Robert Carver, Clark, Christopher A.
INVESTIGATOR: Pettis, G. S.; Clark, C. A.; LaBonte, D. L.
PLANT PATHOLOGY & CROP PHYSIOL
LOUISIANA STATE UNIVERSITY
BATON ROUGE, LOUISIANA 70893
PRACTICAL APPLICATIONS AND ANALYSIS OF PATHOGENICITY AND GROWTH INHIBITION OF THE BACTERIAL SWEET POTATO PATHOGEN STREPTOMYCES IPOMOEAE.
CLASSIFICATION HEADINGS: R201 . Plant Genome, Genetics, and Genetic Mechanisms; S1450 . Sweet potato; F1040 . Molecular biology; R212 . Pathogens and Nematodes Affecting Plants; F1100 . Bacteriology; R215 . Biological Control of Pests Affecting Plants
NON-TECHNICAL SUMMARY: Little information is known as to how the bacterium Streptomyces ipomoeae causes soil rot disease of sweet potatoes. Meanwhile, sweet potato production in Louisiana and elsewhere is solely dependent on the use of soil-rot-resistant cultivars, whose resistance phenotype remains uncharacterized and which are identified only after lengthy field studies. Here, the mechanism(s) by which S. ipomoeae causes soil rot will be analyzed, and useful applications will be developed, including an efficient method of screening for soil-rot-resistant cultivars and creation of avirulent S. ipomoeae mutants that offer potential benefits to sweet potato growth. Sweet potato genes involved in soil-rot-resistance will also be identified, and an alternative method of soil rot prevention will be developed.
OBJECTIVES: We will construct stable S. ipomoeae mutants using recently cloned loci believed to be essential for production of the phytotoxin thaxtomin C. The mutants will be tested for thaxtomin C production, for virulence on sweet potato storage and fibrous roots, and, if found to be avirulent, for potentially beneficial nonpathogenic colonization of sweet potato plants. We will also develop a reliable, fast and cost-effective in vitro screening method for soil rot resistance. Candidate genes of potential importance to soil rot resistance in storage and/or fibrous roots of sweet potato will be identified by using microarray analysis. Finally, we will isolate a second inhibitory substance that is specific for sensitive S. ipomoeae strains as well as clone the cognate genes for this inhibitor.
APPROACH: We have recently cloned and sequenced the biosynthetic gene cluster that is likely to be responsible for production of the phytotoxin thaxtomin C from S. ipomoeae. To construct specific mutants of this gene cluster, which is the basis of our first objective, individual genes will be replaced with an antibiotic resistance gene cassette, and these constructs will be introduced back into S. ipomoeae. Following appropriate recombination, genetically stable mutants will be tested for thaxtomin production and virulence on fibrous and storage roots of sweet potatoes. If the S. ipomoeae mutants are avirulent, we will explore the possibility that they can still colonize sweet potato roots and thus potentially contribute beneficially to the growth of sweet potatoes. A plasmid encoding a modified form of green fluorescent protein (GFP) will be introduced into the mutants, and the roots of sweet potato plants that have been exposed to S. ipomoeae mutants carrying GFP plasmid sequences will be analyzed by using laser scanning confocal microscopy. For development of an in vitro screening assay for soil rot resistance (i.e., our second objective), plantlets from a variety of resistant or sensitive sweet potato cultivars will be grown on agar media containing various concentrations of thaxtomin C, which we will purify either from S. ipomoeae itself or possibly from a heterologous streptomycete host that contains the cloned thaxtomin C gene cluster. As an alternative, we will also test the growth of plantlets on agar media that has been seeded with individual S. ipomoeae strains. For our third objective, we will use the technology of microarray analysis to identify genes important for soil rot resistance in sweet potatoes. The fibrous and storage roots of resistant and sensitive cultivars will be exposed to pathogenic S. ipomoeae bacteria, and RNA will be isolated from relevant plant tissue samples and used to make fluorescently labeled complementary DNA (cDNA). A given microarray hybridization will involve a single sweet potato cultivar (resistant or sensitive), and will compare RNA isolated from plants that were exposed to S. ipomoeae versus control plants that were not exposed. The ratios of induction or repression for a particular cultivar will then be compared to those of other cultivars, and statistically significant differences in expression between sensitive and resistant cultivars will be evaluated. The fourth and final objective will involve isolation of a substance that is produced by some S. ipomoeae strains which is inhibitory to other strains of this same bacterial species. We previously isolated a separate interstrain inhibitory substance and, based on our previous analysis of S. ipomoeae interstrain reactions, these compounds together will inhibit the growth of potentially all S. ipomoeae strains. In addition to isolation of the additional inhibitor we will also clone the genes responsible for its production either by using colony blot hybridization of a relevant S. ipomoeae genomic cosmid library, or by screening that library for inhibitor production using a heterologous streptomycete host.
