Genomic Diversity of Erwinia carotovora subsp. carotovora and Its Correlation with Virulence

We used genetic and biochemical methods to examine the genomic diversity of the enterobacterial plant pathogen Erwinia carotovora subsp. carotovora. The results obtained with each method showed that E. carotovora subsp. carotovora strains isolated from one ecological niche, potato plants, are surprisingly diverse compared to related pathogens. A comparison of 23 partial mdh sequences revealed a maximum pairwise difference of 10.49% and an average pairwise difference of 2.13%, values which are much greater than the maximum variation (1.81%) and average variation (0.75%) previously reported for Escherichia coli. Pulsed-field gel electrophoresis analysis of I-CeuI-digested genomic DNA revealed seven rrn operons in all E. carotovora subsp. carotovora strains examined except strain WPP17, which had only six copies. We identified 26 I-CeuI restriction fragment length polymorphism patterns and observed significant polymorphism in fragment sizes ranging from 100 to 450 kb for all strains. We detected large plasmids in two strains, including the model strain E. carotovora subsp. carotovora 71. The two least virulent strains had an unusual chromosomal structure, suggesting that a particular pulsotype is correlated with virulence. To compare chromosomal organization of multiple enterobacterial genomes, several genes were mapped onto I-CeuI fragments. We identified portions of the genome that appear to be conserved across enterobacteria and portions that have undergone genome rearrangements. We found that the least virulent strain, WPP17, failed to oxidize cellobiose and was missing several hrp and hrc genes. The unexpected variability among isolates obtained from clonal hosts in one region and in one season suggests that factors other than the host plant, potato, drive the evolution of this common environmental bacterium and key plant pathogen.     

Genomic insights into the plant-associated lifestyle of Kosakonia radicincitans MUSA4, a diazotrophic plant-growth-promoting bacterium

The process of nitrogen (N) fixation by plant-associated bacteria plays an indispensable role in the development of novel agricultural solutions worldwide. In this sense, it is of extreme importance to identify and understand the properties of efficient plant-growth-promoting bacteria (PGPB) that are able to fix N. In this study, the characterization and detailed genomic analysis of the diazotrophic bacterium Kosakonia radicincitans MUSA4, isolated from the internal leaf tissues of a banana tree in Brazil, were undertaken.K. radicincitans MUSA4 presented several plant-growth-promoting traits, including indoleacetic acid, siderophore, acetoin and polyamine biosynthesis, phosphate solubilization, and nitrogen fixation. The strain was able to increase cucumber plant growth significantly, demonstrating its potential in beneficial interactions with plant hosts. Detailed genomic analysis of strain MUSA4 revealed the abundant presence of genes involved in plant colonization, stress resistance and plant-growth-promoting abilities. Moreover, the genome harbored the nif and anf gene clusters, encoding the Fe-Mo nitrogenase and Fe-Fe nitrogenase systems, respectively. Comparative genomic analysis also showed that strain MUSA4 possessed several strain-specific genes, which could be related to its evolutionary history in Brazilian mangrove environments.The results obtained in the present study revealed the plant beneficial role and biotechnological potential of K. radicincitans MUSA4, and provided new insights into plant colonization and plant growth promoting mechanisms employed by diazotrophic Kosakonia.     

Comparative genomic analysis of Pseudomonas amygdali pv. lachrymans NM002: Insights into its potential virulence genes and putative invasion determinants

Pseudomonas amygdali pv. lachrymans is currently of important plant pathogenic bacteria that causes cucumber angular leaf spot worldwide. The pathogen has been studied for its roles in pathogenicity and plant inheritance resistance. To further delineate traits critical to virulence, invasion and survival in the phyllosphere, we reported the first complete genome of P. amygdali pv. lachrymans NM002. Analysis of the whole genome in comparison with three closely-related representative pathovars of P. syringae identified the conservation of virulence genes, including flagella and chemotaxis, quorum-sensing systems, two-component systems, and lipopolysaccharide and antiphagocytosis. It also revealed differences of invasion determinants, such as type III effectors, phytotoxin (coronatine, syringomycin and phaseolotoxin) and cell wall-degrading enzyme, which may contribute to infectivity. The aim of this study was to derive genomic information that would reveal the probable molecular mechanisms underlying the virulence, infectivity and provide a better understanding of the pathogenesis of the P. syringae pathovars.     

