LEAFY GALL FORMATION BY RHODOCOCCUS FASCIANS Koen ...

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Annu. Rev. Phytopathol. 2001. 39:27–52 c 2001 by Annual Reviews. All rights reserved Copyright °

LEAFY GALL FORMATION BY RHODOCOCCUS FASCIANS Koen Goethals, Danny Vereecke, Mondher Jaziri1, Marc Van Montagu, and Marcelle Holsters Vakgroep Moleculaire Genetica & Departement Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium 1 Laboratoire de Biotechnologie Végétalé, Université Libre de Bruxelles, Chaussée de Wavre 1850, B-1160 Brussels, Belgium; e-mail: [email protected], [email protected], [email protected], [email protected], [email protected]

Key Words colonization, cytokinin, endophyte, fasciation, meristem ■ Abstract Rhodococcus fascians infects a wide range of plants, initiating the formation of leafy galls that consist of centers of shoot amplification and shoot growth inhibition. R. fascians is an epiphyte but it also can establish endophytic populations. Bacterial signals involved in symptom development initiate de novo cell division and shoot meristem formation in differentiated tissues. The R. fascians signals exert activities that are distinct from mere cytokinin effects, and the evidence points to a process that adopted cytokinin biosynthetic enzymes to form derivatives with unique activity. Genes implicated in leafy gall formation are located on a linear plasmid and are subject to a highly controlling, complex regulatory network, integrating autoregulatory compounds and environmental signals. Leafy galls are considered as centers with specific metabolic features, a niche where populations of R. fascians experience a selective advantage. Such “metabolic habitat modification” might be universal for gall-inducing bacteria.

CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THE CAUSAL AGENT OF LEAFY GALL FORMATION . . . . . . . . . . . . . . . . . . . . SYMPTOM DEVELOPMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . COLONIZATION OF PLANTS BY R. FASCIANS . . . . . . . . . . . . . . . . . . . . . . . . . . . THE ROLE OF PHYTOHORMONES IN LEAFY GALL FORMATION . . . . . . . . . Cytokinins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Auxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Plant Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MOLECULAR GENETICS OF R. FASCIANS VIRULENCE GENES . . . . . . . . . . . . Sequence of the fas Locus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0066-4286/01/0901-0027$14.00

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GOETHALS ET AL Regulation of fas Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Function of the fas Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The hyp and the att Loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . METABOLIC COLONIZATION: THE FATE OF A LEAFY GALL . . . . . . . . . . . . .

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INTRODUCTION In 1927, Brown (16) described fasciations on the crown of sweet pea (Lathyrus odoratus) as bunches of short, fleshy stems carrying misshaped and aborted leaves and she proposed that this was a form of crown gall, induced by a weak or specialized form of Agrobacterium tumefaciens. Currently, the bacterium responsible for these fasciations is known as Rhodococcus fascians, a Gram-positive bacterium that infects diverse plant families and genera and provokes a variety of disease symptoms, the most dramatic being the formation of leafy galls as centers of abundant shoot meristem amplification. Starting from its originally alleged connection with crown galls, leafy gall formation was considered as another plant disease caused by bacterially produced phytohormones. As parallelism with crown gall formation had been ruled out, research was oriented toward the role of plant hormones in the galling syndrome. Molecular analysis of the pathogenicity determinants of R. fascians indicates that leafy gall formation is based on some elements common to Gram-negative galling bacteria and on more specific elements that may relate to the highly particular nature of leafy galls. In this sense, R. fascians should not be regarded as a bacterium that acquired its tumorigenic properties from well-described, Gram-negative bacteria. Molecular studies of different gall-inducing bacteria show no recurring strategies for plant gall formation. It would be erroneous to assume that all galls are alike, an idea refuted by the existing literature. Although various gall-forming bacteria employ some common elements during infection and symptom development, different bacteria use distinctive systems to produce galls. R. fascians and leafy galls are no exception to this rule.

THE CAUSAL AGENT OF LEAFY GALL FORMATION Ten years after Brown’s proposition, research by Tilford (139) on the causal agent of the sweet pea fasciations led to the isolation of the pathogen: a Gram-positive bacterium that was named Phytomonas fascians. Although there was an agreement on the distinct nature of the fasciating bacterium at that time, just three years later Lacey (83) renamed the bacterium Bacterium fascians. The unclear taxonomic status was resolved by classifying the pathogen as an Actinomycete, coryneform bacterium, and the species name Corynebacterium fascians was proposed (38), a name that was retained until 1984.

