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MAX2 signalling confers competence to root explants for callus formation and shoot regeneration. Transcriptome analysis of max2reveals misregulation of LBDand light-responsive genes early in incubation on callus-inducing medium. Although the division of the pericycle cells initiates both lateral root development and root-derived callus formation, these developmental processes are affected differently in the strigolactone and karrikin/KARRIKIN INSENSITIVE 2 (KAI2) ligand signalling mutant more axillary growth 2 (max2). Whereas max2 produces more lateral roots than the wild type, it is defective in the regeneration of shoots from root explants. We suggest that the decreased shoot regeneration of max2 originates from delayed formation of callus primordium, yielding less callus material to regenerate shoots. Indeed, when incubated on callus-inducing medium, the pericycle cell division was reduced in max2 and the early gene expression varied when compared with the wild type, as determined by a transcriptomics analysis. Furthermore, the expression of the LATERAL ORGAN BOUNDARIES DOMAIN genes and of callus-induction genes was modified in correlation with the max2 phenotype, suggesting a role for MAX2 in the regulation of the interplay between cytokinin, auxin, and light signalling in callus initiation. Additionally, we found that the in vitro shoot regeneration phenotype of max2 might be caused by a defect in KAI2, rather than in DWARF14, signalling. Nevertheless, the shoot regeneration assays revealed that the strigolactone biosynthesis mutants max3 and max4 also play a minor role.

Rhodococcus fascians is a gram-positive phytopathogen that infects a wide range of plant species. The actinomycete induces the formation of neoplastic growths, termed leafy galls, that consist of a gall body covered by small shoots of which the outgrowth is arrested due to an extreme form of apical dominance. In our previous work, we demonstrated that in the developing gall, auxin drives the transdifferentiation of parenchyma cells into vascular elements. In this work, with the use of transgenic Arabidopsis thaliana plants carrying molecular reporters for cell division (pCYCB1;1:GUS) and meristematic activity (pSTM:GUS), we analyzed the fate of cells within the leafy gall. Our results indicate that the size of the gall body is determined by ongoing mitotic cell divisions as illustrated by strong CYCB1;1 expression combined with the de novo formation of new meristematic areas triggered by STM expression. The shoot meristems that develop in the peripheral parts of the gall are originating from high ectopic STM expression. Altogether the presented data provide further insight into the cellular events that accompany the development of leafy galls in response to R. fascians infection.

Rhizospheric microorganisms can alter plant physiology and morphology in many different ways including through the emission of volatile organic compounds (VOCs). Here we demonstrate that VOCs from beneficial root endophytic Serendipita spp. are able to improve the performance of in vitro grown Arabidopsis seedlings, with an up to 9.3-fold increase in plant biomass. Additional changes in VOC-exposed plants comprised petiole elongation, epidermal cell and leaf area expansion, extension of the lateral root system, enhanced maximum quantum efficiency of photosystem II (Fv/Fm), and accumulation of high levels of anthocyanin. Notwithstanding that the magnitude of the effects was highly dependent on the test system and cultivation medium, the volatile blends of each of the examined strains, including the references S. indica and S. williamsii, exhibited comparable plant growth-promoting activities. By combining different approaches, we provide strong evidence that not only fungal respiratory CO2 accumulating in the headspace, but also other volatile compounds contribute to the observed plant responses. Volatile profiling identified methyl benzoate as the most abundant fungal VOC, released especially by Serendipita cultures that elicit plant growth promotion. However, under our experimental conditions, application of methyl benzoate as a sole volatile did not affect plant performance, suggesting that other compounds are involved or that the mixture of VOCs, rather than single molecules, accounts for the strong plant responses. Using Arabidopsis mutant and reporter lines in some of the major plant hormone signal transduction pathways further revealed the involvement of auxin and cytokinin signaling in Serendipita VOC-induced plant growth modulation. Although we are still far from translating the current knowledge into the implementation of Serendipita VOCs as biofertilizers and phytostimulants, volatile production is a novel mechanism by which sebacinoid fungi can trigger and control biological processes in plants, which might offer opportunities to address agricultural and environmental problems in the future.

Kiwiberry [Actinidia arguta (Siebold et Zucc.) Planch. ex Miq.] is – just like the green (Actinidia chinensis var. deliciosa) and yellow (A. chinensis var. chinensis) kiwifruit – a fast-growing, deciduous climbing plant that requires adequate fertilizer management for optimum orchard production. However, compared with kiwifruit, very little is known about the fertilization needs of this crop. Since kiwiberry is gaining importance worldwide, a project was started at four mature orchards in Belgium and the Netherlands with a focus on the mineral status of kiwiberry plants. During two growing seasons, visual deficiency symptoms were recorded and the mineral composition of the first basal leaves on fruiting canes was monitored for three cultivars, ‘Geneva’, ‘Weiki’ and ‘Issai’. We then assessed how these data correlated with each other and whether they were related to soil parameters. Additionally, the mineral composition of the fruits was determined and linked to fruit quality, which was evaluated by storage tests. Sufficiency ranges were set up for kiwiberry leaf composition based on hundreds of foliar analyses for three different timeframes during the growing season: pre-flowering, from flowering until 50 days after full flowering, and from 50 days after full flowering until harvest. The data showed that, in comparison with green and yellow kiwifruit, kiwiberry has a tendency to have higher B, higher Cl, and lower Zn levels in its leaves. Moreover, kiwiberry appeared to be very sensitive to Mn deficiency. Clear varietal differences were observed in the leaves for Mg and P. Sufficiency ranges are important to evaluate foliar analyses and, by consequence, the mineral status of kiwiberry orchards. Although the mineral fruit composition at harvest varied significantly depending on the location, Ca consistently played a key role in the storability of the berries. However, besides Ca, the ratios with, inter alia, K, Mg and N were also important.

