The Plant Pathogen Pseudomonas syringae pv. tomato Is Genetically Monomorphic and under Strong Selection to Evade Tomato Immunity

Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today's Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain

The identification of genes important in pseudomonas syringae pv. phaseolicola plant colonisation using in vitro screening of transposon libraries

The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph) colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms) around plant cells. If the pathogen can suppress the plant’s natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn) mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP) genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interactio

Evolutionary and Experimental Assessment of Novel Markers for Detection of Xanthomonas euvesicatoria in Plant Samples

BACKGROUND: Bacterial spot-causing xanthomonads (BSX) are quarantine phytopathogenic bacteria responsible for heavy losses in tomato and pepper production. Despite the research on improved plant spraying methods and resistant cultivars, the use of healthy plant material is still considered as the most effective bacterial spot control measure. Therefore, rapid and efficient detection methods are crucial for an early detection of these phytopathogens. METHODOLOGY: In this work, we selected and validated novel DNA markers for reliable detection of the BSX Xanthomonas euvesicatoria (Xeu). Xeu-specific DNA regions were selected using two online applications, CUPID and Insignia. Furthermore, to facilitate the selection of putative DNA markers, a customized C program was designed to retrieve the regions outputted by both databases. The in silico validation was further extended in order to provide an insight on the origin of these Xeu-specific regions by assessing chromosomal location, GC content, codon usage and synteny analyses. Primer-pairs were designed for amplification of those regions and the PCR validation assays showed that most primers allowed for positive amplification with different Xeu strains. The obtained amplicons were labeled and used as probes in dot blot assays, which allowed testing the probes against a collection of 12 non-BSX Xanthomonas and 23 other phytopathogenic bacteria. These assays confirmed the specificity of the selected DNA markers. Finally, we designed and tested a duplex PCR assay and an inverted dot blot platform for culture-independent detection of Xeu in infected plants. SIGNIFICANCE: This study details a selection strategy able to provide a large number of Xeu-specific DNA markers. As demonstrated, the selected markers can detect Xeu in infected plants both by PCR and by hybridization-based assays coupled with automatic data analysis. Furthermore, this work is a contribution to implement more efficient DNA-based methods of bacterial diagnostics

Complete Genome Sequence of the N2-Fixing Broad Host Range Endophyte Klebsiella pneumoniae 342 and Virulence Predictions Verified in Mice

We report here the sequencing and analysis of the genome of the nitrogen-fixing endophyte, Klebsiella pneumoniae 342. Although K. pneumoniae 342 is a member of the enteric bacteria, it serves as a model for studies of endophytic, plant-bacterial associations due to its efficient colonization of plant tissues (including maize and wheat, two of the most important crops in the world), while maintaining a mutualistic relationship that encompasses supplying organic nitrogen to the host plant. Genomic analysis examined K. pneumoniae 342 for the presence of previously identified genes from other bacteria involved in colonization of, or growth in, plants. From this set, approximately one-third were identified in K. pneumoniae 342, suggesting additional factors most likely contribute to its endophytic lifestyle. Comparative genome analyses were used to provide new insights into this question. Results included the identification of metabolic pathways and other features devoted to processing plant-derived cellulosic and aromatic compounds, and a robust complement of transport genes (15.4%), one of the highest percentages in bacterial genomes sequenced. Although virulence and antibiotic resistance genes were predicted, experiments conducted using mouse models showed pathogenicity to be attenuated in this strain. Comparative genomic analyses with the presumed human pathogen K. pneumoniae MGH78578 revealed that MGH78578 apparently cannot fix nitrogen, and the distribution of genes essential to surface attachment, secretion, transport, and regulation and signaling varied between each genome, which may indicate critical divergences between the strains that influence their preferred host ranges and lifestyles (endophytic plant associations for K. pneumoniae 342 and presumably human pathogenesis for MGH78578). Little genome information is available concerning endophytic bacteria. The K. pneumoniae 342 genome will drive new research into this less-understood, but important category of bacterial-plant host relationships, which could ultimately enhance growth and nutrition of important agricultural crops and development of plant-derived products and biofuels

Functional and Computational Analysis of Amino Acid Patterns Predictive of Type III Secretion System Substrates in Pseudomonas syringae

Bacterial type III secretion systems (T3SSs) deliver proteins called effectors into eukaryotic cells. Although N-terminal amino acid sequences are required for translocation, the mechanism of substrate recognition by the T3SS is unknown. Almost all actively deployed T3SS substrates in the plant pathogen Pseudomonas syringae pathovar tomato strain DC3000 possess characteristic patterns, including (i) greater than 10% serine within the first 50 amino acids, (ii) an aliphatic residue or proline at position 3 or 4, and (iii) a lack of acidic amino acids within the first 12 residues. Here, the functional significance of the P. syringae T3SS substrate compositional patterns was tested. A mutant AvrPto effector protein lacking all three patterns was secreted into culture and translocated into plant cells, suggesting that the compositional characteristics are not absolutely required for T3SS targeting and that other recognition mechanisms exist. To further analyze the unique properties of T3SS targeting signals, we developed a computational algorithm called TEREE (Type III Effector Relative Entropy Evaluation) that distinguishes DC3000 T3SS substrates from other proteins with a high sensitivity and specificity. Although TEREE did not efficiently identify T3SS substrates in Salmonella enterica, it was effective in another P. syringae strain and Ralstonia solanacearum. Thus, the TEREE algorithm may be a useful tool for identifying new effector genes in plant pathogens. The nature of T3SS targeting signals was additionally investigated by analyzing the N-terminus of FtsX, a putative membrane protein that was classified as a T3SS substrate by TEREE. Although the first 50 amino acids of FtsX were unable to target a reporter protein to the T3SS, an AvrPto protein substituted with the first 12 amino acids of FtsX was translocated into plant cells. These results show that the T3SS targeting signals are highly mutable and that secretion may be directed by multiple features of substrates

Construction and Use of a Nonradioactive DNA Hybridization Probe for Detection of Pseudomonas syringae pv. Tomato on Tomato Plants

Pseudomonas syringae pv. tomato, the causal agent for bacterial speck of tomato, produces the phytotoxin coronatine. A 5.3-kilobase XhoI fragment from the chromosomal region controlling toxin production was cloned into the plasmid pGB2, and the resulting recombinant plasmid, pTPR1, was tested for its ability to serve as a diagnostic probe for P. syringae pv. tomato. In a survey of 75 plant-associated bacteria, pTPR1 hybridized exclusively to those strains that produced coronatine. The detection limit for this probe, which was labeled with the Chemiprobe nonradioactive reporter system, was approximately 4 × 10(3) CFU of lesion bacteria. During the 1989 growing season, a total of 258 leaf and fruit lesions from nine tomato fields were screened for P. syringae pv. tomato by using pTPR1 and the culture method of detection. The best agreement between the two methods, 90%, occurred early in the season with samples taken from relatively young (5-week-old) plants. Young plants also had a higher percentage of P. syringae pv. tomato-positive lesions. P. syringae pv. tomato was the only coronatine producer recovered from the nine tomato fields. All 244 P. syringae pv. tomato strains isolated during this study reacted strongly with the probe. The P. syringae pv. tomato population of healthy field tomato leaves was determined by a pTPR1 colony hybridization procedure. Every probe-positive colony that was isolated and characterized was identified as P. syringae pv. tomato. The pTPR1 probe should expedite disease diagnosis and facilitate epidemiological studies of this pathogen. It also should aid in screening transplant seedlings for bacterial speck infestation