacterium tumefaciens T - D N A gene 6 b is an onc gene P. J. J. Hooykaas, H. den Dulk-Ras and R. A. Schilperoort
Department of Plant Molecular Biology, Leiden University, Wassenaarseweg 64, 2333 A L Leiden, Netherlands Received 29 June 1988; accepted in revised form 12 September 1988
Key words." Agrobacterium tumefaciens, t u m o r induction, onc gene, T-DNA Abstract In this article it is shown that the T-DNA of Agrobacterium tumefaciens contains besides the well-known cyt and aux genes another gene with an oncogenic effect in plants. The gene in question is called 6 b and causes the formation of small tumors in plant species such as Nicotiana glauca and Kalanchoe tubiflora.
Introduction The soil bacterium Agrobacterium tumefaciens is a phytopathogen by virtue of its ability to transform normal plant cells into t u m o r cells. This eventually results in t u m o r formation in dicotyledonous plants. The molecular mechanism underlying this bacterium-plant interaction has been described in some detail (for a recent review see [11]). Transfer of a piece of oncogenic DNA, the T-DNA, originating from the large, bacterial Ti plasmid, to plant cells at the infection sites is the key event of the process. The TD N A contains onc genes, the expression of which is responsible for the tumorous character of the transformed plant cells. Mutational analysis has shown that three one genes are of prime importance, viz. the genes cyt (ipt), auxl (iaaM) and aux2 (iaaH). These genes code for enzymes involved in the biosynthesis of the cytokinin isopentenyl-AMP (cyt) and the auxin indole acetic acid (auxl, aux2). Mutations in Agrobacterium T-DNA genes other than cyt, auxl and aux2 did not lead to avirulence [4]. Therefore, it is questionable whether any of the other genes that naturally are present in the T-DNA, are involved in tumorigenesis. Only some indirect data point to a possible accessory or regulatory role of some of these
genes (e.g. T-DNA genes 5 and 6 a, 6 b) in tumorigenicity. Mutations in the area covering genes 6 a and 6 b of the wild-type octopine Ti plasmid were found to lead to an increased size o f tumors induced on kalanchoe [4] but similar mutations in the nopaline Ti plasmid had no such effect [9]. Recent data showed that gene 6 a determines a permease system for the excretion of octopine and related opines from transformed plant cells [12], which makes a role of this gene in determining t u m o r size difficult to understand. In this paper we show that T-DNA gene 6 b is an onc gene by itself, capable of inducing t u m o r formation in a limited set of plant species.
Materials and methods
Bacterial strains and plasmids Escherichia coli strain K M B L l I 6 4 thi pro lac (P, van de Putte) was used as a recipient for plasmid constructs in transformation experiments. Transformed plasmids were subsequently transferred to Agrobacterium via conjugation in triparental matings with HB101 (pRK2013). The binary vector pBinl9, which contains a K m r marker, was described by Bevan [2].
792
Ba m H
Agrobacterium strain LBA4404 [6] was used as a recipient for the b i n a r y constructs. S t r a i n LBA1834 is a cyt aux d o u b l e m u t a n t [5], while LBA4443 is a derivative o f LBA4404 c o n t a i n i n g a b i n a r y vector, which still c o n t a i n s o c t o p i n e Ti T - D N A genes 5, 6 b a n d 3 [7]. S t r a i n LBA1010 c o n t a i n s the w i l d - t y p e Ti p l a s m i d a n d is used as a c o n t r o l in virulence assays.
Conjugation, cloning, D N A isolation C l o n i n g experiments, D N A i s o l a t i o n s etc. were d o n e a c c o r d i n g to the p r o t o c o l s in the l a b o r a t o r y m a n u a l o f M a n i a t i s et al. [10]. C o n j u g a t i o n s were p e r f o r m e d o n S a r t o r i u s filters as d e s c r i b e d previously [8]. Transc o n j u g a n t s were selected by the r i f a m p i c i n resistance (20 # g / m l ) o f the Agrobacterium recipient L B A 4 4 0 4 a n d the k a n a m y c i n resistance ( 5 0 / ~ g / m l ) o f the p B i n l 9 vector o n SM m i n i m a l m e d i u m plates [8].