Pistachio Bushy Top Syndrome (PBTS) is a recently emerged disease that has strongly impacted the pistachio industry in California, Arizona, and New Mexico. The disease is caused by two bacteria, designated PBTS1 that is related to Rhodococcus corynebacterioides and PBTS2 that belongs to the species R. fascians. Here, we assessed the pathogenic character of the causative agents and examined their chromosomal sequences to predict the presence of particular functions that might contribute to the observed co-occurrence and their effect on plant hosts. In diverse assays, we confirmed the pathogenicity of the strains on “UCB-1” pistachio rootstock and showed that they can also impact the development of tobacco species, but concurrently inconsistencies in the ability to induce symptoms were revealed. We additionally evidence that fas genes are present only in a subpopulation of pure PBTS1 and PBTS2 cultures after growth on synthetic media, that these genes are easily lost upon cultivation in rich media, and that they are enriched for in an in planta environment. Analysis of the chromosomal sequences indicated that PBTS1 and PBTS2 might have complementary activities that would support niche partitioning. Growth experiments showed that the nutrient utilization pattern of both PBTS bacteria was not identical, thus avoiding co-inhabitant competition. PBTS2 appeared to have the potential to positively affect the habitat fitness of PBTS1 by improving its resistance against increased concentrations of copper and penicillins. Finally, mining the chromosomes of PBTS1 and PBTS2 suggested that the bacteria could produce cytokinins, auxins, and plant growth-stimulating volatiles and that PBTS2 might interfere with ethylene levels, in support of their impact on plant development. Subsequent experimentation supported these in silico predictions. Altogether, our data provide an explanation for the observed pathogenic behavior and unveil part of the strategies used by PBTS1 and PBTS2 to interact with plants.

Members of the recently proposed Serendipitaceae family (Sebacinales) have been subjected to multiple taxonomic revisions and several uncertainties at the genus and species level remain to be resolved. This volatile classification can largely be attributed to the limited number of available axenic cultures and to the scarcity of useful micro- and macromorphological traits. In the current study, we attempted to discover taxonomic markers which do not rely on DNA sequences, but have enough resolving power to distinguish the members of our unique Congolese collection of endophytic Serendipita isolates and the reference strains S. indica (syn. Piriformospora indica) and S. williamsii (syn. P. williamsii). Therefore, we first examined the nuclear distribution across their hyphal cells and determined their genome sizes by flow cytometry using optimized protocols, but these characteristics appeared to be of little systematic value to differentiate among the closely related sebacinoids. Additionally, we investigated the ultrastructure of the dolipore septa by transmission electron microscopy (TEM) and systematically took a series of measurements on the septal pore swellings, the associated parenthesomes, and the attached endoplasmic reticulum (ER), while qualitatively scoring several other dolipore-related features, such as the presence of annulus-like material in the pore channel. Subsequent multivariate analysis of these parameters revealed that subtle differences in ultrastructural characteristics, especially those linked to the parenthesome and the connected ER network, can be used to delineate isolate groups, indicating that quantification of these features is relevant when studying taxonomically challenging fungi. Moreover, the observed clustering pattern showed that there might be more diversity amongst the Congolese isolates within the S. ‘williamsii’ species complex than previously anticipated based on the molecular data. Altogether, our results provide novel perspectives on the use of an integrative approach to sebacinoid and Serendipitaceae taxonomy.

The unique ecosystem of the Congolese rainforest has only scarcely been explored for its plant–fungal interactions. Here, we characterized the root fungal communities of field-grown maize and of Panicum from adjacent borders in the Congo Basin and assessed parameters that could shape them. The soil properties indicated that comparable poor soil conditions prevailed in fields and borders, illustrating the low input character of local subsistence farming. The rhizosphere fungal communities, dominated by ascomycetous members, were structured by plant species, slash-and-burn practices and soil P, pH and C/N ratio. Examining fungi with potential plant growth-promoting abilities, the glomeromycotan communities appeared to be affected by the same parameters, whereas the inconspicuous symbionts of the order Sebacinales seemed less susceptible to environmental and anthropogenic factors. Notwithstanding the low abundances at which they were detected, sebacinoids occurred in 87% of the field samples, implying that they represent a consistent taxon within indigenous fungal populations across smallholder farm sites. Pending further insight into their ecosystem functionality, these data suggest that Sebacinales are robust root inhabitants that might be relevant for on-farm inoculum development within sustainable soil fertility management in the Sub-Saharan region.

Rhodococcus fascians, causative agent of the leafy gall syndrome, produces a mixture of cytokinins to modify the hormone landscape of its broad range of plant hosts leading to tissue deformations and developmental alterations. Recent developments indicate that the pathogenic nature of these bacteria is superimposed on its plant growth-promoting effect. In the last two decades, its unique position as the only species within the genus able to interact with plants has been overthrown. Indeed, Pistachio Bushy Top Syndrome is an emerging disease linked to the presence of two Rhodococcus species, R. fascians and R. corynebacterioides. Both bacteria would act synergistically to cause the symptoms, giving the prospect of virulence strategies that differ from those of the leafy gall inducers. Additionally, as a result of microbiome research, it is clear that many Rhodococcus species live in close association with plants, and several of them exhibit plant growth-promoting activities. Finally, genome analyses of a collection of R. fascians isolates imply that the taxonomic position of this group of bacteria within the genus will have to be reevaluated, and likely a new genus consisting of several species will be proposed soon.

Highlight To antagonize the developmental process initiated by Rhodococcus fascians and in response to the bacterial cytokinins, Arabidopsis activates its strigolactone response, partially suppressing shoot branching in the rosette., Leafy gall syndrome is the consequence of modified plant development in response to a mixture of cytokinins secreted by the biotrophic actinomycete Rhodococcus fascians. The similarity of the induced symptoms with the phenotype of plant mutants defective in strigolactone biosynthesis and signalling prompted an evaluation of the involvement of strigolactones in this pathology. All tested strigolactone-related Arabidopsis thaliana mutants were hypersensitive to R. fascians. Moreover, treatment with the synthetic strigolactone mixture GR24 and with the carotenoid cleavage dioxygenase inhibitor D2 illustrated that strigolactones acted as antagonistic compounds that restricted the morphogenic activity of R. fascians. Transcript profiling of the MORE AXILLARY GROWTH1 (MAX1), MAX2, MAX3, MAX4, and BRANCHED1 (BRC1) genes in the wild-type Columbia-0 accession and in different mutant backgrounds revealed that upregulation of strigolactone biosynthesis genes was triggered indirectly by the bacterial cytokinins via host-derived auxin and led to the activation of BRC1 expression, inhibiting the outgrowth of the newly developing shoots, a typical hallmark of leafy gall syndrome. Taken together, these data support the emerging insight that balances are critical for optimal leafy gall development: the long-lasting biotrophic interaction is possible only because the host activates a set of countermeasures—including the strigolactone response—in reaction to bacterial cytokinins to constrain the activity of R. fascians.

