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Mitigating the Effect of Plant Disease Through an Understanding of Bacterial Soft Rot

Dumenyo, C Korsi
Tennessee State University
Start date
End date


1. Purify the inducer of bacterial virulence from host extracts.

2. Identify bacterial genes responding to host signals and therefore important for disease.

Expected Outcomes:

1. Knowledge of the chemicals or class of chemicals produced by the host which serve as inducers of pathogen virulence.

2. The establishment of a relatively simple method for testing different plants species and their products for the production of the chemicals that induce bacterial virulence.

3. A list of cultivated plant species that produce the inducer.

4. A mutant library of Pectobacterium carotovorum with reporter fusions to all possible genes.

5. Collection of bacterial mutants in genes involved in the responses to host signals.

6. Knowledge of the effects of induced genes on bacterial virulence.

More information

Plant diseases cost the US economy billions of dollars annually in lost yield, diseases control and degradation to our environment due to the use of chemical control agents. Blackleg and soft rot are seed-borne diseases of many annual crops caused by the soft rot bacteria. The major pathogen species, Pectobacterium and Dickeya infect over 80 cultivated plants. The key to controlling this disease complex could lie in our understanding of how the pathogen interacts with the host. However, there is still a lot we do not understand about this process. Most importantly, we do not understand how the pathogen invades the host and the function of many bacterial genes. The goal in this project is to dissect into the pathogenesis of soft rot disease using Pectobacterium species to identify the bacterial genes whose expression is important for pathogenesis in the host and to further study how these bacteria respond to the different environmental conditions in which the bacterium finds itself during the disease cycle. Specifically, the project addresses the interaction of the pathogen with the host, induction of pathogen gene expression by signals from both the host and nonhost, assigning functions to the pathogen genes and investigation into the chemical nature of the host-produced pathogen virulence inducer. This project to characterize the mechanisms of pathogenicity by soft rot pathogens addresses NIFA strategic goals of Global food security, and Food safety. The project will increase our understanding of not only the biological role of genes in the sequenced genome of important plant pathogens but also how these genes are used to sense the presence of a host in the vicinity of the pathogen. Additionally, the project will contribute to narrow the ever widening gap between the available pathogen gene sequences and known functions for those genes. The findings from this project will give us the information we need to possibly start on the path of manipulating the hosts to make them less susceptible to this deadly group of pathogens.

The goals here are to purify the plant metabolite that is responsible for inducing pathogen virulence and identify bacterial genes which respond to the presence of the host signal. We shall begin with the physical characterization of the inducer in the host extract. This will compost of confirmation of induction, characterization of the conditions of induction and physical characterization of the inducer. Comparison will be made of the responses of P. carotovorum (Pc) to (PGA), potato extract (PE) and celery extract (CE) in minimal medium. The kdgR mutant of Pc, which no longer responds PGA will also be tested for its response to the host extracts. The conditions governing the induction such as the effect of pH of the medium on the induction will also be tested. Different hosts will be survey for their production of the inducer. Before any purification attempts, we shall do studies such as dosage effect, different extraction methods, the effect of dialysis, acid and alkaline shock of the extract to use in characterizing the inducer. Following the initial characterization, we shall run the extract on thin layer chromatography using different solvents. The solvent in which the content will be resolved well shall be used for possible column chromatography. We shall use the transposon pEVS168 with a promoter-less gfp gene to mutagenize Pc Ecc71N, a spontaneous Nal-resistant strain in bi-parental mating. The mating mixture will be selected on LB broth supplemented with nalidixic acid (Nal) and chloramphenicol (Km) and sub-cultured continuously on the same selection medium until the pool is made up of only Pectobacterium mutant population. After 6-8 sub-culturing, a sample of the mutant library culture will be diluted and plated on MM agar+host extract + chloramphenicol and high GFP expressers will be selected and double patched on same medium without host extract. Colonies which express high GFP on host extract but not on medium without extract will be selected for further analysis of the effect on host extract on the expression of the truncated genes and the role of the genes on virulence of the pathogen.

PROGRESS: 2013/01 TO 2013/09
Target Audience: Scientific Research community Graduate and undergraduate students from underrepresented minority groups.

