Progress 06/15/20 to 06/14/24
Outputs
Target Audience:We presented results to regional, national and international audiences that included academic, government, and industry scientists. We introduced 25 middle and high school teachers to bacterial plant pathology concepts and classroom resources and introduced high school students to the study of plant pathogens, including Xanthomonas. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three postdocs, two graduate students, and two undergraduates were trained on the project. How have the results been disseminated to communities of interest?Our work was disseminated to academic communities via publication in peer-reviewed journals and presentations at local, national, and international conferences, including Plant Health and the International Conference for Plant Pathogenic Bacteria. Co-PI Vallad regularly communicates research results to tomato growers. We trained members of the plant pathology community through our online workshop,"Basic bioinformatics and command-line tools for phytopathologists: How to handle, explore, and organize big biological data". This workshop was taught three times through the American Phytopathological Society and was aimed at beginners with little experience working on the command line and writing scripts for bioinformatic analyses. Weheld two "Plants Get Sick Too!" workshops for north central Florida K-12 teachers. We introduced 25 teachers, many from Title I schools, to the field of plant pathology, including plant diseases like bacterial spot that affect Florida agriculture. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: We published our work on the mutations that occur inX. perforans(syn.X. euvesicatoriapv.perforans)during a tomato field season in the journal MPMI. Our experiments show estimates of 0.3-0.5 mutations per genome per week in tomato fields, a typical mutation rate for bacteria, which revealed that the diversity we see inX. perforansis due to horizontal gene transfer and recombination that may be relatively rare but impactful on pathogen evolution. In three separate field experiments, we did not detect plasmid movement into target strains during a growing season. This negative result combined with measurement ofconjugation rates among distinct genetic lineages in vitro, suggests more complexity in horizontal gene transfer than inX. euvesicatoria, for which experiments conducted decades ago showedrampant plasmid movement during a tomato growing season. Analyses of plasmid diversity and distribution in genomes ofX. perforansstrains collected from Florida revealed potential incompatibility among plasmids that may explain our lack of detection of plasmid movement in field experiments. Characterization of plasmid distribution and compatibility inX. perforanswill continue. In the course of examining plasmid content, we found that mostX. perforansstrains examined did not contain the copper resistance (cop) gene cluster on a large plasmid, as has been described for otherXanthomonas. Our preliminary finding that a genomic island containing the cop genes was located on the chromosome in many strains was published in Phytopathology. A second manuscript is submitted that reports details of the distribution of copper resistance on plasmid and/or chromosome in theX. perforanspopulation relative to core gene phylogeny, the apparent selective sweep of one haplotype of the cop cluster in the US population, and comparison to cop genes inX. euvesicatoria. We also report that copper tolerance may vary with cop gene copy number. Objective 2: Experiments that carefully examined rates of ingress and egress by strains only differing in the presence of the gene for effector XopJ2 (syn. AvrBsT), showed that the effect of XopJ2 is on ingress. We found that when bacteria are sprayed on tomato leaves, approximately twice as many lesions are produced by the XopJ2+ strain than the XopJ2- strain. In contrast, when leaves are infiltrated with bacterial suspensions, egress of bacteria is not significantly differentbetween strains. This suggests that XopJ2 affects the efficacy of infection, which is expected to translate to more infections of a given leaf, higher bacterial populations across the leaf, and more leaf-to-leaf transmission than strains that lack XopJ2. To investigate how the XopJ2 effector facilitates ingress, we are conducting experiments with GFP-labeled isogenic strains to examine ingress of strains through stomatal openings. A graduate student will continue this work beyond the end of the grant period. As part of our work on XopJ2, we identified a homolog that shares 70% amino acid identity with XopJ2 but is located on a different plasmid. In planta experiments using pepper, tomato carrying the PTR1 gene from Solanum lycopersicoides, and Arabidopsis thaliana ecotype Pi-0 suggested that the homolog is recognized by the same host receptors as XopJ2 in these incompatible genotypes. Protein modeling indicated that both XopJ2 and it's homologare structurally similar to PopP2, a type III effector from Ralstonia solancearum withacetyltransferase activity. In addition, the YopJcatalytic residues are spatially conserved in both proteins and are predicted to have comparable binding affinities to IP6, which is required for acetylation of XopJ2 and PopP2. Because the homolog seems to have a similar function to XopJ2, we designated it as XopJ2b. XopJ2b is found in more tomato production regions worldwidethan XopJ2, which is more frequent inX. perforanspopulations in Florida. This work shows convergent acquisition of XopJ2 family effectors byX. perforans. The presence of XopJ2 effectors in other Xanthomonads suggests the gene may confer a fitness benefit in other species. Objective 3: We found that X. perforans carries