A “plant gene vector cassette” to be used in combination with various Escherichia coli gene-cloning vectors was constructed. This cassette contains a replication and mobilization unit which allows it to be maintained and to be transferred back and forth between E. coli and Agrobacterium tumefaciens hosts provided these hosts contain plasmid RK2 replication and mobilization helper functions. The cassette also harbors a transferable DNA unit with plant selectable marker genes and cloning sites which can be combined with different bacterial replicons, thus facilitating the reisolation of transferred DNA from transformed plants in E. coli. The vector cassette contains two different promoters derived from the T-DNA-encoded genes 5 and nopaline synthase (NOS). By comparing the levels of expression of the marker enzymes linked to each of these promoter sequences, it was found that the gene 5 promoter is active in a tissue-specific fashion whereas this is not the case for the NOS promoter. This observation provides the first documented instance of a gene derived from a procaryotic host the expression of which is apparently regulated by plant growth factors.

https://link.springer.com/content/pdf/10.1007/BF00331014.pdf

The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector

Studies of the molecular genetics, mechanisms of activation and functional connections between general phenylpropanoid metabolism and certain branch pathways in parsley, bean, potato plants and cell cultures

Physiology and Molecular Biology of Phenylpropanoid Metabolism

Production of the phytohormone indole-3-acetic acid (IAA) is widespread among bacteria that inhabit the rhizosphere of plants. Several different IAA biosynthesis pathways are used by these bacteria, with a single bacterial strain sometimes containing more than one pathway. The level of expression of IAA depends on the biosynthesis pathway; the location of the genes involved, either on chromosomal or plasmid DNA, and their regulatory sequences; and the presence of enzymes that can convert active, free IAA into an inactive, conjugated form. The role of bacterial IAA in the stimulation of plant growth and phytopathogenesis is considered.

Bacterial biosynthesis of indole-3-acetic acid

We used a binary-vector strategy to study the hypervirulence of Agrobacterium tumefaciens A281, an L,L-succinamopine strain. Strain A281 is hypervirulent on several solanaceous plants. We constructed plasmids (pCS65 and pCS277) carrying either the transferred DNA (T-DNA) or the remainder of the tumor-inducing (Ti) plasmid (pEHA101) from this strain and tested each of these constructs in trans with complementary regions from heterologous Ti plasmids. Hypervirulence on tobacco could be reconstructed in a bipartite strain with the L,L-succinamopine T-DNA and the vir region on separate plasmids. pEHA101 was able to complement octopine T-DNA to hypervirulence on tobacco and tomato plants. Nopaline T-DNA was complemented better on tomato plants by pEHA101 than it was by its own nopaline vir region, but not to hypervirulence. L,L-Succinamopine T-DNA could not be complemented to hypervirulence on tobacco and tomato plants with either heterologous vir region. From these results we suggest that the hypervirulence of strain A281 is due to non-T-DNA sequences on the Ti plasmid.

The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA.

Agrobacterium rhizogenes, which induces hairy root disease of dicotyledonous plants1, is closely related to Agrobacterium tumefaciens, the causative agent of crown gall disease1–3. Virulence in both species is conferred by large plasmids4–7. Infected plant tissue synthesizes novel metabolites, opines8–11, that are not found in normal plant tissues. The pattern of opines synthesized is determined by the type of virulence plasmid in the bacterium, and in general virulence plasmids confer on the host bacterium the ability to catabolize the same opines (refs 8,12, 13 and A.P. et al., in preparation). Opine synthesis persists when the affected plant tissue is cultivated in vitro in the absence of the pathogenic bacterium9–11,14,15, which in the case of crown gall tumours is a consequence of gene transfer from the pathogen to the plant cells. A small specific part of the tumour-inducing (Ti) plasmid of A. tumefaciens, termed T-DNA (transferred DNA), is incorporated into host plant nuclear DNA16–21 and transcribed into mRNA18,22–25. A specific region of T-DNA confers the ability to synthesize the characteristic opine26,27. Synthesis of opines is thus a natural example of genetic engineering in which the agent is the Ti plasmid of,A. tumefaciens. The discovery of opines in roots induced by A. rhizogenes (ref. 11 and A.P. et al., in preparation) suggested that they too might contain T-DNA derived from the virulence plasmid of the pathogen. We report here DNA hybridization studies that confirm this hypothesis.

Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells

Gene 2 from the T region of Ti plasmids appears to be expressed both in eucaryotic and in procaryotic systems. In transformed plant cells it participates in auxin-controlled growth and differentiation, and in bacteria it is expressed into a defined protein of Mr 49000. We investigated the possibility that it codes for an enzyme involved in auxin biosynthesis.



