DOI: 10.5061/dryad.w9ghx3g0m
title: Data from: Managing friends and foes: Sanctioning mutualists in mixed‐infection nodules trades off with defense against antagonists

description: Successful plant growth requires plants to minimize harm from antagonists
 and maximize benefit from mutualists. However, these outcomes may be
 difficult to achieve simultaneously, since plant defenses activated in
 response to antagonists can compromise mutualism function, and plant
 resources allocated to defense may trade off with resources allocated to
 managing mutualists. Here, we investigate how antagonist attack affects
 plant ability to manage mutualists with sanctions, in which a plant
 rewards cooperative mutualists and/or punishes uncooperative mutualists.
 We studied interactions among wild and domesticated pea plants, pea
 aphids, an aphid‐vectored virus (Pea enation mosaic virus, PEMV), and
 mutualistic rhizobial bacteria that fix nitrogen in root nodules. Using
 isogenic rhizobial strains that differ in their ability to fix nitrogen
 and express contrasting fluorescent proteins, we found that peas
 demonstrated sanctions in both singly‐infected nodules and mixed‐infection
 nodules containing both strains. However, the plant's ability to
 manage mutualists in mixed‐infection nodules traded off with its ability
 to defend against antagonists: when plants were attacked by aphids, they
 stopped sanctioning within mixed‐infection nodules, and plants that
 exerted stricter sanctions within nodules during aphid attack accumulated
 higher levels of the aphid‐vectored virus, PEMV. Our findings suggest that
 plants engaged in defense against antagonists suffer a reduced ability to
 select for the most beneficial symbionts in mixed‐infection tissues.
 Mixed‐infection tissues may be relatively common in this mutualism, and
 reduced plant sanctions in these tissues could provide a refuge for
 uncooperative mutualists and compromise the benefit that plants obtain
 from mutualistic symbionts during antagonist attack. Understanding the
 conflicting selective pressures plants face in complex biotic environments
 will be crucial for breeding crop varieties that can maximize benefits
 from mutualists even when they encounter antagonists.
description: Overview
 of experiment. This dataset was collected from a greenhouse
 experiment performed at Washington State University, Vancouver in Fall
 2020-Winter 2021. We grew different varieties of pea plants in individual
 pots, inoculated young plants with different strains of rhizobial bacteria
 (2 weeks post-sowing), exposed plants to different aphid treatments (6
 weeks post-sowing), and then harvested plants (8 weeks post-sowing). The
 experiment followed a full-factorial design with 3 pea varieties x 5 types
 of rhizobial inoculum x 3 types of aphid exposure x 6 block replicates,
 for a total of 270 plants in the experiment. Description of
 treatments. The 3 pea varieties were 1) a non-nodulating
 domesticated variety, 2) a nodulating domesticated variety, and 3) a wild
 variety. For rhizobial inocula we fluorescently labeled a nitrogen-fixing
 (Fix+) and non-nitrogen-fixing (Fix-) strain, reciprocally labeling each
 strain with either a red or green fluorescent marker. The 5 rhizobial
 inocula were as follows: 1) uninoculated control, 2) red Fix- and green
 Fix-, 3) red Fix+ and green Fix+, 4) red Fix+ and green Fix-, and 5) green
 Fix+ and red Fix-. Thus, inoculations #2 and #3 were "single
 inoculations" in which plants could only form one type of nodule
 (Fix- or Fix+, respectively), and inoculations #4 and #5 were
 "co-inoculations" in which plants could form Fix+ nodules, Fix-
 nodules, or "mixed-infection" nodules containing both strains.
 The 3 aphid treatments were 1) no aphid exposure, 2) exposure to
 virus-free aphids, and 3) exposure to aphids carrying the <em>Pea
 enation mosaic virus</em> (PEMV), which can be transmitted to pea
 plants. Types of data
 collected. We measured shoot mass of harvested plants,
 assayed shoot tissue for accumulation of the aphid-transmitted plant virus
 (PEMV), photographed root systems with a fluorescence microscope to
 determine the number and color of individual nodules, and cultured
 rhizobia from a subset of nodules to check that the external color of
 nodules corresponded with the type of strain occupying the nodule. From
 the fluorescence photographs, we used ImageJ to calculate the size and
 color of each nodule on each plant's root system. We collated these
 data into three datasheets which are archived here.
