A large, mobile pathogenicity island confers plant pathogenicity on Streptomyces species

Potato scab is a globally important disease caused by polyphyletic plant pathogenic Streptomyces species. Streptomyces acidiscabies, Streptomyces scabies and Streptomyces turgidiscabies possess a conserved biosynthetic pathway for the nitrated dipeptide phytotoxin thaxtomin. These pathogens also possess the nec1 gene which encodes a necrogenic protein that is an independent virulence factor. In this article we describe a large (325-660 kb) pathogenicity island (PAI) conserved among these three plant pathogenic Streptomyces species. A partial DNA sequence of this PAI revealed the thaxtomin biosynthetic pathway, nec1, a putative tomatinase gene, and many mobile genetic elements. In addition, the PAI from S. turgidiscabies contains a plant fasciation (fas) operon homologous to and colinear with the fas operon in the plant pathogen Rhodococcus fascians. The PAI was mobilized during mating from S. turgidiscabies to the non-pathogens Streptomyces coelicolor and Streptomyces diastatochromogenes on a 660 kb DNA element and integrated site-specifically into a putative integral membrane lipid kinase. Acquisition of the PAI conferred a pathogenic phenotype on S. diastatochromogenes but not on S. coelicolor. This PAI is the first to be described in a Gram-positive plant pathogenic bacterium and is responsible for the emergence of new plant pathogenic Streptomyces species in agricultural systems.     

What does it take to be a plant pathogen: genomic insights from <Emphasis Type="Italic">Streptomyces</Emphasis> species

Plant pathogenicity is rare in the genus Streptomyces, with only a dozen or so species possessing this trait out of the more than 900 species described. Nevertheless, such species have had a significant impact on agricultural economies throughout the world due to their ability to cause important crop diseases such as potato common scab, which is characterized by lesions that form on the potato tuber surface. All pathogenic species that cause common scab produce a family of phytotoxins called the thaxtomins, which function as cellulose synthesis inhibitors. In addition, the nec1 and tomA genes are conserved in several pathogenic streptomycetes, the former of which is predicted to function in the suppression of plant defense responses. Streptomyces scabies is the oldest plant pathogen described and has a world-wide distribution, whereas species such as S. turgidiscabies and S. acidiscabies are believed to be newly emergent pathogens found in more limited geographical locations. The genome sequence of S. scabies 87-22 was recently completed, and comparative genomic analyses with other sequenced microbial pathogens have revealed the presence of additional genes that may play a role in plant pathogenicity, an idea that is supported by functional analysis of one such putative virulence locus. In addition, the availability of multiple genome sequences for both pathogenic and nonpathogenic streptomycetes has provided an opportunity for comparative genomic analyses to identify the Streptomyces pathogenome. Such genomic analyses will contribute to the fundamental understanding of the mechanisms and evolution of plant pathogenicity and plant-microbe biology within this genus.     

Production of thaxtomin a by two species of Streptomyces causing potato scab

A total of nine isolates of streptomycetes were isolated from scab lesions on potato tubers. Five out of them were pathogenic on potato minitubers and four of the pathogenic isolates produced thaxtomin A in infected tubers tissues. The lesion surface areas induced by thaxtomin A were highest in treatment of the minitubers with extract of OMB inoculated with S-6 and S-7, intermediate with that inoculated with S-4 and lowest with S-3. The pathogenic isolates were identified by their colour of aerial mycelia, melanin pigment productivity (+ or -), the type of spore chains morphology and carbon utilization as either S. scabies strains S-3, S-4 and S-8, or S. acidiscabies strains S-6 and S-7. S-3 and S-4 produced 0.65 and 1.60 micrograms thaxtomin A per milliliter of OMB, respectively, whereas S-6 and S-7 produced similar amounts of thaxtomin A, 2.36 and 2.10 micrograms per ml of OMB, respectively. The optimal temperature for production of thaxtomin A by S. scabies and S. acidiscabies was 28 degrees C. Production of thaxtomin A by S. scabies strain S-4 and S. acidiscabies strain S-6 was suppressed at least 50-fold at 0.5 and 0.3% of glucose, respectively. Fructose enhanced the production of thaxtomin A by both S. scabies and S. acidiscabies.     

