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Virulence mechanisms of Gram-positive plant pathogenic bacteria
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Virulence mechanisms of Gram-positive plant pathogenic bacteria Saskia A Hogenhout1 and Rosemary Loria2 Actinobacteria and Firmicutes comprise a group of highly divergent prokaryotes known as Gram-positive bacteria, which are ancestral to Gram-negative bacteria. Comparative genomics is revealing that, though plant virulence genes are frequently located on plasmids or in laterally acquired gene clusters, they are rarely shared with Gram-negative bacterial plant pathogens and among Gram-positive genera. Grampositive bacterial pathogens utilize a variety of virulence strategies to invade their plant hosts, including the production of phytotoxins to allow intracellular and intercellular replication, production of cytokinins to generate gall tissues for invasion, secretion of proteins to induce cankers and the utilization and manipulation of sap-feeding insects for introduction into the phloem sieve cells. Functional analysis of novel virulence genes utilized by Actinobacteria and Firmicutes is revealing how these ancient prokaryotes manipulate plant, and sometimes insect, metabolic processes for their own benefit. Addresses 1 Department of Disease and Stress Biology, John Innes Centre, Norwich Research Park, Colney Lane, Colney, Norwich NR4 7UH, United Kingdom 2 Department of Plant Pathology and Plant-Microbe Biology, 360 Plant Science Building, Cornell University, Ithaca, NY 14853, USA Corresponding author: Hogenhout, Saskia A ([email protected]) and Loria, Rosemary ([email protected])
Current Opinion in Plant Biology 2008, 11:449–456 This review comes from a themed issue on Biotic Interactions Edited by Murray Grant and Sophien Kamoun Available online 17th July 2008 1369-5266/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2008.05.007
Introduction Research on Gram-positive bacterial plant pathogens has resulted in the discovery and characterization of novel virulence determinants. Phylogenetic distance (Figure 1) and fundamental differences in cell structure between Gram-negative and Gram-positive bacteria predict that these bacteria do not share vertically transferred plant pathogenicity genes. Furthermore, the two clades that compose the Gram-positive bacteria, the Actinobacteria (high G + C) and the Firmicutes (low G + C), diverged very early in evolutionary history [1] (Figure 1), suggesting that these phyla are unlikely to share ancestral www.sciencedirect.com
pathogenicity loci either. Recent progress in molecular genetics and genomics of plant pathogenicity in the actinobacterial genera Clavibacter [2], Streptomyces [3], Leifsonia [4] and Rhodococcus [5] and in the Firmicute genera Spiroplasma [6] and Candidatus Phytoplasma [7], both of which lie within the class Mollicutes [8,9], support a model of phyla-specific virulence genes enhanced through horizontal gene transfer (HGT). Representative genomes of plant pathogenic Firmicutes and Actinobacteria have been sequenced to completion [2,10–16], revealing genes encoding authentic and putative adhesins, toxins, plant hormones and secreted virulence proteins. Here we compare pathogenicity strategies in a few of the diverse bacteria gathered under the Gram-positive ‘umbrella’, highlighting representative plant pathogens in both the Firmicutes and Actinobacteria.
Host colonization and symptomology Plant pathogenic Actinobacteria in the genera Streptomyces and Rhodococcus have very wide host ranges, including economically important crops and model plants. Streptomyces scabies is the most widely distributed and best studied of at least 10 species that cause scab diseases of potato and other laterally expanding underground plant tissues and share virulence genes (reviewed in [3]). However, these pathogens readily infect monocot and dicot seedlings, causing root rot and seedling death; the lack of host specificity corresponds to the inability of scab-causing streptomycetes to produce the hypersensitive response (Loria unpublished). Regardless of plant host or tissue, entry occurs through expanding plant tissues without the requirement for natural openings or wounds, and pathogen growth proceeds both intracellularly and intercellularly (Figure 2). Rhodococcus fascians also infects both monocot and dicot hosts, many of which are economically important [17]. Extensive epiphytic growth precedes intercellular invasion through stomata. Leaf deformation, witches’ broom symptoms, fasciations and leafy galls are caused by hyper induction of shoots through activation of dormant axillary meristems and de novo meristem formation. Symptoms are believed to be the result of elaborate manipulation of host hormone balances and pathogen derived auxin and cytokinin [18,19]. In contrast to Streptomyces and Rhodococcus, plant pathogenic species in the Actinobacteria genera Clavibacter and Leifsonia are host-specific at the species or subspecies level. Clavibacter michiganensis is composed of a number of host-specific subspecies all of which colonize the xylem. Current Opinion in Plant Biology 2008, 11:449–456
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Figure 1
The Actinobacteria, Firmicutes and Proteobacteria diverged early in evolutionary history. The 16S ribosomal RNA gene sequences of representative species were aligned in ClustalX v. 1.83 [50]. The alignment served as input for generating a phylogenetic tree using the Neighbour Joining (NJ) method [51] of ClustalX. Positions with gaps were excluded from the tree calculations. Confidence values of a 1000 bootstrap trials are indicated at the nodes. The tree was visualized in NJ Plot [52] and Dendroscope [53] software. GenBank accession numbers: Acholeplasma laidwadii (NC_010163); Agrobacterium tumefaciens (NC_003062); Aster Yellows phytoplasma strain witches’ broom (AY-WB) (NC_007716); Bacillus subtilis (NC_000964); Clavibacter michiganensis subsp. michiganensis (NC_009480); Erwinia carotovora subsp. atroseptica (NC_004547); Escherichia coli K12 (NC_000913); Leifsonia xyli (NC_006087); Mycobacterium tuberculosis (NC_009525); Mycoplasma genitalium (NC_000908); Pseudomonas syringae pv. tomato (NC_004578); Rhizobium etli (NC_007761); Rhodococcus fascians (AB211229); Spiroplasma citri (X63781); Streptococcus pneumoniae (NC_008533); Streptomyces coelicolor (NC_03888); Streptomyces turgidiscabies (AB026221); Sulfolobus solfataricus (NC_002754); Xanthomonas campestris pv. vesicatoria (NC_007508); Xylella fastidiosa Temecula 1 (NC_004556).