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This proposal was not without controversy, because from the early 1940s until the early 1980s discussion on the exact taxonomic position within the Actinomycetes tossed the bacterium between the Nocardiae (84), Corynebacteriae (62, 159), and the Rhodococci (58, 59). The controversy ended when Corynebacterium fascians was reclassified as Rhodococcus fascians, based on chemical, genetic, and phenotypic data (57). Based on cell wall composition, DNA base composition (61–67% GC), and the presence of tuberculostearic and mycolic acids, R. fascians belongs to the Actinomycetes and is classified within the mycolic acid-containing genera (45). Analysis of the 5S and 16S rRNA sequences (91, 121) and a study of the rRNA operon structure (116) confirmed this taxonomic position. Rhodococcus, together with the genera Gordona, Tsukamurella, Mycobacterium, and Nocardia, is now classified into the family Mycobacteriaceae (23). R. fascians is aerobic and pleiomorphic and can form branched hyphae that soon break up in rods and cocci (nocardioform) (87). It is nonmotile, nonspore-forming and produces moist cream to orange colonies that can be smooth or rough, a feature that does not affect pathogenicity (14, 43, 44, 57, 139). Within the genus Rhodococcus, R. fascians is the only known phytopathogen, although animal pathogens and symbionts have been described (11). For an overview of the genus Rhodococcus, we refer to Finnerty (47) and Bell et al (11). After the bacterium had been isolated from sweet pea, in which it severely reduced flower setting (139), it was later found in “cauliflower” strawberry plants, which carry a large number of dwarfed buds, and in carnation, chrysanthemum, and asparagus (81, 82). Since then, the effects of R. fascians have been noticed primarily in ornamental plants because infection usually results in decreased flower production (98, 110, 143–145) or deformed flowers (160). The disease is spread by infected plant material and infested seeds or soils (7, 83, 110, 139). Dicotyledonous as well as monocotyledonous plants respond to R. fascians infection and the host range encompasses 39 plant families and 86 genera (134, 150; our own observations). In its broad host range, the bacterium resembles Agrobacterium and differs from other gall-forming bacteria, such as Pseudomonas savastanoi and Erwinia herbicola, which have a very restricted host range (19, 51, 52).

SYMPTOM DEVELOPMENT Depending on different parameters, the morphological changes induced upon R. fascians infection range from leaf deformation, witches’ broom formation, and fasciation to the initiation of leafy galls. In addition to the plant species or cultivar, the age of the plant (46), the bacterial strain (40), and the bacterial growth conditions (46) affect symptom development. The inoculation method can markedly influence the pathogenesis and, albeit no absolute prerequisite, wounding will enhance symptoms (7, 83, 125, 150).

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Leaf deformation in tobacco is accompanied by a wrinkling of the lamina and a swelling of the petioles and veins, resulting from enlarged parenchyma cells and secondary growth of the vascular tissues. Patches of “green islands” occur on the lamina, possibly representing sites with increased chloroplast content (150). A witches’ broom is characterized by a bunch of fleshy stems with misshapen and aborted leaves that develop on the crown of the host plant. In peas, this is accompanied by initial stimulation of epicotyl growth (126), but soon growth of the terminal bud is blocked, and the plants become dwarfed and produce fewer blossoms (7, 16, 82, 139). This inhibition of the main shoot is not a consequence of trophic competition with the lateral shoots but is endogenous and directly caused by the bacteria (126, 127). The hypertrophied shoots of witches’ brooms contain regions with actively dividing, meristematic cells, enlarged cortical parenchyma cells, and multiple vascular bundles located at the center (81, 125). R. fascians– induced fasciations are formed when several hypertrophied shoots coalesce, carrying malformed small leaves with thickened petioles and veins (125). Leafy galls represent the most severe symptom and originate from a local amplification of multiple buds that are inhibited for outgrowth. They form exclusively at the site of bacterial inoculation and no secondary leafy gall formation at noninfected sites has been described. The presence of bacteria is required for the multiplication of the shoots and also for gall persistence (126, 150). The inhibition of shoot outgrowth has been interpreted as an extreme form of apical dominance whereby the shoot primordia mutually exclude their further development provoking the formation of the densely packed gall. By inhibiting the bacterial activity in isolated leafy galls, shoot outgrowth is activated. Eventually, these shoots develop roots and grow into normal, fertile plants (55, 125, 126, 150). Thus, in contrast to crown galls that rely on the transgenic production of phytohormones, leafy galls are nonautonomous structures, and gall maintenance depends on the presence activity of R. fascians. At first, buds and meristematic tissue seemed to be the only sites of the plant affected by R. fascians (7, 83, 88, 125) until leafy galls were found to also originate from leaf margins and veins (150). Local infection of tobacco shoot axils revealed that R. fascians induces cell division and de novo meristem formation in subepidermal cells. The reactivation of cortical cell division was correlated with induction of the CYCB1;1 promoter, a mitotic marker, and with increased transcription of the CYCD3;2 gene, involved in the regulation of the G1-to-S transition of the cell cycle (92). These data show that R. fascians can reprogram cells to re-enter the cell cycle in a controlled way because the outcome is the formation of shoot meristems. The results demonstrate that leafy galls consist of shoot primordia that originate both from amplification of existing meristems and from the formation of adventitious meristems from differentiated tissues. Although the root system is generally not affected by R. fascians, severe infection of plants can result in a thickened main root and the absence of secondary

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roots (46, 81, 125, 139). Inoculation of germinating seedlings leads to a complete inhibition of root growth, a thickening of the hypocotyl, and seedling growth inhibition (150). By following the expression of cell cycle genes that are correlated with active cell division and/or competence to divide, a significant decrease of the meristematic activity of Arabidopsis thaliana roots upon R. fascians infection is observed, accompanied by a diminished root number and length. Interestingly, such infections also stimulate lateral root initiation at the pericycle; however, growth of these root initials is arrested (150). Analysis of the phenolic and amino acid content shows that leafy galls are distinct from surrounding plant tissues (42, 149, 152). In tobacco leafy galls, the relative concentrations of phenolics differ from those in noninfected plant parts, but no phenolics that are characteristic for a defense response have been detected (152). Leafy galls are clearly distinct in origin and formation from the knots, blisters, and wart-like formations induced by P. savastanoi (51, 52) and the galls produced by E. herbicola that consist of parenchymatous cells carrying necrotic centers (25, 49). The leafy galls are active centers of ongoing shoot meristem production and growth repression. They do not become necrotic, but remain green long after the mother plant is senescent (150). This characteristic has been interpreted as a result of delayed senescence.