Characterization of an insertion sequence element associated with genetically diverse plant pathogenic Streptomyces spp

Streptomycetes are common soil inhabitants, yet few described species are plant pathogens. While the pathogenicity mechanisms remain unclear, previous work identified a gene, nec1, which encodes a putative pathogenicity or virulence factor. nec1 and a neighboring transposase pseudogene, ORFtnp, are conserved among unrelated plant pathogens and absent from nonpathogens. The atypical GC content of nec1 suggests that it was acquired through horizontal transfer events. Our investigation of the genetic organization of regions adjacent to the 3' end of nec1 in Streptomyces scabies 84.34 identified a new insertion sequence (IS) element, IS1629, with homology to other IS elements from prokaryotic animal pathogens. IS1629 is 1,462 bp with 26-bp terminal inverted repeats and encodes a putative 431-amino-acid (aa) transposase. Transposition of IS1629 generates a 10-bp target site duplication. A 77-nucleotide (nt) sequence encompassing the start codon and upstream region of the transposase was identified which could function in the posttranscritpional regulation of transposase synthesis. A functional copy of IS1629 from S. turgidiscabies 94.09 (Hi-C-13) was selected in the transposon trap pCZA126, through its insertion into the lambda cI857 repressor. IS1629 is present in multiple copies in some S. scabies strains and is present in all S. acidiscabies and S. turgidiscabies strains examined. A second copy of IS1629 was identified between ORFtnp and nec1 in S. acidiscabies strains. The diversity of IS1629 hybridization profiles was greatest within S. scabies. IS1629 was absent from the 27 nonpathogenic Streptomyces strains tested. The genetic organization and nucleotide sequence of the nec1-IS1629 region was conserved and identical among representatives of S. acidiscabies and S. turgidiscabies. These findings support our current model for the unidirectional transfer of the ORFtnp-nec1-IS1629 locus from IS1629-containing S. scabies (type II) to S. acidiscabies and S. turgidiscabies.     

Horizontal transfer of the plant virulence gene, nec1, and flanking sequences among genetically distinct Streptomyces strains in the Diastatochromogenes cluster

Evidence for the horizontal transfer of a pathogenicity island (PAI) carrying the virulence gene nec1 and flanking sequences among Streptomyces strains in the Diastatochromogenes cluster is presented. Plant-pathogenic, thaxtomin-producing Streptomyces strains, previously classified as S. scabiei based on the conventionally used phenotypic characteristics, were found to be genetically distinct from the type strain of S. scabiei based on DNA relatedness and 16S rDNA sequence analysis. Pairwise DNA-DNA hybridizations between some of these strains and the S. scabiei type strain were as low as 36%, a value much below what is conventionally accepted for species identity (70%). The sequence of the nec1 gene, however, was identical in all the S. scabiei and S. scabiei-like strains tested, irrespective of their DNA relatedness to the type strain of S. scabiei, their geographic origin, or the isolation host. Furthermore, a 26-kb DNA fragment including and flanking nec1 was also conserved among these strains based on restriction and Southern analyses. These data indicate that the etiology of potato scab is more complex than previously recognized; this result has important implications for potato scab management strategies. Previous research has suggested that horizontal transfer of a PAI was the mechanism for evolution of pathogenicity in S. acidiscabies and S. turgidiscabies, species that lie outside of the Diastatochromogenes cluster. Data presented here support this model and indicate that PAI transfer also has occurred frequently in species closely related to S. scabiei.     

Changes in race-specific virulence in Pseudomonas syringae pv. phaseolicola are associated with a chimeric transposable element and rare deletion events in a plasmid-borne pathogenicity island

Virulence for bean and soybean is determined by effector genes in a plasmid-borne pathogenicity island (PAI) in race 7 strain 1449B of Pseudomonas syringae pv. phaseolicola. One of the effector genes, avrPphF, confers either pathogenicity, virulence, or avirulence depending on the plant host and is absent from races 2, 3, 4, 6, and 8 of this pathogen. Analysis of cosmid clones and comparison of DNA sequences showed that the absence of avrPphF from strain 1448A is due to deletion of a continuous 9.5-kb fragment. The remainder of the PAI is well conserved in strains 1448A and 1449B. The left junction of the deleted region consists of a chimeric transposable element generated from the fusion of homologs of IS1492 from Pseudomonas putida and IS1090 from Ralstonia eutropha. The borders of the deletion were conserved in 66 P. syringae pv. phaseolicola strains isolated in different countries and representing the five races lacking avrPphF. However, six strains isolated in Spain had a 10.5-kb deletion that extended 1 kb further from the right junction. The perfect conservation of the 28-nucleotide right repeat of the IS1090 homolog in the two deletion types and in the other 47 insertions of the IS1090 homolog in the 1448A genome strongly suggests that the avrPphF deletions were mediated by the activity of the chimeric mobile element. Our data strongly support a clonal origin for the races of P. syringae pv. phaseolicola lacking avrPphF.     