The genomes of C. michiganensis subsp. michiganensis (Cmm), a tomato pathogen and C. michiganensis subsp. sepidonicus (Cms), a potato pathogen, are sequenced [20,21]. Genome comparisons show substantial gene content diversity within the species, apparently driven by differences in the ecology of the subspecies; Cms lives as an endophyte in a vegetatively propagated crop while Cmm also grows epiphytically and infects seeds and epidermal tissue. The genus Leifsonia includes xylemlimited, fastidious bacterial pathogens. The best known of these pathogens is L. xyli subsp. xyli, (Lxx) the causative agent of a systemic disease called ratoon stunting of sugarcane. Plant growth inhibition, the hallmark of this disease, may be due to a putative fatty acid desaturase that modifies the carotenoid biosynthesis pathway to produce abscisic acid, a growth inhibitor [4,13]. Phytoplasmas and two Spiroplasma spp., Spiroplasma citri and Spiroplasma kunkelii, parasitize both plants and insects Current Opinion in Plant Biology 2008, 11:449–456
(Figure 3), a feat that requires manipulation of phylogenetically diverse eukaryotic cells. In plants, these fastidious Firmicutes are largely restricted to the cytoplasm of phloem cells (Figure 3b), but some have very broad plant host ranges (reviewed in [7]). S. citri is an economically important pathogen of Citrus spp. that also infects many other plants including Arabidopsis thaliana. By contrast, S. kunkelii only infects maize. Some phytoplasma groups, particularly the AY (Aster Yellows) and STOL (stolbur) group phytoplasmas, have very broad host ranges. Aster Yellows phytoplasma strain witches’ broom (AY-WB) alone can infect China aster, lettuce, Nicotiana benthamiana, tomato, A. thaliana and maize [7]. In insects, phytoplasmas and spiroplasmas have to cross several barriers (Figure 3a), including for instance the gut epithelial cell layer (Figure 3c). Interactions of the phytoplasma surface exposed Antigenic membrane proteins (Amps) with the microfilaments of the insect intestinal tract play a major role in phytoplasma transmission by insects [22,23]. www.sciencedirect.com
Virulence of Gram-positive bacteria Hogenhout and Loria 451
Figure 2
Streptomyces turgidiscabies is among the streptomycete species that infect subterranean plant structures, causing scab diseases and necrosis and stunting of fibrous roots. Confocal micrographs showing S. turgidiscabies, expressing enhanced green fluorescent protein (EGFP), colonizing a radish seedling root at 15 days post-inoculation. (a) Extensive intercellular and intracellular colonization. (b) Intracellular colonization of a radish trichoblast. Image credit Simon Moll.
Plants infected with phytoplasmas and spiroplasmas are frequently stunted, have discoloured leaves and produce abnormal flowers, fruits or seeds. Phytoplasmas also interfere with plant development as they induce, for instance, phyllody (retrograde metamorphosis of floral organs to the condition of leaves), witches’ broom (growth of a dense mass of shoots from a single point), virescence (green
coloration of non-green flower parts) and bolting (growth of elongated stalks). Because the majority of insect vectors of phytoplasmas lay eggs in young green plant tissues, these plant symptoms may increase insect vector progeny numbers and phytoplasma infection that, in turn, promotes phytoplasma dispersal in nature. Indeed, the longevity and reproduction of the leafhopper vector
Figure 3
The phytoplasma life cycle involves replication in plants and insects. (a) Schematic illustration of the different stages of phytoplasma movement through the leafhopper and plant hosts. Phytoplasmas are indicated as yellow dots and phytoplasma movement is indicated with yellow block arrows. Leafhoppers acquire phytoplasmas from the plant phloem. Phytoplasmas are ingested with plant sap and move through the stylet’s food canal to the midgut. Phytoplasmas cross the basal lamina to enter the hemolymph, from where they can move to the salivary gland. They enter the secretory salivary gland cells and are then transported along with the saliva into the plant phloem tissue during leafhopper feeding. The latent period, that is, the time between initial acquisition of the phytoplasmas by the insect vector from plants and the ability for the insect to introduce phytoplasmas back into plants, can vary between 7 and 80 days, depending on the insect vector and plant host species. (b and c) Electron micrographs of phytoplasmas in the plant phloem and in leafhopper midgut as indicated in (a). (b) Phytoplasmas (arrowheads) in adjacent sieve elements (se1) close to the nucleus (n), in a China aster leaf; the arrow indicates a sieve pore between the sieve plates (sp), and asterisks indicate possibly dividing phytoplasma cells. (c) Accumulations of phytoplasmas (arrowheads) in the cytoplasm of a cell near the nucleus (n) in the muscle layer of the midgut. Scale bars, 1 mm. Credit for electron micrographs shown in b and c, El-Desouky Ammar. www.sciencedirect.com
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Macrosteles quadrilineatus is greater on AY phytoplasmainfected plants than on healthy plants [24]. Thus, AY-WB phytoplasmas have a marked effect on plant–insect interactions, that is, they convert plants into better hosts for phloem-feeding insects. S. citri symptom development is linked to carbohydrate utilization preference in the phloem. Fructose uptake through the phosphoenolpyruvate:fructose phosphotransferase system (fructose PTS) by S. citri induces severe symptoms [25], whereas glucose uptake through the glucose PTS does not [26]. It is likely that in S. citri infected plants the activity of invertase, which converts sucrose to fructose and glucose in phloem companion cells, increases in response to low fructose concentrations [26]. This, in turn, leads to abnormally high glucose concentrations in the phloem, resulting in the chlorosis and internode shortening typical of S. citri infected plants [26]. The genome sequences of OY-M and AY-WB have revealed that phytoplasmas do not possess characterized sugar PTSs but have ATP-binding cassette (ABC) trans-
porters for uptake of maltose, trehalose, sucrose and palatinose [10,14]. The utilization of one or more of these sugars in the phloem may be responsible for induction of symptoms in phytoplasma-infected plants. However, the phyllody, witches’ broom, virescence and bolting are probably induced through other mechanisms. Since phytoplasma genomes lack identifiable plant hormone biosynthesis pathways, one or more of the 56 secreted virulence proteins of AY-WB are probably responsible for symptom induction (reviewed in [7]). Of these 56 secreted proteins, 51 are predicted to be <40 kDa and therefore may be able to traverse the plasmodesmata that connect phloem cells with other cell in developing, but not mature, plant tissues (Figure 4a). Indeed, symptoms in phytoplasma-infected plants are most prevalent in developing tissues (Figure 4b).