Nothing Reported What opportunities for training and professional development has the project provided? The following students were trained under this project resulting in their theses and dissertations for their MS and PhD degree, respectively. Paul Agyemang, PhD in Biological Sciences, May 2011. Project Title: Interaction between soft rot erwinia and host signals. Caleb Kersey, PhD in Biological Sciences, May 2011. Project title: Regulation of Pectobacterium virulence by membrane transporters. Sean Taylor, MS in Agricultural Sciences Dec 2012. Thesis Title: Genetic and physiological characterization of Erwinia tracheiphilla, the pathogen that causes bacterial wilt of cucumber. Roodie Johnson, MS in Agricultural Sciences, May 2013. Project Title: Host signal regulation of soft rot virulence. How have the results been disseminated to communities of interest? Publication: Kersey, C. M., Agyemang, P. A. and Dumenyo, C.K. 2012. CorA, the magnesium/nickel/cobalt transporter, affects virulence and extracellular enzyme production in the soft rot pathogen Pectobacterium carotovorum. Molecular Plant Pathology 13(1): 58-71 Conference Presentations Dumenyo, CK, Kersey, CM, Agyemang, PA, and Hageman, BH. 2011. Bacterial soft rot disease: Identification of the players involved in Pectobacterium-host interactions. Abstract # O-SP-S-5. ARD Symposium, Atlanta, GA. April 9-13, 2011. Kersey, CM and Dumenyo, CK. 2011. The Magnesium, Nickel, Cobalt transporter, CorA affects exoenzyme production and virulence in the soft rot pathogen, Pectobacterium carotovorum. Abstract # P-FS-G-11. ARD Symposium, Atlanta, GA. April 9-13, 2011. Kersey, C. & Dumenyo, C. K. 2010. CorA, a magnesium/nickel/cobalt transporter is required for full virulence in the soft rot pathogen, Pectobacterium carotovorum. Poster # 532-P. Annual Meeting of the American Phytopaythological Society, 7-11 August 2010, Charlotte, NC. Phytopathology 100:S61 Agyemang, P. and Dumenyo, C. K. 2010. Isolation and characterization of Pectobacterium carotovorum mutnats in host signal-responsive genes. Poster # 164-P. Annual Meeting of the American Phytopaythological Society, 7-11 August 2010, Charlotte, NC. Phytopathology 100:S3 Agyemang, P., Kersey, C. and Dumenyo, C. K. 2010. Initial characterization of plant metabolites inducing virulence in the soft rot pathogens, Pectobacterium spp. Poster # TP14, 32nd Annual University-wide research symposium. Tenn. State University. March 15-19, 2010 Kersey, C. and Dumenyo, C. K. 2010. Characterization of a Tn5 mutant of the soft rot bacterium, Pectobacterium carotovorum. Poster # TA8, 32nd Annual University-wide research symposium. Tenn. State University. March 15-19, 2010 Dumenyo, C. Korsi. 2009. Flow cytometric analysis of gene expression in the pathogenic bacterium, Erwinia carotovora subspecies atroseptica. Oral Presentation at the 31st Annual University Wide Research Symposium. TSU Dumenyo, C.K. Verbene-Sutton, S. and Ghazi, S. 2009. Flow cytometric analysis and sorting of host signal inducible transposon insertion mutants of Erwinia carotovora subsp. atroseptica. 15th Biennial ARD Research Symposium. Atlanta, GA. Dissertations/Theses 1. Kersey, Caleb M. 2011. Regulation of Pectobacterium virulence by membrane transporters. PhD Dissertation, Tennessee State University, Nashville, TN, USA. 2. Agyemang, Paul A. 2011. Interaction between soft rot erwinia and host signals. PhD. Dissertation. Tennessee State University, Nashville, TN, USA. 3. Johnson, Roodie 2013. Isolation of host extract induced mutants of Pectobacterium carotovorum by transposon mutagenesis. MS Thesis. Tennessee State University, Nashville, TN, USA What do you plan to do during the next reporting period to accomplish the goals? This is final Report