COLONIZATION OF PLANTS BY R. FASCIANS Failure to reproducibly demonstrate any organism in the interior of the infected tissue (85) suggested that most bacteria are present on the exterior of the fasciations (7, 81, 125). Only occasionally were the bacteria shown to invade epidermal cells and one or two underlying cell layers, causing death of the invaded cells and intercellular growth up to six cell layers deep (81). Until recently, no detailed studies on host infection by R. fascians were available. The behavior of R. fascians on tobacco and Arabidopsis has been re-evaluated (28, 29). The bacteria were found to colonize the leaf surface and, to a much lesser extent, the stem and the crown. They spread by growth with occasional hyphal formation and are embedded in a slime layer. The bacteria are often located in cavities and at epidermal cell wall junctions, which might represent a way to avoid environmental stress (10). Plant parts that are heavily colonized by R. fascians do not necessarily have symptoms, indicating that the production and/or activity of bacterial signals implicated in fasciation is restricted. Although stomata can be colonized, there is no evidence that they represent a preferred entryway for the bacteria. Instead, massive ingressions in leaf and stem tissues are observed, accompanied by a collapse of plant cells. These penetration sites do not show necrosis, although some cells in the vicinity have an increased autofluorescence.

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By fluorescence in situ hybridization (FISH) (4, 5, 28, 29), bacteria are traced in the inner tissues of leaves and leafy galls in which they mainly occupy the intercellular spaces. The bacteria are found throughout the tissues but their occurrence decreases in the deeper cell layers, and, in contrast to other phytopathogens such as Ralstonia solanacearum, A. tumefaciens, P. savastanoi, and E. herbicola (32, 49, 97, 146, 158), R. fascians has never been detected in vascular tissues. Symptom development precedes the occurrence of endophytic forms. Penetration and colonization of plant tissues represent a phase in the infection cycle that follows the onset of leafy gall formation. On several occasions, we have observed bacteria surrounded by the cell wall of a healthy living plant cell. These data suggest intracellular forms of the bacterium, the existence of which has been corroborated in infected BY-2 tobacco cell cultures. The intracellular nature needs further confirmation by electron microscopy (28, 29). Lacey (85) showed that inoculation of gelatin medium with gall filtrates led initially to the isolation of amorphous particles that could convert to nonpathogenic R. fascians rods. These results were interpreted as evidence that bacterial L-forms, characterized by the absence of a cell wall, are a form of R. fascians inhabiting the inner tissues of the infected plant (85, 88). Immunolocalization using an R. fascians–derived polyclonal antibody confirmed the epiphytic location of the bacteria, but could not detect the endophytic forms described by FISH (28, 29). Perhaps the endophytic bacteria have no or an altered cell wall structure. The different forms of R. fascians found on and in plants might be dedicated to different aspects of the infection cycle. Epiphytic bacteria initiate shoot meristem formation, given the right conditions and the presence of susceptible target tissues. The role of the endophytic forms is still enigmatic. At least two types of endophytic populations are detected, intercellular bacteria and bacteria that are surrounded by plant cell walls. They are implicated in the maintenance of a leafy gall and/or might represent populations that utilize the newly formed tissues as a specific niche for survival. This hypothesis relates to a fundamental question concerning leafy gall formation and gall or hyperplasia formation per se: What selective advantage does a pathogen experience by inducing the amplification of tissues and organs?

THE ROLE OF PHYTOHORMONES IN LEAFY GALL FORMATION The symptoms following infection by R. fascians clearly result from a perturbation in the hormonal balance of the host plant, but the exact nature of the responsible signals has not been elucidated. Whatever the compounds involved, they do not diffuse a great distance (125, 160); for instance, grafting of normal plants with symptomatic scions does not result in symptom transmission (160). Furthermore, the compounds need to be continuously provided by the bacteria for a leafy gall to persist (81, 82, 125, 126, 152).