Virulence factors Secreted proteins that act inside the plant cell are central to microbial pathogenesis [27]. The lack of a type III protein secretion system (TTSS) in Firmicutes and Actinobacteria immediately raises questions about delivery of virulence proteins across the plant cell wall
Figure 4
Aster Yellows phytoplasma strain witches’ broom (AY-WB) induces severe symptoms in young plant tissues. (a) Schematic illustration showing systemic movement of secreted AY-WB proteins (SAPs) young plant tissues. AY-WB phytoplasmas secrete various SAPs, including SAP11, into the phloem. These SAPs then unload from phloem cells into mesophyll and other cell types of young (sink) plant parts where they induce witches’ broom, chlorosis and other aberrations. (b) AY-WB symptoms are most obvious in young growing points (the crown) of Nicotiana benthamiana plants consistent with the systemic movement of SAPs (as shown in a). The inset shows a close-up of the crown of one of the AY-WB-infected plants. Current Opinion in Plant Biology 2008, 11:449–456
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and membrane—questions that largely remain unanswered. S. scabies, Streptomyces turgidiscabies and S. acidiscabies directly penetrate plant cell walls with the help of thaxtomin, a phytotoxin that inhibits cellulose biosynthesis (reviewed in [3]), presumably allowing secretion of proteins at the host cell membrane. These pathogens secrete Nec1, a novel protein that is proposed to suppress host defence through an unknown mechanism [28]. S. turgidiscabies nec1 deletion mutants that produced wild-type levels of thaxtomin were unable to colonize radish roots under conditions in which the wild-type strain ramified apical and subapical cells within 24 h. Nec1 has an unknown target but is secreted via an N-terminal signal sequence and is expected to interact with host proteins in the cytoplasm [28]. By contrast, proteins that manipulate plant defence in Gram-negative bacteria typically cross bacterial and host membranes via the TTSS [29]. By contrast, spiroplasmas and phytoplasmas are injected into the cytoplasm of phloem cells by their insect vectors (reviewed in [7]). These pathogens secrete proteins into the phloem cell cytoplasm allowing movement of these proteins to other plant cells, for example, to developing (sink) tissue cells through plasmodesmata (Figure 4). The AY-WB secreted protein, SAP11, has been characterized [7]. SAP11 is 11 kDa and contains a bipartite nuclear localization signal (NLS) that is functional in plant cells. Nuclear localization of SAP11 depends on the plant host factor importin a. SAP11 also moves out of the phloem as it is detected in nuclei of mesophyll and other cells of young tissues in AY-WB-infected plants. Finally, there is evidence that SAP11 differentially regulates several plant genes. Thus, SAP11 appears to be a virulence factor although the mechanism of action remains to be determined. Secreted proteases and cellulases appear to be crucial to virulence in Cms and Cmm. Cms secretes an endo-b-1, 4-glucanase, CelA that consists of a cellulose-binding domain, a catalytic domain, and a C-terminal expansinlike domain; CelA is required for wilt induction by this pathogen [30]. Homologues of CelA that lack the expansin-like domain are present in other xylem colonizing plant pathogens, including Cms and Lxx, suggesting a role in degradation of xylem cell walls. Serine proteases, encoded by the pat-1 and multiple chp genes, are pathogenicity determinants in Cmm. There is a single Chp homologue in Lxx, but Cms contains 11 members of the Chp family [13,20,21]. It has been speculated that these proteins might modulate plant defences, as do some cysteine proteases in Proteobacteria [29]. Pathogen-derived small molecules play crucial roles in pathogenicity through manipulation of plant metabolism and signaling. The best-characterized toxin produced by Gram-positive plant pathogens is thaxtomin A, a nitrated www.sciencedirect.com
dipeptide required for virulence in S. scabies and other scab-causing streptomycetes. Biosynthesis is via a nonribosomal peptide pathway and includes a nitric oxide (NO) synthase; NO is utilized in the nitration of the tryptophan moiety [31] and NO is released at the host– pathogen interface where it is available to modulate host signaling [32]. Thaxtomin A inhibits the synthesis of cellulose in expanding plant cells via an unknown mechanism that appears to be conserved among higher plants [33,34]. Thaxtomin A and other members of the thaxtomin family are produced exclusively by scab-causing streptomycetes, an example of a novel pathogenicity determinant, and the biosynthetic pathway is carried on a PAI, mobilization of which is responsible for emergence of pathogenic species in agricultural systems [35]. Thaxtomin and NO are induced by cellobiose, which is a ligand for the AraC/XylE family regulator TxtR [36]; apparently cellobiose is a signal for expanding plant tissue. The fas operon in R. fascians is an interesting example of a highly regulated pathway that produces a modified cytokinin and that is absolutely required for virulence in this pathogen. Interestingly, the fas operon also exists on the PAI of S. turgidiscabies; it confers on this soil borne pathogen the ability to produce leafy galls on aerial plant parts [37].
Horizontal gene transfer: a short cut to virulence Genome sequences reveal the importance of lateral gene transfer (HGT) in evolution of virulence in both Actinobacteria and Firmicutes. The pat-1 gene, which is required for virulence in Cmm is plasmid borne [21,38]. The tomA gene in Cmm [39] lies on a 129-kb region with a lower G + C content than the rest of the genome [21]. This region also encodes several serine proteases that are required for virulence [21]. The tomA gene, which is believed to be involved suppression of plant defence responses, has a homologue on the PAI of S. turgidiscabies. This PAI resides on a 660 kb integrative conjugative element that also encodes the thaxtomin biosynthetic pathway, nec1 and the fas operon, and is capable of mobilizing to other Streptomyces spp. [35]. The fas operon on this PAI is homologous and colinear and with the fas operon of R. fascians [37,40]. The Lxx genome has four HGT regions encoding pectinases and polygalacturonases and a 50-kb HGT region harbouring a celA homologue and the gene suggested to be involved in the biosynthesis of the plant hormone abscissic acid [13]. Phytoplasma genomes harbour many repeated sequences that are organized in clusters resembling composite transposons named potential mobile units (PMUs) [10]. The PMUs are up to 20 kb in size and contain genes for a specialized transcription factor (sigF), DNA replication (dnaB, dnaG), DNA synthesis (tmk), DNA recombination (ssb, himA), membrane-targeted proteins, and tra5 that is a Current Opinion in Plant Biology 2008, 11:449–456
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IS3 family insertion sequence encoding a full-length ORFAB fusion transposase [10]. Phytoplasma genomes have many copies of PMUs and PMU-like clusters [14,41]. The latter appear to be degenerate versions of PMUs [10]. PMU and PMU-like clusters are prevalent in 500 kb of the phytoplasma chromosomes but completely absent from 250-kb chromosome regions harbouring predominantly genes for important metabolic functions in phytoplasmas [10]. One PMU, PMU1, in the genome of Aster Yellows strain witches’ broom (AY-WB) phytoplasma contains full-length versions of all genes with known functions and is also flanked by 328-bp inverted repeats upstream of sigB (first gene of PMU1) and downstream of tra5 (last gene of PMU1) [10]. Recent results suggest that the AY-WB genome contains a second, probably circular, copy of PMU1 [7]. Some PMU-like regions encode small secreted proteins of which one is the candidate virulence protein SAP11 [7]. Phytoplasmas can have two to four plasmids that vary in size [10,42,43]. The plasmids contain genes for replicases (Rep) similar to those found in geminiviruses (plant viruses) and circoviruses (animal viruses) [44]. Many of the plasmid genes are predicted to encode secreted proteins and several spontaneous mutants of OY-M were not insect transmissible [44] and lacked several plasmid genes [42], suggesting a relationship between the plasmids and phytoplasma insect transmissibility [42]. Plasmids of spiroplasmas are also involved in insect transmission. These plasmids harbour adhesins and components of type IV translocation systems [45–47]. In addition, plasmid pSci6 contains P32, which is a hydrophilic protein of unknown function that is associated with insect-transmissible S. citri strains [48,49].
pathogens is that they all are under explored, fascinating pathosystems.