description: # Managing Friends and Foes: Sanctioning Mutualists in Mixed‐Infection
 Nodules Trades off With Defense Against Antagonists
 [https://doi.org/10.5061/dryad.w9ghx3g0m](https://doi.org/10.5061/dryad.w9ghx3g0m) ## Description of the data and file structure ### Files and variables #### File: 1\_plant\_data\_2024-12-18.csv **Description:** Plant-level data from the experiment. Each line contains data from one plant.  ##### Variables * Unique.ID: Unique identifier for the plant, ranges A001-A270  * Block: Experimental block to which plant was assigned, ranges Block 1-6  * Accession.label: Informal name of pea accession (Wild pea, Domesticated pea, Nod minus pea)  * Accn: Abbreviated informal name of pea accession (Wild, Domest., Non-nod.)  * Inoculum: Full name of rhizobial inoculum, indicating Rhizobium strain (3841 or 727) and red (R) or green (G) fluorescent label. Note, 3841 is Fix+ or “beneficial” strain, and 727 is Fix- or “ineffective” strain. Inocula are: 3841R_727G, 3841G_727R, 727G_727R, 3841G_3841R, and neg.  * Inoculum.label: Descriptive name of rhizobial inoculum (Coinoc mix 1, Coinoc mix 2, Fix minus, Fix plus, Neg)  * Inoc.pooled: Same as “Inoculum.label”, but pools Coinoc mix 1 and Coinoc mix 2 into “Coinoc”  * Aphid: Aphid treatment (sham.aphids, PEMV.aphids, no.aphids)  * No.germ: y = indicates that seed failed to germinate in this pot. NA = seed grew.  * Replanted: y = seed was resown in this pot after start of experiment. NA = pot was not resown.  * EarlySPAD: SPAD value measured from leaves at early timepoint in experiment (15 January 2021). NA = no data collected.  * LateSPAD: SPAD value measured from leaves at later timepoint in experiment (29 January 2021). NA = no data collected.  * Harvest.date: Date when shoot was cut. NA = no data collected.  * ShootMass.G: Fresh mass of shoot in grams at time of harvest. NA = no data collected.  * PEMV.raw: Band intensity of PEMV amplicon in gel image of PCR product. NA = no data collected.  * TotNodCount: Total number of nodules on the plant, estimated by ImageJ macro (NodCounts_v3.ijm). NA = no data collected.  * PureGreen.nods: Total number of pure green nodules (i.e., singly infected by the green-labeled strain) on the plant, estimated by ImageJ macro (NodCounts_v3.ijm). NA = no data collected.  * PureRed.nods: Total number of pure red nodules (i.e., singly infected by the red-labeled strain) on the plant, estimated by ImageJ macro (NodCounts_v3.ijm). NA = no data collected.  * Mixed.nods: Total number of mixed nodules (i.e., infected by both the red- and the green-labeled strain) on the plant, estimated by the ImageJ macro (NodCounts_v3.ijm). NA = no data collected.  * Green.nods.manual:Total number of pure green nodules (i.e., singly infected by the green-labeled strain) on the plant, estimated by manually counting nodules from root image. NA = no data collected.  * Red.nods.manual: Total number of pure red nodules (i.e., singly infected by the red-labeled strain) on the plant, estimated by manually counting nodules from root image. NA = no data collected.  * Mixed.nods.manual: Total number of mixed nodules (i.e., infected by both the red- and the green-labeled strain) on the plant, estimated by manually counting nodules from root image. NA = no data collected.  * Mixed.pct.red: Mean percentage of mixed nodules (i.e., infected by both the red- and the green-labeled strain) occupied by the red-labeled strain on the plant, estimated by the ImageJ macro (NodCounts_v3.ijm). NA = no data collected.  * Mixed.pct.green: Mean percentage of mixed nodules (i.e., infected by both the red- and the green-labeled strain) occupied by the green-labeled strain on the plant, estimated by the ImageJ macro (NodCounts_v3.ijm). NA = no data collected.  * PureGreen.mm2: Mean cross-sectional area in mm2 of pure green nodules (i.e., singly infected by the green-labeled strain) on the plant, estimated by the ImageJ macro (NodCounts_v3.ijm). NA = no data collected.  * PureRed.mm2: Mean cross-sectional area in mm2 of pure red nodules (i.e., singly infected by the red-labeled strain) on the plant, estimated by the ImageJ macro (NodCounts_v3.ijm). NA = no data collected.  * Mixed.mm2: Mean cross-sectional area in mm2 of mixed nodules (i.e., infected by both the red- and the green-labeled strain) on the plant, estimated by the ImageJ macro (NodCounts_v3.ijm). NA = no data collected.  #### File: 2\_cfu\_data\_2024-12-18.csv **Description:** Colony-forming unit (CFU) data from nodules cultured in the experiment. Each line contains data from one nodule.  ##### Variables * Unique.ID: Same as in 1_plant_data_2024-12-18.csv  * NodID: Unique identifier for cultured nodule, named for its position in a single 96-well plate. Note: this is different from “NodID” in the ImageJ nodule dataset (3_imagej_2024-12-18.csv).  * Block: Same as in 1_plant_data_2024-12-18.csv  * Accession.label: Same as in 1_plant_data_2024-12-18.csv  * Accn: Same as in 1_plant_data_2024-12-18.csv  * Inoculum: Same as in 1_plant_data_2024-12-18.csv  * Inoc.pooled: Same as in 1_plant_data_2024-12-18.csv  * Aphid: Same as in 1_plant_data_2024-12-18.csv  * CFU: Colony forming units of rhizobia estimated in the nodule. NA = no data collected.  * NodColor: Color of the nodule when removed from the plant (red, mixed, green, nonglowing)  * Colony.colors: Fluorescent color(s) of colonies cultured from the nodule (red, mixed, green, no growth). NA = no data collected.  * NodOccupant: Strain occupant(s) of the nodule (Fix plus, Fix minus, Both strains). NA = no data collected.   * Total.pixels: Total cross-sectional area of the nodule in pixels, estimated by tracing nodule photo in ImageJ  * Tot.mm2: Total cross-sectional area of the nodule in mm2, taking into account the zoom level of the nodule image  #### File: 3\_imagej\_data\_2024-12-18.csv **Description:** Nodule color and size data estimated by ImageJ macro from images of fluorescent strains in root nodules. Each line contains data from one nodule.  ##### Variables * Unique.ID: Same as in 1_plant_data_2024-12-18.csv  * NodID: Unique identifier for the nodule on the plant based on ImageJ analysis; NodIDs are re-used from one plant to the next (i.e., plant A001 has NodID 1, 2, 3, and plant A002 also has Nod ID 1, 2, 3, etc.). Note: this is different from NodID in the cultured nodule dataset (2_cfu_data_2024-12-18.csv).  * Block: Same as in 1_plant_data_2024-12-18.csv  * Accession.label: Same as in 1_plant_data_2024-12-18.csv  * Accn: Same as in 1_plant_data_2024-12-18.csv  * Inoculum.label: Same as in 1_plant_data_2024-12-18.csv  * Inoc.pooled: Same as in 1_plant_data_2024-12-18.csv  * Inoculum: Same as in 1_plant_data_2024-12-18.csv  * Aphid: Same as in 1_plant_data_2024-12-18.csv  * TotPixels: Total cross-sectional area of the nodule in pixels  * Pixels.per.1mm2: Pixels per mm2 resolution of the plant’s image (determined for each UniqueID by measuring the number of pixels in a region of the darkfield image of the plant, which had a 1cm2 grid in the background)  * Area.mm2: Cross-sectional area of the nodule in mm2, determined from the size in pixels and the pixels per mm2 resolution  * Percent.red: Percentage of the nodule area that has any red fluorescence (note, some or all of this area may also be green)  * Percent.green: Percentage of the nodule area that has any green fluorescence (note, some or all of this area may also be red)  * Percent.yellow: Percentage of the nodule area that has both red and green fluorescence  * NodType: Type of nodule: “mixed” nodules have both red and green fluorescence; “pure green” nodules have only green and no red fluorescence; “pure red” nodules have only red and no green fluorescence  * Ben.color: Color of the beneficial (Fix+) strain in the inoculum for this plant. R = red; G = green.  * Ineff.color: Color of the ineffective (Fix-) strain in the inoculum for this plant. R = red; G = green.  * Occupant: Occupant of the nodule: Ben, Ineff, or Mixed. Ben = Fix+ strain; Ineff = Fix- strain.  * Ben.present: Binary indicator for the presence of the beneficial (Fix+) strain in the nodule; 1 = Fix+ strain is present; 0 = Fix+ strain is absent.  * Ineff.present: Binary indicator for the presence of the ineffective (Fix-) strain in the nodule; 1 = Fix- strain is present; 0 = Fix- strain is absent  * TotPixels.darkfield: The size of the nodule in pixels, determined by manually tracing the darkfield image of the nodule  * NodType.darkfield: Type of nodule determined by comparing ImageJ output to darkfield image of nodule. Options are “single” if the ImageJ output represents a single nodule, “fragment” if the ImageJ output is just a fragment of a nodule, or “multiple” if the ImageJ output represents multiple real nodules.  ## Code/software To recreate analyses and figures, use the R file entitled "Wendlandt-et-al-2025-Evolutionary-Applications-analysis.R." We used R version 4.1.2. The required packages to use in R are described at the beginning of the R code file. To process images of nodulated root systems and generate data on the size and color of each nodule on each plant, we used a custom ImageJ macro file entitled "NodCounts-v3.ijm." We used ImageJ v. 1.53K. We also made use of two other custom ImageJ scripts entitled "NodCounts-v3-darkfield-sizes.ijm" and "Overlays.ijm", whose usage is described in Methods S2 of the accompanying manuscript (DOI: 10.1111/eva.70064).