Annik Petit

Auxin Production Is a Common Feature of Most Pathovars of Pseudomonas syringae

We investigated indole-3-acetic acid (IAA) production by 57 pathovars of Pseudomonas syringae and related species. Most of those analyzed produced IAA, especially in the presence of tryptophan. Eight strains produced high IAA concentrations in the absence of Trp. The iaaM and iaaH genes of P. savastanoi pv. savastanoi were detected in a limited number of strains only, including the eight above-mentioned strains. Thus, IAA synthesis in most assayed strains of P. syringae and related species does not involve genes highly similar to iaaM and iaaH. In contrast, the iaaL gene encoding an IAA-lysine synthase was detected in most pathovars, and was often found on plasmids.

Isolation of Agrobacterium Ti-plasmid regulatory mutants

SummaryCrown gall tumors incited by Agrobacterium tumefaciens synthesize basic amino acid derivatives called opines. Opine production in tumours and opine catabolism by A. tumefaciens are coded by Ti-plasmids which confer oncogenicity on this bacterium. Catabolism of opines is inducible, and a method for isolation of regulatory mutants is described. From octopine-type bacteria, by plating on non-inducing substrates (noroctopine, noroctopine acid, D-histopine) we have isolated regulatory mutants of three types: constitutive, partially constitutive, and fully inducible by the analogue. From nopaline-type bacteria, by plating on octopine (a non inducing substrate) we have isolated analogous regulatory mutants.Synthetic opines, in which the amino acid moiety has been replaced by toxic arginine analogues, are toxic for these regulatory mutants. We isolated mutants resistant to such synthetic opines, and found that some had lost the capacity to utilize octopine. A survey of a large number of such mutants revealed that all of them still incited octopine synthetizing tumors.Mutants constitutive for octopine catabolism are in some instances also constitutive for Ti-plasmid transfer. A simple method for screening regulatory mutants for constitutive Ti-plasmid transfer is described.

Opines and Opine-Like Molecules Involved in Plant-Rhizobiaceae Interactions

The first reports on opines date back to the midfifties, when Morel (1956) and Lioret (1956) independently presented their results on, respectively, the metabolism of arginine and the identification of unusual amino acids in Agrobacterium-induced crown gall tumors, at a meeting of the French Society for Plant Physiology. These tumor compounds were later purified and identified (Figure 1) as lysopine (Biemann et al., 1960), octopine (Menage and Morel, 1964), octopinic acid (Menage and Morel, 1965), nopaline (Goldmann et al., 1969) and collectively termed opines (reviews: Tempe and Schell, 1977; Tempe and Goldmann, 1982). The significance of the metabolic perturbation undergone by the crown gall cells remained unclear for several years mostly because the specificity of opines as markers of these tissues was long debated (reviewed by Tempe and Goldmann, 1982). Three observations, however, were milestones in the understanding of the role of opines in the interaction: 1) Agrobacterium can degrade opines (Lejeune and Jubier, 1968); 2) the nature of opines synthesized in a tumor depends on the inciting Agrobacterium tumefaciens strains, not on the plant as it had been initially proposed (Goldmann et al., 1968; Petit et al., 1970); and 3) the correlation between opine degradation by agrobacteria and opine synthesis in plant cells is strict: that is, a given A. tumefaciens strain can only degrade the opines synthesized by the tumors it induced (Petit et al., 1970; reviewed by Tempe and Petit, 1983). Interestingly, these features were immediately interpreted as an indication of a possible gene transfer by Petit and Tourneur (1972) — a hypothesis that was first suggested by Braun (1947) and Klein (1954) (reviewed in Braun, 1982 and in Tempe and Petit, 1983) — whereas it was understood only years later that the production of opines by the crown gall cells provided the pathogen with a selective growth advantage (Schell et al., 1979; Tempe et al., 1979). This understanding resulted also from the discovery of the Ti plasmids (Zaenen et al., 1974; Van Larebeke et al., 1974; Watson et al., 1975), the elucidation of the tumorigenesis mechanism (Chilton et al., 1977; De Beuckeleer et al., 1978; Thomashow et al., 1980; Willmitzer et al., 1980, see also other chapters in this book), the localization of the genes involved in opine synthesis and degradation on the Ti plasmids (Bomhoff et al., 1976; Montoya et al., 1977) and the demonstration of the opine-induced, conjugal activity of these plasmids (Kerr et al., 1977; Petit et al., 1978a). All these data were combined and the fundamental role of opines in the Agrobacterium-plant interaction was rationalized by the originators of “the opine concept” (Tempe et al., 1979) and “the genetic colonization theory” (Schell et al., 1979) which define opines as follows. Opines are small-size molecules, the presence of which in the crown gall tumor is triggered by the pathogen to support its multiplication and to promote the dissemination of its virulence determinants (previous reviews on this topics: Dessaux et al., 1992, 1993; Gelvin, 1992).

Chemistry and biochemistry of opines, chemical mediators of parasitism

Abstract Opines consititute a class of substances that are characteristic of crown gall and hairy root, two proliferative plant diseases caused by Agrobacterium tumefaciens and A. rhizogenes. About 20 of these compounds have been described. Being produced by plant cells and used by the pathogen as growth substrates, they can be described as chemical mediators of parasitism. This plant-bacteria interaction is a natural instance of genetic manipulation since the genes encoding opine synthesis in plant cells are transferred from a bacterial plasmid into the plant genome. The current knowledge on opines is presented, as well as some biological aspects of this unique system.