Acknowledgements A portion of the research described here was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2002-35600-12752 to Saskia Hogenhout and 2005-35319-15289 to Rosemary Loria. Assistance with Figure 2 by Simon Moll is gratefully acknowledged. We thank El-Desouky Ammar for providing the electron micrographs in Figure 3, and Heather Kingdom for generating the AY-WB-infected N. benthamiana plants shown in Figure 4b.
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Conclusions Actinobacteria and Firmicutes have evolved exclusive strategies for plant pathogenicity independent of Proteobacteria. Novel virulence factors, including the cellulose biosynthesis inhibitor thaxtomin, a highly modified cytokinin produced by the fas operon, and a family of serine proteases have been characterized. There is also abundant evidence for acquisition of virulence genes through horizontal gene transfer and there are several examples of homologous virulence genes in multiple genera. Movement of PAIs and PMUs among species occurs and in at least some cases may be sufficient for emergence of new pathogenic species. Research on actinobacterial plant pathogens and their fastidious relatives, the phytoplasmas and spiroplasmas, has revealed novel toxins, secreted proteins and plant hormones, which serve to manipulate plant metabolic and defence pathways, largely by unknown mechanisms. The most important commonality among these extremely diverse Gram-positive plant Current Opinion in Plant Biology 2008, 11:449–456
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32. Johnson EG, Sparks JP, Dzikovski B, Crane BR, Gibson DM, Loria R: Plant-pathogenic Streptomyces species produce nitric oxide synthase-derived nitric oxide in response to host signals. Chem Biol 2008, 15:43-50. Nitric oxide is a potent signaling molecule in plants and animals. This paper demonstrates for the first time that bacterial nitric oxide synthases produce nitric oxide at the host–pathogen interface where it is available to modulate plant metabolism. 33. Fry BA, Loria R: Thaxtomin A: evidence for a plant cell wall target. Phys Mol Plant Pathol 2002, 60:1-8. 34. Scheible WR, Fry BA, Kochevenko A, Schindelasch D, Zimmerli L, Somerville S, Loria R, Somerville CR: An Arabidopsis mutant resistant to thaxtomin A, a cellulose synthesis inhibitor from Streptomyces species. Plant Cell 2003, 15:1781-1794. 35. Kers JA, Cameron KD, Joshi MV, Bukhalid RA, Morello JE, Wach MJ, Gibson DM, Loria R: A large, mobile pathogenicity island confers plant pathogenicity on Streptomyces species. Mol Microbiol 2005, 55:1025-1033. 36. Joshi MV, Bignell DR, Johnson EG, Sparks JP, Gibson DM, Loria R: The AraC/XylS regulator TxtR modulates thaxtomin biosynthesis and virulence in Streptomyces scabies. Mol Microbiol 2007, 66:633-642. 37. Joshi MV, Loria R: Streptomyces turgidiscabies possesses a functional cytokinin biosynthetic pathway and produces leafy galls. Mol Plant Microbe Interact 2007, 20:751-758. 38. Dreier J, Meletzus D, Eichenlaub R: Characterization of the plasmid encoded virulence region pat-1 of phytopathogenic Clavibacter michiganensis subsp. michiganensis. Mol Plant Microbe Interact 1997, 10:195-206. 39. Kaup O, Grafen I, Zellermann EM, Eichenlaub R, Gartemann KH: Identification of a tomatinase in the tomato-pathogenic actinomycete Clavibacter michiganensis subsp. michiganensis NCPPB382. Mol Plant Microbe Interact 2005, 18:1090-1098. 40. Cornelis K, Maes T, Jaziri M, Holsters M, Goethals K: Virulence genes of the phytopathogen Rhodococcus fascians show specific spatial and temporal expression patterns during plant infection. Mol Plant Microbe Interact 2002, 15:398-403. 41. Jomantiene R, Zhao Y, Davis RE: Sequence-variable mosaics: composites of recurrent transposition characterizing the genomes of phylogenetically diverse phytoplasmas. DNA Cell Biol 2007, 26:557-564. 42. Nishigawa H, Oshima K, Kakizawa S, Jung HY, Kuboyama T, Miyata S, Ugaki M, Namba S: A plasmid from a non-insecttransmissible line of a phytoplasma lacks two open reading frames that exist in the plasmid from the wild-type line. Gene 2002, 298:195-201. Current Opinion in Plant Biology 2008, 11:449–456
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43. Liefting LW, Shaw ME, Kirkpatrick BC: Sequence analysis of two plasmids from the phytoplasma beet leafhopper-transmitted virescence agent. Microbiology 2004, 150:1809-1817. 44. Oshima K, Shiomi T, Kuboyama T, Sawayanagi T, Nishigawa H, Kakizawa S, Miyata S, Ugaki M, Namba S: Isolation and characterization of derivative lines of the onion yellows phytoplasma that do not cause stunting or phloem hyperplasia. Phytopathology 2001, 91:1024-1029. 45. Bai X, Fazzolari T, Hogenhout SA: Identification and characterization of traE genes of Spiroplasma kunkelii. Gene 2004, 336:81-91. 46. Davis RE, Dally EL, Jomantiene R, Zhao Y, Roe B, Line SP, Shao J: Cryptic plasmid pSKU146 from the wall-less plant pathogen Spiroplasma kunkelii encodes an adhesin and components of a type IV translocation-related conjugation system. Plasmid 2005, 53:179-190. 47. Joshi BD, Berg M, Rogers J, Fletcher J, Melcher U: Sequence comparisons of plasmids pBJS-O of Spiroplasma citri and pSKU146 of Spiroplasma kunkelii: implications for plasmid evolution. BMC Genomics 2005, 6:. 48. Berho N, Duret S, Danet JL, Renaudin J: Plasmid pSci6 from Spiroplasma citri GII-3 confers insect transmissibility to the
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non-transmissible strain S. citri 44. Microbiology-Sgm 2006, 152:2703-2716. The authors show genetic evidence that a plasmid carries the determinant(s) required for insect transmission. 49. Killiny N, Batailler B, Foissac X, Saillard C: Identification of a Spiroplasma citri hydrophilic protein associated with insect transmissibility. Microbiology-Sgm 2006, 152:1221-1230. 50. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997, 25:4876-4882. 51. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987, 4:406-425. 52. Perriere G, Gouy M: WWW-Query: an on-line retrieval system for biological sequence banks. Biochimie 1996, 78:364-369. 53. Huson DH, Richter DC, Rausch C, Dezulian T, Franz M, Rupp R: Dendroscope—an interactive viewer for large phylogenetic trees. BMC Bioinformatics 2007, 8:460.