Extensive de novo vascularization of leafy galls emerging upon Rhodococcus fascians infection is achieved by fascicular/interfascicular cambium activity and transdifferentiation of parenchyma cells correlated with increased auxin signaling. A leafy gall consisting of fully developed yet growth-inhibited shoots, induced by the actinomycete Rhodococcus fascians, differs in structure compared to the callus-like galls induced by other bacteria. To get insight into the vascular development accompanying the emergence of the leafy gall, the anatomy of infected axillary regions of the inflorescence stem of wild-type Arabidopsis thaliana accession Col-0 plants and the auxin response in pDR5:GUS-tagged plants were followed in time. Based on our observations, three phases can be discerned during vascularization of the symptomatic tissue. First, existing fascicular cambium becomes activated and interfascicular cambium is formed giving rise to secondary vascular elements in a basipetal direction below the infection site in the main stem and in an acropetal direction in the entire side branch. Then, parenchyma cells in the region between both stems transdifferentiate acropetally towards the surface of the developing symptomatic tissue leading to the formation of xylem and vascularize the hyperplasia as they expand. Finally, parenchyma cells in the developing gall also transdifferentiate to vascular elements without any specific direction resulting in excessive vasculature disorderly distributed in the leafy gall. Prior to any apparent anatomical changes, a strong auxin response is mounted, implying that auxin is the signal that controls the vascular differentiation induced by the infection. To conclude, we propose the “sidetracking gall hypothesis” as we discuss the mechanisms driving the formation of superfluous vasculature of the emerging leafy gall.

Plant pathogenic bacteria can have devastating effects on plant productivity and yield. Nevertheless, because these often soil-dwelling bacteria have evolved to interact with eukaryotes, they generally exhibit a strong adaptivity, a versatile metabolism, and ingenious mechanisms tailored to modify the development of their hosts. Consequently, besides being a threat for agricultural practices, phytopathogens may also represent opportunities for plant production or be useful for specific biotechnological applications. Here, we illustrate this idea by reviewing the pathogenic strategies and the (potential) uses of five very different (hemi)biotrophic plant pathogenic bacteria: Agrobacterium tumefaciens, A. rhizogenes, Rhodococcus fascians, scab-inducing Streptomyces spp., and Pseudomonas syringae.

The Actinomycete Rhodococcus fascians causes the leafy gall syndrome, an infectious plant disease that affects a wide range of plants, primarily dicotyledonous herbs. The syndrome is associated with delayed senescence, loss of apical dominance, activation of dormant axillary meristems, and formation of multiple inflorescences, leading to a stunted and bushy plant appearance. A major breakthrough in the elucidation of the virulence strategy of this pathogen was the discovery of a linear virulence plasmid, pFiD188 for R. fascians strain D188. Upon perception of a compatible host plant, an autoregulatory mechanism mediated by the att operon directs a switch in the bacterial life style from a harmless epiphyte into a pathogenic endophyte and, concomitantly, activates gene expression of the fas operon that encodes a cytokinin biosynthesis pathway. A mixture of five cytokinins determines the cytokinin activity of R. fascians that directly affects plant responses and development. Moreover, the bacterial cytokinins stimulate the host to produce auxins and polyamines, that function as accessory signals to aid in symptom development. The plant reacts against the developmental hijacking by R. fascians by activating a set of counteracting measures that ultimately results in a delicate balance, allowing a long-lasting biotrophic interaction.

In the last decade, there has been an increasing focus on the implementation of plant growth-promoting (PGP) organisms as a sustainable option to compensate for poor soil fertility conditions in developing countries. Trap systems were used in an effort to isolate PGP fungi from rhizospheric soil samples collected in the region around Kisangani in the Democratic Republic of Congo. With sudangrass as a host, a highly conducive environment was created for sebacinalean chlamydospore formation inside the plant roots resulting in a collection of 51 axenically cultured isolates of the elusive genus Piriformospora (recently transferred to the genus Serendipita). Based on morphological data, ISSR fingerprinting profiles and marker gene sequences, we propose that these isolates together with Piriformospora williamsii constitute a species complex designated Piriformospora (= Serendipita) 'williamsii.' A selection of isolates strongly promoted plant growth of in vitro inoculated Arabidopsis seedlings, which was evidenced by an increase in shoot fresh weight and a strong stimulation of lateral root formation. This isolate collection provides unprecedented opportunities for fundamental as well as translational research on the Serendipitaceae, a family of fungal endophytes in full expansion.

In vitro shoot regeneration is implemented in basic plant research and commercial plant production, but for some plant species, it is still difficult to achieve by means of the currently available cytokinins and auxins. To identify novel compounds that promote shoot regeneration, we screened a library of 10,000 small molecules. The bioassay consisted of a two-step regeneration protocol adjusted and optimized for high-throughput manipulations of root explants of Arabidopsis (Arabidopsis thaliana) carrying the shoot regeneration marker LIGHT-DEPENDENT SHORT HYPOCOTYLS4. The screen revealed a single compound, the cytokinin-like phenyl-adenine (Phe-Ade), as a potent inducer of adventitious shoots. Although Phe-Ade triggered diverse cytokinin-dependent phenotypical responses, it did not inhibit shoot growth and was not cytotoxic at high concentrations. Transcript profiling of cytokinin-related genes revealed that Phe-Ade treatment established a typical cytokinin response. Moreover, Phe-Ade activated the cytokinin receptors ARABIDOPSIS HISTIDINE KINASE3 and ARABIDOPSIS HISTIDINE KINASE4 in a bacterial receptor assay, albeit at relatively high concentrations, illustrating that it exerts genuine but weak cytokinin activity. In addition, we demonstrated that Phe-Ade is a strong competitive inhibitor of CYTOKININ OXIDASE/DEHYDROGENASE enzymes, leading to an accumulation of endogenous cytokinins. Collectively, Phe-Ade exhibits a dual mode of action that results in a strong shoot-inducing activity.