Phylogenetic position and virulence apparatus of the pear flower necrosis pathogen Erwinia piriflorinigrans CFBP 5888 as assessed by comparative genomics

Erwinia piriflorinigrans is a necrotrophic pathogen of pear reported from Spain that destroys flowers but does not progress further into the host. We sequenced the complete genome of the type strain CFBP 5888^T clarifying its phylogenetic position within the genus Erwinia, and indicating a position between its closest relative, the epiphyte Erwinia tasmaniensis and other plant pathogenic Erwinia spp. (i.e., the fire blight pathogen E. amylovora and the Asian pear pathogen E. pyrifoliae). Common features are the type III and type VI secretion systems, amylovoran biosynthesis and desferrioxamine production. The E. piriflorinigrans genome also provided the first evidence for production of the siderophore chrysobactin within the genus Erwinia sensu stricto, which up to now was mostly associated with phytopathogenic, soft-rot Dickeya and Pectobacterium species. Plasmid pEPIR37, reported in this strain, is closely related to small plasmids found in the fire blight pathogen E. amylovora and E. pyrifoliae. The genome of E. piriflorinigrans also gives detailed insights in evolutionary genomics of pathoadapted Erwinia.     

Production of the Catechol Type Siderophore Bacillibactin by the Honey Bee Pathogen Paenibacillus larvae

The Gram-positive bacterium Paenibacillus larvae is the etiological agent of American Foulbrood. This bacterial infection of honey bee brood is a notifiable epizootic posing a serious threat to global honey bee health because not only individual larvae but also entire colonies succumb to the disease. In the recent past considerable progress has been made in elucidating molecular aspects of host pathogen interactions during pathogenesis of P. larvae infections. Especially the sequencing and annotation of the complete genome of P. larvae was a major step forward and revealed the existence of several giant gene clusters coding for non-ribosomal peptide synthetases which might act as putative virulence factors. We here present the detailed analysis of one of these clusters which we demonstrated to be responsible for the biosynthesis of bacillibactin, a P. larvae siderophore. We first established culture conditions allowing the growth of P. larvae under iron-limited conditions and triggering siderophore production by P. larvae. Using a gene disruption strategy we linked siderophore production to the expression of an uninterrupted bacillibactin gene cluster. In silico analysis predicted the structure of a trimeric trithreonyl lactone (DHB-Gly-Thr)₃ similar to the structure of bacillibactin produced by several Bacillus species. Mass spectrometric analysis unambiguously confirmed that the siderophore produced by P. larvae is identical to bacillibactin. Exposure bioassays demonstrated that P. larvae bacillibactin is not required for full virulence of P. larvae in laboratory exposure bioassays. This observation is consistent with results obtained for bacillibactin in other pathogenic bacteria.     

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.     

The Complete Genome Sequence of the Plant Growth-Promoting Bacterium Pseudomonas sp. UW4

The plant growth-promoting bacterium (PGPB) Pseudomonas sp. UW4, previously isolated from the rhizosphere of common reeds growing on the campus of the University of Waterloo, promotes plant growth in the presence of different environmental stresses, such as flooding, high concentrations of salt, cold, heavy metals, drought and phytopathogens. In this work, the genome sequence of UW4 was obtained by pyrosequencing and the gaps between the contigs were closed by directed PCR. The P. sp. UW4 genome contains a single circular chromosome that is 6,183,388 bp with a 60.05% G+C content. The bacterial genome contains 5,423 predicted protein-coding sequences that occupy 87.2% of the genome. Nineteen genomic islands (GIs) were predicted and thirty one complete putative insertion sequences were identified. Genes potentially involved in plant growth promotion such as indole-3-acetic acid (IAA) biosynthesis, trehalose production, siderophore production, acetoin synthesis, and phosphate solubilization were determined. Moreover, genes that contribute to the environmental fitness of UW4 were also observed including genes responsible for heavy metal resistance such as nickel, copper, cadmium, zinc, molybdate, cobalt, arsenate, and chromate. Whole-genome comparison with other completely sequenced Pseudomonas strains and phylogeny of four concatenated “housekeeping” genes (16S rRNA, gyrB, rpoB and rpoD) of 128 Pseudomonas strains revealed that UW4 belongs to the fluorescens group, jessenii subgroup.     