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Cytokinins Several disease symptoms, such as wrinkling of leaves, formation of green islands, shoot proliferation, delayed senescence, and root inhibition, are reminiscent of cytokinin effects. Some symptoms can be mimicked by the addition of cytokinins (77, 111), and exogenous cytokinins have a similar, albeit less pronounced, effect as R. fascians on shoot meristems and cell cycle gene expression (92, 152). Several cytokinins have been isolated from the culture supernatant of the bacterium (6, 41, 66, 77, 123). A correlation between the virulence of strains and cytokinin secretion has been suggested (103), but could not be confirmed (30, 41, 129). The primary source of cytokinins released by R. fascians has been proposed to be the tRNA (95, 103), but the increased quantity of nonhydroxylated cytokinins in virulent strains when compared to nonpathogenic strains was interpreted as involving an alternative biosynthetic pathway (103). Although cytokinin residues in tRNA may serve as precursors of free cytokinin, tRNA-mediated cytokinin release is probably not biologically significant in any organism (20, 100). Assigning a role to cytokinins in pathogenicity is further complicated by the secreted amounts. The concentration of trans-zeatin and isopentenyl adenine found in cultures of A. tumefaciens and P. savastanoi is 100-fold higher than that of virulent R. fascians cultures (2, 103, 117). Furthermore, these Gram-negative phytopathogens do not induce shoots at the infection sites. Therefore, the amounts of secreted cytokinins are considered too low to account for the shoot proliferation provoked by R. fascians. However, once in the plant, the bacteria can continuously apply cytokinins directly to the target cells or cytokinin production by the pathogen can greatly increase in close proximity to its host (30, 41). Bacterial cytokinin production has been suggested to be of secondary importance. Instead, cytokinin production by the plant could be stimulated upon infection (138). In these cases, elevated levels of cytokinins could be expected in diseased tissues when compared to noninfected plants or to plants infected with a nonvirulent strain. Although Balázs & Sziráki (8) showed that the cytokinin activity in R. fascians–infected Pelargonium zonale plants was considerably higher than that of healthy plants, similar observations could not be made by others (41, 92, 152). The nature of the R. fascians signal molecules implicated in leafy gall formation is unknown, but evidence points to the involvement of a cytokinin-like compound. Crespi et al (30) showed the presence of an isopentenyl transferase (ipt) gene in R. fascians strain D188 that is absolutely required for virulence. Isopentenyl transferases catalyze the first dedicated step of cytokinin biosynthesis, the formation of isopentenyl adenine from 50 AMP and dimethylallyl pyrophosphate, and have been isolated from plants and other organisms (20). First, ipt genes were described in Agrobacterium and P. savastanoi and later in E. herbicola, in which they are involved in virulence (65, 89, 90, 118). Although ipt genes contribute to the type or degree of symptom development in these bacteria, they are not necessary for virulence. Mutants in the T-DNA-encoded ipt

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gene of Agrobacterium sp. still induce crown galls, although the tumors have an altered morphology with the formation of multiple roots (1, 9, 17). In P. savastanoi, ipt mutants produce galls that are smaller and have a changed anatomy (71), and in E. herbicola, mutation of an ipt gene essential for cytokinin production caused a reduction in gall size (89, 90). These observations show that cytokinin production by these bacteria contributes to, but is not indispensable for, gall formation. In P. savastanoi, auxin is the major pathogenicity factor, stimulating cambium cell division and the proliferation of disorganized tissue (71). Furthermore, the relation between different P. savastanoi strains and particular plant hosts of the Oleaceae family can in part be correlated with different levels of auxins produced by the pathogen (54, 70, 124, 132). In E. herbicola, auxins are also involved in pathogenicity but, as for cytokinins, they are not indispensable for gall formation (24, 89, 90). Instead, an hrp gene cluster has been identified in this bacterium that is essential for gall formation (99, 106, 107, 142). The hrp genes have been described in biotrophic, Gramnegative bacteria in which they are implicated in the formation of a type III delivery system that injects protein virulence factors directly into the host cells (27, 50). Perhaps both auxin and cytokinin produced by E. herbicola boost a gall induction process that is initiated by one or more proteinaceous factors delivered to the host cells. These data are in contrast with the absolute requirement of the R. fascians D188 ipt gene in leafy gall formation. The significance of this gene for pathogenicity has been confirmed by a strict correlation with virulence in different R. fascians isolates (134). For the R. fascians D188 ipt gene, the enzymatic activity of its gene product has been demonstrated, but at relatively low efficiency (30). The low overall sequence identity of the enzyme with isopentenyl transferases of P. savastanoi, E. herbicola, and Agrobacterium species (ranging from 20% to 26%) (30, 135) may reflect the acquisition of different enzymatic specificities. Cytokinin activity as detected in the supernatant of R. fascians D188 cultures using a standard cytokinin bioassay could not be correlated with the expression of the ipt gene and an ipt mutant could not be complemented by the exogenous application of isopentenyl adenine (135). These data suggest that the function of the R. fascians ipt gene is distinct from the production of classical cytokinins.

Auxins An auxin-degrading capacity has been reported for R. fascians (75, 125) that can, in combination with the simultaneous secretion of cytokinins, rapidly increase the cytokinin/auxin ratio leading to symptom development (8). On the other hand, cell swelling, secondary differentiation of vascular tissue, and induction of lateral root initiation are typical auxin effects. In plants carrying a leafy gall, a significantly higher in the indole-3-acetic acid (IAA) level than in normal tissues has been detected (92, 152). Recent experiments show that R. fascians can produce IAA in culture (M Jaziri, unpublished results). In addition to a

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possible direct role of auxin in the initiation of leafy galls, a function in the epiphytic survival of the bacterium, as shown for E. herbicola, should be considered (15, 94).

Other Plant Hormones In peas, gibberellic acid was reported to counteract the effects of R. fascians infection (126), suggesting a direct involvement of gibberellins in fasciation. Interestingly, a combined treatment with cytokinins and imidazole-type fungicides that block the activity of P450 cytochromes (61, 119), results in symptoms on Aracea that are very similar to R. fascians–induced phenotypes (155). This effect can be counteracted by the addition of gibberellin, indicating that a combined activity of cytokinins and a decrease in gibberellin content phenocopy leafy gall induction (156). Nevertheless, gibberellin treatments of R. fascians–infected tobacco and Arabidopsis wild-type and gibberellic acid mutants do not significantly alter gall formation (135; our unpublished data), indicating a more complex mechanism than the mere alteration of endogenous gibberellin concentrations. An array of A. thaliana mutants that are defective in ethylene, cytokinin, auxin, gibberellin, and abscisic acid production and sensitivity have been infected with R. fascians D188 without significant differences in symptom production. Similarly, treatment of R. fascians–infected Arabidopsis with different hormones or hormone inhibitors does not affect the disease symptoms (our unpublished observations). These results suggest that endogenous hormone levels do not play a central role in leafy gall formation, but that R. fascians–produced signals act autonomously from the existing hormone landscape in the host during initiation and development of leafy galls.