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Virulence mechanisms of Gram-positive plant pathogenic bacteria Saskia A Hogenhout1 and Rosemary Loria2 Actinobacteria and Firmicutes comprise a group of highly divergent prokaryotes known as Gram-positive bacteria, which are ancestral to Gram-negative bacteria. Comparative genomics is revealing that, though plant virulence genes are frequently located on plasmids or in laterally acquired gene clusters, they are rarely shared with Gram-negative bacterial plant pathogens and among Gram-positive genera. Grampositive bacterial pathogens utilize a variety of virulence strategies to invade their plant hosts, including the production of phytotoxins to allow intracellular and intercellular replication, production of cytokinins to generate gall tissues for invasion, secretion of proteins to induce cankers and the utilization and manipulation of sap-feeding insects for introduction into the phloem sieve cells. Functional analysis of novel virulence genes utilized by Actinobacteria and Firmicutes is revealing how these ancient prokaryotes manipulate plant, and sometimes insect, metabolic processes for their own benefit. Addresses 1 Department of Disease and Stress Biology, John Innes Centre, Norwich Research Park, Colney Lane, Colney, Norwich NR4 7UH, United Kingdom 2 Department of Plant Pathology and Plant-Microbe Biology, 360 Plant Science Building, Cornell University, Ithaca, NY 14853, USA Corresponding author: Hogenhout, Saskia A ([email protected]) and Loria, Rosemary ([email protected])
Current Opinion in Plant Biology 2008, 11:449–456 This review comes from a themed issue on Biotic Interactions Edited by Murray Grant and Sophien Kamoun Available online 17th July 2008 1369-5266/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2008.05.007
Introduction Research on Gram-positive bacterial plant pathogens has resulted in the discovery and characterization of novel virulence determinants. Phylogenetic distance (Figure 1) and fundamental differences in cell structure between Gram-negative and Gram-positive bacteria predict that these bacteria do not share vertically transferred plant pathogenicity genes. Furthermore, the two clades that compose the Gram-positive bacteria, the Actinobacteria (high G + C) and the Firmicutes (low G + C), diverged very early in evolutionary history [1] (Figure 1), suggesting that these phyla are unlikely to share ancestral www.sciencedirect.com
pathogenicity loci either. Recent progress in molecular genetics and genomics of plant pathogenicity in the actinobacterial genera Clavibacter [2], Streptomyces [3], Leifsonia [4] and Rhodococcus [5] and in the Firmicute genera Spiroplasma [6] and Candidatus Phytoplasma [7], both of which lie within the class Mollicutes [8,9], support a model of phyla-specific virulence genes enhanced through horizontal gene transfer (HGT). Representative genomes of plant pathogenic Firmicutes and Actinobacteria have been sequenced to completion [2,10–16], revealing genes encoding authentic and putative adhesins, toxins, plant hormones and secreted virulence proteins. Here we compare pathogenicity strategies in a few of the diverse bacteria gathered under the Gram-positive ‘umbrella’, highlighting representative plant pathogens in both the Firmicutes and Actinobacteria.
Host colonization and symptomology Plant pathogenic Actinobacteria in the genera Streptomyces and Rhodococcus have very wide host ranges, including economically important crops and model plants. Streptomyces scabies is the most widely distributed and best studied of at least 10 species that cause scab diseases of potato and other laterally expanding underground plant tissues and share virulence genes (reviewed in [3]). However, these pathogens readily infect monocot and dicot seedlings, causing root rot and seedling death; the lack of host specificity corresponds to the inability of scab-causing streptomycetes to produce the hypersensitive response (Loria unpublished). Regardless of plant host or tissue, entry occurs through expanding plant tissues without the requirement for natural openings or wounds, and pathogen growth proceeds both intracellularly and intercellularly (Figure 2). Rhodococcus fascians also infects both monocot and dicot hosts, many of which are economically important [17]. Extensive epiphytic growth precedes intercellular invasion through stomata. Leaf deformation, witches’ broom symptoms, fasciations and leafy galls are caused by hyper induction of shoots through activation of dormant axillary meristems and de novo meristem formation. Symptoms are believed to be the result of elaborate manipulation of host hormone balances and pathogen derived auxin and cytokinin [18,19]. In contrast to Streptomyces and Rhodococcus, plant pathogenic species in the Actinobacteria genera Clavibacter and Leifsonia are host-specific at the species or subspecies level. Clavibacter michiganensis is composed of a number of host-specific subspecies all of which colonize the xylem. Current Opinion in Plant Biology 2008, 11:449–456
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Figure 1
The Actinobacteria, Firmicutes and Proteobacteria diverged early in evolutionary history. The 16S ribosomal RNA gene sequences of representative species were aligned in ClustalX v. 1.83 [50]. The alignment served as input for generating a phylogenetic tree using the Neighbour Joining (NJ) method [51] of ClustalX. Positions with gaps were excluded from the tree calculations. Confidence values of a 1000 bootstrap trials are indicated at the nodes. The tree was visualized in NJ Plot [52] and Dendroscope [53] software. GenBank accession numbers: Acholeplasma laidwadii (NC_010163); Agrobacterium tumefaciens (NC_003062); Aster Yellows phytoplasma strain witches’ broom (AY-WB) (NC_007716); Bacillus subtilis (NC_000964); Clavibacter michiganensis subsp. michiganensis (NC_009480); Erwinia carotovora subsp. atroseptica (NC_004547); Escherichia coli K12 (NC_000913); Leifsonia xyli (NC_006087); Mycobacterium tuberculosis (NC_009525); Mycoplasma genitalium (NC_000908); Pseudomonas syringae pv. tomato (NC_004578); Rhizobium etli (NC_007761); Rhodococcus fascians (AB211229); Spiroplasma citri (X63781); Streptococcus pneumoniae (NC_008533); Streptomyces coelicolor (NC_03888); Streptomyces turgidiscabies (AB026221); Sulfolobus solfataricus (NC_002754); Xanthomonas campestris pv. vesicatoria (NC_007508); Xylella fastidiosa Temecula 1 (NC_004556).