Virulence assays These were d o n e using Kalanchoe tubiflora a n d Nicotiana glauca p l a n t s as d e s c r i b e d p r e v i o u s l y [8].
Results Several years ago the Ti p l a s m i d was d i s a r m e d in several l a b o r a t o r i e s i n c l u d i n g o u r own via the inactiv a t i o n o f the T-cyt gene as well as o n e o r b o t h o f the T-aux genes. Agrobacterium strains c a r r y i n g such p l a s m i d s were said to be d i s a r m e d , b e c a u s e they were no l o n g e r c a p a b l e o f i n d u c i n g t u m o r s on p l a n t species such as t o b a c c o a n d t o m a t o . However, w h e n a n a l y z i n g such strains o n a wider range o f p l a n t species we a n d others [14] f o u n d u n e x p e c t e d l y t h a t s o m e were able to f o r m small t u m o r s o n Kalanchoe tubiflora a n d Nicotiana glauca. A s a n e x a m p l e strain LBA1834 c a n be m e n t i o n e d . This strain contains a derivative o f the o c t o p i n e Ti p l a s m i d with ina c t i v a t e d genes aux2, cyt a n d 6 a [5]. Therefore, in this case any o f the T - D N A genes auxl, 5, 7, 6 b o r 3 m i g h t be r e s p o n s i b l e for o n c o g e n i c i t y on the two p l a n t species m e n t i o n e d . A n o t h e r example c o n c e r n s strain LBA4443, which is a derivative o f the h e l p e r vi-
BP
P
I I D
117 a
S
B
t t ~-\\\\~ I p
I.
11
6a 6b Fig. 1. On the map of the Bam HI fragment 17a (coordinates 9062-13774) of the octopine Ti plasmid [2, 3], the cleavage sites for the restriction enzymes Bam HI (B), Pst I (P) - coordinates 9211 and 10069 - and Sma I (S) - coordinate 11207 - are indicated according to Barker et aL [1]. The symbols p and t denote promoter and terminator, respectively.
rulence strain LBA4404 with a b i n a r y vector e m b r a c ing the T - D N A genes 5, 6 b a n d 3 [7]. In o r d e r to find o u t directly w h e t h e r gene 6 b h a d a n y t h i n g to do with this p h e n o m e n o n , we c l o n e d the Bam H I fragm e n t 17 a, which c o n t a i n s genes 6 a a n d 6 b (Fig. 1) f r o m the w i l d - t y p e o c t o p i n e Ti p l a s m i d in b o t h o r i e n t a t i o n s into the b i n a r y vector pBinl9. A f t e r transfer to the helper virulence strain LBA4404 we assayed the resulting strains LBA4463 a n d LBA4464 for virulence a n d f o u n d t h a t b o t h were able to induce small t u m o r s on K. tubiflora a n d N. glauca indeed (Fig. 2). This shows t h a t either gene 6 a o r 6 b or b o t h are onc genes by themselves. In o r d e r to determ i n e w h e t h e r gene 6 b was solely responsible for t u m o r i g e n i c i t y , we deleted a Pst I segment - c o o r d i n a t e s 9211-10069 [1] f r o m f r a g m e n t Bam H I 17 a (Fig. 1) in vector pACYC184, recloned the deleted Bam H I 17 a fragment, which carries gene 6 b b u t n o t 6 a, into p B i n l 9 in b o t h o r i e n t a t i o n s a n d transferred the resulting p l a s m i d s to LBA4404. T h e resulting strains called LBA4465 a n d LBA4466 were i n d e e d able to i n d u c e small t u m o r s on N. glauca a n d K. tubiflora whereas the virulence helper strain (with o r w i t h o u t the p B i n l 9 vector) did n o t i n d u c e any p r o l i f e r a t i o n at all. This latter result proves t h a t in fact gene 6 b is an onc gene with a t u m o r i g e n i c effect o n l y o n p a r t i c u l a r p l a n t species such as N. glauca a n d K. tubiflora.