MOLECULAR GENETICS OF R. FASCIANS VIRULENCE GENES Plasmids often are associated with virulence and phytohormone biosynthesis in hyperplasia-inducing bacteria. The involvement of plasmids in gall formation by R. fascians was proposed (60, 103) but was later contested due to the observation that all R. fascians strains carry circular plasmids, irrespective of their pathogenicity (86). Before the 1990s, reports on the genetics of the interaction were restricted to a publication on the effects of pigment and auxotrophic mutants on pathogenicity (72). Studies on the underlying mechanisms of the interaction were mainly hampered by the absence of molecular tools to study the bacterium. In the early 1990s, plasmid isolation and conjugation methods (34), a transformation protocol and shuttle vectors (35), and a random mutagenesis procedure (33) were developed. Reporter genes useful for the molecular study of R. fascians were characterized (36).

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Crespi et al (30) showed the presence of a large (180 kb) conjugative linear plasmid pFiD188 that is essential for pathogenesis in R. fascians strain D188. Although a linear plasmid-free strain can still penetrate plants, the efficiency of endophytic colonization is lower than that of the wild-type bacteria (28). In addition to pFiD188, R. fascians strain D188 also carries a circular plasmid that is involved in cadmium resistance but plays no role in pathogenesis (34). The occurrence of a linear virulence plasmid explains the earlier contesting results on a linkage between pathogenicity and plasmids, because linear plasmids behave differently during gel electrophoresis when compared to circular replicons. The presence of a linear plasmid as a major determinant of pathogenicity has been expanded to other R. fascians strains (134), and a strict correlation has been shown between the presence of linear plasmids and fasciation, with only one R. fascians strain carrying virulence genes on a circular replicon (134). Linear replicons have been described in Streptomyces, Rhodococcus, and Mycobacterium where they are linked with diverse processes (67, 113). The chromosome of Streptomyces strains is also linear (21). In Actinomycetes, linear plasmids carry termini that are covalently bound by proteins at the 50 end (67). The structure of the pFiD188 ends complies with the typical actinomycete linear plasmids (our unpublished results). Although the chromosome of R. fascians was thought to be linear (30), later data have proven the genome to be circular (115). Random mutagenesis of R. fascians D188 resulted in the isolation of different virulence mutants that were mapped on pFiD188 (30). Three types of mutants were described: a nonpathogenic fas mutant, an att mutant showing attenuated virulence, and a hyp mutant with a hypervirulent phenotype.

Sequence of the fas Locus The fas locus carries a large operon with six open reading frames (ORFs) including the ipt homologue (ORF4). Mutants in ORF1 and ORF4 are nonvirulent (30, 31, 55, 135). At least one ORF downstream of ipt (ORF5 or ORF6) is not required for full virulence on seedlings, but is involved in repression of shoot outgrowths in leafy galls. This observation suggests that the fas-encoded proteins are required for the production of a signal molecule that mediates both shoot initiation and growth inhibition and that ORF5 and/or ORF6 are important for the shoot inhibition process. ORF5 is homologous to cytokinin oxidase genes from maize and A. thaliana (68, 101, 135). Cytokinin oxidases irreversibly inactivate cytokinins by catalyzing the cleavage of the N6 side chain (157). The presence of a cytokinin oxidase gene downstream of ipt, involved in cytokinin biosynthesis, raises the question as to why a bacterium would spend energy to produce cytokinin that is degraded subsequently. Cytokinin oxidases act via the formation of an intermediate with an activated N6 atom and hydrolysis of this intermediate releases the N6 side chain. When another molecule reacts with the activated N6 atom, the reaction product would

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be an adenine carrying another side chain at the N6 position. Alternatively, the isopentenyl transferase-produced side chain would not be cleaved, but an adenine derivative carrying two side chains would be formed. In either case, the molecule may be more active than the initial isopentenyl transferase-produced cytokinin, allowing fasciation of both young and older plants. The ORF6 gene product shows similarity to glutathione S-transferases (GSTs) of the θ class with conservation of particular amino acids involved in glutathionebinding and catalytic activity (39, 134, 154). Whereas the functions of most bacterial GSTs remain to be established (154), eukaryotic GSTs are involved in the detoxification of exogenous substances and endogenous reactive products of cellular metabolism (26). The similarity of the ORF6 protein to such enzymes suggests its involvement in transfer of glutathione, or another peptide residue, to the N6 atom activated by the ORF5 gene product. Two ORFs that are located upstream of the ipt gene are homologous with P450 cytochromes (ORF1) and the ancillary ferredoxins (ORF2) found in biodegradation and biosynthesis pathways of Actinomycetes (31, 109, 128). No homologies with ferredoxin reductase, the third component of these oxido-reduction systems, could be detected. However, the ORF2 product carries a carboxyl-terminal domain, which resembles the α subunit of pyruvate dehydrogenase (18, 64, 134). Pyruvate dehydrogenases catalyze the oxidative decarboxylation of pyruvate, requiring thiamine pyrophosphate (TPP) as a cofactor. The proteins are composed of two α and two β subunits (12, 105) and, interestingly, ORF3 encodes a protein similar to the β subunit. Regions involved in TPP binding (102) are also found in ORF2 and ORF3. The fusion between a ferredoxin-like domain and a TPP-binding domain in the ORF2-encoded protein is reminiscent of the pyruvate:ferredoxin oxidoreductases that are responsible for the oxidation of pyruvate in several bacteria (80, 114). These enzymes can use the high reducing power of pyruvate for reactions requiring strong reductants (76). The presence of such an enzyme in the fas locus may be relevant to the action of the cytochrome P450 encoded by ORF1. In most bacterial cytochrome P450 systems, the reducing power is generated by the oxidation of NAD(P)H through the action of a ferredoxin reductase. The electrons are subsequently delivered to the cytochrome P450 by a ferredoxin (108). The fas-encoded cytochrome P450 system could be unique in its ability to use pyruvate or another α-keto acid as an electron donor. The highenergy electrons, generated from the oxidative decarboxylation of these molecules by the action of the ORF2- and ORF3-encoded proteins, could be delivered directly to cytochrome P450 via the ferredoxin-like domain of the ORF2 product (55, 135). Cytochromes P450 have been described in other plant-pathogenic microorganisms such as A. tumefaciens and the fungus Nectria haematococca, where they enhance virulence by detoxification of phytoalexins (74, 96). In R. fascians, nonpolar mutants in ORF1 are completely nonvirulent, and a role for the gene product in phytoalexin degradation is not very probable. Like the fas locus of R. fascians, the cytochrome P450 gene cluster from Bradyrhizobium japonicum also comprises

