LAB93806 - Practical Applications and Analysis of Pathogenicity and Growth Inhibition of the Bacterial Sweet Potato Pathogen Streptomyces Ipomoeae
ACCESSION NO: 0206883 SUBFILE: CRIS
PROJ NO: LAB93806 AGENCY: CSREES LA.B
PROJ TYPE: HATCH PROJ STATUS: NEW
START: 01 JUL 2006 TERM: 30 JUN 2011 FY: 2007
INVESTIGATOR: Pettis, G. S.; Clark, C. A.; LaBonte, D. L.
PERFORMING INSTITUTION:
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
| KA |
Subject |
Science |
Pct |
| 201 |
1450 |
1040 |
25 |
| 212 |
1450 |
1100 |
50 |
| 215 |
1450 |
1100 |
25 |
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
BASIC 50% APPLIED 50% DEVELOPMENTAL 000%
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 non-pathogenic 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: 2007/01 TO 2007/12
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 currect 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 TO 2007/12
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 (not previously reported): 2007/01 TO 2007/12
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
PROJECT CONTACT:
Name: Pettis, G. S.
Phone: 225-578-2798
Fax: 225-578-2597
Email: GPettis@agcenter.lsu.edu
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