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Auxin-autonomous growth in vitro may be related to the integration and expression of the aux and rol genes from the root-inducing (Ri) plasmid in plant cells infected by agropine-type Agrobacterium rhizogenes. To elucidate the functions of the aux and rol genes in plant cell division, plant cell lines transformed with the aux1 and aux2 genes or with the rolABCD genes were established using tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells. The introduction of the aux1 and aux2 genes enabled the auxin-autonomous growth of BY-2 cells, but the introduction of the rolABCD genes did not affect the auxin requirement of the BY-2 cells. The results clearly show that the aux genes are necessary for auxinautotrophic cell division, and that the rolABCD genes are irrelevant in auxin autotrophy.
The plant pathogen A. rhizogenes has a large Ri plasmid and induces hairy root disease, causing root proliferation from the infection site. When A. rhizogenes infects plants, a portion of the Ri plasmid, the T-DNA, is transferred to the plant cell, integrated into the plant genome, and expressed as polyadenylated mRNA.1–4 The T-DNA is defined by left-border and right-border sequences, which are cis-acting 25-bp direct repeats.5–7
The most studied A. rhizogenes strains, which belong to the group of agropine-type strains, have two T-DNA regions on their Ri plasmid. These regions, designated the TL-DNA and TR-DNA, can be independently transferred to the nuclear genome of infected plant cells.3,8–10 A. rhizogenes strains 15834 and A4 are typical agropinetype strains, and contain the essentially identical large plasmids pRi15834 and pRiA4, respectively.4
The Ri TL-DNA carries approximately 18 potential genes,11 of which four genes, rol A, B, C and D, have a fundamental role in the induction of hairy root syndrome in tobacco,10,12–16 although nothing is known about the functions of the proteins encoded by these genes. The Ri TR-DNA carries the genes responsible for opine synthesis17 and the genes representing two steps of auxin biosynthesis, referred to as aux1 and aux2 (also referred to as tms1 and tms2, respectively).8,14,18–22 The Ri TR-DNA also contains a rolB homolog, but the function of the rolB (TR) gene product is unknown.23
Hairy roots induced by A. rhizogenes infection can grow in phytohormone-free medium in vitro after the elimination of bacteria.24–26 Phenotype variations among hairy roots have been attributed to differences in the genes from the TL-DNA and TR-DNA regions that have been integrated into the plant genome.24,25,27,28 In one example, the TR-DNA from the Ri plasmid bearing the aux1 and aux2 genes was never integrated in the cells of coffee hairy roots, even though the TL-DNA bearing the rol genes was always transferred.29 Various experiments, performed by different researchers, have demonstrated that the aux genes do not play a major role in hairy root disease, whereas the rol genes are necessary and sufficient to induce hairy roots.13,15,30 However, there have been contradictory reports of the contributions of aux1 and aux2 to plant tumorigenicity; some have suggested that these aux genes are important for hairy root formation.14,19
Auxin-autonomous growth may be related to the integration and expression in transformed cells of aux and rol genes from the Ri plasmid that enable the production of endogenous auxin and/or trigger auxin signal transduction. To elucidate the functions of the aux and rol genes in plant cell division, we generated transformed plant cell lines by infecting BY-2 cells with A. rhizogenes strain 15834.31 Tobacco BY-2 cells require only auxin for growth, and are usually cultured in Linsmaier-Skoog (LS) medium32 containing 0.2 µg mL−1 of 2,4-dichlorophenoxyacetic acid (2,4-D) (LSD medium).
The transgenic cell lines, established by further culturing through seven passages in phytohormone-free medium, were named BYHR-1, 3, 5, 7, 8 and 9. To determine the presence or absence of the T-DNA in the genomes of the transgenic lines, PCR analysis was performed using the primer sets TL-F1/TL-R1 and TR-F7/TR-R5,31 which cover the rolA and rolB (TL) loci (3,244 bp) in the TL-DNA and the aux1 and rolB (TR) loci (3,405 bp) in the TR-DNA, respectively. In a previous study, we showed that BYHR-7 is transgenic for both the TR- and TL-DNAs, whereas BYHR-3 is only transgenic for the TR-DNA.31 Figure 1 shows that the aux1 and rolB (TR) loci were detected in the genomes of all of the transgenic lines, but the rolA and rolB (TL) loci were detected only in the genomes of transgenic lines BYHR-1 and BYHR-7. These results show that BYHR-1 and BYHR-7 are transgenic for both the TR- and TL-DNAs, whereas the other lines are possibly only transgenic for the TR-DNA.
The rolABCD genes, along with their own promoter region, were isolated from pRi15834 by PCR using Phusion® High-Fidelity DNA Polymerase (NEB) with the forward primer TL-FA (5′-GCC ACG CAA CTC GAG ATA TTC CCA C-3′) and the reverse primer TL-RD (5′-GCG ACG AAT TCA AAC AAG CCC GTC T-3′), and subcloned into the EcoRV site of pBluescript II SK+ (Stratagene). This construct was digested with Acc65I and XbaI, and inserted into the binary vector pGAH33 that had been digested with Acc65I and XbaI. The resulting plasmid, pGABCD, was introduced into A. tumefaciens GV3101 (pMP90). To elucidate the functions of the rol genes in plant cell division, we established 125 transgenic BY-2 cell lines transformed with the rolABCD genes through selection based on the vector-derived kanamycin and hygromycin resistance markers in the presence of auxin. None of the transgenic cell lines was able to multiply without auxin. These results imply that the rolABCD genes are irrelevant in auxin-autotrophic cell division.
To elucidate the functions of the aux genes in plant cell division, we established another type of transgenic BY-2 cell line. The aux2, aux1 and rolB (TR) genes with their own promoter regions and the additional region Z were isolated from pRi15834 by PCR using PfuTurbo® DNA Polymerase (Stratagene) with the forward primer TR-F21 (5′-TCG ATT GTA CTG AAT CGG ATT TTC AAG GGT-3′) and the reverse primer TR-R21 (5′-CAC CAG GGC GAG ATA AGC TTT CAG GTC A-3′), and inserted into the EcoRV site of pER1A.31 This construct served as the entry vector to transfer the aux2, aux1 and rolB (TR) genes and the region Z into the binary destination vector pBCR-112 (Seki et al. unpublished data) via the Gateway system (Invitrogen). The resulting plasmid pF21BZ was modified to construct the plasmids pF21B, which contains the aux2, aux1 and rolB (TR) genes; pF21, which contains the aux2 and aux1 genes; and pFZ, which contains the region Z (Fig. 2). These plasmids were introduced into A. tumefaciens GV3101 (pMP90), and BY-2 cells were infected with the resulting A. tumefaciens strains for transformation.
The transgenic cell lines grown on kanamycin-containing LSD medium were tested to examine their auxin-autotrophic characteristics. Among the transgenic cell lines, the percent of auxin-autotrophic cell lines was 11% when inoculating GV3101 containing pF21BZ, 15% for pF21B, 13% for pF21 and 0% for pFZ. These results show that the aux1 and aux2 genes are essential for plant cell division in vitro.
In conclusion, these results clearly show that the aux genes are necessary for auxin-autotrophic cell division, and that the rolABCD genes are irrelevant in auxin autotrophy.
Previously published online: www.landesbioscience.com/journals/psb/article/9904