Identification of tomato plant as a novel host model for Burkholderia pseudomallei
© Lee et al; licensee BioMed Central Ltd. 2010
Received: 10 September 2009
Accepted: 29 January 2010
Published: 29 January 2010
Burkholderia pseudomallei is the causative agent for melioidosis, a disease with significant mortality and morbidity in endemic regions. Its versatility as a pathogen is reflected in its relatively huge 7.24 Mb genome and the presence of many virulence factors including three Type Three Secretion Systems known as T3SS1, T3SS2 and T3SS3. Besides being a human pathogen, it is able to infect and cause disease in many different animals and alternative hosts such as C. elegans.
Its host range is further extended to include plants as we demonstrated the ability of B. pseudomallei and the closely related species B. thailandensis to infect susceptible tomato but not rice plants. Bacteria were found to multiply intercellularly and were found in the xylem vessels of the vascular bundle. Disease is substantially attenuated upon infection with bacterial mutants deficient in T3SS1 or T3SS2 and slightly attenuated upon infection with the T3SS3 mutant. This shows the importance of both T3SS1 and T3SS2 in bacterial pathogenesis in susceptible plants.
The potential of B. pseudomallei as a plant pathogen raises new possibilities of exploiting plant as an alternative host for novel anti-infectives or virulence factor discovery. It also raises issues of biosecurity due to its classification as a potential bioterrorism agent.
Burkholderia pseudomallei is a Gram-negative bacterium that is the causative agent for melioidosis, a disease endemic in Southeast Asia and Northern Australia with significant morbidity and mortality [1, 2]. The bacterium exhibits broad host range and has been shown to cause disease in cattle, pigs, goats, horses, dolphins, koalas, kangaroos, deers, cats, dogs and gorillas . Acquisition of the bacterium could be through inhalation of aerosol, ingestion of contaminated water and inoculation through open skin . In humans, the disease could present with varied manifestations ranging from asymptomatic infection, localized disease such as pneumonia or organ abscesses to systemic disease with septicemia . The disease could be acute or chronic, and relapse from latency is possible .
The versatility of B. pseudomallei as a pathogen is reflected in its huge 7.24 Mb genome organized into two chromosomes . One of the most important virulence factors that has been partially characterized in B. pseudomallei is its Type Three Secretion Systems (T3SS), of which it has three [8, 9]. Each T3SS typically consists of a cluster of about 20 genes encoding structural components, chaperones and effectors which assemble into an apparatus resembling a molecular syringe that is inserted into host cell membrane for the delivery of bacterial effectors into host cell cytosol. One of the B. pseudomallei T3SS known as Bsa or T3SS3 resembles the inv/mxi/spa T3SS of Salmonella and Shigella, and has been shown to be important for disease in animal models . The other two T3SS (T3SS1 and 2) resemble the T3SS of plant pathogen Ralstonia solanacearum  and do not contribute to virulence in mammalian models of infection . Being a soil saprophyte and having the plant pathogen-like T3SS raise the possibility that B. pseudomallei could also be a plant pathogen. As B. pseudomallei is a risk group 3 agent with specific requirements for containment, we first test this hypothesis using the closely related species B. thailandensis as a surrogate model especially in experiments where risk of aerosolization is high, before we verify key experiments with B. pseudomallei. B. thailandensis is considered largely avirulent in mammalian hosts unless given in very high doses [13, 14]. We infected both tomato as well as rice plants with B. pseudomallei to determine their susceptibility to disease. Furthermore, the role of the three B. pseudomallei T3SS in causing plant disease is evaluated and the implication of the ability of B. pseudomallei to infect plants is discussed.
Bacterial strains, plasmids and growth conditions
All bacterial strains, plasmids used and constructed.