Rhodococcus fascians , a phytopathogen that alters plant development, inflicts significant losses in plant production around the world. We report here the complete genome sequence of R. fascians D188, a well-characterized model isolate, and Rhodococcus species PBTS (pistachio bushy top syndrome) 1 and 2, which were shown to be responsible for a disease outbreak in pistachios.

SUMMARYThe biotrophic phytopathogen Rhodococcus fascians has a profound impact on plant development, mainlythrough its principal virulence factors, a mix of synergistically acting cytokinins that induce shoot formation.Expression profiling of marker genes for several auxin biosynthesis routes and mutant analysis demonstratedthat the bacterial cytokinins stimulate the auxin biosynthesis of plants via specific targeting of the indole-3-pyruvic acid (IPA) pathway, resulting in enhanced auxin signaling in infected tissues. The double mutanttryptophan aminotransferase 1-1 tryptophan aminotransferase related 2-1 (taa1-1 tar2-1) of Arabidopsis(Arabidopsis thaliana), in which the IPA pathway is defective, displayed a decreased responsiveness towardsR. fascians infection, although bacterial colonization and virulence gene expression were not impaired. Theseobservations implied that plant-derived auxin was employed to reinforce symptom formation. Furthermore,theincreasedauxinproductionand,possibly,theaccumulatingbacterialcytokininsininfectedplantsmodifiedthe polar auxin transport so that new auxin maxima were repetitively established and distributed, a processthat is imperative for symptom onset and maintenance. Based on these findings, we extend our model of themode of action of bacterial and plant signals during the interaction between R. fascians and Arabidopsis.Keywords: Rhodococcus fascians, Arabidopsis thaliana, TAA1, polar auxin transport, gall formation.INTRODUCTIONInfection with the phytopathogenic bacterium Rhodococcusfascians modifies the hormone landscape of the host, lead-ing to malformations on a wide range of herbaceous andwoody plants. Infected plants develop leafy galls, whichconsist of small shoots that arise through the activation ofexisting meristems in leaf axils combined with the initiationof new meristems (Stes et al., 2011b). The shoot aspect ofthesymptomsprovokedbyR. fasciansinfectionimpliesthatcytokinins play an important role in the pathology. Indeed,the production of pathological levels of a mix of cytokininsby R. fascians strain D188, encoded by the fas operonlocated on a linear virulence plasmid pFiD188, is essential forthe establishment of symptoms. In contrast, the basal levelof cytokinins chromosomally produced by strain D188-5, thenon-pathogenic,plasmid-freederivativeofstrainD188,doesnot affect plant development (Pertry et al., 2009, 2010). Fur-thermore, the fas-derived cytokinins must be perceived bythe Arabidopsis (Arabidopsis thaliana) cytokinin receptorsARABIDOPSIS HISTIDINE KINASE 3 (AHK3) and AHK4 (Per-try et al., 2009). Despite the establishment of multiplehomeostasis mechanisms, such as the repression of severalplant ISOPENTENYL TRANSFERASE genes and the activa-tionofallCYTOKININOXIDASE/DEHYDROGENASEgenes,asubset of bacterial cytokinins accumulates in infectedtissues and steers an extensive transcriptional modulation(Depuydt et al., 2009b). Importantly, the activation of cellcycle regulators, such as the D3-type cyclin CYCD3 and theB-type cyclin-dependent kinase CDKB1;1, allows mitotic celldivisions to continue, generating meristematic tissues thatnever seem to mature (Depuydt et al., 2009a). Moreover, thecytokinin-induced accumulation of putrescine results in anamplification of the cytokinin-activated signal transductionin the plant, supporting symptom formation (Stes et al.,2011b). Finally, the morphogenic effects are also accompa-nied by alterations in other hormones, such as abscisic acid,gibberellins and auxins (Simo´n-Mateo et al., 2006; Depuydt

The phytopathogenic Actinomycete Rhodococcus fascians induces leafy galls on a wide range of hosts, causing major economical losses in the ornamentals industry. Although differences in the responsivity occur within species, no plant tested so far could be considered resistant to R. fascians strain D188 infection. Here, we observed that members of the genus Dalbergia, which belong to the Fabaceae, did not develop leafy galls when challenged with R. fascians and we set out to unravel the mechanism of this recalcitrance. Whereas organic extracts of Dalbergia tissues exhibited toxicity towards the bacteria, more importantly, dichloromethane bark extracts inhibited the induction of bacterial virulence gene expression without any apparent loss of viability, illustrating that resistance is likely multifactorial. The virulence quencher was identified as a new prenylated isoflavanone, termed perbergin, and specifically targeted the AttR regulon (a LysR-type transcriptional regulator) which is imperative for the switch of R. fascians from an epiphytic to a pathogenic lifestyle. The mode of action of perbergin demonstrated that just like in Gram-negative host-microbe interactions, also in Gram-positive phytopathogens autoregulation is being targeted by the plant as an efficient means of defence. Moreover, the identification of perbergin opens the path to disease control in affected nurseries.

The phytopathogenic actinomycete Rhodococcus fascians induces neoplastic shooty outgrowths on infected hosts. Upon R. fascians infection of Arabidopsis (Arabidopsis thaliana), leaves are formed with small narrow lamina and serrated margins. These symptomatic leaves exhibit reduced tissue differentiation, display more but smaller cells that do not endoreduplicate, and accumulate in the G1 phase of the cell cycle. Together, these features imply that leaf growth occurs primarily through mitotic cell division and not via cell expansion. Molecular analysis revealed that cell cycle gene expression is activated continuously throughout symptomatic leaf development, ensuring persistent mitotic cycling and inhibition of cell cycle exit. The transition at the two major cell cycle checkpoints is stimulated as a direct consequence of the R. fascians signals. The extremely reduced phenotypical response of a cyclind3;1-3 triple knockout mutant indicates that the D-type cyclin/retinoblastoma/E2F transcription factor pathway, as a major mediator of cell growth and cell cycle progression, plays a key role in symptom development and is instrumental for the sustained G1-to-S and G2-to-M transitions during symptomatic leaf growth.

