Christophe Robaglia

Transformation of Pakchoi (Brassica rapa L. ssp. chinensis) by Agrobacterium infiltration

Transgenic pakchoi (Brassica rapa L. ssp. chinensis) plants were obtained in the progeny of plants infiltrated by an Agrobacterium tumefaciens strain carrying a gene for resistance to the herbicide phosphinotricin (Basta). Genetic analysis demonstrates the transmission of the herbicide resistant trait to the progeny. Molecular analyses show that the transgene was inserted in the plant genome and expressed. This work demonstrates that the infiltration transformation method originally devised for Arabidopsis thaliana can be adapted for other crucifer species and opens up the possibility of genetic engineering of pakchoi, an important vegetable plant.

Molecular characterization of potato virus YN isolates by PCR-RFLP : Differentiation of PVYN isolates by PCR-RFLP

Based on the sequence polymorphism in the 5′ terminal part of the viral genome, a range of PVYN isolates were characterized by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP). Three pairs of primers selected in the 5′ non-translated and P1 protein region were tested. Two of them yielded PCR products of about 1Kb from all isolates tested. Restriction analysis of the PCR products gave two distinct electrophoretic patterns, whichever of the three enzymes was used. In this way, the 18 isolates were separated into two easily identifiable subgroups. All tuber necrosing isolates (PVYNTN) were clustered in the same subgroup.

RFLP mapping of the whole genome of ten viral isolates representative of different biological groups of potato virus Y.

SummaryTen PVY isolates representative of four PVY groups (YN, YNTN, YN-W, Y O), differing by their ability to induce reactions of vein necrosis on tobacco and tuber necrosis on potato, were studied in order to research the regions of the viral genome involved in these necrosis phenomena. The whole genome of these isolates was amplified in two fragments (4u2009063 and 5u2009670 nucleotides) and was subjected to a restriction fragment length polymorphism (RFLP) study. In the first 4u2009063 nucleotides of the PVY genome, a phenetic analysis of RFLP data resulted in a clustering of our PVY isolates into three groups: PVYN isolates (groupu2009A); PVYNTN and PVYN-W isolates (groupu2009B) and PVYO isolates (groupu2009C). In the last 5u2009670 nucleotides, two groups were found: PVYN and PVYNTN isolates (groupu2009D) and PVYO and PVYN-W isolates (group E). From this clustering and the necrosing properties known for theseisolates, the tobacco necrosis determinants seem more likely located inthe 5′ than in the 3′ half part of the viral RNA, whereas it would be theopposite situation for the determinants of the necrosis on potato tubers.Moreover a recombination event seemed to have occurred in the genome ofthe PVYN-W isolates.

Plant lipoxygenase 2 is a translation initiation factor-4E-binding protein

The eukaryotic initiation factor 4E (eIF4E) emerged recently as a target for different types of regulation affecting translation. In animal and yeast cells, eIF4E-binding proteins modulate the availability of eIF4E. A search for plant eIF4E-binding proteins from Arabidopsis thaliana using the yeast genetic interaction system identified a clone encoding a lipoxygenase type 2 (AtLOX2). Inxa0vitro and inxa0vivo biochemical assays confirm an interaction between AtLOX2 and plant eIF4E(iso) factor. A two-hybrid assay revealed that AtLOX2 is also able to interact with both wheat initiation factors 4E and 4E(iso). Deletion analysis maps the region of AtLOX2 involved in interaction with AteIF(iso)4E between amino acids 175 and 232. A sequence related to the conserved motif present in several eIF4E-binding proteins was found in this region. Furthermore, the wheat p86 subunit, a component of the plant translation eIF(iso)4F complex, was found to interfere with the AteIF(iso)4E-AtLOX2 interaction suggesting that p86 and AtLOX2 compete for the same site on eIF(iso)4E. These results may reflect a link between eIF4Es factors mediating translational control with LOX2 activity, which is probably conserved throughout the plant kingdom.

