Henk Joos

Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity.

A Ti plasmid mutant was constructed in which all the on‐cogenic functions of the T‐DNA have been deleted and replaced by pBR322. This Ti plasmid, pGV3850, still mediates efficient transfer and stabilization of its truncated T‐DNA into infected plant cells. Moreover, integration and expression of this minimal T‐DNA in plant cells does not interfere with normal plant cell differentiation. A DNA fragment cloned in a pBR vector can be inserted in the pGV3850 T‐region upon a single recombination event through the pBR322 region of pGV3850 producing a co‐integrate useful for the transformation of plant cells. Based upon these properties, pGV3850 is proposed as an extremely versatile vector for the introduction of any DNA of interest into plant cells.

Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens.

Transmission of ColE1/pMB1‐derived plasmids, such as pBR322, from Escherichia coli donor strains was shown to be an efficient way to introduce these plasmids into Agrobacterium. This was accomplished by using E. coli carrying the helper plasmids pGJ28 and R64drd11 which provide the ColE1 mob functions and tra functions, respectively. For example, the broad host‐range replication plasmid, pGV1150, a co‐integrate plasmid between pBR322 and the W‐type mini‐Sa plasmid, pGV1106, was transmitted from E. coli to A. tumefaciens with a transfer frequency of 4.5 x 10(‐3). As pBR322 clones containing pTiC58 fragments were unable to replicate in Agrobacterium, these clones were found in Agrobacterium only if the acceptor carried a Ti plasmid, thus allowing a co‐integration of the pBR322 clones with the Ti plasmid by homology recombination. These observations were used to develop an efficient method for site‐specific mutagenesis of the Ti plasmids. pTiC58 fragnents, cloned in pBR322, were mutagenized in vitro and transformed into E. coli. The mutant clones were transmitted from an E. coli donor strain containing pGJ28 and R64drd11 to an Agrobacterium containing a target Ti plasmid. Selecting for stable transfer of the mutant clone utilizing its antibiotic resistance marker(s) gave exconjugants that already contained a co‐integrate plasmid between the mutant clone and the Ti plasmid. A second recombination can dissociate the co‐integrate plasmid into the desired mutant Ti plasmid and a non‐replicating plasmid formed by the vector plasmid pBR322 and the target Ti fragment. These second recombinants lose the second plasmid and they are identified by screening for the appropriate marker combination.

Genetic analysis of T-DNA transcripts in nopaline crown galls

Plant crown gall tumor cells result from the insertion and expression of a defined DNA sequence, called T-DNA, which is derived from the Ti plasmid, harbored by Agrobacterium tumefaciens strains. To study the function of the genes of the T-DNA of the nopaline Ti plasmid, pTiC58, a collection of mutants was isolated so that T-DNA genes are inactivated either separately or in various combinations. It was found that no single T-DNA gene or T-region border is absolutely essential for stable tumor formation. We have identified the gene responsible for synthesis in transformed cells of the phosphorylated sugar, agrocinopine, and at least three additional genes controlling the morphology of plant tumors. Two of these latter genes work together to inhibit shoot formation and ensure efficient tumorous growth. Inactivation of these genes can be suppressed by the addition of auxins. The third gene inhibits root formation and appears to play a role in the cytokinin-independent growth of transformed cells. Mutants missing all three genes do not induce tumors, nor shoot or root formation, although the mutant T-DNA sequence is transferred to plant cells.

Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii

Two plasmids, pLAB1000 and pLAB2000 (3.3 and 9.1 kb, respectively), have been isolated from a grass silage strain of Lactobacillus hilgardii. Both plasmids were cloned in Escherichia coli and characterized through restriction mapping. A 1.6-kb XbaI-SacI fragment of pLAB1000 appeared to be sufficient for autonomous replication in Lactobacillus plantarum and in Bacillus subtilis. Different shuttle vectors for E. coli and gram-positive bacteria were developed using the pLAB1000 plasmid. These could stably be maintained in Lactobacillus, Enterococcus, and Bacillus under selective conditions. Plasmids sharing DNA homologies with pLAB1000 have been observed in different strains of the related species L. plantarum.

Fundamental aspects of rhizobacterial plant growth promotion research

Abstract The widescale application and commercialization of the use of inocula of free-living rhizobacteria for plant growth promotion (PGP) is impeded by inconsistent field performance. Despite a decade of intensive research there is, more than ever, a need for fundamental research in order to develop effective and reliable inocula.

