DOI: 10.5061/dryad.pnvx0k6hb
title: A select-and-resequence approach reveals strain-specific effects of Medicago nodule-specific PLAT-domain genes

description: 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.
description: This repository includes supplemental material and analysis for a
 'Select and resequence' experiment conducted on
 <em>Medicago truncatula</em> and a mixed inoculum of 68
 strains of <em>Ensifer meliloti</em> (also known as
 <em>Sinorhizobium meliloti</em>).  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
 <em>npd2</em>, WT minus <em>npd12345</em>, and
 <em>npd2</em> minus <em>npd12345</em>). I also
 included files for analyses of strain fitness in nodules for each host
 genotype individually (WT, <em>npd2</em>, and
 <em>npd12345</em>). 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 <em>Ensifer meliloti</em>, Dryad, Dataset, https://doi.org/10.5061/dryad.tn6652t 
description: Each tab-delimited text file includes hashed out ReadMe
 information at the top of the file 
 <em>Host germplasm:</em> The host mutants
 have different combinations of NPD family genes disrupted via
 Crisper.  Wildtype throughout is HM340 (R108)<br>
 <em>npd2</em> is trna18-1:    1 gene knockout<br>
 <em>npd2/4 </em>is Gm12-7:   2 gene knockout<br>
 <em>npd1/2/4 </em>is<em> </em>Gm8-5:    3 gene
 knockout<br> <em>npd2/4/5</em> is Gm26-1:  3 gene
 knockout<br> <em>npd1/2/4/5</em> is Gm20-2:  4 gene
 knockout<br> <em>npd1/2/3/4/5</em> is MPC12:  5 gene
 knockout (called <em>npd1-5</em> in the paper) 
 Disruptions
 in <i>NPD1</i> (<i>Medtr2g103303</i>); <i>NPD2</i> (<i>Medtr2g103307</i>); <i>NPD3</i> (<i>Medtr2g103313</i>); <i>NPD4</i> (<i>Medtr2g103330</i>); <i>NPD5</i> (<i>Medtr2g103360</i>) 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 <i>Medicago</i> lineage and required for nodulation. New Phytol, 222: 1538-1550. doi:10.1111/nph.15697 <em>Strain information:</em> The 68<em> Ensifer</em> 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 <em>Ensifer meliloti.</em> 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 <em>Ensifer meliloti</em> strains offer insights into intra- and inter-replicon gene transfer, transposon-associated loci, and repeat elements. <i>Microbial genomics</i>, <i>4</i>(5), e000174. DOI:10.1099/mgen.0.000174