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a ferredoxin gene and a prenyltransferase (141). This operon is thought to encode a pathway for the synthesis of gibberellic acid in bacteroids, but no role in symbiotic properties has been assigned (140). The linked presence of P450 monooxygenase, an ancillary electron transfer system, and an isopentenyl transferase in the fas locus could indicate a functional relationship between these enzymes by which isopentenyl adenine—generated by the isopentenyl transferase—is oxidized to trans- or cis-zeatin. These latter cytokinins have been detected in R. fascians supernatants (2, 41, 77, 129), but have not been correlated with virulence. Considering the proposed role of the ORF5and ORF6-encoded proteins in the attachment of another side chain at the activated N6 position, hydroxylation of this moiety by cytochrome P450 is equally possible. However, a function in modifying the side chain added by the ORF6 product is not consistent with the observed phenotype of the ORF1 and ORF5 mutants. If the essential role of the P450 monooxygenase was to act on this side chain, the ORF5 mutant in which the completed fas product cannot be formed, should be nonvirulent. In contrast, the ORF5 mutant has been shown to be virulent (31). The role of cytochrome P450 remains to be established. Possibly it is involved in the hydroxylation at an unorthodox site of a cytokinin-like substrate with properties that differ from those of well-known cytokinins.

Regulation of fas Genes The expression of the fas genes is high in plants. Optimal expression in cultures requires a defined medium and pH 5 (136). Furthermore, gene expression in culture has been detected in the presence of gall extracts, but not of extracts of noninfected plants (30, 136). In other pathogens, virulence gene expression is often correlated with the pH conditions met in the host (93, 120, 133). The nature of the compound inducing fas gene expression is unknown, but inducing activity could already be detected 24 hours after infiltration of tobacco plants by R. fascians, long before symptoms are visible. Inducing activity has been detected in noninfected parts of the plant and the surrounding medium, suggesting the involvement of diffusible factors (149). There is now evidence that the factor is produced by the bacterium through the action of the att locus (our unpublished data). In a working model, att-produced compounds build up during plant colonization and act as autoregulatory molecules inducing the expression of both the att and fas genes. Initial induction of the att genes requires no plant-derived signal molecules; instead confinement of the bacteria under specific conditions during plant infection creates the settings necessary to build up a critical amount of attderived inducer, resulting—via a positive feedback loop—in inducer quantities that meet the demands for fas gene expression. Interestingly, histidine has been shown to induce fas gene expression in a defined medium (136, 153). Histidine levels remain unchanged in leafy galls and the levels necessary to induce gene expression are relatively high (2–5 mM). It can be hypothesized that such levels in the media somehow mimic the conditions in and

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on the plant that are compatible with an initial att gene induction. Alternatively, histidine could resemble the native inducer. The regulatory gene, fasR, which belongs to the AraC family of transcriptional regulators and is essential for both fas gene expression and leafy gall formation, is located upstream of the fas locus (136). Curiously, fas gene expression is regulated at the posttranscriptional level. A posttranscriptional regulator has been proposed to be encoded by the linear plasmid and its transcription to be regulated by the fasR product. The induction of the fas genes would then be mediated through an interaction of an inducer with the posttranscriptional regulatory protein or with the fasR product. The complex and tight regulation of the R. fascians fas gene expression probably reflects the importance of a controlled release of signal molecules. An uncontrolled production of signals might lead to unfavorable side effects on the plant host that could counteract a successful interaction or interfere with a stringent control of shoot primordium induction. In P. savastanoi, expression of the auxin and cytokinin biosynthetic genes is constitutive (48, 118). However, the production of an active IAA pool is controlled by the endogenous tryptophan concentration (79), feedback inhibition of the biosynthesis (69), and the formation of IAA-lysine conjugates (53, 112, 124). The interpretation of these results is that a controlled production of IAA is important for optimal colonization and symptom development in the host. For E. herbicola, expression of the IAA biosynthetic genes is relatively low in cultures and on the plant surface but is highly induced in plants (94). The cytokinin biosynthetic genes are constitutively expressed, although some regulation in function of the growth cycle of the bacterium has been observed (89, 90). The regulatory requirements of cytokinin production by P. savastanoi and E. herbicola seem less relevant for controlled pathogenicity, in contrast to the tightly controlled expression to which the fas locus is subjected. This suggests a distinctive role of the fas locus in the synthesis of an active signal that differs both in activity and structure from the classical cytokinins described in relation to hyperplasia formation.

