Erno Szegedi

Identification of an Agrobacterium tumefaciens pTiB6S3 vir region fragment that enhances the virulence of pTiC58

SummaryThe Agrobacterium tumefaciens octopine strain B6S3 and the nopaline strain C58 were compared for their ability to induce opine synthesis on Kalanchoe daigremontiana stem fragments. Whereas B6S3 induced high levels of octopine synthesis, C58 induced only low levels of nopaline synthesis. However, C58-induced nopaline synthesis was greatly increased by mixed infection with B6S3. This effect (called “helper-effect”) was shown to be due to the activity of a 5 kb fragment from the virulence region of the B6S3 Ti plasmid, since incorporation of this fragment into the C58 plasmid enabled C58 to induce high levels of nopaline synthesis in the absence of a helper strain. The 5kb region contains the vir F locus, as defined earlier (Hooykaas et al. 1984b). A possible correlation between the helper function and vir F is discussed. Our results show that large differences in virulence on particular host plants exist between natural Agrobacterium strains and can be overcome by mixed infections.

Nucleotide sequence, evolutionary origin and biological role of a rearranged cytokinin gene isolated from a wide host range biotype III Agrobacterium strain

SummaryA DNA fragment with homology to the cytokinin (ipt) gene from biotype I Agrobacterium tumefaciens strain Ach5 was cloned from the Ti plasmid of the wide host range biotype III Agrobacterium strain Tm-4 and sequenced. The fragment contains an intact ipt coding sequence. However, the 3′ non-coding region of this ipt gene is rearranged due to a 0.9 kb deletion fusing it to the 3′ coding region of the neighbouring gene 6a, most of which was found to be deleted. The Tm-4 ipt gene is strongly related to the partially deleted ipt gene of the limited host range biotype III strain Ag162. To test its biological activity, the Tm-4 ipt gene was inserted into a specially constructed, disarmed Ti vector lacking tzs and tested on tobacco, where the rearranged ipt gene induced shoot formation. The cloned Tm-4 ipt gene was mutated with Tn5 and the intact gene on the wild-type Tm-4 Ti plasmid was replaced by the mutated gene. The resulting strain was avirulent on tobacco but normally virulent on the natural host of the wild-type strain Tm-4, grapevine. As the biotype 1 6b gene diminishes the effect of a corresponding ipt gene, a larger Tm-4 fragment carrying both the ipt gene and an adjacent 6b-like gene was also tested on tobacco and compared with the Tm-4 ipt fragment alone and with an ipt and 6b/ipt fragment derived from Ach5. The Tm-4 6b gene diminishes the effect of the Tm-4 ipt gene, showing the Tm-4 6b gene to be active as well. The Tm-4 6b/ipt combination is less effective than the Ach5 combination. These results provide further insight into the molecular basis of the host range differences between limited host range and wide host range biotype III Agrobacterium strains and show that the WHR cytokinin gene, although active, does not significantly contribute to tumour formation on the natural host of the WHR biotype III strains, grapevine.

Opines in crown gall tumours induced by biotype 3 isolates of Agrobacterium tumefaciens.

Pathogenic properties and opine markers of biotype 3 (grapevine) isolates of Agrobacterium tumefaciens (Smith and Townsend) Conn were compared with those of biotypes 1 and 2. By contrast with biotypes 1 and 2, grapevine isolates induced only small tumours on Kalanchoe daigremontiana Hamet and Perrier stems but produced large organogenic galls on decapitated shoot tips of this plant species. Grapevine agrobacteria were classified according to their opine catabolic properties and the type of opine they induced in Kalanchoe tubiflora Hamet and grapevine ( Vitis vinifera L.) tumours. Crown gall tissues obtained after infection with octopine-utilizing grapevine isolates contained octopine in very large amounts compared to biotype 1 tumours. A similar quantitative difference was not observed when nopaline production of tumours incited by biotypes 1 and 2 and by grapevine isolates was examined. Tumours formed by a third class of grapevine isolates synthesized a novel opine, for which we propose the trivial name vitopine. Octopine, nopaline, agropine and vitopine were also detected in healthy tissues of plants infected with the appropriate Agrobacterium isolates, indicating that these opines can selectively promote the colonization of the whole plant by agrobacteria. Biotype 3 octopine isolates induced octopine synthesis at the infection sites of a crown gall-resistant grapevine hybrid, showing that pathogenic agrobacteria can transform plants without tumour formation.

