Patent Grant
8227186
Exemplary embodiments relates to compositions and methods for analyzing bacterial pathogens. More particularly, embodiments relate to bioluminescent reporter bacteria for studying plant pathogens.
Tomato, one of the major vegetables, is widely cultivated in the world. In 2004, the production of tomato worldwide reached 120 million metric tons. The United States ranks second in tomato production with 428,900 acre of planted area, and total value was over 2 billion dollars in 2006. In the US, Ohio ranks third for both processing (6,400 acres) and fresh tomato production (6,700 acres) with total value of 125 million dollars in 2006. Clavibacter michiganensis subsp. michiganensis (Cmm), the bacterial pathogen of tomato canker, causes severe economic losses in commercial tomato production in the US and worldwide. Loss of yield is mainly due to defoliation, wilting and death of plants, reduced fruit size, and lesions on fruit. The disease was first discovered in a Michigan greenhouse in 1909 and many occurrences have been reported since then in North America. Bacterial canker is considered by the greenhouse tomato industry, which produces about 35% of all tomatoes sold in supermarkets and similar venues in North America, to be the most important and costly disease it must manage. Cmm is a quarantined pathogen in Europe and infected greenhouse and field plantings must be destroyed. Severe epidemics may cause 80% yield loss and according to the 2005 EPPO report tomato canker is present almost in all tomato production areas around the world. Bacterial canker is very difficult to control once it has been established in a planting, and to date there are no antibiotics or bactericides available to kill Cmm in the plant and prevent its spread to other plants.
An important means of disease spread is through infected seed and seedlings. Even a low transmission rate (0.01%) from seed to seedling can cause an epidemic of the disease under favorable conditions. Tomato seedlings are usually kept in a nursery for 4-6 weeks before transplanting to either greenhouse or field. As the density of seedlings is very high, the bacteria are easily spread through irrigation and leaf-to-leaf contact. Some infected young plants die soon after transplanting but many survive with epiphytic populations of Cmm that may spread to other plants. The mechanism of seed to seedling transmission of Cmm and the significance of its epiphytic phase in the ecology and epidemiology of the disease are still not fully understood.
This and other unmet advantages are provided by the methods and compositions described and shown in more detail below.
Exemplary embodiments comprise a method of studying pathogen transmission in a plant using a bioluminescent reporter bacteria, the method comprising the following steps: a) obtaining pathogenic bacteria from an infected plant; b) introducing into the bacteria a nucleic acid that encodes a bioluminescent reporter protein under conditions whereby the nucleic acid encoding the bioluminescent reporter protein is taken up by, stably integrated into the genome of, and expressed in the bacteria, wherein the bioluminescent reporter protein does not naturally occur in the bacteria; c) contacting a plant or plant seed with bioluminescent reporter bacteria obtained by steps a) and b); and d) monitoring bacterial colonization of the plant or plant seed in real-time by detecting in vivo luminescence signals on the plant or plant seed; wherein an amount of photons emitted correlates with a biomass of living pathogenic bacteria.
In various embodiments, the gram-positive pathogenic bacterium is a Clavibacter strain. Preferrably, the strain is a Clavibacter michiganensis subsps. michiganensis. The plant or plant seed may be a fully developed plant or plant part, a seedling, and/or seed. In a preferred embodiment, the bioluminescent reporter protein is constitutively expressed.
Embodiments also include methods for identifying compounds that inhibit the growth of pathogenic Clavibacter bacteria. Exemplary embodiments include a method of identifying a compound that inhibits pathogenic bacteria, the method comprising: a. providing a plant or seed colonized by bioluminescent reporter bacteria that expresses a bioluminescent reporter protein; b. contacting the plant or seed with a test compound; c. imaging the plant or seed to detect luminescence; and d. correlating a reduction in luminescence with an antibacterial effect of the test compound; wherein the bioluminescent reporter bacteria are mutants of the pathogenic bacteria. In various embodiments, the pathogenic bacteria are a Clavibacter michiganensis strain.
Various embodiments comprise mutant Clavibacter michiganensis bacteria. The mutant bacteria comprise an integrated nucleic acid that encodes a bioluminescent reporter protein. Preferrably, the nucleic acid that encodes the reporter protein is a lux construct. In exemplary embodiments, the reporter protein is constitutively expressed. In at least one embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 2 or a degenerate variant of SEQ ID NO: 2.