Function of the fas Genes Culture supernatant extracts from R. fascians strain D188 display a very low cytokinin activity of approximately 5 ng/ml in kinetin equivalents (135). Other highly virulent strains of R. fascians also secrete low amounts of isopentenyl adenine (24 ng/ml in kinetin equivalents) (103) or zeatin (2 ng/ml) (2). The cytokinins detected in R. fascians culture media must be of chromosomal origin because they are also present in extracts derived from a plasmid-free strain (135). They may support cell division and/or render the plant cells more sensitive to the activity of the fas molecule. High amounts of trans-zeatin and isopentenyl adenine are found in cultures of several P. savastanoi strains (400 ng/ml for EW 1006) (117), E. herbicola

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(200–300 ng/ml) (89, 90), and A. tumefaciens C58 (500 ng/ml) (2). Nevertheless, infection of plants with these phytopathogens never leads to the formation of leafy galls, indicating that virulence of R. fascians is not due to the secretion of typical cytokinins alone. Partly purified supernatant or bacterial extracts of D188 cultures induced for fas expression do not contain additional cytokinin activity as measured using an Amaranthus caudatus betacyanin bioassay, implying that fas-derived molecules have only weak activity, if any, in this assay (135). Such extracts reduce the internode length and the leaf size when applied to plants, but formation of leafy galls has never been observed. Similarly, leaf and root development of seedlings are seriously reduced by the extracts. Such effects are not seen when extracts of noninduced or fas mutant cells are applied (135). BY-2 tobacco cell cultures synchronized for cell division were treated with R. fascians, preinduced for fas gene expression (135a). In this assay, cytokinins increase the proportion of cells in mitosis. In contrast, preinduced R. fascians cells partly delay the prophase of mitosis of BY-2 cells. This effect depends on an intact and induced fas locus and has been shown to be dominant over the effects exerted by cytokinins. Furthermore, partly purified extracts provoke the same effects, also relying on an intact and induced fas locus. Whatever the biological relevance of these observations might be, they suggest that fas-derived signals have a different action on the plant cell than do cytokinins. These results indicate that the fas locus is involved in the synthesis of signals that affect the growth and development of plants in a particular way. The fas genes have been proposed to be involved in the synthesis of a cytokinin analogue, employing the basic biochemistry for cytokinin biosynthesis but with particular substrates and modifications.

The hyp and the att Loci Upon infection of decapitated plants, mutants in the hyp locus cause the development of larger leafy galls (30). The hypervirulent phenotype suggests the involvement of the hyp locus in the reduction of symptoms by modifying the bacterial signals to a less active form or by repressing the expression of virulence genes. Sequence analysis of the hyp locus showed the presence of an ORF whose deduced amino acid sequence is homologous to the “D-E-A-D box” family of RNA helicases (135, 153). These proteins are important in the regulation of mRNA expression and translation initiation (131). The hyp locus could encode a regulatory protein involved in the posttranscriptional control of the fas gene expression. However, the evidence is still scanty because it currently relies on the sequence analysis only. The att locus is closely linked to the fas locus on pFiD188 (30). Infection of plants with att mutant bacteria results in a range of phenotypes. Most plants develop less compact and smaller leafy galls, and seedlings infected with the att mutant grow to an intermediate height. The att gene product(s) is unknown, but judging from the attenuated phenotype provoked by the mutant, several modes of

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action can be hypothesized (153; our unpublished data). The intermediary phenotype could be the result of impaired attachment or growth of the mutant, but this is contradicted by the mutant’s capacity to successfully colonize Arabidopsis thaliana plants (our unpublished data). Another function could be the production of one or more phytotoxins that weaken the plant’s surface barrier, facilitating infection by R. fascians. The fact that wounding, although not necessary for virulence of R. fascians, greatly enhances symptom development is in agreement with this hypothesis. Furthermore, wounding is necessary for symptom development by att mutant bacteria on detached tobacco leaves (our unpublished data). However, leafy gall and surrounding plant tissues never show signs of chlorosis or deterioration. If a toxin is involved, it does not cause symptoms typically associated with phytotoxins. The att product could directly interact with the fas product by enhancing the plant’s sensitivity to cytokinins or, alternatively, both products could act in concert and give rise to full symptom development. However, coinfection of plants with att and fas mutant bacteria does not result in complementation, but instead gives rise to typical att mutant symptoms (our unpublished data). Expression of the att genes is induced by a gall-derived factor and histidine (153), and in galls incited by att mutants, the production of such a factor is absent (our unpublished data). Therefore, the att locus may form an autoregulatory compound that also induces expression of the fas locus. The attenuated phenotype could be explained by decreased production of the fas product because of an inefficient expression due to lack of an att-encoded inducer. Such a function does not exclude the possibility that att serves other functions in gall formation. Recently, the expression of fas and att genes has been evaluated during infection of tobacco plants (28). While expression of the att genes gradually decreases during infection and only occurs in bacteria present at the plant surface, the fas genes are expressed both at the surface and the interior of the plant. Furthermore, fas expression remains high over a longer time period than att expression. These data suggest that fas gene regulation is very complex and involves more than the mere presence of att-encoded inducer molecules. Possibly, the att-encoded inducer initiates fas gene expression, resulting in the onset of leafy gall formation. At later stages, when the bacteria penetrate the plant’s surface, the role of att-derived inducer may be secondary or even superfluous and other regulatory elements may abound, precluding att gene expression and allowing further fas gene expression. Because fas and att mutant bacteria fail to complement one another, other cell-contained factors that depend on att and/or fas activity must play a role in disease development. Although important, the loci affected by the insertions in the fas and att mutants are not sufficient for pathogenicity because a pFiD188-cured strain carrying a cosmid spanning these loci is nonvirulent (30). More recently, another pFiD188 locus has been isolated that is necessary for full virulence (our unpublished data). This locus could be involved in penetration, regulation of gene expression in planta, or the synthesis of accessory signals or compounds that affect colonization of the plant’s interior. Whatever their activities, functions associated with additional