Linking Sequence to Function in Soil Bacteria: Sequence-Directed Isolation of Novel Bacteria Contributing to Soilborne Plant Disease Suppression

Microbial community profiling of samples differing in a specific ecological function, i.e., soilborne plant disease suppression, can be used to mark, recover, and ultimately identify the bacteria responsible for that specific function. Previously, several terminal restriction fragments (TRF) of 16S rRNA genes were statistically associated with damping-off disease suppression. This work presents the development of sequence-based TRF length polymorphism (T-RFLP)-derived molecular markers to direct the identification and isolation of novel bacteria involved in damping-off pathogen suppression. Multiple sequences matching TRF M139 and M141 were cloned and displayed identity to multiple database entries in the genera incertae sedis of the Burkholderiales. Sequences matching TRF M148, in contrast, displayed greater sequence diversity. A sequence-directed culturing strategy was developed using M139- and M141-derived markers and media reported to be selective for the genera identified within this group. Using this approach, we isolated and identified novel Mitsuaria and Burkholderia species with high levels of sequence similarity to the targeted M139 and M141 TRF, respectively. As predicted, these Mitsuaria and Burkholderia isolates displayed the targeted function by reducing fungal and oomycete plant pathogen growth in vitro and reducing disease severity in infected tomato and soybean seedlings. This work represents the first successful example of the use of T-RFLP-derived markers to direct the isolation of microbes with pathogen-suppressing activities, and it establishes the power of low-cost molecular screening to identify and direct the recovery of functionally important microbes, such as these novel biocontrol strains.     

Linear fusigen as the major hydroxamate siderophore of the ectomycorrhizal Basidiomycota Laccaria laccata and Laccaria bicolor

A screening for siderophores produced by the ectomycorrhizal fungi Laccaria laccata and Laccaria bicolor in synthetic low iron medium revealed the release of several different hydroxamate siderophores of which four major siderophores could be identified by high resolution mass spectrometry. While ferricrocin, coprogen and triacetylfusarinine C were assigned as well as other known fungal siderophores, a major peak of the siderophore mixture revealed an average molecular mass of 797 for the iron-loaded compound. High resolution mass spectrometry indicated an absolute mass of m/z = 798.30973 ([M + H]⁺). With a relative error of Δ = 0.56 ppm this corresponds to linear fusigen (C₃₃H₅₂N₆O₁₃Fe; MW = 797.3). The production of large amounts of linear fusigen by these basidiomycetous mycorrhizal fungi may possibly explain the observed suppression of plant pathogenic Fusarium species. For comparative purposes Fusarium roseum was included in this study as a well known producer of cyclic and linear fusigen.     

Suppression of fusarium wilts by fluorescent pseudomonads: Mechanisms and applications

Fluorescent pseudomonads are involved in the natural suppressiveness of some soils to fusarium wilts. These bacteria have been applied successfully to suppress fusarium wilts of various plant species grown in conducive soils and growing substrates. Suppression of fusarium wilts by fluorescent pseudomonads can be ascribed to direct and indirect effects against pathogenic Fusarium oxysporum. Direct effects are expressed by a reduction of the saprophytic growth of the pathogen leading to delay and reduction of root infections. This antagonism was demonstrated to be related to siderophore‐mediated iron competition. Iron competition was also shown to enhance the antagonistic effect of non‐pathogenic F. oxysporum by making the pathogen more susceptible to fungal competition for carbon. Indirect effects of fluorescent pseudomonads against pathogenic F. oxysporum are mainly related to the induction of host plant resistance which can be associated with the bacterial lipopolysaccharides. Suppression of fusarium wilts by some fluorescent pseudomonads could also be related to their ability to detoxify fungal metabolites such as fusaric acid. Association of different mechanisms of suppression increases the efficacy and consistency of the biological control under various experimental conditions. Increased knowledge of mechanisms of suppression now enables management of the environment, to some extent, to favour expression of the beneficial activities. These activities are only expressed if the bacterial density is sufficiently high.     

Disease suppression and crop improvement in moong beans (<Emphasis Type="Italic">Vigna radiata</Emphasis>) through <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Burkholderia</Emphasis> strains isolated from semi arid region of Rajasthan,India

Pseudomonas fluorescens BAM-4, Burkholderia cepacia BAM-6 and B. cepacia BAM-12 isolated from the rhizosphere of moong bean (Vigna radiata L.) showed significant growth-inhibitory activity against a range of phytopathogenic fungi. Light and scanning electron microscopic (SEM) studies showed morphological abnormalities such as fragmentation, swelling, perforation and lysis of hyphae of pathogens by Pseudomonas and Burkholderia. Two of the strains (BAM-4 and BAM-6) produced siderophore in CAS agar plates, whereas all three strains produced chitinase. Bacterization of seeds of moong bean with pseudomonads has been reported as a potential method for enhancing plant growth and yield, and for providing protection against Macrophomina phaseolina. Seed bacterization with these plant growth-promoting rhizobacteria (PGPR) showed a significant increase in seed germination, shoot length, shoot fresh and dry weight, root length, root fresh and dry weight, leaf area and rhizosphere colonization. Yield parameters such as pods, number of seeds, and grain yield per plant also enhanced significantly in comparison to control. The disease suppression and plant growth enhancement along with the positive rhizosphere colonization by these strains indicate their possible use as PGPR/biocontrol agents against charcoal rot.     