At least one embodiment is directed to an isolated nucleic acid vector comprising the nucleotide sequence of SEQ ID NO: 1 or a degenerate variant of SEQ ID NO: 1.
A better understanding of the present invention will be obtained from a reading of the following detailed description and the accompanying drawings wherein identical reference characters refer to identical parts and in which:
The following sequences conform with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. 1.822.
aRestriction sites added in the original sequences are underlined.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Clavibacter michiganensis subsp. michiganensis (Cmm) is an important Gram-positive-bacterial pathogen that infects tomato plants causing wilting and cankers and leading to severe economic losses in commercial tomato production worldwide. An important means of spreading disease is through infected seeds and seedlings. However, little is known regarding the mechanism of seed and seedling infection. In order to visualize the infection process of Cmm in germinating seeds, bioluminescent Cmm strains were constructed by transforming the lux reporter gene into the bacterium. A vector pXX2 (SEQ ID NO: 1) was constructed by inserting a modified promoterless lux-operon to a Cmm transposon mutagenesis vector, pKGT452Cβ. After electroporation, pXX2 (SEQ ID NO: 1) carrying the Cmxr::luxABCDE::Tn1409 cassette resulted in insertion of lux-operon into the Cmm chromosome.
In total, 19 bioluminescent Cmm mutants were obtained. The mutants varied in light production, but all tested strains induced hypersensitive response with Mirabilis jalapa (four o'clock plant) and caused wilting in tomatoes. The virulent, stable, constitutively bioluminescent strain BL-Cmm17 was selected for further characterization of growth properties and inoculation on tomato seeds.
Using this bioluminescent strain, Cmm colonization dynamics of germinating seeds were monitored in real-time using the in vivo imaging system (IVIS). Disclosed Cmm strains colonized the seed's hypocotyl and the cotyledon at an early stage of germination. Importantly, the novel embodiments using bioluminescent Cmm will help elucidate the dynamics of plant infections with this and other important and difficult-to-control pathogens.
Because the lux transposon vector embodied herein may be useful in the other subspecies of agriculturally important C. michiganensis as well as other members of actinomycetes, embodiments may have significant impact in understanding pathogenesis of disease caused by other pathogens, for example, actinomycetes.
The following definitions and abbreviations are to be used for the interpretation of the claims and the specification.
The term “gene” refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.
As used herein the term “coding sequence” refers to a DNA sequence that codes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.
The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation
The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.
As used herein the term “transformation” refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.
The terms “plasmid” and “vector” refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Transformation vector” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell.
As used herein the term “codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
The term “codon-optimized” as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA.
Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Bacteria Strains and Growth Conditions
Bacterial strains and plasmids used in the examples are listed in Table 3.
Clavibacter
michiganensis
michiganensis
C.
michiganensis
michiganensis
E.
coli DH5α
E.
coli ER2925
P.
luminescence Amp; 8.7 Kb
C. michiganensis subsp. michiganensis strains A300 and C290 were isolated from infected plants in Ohio and belonged to BOX-PCR fingerprint type A and type C, respectively (Louws et al, 1998). Depending on the assay, Cmm strains were grown in yeast dextrose calcium carbonate broth (YDC) (Bacto, Franklin Lakes, N.J.), nutrient-broth yeast extract broth (NBY) (Bacto, Franklin Lakes, N.J.), or tryptone broth with yeast (TBY, containing 10 g/liter of tryptone, 5 g/liter of yeast extract, and 5 g/liter of NaCl; pH7.5). E. coli strains (DH5a and ER2925) were grown on LB at 37° C. Antibiotics Ampicillin (150 μg/ml), chloramphenicol (20 μg/ml), and kanamycin (50 μg/ml) were added to the media as necessary.