Discussion In this p a p e r we have shown t h a t the T - D N A o f the Ti p l a s m i d contains, besides the three well-
793
Fig. 2. Tumor formation on Nicotiana glauca by LBA4404 (left: no tumors formed), LBA1010 (middle: large tumors formed) and LBA4463, LBA4464 (right: small tumors induced). LBA4404 only contains the virulence helper plasmid; LBA1010contains the wild-type Ti plasmid; LBA4463, LBA4464 are derived from LBA4404 and have the pBinl9 vector with the Barn HI 17a fragment in two orientations.
c h a r a c t e r i z e d onc genes cyt, a u x l a n d aux2, at least o n e m o r e gene called 6 b with a n o n c o g e n i c effect o n c e r t a i n p l a n t species. In the literature certain m o r p h o g e n i c effects have b e e n a s c r i b e d to gene 6 b. M u t a t i o n s in the a r e a covering genes 6 a a n d 6 b o f the o c t o p i n e Ti p l a s m i d were r e p o r t e d to result in the f o r m a t i o n o f larger t u m o r s o n k a l a n c h o e [4], a l t h o u g h a similar effect was n o t seen in the n o p a l i n e Ti system [9]. A n y i n h i b i t o r y role o f gene 6 b o n t u m o r f o r m a t i o n o n k a l a n c h o e is difficult to reconcile with its o n c o g e n i c effect p e r se on the s a m e p l a n t species as s h o w n in this article. D o u b l e m u t a n t s with a m u t a t i o n in gene cyt as well as in 6 b i n d u c e t u m o r s o n K. tubiflora f r o m which m o r e r o o t s proliferate t h a n f r o m t u m o r s f o r m e d v i a cyt m u t a n t s [14]. R e a m et al. [14] have s h o w n also t h a t d o u b l e m u t a n t s with m u t a t i o n s in a u x a n d the 6 b gene cause t u m o r s t h a t are less p r o n e to s h o o t s f o r m a t i o n as c o m p a r e d to a u x m u t a n t s . These l a t t e r d a t a reveal t h a t gene 6 b has a n i n h i b i t o r y effect o n r o o t f o r m a t i o n a n d s t i m u l a t e s s h o o t i n g . Such effects c o u l d be e x p l a i n e d
if the p r o d u c t s d e t e r m i n e d by gene 6 b led to breakdown or inactivation of auxin or modify a cytokinin into a m o r e p o t e n t form. Alternatively, the gene m i g h t m a k e t r a n s f o r m e d p l a n t cells m o r e sensitive to c y t o k i n i n o r less sensitive to auxin. T h e fact t h a t gene 6 b acts as a n onc gene by itself shows t h a t if the p r o d u c t s o f gene 6 b are involved in the m o d i f i c a t i o n o f a class o f p h y t o h o r m o n e s , the target m o l e c u l e s are n o t (only) the p l a n t g r o w t h regulators f o r m e d via the T - D N A genes, b u t rather c o m p r i s e t h o s e f o r m e d by the p l a n t cells naturally. G e n e 6 b is n o t o n l y present in the o c t o p i n e Ti p l a s m i d T - D N A , b u t also in t h a t o f the n o p a l i n e Ti p l a s m i d [9, 15]. Moreover, h o m o l o g o u s sequences have also been detected in the T - D N A s o f the limited h o s t range t y p e Ti p l a s m i d pTiAG162 [16]. T h e wides p r e a d o c c u r r e n c e o f these 6b-like sequences suggests t h a t the gene plays a role in the i n t e r a c t i o n o f m a n y different a g r o b a c t e r i a a n d p l a n t cells. A l t h o u g h n o t essential for t u m o r f o r m a t i o n o n the p l a n t species s t u d i e d thus far, it m i g h t be t h a t the
794 gene has a much more prominent role in tumorigenicity on some plant species. Even on plant species where no direct oncogenic
7.