Source or Reference
oriT; KmR; sacB gene
pGEM containing a tetracycline resistance cassette, TetR, AmpR
Y. Chen, unpublished
pGEM containing a zeocin resistance cassette, ZeoR, AmpR
Y. Chen, unpublished
pK18mobsac B containing upstream and downstream of TTSS1 flanking a tet cassette, KmR, TetR
pK18mobsac B containing upstream and downstream of TTSS2 flanking a tet cassette, KmR, TetR
pK18mobsac B containing upstream and downstream of TTSS3 flanking a zeo cassette, KmR, ZeoR
Eu Hian Yap, unpublished
Eu Hian Yap, unpublished
Eu Hian Yap, unpublished
Wild-type parental strain, clinical isolate, KmS
BPSS1386-1411 region was replaced with tet cassette, TetR, KmS
BPSS1592-1629 region was replaced with tet cassette, TetR, KmS
BPSS1520-1552 region was replaced with zeo cassette, ZeoR, KmS
Tomato seeds of the Solanum lycopersicum variety Season Red F1 Hybrid (Known-You Seeds Distribution (S.E.A) Pte Ltd) and Arabidopsis thaliana (Loh Chiang Shiong, NUS) were surface sterilized with 15% bleach solution for 15 minutes with vigorous shaking. The seeds were rinsed in sterile distilled water and germinated in MS agar medium. The seedlings were cultivated with a photoperiod of 16 hour daylight and 8 hour darkness. One month old plantlets were used for infection. Tomato plantlets were transferred into 50 mL Falcon tubes with 5 mL of liquid MS medium for infection while 1 mL of MS medium was used for Arabidopsis. Rice seeds (Japonica nipponbare) were obtained from Dr Yin Zhong Zhao (Temasek Life Sciences Laboratories, Singapore). Seeds were surface sterilized as described above. The seeds were rinsed in sterile distilled water and germinated in N6 agar medium. The germinated seedlings were placed on N6 agar supplemented with 2 mg/mL of 2, 4-dichlorophenyoxyacetic acid (2, 4-D) in the dark to induce callus production. The callus were regenerated on N6 medium supplemented with 2 mg/mL Benzylaminopurine (BA), 1 mg/mL Naphthylacetic Acid (NAA), 1 mg/mL Indole-3-acetic acid (IAA) and 1 mg/mL Kinetin under 16 hour daylight and 8 hour dark photoperiod. Rice plantlets were transferred and maintained in MS agar medium. The plantlets were transferred into 50 mL Falcon tubes with 5 mL of liquid MS medium for infection. Some plantlets were also wounded by cutting off the roots before being transferred.
Tomato, rice and Arabidopsis plantlets were infected with log phase cultures at the concentration of 1 × 107 colony forming units (cfu)/5 mL medium by immersing only the roots of the plantlets in the inoculum in a 50 mL tube. The plantlets were maintained at 24-25°C, shaking at 100 rpm. The plantlets were observed for symptoms such as yellowing of leaves, blackening of the leaf veins, wilting and necrosis daily over 7 days. Each plantlet was scored daily on a disease index score of 1 to 5 based on how extensive the symptoms were as calculated by the percentage of the plant with symptoms (1: no symptoms; 2: 1 to 25% of the plant showed symptoms; 3: 26 to 50% of the plant showed symptoms; 4: 51 to 75% of the plant showed symptoms; 5: 76 to 100% of the plant showed symptoms or the plant was dead) . Each experiment included at least 12 to 20 plantlets infected with bacteria except for experiments with rice and Arabidopsis plantlets where 6 plantlets were used. All experiments were repeated at least twice.
Multiplication of B. thailandensis in tomato plantlets and leaves
Tomato plantlets were infected with bacteria through unwounded roots and three leaves from each plantlet were excised at day 1, 3, 5 and 7 after infection. The leaves were macerated in 1 mL PBS with a micro-pestle, serially diluted and plated on TSA plates in duplicates. Tomato leaves were infected by cutting with a pair of scissors dipped in 1 × 109 cfu/mL of B. thailandensis. Five plantlets were used in each experiment. At days 1 and 3 after infection, one infected leaf from each plantlet was excised, washed with 10% bleach solution for 1 min and rinsed with sterile water. The leaf was blotted dry on sterile filter paper and imprinted on TSA agar plates to determine if there were any bacteria on the surface of the leaves. The imprinted plates were incubated at 37°C for 24 hours before checking for any bacteria growth. The leaves were then weighed and macerated in 1 mL PBS with a micro-pestle, serially diluted and plated on TSA plates in duplicates. Only leaf samples which did not show any bacteria growth on the imprinted plates will be counted to avoid counting contaminating bacteria from leaf surfaces.