Construction of Plasmids pXX1, pXX2 and pXX3
Both pXen5 and pXen13 (Xenogen Corporation, Alameda, Calif.) carry promoterless lux-operon (luxCDABE) originally isolated from Photorhabdus luminescence. The lux-operon in pXen5 had been modified by adding an upstream Gram-positive ribosome binding site (AGGAGG) upstream of each lux genes for optimal expression of lux genes in Gram positive bacteria (Francis et al. 2001). To construct pXX1, the lux-operon and kanamycin resistance gene from pXen5 were amplified by a long range, high fedility PCR using Herculase Polymerase (Stratagene, Calif.) and the LuxpXen5F/LuxpXen5R primers. Appropriate restriction sites (EcoRI and SalI) were inserted in the primers to facilitate cloning. Oligonucleotides were designed using Vector NTI® software and commercially synthesized by Integrated DNA Technologies (Skokie, Ill.). All the oligonucleotides used in the examples are listed in Table 2. Appropriate restriction sites were included in the primers to facilitate cloning. The amplified PCR product was digested and ligated to a similarly digested Tn5 transposon construction vector pMod 3 (Epicentre, Madison, Wis.) in order to generate the plasmid pXX1. Consequently, pXX1 carries EZ::TN<lux-kan> cassette insert, which is flanked by the Tn5 transposon mosaic ends.
pXX2 (SEQ ID NO: 1) was constructed by amplifying the lux-operon from pXen-5 with primers LuxpXen5 F2/LuxpXen5R2. Inverse PCR was performed on the Cmm mutangensis vector pKGT452Crβ (Kirchner et al, 2001) with pKGT1 F and pKGT1R primers that were designed to amplify all but the region of lux insertion. Appropriate restriction sites (NotI and XhoI) were added to the PCR primers to facilitate cloning (Table 2). The inverse PCR product was digested and ligated to the similarly digested lux-operon (SEQ ID NO: 2) from pXen5, generating pXX2 (SEQ ID NO: 1), which carries Gram-positive Lux (Cmxr:: luxABCDE::Tn1409).
To construct pXX3, the lux-operon was obtained by digesting pXen13 with NotI and XhoI and ligated to similarly digested inverse PCR product of the plasmid pKGT452Crβ amplified using the pKGT2F/pKGT2R primers. The pXX3 carries Gram-negative Lux (Cmxr:: luxABCDE::Tn1409). Recombinant plasmids pXX1, pXX2 (SEQ ID NO: 1) and pXX3 were transformed to E. coli DH5a and subsequently propagated in the dam and dcm deficient E. coli strain, ER2925, (New England Biolabs, Beverly, Mass., U.S.A.), since the use of unmethylated DNA improves transformation efficiency in Cmm (Kirchner et al, 2001).
Mutagenesis and Isolation of Bioluminescent Cmm
Bioluminescent Cmm strains were generated by electroporation of either the modified EZ::TN transposomes from pXX1, containing the aforementioned Gram-positive promoterless luxABCDE genes (G+ lux) or pUWGR4, containing the Gram-negative promoterless luxCDABE (Gram− lux) (Rajashekara et al, 2005) or by directly electroporating the suicide vectors containing the modified transposon Tn1409 (pXX2 and pXX3). Before electroporation, the EZ::TN transposome was prepared as described by the manufacturer (Epicentre, Madison, Wis.), while pUWGR4 and pXX1 were digested with PvuII and the resulting 6.8 kb and 7.3 kb linear DNA fragments, respectively containing the Gram− lux and Gram+ lux with kanR genes, were gel purified using QIAquick gel extraction kit (Qiagen, Valencia, Calif.). Two μl of each purified DNA (100 ng/μl) were mixed separately with 4 μl of EZ::TN transposase (Epicentre, Madison, Wis.) and 2 μl of 100% glycerol, achieving a total volume of 8 μl per reaction. Following incubation for 30 min at room temperature, the transposome complexes were stored at −20° C. until further use.