Resorcylic acid lactones from the ginseng pathogen Ilyonectria mors-panacis

Ilyonectria mors-panacis, previously Cylindrocarpon destructans, is the main plant pathogen responsible for the fungal disease ginseng root rot. This economically important disease, also called disappearing root rot, reduces crop yields by an average of 30% at harvest. While the disease is well studied from ecological and genomic perspectives, the role of I. mors-panacis secondary metabolites in the disease process is not well understood. Our previous metabolomics study showed Ilyonectria strains that cause ginseng root rot produce mixtures of putative resorcylic acid lactones, whereas avirulent strains did not, and collectively synthesize fewer metabolites. To confirm these metabolomics findings, we isolated and characterized the secondary metabolites from I. mors-panacis DAOMC 251601, a strain that causes ginseng root rot. From its EtOAc soluble culture filtrate extract, eight resorcylic acid lactones (1-8), including chlorinated and non-chlorinated congeners, were characterized by HRMS and spectroscopic approaches (NMR, OR, UV). The structure of one new metabolite, named radicicol E (1), was elucidated and additional spectroscopic data for the known compound nordinonediol (2) are reported. Further, radicicol (9) production was confirmed by comparison to a standard. The roles that resorcylic acid lactones and the siderophore N,N′,N” triacetylfusarine C have in promoting Ilyonectria ginseng root rot are also discussed.     

The Siderophore Metabolome of Azotobacter vinelandii

In this study, we performed a detailed characterization of the siderophore metabolome, or “chelome,” of the agriculturally important and widely studied model organism Azotobacter vinelandii. Using a new high-resolution liquid chromatography-mass spectrometry (LC-MS) approach, we found over 35 metal-binding secondary metabolites, indicative of a vast chelome in A. vinelandii. These include vibrioferrin, a siderophore previously observed only in marine bacteria. Quantitative analyses of siderophore production during diazotrophic growth with different sources and availabilities of Fe showed that, under all tested conditions, vibrioferrin was present at the highest concentration of all siderophores and suggested new roles for vibrioferrin in the soil environment. Bioinformatic searches confirmed the capacity for vibrioferrin production in Azotobacter spp. and other bacteria spanning multiple phyla, habitats, and lifestyles. Moreover, our studies revealed a large number of previously unreported derivatives of all known A. vinelandii siderophores and rationalized their origins based on genomic analyses, with implications for siderophore diversity and evolution. Together, these insights provide clues as to why A. vinelandii harbors multiple siderophore biosynthesis gene clusters. Coupled with the growing evidence for alternative functions of siderophores, the vast chelome in A. vinelandii may be explained by multiple, disparate evolutionary pressures that act on siderophore production.     

Social evolution of toxic metal bioremediation in Pseudomonas aeruginosa

Bacteria are often iron-limited, and hence produce extracellular iron-scavenging siderophores. A crucial feature of siderophore production is that it can be an altruistic behaviour (individually costly but benefitting neighbouring cells), thus siderophore producers can be invaded by non-producing social ‘cheats’. Recent studies have shown that siderophores can also bind other heavy metals (such as Cu and Zn), but in this case siderophore chelation actually reduces metal uptake by bacteria. These complexes reduce heavy metal toxicity, hence siderophore production may contribute to toxic metal bioremediation. Here, we show that siderophore production in the context of bioremediation is also an altruistic trait and can be exploited by cheating phenotypes in the opportunistic pathogen Pseudomonas aeruginosa. Specifically, we show that in toxic copper concentrations (i) siderophore non-producers evolve de novo and reach high frequencies, and (ii) producing strains are fitter than isogenic non-producing strains in monoculture, and vice versa in co-culture. Moreover, we show that the evolutionary effect copper has on reducing siderophore production is greater than the reduction observed under iron-limited conditions. We discuss the relevance of these results to the evolution of siderophore production in natural communities and heavy metal bioremediation.     