e f f e c t o f 6 b is s e e n s u c h a s t o b a c c o , its p r e s e n c e leads
probably
to
growth
aberrations.
plants containing a truncated
Tobacco
T-DNA comprising
8.
only genes 6b and 3 (octopine synthase), form a poor root system and after flowering show premat u r e l e a f a b s c i s s i o n b e s i d e s o t h e r a b n o r m a l i t i e s [13]. F r o m all t h e s e r e s u l t s it is clear, t h e r e f o r e , t h a t t h e
9.
presence of gene 6b must be avoided in plant vectors which are to be applied for the improvement
of
c r o p s . F u r t h e r s t u d i e s will h o p e f u l l y r e v e a l t h e p r e -
10.
cise f u n c t i o n d e t e r m i n e d b y g e n e 6 b. 11.
References 12. 1. Barker RF, Idler KB, Thompson DV, Kemp JD: Nucleotide sequence of the T-DNA region from Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol Biol 2: 335-350 (1983). 2. Bevan M: Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12:8711-8721 (1984). 3. De Vos G, De Beuckeleer M, Van Montagu M, Schell J: Restriction endonuclease mapping of the octopine tumorinducing plasmid pTiAch5 of Agrobacterium tumefaciens. Plasmid 6 : 2 4 9 - 2 5 3 (1981). 4. Garfinkel DJ, Simpson RB, Ream LW, White FF, Gordon MP, Nester EW: Genetic analysis of crown gall; fine structure map of the T-DNA by site-directed mutagenesis. Cell 27: 143-153 (1981). 5. Hille J, Wullems G, Schilperoort R: Non-oncogenic T-region mutants of Agrobacterium tumefaciens do transfer T-DNA into plant cells. Plant Mol Biol 2:155-163 (1983). 6. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA: A binary plant vector strategy based on separation of vir and
13.
14.
15.
16.
T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179-180 (1983). Hoekema A, Van Haaren M J J, Fellinger A J, Hooykaas PJJ, Schilperoort RA" Non-oncogenic plant vectors for use in the Agrobacterium binary system. Plant Mol Biol 5 : 8 5 - 8 9 (1985). Hooykaas PJJ, Hofker M, den Dulk-Ras H, Schilperoort RA: A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants. Plasmid 11:195-205 (1984). Joos H, Caplan A, Sormann M, Van Montagu M, Schell J: Genetic analysis of T-DNA transcripts in nopaline crown galls. Cell 32:1057-1067 (1983). Maniatis T, Fritsch EF, Sambrook J: Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). Melchers LS, Hooykaas PJJ: Virulence of Agrobacterium. In: Miflin BJ (ed) Oxford Surveys of Plant Molecular and Cell Biology, Vol 4 pp 167-220. Oxford University Press, London (1987). Messens E, Lenaerts A, Van Montagu M, Hedges RW: Genetic basis for opine secretion from crown gall tumour cells. Mol Gen Genet 199:344-348 (1985). Peerbolte R, Leenhouts K, Hooykaas-Van Slogteren GMS, Hoge JHC, Wullems GJ, Schilperoort RA: Clones from a shooty tobacco crown gall tumor I: deletions, rearrangements and amplifications resulting in irregular T-DNA structures and organizations. Plant Mol Biol 7 : 2 6 5 - 2 8 4 (1986). Ream LW, Gordon MP, Nester EW: Multiple mutations in the T region of the Agrobacteriurn tumefaciens tumor-inducing plasmid. Proc Natl Acad Sci USA 80:1660-1664 (1983). Vanderleyden J, Desair J, De Meirsman C, Michiels K, Van Gool A, Jen G, Chilton M-D: Nucleotide sequence of the TDNA region encoding transcripts 6a and 6b of the pTiT37 nopaline Ti plasmid. Plant Mol Biol 7 : 3 3 - 4 1 (1986). Yanofsky M, Montoya A, Knauf V, Lowe B, Gordon M, Nester E: Limited-host-range plasmid of Agrobacterium tumefaciens: molecular and genetic analysis of transferred DNA. J Bact 163:341-348 (1985).