Electrocompetent Cmm 290 aliquots were prepared as described previously (Kirchner et al, 2001). Briefly, bacteria were grown to OD600 0.5-0.7 in TBY broth at 25° C. Cells were then pretreated with 2.5% glycine for 2 h and harvested by centrifugation at 9,000×g for 10 min, which was followed by washing with sterile ice cold distilled water for three times and 10% glycerol twice. The cell pellet was then resuspended in 15% glycerol to 1/250th of the original volume. Transformation was achieved by mixing 100 μl of electrocompetent Cmm cells with 1-2 μl of the EZ::TN transposome complex or suicide plasmids pXX2 or pXX3 (1 μg each) and electroporation was performed in a 0.2-cm cuvette, using a Gene Pulser Xcell electroporator (BioRad, Hercules, Calif.) with the following setting: Voltage, 2.5 kV; capacitance, 25 μF; resistance, 600Ω. Immediately after electroporation, the cells were mixed with 0.6 ml of SB medium, (tryptone at 10 g/liter, yeast extract at 5 g/liter, NaCl at 4 g/liter, 91.1 g sorbitol, 0.4 g MgCl2, 0.3 g CaCl2, pH 7.5), transferred into a 10 ml sterile disposable tube and incubated shaking (140 rpm) for 3 h at 28° C. Finally, the cells were spread on SB agar plates containing kanamycin (50 μg/ml) or chloramphenicol (10 μg/ml) and incubated for 4 to 7 days at 28° C. The kanR or cmR colonies were recovered and streaked onto NBY plates containing appropriate antibiotics and screened for bioluminescence using a sensitive charge-coupled device (CCD) camera (Xenogen Corporation, Alameda, Calif.). The bioluminescent colonies were purified and stored for further analysis.
PCR Analysis of Bioluminescent Cmm Strains
To confirm the integration of the target DNA, PCR analysis was performed on all recovered colonies. After electroporation with pXX1, colonies were tested using the KanF/KanR primers that target part of the kananmycin resistant gene. Additionally, colonies recovered after electroporation with pXX2 (SEQ ID NO: 1) or pXX3 were tested for the presence of the chloramphenicol gene using primers CmxF/cmxR. PCR was performed on genomic DNA obtained from mutants using Taq PCR Master Mix kit (Qiagen, Valencia, Calif.).
Analysis of Bioluminescence
To quantify the amount of bioluminescence emitted by the transformed Cmm, isolates were grown in NBY broth containing the appropriate antibiotics with aeration at 28° C. for 48 h. Three replicates (100 μl) of each culture were then transferred to a black 96-well plate and bioluminescence was quantified using In vivo imaging system (IVIS) (Xenogen Corporation, Alameda, Calif.). The photon amount of each well (photons/s) was normalized for background luminescence by subtracting the value of a control well, containing NBY broth only. The O.D. was measured and the photon amount was divided by the O.D. to normalize for culture cell density. Highly bioluminescent colonies were selected for further studies.
The Hypersensitive Response (HR) Assay
The bioluminescent strains were tested for virulence in four o'clock (Mirabilis jalapa) plants (HR test). Briefly, four o'clock plants were grown in clay pots. Bacterial suspensions (108 cfu/ml) were prepared in sterile water and injected into fully expanded leaves of four o'clock plants using needle-less syringes, ensuring that the bacteria would infiltrate into intercellular spaces. Typically, the wild type virulent Cmm produces HR-characteristic necrotic lesions in the infiltrated areas within 36-48 hours (Gitaitis, 1990). For each strain, four injections were applied on one leaf and inoculated plants were placed for up to 48 hours in a greenhouse with a 14 hour light/10 hour dark photoperiod. The wild type Cmm strain C290 and sterile water were used as positive and negative controls, respectively.
Pathogenicity Assay
The inoculation of tomato seedlings with bioluminescent Cmm strains was performed as previously described by Poysa (1993). The cotyledons of tomato seedlings were clipped using dissecting scissors that had been dipped in a Cmm suspension (108 cfu/ml). Tomato seedlings (n=3) were inoculated with each strain and monitored for symptoms for three weeks. Furthermore, virulence was assessed by determining the number of plants that exhibited wilting symptoms within three weeks after inoculation. To quantify the bacterial numbers in infected tomato plants, stem samples from three plants were taken 3 cm above the cotyledons. Samples were ground in 3 ml phosphate buffer (pH=7.4, 10 mM) and centrifuged at 1000 rpm for 3 min. The supernatant was serially diluted and plated onto NBY agar plates that were supplemented with chloramphenicol (10 μg/ml). For samples inoculated with C290, serial dilutions were spread onto D2ANX (a semi-selective medium for Cmm). Colony counts were recorded on the fifth day. The bacterial numbers (cfu/g) were calculated as number of colonies*dilution*3 ml/weight of sample tissue. The HR and pathogenicity assays were repeated twice.