This paper will exemplify molecular communications in the rhizosphere, especially between plants and bacteria, and between bacteria and bacteria. More specifically, we describe signalling pathways that allow bacteria to sense a wide diversity of plant signals, plants to respond to bacterial infection, and bacteria to coordinate gene expression at population and community level. Thereafter, we focus on mechanisms evolved by bacteria and plants to disturb bacterial signalling, and by bacteria to modulate hormonal signalling in plants. Finally, the dynamics of signal exchange and its biological significance we elaborate on the cases of Rhizobium symbiosis and Agrobacterium pathogenesis.

The ability of Rhodococcus fascians strain D188 to provoke leafy gall formation on a variety of plant species is correlated with the linear plasmid pFiD188, on which different pathogenicity loci were identified. The att locus affects the severity of symptom development on tobacco, whereas the fas locus is essential for virulence. To gain insight into the function of the att locus, sequence and expression analyses were performed. The att locus contains nine open reading frames homologous to arginine and beta -lactam biosynthetic genes. aft gene expression is transcriptionally induced by leafy gall extracts, but not by extracts of uninfected plants, and depends on the attR gene that encodes a LysR-type transcriptional regulator. The att locus proves to be essential for the formation of inducing factors (IFs) that are present in gall extracts. Because the induction of the las locus also requires the presence of IFs in gall extracts, the att locus is proposed to play an important role in regulating the expression of the virulence loci of R. fascians.

* To characterize plant cell cycle activation following Rhodococcus fascians infection, bacterial impact on cell cycle progression of tobacco BY-2 cells was investigated. * S-phase-synchronized BY-2 cells were cocultivated with R. fascians and cell cycle progression was monitored by measuring mitotic index, cell cycle gene expression and flow cytometry parameters. Cell cycle alteration was further investigated by cDNA-AFLP (amplified fragment length polymorphism). * It was shown that cell cycle progression of BY-2 cells was accelerated only upon infection with bacteria whose virulence gene expression was induced by a leafy gall extract. Thirty-eight BY-2 genes showed a differential expression within 6 h post-infection. Among these, seven were previously associated with specific plant cell cycle phases (in particular S and G2/M phases). Several genes also showed a differential expression during leafy gall formation. * R. fascians-infected BY-2 cells provide a simple model to identify plant genes related to leafy gall development. R. fascians can also be regarded as a useful biotic agent to alter cell cycle progression and, thereby, gain a better understanding of cell cycle regulation in plants.

Rhodococcus fascians is a phytopathogenic Gram-positive Actinomycete with a very broad host range encompassing especially dicotyledonous herbaceous perennials, but also some monocots, such as the Liliaceae and, recently, the woody crop pistachio. The pathogenicity of R. fascians strain D188 is known to be encoded by the linear plasmid pFiD188 and to be dictated by its capacity to produce a mixture of cytokinins. Here, we show that D188-5, the nonpathogenic plasmid-free derivative of the wild-type strain D188 actually has a plant growth-promoting effect. With the availability of the genome sequence of R. fascians, the chromosome of strain D188 was mined for putative plant growth-promoting functions and the functionality of some of these activities was tested. This analysis together with previous results suggests that the plant growth-promoting activity of R. fascians is due to production of plant growth modulators, such as auxin and cytokinin, combined with degradation of ethylene through 1-amino-cyclopropane-1-carboxylic acid deaminase. Moreover, R. fascians has several functions that could contribute to efficient colonization and competitiveness, but there is little evidence for a strong impact on plant nutrition. Possibly, the plant growth promotion encoded by the D188 chromosome is imperative for the epiphytic phase of the life cycle of R. fascians and prepares the plant to host the bacteria, thus ensuring proper continuation into the pathogenic phase.

SUMMARY Rhodococcus fascians is a Gram-positive bacterium that interacts with many plant species and induces multiple shoots through a combination of activation of dormant axillary meristems and de novo meristem formation. Although phenotypic analysis of the symptoms of infected plants clearly demonstrates a disturbance of the phytohormonal balance and an activation of the cell cycle, the actual mechanism of symptom development and the targets of the bacterial signals are unknown. To elucidate the molecular pathways that are responsive to R. fascians infection, differential display was performed on Nicotiana tabacum as a host. Four differentially expressed genes could be identified that putatively encode a senescence-associated protein, a gibberellin 2-oxidase, a P450 monooxygenase and a proline dehydrogenase. The differential expression of the three latter genes was confirmed on infected Arabidopsis thaliana plants by quantitative reverse transcription polymerase chain reactions, supporting their general function in R. fascians-induced symptom development. The role of these genes in hormone metabolism, especially of gibberellin and abscisic acid, in breaking apical dominance and in activating axillary meristems, which are processes associated with symptom development, is discussed.

To better characterise at the molecular level the nature of plant responses to infection by Rhodococcus fascians PCR-based differential display patterns of Atropa belladonna leafy gall (LG) and non-infected plant tissues were compared. Six differentially expressed genes were identified and their altered expression was confirmed by RT-PCR. Three of them corresponded to up-regulated genes which encode proteins involved in plant defence. The three remaining cDNA fragments which correspond to down-regulated genes in LG, encoded proteins with similarity to a multicystatin, a miraculin and a methallothionein-like protein, respectively. Upon elimination of the bacteria from infected plant tissue, the expression of up-regulated genes was maintained, whereas expression of down-regulated genes resumed suggesting a potential role of these up-regulated genes in plant growth and development.