KEYWORDS: crop disease; commodity production; plant resistance; plant pathogen; gene expression; vegetable; sweet potato; in vitro assay; microarrays; phytotoxin; thaxtomin; streptomyces ipomoeae
PROGRESS: 2010/01 TO 2010/12
OUTPUTS: The project produced three outputs during this calendar year. The first output was a presentation at a national meeting, which is now being prepared for publication. The other two outputs consisted of two new mutants of S. ipomoeae that were constructed. The mutants are deleted for separate genes involved in synthesis of the phytotoxin thaxtomin C. This brings the total number of thaxtomin mutants constructed so far during the project to four. These mutants are helping to unravel the mechanisms used by S. ipomoeae to cause soil rot disease of sweet potato. PARTICIPANTS: The PI (Pettis) directed the project and trained Dongli Guan, a Ph.D. graduate student who performed the majority of the research described in this report. Co-investigator Clark also provided training to Ms. Guan for experiments involving sweet potato plants. For the manuscript being prepared for publication, Ms. Guan will be first author and a former Ph.D. student (Dr. Brenda Grau), who was also trained by the PI, will be second author. The project has provided extensive training in the area of plant-pathogenic microbe interactions for several graduate students. TARGET AUDIENCES: Experimental results, interpretations of those results, and publications generated from the project are shared with LSU AgCenter and other sweet potato researchers and extension personnel at annual meetings. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
IMPACT: 2010/01 TO 2010/12
One of the mutants was constructed by deleting a gene known as txtR, which encodes a transcriptional activator of the thaxtomin gene cluster. In Streptomyces potato pathogens, the TxtR protein binds cellobiose (the smallest subunit of the plant cell wall constituent cellulose), and this complex then activates thaxtomin gene transcription. We found that the txtR mutant of S. ipomoeae was unable to infect fibrous roots of sweet potato (which appears to be the entry point for sweet potato infection) but was still able to cause necrosis on sweet potato storage-root tissue. These virulence results are consistent with those of our previous two thaxtomin mutants and indicate that thaxtomin is required for the initial infection of sweet potato. After invasion, however, S. ipomoeae then has other virulence factor(s), which cause necrosis. During this calendar year we also found evidence that cellobiose is not the molecule to which S. ipomoeae TxtR protein binds to activate thaxtomin gene transcription. Therefore, it appears that a novel molecule is involved, and it may be specific somehow to sweet potato and related Convolvulaceae plant species that comprise the host range of S. ipomoeae. The other mutant was constructed by deleting a gene known as txtH, which appears to be involved in synthesis of certain secondary metabolites in bacteria, including thaxtomins. Interestingly, the txtH mutant was still able to infect fibrous roots. It also still caused necrosis on storage-root tissue. Based on similar analysis performed in other bacteria, we suspect that there exists at least one other txtH paralog in the S. ipomoeae genome which can complement the txtH deletion. The other gene is likely in a biosynthesis cluster for a different secondary metabolite. Overall, our results are providing a clearer picture of the role of thaxtomin in the infection of sweet potato by S. ipomoeae. This information may also provide insights as to how other pathogens attack sweet potato and so may ultimately help reduce disease incidence. A practical outcome of this research will be the development of an in vitro screening method for soil-rot-resistant sweet potato cultivars, which would replace the current laborious and inefficient field testing method. Our results indicate that since thaxtomin C is required for initial infection by S. ipomoeae, sweet potato germplasm could be screened for resistance to purified thaxtomin C in petri dishes. This method would be faster, allow much more germplasm to be screened and so will greatly improve our ability to identify cultivars with other desirable growth characteristics in addition to soil rot resistance. This endpoint would be beneficial to both farmers and consumers.