Transmission Electron Microscope (TEM)
Tomato leaf and rice blade were infected by cutting with a pair of scissors dipped in 1 × 109 cfu/mL of B. pseudomallei strain KHW or B. thailandensis. One day after infection, the infected tomato leaf and rice blade were excised for TEM. One millimeter from the infected leaf/blade edge were cut and discarded to avoid contamination from extracellular bacteria at the infection site. A further two millimeter from the infected leaf/blade edge were then cut and sliced into smaller sections and fixed with 4% glutaraldehyde in 0.1 M phosphate buffer under vacuum for 4 hours. It was post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer for 1 hour at 4°C. Samples were dehydrated sequentially through 30%, 50%, 70%, 90%, 100% ethanol, and finally in propylene oxide prior to infiltration with Spurr resin . Samples were embedded in 100% spur resin and polymerized at 70°C overnight. Ultra-thin sections were cut on a Leica Ultracut UCT ultra-microtome and examined with a transmission electron microscope (JEM1230, JEOL, Japan) at 120 kV.
Growth of bacteria in different media
Overnight cultures were used to inoculate 5 mL of LB and Murashige and Skoog (MS)  medium to a starting optical density at 600 nm of 0.1. The cultures were incubated at 37°C for LB medium and 25°C for MS medium. Optical density at 600 nm for all cultures was measured at 0, 2.5, 6 and 24 hours. All experiments were repeated twice with duplicates.
Generation of B. pseudomallei T3SS1, T3SS2 and T3SS3 mutants
Approximate one kb fragments upstream and downstream of the T3SS1, T3SS2 or T3SS3 locus were amplified from B. pseudomallei KHW genomic DNA and subsequently cloned into pK18mobsacB. The tet cassette from pGEM-tet or zeo cassette (kindly provided by Dr Herbert Schweizer, Colorado State University, USA) from pCLOXZ1 was inserted between the upstream and downstream fragments resulting in pT3SS1/upstream/downstream/tet, pT3SS2/upstream/downstream/tet, and pT3SS3/upstream/downstream/zeo. The plasmids were electroporated into SM10 conjugation host and conjugated into B. pseudomallei strain KHW. Homologous recombination was selected for retention of antibiotic marker (Tet or Zeo) linked to the mutation and loss of the plasmid marker (Km) to generate KHWΔT3SS1, KHWΔT3SS2 and KHWΔT3SS3. Each mutant was confirmed by PCR for the loss of a few representative T3SS genes in the locus.
Cytotoxicity assay on THP-1 cells
Average disease scores with standard deviation were calculated based on at least 100 tomato plantlets infected with each strain of bacteria or mutant. Data were analyzed using repeated measure analysis of variance . All statistical analyses were performed using SPSS version 17 software (SPSS Inc). A p value of less than 0.001 is considered significant.
Using B. thailandensis infection of tomato plantlets as a model
For a phytopathogen to successfully colonize the plant, it must be able to replicate intercellularly . To determine whether bacteria are able to replicate intercellularly, we sampled leaves from two representative plantlets which had been inoculated with bacteria via unwounded roots at 1, 3, 5 and 7 days post-inoculation. Three leaves were sampled at each time-point per plantlet. Both plantlets showed a progressive increase in bacterial load in their leaves over time (Fig 1D).
Susceptibility of tomato plantlets to B. pseudomallei infection
Localization of bacteria at site of infection
The role of T3SS in plant infection
Susceptibility of rice and Arabidopsis plantlets to B. pseudomallei and B. thailandensis infection
B. cepacia, the important opportunistic pathogen often associated with cystic fibrosis and chronic granulomatous disease patients , was originally described as a phytopathogen causing soft rot in onions . Subsequently, many strains from various B. cepacia complex were shown to be able to cause disease in the alfalfa infection model as well as in the rat agar bead model . In this study, we show that B. pseudomallei and B. thailandensis are also potential plant pathogens. They are capable of infecting susceptible plants such as tomato.
