The non-conserved region of cucumopine-type Agrobacterium rhizogenes T-DNA is responsible for hairy root induction

Abstract

The T-DNA regions of three strains of Ri plasmids 1855, 8196, 2659 (agropine, mannopine and cucumopine type respectively) share two highly conserved regions flanking a non-homologous central part. We have cloned segments of the cucumopine Ri plasmid 2659 T-DNA in the binary vector system Bin 19 and infected carrot discs with recombinant Agrobacterium strains. We show here that the central non-conserved region is crucial in hairy root induction as it is sufficient to induce rooting on the apical (auxin-rich) surface of carrot discs; in order to observe rooting on the basal (auxin-depleted) side of the discs, a longer T-DNA fragment, also encompassing part of the right conserved region, had to be utilized in conjunction with a Agrobacterium strain carrying aux genes. Differences of growth properties in culture are exhibited by roots transformed with different fragments of pRi 2659 T-DNA, although all transformed roots show the plagiotropic behaviour typical of hairy roots.

rol genes of Agrobacterium rhizogenes cucumopine strain: sequence, effects and pattern of expression

“…Standard techniques were used for the construction of recombinant plasmids, pSK18, encompassing rol~, was constructed by inserting Eco RI/ KpnI fragment from pUCSSpl8 [16] (sites located on rol~ promoter and pUC 18 respectively) into the Eco RI/Kpn I site of the binary vector Binl9 [4]. An Nci I 18 fragment, containing rolfl, was excised from pUCSSpl8, treated with Klenow polymerase and cloned into the Sma I site of pUC18; the construct was digested with Eco RI/ Hind III and the fragment cloned in the polylinker of Binl9 [4].…”

“…An Nci I 18 fragment, containing rolfl, was excised from pUCSSpl8, treated with Klenow polymerase and cloned into the Sma I site of pUC18; the construct was digested with Eco RI/ Hind III and the fragment cloned in the polylinker of Binl9 [4]. Recombinant plasmids were transferred to Agrobacterium LBA4404 as previously described [ 16].…”

“…All the genes relevant for hairy root induction, denominated rolA, B and C reside in this central, less conserved region of the agropine TL-DNA [10,27]. In a previous work, we demonstrated that the central region is crucial also for the cucumopine-type T-DNA [ 16], in that a clone encompassing a 4 kb Hpal8 fragment of this core T-DNA was shown to be capable of hairy root induction on carrot discs. Some differences were, however, observed in the auxin required for root induction by the two core T-DNAs.…”

“…Some differences were, however, observed in the auxin required for root induction by the two core T-DNAs. On carrot discs, agropine rol genes require exogenous auxin, while the central region of the cucumopine T-DNA is capable of stimulating root formation without any further auxin supply [7,8,16].…”

“…Furthermore, roots containing the rolA + B + C genes show a geotropic growth pattern in hormone-free medium, while auxin addition causes loss of the geotropic response; in contrast, roots transformed by the core of the cucumopine T-DNA grow plagiotropically also in the absence of exogenous auxin [ 10,16].…”