PUBLICATIONS (not previously reported): 2010/01 TO 2010/12
Guan D., Grau B.L., Clark C.A., Loria R., and Pettis G.S. 2010. Cloning and Characterization of the Thaxtomin C Biosynthetic Gene Cluster of the Bacterial Sweet Potato Pathogen Streptomyces ipomoeae. 110th General Meeting of the American Society for Microbiology Abstracts (ISBN 978-1-55581-623-0; 2010:B-584), San Diego, CA, May 2010.
PROGRESS: 2009/01/01 TO 2009/12/31
OUTPUTS: Streptomyces ipomoeae is the causative agent of soil rot disease of sweet potatoes. The mechanisms by which the organism causes this disease are largely unknown, but we are making progress toward their identification. Last year we developed a method to genetically manipulate the bacterium (this is the subject of the publication listed below) and used the method to construct a targeted S. ipomoeae mutant that we anticipated would show less virulence. This year we used the method to construct an additional targeted mutant that we also anticipated would show less virulence and we also fully characterized the two mutants for virulence function (see next section). The other output for this reporting period is that we developed a PCR-based technique for monitoring gene expression of S. ipomoeae in situ during infection of sweet potato storage roots. PARTICIPANTS: The PI (Pettis) directed the project, including training and working with several Ph.D. graduate students (e.g., Dongli Guan, Jing Wang, Brenda Grau), who are not supported by the project but who performed much of the research described. Co-investigator Clark also provided training and direction to students for experiments involving sweet potato plants. We are currently preparing a manuscript based on our thaxtomin C mutant studies with Dongli as first author and Brenda as a co-author. The project has thus provided an extensive training ground in the area of plant-microbe interactions for several graduate students. TARGET AUDIENCES: Results from the lab, including publications generated, are shared with other LSU Ag Center sweet potato researchers and extension personnel at annual meetings. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
IMPACT: 2009/01/01 TO 2009/12/31
Both of our targeted mutants lacked portions of the gene cluster that is involved in production of a phytotoxin known as thaxtomin C. The mutants were unable to infect sweet potato seedlings via their fibrous feeder roots. Wild type S. ipomoeae causes necrotic destruction of the fibrous roots and thereby reduces sweet potato yield. The two mutants showed no necrosis on the feeder roots, a result which indicates that penetration and infection of sweet potato fibrous roots by S. ipomoeae is thaxtomin C-dependent. The two mutants did, however, cause necrosis on storage root slices which was indistinguishable from that caused by the wild-type bacterium. This result indicates that thaxtomin C is dispensable for the ability of S. ipomoeae to cause infection at wound sites on storage roots. Such infection leads to lesions on the storage roots, which reduce their market value. We will continue our efforts to identify other virulence factors of S. ipomoeae which contribute to this latter form of necrosis. The outcome of our PCR technique to monitor S. ipomoeae gene expression on sweet potato storage roots is that we were able to demonstrate in situ expression of a thaxtomin C biosynthetic gene, which was not expressed in the S. ipomoeae spores used to inoculate the storage roots. In our studies of S. ipomoeae virulence, we can now use this rapid and inexpensive method as an indicator of in situ thaxtomin C production rather than having to perform the more lengthy and costly method of purifying toxin from the host plant. Overall, our results here confirm that thaxtomin C is an important virulence factor in the development of soil rot. The impact of these findings is that they provide a basis for further analysis of thaxtomin C biosynthesis, including identifying a putative inducer molecule(s) produced by the sweet potato host. This in turn will allow us to purify thaxtomin C in sufficient quantities so that it can be included in an efficient in vitro screening method for identifying sweet potato cultivars that are soil-rot-resistant. Such an in vitro method will replace the current lengthy, inefficient and labor-intensive field method of screening and thus improve our ability to identify cultivars with other desirable growth characteristics. This overall goal will benefit farmers and consumers by providing significant improvements in new variety development, as well as yield and market value of sweet potatoes.