multiple bacteriocins that allow it to out-compete X. euvesicatoria even in the absence of bacteriocin bcnA. The activity of these other bacteriocins may explain why X. euvesicatoria is not found on tomato even with the loss of bcnA in a major lineage of X. perforans in Florida. Thus the bcA- strains are still effective competitors. The large effect of a novel bacteriocin inX. perforanson bacterial spot species interactions shifted our attention to the nature of induction of this bacteriocin. Specifically, we found that the bacteriocin is active againstX. euvesicatoriaunder high pH in nutrient agar, but not on buffered agar that retains pH 7 even as growing bacterial colonies are producing ammonia. This suggests that the ammonia naturally produced by bacteria may trigger the bacteriocin activity. BecauseX. perforansis resistant to this bacteriocin, and appears more tolerant to high pH than X. euvesicatoria, it may allowX. perforansto outcompeteX. euvesicatoriawhen pH is high. During in vitro experiments, we identified mutants of X. euvesicatoria that are resistant to activity of the pH-dependent bacteriocin in X. perforans. However, these mutants have reduced growth in tomato, which suggests that there is a fitness tradeoff between resistance and virulence that has prevented evolution of resistance by X. euvesicatoria to the X. perforans bacteriocin. Together, our work revealed that multiple genetic factors, including Type III effectors, copper resistance genes, and bacteriocins involved in strain-strain competition, differ amongX. perforansstrains from US, strains from other global production regions, and other bacterial spot pathogens. Differences in the evolutionary dynamics of these fitness factors appear to contribute to the aggressiveness of X. perforanson tomato in Florida production systems.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Timilsina, S, Kaur, A, Sharma, A, Ramamoorthy, S, Vallad, GE, Wang, N, White, FF, Potnis, N, Goss, EM, and Jones, JB (2024). Xanthomonas as a model system for studying pathogen emergence and evolution. Phytopathology 114, 1433-1446. 10.1094/phyto-03-24-0084-rvw
- Type:
Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
Sharma, A, Iruegas-Bocardo, F, Bibi, S, Chen, Y-C, Kim, J-G, Abrahamian, P, Minsavage, GV, Hurlbert, JC, Vallad, GE, Mudgett, MB, Jones, JB, and Goss, EM (2024). Multiple acquisitions of XopJ2 effectors in populations of Xanthomonas perforans. Molecular Plant-Microbe Interactions. 10.1094/MPMI-05-24-0048-R
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Kaur, Amandeep, Minsavage, Gerald V, Potnis, Neha, Jones, Jeffrey B, Goss, Erica M. Evolution of copper resistance in Xanthomonas euvesicatoria pv. perforans population. Under review at mSystems.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
McDuffee, Sophia, Mutisya, Trisha, Goss, Erica, Sharma, Anuj, Jones, Jeffrey B. Ammonia-Triggered Effect on X. perforans Density-Dependent Bacteriocin Activity. Abstract submitted to the International Conference on Plant Pathogenic Bacteria & Biocontrol 2024.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Kaur, Amandeep, Minsavage, Gerald V, Potnis, Neha, Jones, Jeffrey B, Goss, Erica M. Evolution of copper resistance in Xanthomonas euvesicatoria pv. perforans population. Abstract submitted to the International Conference on Plant Pathogenic Bacteria & Biocontrol 2024, Blacksburg, VA, July 7-12, 2024.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Kaur, Amandeep, Minsavage, Gerald V, Potnis, Neha, Jones, Jeffrey B, Goss, Erica M. Evolutionary insights into chromosomally encoded copper resistance. Abstract submitted to Plant Health 2024, Memphis, TN, July 27-30, 2024.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Sharma, Anuj, Chen, Yun-Chu, Minsavage, Gerald V, Iruegas-Bocardo, Fernanda, Bibi, Shaheen, Kim, Jung-Gun, Abrahamian, Peter, Vallad, Gary E, Hurlbert, Jason C, Goss, Erica M, Mudgett, Mary B, Jones, Jeffrey B. Characterization of a XopJ2 variant in Xanthomonas perforans. Abstract submitted to Plant Health 2024, Memphis, TN, July 27-30, 2024.
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Progress 06/15/22 to 06/14/23
Outputs
Target Audience:We presented project results to the Florida plant pathology community at the 2023 Florida Phytopathological Society meeting and to the international research community at the 2023 International Societyfor Plant-Microbe Interactions Congress, heldin Rhode Island. For the third year, we co-organized the workshop, "Basic bioinformatics and command-line tools for phytopathologists: How to handle, explore, and organize big biological data"as a stand-alone virtual workshop hosted by the American Phytopathological Society, aimed at beginners with little experience working on the command line and writing scripts for bioinformatic analyses. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We organized a bioinformatics workshop for beginners, described under Target Audience. This year there were two postdocs on the project, two PhD students conducting project experiments, and two undergraduates trained in bioinformatics and bacterial genomics. How have the results been disseminated to communities of interest?In May 2023, weheld a "Plants Get Sick Too!" workshop for north central Florida K-12 teachers, with 7 attendees. Teachers were introducedto the field of plant pathology, including plant diseases like bacterial spot that affect Florida agriculture. What do you plan to do during the next reporting period to accomplish the goals?In the last year of the project, we will conduct experiments further examining the effect of the XopJ2 effector on infection efficiency and the effectsof bacteriocins on strain fitness. Results from Objective 1 will be prepared for publication.