Identification of expressed genes during infection of Chinese cabbage (Brassica rapa subsp. pekinensis) by Plasmodiophora brassicae

Plasmodiophora brassicae is an obligate, biotrophic pathogen causing the club-root disease of crucifers. Despite its importance as a plant pathogen, little is known about P. brassicae at the molecular level as most of its life cycle takes place inside the plant host, and axenic culturing is impossible. Discovery of genes expressed during infection and gene organization are the first steps toward a better understanding of the pathogen-host interaction. Here, suppression subtractive hybridization was used to search for the P. brassicae genes expressed during plant infection. One-hundred and forty ESTs were found of which 49% proved to be P. brassicae genes. Ten novel P. brassicae genes were identified, and the genomic sequences surrounding four of the ESTs were acquired using genome walking. Alignment of the ESTs and the genomic DNA sequences confirmed that P. brassicae genes are intron rich and that the introns are small. These results show that it is possible to discover new P. brassicae genes from a mixed pool of both plant and pathogen cDNA. The results also revealed that some of the P. brassicae genes expressed in Chinese cabbage (Brassica rapa subsp. pekinensis) were identical to the genes expressed in the infection of Arabidopsis plants, indicating that these genes play an important role in P. brassicae infection.     

Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars

We have isolated 576 endophytic bacteria from the leaves, stems, and roots of 10 rice cultivars and identified 12 of them as diazotrophic bacteria using a specific primer set of nif gene. Through 16S rDNA sequence analysis, nifH genes were confirmed in the two species of Penibacillus, three species of Microbacterium, three Bacillus species, and four species of Klebsiella. Rice seeds treated with these plant growth-promoting bacteria (PGPB) showed improved plant growth, increased height and dry weight and antagonistic effects against fungal pathogens. In addition, auxin and siderophore producing ability, and phosphate solubilizing activity were studied for the possible mechanisms of plant growth promotion. Among 12 isolates tested, 10 strains have shown higher auxin producing activity, 6 isolates were confirmed as strains with high siderophore producing activity while 4 isolates turned out to have high phosphate-solubilizing activity. These results strongly suggest that the endophytic diazotrophic bacteria characterized in this study could be successfully used to promote plant growth and inducing fungal resistance in plants.     

Genome subtraction for novel target definition in Salmonella typhi

Large genomic sequencing projects of pathogens as well as human genome leads to immense genomic and proteomic data which would be very beneficial for the novel target identification in pathogens. Subtractive genomic approach is one of the most useful strategies helpful in identification of potential targets. The approach works by subtracting the genes or proteins homologous to both host and the pathogen and identify those set of gene or proteins which are essential for the pathogen and are exclusively present in the pathogen. Subtractive genomic approach is employed to identify novel target in salmonella typhi. The pathogen has 4718 proteins out of which 300 are found to be essential (“ indispensable to support cellular life”) in the pathogen with no human homolog. Metabolic pathway analyses of these 300 essential proteins revealed that 149 proteins are exclusively involved in several metabolic pathway of S. typhi. 8 metabolic pathways are found to be present exclusively in the pathogen comprising of 27 enzymes unique to the pathogen. Thus, these 27 proteins may serve as prospective drug targets. Sub-cellular localization prediction of the 300 essential proteins was done which reveals that 11 proteins lie on the outer membrane of the pathogen which could be probable vaccine candidates.     

Bacterially Induced Weathering of Ultramafic Rock and Its Implications for Phytoextraction

The bioavailability of metals in soil is often cited as a limiting factor of phytoextraction (or phytomining). Bacterial metabolites, such as organic acids, siderophores, or biosurfactants, have been shown to mobilize metals, and their use to improve metal extraction has been proposed. In this study, the weathering capacities of, and Ni mobilization by, bacterial strains were evaluated. Minimal medium containing ground ultramafic rock was inoculated with either of two Arthrobacter strains: LA44 (indole acetic acid [IAA] producer) or SBA82 (siderophore producer, PO₄ solubilizer, and IAA producer). Trace elements and organic compounds were determined in aliquots taken at different time intervals after inoculation. Trace metal fractionation was carried out on the remaining rock at the end of the experiment. The results suggest that the strains act upon different mineral phases. LA44 is a more efficient Ni mobilizer, apparently solubilizing Ni associated with Mn oxides, and this appeared to be related to oxalate production. SBA82 also leads to release of Ni and Mn, albeit to a much lower extent. In this case, the concurrent mobilization of Fe and Si indicates preferential weathering of Fe oxides and serpentine minerals, possibly related to the siderophore production capacity of the strain. The same bacterial strains were tested in a soil-plant system: the Ni hyperaccumulator Alyssum serpyllifolium subsp. malacitanum was grown in ultramafic soil in a rhizobox system and inoculated with each bacterial strain. At harvest, biomass production and shoot Ni concentrations were higher in plants from inoculated pots than from noninoculated pots. Ni yield was significantly enhanced in plants inoculated with LA44. These results suggest that Ni-mobilizing inoculants could be useful for improving Ni uptake by hyperaccumulator plants.     

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.