Changes in Race-Specific Virulence in Pseudomonas syringae pv. phaseolicola Are Associated with a Chimeric Transposable Element and Rare Deletion Events in a Plasmid-Borne Pathogenicity Island

Virulence for bean and soybean is determined by effector genes in a plasmid-borne pathogenicity island (PAI) in race 7 strain 1449B of Pseudomonas syringae pv. phaseolicola. One of the effector genes, avrPphF, confers either pathogenicity, virulence, or avirulence depending on the plant host and is absent from races 2, 3, 4, 6, and 8 of this pathogen. Analysis of cosmid clones and comparison of DNA sequences showed that the absence of avrPphF from strain 1448A is due to deletion of a continuous 9.5-kb fragment. The remainder of the PAI is well conserved in strains 1448A and 1449B. The left junction of the deleted region consists of a chimeric transposable element generated from the fusion of homologs of IS1492 from Pseudomonas putida and IS1090 from Ralstonia eutropha. The borders of the deletion were conserved in 66 P. syringae pv. phaseolicola strains isolated in different countries and representing the five races lacking avrPphF. However, six strains isolated in Spain had a 10.5-kb deletion that extended 1 kb further from the right junction. The perfect conservation of the 28-nucleotide right repeat of the IS1090 homolog in the two deletion types and in the other 47 insertions of the IS1090 homolog in the 1448A genome strongly suggests that the avrPphF deletions were mediated by the activity of the chimeric mobile element. Our data strongly support a clonal origin for the races of P. syringae pv. phaseolicola lacking avrPphF.     

Friends and foes: streptomycetes as modulators of plant disease and symbiosis

The ecological role of soil streptomycetes within the plant root environment is currently gaining increased attention. This review describes our recent advances in elucidating the complex interactions between streptomycetes, plants, pathogenic and symbiotic microorganisms. Streptomycetes play diverse roles in plant-associated microbial communities. Some act as biocontrol agents, inhibiting plant interactions with pathogenic organisms. Owing to the antagonistic properties of streptomycetes, they exert a selective pressure on soil microbes, which may not always be for plant benefit. Others promote the formation of symbioses between plant roots and microbes, and this is in part due to their direct positive influence on the symbiotic partner, expressed as, e.g., promotion of hyphal elongation of symbiotic fungi. Recently, streptomycetes have been identified as modulators of plant defence. By repressing plant responses to pathogens they facilitate root colonisation with pathogenic fungi. In contrast, other strains induce local and systemic resistance against pathogens or enhance plant growth. In conclusion, while streptomycetes have a clear potential of acting as biocontrol agents, care has to be taken to avoid strains that select for virulent pathogens or enhance disease development. We argue towards the use of an integrated screening approach in the search for efficient biocontrol agents, including assays on in vitro antagonism, plant growth, and disease suppression.     

Characterization of FP22, a large streptomycete bacteriophage with DNA insensitive to cleavage by many restriction enzymes

Bacteriophage FP22 has a very broad host range within streptomycetes and appeared to form lysogens of Streptomyces ambofaciens ATCC 15154. FP22 shared strong cross-immunity and antibody cross-reactivity with bacteriophage P23, but not with seven other streptomycete bacteriophages. FP22 particles had a head diameter of 71 nm and a tail length of 307 nm. The FP22 genome was 131 kb, which is the largest bacteriophage genome reported for streptomycetes. The G + C content of the genome was 46 mol% and restriction mapping indicated that FP22 DNA had discrete ends. NaCl- and pyrophosphate-resistant deletion mutants were readily isolated and the extent of the deletions defined at least 23 kb of dispensable DNA in two regions of the genome. The DNA was not cleaved by most restriction endonucleases (or isoschizomers) which have been identified in the streptomycetes, including the tetranucleotide cutter MboI (GATC).     

Genome-wide identification of Phytophthora sojae SNARE genes and functional characterization of the conserved SNARE PsYKT6

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central components of the machinery mediating membrane fusion and key factors for vesicular trafficking in all eukaryotic cells. Taking advantage of the available whole genome sequence of the oomycete plant pathogen Phytophthora sojae, 35 genes encoding putative SNARE proteins were identified in the genome of this organism. PsYKT6, one of the most conserved SNARE proteins, was functionally characterized by homology-dependent gene silencing. The phenotype analysis showed that PsYKT6 is important for proper asexual development, sexual reproduction, and pathogenesis on host soybean cultivars.     

Molecular diagnosis of Colletotrichum gloeosporioides causing Anthracnose/Dieback disease in Greater Yam (Dioscorea alata L.)

The Greater Yam (Dioscorea alata L.), a significant tropical tuber crop is highly affected by the anthracnose/dieback disease caused by Colletotrichum gloeosporioides, which greatly reduces the yield as well as market acceptability of the tubers. The different methods that could be used for proper identification of the pathogen by PCR were investigated. The studies indicate that species specific polymerase chain reaction assay based on highly conserved regions in ITS in the genome of the pathogen can be the best strategy for detection of this pathogen at species level. The use of genus specific primers was also successful in detection of Colletotrichum spp. The cloning and sequencing of evolutionarily conserved regions such as ITS, PelB and paralleling them with the available sequences in NCBI database is also costly but reliable approach. The various methods are elaborately tested and their use in diagnosis is discussed in detail.     