Impacts
What was accomplished under these goals?
Objective 1: We prepared a manuscript describing the mutations that occur inX. perforansduring a tomato field season. This is reported under Publications. After fall field experiments were destroyed by two hurricanes, a spring experiment to measure plasmid transfer in the field was completed. Based on the results of repeated field experiments, we have refocused our efforts on investigating the reasons for lack of plasmid transfer inX. perforanswhen this appeared common in the closely related pathogen,X. euvesicatoria. As part of this effort, weare characterizing the distribution of plasmids in our genome database ofX. perforansstrains. We found that a genomic island conferring copper tolerance, found in a plasmid in some strains and otherXanthomonasspecies, has been integrated into the chromosome in most Florida strains. Objective 2: To investigate the behavior of isogenic bacteria with and without the XopJ2 effector on tomato leaves during infection, we created strains expressing GFP. Experiments are ongoing. Objective 3: We continue to examine the effect of bacteriocin bcnA onX. perforansgrowth and competition in vitro. Wildtype strains with and without bcnA competed equally well with X. euvesicatoria in culture, in partbecause ofadditional bacteriocins carried by strains. New mutants were created that lack activity for all known bacteriocins against our test strain ofX. euvesicatoria in order to isolate the effect of the bcnA polymorphism. We are also investigating a novel bacteriocin, including the conditions under which it is active and resistanceinX. euvesicatoria.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2023
Citation:
Bibi, S, Weis, K, Kaur, A, Bhandari, R, Goss, EM, Jones, JB, Potnis, N (2023) A brief evaluation of copper resistance mobile genetic island in the bacterial leaf spot pathogen, Xanthomonas euvesicatoria pv. perforans Phytopathology. https://doi.org/10.1094/PHYTO-02-23-0077-SC
- Type:
Journal Articles
Status:
Submitted
Year Published:
2023
Citation:
Sharma, A, Timilsina, S, Abrahamian, P, Minsavage, GV, Jones, JB, Vallad, GE, Goss, EM. Bacterial mutation during seasonal epidemics.
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Progress 06/15/21 to 06/14/22
Outputs
Target Audience:We presented initial results to the plant pathology community in a poster at the Plant Health 2020 virtual conference and more developed results to the Xanthomonas genomics research community at the 7thXanthomonas Genomics Conference. For the second year, we co-organized the workshop, "Basic bioinformatics and command-line tools for phytopathologists: How to handle, explore, and organize big biological data"as part of the 2021 virtual Plant Health conference held by the American Phytopathological Society, aimed at beginners with little experience working on the command line and writing scripts for bioinformatic analyses. Changes/Problems:Co-PISujan Timilsina left the project for a position in industry, as previouslyreported to NIFA.Two postdocs have taken over his work on the project. What opportunities for training and professional development has the project provided?We organized a bioinformatics workshop for beginners, described under Target Audience. The project has also trained a PhD student, two postdocs, and undergraduate assistant in bacteriology, epidemiology, plant pathology,and bioinformatics. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: We have been analyzing mutations accumulated in markedX. perforansthat were released and re-isolated over two 10-week long field experiments. Illumina reads from 300 recaptured strains were aligned against complete genomes to identify mutations. We found up to 0.9 SNPs per genome, on average, in the 2017 experiment and 0.5 SNPs per genome in the 2020 experiment, leading to estimates of 0.3-0.5 mutations per genome per week. When we convert this rate to mutation rate per site per year, our estimate is very similar to the rate obtained forE. coli. We found a few genes that showed multiple mutations and mutations that were present in 10-20 recovered strains. These results provide an important point of reference for understanding the genetic variation observed inX. perforanslocally and globally. We also conducted two field experiments to quantify plasmid transfer. The first experiment required strains to move between rows of tomato plants and was not successful due to especially dry weather. The second experiment did not require movement and used supplemental irrigation. However, we did not detect plasmid transfer. We are now investigating the reasons for this finding in laboratory experiments, based on initial results that conjugations rates vary dramatically between different genotypes ofX. perforans. Objective 2: We conducted multiple experiments examining rates of ingress and egress by strains only differing in the presence of the gene for effector XopJ2. We found that when bacteria are sprayed on tomato leaves, approximately twice as many lesions are produced by the XopJ2+ strain. This suggests that XopJ2 affects the efficacy of infection. Objective 3: We created BCNA+ and BCNA- isogenic strains. Given the challenges we faced in measuring plasmid movement in the field, we designed and are conducting in vitro experiments to examine the effect of the bacteriocin BCNA on intra- and inter-specific competition before taking this question to the field. These experiments are ongoing.?
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
S Sacrist�n, EM Goss, S Eves-van den Akker. (2021) How do pathogens evolve novel virulence activities? Molecular Plant-Microbe Interactions 34 (6), 576-586.
Molecular Plant-Microbe Interactions 34 (6), 576-586
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