Dryad
Published August 8, 2019 | Version v1
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Data from: Phage resistance evolution in vitro is not reflective of in vivo outcome in a plant-bacteria-phage system

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The evolution of resistance to parasites is fundamentally important to disease ecology, yet we remain unable to predict when and how resistance will evolve. This is largely due to the context-dependent nature of host-parasite interactions, as the benefit of resistance will depend on the abiotic and biotic environment. Through experimental evolution of the plant pathogenic bacterium Pseudomonas syringae and two lytic bacteriophages across two different environments (high-nutrient media and the tomato leaf apoplast), we demonstrate that de novo evolution of resistance is negligible in planta despite high levels of resistance evolution in vitro. We find no evidence supporting the evolution of phage resistance in planta despite multiple passaging experiments, multiple assays for resistance, and high multiplicities of infection. Additionally, we find that phage-resistant mutants (evolved in vitro) did not realize a fitness benefit over phage-sensitive cells when grown in planta in the presence of phage, despite reduced growth of sensitive cells, evidence of phage replication in planta, and a large fitness benefit in the presence of phage observed in vitro. Thus, this context-dependent benefit of phage resistance led to different evolutionary outcomes across environments. These results underscore the importance of studying the evolution of parasite resistance in ecologically relevant environments.

Notes

Data analysis code
Code for all data analysis in the manuscript (main text and supplement), split by figure number.
BPIPallstats_forDryad.R
Figure generation code
Code for generation of all figures in the manuscript (main text and supplement), split by figure number.
BPIPallfigures_forDryad.R
Bacterial densities in the six-passage experiment
Bacterial densities in colony forming units (CFUs) after each passage in vitro and in vivo. "CFUperml" has been calculated as the CFU per ml culture (in vitro lines) or CFU per 1 mm2 tissue (in planta lines), and any values below the limit of detection were recorded as being at that limit (as a conservative estimate of density). "Sample" is the evolution line identifier, "Treatment" is passaging treatment, "Environment" is the passaging environment, "Phage" indicates whether the inoculum for that sample had phage present, and "Week" is the passage number. Results are presented in Figure 1.
BPIPCFUforR_forDryad.txt
Amplified phage densities in the six-passage experiment
Phage densities in plaque forming units (PFUs) after amplification for each passage in vitro and in vivo. "PFUperml_limitadjusted" has been calculated as the PFU per ml culture (in vitro lines) or PFU per 1 mm2 tissue (in planta lines), and any values below the limit of detection were recorded as being at that limit (as a conservative estimate of density). "Line" is the evolution line identifier, "Treatment" is passaging treatment, "Environment" is the passaging environment, and "Week" is the passage number. Results are presented in Supplementary Figure 1.
AmplifiedPFUsforR_forDryad.txt
Number and proportions of resistant colonies in the six-passage experiment
Number and proportions of resistant, sensitive, and moderately resistant colonies from passage three and passage six evolved populations. "Prop_notS" and "Prop_S" are the proportion of non-sensitive (resistant and moderately resistant colonies) and proportion of sensitive colonies in the assayed populations, respectively. "Line" is the evolution line identifier, "Bweek" is the passage number that bacteria were isolated from, "Phage" is the type of phage that the colonies were streaked against to determine resistance, "Treatment" is the passaging treatment of the bacterial population, "Environment" is the original environment of the bacterial population. Results are presented in Figure 2.
BPIPresfreqonly_forDryad.txt
Bacterial densities in the resistance in planta experiment
Bacterial densities (CFU values) of the leaf samples analyzed in the resistance in planta assay. "AvgCFUperml" is the average value of the CFU per 1 mm2 leaf tissue, and any values below the limit of detection were recorded as being at that limit (as a conservative estimate of density). "Line" is the evolution line identifier, "Treatment" is passaging treatment of the bacterial populations, "Time" is number of hours post-inoculation into leaves, and "Phage" is whether ancestral phage was inoculated onto that same plant. Results are presented in Figure 3.
Avgdleaf_res3_feb2018_forDryad.txt
Bacteria and phage densities in the resistance in planta experiment using ddPCR
ddPCR bacteria and phage concentrations from the second resistance in planta assay. "AvgB_limitadjusted" and "AvgP_limitadjusted" are the number of target copies of bacteria and phage (respectively) per 1 mm2 leaf tissue, and any values below the limit of detection were recorded as being at that limit (as a conservative estimate of density). "Sample" is the evolution line identifier, "Time" is number of hours post-inoculation into leaves, "Treatment" is passaging treatment of the bacterial populations, and "Phage" indicates whether ancestral phage was present in the inoculum. Results are presented in Figure 4.
resinvivo2_forR_forDryad.txt
Bacterial densities and proportion resistant in the phage cocktail passaging experiment
Bacterial densities and proportion of resistant colonies (as determined via plating on "phage agar") after each passage of the phage cocktail passaging experiment. "CFUperml" has been calculated as the CFU per ml culture (in vitro lines) or CFU per 1 mm2 tissue (in planta lines), and any values below the limit of detection were recorded as being at that limit (as a conservative estimate of density). "Propres" is the proportion of colonies that grew on the "phage agar" plate relative to the non-phage plate, and "Propres_minusPT23prop" has had the proportion of colonies from the control plate subtracted from the sample proportions. "Sample" is the evolution line identifier, "Treatment" is passaging treatment, "Environment" is the passaging environment, "Week" is the passage number, "Phage" indicates whether phage was present on the CFU plate, and "Week" is the passage number. Results are presented in Figure 5.
Fall 2017 Phage cocktail plant data for R_forDryad.txt
Bacteria and phage densities in the benefits of resistance experiment
Bacterial densities (CFUs) and ddPCR bacteria and phage densities for the benefits of resistance experiment in vitro and in planta. "CFUperml_limitadjusted", "AvgB_limitadjusted", and "AvgP_limitadjusted" are the bacterial densities in CFUs, bacterial densities in ddPCR copy number, and phage densities in ddPCR copy number, respectively. They each were calculated as the concentration per ml culture (in vitro samples) or per 1 mm2 tissue (in planta samples), and any values below the limit of detection were recorded as being at that limit (as a conservative estimate of density). "ID" is the sample identifier, "Colony" is the colony identifier, "Plant" specifies the plant that the colony was inoculated on, "Res_category" indicates whether the colony was resistant or sensitive, "Phage" indicates whether phage was present in the inoculum, "Environment" is the growth environment, and "Time" is the number of hours post-inoculation. Results are presented in Figure 6, Supplementary Figure 2, and Supplementary Figure 3.
RS_FRS_invivoinvitro_cleanedJan2019_forRforDryad.txt

Funding provided by: National Science Foundation
Crossref Funder Registry ID: http://dx.doi.org/10.13039/100000001
Award Number: DGE 1106400; DGE 1752814

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10.1111/evo.13833 (DOI)