The glyoxylate cycle and the glycine cleavage system are part of conserved metabolic pathways involved in the chronic persistence of microorganisms in animal hosts. In the chromosome of the plant pathogen Rhodococcus fascians, the vic locus has been identified as a region containing genes essential for persistence inside induced leafy galls. Sequence analysis showed that this 18-kb locus is syntenic with chromosomal regions of Mycobacterium species that encompass the 'persistence' loci of these mammalian pathogens. Hence, the ability to switch diet inside the host appears to be governed by 'persistence' enzymes that are conserved between pathogens of animals and plants.

De novo shoot organogenesis (i.e., the regeneration of shoots on nonmeristematic tissue) is widely applied in plant biotechnology. However, the capacity to regenerate shoots varies highly among plant species and cultivars, and the factors underlying it are still poorly understood. Here, we evaluated the shoot regeneration capacity of 88 Arabidopsis thaliana accessions and found that the process is blocked at different stages in different accessions. We show that the variation in regeneration capacity between the Arabidopsis accessions Nok-3 and Ga-0 is determined by five quantitative trait loci (QTL): REG-1 to REG-5. Fine mapping by local association analysis identified RECEPTOR-LIKE PROTEIN KINASE1 (RPK1), an abscisic acid-related receptor, as the most likely gene underlying REG-1, which was confirmed by quantitative failure of an RPK1 mutation to complement the high and low REG-1 QTL alleles. The importance of RPK1 in regeneration was further corroborated by mutant and expression analysis. Altogether, our results show that association mapping combined with linkage mapping is a powerful method to discover important genes implicated in a biological process as complex as shoot regeneration.

Plant regeneration through de novo shoot organogenesis in tissue culture is a critical step in most plant transformation and micropropagation procedures. Establishing an efficient regeneration protocol is an empirical process and requires optimization of multiple factors that influence the regeneration capacity. Here, we review the molecular process of shoot induction in a two-step regeneration protocol and focus on the role of auxins and cytokinins. First, during incubation on an auxin-rich callus induction medium (CIM), organogenic callus is produced that exhibits characteristics of a root meristem. Subsequent incubation on a cytokinin-rich shoot induction medium (SIM) induces root to shoot conversion. Through a detailed analysis of the different aspects of shoot regeneration, we try to reveal hinge points and novel candidate genes that may be targeted to increase shoot regeneration capacity in order to improve transformation protocols.

Though plant metabolic changes are known to occur during interactions with bacteria, these were rarely challenged for pharmacologically active compounds suitable for further drug development. Here, the occurrence of specific chemicals with antiproliferative activity against human cancer cell lines was evidenced in hyperplasia (leafy galls) induced when plants interact with particular phytopathogens, such as the Actinomycete Rhodococcus fascians., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

An isocitrate lyase (Icl)-encoding gene (icl) from the Gram+ plant pathogen Rhodococcus fascians was identified serendipitously as part of a scrambled fragment after shotgun cloning in the promoter probe vector, pDP1. The Icl protein is 429 amino acids long (47.11 kDa) and has a predicted pI of 4.84; it is 54% similar to the Escherichia coli Icl and 24–27% to eukaryotic homologues. Comparison of the prokaryotic and eukaryotic Ici confirms the earlier proposal of Matsuoka and McFadden [J. Bacteriol. 143 (1988) 4528–4536] that the enzyme has enlarged during evolution.

The biotrophic phytopathogen Rhodococcus fascians has a profound impact on plant development, mainly through its principal virulence factors, a mix of synergistically acting cytokinins that induce shoot formation. Expression profiling of marker genes for several auxin biosynthesis routes and mutant analysis demonstrated that the bacterial cytokinins stimulate the auxin biosynthesis of plants via specific targeting of the indole-3-pyruvic acid (IPA) pathway, resulting in enhanced auxin signaling in infected tissues. The double mutant tryptophan aminotransferase 1-1 tryptophan aminotransferase related 2-1 (taa1-1 tar2-1) of Arabidopsis (Arabidopsis thaliana), in which the IPA pathway is defective, displayed a decreased responsiveness towards R. fascians infection, although bacterial colonization and virulence gene expression were not impaired. These observations implied that plant-derived auxin was employed to reinforce symptom formation. Furthermore, the increased auxin production and, possibly, the accumulating bacterial cytokinins in infected plants modified the polar auxin transport so that new auxin maxima were repetitively established and distributed, a process that is imperative for symptom onset and maintenance. Based on these findings, we extend our model of the mode of action of bacterial and plant signals during the interaction between R. fascians and Arabidopsis.

Rhodococcus fascians is a gram-positive phytopathogen that induces differentiated galls, known as leafy galls, on a wide variety of plants, employing virulence genes located on a linear plasmid. The pathogenic strategy consists of the production of a mixture of six synergistically acting cytokinins that overwhelm the plant's homeostatic mechanisms, ensuring the activation of a signaling cascade that targets the plant cell cycle and directs the newly formed cells to differentiate into shoot meristems. The shoots that are formed upon infection remain immature and never convert to source tissues resulting in the establishment of a nutrient sink that is a niche for the epiphytic and endophytic R. fascians subpopulations. Niche formation is accompanied by modifications of the transcriptome, metabolome, physiology, and morphology of both host and pathogen. Here, we review a decade of research and set the outlines of the molecular basis of the leafy gall syndrome.

The phytopathogenic actinomycete Rhodococcus fascians drives its host to form a nutrient-rich niche by secreting a mixture of cytokinins that triggers plant cell division and shoot formation. The discrepancy between the relatively low amount of secreted cytokinins and the severe impact of R. fascians infection on plant development has puzzled researchers for a long time. Polyamine and transcript profiling of wild-type and cytokinin receptor mutant plants revealed that the bacterial cytokinins directly stimulated the biosynthesis of plant putrescine by activating arginine decarboxylase expression. Pharmacological experiments showed that the increased levels of putrescine contributed to the severity of the symptoms. Thus, putrescine functions as a secondary signal that impinges on the cytokinin-activated pathway, amplifying the hormone-induced changes that lead to the formation of a leafy gall. Exogenous putrescine and treatment with polyamine biosynthesis inhibitors combined with transcript and polyamine analyses of wild-type and mutant plants indicated that the direct target of both the bacterial cytokinins and plant putrescine was the expression of D3-type cyclins. Hence, the activated d-type cyclin/retinoblastoma/E2F transcription factor pathway integrates both external and internal hormonal signals, stimulating mitotic cell divisions and inducing pathological plant organogenesis.