PUBLICATIONS: 2009/01/01 TO 2009/12/31
Guan D. and Pettis, G.S. (2009). Intergeneric conjugal gene transfer from Escherichia coli to the sweet potato pathogen Streptomyces ipomoeae. Lett. Appl. Microbiol. 49: 67-72.
PROGRESS: 2008/01/01 TO 2008/12/31
OUTPUTS: Streptomyces ipomoeae causes the devastating disease known as soil rot of sweet potatoes. The disease reduces both the yield and market value of susceptible sweet potato varieties; however, the mechanisms by which this bacterium causes sweet potato disease remain unknown. Toward elucidation of these mechanisms, two main outputs were generated during this project period. To identify S. ipomoeae genes important for its virulence, a method to genetically manipulate this pathogen needed to be developed. The first output was to develop such a technique. This technique was then used to generate the second significant output, which was the construction of a targeted S. ipomoeae mutant whose virulence has been compromised. It is important to note that prior to these studies no genetic manipulation of this sweet potato pathogen had ever been accomplished. PARTICIPANTS: The PI (G.S. Pettis) directed the project, including working with several Ph.D. graduate students (D. Guan, J. Wang, K.S. Schully, and B.L. Grau), who were not supported by this project but who nevertheless participated by performing the research described. Co-investigator Dr. C. Clark also provided training for the graduate students in pathogenicity and bioassay protocols, including interpretation of results. The construction of thaxtomin mutants of S. ipomoeae was facilitated by cloning of the thaxtomin biosynthetic gene cluster, which was performed by the PI during sabbatical in Dr. Rosemary Loria's lab at Cornell University. Upon completion of our thaxtomin mutant experiments, the results will be submitted for publication to a high-quality journal with one of the graduate students (D. Guan) as lead author along with C.A. Clark, B.L. Grau (another graduate student), R. Loria and the PI. The project has thus provided an extensive training ground in the area of plant-microbe interactions and related areas of microbiology for several graduate students. TARGET AUDIENCES: Results from the lab, including publications generated, are shared with other sweet potato researchers and extension personnel at the annual conference of the Louisiana State University Agricultural Center. In November 2008, the PI also presented a seminar to the Department of Plant Pathology and Crop Physiology (part of the LSU Ag Center) entitled "Construction and characterization of thaxtomin C mutants of the sweet potato pathogen Streptomyces ipomoeae." PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
IMPACT: 2008/01/01 TO 2008/12/31
The first outcome of this project period is that we were able to genetically manipulate S. ipomoeae. The technique involves transferring mutated versions of cloned S. ipomoeae genes from a separate genetically manipulatable bacterial species, Escherichia coli, back into S. ipomoeae by the process known as conjugation. Various experimental parameters affecting the efficiency of this conjugation method were also optimized, and our results were submitted for publication (a revised version of this manuscript that incorporated minor modifications suggested by the reviewers has just been completed). We then used this technique to create a mutant of S. ipomoeae that is defective in production of a likely phytotoxin known as thaxtomin C. Our preliminary analysis so far is that this thaxtomin mutant of S. ipomoeae shows significantly lower virulence on sweet potatoes; however, it is possible that the mutant can still colonize surfaces of the plant without causing disease. The direct impact of these results is that we have demonstrated that S. ipomoeae can be genetically manipulated, and, in so doing, we have likely identified one important virulence factor for this sweet potato pathogen. An indirect impact of this work is that it provides impetus for the eventual large-scale purification of thaxtomin C for its inclusion in an efficient in vitro screening method for soil-rot-resistant sweet potato germplasm. This method would supersede the current lengthy and labor-intensive field method of screening and would improve our ability to isolated sweet potato cultivars with more overall desirable growth and resistance characteristics. If our S. ipomoeae thaxtomin mutant can still colonize the surface of sweet potatoes (without causing disease), another indirect impact of this work is that the mutant may actually offer potential growth benefits to sweet potato plants (e.g., protection against other pathogenic S. ipomoeae strains via interstrain inhibition, protection against pathogenic fungi via antibiotic production, promotion of the mycorrhiza etc.). A final outcome of this reporting period is that we determined the production kinetics of a substance (the bacteriocin known as ipomicin) which is produced by certain strains of S. ipomoeae and which kills other strains of this same bacterial species. The direct impact of this finding is that it improves our ability for large-scale purification of ipomicin so that we can study its use as a potential biocontrol agent for preventing infection of sweet potatoes by S. ipomoeae. Taken together, all of these direct and indirect impacts have the potential to offer farmers and ultimately consumers significant improvements in new variety development, yield and market value of sweet potatoes.