The genomes of C. michiganensis subsp. michiganensis (Cmm), a tomato pathogen and C. michiganensis subsp. sepidonicus (Cms), a potato pathogen, are sequenced [20,21]. Genome comparisons show substantial gene content diversity within the species, apparently driven by differences in the ecology of the subspecies; Cms lives as an endophyte in a vegetatively propagated crop while Cmm also grows epiphytically and infects seeds and epidermal tissue. The genus Leifsonia includes xylemlimited, fastidious bacterial pathogens. The best known of these pathogens is L. xyli subsp. xyli, (Lxx) the causative agent of a systemic disease called ratoon stunting of sugarcane. Plant growth inhibition, the hallmark of this disease, may be due to a putative fatty acid desaturase that modifies the carotenoid biosynthesis pathway to produce abscisic acid, a growth inhibitor [4,13]. Phytoplasmas and two Spiroplasma spp., Spiroplasma citri and Spiroplasma kunkelii, parasitize both plants and insects Current Opinion in Plant Biology 2008, 11:449–456
(Figure 3), a feat that requires manipulation of phylogenetically diverse eukaryotic cells. In plants, these fastidious Firmicutes are largely restricted to the cytoplasm of phloem cells (Figure 3b), but some have very broad plant host ranges (reviewed in [7]). S. citri is an economically important pathogen of Citrus spp. that also infects many other plants including Arabidopsis thaliana. By contrast, S. kunkelii only infects maize. Some phytoplasma groups, particularly the AY (Aster Yellows) and STOL (stolbur) group phytoplasmas, have very broad host ranges. Aster Yellows phytoplasma strain witches’ broom (AY-WB) alone can infect China aster, lettuce, Nicotiana benthamiana, tomato, A. thaliana and maize [7]. In insects, phytoplasmas and spiroplasmas have to cross several barriers (Figure 3a), including for instance the gut epithelial cell layer (Figure 3c). Interactions of the phytoplasma surface exposed Antigenic membrane proteins (Amps) with the microfilaments of the insect intestinal tract play a major role in phytoplasma transmission by insects [22,23]. www.sciencedirect.com
Virulence of Gram-positive bacteria Hogenhout and Loria 451
Figure 2
Streptomyces turgidiscabies is among the streptomycete species that infect subterranean plant structures, causing scab diseases and necrosis and stunting of fibrous roots. Confocal micrographs showing S. turgidiscabies, expressing enhanced green fluorescent protein (EGFP), colonizing a radish seedling root at 15 days post-inoculation. (a) Extensive intercellular and intracellular colonization. (b) Intracellular colonization of a radish trichoblast. Image credit Simon Moll.
Plants infected with phytoplasmas and spiroplasmas are frequently stunted, have discoloured leaves and produce abnormal flowers, fruits or seeds. Phytoplasmas also interfere with plant development as they induce, for instance, phyllody (retrograde metamorphosis of floral organs to the condition of leaves), witches’ broom (growth of a dense mass of shoots from a single point), virescence (green
coloration of non-green flower parts) and bolting (growth of elongated stalks). Because the majority of insect vectors of phytoplasmas lay eggs in young green plant tissues, these plant symptoms may increase insect vector progeny numbers and phytoplasma infection that, in turn, promotes phytoplasma dispersal in nature. Indeed, the longevity and reproduction of the leafhopper vector
Figure 3
The phytoplasma life cycle involves replication in plants and insects. (a) Schematic illustration of the different stages of phytoplasma movement through the leafhopper and plant hosts. Phytoplasmas are indicated as yellow dots and phytoplasma movement is indicated with yellow block arrows. Leafhoppers acquire phytoplasmas from the plant phloem. Phytoplasmas are ingested with plant sap and move through the stylet’s food canal to the midgut. Phytoplasmas cross the basal lamina to enter the hemolymph, from where they can move to the salivary gland. They enter the secretory salivary gland cells and are then transported along with the saliva into the plant phloem tissue during leafhopper feeding. The latent period, that is, the time between initial acquisition of the phytoplasmas by the insect vector from plants and the ability for the insect to introduce phytoplasmas back into plants, can vary between 7 and 80 days, depending on the insect vector and plant host species. (b and c) Electron micrographs of phytoplasmas in the plant phloem and in leafhopper midgut as indicated in (a). (b) Phytoplasmas (arrowheads) in adjacent sieve elements (se1) close to the nucleus (n), in a China aster leaf; the arrow indicates a sieve pore between the sieve plates (sp), and asterisks indicate possibly dividing phytoplasma cells. (c) Accumulations of phytoplasmas (arrowheads) in the cytoplasm of a cell near the nucleus (n) in the muscle layer of the midgut. Scale bars, 1 mm. Credit for electron micrographs shown in b and c, El-Desouky Ammar. www.sciencedirect.com
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Macrosteles quadrilineatus is greater on AY phytoplasmainfected plants than on healthy plants [24]. Thus, AY-WB phytoplasmas have a marked effect on plant–insect interactions, that is, they convert plants into better hosts for phloem-feeding insects. S. citri symptom development is linked to carbohydrate utilization preference in the phloem. Fructose uptake through the phosphoenolpyruvate:fructose phosphotransferase system (fructose PTS) by S. citri induces severe symptoms [25], whereas glucose uptake through the glucose PTS does not [26]. It is likely that in S. citri infected plants the activity of invertase, which converts sucrose to fructose and glucose in phloem companion cells, increases in response to low fructose concentrations [26]. This, in turn, leads to abnormally high glucose concentrations in the phloem, resulting in the chlorosis and internode shortening typical of S. citri infected plants [26]. The genome sequences of OY-M and AY-WB have revealed that phytoplasmas do not possess characterized sugar PTSs but have ATP-binding cassette (ABC) trans-
porters for uptake of maltose, trehalose, sucrose and palatinose [10,14]. The utilization of one or more of these sugars in the phloem may be responsible for induction of symptoms in phytoplasma-infected plants. However, the phyllody, witches’ broom, virescence and bolting are probably induced through other mechanisms. Since phytoplasma genomes lack identifiable plant hormone biosynthesis pathways, one or more of the 56 secreted virulence proteins of AY-WB are probably responsible for symptom induction (reviewed in [7]). Of these 56 secreted proteins, 51 are predicted to be <40 kDa and therefore may be able to traverse the plasmodesmata that connect phloem cells with other cell in developing, but not mature, plant tissues (Figure 4a). Indeed, symptoms in phytoplasma-infected plants are most prevalent in developing tissues (Figure 4b).