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pathogenicity genes can be predicted to be integrated into a regulatory and functional web involving both the fas and att genes.

METABOLIC COLONIZATION: THE FATE OF A LEAFY GALL An R. fascians D188 chromosomal mutant has been isolated that carries an insertion in a malate synthase homolog. The mutant cannot grow on acetate and grows less well than wild type on leafy gall extracts and accumulates glyoxylate. The mutant bacteria also show a reduced endophytic colonization of symptomatic tissues (28) and exhibit strongly reduced pathogenicity (149, 151, 153). To understand how a gene of the glyoxylate shunt (78) is involved in virulence, it was hypothesized that the locus is involved in the metabolism of plant components that are used as nutrients during the interaction. The catabolism of these compounds would be mediated through glyoxylate, requiring the action of a malate synthase. During in planta growth, the malate synthase mutant is poisoned by accumulating glyoxylate and becomes less virulent. Nutritional requirements push soil bacteria to interact more or less intimately with plants. From an evolutionary point of view, this intimacy for nutritional sequestering probably occurred gradually. The most primitive way for a soil bacterium to profit from the vast amount of nutritional sources that plants represent is to colonize the rhizosphere and utilize the exuded nitrogen and carbon sources or to attack a host, stimulating it to release nutrients, the strategy of necrogenic plant pathogens (3). These approaches give little selective advantage because common compounds are readily utilized by all soilborne microorganisms. A more advanced strategy is to specialize in the metabolism of specific exudates from the plant roots that are catabolized by a limited amount of soil bacteria. Calystegin (137), certain flavonoids (63), proline (73), L-homoserine, and betaines (13, 56) are produced by many plants, but are rather specifically used by certain Rhizobium species. Another approach is to divert photosynthate of the plant to compounds that are specifically used by the inciting bacteria. Approximately 10% of all Rhizobium meliloti and R. leguminosarum pv viciae strains are known to use this strategy in which modified bacteria, bacteroids, produce rhizopines derived from plant metabolites that are used by the free-living rhizobia (122). Only one type of rhizopine has been identified to date, but it has been predicted that other types with similar functions are produced analogously (104). The ultimate step in specialization for the use of nutritional mediators is represented by Agrobacterium strains. Genetic colonization of the host leads to a free production of opines that are almost exclusively used by the crown gall–inducing bacteria that carry the respective catabolic genes (37, 130). An appealing hypothesis on the origin of the opine concept has been formulated by Vaudequin-Dransart et al (147, 148). Primitive agrobacteria evolved to acquire genes for the catabolism

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of an ancestral opine produced by uninfected plants at only a certain stage in their development. These catabolic genes were the first to be incorporated into the T-DNA and upon integration into the plant cell nucleus, their activity shifted toward the biosynthesis of the ancestral opine. Later, other opine biosynthetic genes would have evolved from these ancestors. Deoxy-fructosyl-glutamine is an opine of the chrysopine family (22). It is produced in uninfected plants upon ageing, can be used by a wide range of agrobacteria, irrespective of their pathogenicity, and is an intermediate in the biosynthesis of several other opines. Deoxyfructosyl-glutamine is therefore a good candidate for being an ancestral opine, but it is not unreasonable to assume that other plant metabolites could fit this profile. In this context, R. fascians has been proposed to use plant carbon sources that are only present at low concentrations in the plant. However, because of the proliferation of specific plant tissues—meristems and shoots—during leafy gall formation, the concentration of these compounds would increase to levels that could serve as specialized nutrients for the bacteria. As such, a leafy gall would represent a specific niche for R. fascians in which the bacteria experience a selective advantage over other plant-inhabiting bacteria. The occurrence of a nutrient pool inaccessible to others is the driving force for such biotrophic pathogens. We feel that this concept of “metabolic habitat modification” may fit many, if not all, plant-galling bacteria.

ACKNOWLEDGMENTS The authors thank Tania Maes and Tita Ritsema for allowing the insertion of some of their unpublished data and Martine De Cock for critical reading of the manuscript and help in preparing it. This work was supported by a grant from the Interuniversity Poles of Attraction Programme (Belgian State, Prime Minister’s Office, Federal Office for Scientific, Technical and Cultural Affairs; P4/15). DV is indebted to the Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie for a postdoctoral fellowship. Visit the Annual Reviews home page at www.AnnualReviews.org

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