Plant pathogenic bacteria have been shown to express a large number of T3SS effectors capable of interfering with plant basal defense triggered by bacterial pathogen-associated molecular patterns (PAMPs) as well as Resistance (R) protein-mediated immunity typically characterized by the Hypersensitive Response (HR) [24–26]. The outcome of the interaction with susceptible hosts for these successful pathogens would be disease. We found that the virulence of B. pseudomallei in tomato is contributed significantly by T3SS1 and T3SS2, but to a much lesser extent by T3SS3. T3SS1 and T3SS2 are likely non-redundant to each other in causing disease because each mutant demonstrates significant attenuation, possibly because both T3SS1 and T3SS2 are co-ordinately involved in pathogenesis. This is the first time that a role has been defined for T3SS1 and T3SS2 in B. pseudomallei, showing that they are functional and not simply vestiges of evolution. The role of T3SS3 could be due to its contribution of a structural component or chaperone to the other two T3SS or an effector which could also interfere with plant cell physiology albeit less efficiently than with mammalian cells. Nevertheless, our study shows the important role played by T3SS in B. pseudomallei pathogenesis in tomato plants.
In contrast to tomato, we found that both B. pseudomallei and B. thailandensis are non-adapted for rice. This is not surprising as B. pseudomallei are routinely recovered from rice paddy fields in regions of endemicity such as Thailand and have never been reported to cause any disease in rice plants. It is possible that PAMPs from B. pseudomallei and B. thailandensis are able to trigger an effective basal defence from rice to halt bacterial colonization, a common means of plant resistance against non-adapted microorganisms [24–26]. Another intriguing possibility is that compounds secreted by rice plants may inhibit the growth of B. thailandensis and B. pseudomallei. The presence of secondary metabolites induced by B. pseudomallei infection in plants with differential susceptibility to disease could reveal novel anti-infective compounds against melioidosis to counter the problem of extensive antibiotic resistance in this bacterium.
Thus, B. pseudomallei joins a growing list of human pathogens which have been found to be able to infect plants , the first of which to be described was P. aeruginosa . The plant host model has been used to perform large scale screening of a library of P. aeruginosa mutants to identify novel virulence factors  as some virulence factors encoded by genes such as toxA, plcS and gacA were shown to be important for bacterial pathogenesis in both plants and animals . Given the evidence that B. pseudomallei T3SS3 may be capable of interacting with both mammalian and plant hosts, and the ability of B. pseudomallei to infect tomato, one could develop susceptible plants as alternative host models for large scale screening of B. pseudomallei mutants to aid in novel virulence factor discovery, similar to what had been done for P. aeruginosa.
Previously, B. pseudomallei has been shown to infect C. elegans  and Acanthamoeba species  and C. elegans could be used as an alternative host model for large scale screening and identification of B. pseudomallei virulence factors . Our current finding reveals the additional versatility of B. pseudomallei as a pathogen and further research would likely uncover novel bacterial mechanisms capable of interacting with its varied hosts. Much more work is needed to define the susceptibility of various plant species to B. pseudomallei to find a suitable plant host for virulence factor discovery. It remains to be seen if B. pseudomallei is a natural pathogen for crops such as tomatoes.
In summary, we identified B. pseudomallei as a plant pathogen capable of causing disease in tomato but not rice plants. B. pseudomallei T3SS1 and T3SS2 contribute significantly to disease whereas T3SS3 plays a more minor role. Although the significance of B. pseudomallei as a natural plant pathogen in the environment is unknown, one could postulate that certain plants may serve as a reservoir for the bacteria. Since B. pseudomallei is classified as a bioterrorism agent by the US Centers for Disease Control and Prevention http://www.cdc.gov/od/sap, our findings indicate that it may be necessary to re-evaluate whether B. pseudomallei poses threats beyond the animal kingdom and whether plant systems could be used as environmental indicators of the presence of the bacteria either as endemic residents or due to the intentional release by terrorists, a concept that has been previously proposed .
This work was funded by the grant 04/1/21/19/329 from the Singapore Biomedical Research Council (BMRC). We thank Chiang Shiong Loh for providing Arabidopsis seeds. We also thank Seng Kee Tan for technical advice on plant infection. YHL was funded by a stipend from Temasek Polytechnic.
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