Identification of hairy root loci in the T-regions of Agrobacterium rhizogenes Ri plasmids

Abstract

Agrobacterium rhizogenes induces root formation at the wound site of inoculation in plants and inserts a fragment of its plasmid (Ri) into the plant nuclear DNA. Parts of the transferred region (T-region) of the Ri plasmid of A. rhizogenes strain A4 or 8196 are cloned in Escherichia coli. Insertions of the E. coli lacZ coding region into the hybrid plasmids were made in vivo using transduction by miniMu. Twenty insertions localized in the TL-DNA of pRiA4 (or pRi1855) and 2 inserts in the T-DNA of pRi8196 were obtained in E. coli. One of the TL-DNA insertions is saved up because it is linked to an internal T-DNA deletion; the others because they confer a lactose plus phenotype on E. coli; this indicates that the T-DNA harbours sequences that are expressed in E. coli. Fifteen of these T-DNA insertions were transfered to Agrobacterium where they substitute the corresponding wild-type T-DNA of the Ri plasmid by homologous recombination. These strains corresponding to insertion-directed mutagenesis were used to inoculate Daucus carota slices and stems and leaves of Kalanchoe daigremontiana. The two insertions strains obtained in the T-DNA of pRi8196 are avirulent on K. daigremontiana; but their phenotypes differ on D. carota slices, suggesting that insertions affect distinct loci on the T-DNA involved in hairy root formation. Only one insertion out of the twenty obtained in the TL-DNA of pRiA4 (or 1855) induces a loss of virulence on leaves of K. daigremontiana. However the TL-DNA deletion harbouring strain induces a loss of virulence on D. carota and K. daigremontiana (stems and leaves), confirming the importance of the TL-DNA for hairy root induction. re]19850711 rv]19851230 ac]19860114.

rolA locus of the Ri plasmid directs developmental abnormalities in transgenic tobacco plants.

“…Mutations in the tmsl and tmsl genes severely attenuate the root induction response on to bacco stem (Nicotiana tabacum], basal carrot disks [Daucus car Ota), and K. diagremontiana leaves (CardareUi et al 1985;White et al 1985;Boulanger et al 1986;. Four left T-DNA (TL-DNA) loci have been identified by transposon and dele tion mutagenesis, which affects tumor morphology on K. diagremontiana leaves, and these loci have been ten-tatively assigned the names rolA, rolB, rolC, and rolD (White et al 1985;Estramareix et al 1986).…”

A common organization of the T-DNA genes expressed in plant hairy roots induced by different plasmids of Agrobacterium rhizogenes

“…However for our own concern we prefer to also take into account the T-DNA regions that appear to be more loosely related for the following reasons. First, genetic loci relevant to the hairy root process have been mapped in these regions [2,11]. Second, partially divergent DNA sequences may encode for final polypeptide products with the same function.…”

“…Therefore functions other than aux and cyt account for hairy root formation by pRi T-DNAs. It has been shown some equivalence between the T-DNA of mannopine Ri plasmid and the TL-DNA of agropine Ri plasmid, first at genetic level [2,11] and also by sequence homologies [1,6,25]. Nucleotide sequence with putative open reading frames (ORFs) [23] and transcription products [10,18,27] have been described for the agropine TL-DNA.…”

Function of theauxandrolgenes of the Ri plasmid in plant cell division in vitro

“…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.…”