Assessing In Vitro Growth, Constitutive Expression of Bioluminescence, and Stability of Tn1409 Insertion in a Virulent Bioluminescent Cmm Strain
To assess constitutive lux expression, the strongly bioluminescent BL-Cmm17 strain was grown on NBY plates for 48 h at 28° C. and then transferred to 50 ml NBY broth in a 200 ml flask and incubated at 28° C. with shaking (160 rpm). At different time points, the OD600 and total bioluminescent counts were determined. Relative light units were determined by log transformation of average radiance (photons/second/cm2).
Stability of the transposon insertion was verified by growing the BL-Cmm strain without antibiotic selection in 5 ml NBY broth at 28° C. for 72 h, which was repeated for a total of five rounds (with 5 μl for each re-inoculation). After five rounds of growth, the culture was diluted in NBY broth and plated on NBY agar plates without antibiotics and incubated at 28° C. for 72 h. To assess the stability of insertion, 50 colonies from three replicates were randomly selected and patched on to NBY agar plates with chloramphenicol. The growth and bioluminescence of each colony was assessed to determine the stability of insertion.
The in vitro growth of the bioluminescent BL-Cmm17 was determined in both rich NBY medium as well as a Cmm minimal medium (Alarcon, et al, 1998). BL-Cmm17 strain growing on NBY plates for 48 h at 28° C. were inoculated into a 200 ml flask containing 50 ml NBY broth or the Cmm minimal medium and incubated at 28° C. with shaking at 160 rpm for 72 h. The Bacterial growth was monitored by measuring the OD600 from 1 ml culture at different times. C290 was used as a control throughout the experiment.
Mapping of the Tn1409 Insertion Site
Genomic DNA was extracted from both BL-Cmm6 and BL-Cmm17 using a MasterPure Gram Positive DNA purification kit (Epicentre, Madison, Wis.) and the insertion site in each strain was determined by bi-directional sequencing using the primers Xu3F/Xu3R. Sequencing was performed using dye terminators at a DNA sequencing core facility, (The Plant-Microbe Genomic Facility, The Ohio State University). Sequences were compared to the Cmm genome to determine the site of insertion.
Seed Inoculation with Bioluminescent Cmm Strains
The BL-Cmm17 was tested on tomato seeds using different inoculation methods. Healthy tomato seeds (cv. OH9242) were inoculated either by soaking seeds in bacterial suspension (108 cfu/ml) for 5 min under vacuum or by dropping 10 μl of bacterial suspension (108 to 105 cfu/ml) onto each seed. Seeds that were inoculated with sterilized water were used as controls. After inoculation, the seeds were air dried and placed on a moisture filter paper or in a glass tube with water agar (0.7%) or a square petri dish with water agar. The seeds were allowed to germinate in the dark at 28° C. Bioluminescent Cmm colonization of germinating seeds was monitored daily using the IVIS and bacteria were isolated from 5th day seedlings that exhibited luminescence signals.
Isolation of the Bioluminescent Cmm Strain
In order to generate a Cmm strain, we desired stable insertion of lux operon as well as constitutive expression of bioluminescence. Stable insertion is important for in planta real time studies even in the absence of antibiotic selection. Because of the lack of tools for genetic manipulation of Cmm, we tried several approaches to integrate lux genes into Cmm chromosome. Since the lux-operon in pXen13 was originally derived from the Gram-negative bacterium, Photorhabdus luminescence, it might not be expressed efficiently in Cmm, a Gram-positive bacterium. Consequently, the lux-operon was modified by incorporating AGGAGG, a Gram-positive ribosome binding site, upstream of each luxABCDE genes (Francis et al., 2000). The plasmid pXen-5 carries the lux-operon (SEQ ID NO: 2) optimized for expression in Gram positive bacteria. Initially we tried Tn5 based transposon mutagenesis approach that has been successfully used in Corynebacterium matruchotii that is closely related to Cmm. The plasmids, pUWGR4 and pXX1 carried a promoterless Gram negative lux-operon and a modified Gram positive lux-operon, respectively. The mosaic ends flanking these operons can be recognized by Tn5 transposase. After the electroporation of the Tn5:lux complexes into Cmm strains A300 and C290, a total of 84 colonies were recovered on SB plates supplemented with kanamycin (50 μg/ml). However, only about 50% of the recovered colonies grew when introduced to fresh plates and none were bioluminescent when screened by the CCD camera. PCR analysis targeting the kanamycin gene in resulting colonies showed that they lacked the transposon. Further, the negative control, Cmm electroporated without an EZ::TN<lux-Kan> casette, also resulted in colonies that grew on the kanamycin-containing plates, which suggested that Cmm strains used in electroporation might have exhibited spontaneous kanamycin resistance. These results suggest that the Tn5 transposon system is not suitable for Cmm mutagenesis.