PROGRESS: 2012/01/01 TO 2012/12/31
OUTPUTS: We have characterized another mutant from the pool of mutants isolated initially through transposon mutagenesis using mini-Tn lacZ1. This mutant, designated KD200, exhibited an increase in extracellular protease activity. The mutant KD200 was tested for pectolytic activity on polygalacturonate yeast extract agar (PYA) medium and showed a similar increase in enzymatic activities compared to the parent KD100. Increased enzymatic activity of mutant KD200 was further confirmed using semi-quantitative agar plate assays. The levels of pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt) were all notably higher in KD200 as compared to KD100. To accurately measure the increase in enzyme production in mutant KD200, parent KD100 and KD200 were grown in minimal medium and the culture supernatant was assayed for pectate lyase (Pel) activity. The production of Pel in KD200 was approximately 32 times higher than its parent KD100. This level of enzyme activity is one of the highest we have seen and is typically only found in mutants of strong negative regulators of exoenzyme such as rsmA, hexA, and rsmC. To determine how this high enzyme producing mutant would respond to inducers present in host extracts, KD200 and the parent KD100 were grown in celery extract (CE)-supplemented minimal medium (MM+CE) and the culture supernatant was measured for Pel activity. The mutant KD200 was only induced further about 4 fold in the presence of CE while the parent KD100 was induced 16 fold higher than MM. The increased activities of the major exoenzymes in mutant KD200 corresponded with an increase in transcripts of these enzymes, Pel, Peh, Cel, and Prt as measured by semi-quantitative RT-PCR. The differences in transcript levels observed using semi-quantitative RT-PCR assays were further quantified by measuring the expression with a real time RT-qPCR using SYBR Green detection system. Transcript levels of isozymes of pectale lyase, pel-1; polygalacturonase, peh-1; cellulose, celV; and protease, prtW were approximately 8, 24, 3 and 19 times higher in mutant KD200 respectively. These data confirm that the mutation in K200 produced an increase in exoenzyme transcripts and ultimately elevated enzyme activity. Primers were used to identify the gene truncated by insertion of the transposon in KD200 by sequencing across the transposon junction into the flanking genomic sequence. The generated DNA sequence was used as query to search against genomic databases of Pectobacterium and Dickeya spp. Based on the genomic sequence of P. carotovorum PC1 (NC 012917.1), the transposon inserted at base 41363. The transposon junction sequence revealed a 90% identity (with an E-value 1e-58.) with segments of P. carotovora subsp. carotovorum PC1 gene (PC1 0037) and P. carotovora SCRI1043 (ECA 4400). These genes are annotated as an anion transporter and putative sodium: sulfate symporter respectively, thus the gene was designated nssAEcc71 for Na SO4 symporter. PARTICIPANTS: Korsi Dumenyo, Caleb Kersey, Paul Agyemang TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

PROGRESS: 2011/01/01 TO 2011/12/31
OUTPUTS: We have determined the optimum concentration of host extract suitable for induction of extracellular enzyme production in the soft rot pathogens, Pectobacterium spp. Pectate lyase (Pel) activity was measured in P. carotovorum strain Ecc71 and in P. atrosepticum strain SCRI1043 grown in minimal medium supplemented with varying concentrations of juiced and filtered (GJF) or autoclaved (GJA) extract of celery petioles (CE) or potato tubers (PE). The enzymes, Pel and ?-galactosidase activities of test tube-grown cultures were also assayed in Pectobacterium strain KD100 carrying the pel1-lacZ plasmid to determine the response of these bacteria to the extracts. We studied how widely distributed the inducer(s) might be among different host plant species of soft rot with GJF extracts from ten fruits or vegetables which also serve as hosts to soft rot disease. The extracts were supplemented in minimal medium (MM) and activities of Pel were measured from the culture supernatants. Since the chemical identity of the inducer in the host extracts is unknown, we determined if it is the same or different from polygalacturonic acid (PGA) by comparing the responses of a KdgR- mutant, (AC5073) and its parent, Ecc71 to host extracts. The two strains were grown in MM supplemented with PGA (0.2%, wt vol-1), GJF/CE or GJF/PE (30%, vol vol-1) in culture flasks. Following the determination that the inducer molecule in the host extracts is different from PGA, we determined some physical properties of the inducer and characterized the induction process. We also determined the heat-stability of the inducer molecule by heat-treating the extract before assaying for enzyme activities. The extracts and induction reactions were also pH-treated to determine the effects of pH on both.

Korsi Dumenyo, PI; Paul Agyemang, Graduate Student; Caleb Kersey, Graduate Student TARGET AUDIENCES: Agricultural research, education and extension community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

PROGRESS: 2010/01/01 TO 2010/12/31
OUTPUTS: Pectobacterium carotovorum strain KD100 (lacZ mutant, Nalidixic Acid resistant) was mutagenized with mini-Tn5 lacZ1 carrying kanamycin (Km) resistance using biparental mating with E. coli S17-1. The transposon vector creates transcriptional LacZ fusion in the truncated genes if it inserts in the direction of the gene flow. Colonies were selected on different media containing Km and Nal. To isolate mutants in host extract (CE)-induced genes, a total of about 31,122 mutants were screened for high expression of Beta-galactosidase in the presence of celery extract (CE). High LacZ expressing mutants were counter-selected on media without CE and low expressing mutants were isolated. After repeated screening and evaluation of promoter activity of the mutants based on betagalactosidase assays, 98 mutants were obtained of which 83 were induced (> 5-fold) and 15 were repressed ( 2-fold) pectate lyase while one was deficient (IMPACT: 2010/01/01 TO 2010/12/31Through this project, mutants of Pectobacterium have been isolated. The affected genes in these mutants will be identified and their role in disease development will be understood. Thus far, we have implicated the major magnesium transporter, CorA in the pathogenesis of this soft rot erwinia. With more understanding, this information could be used in the design of a better management strategy for the diseases. The findings are contributing to the our understanding of which bacterial genes play a role in disease development.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens
Food Defense and Integrity
Sanitation and Quality Standards