How does the T-DNA of Agrobacterium tumefaciens find its way into the plant cell nucleus?

Biochimie (1993) X,635-638 0 SociM fraqaise de biochimie et biologie mokulaire

/ Elsevier, Paris

635

How does the T-DNA of Agrobacterium tumefaciens find its way into the plant cell nucleus? 2 Koukol&ov&Nicola, B Hohn Friedrich Miescher-Institut, PO Box 2543, CH-4002 Basel, Switzerland (Received 24 December 1992; accepted 12 January 1993)

Summary - Agrobacterium tumefaciens causes the crown gall disease in plants by transferring a piece of DNA, the T-DNA, into the genome of the plant cell. The virulence protein VirD2, tightly linked to the T-DNA, is thought to direct it 1o the plant cell nucleus and to assist it in integration. The VirD2 protein contains two nuclear localization signals (NLS) which are functional both in yeast and in plant cells. One signal is located in the N-terminal part of the protein and resembles a single-cluster typx NLS. The second signal is near the C-terminus and is a bipartite type NLS. The involvement of the C-terminal NLS in the entry of tRe T-DNA into the plant cell nucleus was directly tested in vivo.

Agrotiacferium tumefaciens / T-DNA transfer / nuclear targeting ! agroinfection / transient P-glucuronid*ase expression

Introduction Agrobacterium tumefaciens transfers a segment of its genetic information into plant cells. The transferred DNA (T-DNA) is stably integrated into the plant genome and contains genes coding for enzymes involved ‘in the synthesis of plant hormones. The T-DNA is delimited Fy two almost perfect direct 25bp repeats (borders). It is normally part of the large tumor-inducing (Ti) plasmid but is liberated in response to signal compounds excreted by wounded plant cells. The actual T-DNA transfer is mediated by proteins encoded by the virulence genes, which are located on the Ti plasmid and grouped into at least six operons (virA, -B, -C, -D, -E and -G). ‘ILvo virulence proteins, the VirE2 and VirD2, have been shown to form a complex with free T-DNA molecules in the bacterium and thus are thought to assist the T-DNA on its way to the plant (for reviews see [l-3]. VirE2 is a non-specific DNA-binding protein which attaches to single-stranded T-DNA, the T-strand [4]. VirD2 is involved in nicking at the 25bp repeats and remains very tightly attached to the 5’ terminus of the right T-DNA end [5-9 J. It has been proposed that VirD2 is involved in targeting the T-DNA into the plant cell nucleus [ 10-141 and in T-DNA integration [15, 161.

In this communication we examine the role of the VirD2 protein in the transport of the T-DNA into the plant cell nucleus. Nuclear entrance is tightly regulated Entry inio the nucleus of proteins larger than 60 kDa is a selective process which requires the activation of the nuclear pore complex. This activation is mediated by a nuclear localization signal (NLS) carried either by the protein itself or by an associated helper protein (for reviews, see [ 17-191). Two types of nuclear localization signals have been described. The first consists of a single cluster of positively charged amino acids [20]. The second type is a bipartite signal in which two sequence elements made up of basic amino acids are separated by about ten amino acids [2 1, 221. The recognition system involved in nuclear import is most likely universal. Because of this tight control of nuclear uptake, the T-DNA as such will presumably not enter the nucleus efficiently if it is delivered by Agrobacterium into the cytoplasm of the plant cell. Indeed, microinjection of DNA into the cytoplasm of mammalian cells has yielded stable transformants at a much lower frequency than microinjection directly into the nucleus

636 if naked DNA has difficulty in gaining entrance into the nucleus, then a special mechanism must exist that allows the T-DNA complex to find its way into the plant cell nucleus. [23]. Thus,

Nuclear targeting properties of the free VirD2 protein The VirD2 protein is a good candidate for piloting the T-DNA complex into the plant cell nucleus for at least three reasons. First, VirD2 is tightly linked to the right end of the free T-DNA in the bacterium [5-91. Second, this link may persist in the plant cell since in the integrated T-DNA the last bases of the right end are often retained whereas those of the left end are 1OObp

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Fig 1. Location within the GrD2 gene of the sequences coding for the N-terminal and &‘-terminalNLSs and compilation of different NLSs. a. Schematic representation of the octopinc ~‘raD2gene. The shaded region corresponds to the part of the VirD2 protein that is highly conserved in three different A,q~dm~eriwn strains, whereas the dotted region corresponds to the less well conserved part [ 121. The black bars indicate the sequences coding for the NLSs. b. Sequence comparison of different monopartite and bipartite NLSs; the latter are referred to in Dingwall and Laskey [22]. ThlJ amino acids that are considered to be involved in nuclear targeting are in capital letters and underlined, basic amino acids are in capital letters. The homologies between the different VirD2 C-terminal NLSs are indicated by vertical bars (figure modified after [ 141).