The phytopathogenic Actinomycete Rhodococcus fascians induces leafy galls on a wide range of hosts, causing major economical losses in the ornamentals industry. Although differences in the responsivity occur within species, no plant tested so far could be considered resistant to R. fascians strain D188 infection. Here, we observed that members of the genus Dalbergia, which belong to the Fabaceae, did not develop leafy galls when challenged with R. fascians and we set out to unravel the mechanism of this recalcitrance. Whereas organic extracts of Dalbergia tissues exhibited toxicity towards the bacteria, more importantly, dichloromethane bark extracts inhibited the induction of bacterial virulence gene expression without any apparent loss of viability, illustrating that resistance is likely multifactorial. The virulence quencher was identified as a new prenylated isoflavanone, termed perbergin, and specifically targeted the AttR regulon (a LysR-type transcriptional regulator) which is imperative for the switch of R. fascians from an epiphytic to a pathogenic lifestyle. The mode of action of perbergin demonstrated that just like in Gram-negative host-microbe interactions, also in Gram-positive phytopathogens autoregulation is being targeted by the plant as an efficient means of defence. Moreover, the identification of perbergin opens the path to disease control in affected nurseries.

A sensitive and reliable high-performance liquid chromatographic method with tandem mass spectrometric detection has been developed and used for the determination of 2-methylthio-cytokinin derivatives produced by the phytopathogenic actinomycete Rhodococcus fascians. The cultivation medium containing secreted cytokinins was concentrated and subjected to a solid-phase extraction (C18 and ion-exchange). The purified samples were further separated and analyzed by HPLC-ESI–MS/MS. This allowed to achieve chromatographic resolution of six highly hydrophobic cytokinin species including 2-methylthio-isopentenyladenine, 2-methylthio-isopentenyladenosine, 2-methylthio-trans-zeatin and 2-methylthio-trans-zeatin riboside and their cis-isomers when a reversed-phase chromatographic column (C4) and a mobile phase consisting of acetonitrile and 20 mM ammonium formate, pH 5, were used. Quantification was performed by a standard isotope dilution method using a multiple-reaction monitoring (MRM) mode. In the MRM mode, limits of detection reached 20–30 fmol and linear ranges spanned four orders of magnitude. Recovery values were between 35% and 65% and the analytical accuracy between 95% and 149%. The proposed bioanalytical method, which takes advantage of effective chromatographic separation of six 2-methyltio-derivatives (including isomers of zeatin-type cytokinins) and sensitive mass spectrometric detection, may become useful for plant biologists studying the significance of these substances in plant–microbe interactions.

Rhodococcus fascians is the only described phytopathogen in the genus and it induces a constellation of shooty symptoms on a very large range of plants. The main virulence factors, cytokinins, and functions that mediate more subtle aspects of the interaction with the host are encoded by a linear plasmid that shares a common origin with other rhodococcal linear replicons. The chromosome also plays an important role in the lifestyle of the bacterium. Chromosomal pathways are involved in producing auxin, delivering substrates for cytokinin production, and metabolizing nutrients retrieved from infected plants. The colonization of the plant tissues together with the secretion of various morphogens leads to a cascade of molecular responses that ultimately result in the establishment of disease symptoms, known as leafy galls.

The nucleotide sequence of the chloramphenicol-resistance gene (cmr) of Rhodococcus fascians NCPPB 1675 (located on the conjugative plasmid pRF2) allowed the identification of two possible open reading frames (ORFs), of which ORF1 was consistent with the mutational analysis. Biochemical analysis of cmr revealed that it does not encode an antibiotic-modifying enzyme. The amino acid sequence of ORF1 predicted a hydrophobic protein, with 12 putative membrane-spanning domains, homologous to proteins involved in the efflux of tetracycline across the plasma membrane. Expression of the cmr gene was induced by addition of chloramphenicol to the growth media. The promoter of this gene was restricted to 50 bp upstream from a 200 bp 5'-untranslated mRNA region, the latter containing two inverted repeats. At the amino acid level, the cmr gene is 52% identical to a previously identified chloramphenicol-resistance determinant in Streptomyces lividans, indicating a wider dispersion of this type of cmr gene among the actinomycetes.

Summary Plant growth and development are significantly influenced by the presence and activity of microorganisms. To date, the best-studied plant-interacting microbes are Gram-negative bacteria, but many representatives of both the high and low G+C Gram-positives have excellent biocontrol, plant growth-promoting and bioremediation activities. Moreover, actinorhizal symbioses largely contribute to the global biological nitrogen fixation and many Gram-positive bacteria promote other types of symbioses in tripartite interactions. Finally, several prominent and devastating phytopathogens are Gram-positive. We summarize the present knowledge of the beneficial and detrimental interactions of Gram-positive bacteria with plants to underline the importance of this particular group of bacteria.

Decades ago, the importance of cytokinins (CKs) during Rhodococcus fascians pathology had been acknowledged, and an isopentenyltransferase gene had been characterized in the fas operon of the linear virulence plasmid, but hitherto, no specific CK(s) could be associated with virulence. We show that the CK receptors AHK3 and AHK4 of Arabidopsis thaliana are essential for symptom development, and that the CK perception machinery is induced upon infection, underlining its central role in the symptomatology. Three classical CKs [isopentenyladenine, trans -zeatin, and cis -zeatin (cZ)] and their 2-methylthio (2MeS)-derivatives were identified by CK profiling of both the pathogenic R. fascians strain D188 and its nonpathogenic derivative D188–5. However, the much higher CK levels in strain D188 suggest that the linear plasmid is responsible for the virulence-associated production. All R. fascians CKs were recognized by AHK3 and AHK4, and, although they individually provoked typical CK responses in several bioassays, the mixture of bacterial CKs exhibited clear synergistic effects. The cis - and 2MeS-derivatives were poor substrates of the apoplastic CK oxidase/dehydrogenase enzymes and the latter were not cytotoxic at high concentrations. Consequently, the accumulating 2MeScZ (and cZ) in infected Arabidopsis tissue contribute to the continuous stimulation of tissue proliferation. Based on these results, we postulate that the R. fascians pathology is based on the local and persistent secretion of an array of CKs.