Physical map of the vitopine Ti plasmid pTiS4

Within the Agrobacterium vitis group the vitopine strains represent a special subclass. Vitopine bacteria carry Ti plasmids with little or no homology with the well-characterized T-DNAs of Agrobacterium tumefaciens or Agrobacterium rhizogenes. The 262-kb Ti plasmid of the vitopine strain S4 was cloned and mapped. Homology studies with the octopine Ti plasmid pTiAch5, the nopaline Ti plasmid pTiC58, and the agropine/mannopine Ri plasmid pRiHRI identified several regions of homology. The origin of replication was localized to within 2.5 kb.

Identification of an rsh Gene from a Novosphingobium sp. Necessary for Quorum-Sensing Signal Accumulation

The stringent response is a mechanism by which bacteria adapt to environmental stresses and nutritional deficiencies through the synthesis and hydrolysis of (p)ppGpp by RelA/SpoT enzymes. Alphaproteobacteria and plants contain a single Rsh enzyme (named for RelA/SpoT homolog) that is bifunctional. Here we report the identification of a new species of bacteria belonging to the genus Novosphingobium and characterization of an rsh mutation in this plant tumor-associated isolate. Isolate Rr 2-17, from a grapevine crown gall tumor, is a member of the Novosphingobium genus that produces the N-acyl-homoserine lactone (AHL) quorum-sensing (QS) signals. A Tn5 mutant, Hx 699, deficient in AHL production was found to have an insertion in an rsh gene. The Rsh protein showed significant percent sequence identity to Rsh proteins of alphaproteobacteria. The Novosphingobium sp. rsh gene (rsh(Nsp)) complemented the multiple amino acid requirements of the Escherichia coli relA spoT double mutant by restoring the growth on selection media. Besides QS signal production, the rsh mutation also affects soluble polysaccharide production and cell aggregation. Genetic complementation of the Hx 699 mutant with the rsh(Nsp) gene restored these phenotypes. This is the first discovery of a functional rsh gene in a member of the Novosphingobium genus.

Tartrate Utilization Genes Promote Growth of Agrobacterium spp. on Grapevine

Crown gall on grapevine is mainly caused by Agrobacterium vitis, which metabolizes tartrate. Competition experiments between a tartrate-utilizing strain and its non-utilizing derivative showed that tartrate utilization confers a selective advantage on grapevine.

Further evidence that the vitopine-type pTi's of Agrobacterium vitis Represent a novel group of Ti plasmids

To study the incompatibility properties of the vitopine Ti plasmids of Agrobacterium vitis, pPM739 containing the cloned ori region of pTiS4 was introduced by triparental mating into seven Agrobacterium tumefaciens strains carrying incRh-1, incRh-2, and incAg-1 plasmids and into eight A. vitis strains. All strains containing pPM739 retained their original plasmids and virulence or the ability to grow on tartrate, except for the three vitopine strains S4, Sz1, and NW11. Furthermore, pTiS4 was stably maintained in S4 cells following introduction of the ori/inc clones of the incRh-1, incRh-2, and incRh-3 plasmids. These results show that vitopine Ti plasmids represent a novel incompatibility group for which we propose the name incRh-4.

Types of resistance of grapevine varieties to isolates of Agrobacterium tumefaciens biotype 3

Eight grapevine varieties representing four susceptibility patterns were infected with octopine-,nopaline- and vitopine-inducing isolates of Agrobacterium tumefaciens (Smith and Townsend) Conn biotype 3. These varieties showed three different types of resistance. (I) In the first category the wound calluses contained the appropriate opine following inoculation. (II) Resistant hybrids of the 2819 group also formed wound calluses after infection with nopaline-inducing isolates but nopaline was not detectable in these tissues. (III) Infections with vitopine-inducing isolates resulted in tissue necrosis at the wound sites of certain varieties. Co-inoculations with vitopineand octopine- or nopaline-inducing isolates did not result in tumour formation on varieties Kunleany and A211 which are susceptible to octopine- and/or nopaline-inducing isolates of A. tumefaciens.