Only extracts from Escherichia coli cells expressing gene 2 hydrolyzed indole-3-acetamide into a substance which was unambiguously identified as indole-3-acetic acid. The same reaction was found in Agrobacteria containing gene 2, but not in strains lacking the gene. Extracts from tobacco crown gall cells, but not from non-transformed cells, showed the same enzyme activity, and the reaction product was also identified as indole-3-acetic acid. The results indicate that gene 2 of the T region, which participates in tumorous growth of plant cells, codes both in bacteria and in plants for an amidohydrolase involved in the biosynthesis of the plant hormone indole-3-acetic acid.

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1432-1033.1984.tb07927.x

The T‐region of Ti plasmids codes for an enzyme synthesizing indole‐3‐acetic acid

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2881%2980781-8

The mRNA for lysopine dehydrogenase in plant tumor cells is complementary to a Ti-plasmid fragment

The T-region of Ti-plasmids expresses four proteins (mol. wts. 74,000, 49,000, 28,000 and 27,000) in Escherichia coli minicells. Promoter activities are determined by sequences within the T-region, and the protein-coding regions map in that part of the T-region which is highly conserved in octopine and nopaline plasmids and which is responsible for shoot and root inhibition when expressed in plant cells. Three of the regions expressed in bacteria correlate with three regions which are transcribed in transformed plant cells; the fourth protein-coding region has no corresponding transcript in plants. At least three of the proteins synthesized in E. coli minicells are also expressed in cell-free systems prepared from E. coli and from Agrobacterium tumefaciens; the fourth protein (mol. wt. 49,000) is poorly expressed in both cell-free extracts. The possibility is discussed that the same genes are expressed in Agrobacteria and in transformed plant cells and that in both cases the gene products mediate growth regulatory effects to non-transformed plant cells.

The conserved part of the T-region in Ti-plasmids expresses four proteins in bacteria.

An 8.5-kb EcoRI fragment containing the common nod region of the megaplasmid pRme41b of Rhizobium meliloti was recloned in plasmids of Escherichia coli, and a detailed restriction map was established. The region can express at least eight proteins in E. coli minicells and in an in vitro transcription/translation system, prepared from E. coli. Protein coding regions were determined by subcloning of restriction fragments, deletion mutations and by transposon mutagenesis. The coding regions for at least three polypeptide chains (mol. wts. 23 000, 28 500 and 44 000) were mapped on a 3.3-kb nod gene cluster. The 44 000 mol. wt. protein is expressed from a nod region, which is highly conserved in two Rhizobium species. The protein map of the 8.5-kb fragment was correlated to a map of insertion mutations with Nod- and Fix- phenotypes. The data suggest that the proteins encoded by the nod gene cluster may be involved in early steps of the nodulation process. Nod+ Fix- symbiotic mutations were localized in the coding region for a 33 000 mol. wt. protein, suggesting that this polypeptide might be a fix gene product.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1984.tb02035.x

Mapping of the protein-coding regions of Rhizobium meliloti common nodulation genes.

Fragment EcoRI 7 from Ti-plasmid pTi Ach5, a part of the T-DNA in octopine tumors, was cloned in both orientations into pACYC184 and expressed in E. coli minicells. The cells synthesized four proteins from four different coding regions on EcoRI 7. Two of the proteins (Mr 25.000 and 26.000) were expressed with promoters from the Ti-plasmid fragment, while transcription for the two other proteins (Mr 18.000 and 74.000) started with a promoter on pACYC184. The Mr 18.000 protein represented a fusion product between chloramphenicol acetyltransferase (CAT) on pACYC184 and a part of lysopine dehydrogenase (LpDH), the enzyme synthesizing octopine and lysopine in plant tumor cells. The results suggest that E. coli minicells are a valuable system to study the proteins coded for by the T-region of Ti-plasmids.

/pdf/expression-of-plant-tumor-specific-proteins-in-minicells-of-2a1tt2fnsn.pdf

Expression of plant tumor-specific proteins in minicells of Escherichia coli: a fusion protein of lysopine dehydrogenase with chloramphenicol acetyltransferase

Joachim Schröder

Papers

The T‐region of Ti plasmids codes for an enzyme synthesizing indole‐3‐acetic acid

The mRNA for lysopine dehydrogenase in plant tumor cells is complementary to a Ti-plasmid fragment

The conserved part of the T-region in Ti-plasmids expresses four proteins in bacteria.

Mapping of the protein-coding regions of Rhizobium meliloti common nodulation genes.

Expression of plant tumor-specific proteins in minicells of Escherichia coli: a fusion protein of lysopine dehydrogenase with chloramphenicol acetyltransferase