Engineered Rhizosphere: the Trophic Bias Generated by Opine-Producing Plants Is Independent of the Opine Type, the Soil Origin, and the Plant Species

ABSTRACT In a previous study, we demonstrated that transgenic Lotus plants producing opines (which are small amino acid and sugar conjugates) specifically favor growth of opine-degrading rhizobacteria. The opine-induced bias was repeated and demonstrated with another soil type and another plant species (Solanum nigrum). This phenomenon is therefore independent of both soil type and plant species.

Novel Ti plasmids in Agrobacterium strains isolated from fig tree and chrysanthemum tumors and their opinelike molecules

Galls naturally induced on Fig and chrysanthemum plants by strains of Agrobacterium contained, in addition to other well-characterized opines such as nopaline, three tumor-specific opinelike molecules. These molecules were identified as deoxy-fructosyl-glutamine (dfg), deoxy-fructosyl-5-oxo-proline (dfop), and chrysopine (Chilton et al., unpublished). Strains isolated from Fig tree and chrysanthemum tumors harbored different and unrelated Ti plasmids as judged by hybridization with various vir and T-DNA probes. They also exhibited different opine-catabolic properties. The strains isolated from chrysanthemum plants (Chry strains) and Fig trees degraded chrysopine, but only the Chry strains used dfg and dfop. Remarkably, other strains of Agrobacterium catabolized these two molecules: dfg was degraded by most pathogenic and nonpathogenic Agrobacterium strains, and dfop by all Agrobacterium strains degrading the opine agropinic acid. These results have strong ecological and evolutionary inferences which fit previous speculation on the origin of opine-related functions.

Seasonal Fluctuations and Long-Term Persistence of Pathogenic Populations of Agrobacterium spp. in Soils

ABSTRACT Short- and long-term persistence of pathogenic (i.e., tumor forming) agrobacteria in soil was investigated in six nursery plots with a history of high crown gall incidence. No pathogenic Agrobacterium strains were isolated in soil samples taken in fall and winter in any plots, but such strains were isolated from both bulk soils and weed rhizospheres (over 0.5 × 105 pathogenic CFU/g of bulk soil or rhizosphere) in three out of six plots in spring and summer. PCR amplifications of a vir sequence from DNA extracted from soil confirmed the presence of Ti plasmids in summer and their absence in fall and winter. The results indicate that strains that harbor a Ti plasmid had an unforeseen positive fitness versus Ti plasmid-free strains in soil and rhizosphere in spring and summer in spite of the apparent absence of tumor, and hence of opines. The gain of fitness occurred during a bloom of all cultivable agrobacteria observed only in conducive soils. An evolution of the pathogenic population was recorded during a 4-year period in one particularly conducive soil. In 1990, the pathogenic population in this soil consisted of only biovar 1 strains harboring both octopine- and nopaline-type Ti plasmids. In 1994, it consisted of only nopaline-type Ti plasmids equally distributed among biovar 1 and 2 strains. These results suggest that nopaline-type Ti plasmids conferred a better survival ability than octopine-type Ti plasmids to biovar 2 agrobacteria under the present field conditions.

Opine Utilization by Agrobacterium

Publisher Summary This chapter explains the opine utilization by Agrobacterium. Utilization of opines by bacterial strains has been assayed in different ways: (1) by measuring the uptake of substrates in the bacterial cells, (2) by following the fate of the substrates in the supernatant of incubation mixtures or culture media, and (3) by assessing the ability of a strain to grow on media containing the opine as the sole carbon and/or nitrogen source. The pathway of octopine and lysopine degradation has been characterized as active transport into the bacterial cell, and cleavage of the molecule to the parent amino acid and α-keto acid. Opine oxidase is the enzyme responsible for the cleavage of opine. The degradation of the other opines of the octopine family as well as that of nopaline and nopalinic acid probably follows the same pathway, as mutants unable to utilize octopine fail to degrade lysopine, octopinic acid, and histopine.

The Cryptic Plasmid of Agrobacterium tumefaciens Cointegrates with the Ti Plasmid and Cooperates for Opine Degradation

We crossed the Agrobacterium tumefaciens chrysanthemum strain ANT4, which harbors four plasmids, with the plasmid-free recipient C58.00RS. Transconjugants degrading the Amadori-opines chrysopine and deoxy-fructo-syl-oxo-proline (dfop) harbored the Ti plasmid of ANT4, termed pAtANT4b. Upon transfer to the recipient strain C58.00RS, pAtANT4b (pTiANT4) and pANT4a (the largest of the four plasmids of ANT4) could cointegrate. The cointegration of the two plasmids occurs at various places of the pTiANT4, a feature that may affect several functions of the Ti plasmid (e.g., opine degradation). Transcon-jugants utilizing the opine deoxy-fructosyl-glutamine (dfg) always harbored the large pAtANT4a. Other Agrobacterium strains, including nonpathogenic strains such as C58C1, naturally degraded dfg. Remarkably, strain C58C1 carries a large cryptic plasmid termed pAtC58 that also encodes dfg degradation. A screening of physiological traits additionally revealed that this plasmid allows utilization of octopine as sole n...

The chrysopine family of amadori-type crown gall opines

Abstract Crown gall tumours induced by four groups of Agrobacterium tumefaciens isolates, derived from galls from four different locations in North and South America and Europe, were found to contain mannityl opine-related metabolites derived from condensation of glucose with glutamine followed by Amadori rearrangement. The opines were characterized spectroscopically as N- (1′-deoxy- d -fructos-1′-yl)-5-oxo- l -proline , N α - (1′-deoxy- d -fructos-1′-yl)- l -glutamine and its spiropyranosyl lactone, chrysopine. Tumours induced by A. tumefaciens strain 2788 and by Ficus strains also contained nopaline, while those induced by A. tumefaciens K224, K289 and Chry9 contained l , and an unidentified opine, pseudo-nopaline.