Recently, Gartemann et al. developed an efficient mutagenesis system for Cmm using Tn1409. The Tn1409 containing vector pKGT452Cβ was modified to contain either Gram positive (pXX2) (
To evaluate the amount of bioluminescence in these mutants, light intensity of 108 CFU from each strain was measured from late log phase cultures and divided by OD to normalize for differences due to culture growth (
The Impact of the Lux Insertion on the Virulence of the Bioluminescent Cmm Strains
Since Tn1409 is randomly inserted into the Cmm genome (Kirchner,et al. 2001), the lux-operon might affect genes that are essential for pathogenicity. Therefore, it was necessary to test the bioluminescent strains and verify that the insertion did not affect virulence. For this purpose, the pathogenicity of the bioluminescent strains was tested using four'o clock plants, which exhibits a hypersensitive response (HR) when challenged with Cmm (Gitatis, 1990). Nine bioluminescent strains (BL-Cmm1 to 8, and 17) that emitted a relatively higher and different range of light intensity were selected for this assay and all isolates induced a strong HR on four o'clock plant, indicating that their virulence was similar to that of the wild type strain (Table 4,
a+, Positive for HR reaction, −, Negative for HR reaction;
bNumber of wilting plants/total inoculated plants.
cMean population of bacteria recovered from stem tissue of three replicates samples; no significant difference was observed as tested by T-test at P = 0.05.
To further test pathogenicity, the nine BL-Cmm strains (1 to 8, and 17) were inoculated into tomato seedlings and all isolates caused of plant wilting. However, some strains resulted in a lower number of wilting plants compared to the wild type Cmm 290 (data not shown). In addition, the number of bacteria in the stem tissue of the seedlings was determined to further characterize the colonization ability of the bioluminescent strains. The number of the bioluminescent strains retrieved from the stem tissue was similar to that of the wild type C290, suggesting that the ability of the BL strains to colonize the vascular tissue was not impacted by the lux-insert. Since the bacteria recovered from the stem tissues were still strongly bioluminescent when checked by the IVIS, it was concluded that lux-insert was stable in these strains.
Growth Analysis of Virulent and Bioluminescent Cmm Strains
Because of its relatively strong bioluminescent signals and the similarity of its virulence to C290, BL-Cmm17 was selected for further characterization. The in vitro growth of BL-Cmm17 was similar to that of its parent strain C290, both displaying a duplication time of 4.5 h (
Mapping the Insertion Site of Lux-Operon in Bioluminescent BL-Cmm 17
In order to characterize the insertion site of the lux-operon in the genome of BL-Cmm17, chromosomal DNA was isolated from the strain and sequenced with theXu3F/Xu3R primers. The sequences representing the strain and the pXX2 (SEQ ID NO: 1) vector were highly aligned with over 90% of identical sequences suggesting the whole vector integration into the genome of BL-Cmm17. This is not surprising and was found to happen occasionally when using this mutagenesis system in Cmm (personal communication with Dr. Karl-Heinz Gartemann). Therefore, further analysis is needed to identify the insertion sites. However, since the insertion of the lux-operon did not influence the growth of BL-Cmm 17 and the expression of the lux-operon was stable and constitutive, it can be assumed that the lux-operon was inserted dowmstream of a strong promoter without disturbing any housekeeping or essential virulence genes in the BL-Cmm17 genome.