PUBLICATIONS: 2008/01/01 TO 2008/12/31
Wang J., K.L. Schully, and G.S. Pettis. 2009. Growth-regulated expression of a bacteriocin, produced by the sweet potato pathogen Streptomyces ipomoeae, that exhibits interstrain inhibition. Appl. Environ. Microbiol. In press.
PROGRESS: 2007/01/01 TO 2007/12/31
OUTPUTS: By developing laboratory-medium conditions for S. ipomoeae which allow for production of thaxtomin, we have taken the first step towards large-scale production and purification of this virulence factor. Such large-scale production will be critical for the development of an in vitro screening method for soil rot resistance, which will likely involve testing sweet potato germplasm on an agar medium that contains purified S. ipomoeae thaxtomin as well as possibly other potential virulence factors. This method, which should prove to be significantly more efficient than current field testing, will allow much more germplasm to be screened, and thus will greatly improve our ability to isolate cultivars with more overall desirable growth and resistance traits. Research findings have been submitted for publication. PARTICIPANTS: The PI (G.S. Pettis) directed the project, including working with a Ph.D. graduate student (D. Guan), who is not supported by this project but who nevertheless performed the experiments described herein. The PI and graduate student also prepared the manuscript that was recently submitted for publication. A co-investigator (C.A. Clark) also trained the graduate student in performing the pathogenicity assay, which will be needed to complete objective one of the project. Upon completion of construction and characterization of the S. ipomoeae thaxtomin mutants, a manuscript detailing this work will be prepared and this will involve the PI, the graduate student, and Dr. Clark, as well as Dr. Rosemary Loria of Cornell University, in whose lab the PI first cloned the S. ipomoeae thaxtomin gene cluster while on sabbatical. The project has served as a training ground for the Ph.D. student, who has received extensive training from the PI in the areas of microbial genetics and molecular biology as they pertain to Streptomyces plant pathogens as well as training from Dr. Clark in analyzing host-pathogen interactions between sweet potato and the S. ipomoeae bacterium. TARGET AUDIENCES: Results from the lab are reported to other sweet potato researchers and extension personnel at the annual conference of the Louisiana State University Agricultural Center.