Rhodococcus fascians is a Gram-positive phytopathogen that induces shooty hyperplasia on its hosts through the secretion of cytokinins. Global transcriptomics using microarrays combined with profiling of primary metabolites on infected Arabidopsis (Arabidopsis thaliana) plants revealed that this actinomycete modulated pathways to convert its host into a niche. The transcript data demonstrated that R. fascians leaves a very characteristic mark on Arabidopsis with a pronounced cytokinin response illustrated by the activation of cytokinin perception, signal transduction, and homeostasis. The microarray data further suggested active suppression of an oxidative burst during the R. fascians pathology, and comparison with publicly available transcript data sets implied a central role for auxin in the prevention of plant defense activation. Gene Ontology categorization of the differentially expressed genes hinted at a significant impact of infection on the primary metabolism of the host, which was confirmed by subsequent metabolite profiling. The much higher levels of sugars and amino acids in infected plants are presumably accessed by the bacteria as carbon and nitrogen sources to support epiphytic and endophytic colonization. Hexoses, accumulating from a significantly increased invertase activity, possibly inhibited the expression of photosynthesis genes and photosynthetic activity in infected leaves. Altogether, these changes are indicative of sink development in symptomatic tissues. The metabolomics data furthermore point to the possible occurrence of secondary signaling during the interaction, which might contribute to symptom development. These data are placed in the context of regulation of bacterial virulence gene expression, suppression of defense, infection phenotype, and niche establishment.

Decaprenylphosphoryl-d-arabinose (DPA) has been shown to be the donor of the essential d-arabinofuranosyl residues found in the cell wall of Mycobacterium tuberculosis. DPA is formed from phosphoribose diphosphate in a four-step process. The first step is the nucleophilic replacement of the diphosphate group with decaprenyl phosphate. This reaction is catalysed by the integral membrane protein 5-phospho-alpha-D-ribose-1-diphosphate : decaprenyl-phosphate 5-phosphoribosyltransferase (DPPR synthase). The enzyme is essential for growth and thereby an important target candidate for the development of new tuberculosis drugs. Although membrane proteins are an important subset of targets for current antibacterial agents, details about the structures and the active sites of such proteins are often not readily available by X-ray crystallography. To begin a different approach to the issue, homologues from Mycobacterium smegmatis and Corynebacterium glutamicum were expressed in Escherichia coli and shown to be active DPPR synthases. This was followed by bioinformatic analyses of the aligned sequences and then by site-directed mutagenesis of amino acids identified as likely to be important for activity. The results suggested that the enzymic synthesis of decaprenyl-phosphate 5-phosphoribose (DPPR) occurs on the cytoplasmic side of the plasma membrane. Amino acid substitutions showed that the predicted cytoplasmic N-terminal region and two cytoplasmic loops are involved in substrate binding and/or catalysis along with parts of some adjoining inner membrane regions. The enzyme lacks the classical phosphoribose diphosphate (pRpp) binding site found in nucleic acid precursor enzymes of both prokaryotes and eukaryotes but instead contains a conserved NDxxD motif required for enzymic activity. Thus, it is plausible that this DPPR synthase has a pRpp binding site that is different from that of the classical eukaryotic enzymes, and further work to develop inhibitors against this enzyme is thereby encouraged.

Background Biological nitrogen fixation is a prokaryotic process that plays an essential role in the global nitrogen cycle. Azorhizobium caulinodans ORS571 has the dual capacity to fix nitrogen both as free-living organism and in a symbiotic interaction with Sesbania rostrata. The host is a fast-growing, submergence-tolerant tropical legume on which A. caulinodans can efficiently induce nodule formation on the root system and on adventitious rootlets located on the stem. Results The 5.37-Mb genome consists of a single circular chromosome with an overall average GC of 67% and numerous islands with varying GC contents. Most nodulation functions as well as a putative type-IV secretion system are found in a distinct symbiosis region. The genome contains a plethora of regulatory and transporter genes and many functions possibly involved in contacting a host. It potentially encodes 4717 proteins of which 96.3% have homologs and 3.7% are unique for A. caulinodans. Phylogenetic analyses show that the diazotroph Xanthobacter autotrophicus is the closest relative among the sequenced genomes, but the synteny between both genomes is very poor. Conclusion The genome analysis reveals that A. caulinodans is a diazotroph that acquired the capacity to nodulate most probably through horizontal gene transfer of a complex symbiosis island. The genome contains numerous genes that reflect a strong adaptive and metabolic potential. These combined features and the availability of the annotated genome make A. caulinodans an attractive organism to explore symbiotic biological nitrogen fixation beyond leguminous plants.

The occurrence of plasmids is typically correlated with the acquisition of specialized traits that can be very diverse. Production of secondary metabolites, achievement of complex catabolic reactions, and adaptation to particular habitats are just a few examples of functions that are often encoded by circular or linear extrachromosomal replicons. Although virulence is another classical characteristic that is frequently associated with the presence of plasmids, until now there is only one known case in which the phytopathogenic capacity is encoded by a linear plasmid. The actinomycete Rhodococcus fascians is a wide-spectrum plant pathogen that provokes shooty neoplastic outgrowths on its hosts. The key virulence determinants of this bacterium are located on a linear replicon of the invertron type. Even though the sequence determination of the linear plasmid of R. fascians strain D188 (pFiD188) is not completed, a first comparative analysis shows that is has probably a common origin with catabolic linear plasmids of other Rhodococcus strains. Whereas the colinear regions encode plasmid maintenance functions, the three unique regions of pFiD188 are involved in specific aspects of the interaction.