often lost [ 15, 161. This may in part be due to protec-

tion against exonucleolytic attack by the bound VirD2 protein [8]. Third, the VirD2 protein contains two regions of homology with previously described NLSs (see fig I). One is located in the N-terminal part (monopartite type NLS) and the other in the C-terminal part of the VirD2 protein (bipartite type NLS). In addition, comparison of the amino acid sequence of the VirD2 protein from three different Agrobacterium strains revealed that these sequences are particularly well conserved [ 121. Indeed, Hen-era-Estrella et al [lo] showed that in a transgenic plant expressing the N-terminal 292 amino acids of the nopaline VirD2 protein (including the N-tTrmina1 NLS) fused to E coli P-galactosidase, the enzymatic activity is localized in the nucleus. This result was contradicted by Howard et al [ 131, who found that only the C-terminal NLS from the nopaline VirD2 has nuclear targeting properties. These authors studied the transient production and localization in plant protoplasts of different parts of the VirD2 protein fused to E coli P-glucuronidase. Tinland el crl (141 have tested the nuclear localization potential of the octopine VirD2 protein in stably transformed yeast cells. Different parts of VirD2 were fused to E coii P-galactosidase. Each construct containing either the N-terminal or the C-terminal or both nuclear localization signals was expressed in yeast. The plasmid expressing P-galactosidase alone was used as a negative control. All protein products were localized by indirect immunofluorescence to the yeast nucleus, except in the case of P-galactosidase alone. In the latter case, the enzyme was distributed all over the cell. The nuclear targeting signals were precisely defined in yeast cells as two peptides of 11 and 20 amino acids belonging to the N-terminal and to the C-terminal part of the VirD2 protein, respectively. Each of the peptides, when tested individually, was then shown to target P-galactosidase into the nucleus of plant protoplasts [ 141. Despite some apparent contradictions concerning the N-terminal NLS, which are most probably due to differences in sensitivity of the assays used and possibly to the slight sequence differences between the N-terminal NLSs of the nopaline and octopine VirD2 proteins, the data clearly show that the VirD2 protein is able to target itself and other proteins to the nucleus. However, this does not automatically imply that the VirD2 protein is also able to import the linked T-DNA into the plant cell nucleus. Does VirD2 pilot the T-DNA into the plant cell nucleus? The function of the C-terminal bipartite NLS of the VirD2 protein was directly tested in vivo [24]. We

637 used agroinfection [25] and P-glucuronidase (GUS) expression [26-281 as two different very sensitive transient assays for T-DNA transfer. In agroinfection, a multimer of the cauliflower mosaic viral (CaMV), genome was inserted into the T-DNA. Upon transfer to the plant cell, a full-length copy of the CaMV genome is excised from the T-DNA by recombination or by replication (ie transcription and reverse transcription) [25] and ultimately gives rise to viral symptoms. In the second method, a modified GUS gene linked to plant expression signals and not expressed in Agrobacterium was inserted into the T-DNA. T-DNA transfer was detected by assaying for GUS activity. Since GUS expression requires transcriptir -1 and agroinfection requires transcription or recom,,nation of the T-DNA, both assays detect T-DNA transfer into the plant cell nucleus. Moreover, both are thought to detect T-DNA transfer independently of integration. Two mutant octopine Ti plasmids were compared which differ by the presence or absence of the C-terminal NLS of the VirD2 protein. The mutant plasmid lackin; the NLS is tumorigenic but was estimated to be 10-100 times less efficient than the mutant containir e the NLS in all tests performed: agroinfection, trans; znt GUS expression and stable integration as assay ~11by tumor formation and the formation of calli resisttint to kanamycin. The finding that this mutant is less efficient in the two transient assays supports the hypot;s:sis that the transport of the T-DNA into the plant a!11 nucleus is mediated by the nuclear localization signals of the attached VirD2 protein [ 1O-141. The fiwt that the mutant is not completely negative in transl:er into the plant cell nucleus indicates that the N-terum;nal NLS may be sufficient to transport the TDNA across the nuclear envelope. However, it was recenrly suggested that the N-terminal NLS is not involved in this process [29, 301. It is possible that the N-terminal NLS sequence in the free VirD2 protein is more exposed on the surface than in the protein attached to the T-DNA since this NLS is very close to a tyrosine proposed to be involved in the covalent linkage of VirD2 to the T-DNA [3 11. Thus, the Nterminal NLS may not be functional in vivo. Rossi et al [30] constructed various mutated virD2 genes carrying different point mutations in the N-terminal NLS sequence, in addition to a deletion of the C-terminal NLS. These genes were reintroduced into Agrobacterium and the effect of the different mutations on T-DNA transfer was studied. However, in both cases the original virD2 gene in the Ti plasmid was not fully deleted, thus coding for a truncated VirD2 protein containing a wild-type C-terminal NLS. It cannot be ruled out that this truncated protein, although non-functional in nicking, masks the effect of the mutations studied. The functional deficiency of the mutant lacking the C-terminal NLS may alternatively

be complemented by another virulence protein of the T-DNA complex such as VirE2, which was recently shown to contain active NLSs [32]. It is also possible that a certain proportion of the T-DNA molecules may come into contact with plant chromatin when the nuclear membrane disintegrates during cell division. Import into the nucleus of most ribonucleoprotein particles requires not only a protein, which is bound to the RNA, but also the trimethylguanosine cap (m,GpppN) at the 5’-end of the RNA molecule itself (for a review see [ 191). We do not yet know whether a specific feature of the T-DNA molecule itself is also essential for its import into the plant cell nucleus. Acknowledgments We thank Luca Rossi and Walt Ream for communicating their results prior to publication, and Luigina Beffa and Mylena Wels for typing this manuscript.

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