Monitoring the Colonization of Bioluminescent Cmm in Germinating Tomato Seeds
To assess the usability of the BL-Cmm17 for monitoring in vivo infection processes in real-time, tomato seeds were inoculated with this strain. Over 100 seeds were tested using different geminating supplies, including filter papers in petri dishes, glass tubes containing water agar and square plates with water agar. The pattern of colonization was similar regardless of the germination supplies. Specifically, after inoculating the seeds with BL-Cmm17, the bacteria maintained their activity on the seed coat (
Efficiency of Transmission of Cmm During Propagation and the Influence of Environmental Factors on its Transmission
(Prophetic) (a) Seed to seedling transmission: Seed to seedling transmission of Cmm will be studied using rootstock (‘Maxifort’) and scion (open-pollinated indeterminate variety) seed infested with BL-Cmm17 harvested from plants inoculated by 1) flower inoculation and 2) injection of peduncles of very young fruit. Open-pollinated varieties will be used to minimize variation in progeny of inoculated plants. In our experience 90-100% of seed produced by this method are infected with Cmm. The level (CFU/seed) of infestation of individual seeds will be assessed by measuring luminescence; a standard curve will first be produced plotting photons/s vs. CFU from individual seeds. Seeds (five per treatment/sampling time; each seed is a replication) with estimated low (<102 CFU/seed), medium (102-104 CFU/seed) and high (>104 CFU/seed) BL-Cmm17 loads (non-infested seed will be used as a control) will be germinated under varying temperature and moisture conditions, in germination medium. Care will be taken to prevent seedling-to-seedling contamination. Seed-to-seedling movement/colonization by BL-Cmm17 will be visualized nondestructively daily using IVIS until stem diameters reach 1.5-2 mm (approx. 2 weeks). Five randomly selected seedlings/variable will be extracted individually and the extracts plated on YDC+chloramphenicol (10 ug/ml). Luminescent colonies will be counted. Extracts will also be tested by Cmm ELISA (Agdia, Inc., Elkhart, Ind.).
(Prophetic) (b) Transmission by grafting and topping: The influence of Cmm inoculum concentration and rootstock vs. scion colonization may be evaluated using seedlings generated as described above except that the optimal environmental conditions for Cmm transmission will be used. Preliminary studies will determine the timing of planting the scion and rootstock (rootstock usually requires 2-5 additional days). Rootstock and scion seedlings with the four inoculum loads (0, low, medium and high) will be grafted onto healthy plants. Hands, tools and work area will be sanitized after each graft. Seedlings will be incubated under standard conditions for 2-3 weeks (Rivard and Louws 2006). Colonization of seedlings by BL-Cmm17 will be followed using IVIS daily for 2-3 weeks. Five plants per treatment will be sampled randomly every five days and BL-Cmm17 culturing and ELISA, except that population counts will be made from 5 mm stem segments cut from the base of the plant to 2 cm above the graft union. At least five plants per treatment will be maintained in a greenhouse after graft healing and observed for disease symptoms. Topping studies will be designed to determine the population threshold for Cmm transmission by topping, the extent of spread from individual infected plants, and the influence of cutting tool (vs. hand pinching) on transmission. BL-Cmm17-infected plants will be grown from seed infested with 0, low, medium and high Cmm populations, or will be inoculated by injection with the pathogen at similar population levels. The goal is to obtain plants with different internal population counts. It will likely be necessary to produce a large number of plants (approx. 20-30 at each inoculum level) to achieve this goal. For simplicity, non-grafted 4-6 week-old plants will be topped by hand pinching or knife/blade, then 10 healthy plants will be topped sequentially with disinfecting hands or blades. Plants will be labeled in sequence and the extent of transmission will be determined using IVIS every 2-3 days and by symptoms, culturing and ELISA at the end of the experiment (3-4 weeks later). A 1-cm section from the cut end of the topped portion of each plant will be extracted and the extract will be dilution plated and tested by ELISA. Colony counts will be made after 4-5 days.
Identify Novel Small Molecules Inhibitory to Cmm that can Protect Plants from Bacterial Canker
(Prophetic) (a) High-throughput chemical screen for Cmm inhibitors. A library of >50,000 small molecule candidates will be screened for antimicrobial activity against BL-Cmm17 and naturally occurring strains of Cmm from the OSU culture collection. Compounds will be purchased from ChemBridge Corp. (San Diego, Calif., USA) and Enzo Life Sciences International, Inc. (Plymouth Meeting, Pa.). These libraries contain synthetic drug-like compounds as well as natural products with a molecular size generally in the range 250-500 Da and high chemical structure diversity, with tens of thousands of distinct chemical scaffolds. Antibiotics (ampicillin 150 μg/ml) and kanamycin (50 μg/ml) will be used as positive controls.
