Agrobacterium rhizogenes-Mediated Transformation and Its Biotechnological Applications in Crops - Crop Improvement: New Approaches and Modern Techniques - page 20

ORF8
The ORF8 gene has the longest sequence of T L -DNA and coding for a protein con-
taining 780 amino acids (Slightom et al. 1986 ). The ORF8 protein has one of the
most conserved amino acid sequences (81 % similarity) between different strains
like pRiA4 and pRi2659 (Ouartsi et al. 2004 ).
The protein encoded by the ORF8 gene is a natural fusion protein consisting of
N-terminal domain (NORF8) of 213 amino acids homologous to RolB protein of
the A. rhizogenes strain A4 T-DNA and the C-terminal part (CORF8) of approxi-
mately 506-524 amino acids shows homology to the IaaM proteins of various other
bacteria (Yamada et al. 1985 ; Slightom et al. 1986 ; Levesque et al. 1988 ; Dodueva
2007 ; Shoja 2010 ). iaa M genes that homologues to the coding sequence of CORF8
codes for a tryptophan monooxygenase which catalyzes the formation of indole-3-
acetamide (IAM) from tryptophan (Lemcke et al. 2000 ).
Furthermore, ORF8 possesses a 200 amino acid stretch at its N-terminus that
shows homology with the rol B gene product (33.5 % amino acid identity) (Levesque
et al. 1988 ). The N-terminal part (NORF8) of this protein functions in carbohydrate
metabolism such that when only NORF8 was expressed, transformed plant showed
growth retardation, chlorotic and necrotic leaves and accumulation of high levels of
sugars (glucose, fructose and sucrose) and starch (Otten and Helfer 2001 ).
However, some studies show that the auxin content can be elevated by the
genes found in the T L -DNA region on the T-DNA in some hosts, independent of
the presence of the T R -DNA (Lemcke et al. 2000 ). Presumably this occurs because
of conversion of IAM to IAA in cells expressing only t2m protein (Klee et al. 1987 ;
Prinsen et al. 1990 ). Besides this, as a characteristic functional motif of the t2m pro-
teins that catalyzes decarboxylation of tryptophan to indole-3-acetamide exhibits
23-aminoacid- long a flavin adenine dinucleotide (FAD) binding site was identified
by Levesque et al. ( 1988 ). The experimental data obtained from plants and bacteria
suggest that the gene product of ORF8 of A. rhizogenes T L -DNA has t2m activ-
ity responsible for the increased IAM content in transgenic tissues (Lemcke et al.
2000 ). Moreover, there is a physical connection between N- and C-regions of ORF8
protein required for the emergence of a specific phenotype in transgenic plants con-
sisting ORF8 gene. This suggests a distinct specific function for the whole protein
(Umber et al. 2005 ; Dodueva 2007 ).
ORF13andORF14
The ORF13 and ORF14 genes are found to be highly conserved among A. rhizo-
genes strains (Stieger et al. 2004 ). It has been demonstrated that alone A4- rol ABC
genes carried by an Agrobacterium strain are showed to be incapable of inducing
rooting on carrot disc and aux genes located on the T R -DNA or ORF13 and ORF14
located on T L -DNA are also required for rooting (Cardarelli et al. 1987b ; Capone