Tobacco veinal necrosis determinants are unlikely to be located within the 5@ and 3@ terminal sequences of the potato virus Y genome

SummaryThree potato virus Y isolates, representatives of distinct PVY groups, identified in potato fields in northern Poland were submitted to biological and molecular analysis. Phenotypically, two isolates, PVYN-Ny and PVYN-Wi, belong to the necrotic strain and the third one (PVYO-LW) to the common strain. PVYN-Wi, however, did not react with monoclonal antibodies directed against the necrotic strain isolates which recognise PVYN-Ny. To characterise the isolates, coat protein genes were sequenced and compared with sequences from databases. The necrotic PVYN-Wi isolate showed 99% amino acid homology with the common one–PVYO-LW and significantly differed from the second necrotic isolate (PVYN-Ny). Sequence based homology matrix and phylogenetic analysis lead to classification of PVYN-Ny into group I, encompassing solely necrotic strain isolates, whereas PVYN-Wi falls into a phenotypically heterogeneous group II. The sequence analysis allowed for identification of putative group I–specific epitopes. 3′NTR (non-translated region) sequences were identical for PVYN-Wi and PVYO-LW. The 5′NTR, P1 gene, coat protein gene and 3′NTR sequences of the common (PVYO-LW) and the necrotic (PVYN-Wi) isolates are 99–100% homologous. This suggests that tobacco veinal necrosis determinants are located outside the 3′ and 5′ terminal sequences of the PVY genome.

Coat protein gene-mediated protection in Lactuca sativa against lettuce mosaic potyvirus strains.

Lettuce mosaic potyvirus (LMV) can be very destructive on lettuce crops worldwide. The LMV strain 0 (LMV-0) coat protein (CP) gene was engineered for expression in plants. It was introduced into three susceptible cultivars of Lactuca sativa using an improved procedure for transformation and regeneration of lettuce, by co-cultivation of leaf explants with Agrobacterium tumefaciens. Several transformants accumulated detectable levels of LMV CP. The R1 progeny of twelve R0 transformants (four plants per cultivar) with T-DNA integration at one single locus, was studied for protection against LMV. The progeny from five R0 transformants showed resistance to LMV-0, with the effectiveness of resistance depending on the development stage of the plants at the time of inoculation. The R1 and R2 progeny from one of these R0 transformants, Cocarde-9a, were more extensively analysed. The homozygous but not the hemizygous R1 plants displayed protection to LMV-0. The R2 progeny from one homozygous R1 plant were shown to be resistant to infection by LMV-0 and other LMV strains. As previously observed in other cases of potyvirus sequence-mediated protection, a phenomenon of recovery was observed in some plants, as well as complete resistance. However, this recovery phenotype was not always maintained, as opposed to the previous described cases, leading to a late progression of viral infection.

Transformation of two potato cultivars ‘Spunta’ and ‘Claustar’ (Solanum tuberosum) with lettuce mosaic virus coat protein gene and heterologous immunity to potato virus Y

Abstract The coat protein (CP) gene of the lettuce mosaic potyvirus (LMV) was introduced into two potato cultivars—Spunta and Claustar—via Agrobacterium tumefaciens -mediated transformation. Gene expression was verified by Southern. Northern and Western blot. The regenerated plants have been tested for resistance against potato virus Y (PVY-0) in greenhouse. We observed an immune phenotype in four transgenic lines expressing the CP-LMV protein at different detectable levels. However, in two other transgenic plant lines, symptoms were developed at a frequency of 25 and 60% with a delay in their appearance. Transgene transcripts of the CP-LMV gene were detected by Northern blot in these two lines, but the protein could not be detected by Western blot. The correlation between the presence of the CP protein at a detectable level and the expression of an immune phenotype is discussed.