Virulence factors Secreted proteins that act inside the plant cell are central to microbial pathogenesis [27]. The lack of a type III protein secretion system (TTSS) in Firmicutes and Actinobacteria immediately raises questions about delivery of virulence proteins across the plant cell wall
Figure 4
Aster Yellows phytoplasma strain witches’ broom (AY-WB) induces severe symptoms in young plant tissues. (a) Schematic illustration showing systemic movement of secreted AY-WB proteins (SAPs) young plant tissues. AY-WB phytoplasmas secrete various SAPs, including SAP11, into the phloem. These SAPs then unload from phloem cells into mesophyll and other cell types of young (sink) plant parts where they induce witches’ broom, chlorosis and other aberrations. (b) AY-WB symptoms are most obvious in young growing points (the crown) of Nicotiana benthamiana plants consistent with the systemic movement of SAPs (as shown in a). The inset shows a close-up of the crown of one of the AY-WB-infected plants. Current Opinion in Plant Biology 2008, 11:449–456
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Virulence of Gram-positive bacteria Hogenhout and Loria 453
and membrane—questions that largely remain unanswered. S. scabies, Streptomyces turgidiscabies and S. acidiscabies directly penetrate plant cell walls with the help of thaxtomin, a phytotoxin that inhibits cellulose biosynthesis (reviewed in [3]), presumably allowing secretion of proteins at the host cell membrane. These pathogens secrete Nec1, a novel protein that is proposed to suppress host defence through an unknown mechanism [28]. S. turgidiscabies nec1 deletion mutants that produced wild-type levels of thaxtomin were unable to colonize radish roots under conditions in which the wild-type strain ramified apical and subapical cells within 24 h. Nec1 has an unknown target but is secreted via an N-terminal signal sequence and is expected to interact with host proteins in the cytoplasm [28]. By contrast, proteins that manipulate plant defence in Gram-negative bacteria typically cross bacterial and host membranes via the TTSS [29]. By contrast, spiroplasmas and phytoplasmas are injected into the cytoplasm of phloem cells by their insect vectors (reviewed in [7]). These pathogens secrete proteins into the phloem cell cytoplasm allowing movement of these proteins to other plant cells, for example, to developing (sink) tissue cells through plasmodesmata (Figure 4). The AY-WB secreted protein, SAP11, has been characterized [7]. SAP11 is 11 kDa and contains a bipartite nuclear localization signal (NLS) that is functional in plant cells. Nuclear localization of SAP11 depends on the plant host factor importin a. SAP11 also moves out of the phloem as it is detected in nuclei of mesophyll and other cells of young tissues in AY-WB-infected plants. Finally, there is evidence that SAP11 differentially regulates several plant genes. Thus, SAP11 appears to be a virulence factor although the mechanism of action remains to be determined. Secreted proteases and cellulases appear to be crucial to virulence in Cms and Cmm. Cms secretes an endo-b-1, 4-glucanase, CelA that consists of a cellulose-binding domain, a catalytic domain, and a C-terminal expansinlike domain; CelA is required for wilt induction by this pathogen [30]. Homologues of CelA that lack the expansin-like domain are present in other xylem colonizing plant pathogens, including Cms and Lxx, suggesting a role in degradation of xylem cell walls. Serine proteases, encoded by the pat-1 and multiple chp genes, are pathogenicity determinants in Cmm. There is a single Chp homologue in Lxx, but Cms contains 11 members of the Chp family [13,20,21]. It has been speculated that these proteins might modulate plant defences, as do some cysteine proteases in Proteobacteria [29]. Pathogen-derived small molecules play crucial roles in pathogenicity through manipulation of plant metabolism and signaling. The best-characterized toxin produced by Gram-positive plant pathogens is thaxtomin A, a nitrated www.sciencedirect.com
dipeptide required for virulence in S. scabies and other scab-causing streptomycetes. Biosynthesis is via a nonribosomal peptide pathway and includes a nitric oxide (NO) synthase; NO is utilized in the nitration of the tryptophan moiety [31] and NO is released at the host– pathogen interface where it is available to modulate host signaling [32]. Thaxtomin A inhibits the synthesis of cellulose in expanding plant cells via an unknown mechanism that appears to be conserved among higher plants [33,34]. Thaxtomin A and other members of the thaxtomin family are produced exclusively by scab-causing streptomycetes, an example of a novel pathogenicity determinant, and the biosynthetic pathway is carried on a PAI, mobilization of which is responsible for emergence of pathogenic species in agricultural systems [35]. Thaxtomin and NO are induced by cellobiose, which is a ligand for the AraC/XylE family regulator TxtR [36]; apparently cellobiose is a signal for expanding plant tissue. The fas operon in R. fascians is an interesting example of a highly regulated pathway that produces a modified cytokinin and that is absolutely required for virulence in this pathogen. Interestingly, the fas operon also exists on the PAI of S. turgidiscabies; it confers on this soil borne pathogen the ability to produce leafy galls on aerial plant parts [37].