Functional Analysis of Hyaloperonospora arabidopsidis RXLR Effectors

The biotrophic plant pathogen Hyaloperonospora arabidopsidis produces a set of putative effector proteins that contain the conserved RXLR motif. For most of these RXLR proteins the role during infection is unknown. Thirteen RXLR proteins from H. arabidopsidis strain Waco9 were analyzed for sequence similarities and tested for a role in virulence. The thirteen RXLR proteins displayed conserved N-termini and this N-terminal conservation was also found in the 134 predicted RXLR genes from the genome of H. arabidopsidis strain Emoy2. To investigate the effects of single RXLR effector proteins on plant defense responses, thirteen H. arabidopsidis Waco9 RXLR genes were expressed in Arabidopsis thaliana. Subsequently, these plants were screened for altered susceptibility to the oomycetes H. arabidopsidis and Phytophthora capsici, and the bacterial pathogen Pseudomonas syringae. Additionally, the effect of the RXLR proteins on flg22-triggered basal immune responses was assessed. Multifactorial analysis of results collated from all experiments revealed that, except for RXLR20, all RXLR effector proteins tested affected plant immunity. For RXLR9 this was confirmed using a P. syringae ΔCEL-mediated effector delivery system. Together, the results show that many H. arabidopsidis RXLR effectors have small effects on the plant immune response, suggesting that suppression of host immunity by this biotrophic pathogen is likely to be caused by the combined actions of effectors.     

Identification of a putative pathogenicity island in Shigella flexneri using subtractive hybridisation of the S. flexneri and Escherichia coli genomes

The genetic differences between the human pathogen, Shigella flexneri, and the non-pathogenic Escherichia coli were investigated in an attempt to identify pathogenicity islands (PAIs) in the S. flexneri genome. Genomic subtraction identified a large unique region of DNA which was present in S. flexneri serotype 2a but absent from E. coli K-12. This 42-kb DNA segment was localised to the S. flexneri chromosome and was found to contain a number of elements often associated with PAIs including: insertion sequence elements, bacteriophage genes, and a previously identified Shigella virulence gene (criR). These findings indicate that this region may form a new PAI in the S. flexneri genome.     

Mapping of gene-specific markers on the genetic map of chickpea (<Emphasis Type="Italic"> Cicer arietinum </Emphasis>L.)

With the exception of the fact that it is made up of eight different chromosomes, the physical organization of the 738-Mb genome of the important legume crop chickpea (Cicer arietinum L.) is unknown. In an attempt to increase our knowledge of the basic structure of this genome, we determined the map positions of a series of genes involved in plant defence responses (DR) by genetic linkage analysis. Exploiting the sequence data available in GenBank, we selected genes known to be induced in chickpea and other plants by pathogen attack. Gene-specific primers were designed based on conserved regions, and used to detect the corresponding gene sequences in a segregating population derived from an interspecific cross between Cicer arietinum and C. reticulatum. Forty-seven gene-specific markers were integrated into an existing map based on STMS, AFLP, DAF and other anonymous markers. The potential of this approach is discussed.     

Hydroxamate siderophores produced by Streptomyces acidiscabies E13 bind nickel and promote growth in cowpea (Vigna unguiculata L.) under nickel stress

The siderophore-producing ability of nickel-resistant Streptomyces acidiscabies E13 and the role of the elicited siderophores in promoting plant growth under iron and nickel stress are described. Siderophore assays indicated that S. acidiscabies E13 can produce siderophores. Electrospray ionization mass spectrometry (ESI-MS) revealed that the bacterium simultaneously produces 3 different hydroxamate siderophores. ESI-MS showed that in addition to iron, all 3 siderophores can bind nickel. In vitro plant growth tests were conducted with cowpea (Vigna unguiculata) in the presence and absence of the elicited siderophores. Culture filtrates containing hydroxamate siderophores significantly increased cowpea height and biomass, irrespective of the iron status of the plants, under nickel stress. The presence of reduced iron was found to be high in siderophore-containing treatments in the presence of nickel. Measurements of iron and nickel contents of cowpea roots and shoots indicated that the siderophore-mediated plant growth promotion reported here involves the simultaneous inhibition of nickel uptake and solubilization and supply of iron to plants. We conclude that hydroxamate siderophores contained in culture filtrates of S. acidiscabies E13 promoted cowpea growth under nickel contamination by binding iron and nickel, thus playing a dual role of sourcing iron for plant use and protecting against nickel toxicity.