Expression vectors based on the Agrobacterium rhizogenes Ri plasmid transformation system

This article describes several new expression vectors that capitalize on the ability of Agrobacterium rhizogenes to transfer DNA from its Ri plasmid to the plant nuclear genome. The intermediate vectors described include an expression cassette based on one of the three following promoters: the nopaline synthase promoter, or the cauliflower mosaic virus (CaMV) promoters responsible for transcription of either the 19S or 35S CaMV RNA. The termination and polyadenylation signals are either from the nopaline synthase gene or from CaMV. The expression micro-Ri plasmid described bears a selectable marker gene and an expression cassette cloned between the borders of the TL-region of the Ri plasmid of A. rhizogenes A4. Different strategies for using these vectors to introduce chimeric genes into plants are described, and the advantages and disadvantages of the two types of vectors are discussed.This article describes several new expression vectors that capitalize on the ability of Agrobacterium rhizogenes to transfer DNA from its Ri plasmid to the plant nuclear genome. The intermediate vectors described include an expression cassette based on one of the three following promoters: the nopaline synthase promoter, or the cauliflower mosaic virus (CaMV) promoters responsible for transcription of either the 19S or 35S CaMV RNA. The termination and polyadenylation signals are either from the nopaline synthase gene or from CaMV. The expression micro-Ri plasmid described bears a selectable marker gene and an expression cassette cloned between the borders of the TL-region of the Ri plasmid of A. rhizogenes A4. Different strategies for using these vectors to introduce chimeric genes into plants are described, and the advantages and disadvantages of the two types of vectors are discussed.

Research articleExpression vectors based on the Agrobacterium rhizogenes Ri plasmid transformation systemVecteurs d'expression basés sur le système de transformation du plasmide Ri d'Agrobacterium rhizogenes

This article describes several new expression vectors that capitalize on the ability of Agrobacterium rhizogenes to transfer DNA from its Ri plasmid to the plant nuclear genome. The intermediate vectors described include an expression cassette based on one of the three following promoters: the nopaline synthase promoter, or the cauliflower mosaic virus (CaMV) promoters responsible for transcription of either the 19S or 35S CaMV RNA. The termination and polyadenylation signals are either from the nopaline synthase gene or from CaMV. The expression micro-Ri plasmid described bears a selectable marker gene and an expression cassette cloned between the borders of the TL-region of the Ri plasmid of A. rhizogenes A4. Different strategies for using these vectors to introduce chimeric genes into plants are described, and the advantages and disadvantages of the two types of vectors are discussed.This article describes several new expression vectors that capitalize on the ability of Agrobacterium rhizogenes to transfer DNA from its Ri plasmid to the plant nuclear genome. The intermediate vectors described include an expression cassette based on one of the three following promoters: the nopaline synthase promoter, or the cauliflower mosaic virus (CaMV) promoters responsible for transcription of either the 19S or 35S CaMV RNA. The termination and polyadenylation signals are either from the nopaline synthase gene or from CaMV. The expression micro-Ri plasmid described bears a selectable marker gene and an expression cassette cloned between the borders of the TL-region of the Ri plasmid of A. rhizogenes A4. Different strategies for using these vectors to introduce chimeric genes into plants are described, and the advantages and disadvantages of the two types of vectors are discussed.

Expression of genes in transgenic plants from bicistronic transcriptional units

Abstract Bicistronic mRNAs encoding either the potato virus Y (PVY) coat protein or the Bacillus thuringiensis CryIC protein followed by the selectable marker gene neomycin phosphotransferase II ( npt II) were introduced by electroporation into tobacco protoplasts. The initiation codon for the npt II gene was located 44 or 39 nucleotides downstream of the termination codon of the PVY coat protein or B. thuringiensis cryIC gene, respectively. Plants selected using paromomycin (50 μ g/ml) were shown to express the PVY coat protein by Western blot analysis or the B. thuringiensis CryIC protein by an insect feeding bioassay. Transgenic plants were shown, by Northern analysis, to produce transcripts of either construct without rearrangement. A significantly reduced level of npt II activity was demonstrated in transgenic seedlings of the T1 and T2 generation either by germination on agar containing 75 μ g/ml kanamycin or by npt II activity assays. These results demonstrate that transgenes of interest can be expressed from a bicistronic transcriptional unit and that plants expressing the transgene can be selected by monitoring for activity of a distal marker gene.