Genetic studies of legume symbiosis with nitrogen-fixing rhizobial bacteria have traditionally focused on nodule and nitrogen-fixation phenotypes when hosts are inoculated with a single rhizobial strain. These approaches overlook the potential effect of host genes on rhizobial fitness (i.e., how many rhizobia are released from host nodules) and strain-specific effects of host genes (i.e., genome x genome interactions). Using Medicago truncatula mutants in the recently described Nodule-specific PLAT Domain (NPD) gene family, we show how inoculating plants with a mixed inoculum of 68 rhizobial strains (Ensifer meliloti) via a Select and Resequence (S&R) approach can be used to efficiently assay host mutants for strain-specific effects of late-acting host genes on interacting bacteria. The deletion of a single NPD gene (npd2) or all five members of the NPD gene family (npd1-5) differentially altered the frequency of rhizobial strains in nodules even though npd2 mutants had no visible nodule morphology or N-fixation phenotype. Also, npd1-5 nodules were less diverse and had larger populations of colony-forming rhizobia despite their smaller size. Lastly, NPD mutations disrupt a positive correlation between strain fitness and wild type host biomass. These changes indicate that the effects of NPD proteins are strain-dependent and that NPD gene family members are not redundant with regard to their effects on rhizobial strains. Association analyses of the rhizobial strains in the mixed inoculation indicate that rhizobial genes involved in chromosome segregation, cell division, GABA metabolism, efflux systems, and stress tolerance play an important role in the strain-specific effects of NPD genes.

This repository includes supplemental material and analysis for a 'Select and resequence' experiment conducted on Medicago truncatula and a mixed inoculum of 68 strains of Ensifer meliloti (also known as Sinorhizobium meliloti).  In brief, we inoculated WT and mutant host plants with a mixture of previously sequenced strains of rhizobia. Plants grew and formed nodules with the rhizobia for seven weeks in N-limited conditions in a growth chamber. We harvested plants and measured dry root and shoot biomass and nodule characteristics (size, color). We also collected and pooled all nodules from roots and assessed colony-forming units released and downstream DNA extraction and strain frequency inference via HARP. Full experimental details can be found in the paper.

This data package includes: 

1. Strain frequencies inferred via HARP from whole-genome pooled sequencing of legume WT and NPD mutant nodules inoculated with a mixture of 68 rhizobial strains (tab-delimited text- C68_WT&NPDmutants_nodulestrainfreq.txt) 

2. Plant phenotype measurements for single-strain and community inoculations (tab-delimited text: NPDMutantPhenoytpes_community_DRYAD.txt)

3. A folder of tab-delimited text files of rhizobial GWAS results tabulated for each linkage group (gwas_results_gemma_dryad_XXX_fit) and each variant (XXX.association_output.tsv).  XXX stands for each of the three genotypic fitness contrasts that are the focus of this paper (WT minus npd2, WT minus npd12345, and npd2 minus npd12345). I also included files for analyses of strain fitness in nodules for each host genotype individually (WT, npd2, and npd12345).

4. R Code to recreate downstream analysis and generate the figures in the paper (S&R3_PLATmutantanalysis_Dryad.R) 

5. A tab-delimited text conversion file for gene IDs used in (Burghardt et al. 2018) into locus tags and annotations of those candidates on MAGE (USDA1106reference_annotationV1toV2.txt). This file enables a comparison of these results with the supplemental files from the previous paper.

6. Metadata on sequencing coverage, SRA numbers, and characteristics of each replicate pool (tab-delimited text- R108_NPDmutants_C68_USDA1106_read_counts_dryad.txt)

7. A folder containing the raw figures and table files generated from the R code (Tables&Figures)

Code for the HARP reconstruction of strain frequencies from raw reads deposited in NCBI SR archive can be found in the Dryad repositories for previous papers:

Liana T. Burghardt, Brendan Epstein, Joseph Guhlin, Matt S. Nelson, Margaret R. Taylor, Nevin D. Young, Michael J. Sadowsky, Peter Tiffin. Select and resequence reveals relative fitness of bacteria in symbiotic and free-living environments. Proceedings of the National Academy of Sciences Mar 2018, 115 (10) 2425-2430; DOI: 10.1073/pnas.1714246115  (and associated Dryad repository DOI:10.5061/dryad.fp1bg)

Example code for the GEMMA association analysis can be found in the 'Single-variant Association Analyses' folder in a previous Dryad repository:

Epstein, Brendan et al. (2018), Data from Genome-wide association analyses in the model rhizobium Ensifer meliloti, Dryad, Dataset, https://doi.org/10.5061/dryad.tn6652t 

Each tab-delimited text file includes hashed out ReadMe information at the top of the file

Host germplasm: The host mutants have different combinations of NPD family genes disrupted via Crisper. 

Wildtype throughout is HM340 (R108)
npd2 is trna18-1:    1 gene knockout
npd2/4 is Gm12-7:   2 gene knockout
npd1/2/4 is Gm8-5:    3 gene knockout
npd2/4/5 is Gm26-1:  3 gene knockout
npd1/2/4/5 is Gm20-2:  4 gene knockout
npd1/2/3/4/5 is MPC12:  5 gene knockout (called npd1-5 in the paper) 

Disruptions in NPD1 (Medtr2g103303); NPD2 (Medtr2g103307); NPD3 (Medtr2g103313); NPD4 (Medtr2g103330); NPD5 (Medtr2g103360) were described in detail in Trujillo, D. I., Silverstein, K. A. and Young, N. D. (2019), Nodule‐specific PLAT domain proteins are expanded in the Medicago lineage and required for nodulation. New Phytol, 222: 1538-1550. doi:10.1111/nph.15697

Strain information: The 68 Ensifer strains are mostly from the ARS culture collection (https://nrrl.ncaur.usda.gov/). Detailed information can be found in the 'Strain Information' section in Dryad repository (https://doi.org/10.5061/dryad.tn6652t) that accompanies the following paper:

Brendan Epstein, Reda A. I. Abou-Shanab, Abdelaal Shamseldin, Margaret R. Taylor, Joseph Guhlin, Liana T. Burghardt, Matthew Nelson, Michael J. Sadowsky, Peter Tiffin. 2018. Genome-Wide Association Analyses in the Model Rhizobium Ensifer meliloti. mSphere 3:e00386-18; (DOI: 10.1128/mSphere.00386-18)

These strains are also used in/discussed in:

Liana T. Burghardt, Brendan Epstein, Joseph Guhlin, Matt S. Nelson, Margaret R. Taylor, Nevin D. Young, Michael J. Sadowsky, Peter Tiffin. Select and resequence reveals relative fitness of bacteria in symbiotic and free-living environments. Proceedings of the National Academy of Sciences Mar 2018, 115 (10) 2425-2430; DOI: 10.1073/pnas.1714246115  (and associated Dryad repository DOI:10.5061/dryad.fp1bg)

Nelson. M., Guhlin, J., Epstein, B., Tiffin, P., & Sadowsky, M. J. (2018). The complete replicons of 16 Ensifer meliloti strains offer insights into intra- and inter-replicon gene transfer, transposon-associated loci, and repeat elements. Microbial genomics, 4(5), e000174. DOI:10.1099/mgen.0.000174