Cloning and expression of cellulase and xylanase genes in Lactobacillus plantarum

SummaryEleven cellulase genes from Gram-positive bacteria were cloned in a Lactobacillus plantarum silage inoculum. Eight of these genes were expressed as active enzymes from their original promotors and translation signals. Where tested, the enzymes produced by transformed L.plantarum had the same temperature and pH optimum as enzymes produced in the original host, or in transformed Escherichia coli. Using chloramphenicol acetyltransferase as a cell-internal marker enzyme, it could be demonstrated that at least endoglucanase D from Clostridium thermocellum was actively secreted by transformed L. plantarum. In growing L. plantarum cultures, most of the enzymes were irreversibly inactivated when the pH decreased below 4.5. If the transformed strains were to be applied as an inoculum in silage, this pH inactivation might be useful in preventing overdigestion of the crop fibre.

Genetic analysis of transfer and stabilization of Agrobacterium DNA in plant cells.

In an attempt to elucidate the transfer and integration mechanism of Agrobacterium DNA upon crown gall induction, we translocated a borderless T‐DNA to different sites of the C58 Ti plasmid. As a result of the physical linkage of the T‐DNA onc genes with other Ti plasmid functions, the concerned strain retained tumor‐inducing capacity. However, when the borderless T‐DNA is separated on an independent replicon while all other pTi functions are provided in trans, the strain can no longer induce tumors on plants. We provide evidence that the right T‐DNA border region harbors one or more in cis active functions essential in the transfer and/or stabilization of the T‐DNA into plant cells. The strains used in these experiments allowed us to conclude that some function(s) of the Ti plasmid can induce plant cell proliferations independently of the T‐DNA transformation event. The results described here indicate that other Ti plasmid sequences than solely the T‐region can be transferred to plant cells.

Characterization and heterologous expression of the tetL gene and identification of iso-ISS1 elements from Enterococcus faecalis plasmid pJH1.

The tetracycline-resistance (TcR) determinant of the Enterococcus faecalis plasmid pJH1 has been identified and located on a 2.2-kb RsaI-EcoRI fragment. The fragment was cloned in Escherichia coli, and specified TcR in this host. The nucleotide (nt) sequence of the cloned fragment showed the presence of an open reading frame (ORF) of 1374 bp, designated tetL. The nt sequence of tetL from pJH1 was identical to that of the tetL present on pLS1 from Streptococcus agalactiae. Upstream of the pJH1 tetL, part of another ORF was found that, except for two single-nt substitutions, was identical to an iso-ISS1 element from Lactococcus lactis. Hybridization studies indicated the presence of several ISS1-like elements in plasmid pJH1, but not on the En. faecalis chromosome. To study its usefulness as a marker in Gram+ organisms, the pJH1 tetL was cloned on the broad-host-range plasmid pNZ124, resulting in pNZ280, that was found to give resistance to 40 micrograms Tc/ml in Lc. lactis and Bacillus subtilis.

Ti Plasmids as Gene Vectors for Plants

The formation of so-called “crown gall” tumors on dicotyledonous plants is the direct result of the introduction into the nuclear genome of plant cells of a set of genes that regulate cell and organ development. In other words, in nature, a mechanism exists that not only efficiently introduces foreign genes into the plant nucleus, but also contains a set of genes that regulate plant-cell development and differentiation. As a result of this gene transfer, crown gall cells, unlike untransformed plant tissues, can be cultured under axenic conditions on synthetic media in the absence of growth hormones, i.e., cytokinins and auxins.

Genetic engineering of plants

The soil bacterium Agrobaeterium tumefaciens can infect almost all dicotyledonous plants (Braun 1978, 1982). As a result of the infection, the wound tissue proliferates as a neoplastic growth, commonly referred to as a crown gall tumor. Once induced, the tumor no longer requires the presence of the bacteria to grow, and can be cultivated in vitro as an axenic culture (Braun 1943). Two main properties characterize crown galls: their ability to grow in vitro without the supplement of hormones required by normal plant cells, and their ability to synthesize a set of new metabolites termed opines, which are not present in normal cells. Opines are amino acid or sugar derivatives which can be used as a carbon and nitrogen source by the bacteria responsible for inciting the tumor (Tempe and Petit 1982).