Compounds will be screened for antibacterial activity by bacterial growth-inhibition assays using a customized screening system in 96- or 384-well plates using a liquid-handling robot (Biomek3000; Beckman Coulter, Inc. Fullerton, Calif.). BL-Cmm17 will be cultured in TBY or other suitable medium to stationary phase, and then sub-cultured in the same medium (1:100) until it reaches an OD600 of 0.5-0.7. These bacteria, in exponential growth phase, will be diluted to OD600 of 0.05, and placed 200 μl/well into 96- or 384-well clear flat bottom plates. Compounds will be added at a final concentration of 25 μM. In all plates, the OD600 will be measured before and after 24 h incubations at 28° C. with shaking. Negative controls (1% DMSO vehicle) and positive controls (antibiotics) will be run in each plate. Bacterial growth will be monitored by a CCD camera using IVIS. In addition, we will determine the OD600 using a plate reader (Fisher Scientific). The percentage of bacterial growth inhibition will be determined as the percentage inhibition=100×(ODneg control−ODtest compound)/(ODneg control−ODpos control). EC50 values will be derived from analysis of concentration-response data, with serial dilutions of the active compounds.
To determine the effect of selected small molecules on different strains of Cmm, we will choose the five most promising small molecule inhibitors and evaluate their effect using a bacterial growth-inhibition assay by monitoring the OD600 following exposure to different compounds as described above. Different Cmm strains (at least 200) will be cultured in TBY or other selected medium to stationary phase, and then sub-cultured in the same medium (1:100) until they reached an OD600 of 0.5-0.7. These bacteria, which are in exponential growth phase, will be diluted to OD600 of 0.05, and placed 200 μl/well into 96 or 384-well clear flat bottom plates. Selected compounds will be added at a final concentration of 25 μM. Plates will be incubated for up to 48 h at 28° C. with shaking. Negative controls (1% DMSO vehicle) and positive controls (antibiotics) will be run in each plate. Bacterial growth will be monitored by measuring the OD600 at different times. All compounds will be screened in duplicate. We anticipate identifying small molecules that exhibit activity against all strains of Cmm.
(Prophetic) (b) Effect of selected small molecules on Cmm colonization of tomato seedlings. The effects of selected small molecules on colonization of tomato seedlings by Cmm will also be evaluated. Tomato plants (cv. OH9242-an inbred line) will be inoculated with BL-Cmm17 by spraying flowers or injecting peduncles and infested seed will be harvested. Seeds may also be artificially infested with the bacterium by vacuum infiltration (108 CFU/ml for 5 min) if necessary. Seeds will be distributed into 96-well plates and compounds will be added in a randomized complete block design to eight wells each at a final concentration of 25 μM, with control wells containing 1% DMSO. Plates will be incubated as described above for 5 days. The antibacterial effect of selected compounds will be determined by bioluminescent imaging (IVIS) each day and further confirmed by conventional CFU determination on a subset of four replicate germlings per treatment after 5 days. Another subset of four germlings per treatment, including non-treated and non-inoculated controls, with no visible bacterial colonization (no luminescence) will be transplanted individually into small pots and maintained in a greenhouse under warm, humid conditions for 4-6 weeks and assessed for symptoms. Plants will be drip irrigated individually under constant positive water pressure to avoid splashing and possible bacterial spread.
(Prophetic) (c) Effect of selected small molecules on bacterial canker development in production tomatoes (MSU). Finally, compounds that effectively inhibit bacterial canker symptom development in seedlings will be tested for activity in larger plants produced under conditions simulating commercial environments.
The following documents are hereby incorporated by reference (there is no admission thereby made with respect to whether any of the documents constitute prior art with respect to any of the claims):
It is to be understood that while exemplary embodiments have been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. For example, the vector pXX2 (SEQ ID NO: 1), may also be used for transformation of lux operon into other important pathogens facilitating the study of Clavibacter transmission, control, and plant interactions. Relevant pathogens include other important subspecies of C. michiganensis, including subsp. insidiosus (causes wilt and stunt on alfalfa), subsp. nebraskensis (cause wilt and leaf blight on corn), and subsp. sepedonicus (causes ring rot on potato) (Metzler et al. 1997; Kirchner et al. 2001). Therefore, embodiments apply broadly to the pathogenesis of disease caused by actinomycetes. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of priority to U.S. Provisional Patent Application No. 61/083,783, filed Jul. 25, 2008, which is hereby incorporated by reference in its entirety.
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