IMPACT: 2007/01/01 TO 2007/12/31
Progress was made for the first two objectives of the project. The first objective involves constructing S. ipomoeae mutants that are defective for production of the phytotoxin thaxtomin and then analyzing these mutants for virulence and colonization of plants. It is anticipated that such mutants will be unable to cause disease but will still be able to colonize sweet potato plants and thus offer positive growth effects (e.g., hyperparasitization of pathogenic fungi). To construct S. ipomoeae thaxtomin mutants, we first developed a method for introducing DNA into S. ipomoeae using intergeneric conjugation from E. coli. Optimum media conditions for sporulation of S. ipomoeae and for the actual conjugation process were established, and several other parameters important for the method were evaluated. During our previous Hatch project, we had cloned and sequenced the entire thaxtomin gene cluster of S. ipomoeae. For mutant construction here, we initially inserted the antibiotic-resistance gene aadA into individual cloned thaxtomin genes, and then introduced these constructs into S. ipomoeae using our intergeneric conjugation method in order to replace the functional chromosomal copies of these genes via relevant homologous recombination. Unfortunately, we found that the presence of the aadA gene in S. ipomoeae caused slow growth and genetic instability. As an alternative, we have begun constructing in-frame deletions of individual thaxtomin genes, which will be used to replace the functional chromosomal copies using similar homologous recombination events as before. This method eliminates the need for an antibiotic-resistance marker being present in the interrupted gene and so should prove successful here. The second objective is to develop an in vitro screening process for soil rot resistance. It is envisioned that this will involve a laboratory medium containing one or more purified virulence factors of S. ipomoeae upon which individual sweet potato germplasm will be screened for resistance. The obvious first virulence factor to test here is the phytotoxic thaxtomin produced by S. ipomoeae. Unfortunately, this phytotoxin has historically been purified in only very small amounts from infected sweet potato tissue, and it was not previously found to be produced by S. ipomoeae in various lab culture media. Recently, however, we found media conditions that appear to result in production of thaxtomin by S. ipomoeae. Four-day-old culture supernatants of S. ipomoeae grown in oat bran broth containing 0.7% cellobiose showed the yellowish tint which is typical for thaxtomin production. Following extraction of the supernatant with ethyl acetate, the latter fraction was concentrated, and the presence of thaxtomin was demonstrated by thin layer chromatography (TLC) using purified thaxtomin standard as a control. We should now be able to routinely isolate thaxtomin directly from S. ipomoeae laboratory cultures, a process which will greatly streamline thaxtomin isolation and allow us to begin developing and testing our in vitro soil-rot-resistance screening method.
PUBLICATIONS: 2007/01/01 TO 2007/12/31
No publications reported this period
PROGRESS: 2006/07/01 TO 2006/12/31
Although the project is in its early stages, we have made some important progress with regard to our first objective, which is to construct S. ipomoeae mutants that are defective for production of the phytotoxin thaxtomin and assess them for virulence and colonization of sweet potato plants. During our previous Hatch project, we cloned and sequenced the entire thaxtomin C biosynthetic pathway from Streptomyces ipomoeae strain 91-03. To construct stable thaxtomin mutants, a given cloned thaxtomin gene will be inactivated by insertion of an antibiotic resistance cassette, and the interrupted gene will be introduced back into S. ipomoeae where it will replace the normal functional gene copy via homologous recombination. We originally found that S. ipomoeae was not amenable to several typical methods of DNA introduction which work well in other Streptomyces species. These methods included transformation of protoplasts as well as electroporation of mycelium or germinating spores. Recently, however, we found that DNA could be transferred from Escherichia coli to S. ipomoeae during conjugation, and the incoming DNA could be stably maintained in the recipient. We have worked out the parameters for this method (which we will soon write up for publication) and are currently preparing to use the method to introduce an insertionally inactivated copy of a relevant thaxtomin biosynthetic gene in order to construct a stable mutant. We anticipate that in the next two to three months such a mutant will be constructed, and we will then begin testing its virulence capacity on sweet potato storage and fibrous roots. If it is found to be avirulent, we will then examine its ability to still colonize sweet potatoes and offer potential benefits to their growth (e.g., hyperparasitizing of pathogenic fungi, contributing to formation of the mycorrhiza).
IMPACT: 2006/07/01 TO 2006/12/31
The project is expected to be economically beneficial to sweet potato farmers by improving the methods by which sweet potato varieties possessing desirable growth characteristics are screened and isolated and by creating a stable avirulent form of the normally pathogenic soil rot bacterium which may still interact specifically with sweet potatoes but now offer benefits to their growth.
PUBLICATIONS: 2006/07/01 TO 2006/12/31
No publications reported this period
Name: Pettis, G. S.