Horizontal gene transfer: a short cut to virulence Genome sequences reveal the importance of lateral gene transfer (HGT) in evolution of virulence in both Actinobacteria and Firmicutes. The pat-1 gene, which is required for virulence in Cmm is plasmid borne [21,38]. The tomA gene in Cmm [39] lies on a 129-kb region with a lower G + C content than the rest of the genome [21]. This region also encodes several serine proteases that are required for virulence [21]. The tomA gene, which is believed to be involved suppression of plant defence responses, has a homologue on the PAI of S. turgidiscabies. This PAI resides on a 660 kb integrative conjugative element that also encodes the thaxtomin biosynthetic pathway, nec1 and the fas operon, and is capable of mobilizing to other Streptomyces spp. [35]. The fas operon on this PAI is homologous and colinear and with the fas operon of R. fascians [37,40]. The Lxx genome has four HGT regions encoding pectinases and polygalacturonases and a 50-kb HGT region harbouring a celA homologue and the gene suggested to be involved in the biosynthesis of the plant hormone abscissic acid [13]. Phytoplasma genomes harbour many repeated sequences that are organized in clusters resembling composite transposons named potential mobile units (PMUs) [10]. The PMUs are up to 20 kb in size and contain genes for a specialized transcription factor (sigF), DNA replication (dnaB, dnaG), DNA synthesis (tmk), DNA recombination (ssb, himA), membrane-targeted proteins, and tra5 that is a Current Opinion in Plant Biology 2008, 11:449–456
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IS3 family insertion sequence encoding a full-length ORFAB fusion transposase [10]. Phytoplasma genomes have many copies of PMUs and PMU-like clusters [14,41]. The latter appear to be degenerate versions of PMUs [10]. PMU and PMU-like clusters are prevalent in 500 kb of the phytoplasma chromosomes but completely absent from 250-kb chromosome regions harbouring predominantly genes for important metabolic functions in phytoplasmas [10]. One PMU, PMU1, in the genome of Aster Yellows strain witches’ broom (AY-WB) phytoplasma contains full-length versions of all genes with known functions and is also flanked by 328-bp inverted repeats upstream of sigB (first gene of PMU1) and downstream of tra5 (last gene of PMU1) [10]. Recent results suggest that the AY-WB genome contains a second, probably circular, copy of PMU1 [7]. Some PMU-like regions encode small secreted proteins of which one is the candidate virulence protein SAP11 [7]. Phytoplasmas can have two to four plasmids that vary in size [10,42,43]. The plasmids contain genes for replicases (Rep) similar to those found in geminiviruses (plant viruses) and circoviruses (animal viruses) [44]. Many of the plasmid genes are predicted to encode secreted proteins and several spontaneous mutants of OY-M were not insect transmissible [44] and lacked several plasmid genes [42], suggesting a relationship between the plasmids and phytoplasma insect transmissibility [42]. Plasmids of spiroplasmas are also involved in insect transmission. These plasmids harbour adhesins and components of type IV translocation systems [45–47]. In addition, plasmid pSci6 contains P32, which is a hydrophilic protein of unknown function that is associated with insect-transmissible S. citri strains [48,49].
pathogens is that they all are under explored, fascinating pathosystems.
Acknowledgements A portion of the research described here was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2002-35600-12752 to Saskia Hogenhout and 2005-35319-15289 to Rosemary Loria. Assistance with Figure 2 by Simon Moll is gratefully acknowledged. We thank El-Desouky Ammar for providing the electron micrographs in Figure 3, and Heather Kingdom for generating the AY-WB-infected N. benthamiana plants shown in Figure 4b.
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Conclusions Actinobacteria and Firmicutes have evolved exclusive strategies for plant pathogenicity independent of Proteobacteria. Novel virulence factors, including the cellulose biosynthesis inhibitor thaxtomin, a highly modified cytokinin produced by the fas operon, and a family of serine proteases have been characterized. There is also abundant evidence for acquisition of virulence genes through horizontal gene transfer and there are several examples of homologous virulence genes in multiple genera. Movement of PAIs and PMUs among species occurs and in at least some cases may be sufficient for emergence of new pathogenic species. Research on actinobacterial plant pathogens and their fastidious relatives, the phytoplasmas and spiroplasmas, has revealed novel toxins, secreted proteins and plant hormones, which serve to manipulate plant metabolic and defence pathways, largely by unknown mechanisms. The most important commonality among these extremely diverse Gram-positive plant Current Opinion in Plant Biology 2008, 11:449–456
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31. Kers JA, Wach MJ, Krasnoff SB, Widom J, Cameron KD, Bukhalid RA, Gibson DM, Crane BR, Loria R: Nitration of a peptide phytotoxin by bacterial nitric oxide synthase. Nature 2004, 429:79-82.
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32. Johnson EG, Sparks JP, Dzikovski B, Crane BR, Gibson DM, Loria R: Plant-pathogenic Streptomyces species produce nitric oxide synthase-derived nitric oxide in response to host signals. Chem Biol 2008, 15:43-50. Nitric oxide is a potent signaling molecule in plants and animals. This paper demonstrates for the first time that bacterial nitric oxide synthases produce nitric oxide at the host–pathogen interface where it is available to modulate plant metabolism. 33. Fry BA, Loria R: Thaxtomin A: evidence for a plant cell wall target. Phys Mol Plant Pathol 2002, 60:1-8. 34. Scheible WR, Fry BA, Kochevenko A, Schindelasch D, Zimmerli L, Somerville S, Loria R, Somerville CR: An Arabidopsis mutant resistant to thaxtomin A, a cellulose synthesis inhibitor from Streptomyces species. Plant Cell 2003, 15:1781-1794. 35. Kers JA, Cameron KD, Joshi MV, Bukhalid RA, Morello JE, Wach MJ, Gibson DM, Loria R: A large, mobile pathogenicity island confers plant pathogenicity on Streptomyces species. Mol Microbiol 2005, 55:1025-1033. 36. Joshi MV, Bignell DR, Johnson EG, Sparks JP, Gibson DM, Loria R: The AraC/XylS regulator TxtR modulates thaxtomin biosynthesis and virulence in Streptomyces scabies. Mol Microbiol 2007, 66:633-642. 37. Joshi MV, Loria R: Streptomyces turgidiscabies possesses a functional cytokinin biosynthetic pathway and produces leafy galls. Mol Plant Microbe Interact 2007, 20:751-758. 38. Dreier J, Meletzus D, Eichenlaub R: Characterization of the plasmid encoded virulence region pat-1 of phytopathogenic Clavibacter michiganensis subsp. michiganensis. Mol Plant Microbe Interact 1997, 10:195-206. 39. Kaup O, Grafen I, Zellermann EM, Eichenlaub R, Gartemann KH: Identification of a tomatinase in the tomato-pathogenic actinomycete Clavibacter michiganensis subsp. michiganensis NCPPB382. Mol Plant Microbe Interact 2005, 18:1090-1098. 40. Cornelis K, Maes T, Jaziri M, Holsters M, Goethals K: Virulence genes of the phytopathogen Rhodococcus fascians show specific spatial and temporal expression patterns during plant infection. Mol Plant Microbe Interact 2002, 15:398-403. 41. Jomantiene R, Zhao Y, Davis RE: Sequence-variable mosaics: composites of recurrent transposition characterizing the genomes of phylogenetically diverse phytoplasmas. DNA Cell Biol 2007, 26:557-564. 42. Nishigawa H, Oshima K, Kakizawa S, Jung HY, Kuboyama T, Miyata S, Ugaki M, Namba S: A plasmid from a non-insecttransmissible line of a phytoplasma lacks two open reading frames that exist in the plasmid from the wild-type line. Gene 2002, 298:195-201. Current Opinion in Plant Biology 2008, 11:449–456
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