A single synonymous nucleotide change impacts the male-killing phenotype of prophage WO gene wmk

  1. Jessamyn I Perlmutter  Is a corresponding author
  2. Jane E Meyers
  3. Seth R Bordenstein  Is a corresponding author
  1. Department of Biological Sciences, Vanderbilt University, United States
  2. Department of Molecular Biosciences, University of Kansas, United States
  3. Vanderbilt Microbiome Innovation Center, Vanderbilt University, United States
  4. Department of Pathology, Microbiology, and Immunology, Vanderbilt University, United States
  5. Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University, United States
4 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
Overview of experimental design.

To investigate the genotype-phenotype landscape, we transgenically expressed wmk homologs with varying degrees of genetic changes. These sequences are codon-optimized based on different codon biases …

Figure 1—figure supplement 1
Homologs of wmk tested in this study include variation in native gene and transgene sequence identity as well as host species.

(Top) Bayesian nucleotide phylogeny of insect hosts based on 652 bp of the cytochrome oxidase subunit 1 (COI) gene from D. melanogaster, H. bolina, D. bifasciata, C. cautella, D. innubila, D. …

Figure 2 with 1 supplement
Transgenic expression of closelyrelated wmk homologs causes male-killing and all-killing phenotypes in D. melanogaster.

(A) Schematic of wMel, wSuzi, and wRec wmk native nucleotide sequences. The blue tick mark indicates a non-synonymous nucleotide difference. Black tick marks indicate synonymous nucleotide changes. …

Figure 2—source data 1

Data for sex ratios of closely-related homologs corresponding to Figure 2B.

https://cdn.elifesciences.org/articles/67686/elife-67686-fig2-data1-v2.xlsx
Figure 2—source data 2

Statistical output of Kruskal-Wallis test corresponding to sex ratios of closelyrelated homologs in Figure 2B.

https://cdn.elifesciences.org/articles/67686/elife-67686-fig2-data2-v2.xlsx
Figure 2—source data 3

Data for qPCR of closely-related transgenes corresponding to Figure 2C.

https://cdn.elifesciences.org/articles/67686/elife-67686-fig2-data3-v2.xlsx
Figure 2—source data 4

Statistical output of Kruskal-Wallis test corresponding to qPCR for transgene expression in Figure 2C.

https://cdn.elifesciences.org/articles/67686/elife-67686-fig2-data4-v2.xlsx
Figure 2—source data 5

Data for qPCR of msl-2 expression with transgene expression corresponding to Figure 2D.

https://cdn.elifesciences.org/articles/67686/elife-67686-fig2-data5-v2.xlsx
Figure 2—source data 6

Statistical output of Kruskal-Wallis test corresponding to qPCR for msl-2 expression in Figure 2D.

https://cdn.elifesciences.org/articles/67686/elife-67686-fig2-data6-v2.xlsx
Figure 2—figure supplement 1
wRec and wSuzi transgenes expressed with an alternative start codon lose their transgenic phenotypes.

(Top) Sex ratios of adult flies are shown for expressing (Act5c-Gal4) and non-expressing (CyO) offspring. Each sample point represents the adult offspring (N = 50–120, mean 84) produced by a …

Figure 2—figure supplement 1—source data 1

Data for sex ratios of 5’ alternative start codon transgene expression corresponding to Figure 2—figure supplement 1.

https://cdn.elifesciences.org/articles/67686/elife-67686-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 1—source data 2

Statistical output of Kruskal-Wallis test corresponding to sex ratios of 5’ alternative start codon transgene expression in Figure 2—figure supplement 1.

https://cdn.elifesciences.org/articles/67686/elife-67686-fig2-figsupp1-data2-v2.xlsx
Divergent homologs of wmk from male-killing strains do not induce a biased sex ratio in D. melanogaster.

Sex ratios of adult flies are shown from either expressing (Act5c-Gal4) or non-expressing (CyO) offspring. WT refers to the background insertion line and Control gene refers to the WD0034 control …

Figure 3—source data 1

Data for sex ratios of distantly-related homologs in Figure 3.

https://cdn.elifesciences.org/articles/67686/elife-67686-fig3-data1-v2.xlsx
Figure 3—source data 2

Statistical output of Kruskal-Wallis test corresponding to sex ratios of divergent homologs in Figure 3.

https://cdn.elifesciences.org/articles/67686/elife-67686-fig3-data2-v2.xlsx
Synonymous nucleotide changes in the 16th codon position of wmk alters resulting phenotype.

(A) Sequence alignment of transgenic wmk homologs. The codon farthest on the left is the fourth codon in the sequence, and the highlighted codon is the 16th, with the farthest right representing the …

Figure 4—source data 1

Data for sex ratios from expression of transgenes with single codon changes corresponding to Figure 4C.

Data for qPCR from expression of transgenes with single codon changes corresponding to Figure 4D.

https://cdn.elifesciences.org/articles/67686/elife-67686-fig4-data1-v2.xlsx
Figure 4—source data 2

Data for qPCR from expression of transgenes with single codon changes corresponding to Figure 4D.

https://cdn.elifesciences.org/articles/67686/elife-67686-fig4-data2-v2.xlsx
Figure 4—source data 3

Data for qPCR from expression of transgenes with single codon changes corresponding to Figure 4D.

https://cdn.elifesciences.org/articles/67686/elife-67686-fig4-data3-v2.xlsx
Figure 4—source data 4

Statistical output of Kruskal-Wallis test corresponding to qPCR from expression of transgenes with single codon changes in Figure 4D.

https://cdn.elifesciences.org/articles/67686/elife-67686-fig4-data4-v2.xlsx

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Wolbachia pipientis)WD0626NCBINCBI:WD_RS02815Also known as wmk
(WO-mediated killing)
Genetic reagent (D. melanogaster)Act5c-Gal4/CyOBloomington Drosophila Stock CenterBDSC:3953; FlyBase FBti0012290P{AyGAL4}25
Genetic reagent (D. melanogaster)WT (y1w67c23; P[CaryP]P2)Bloomington Drosophila Stock CenterBDSC:8622; FlyBase FBti0040535WT strain used in this
study; P{CaryP}attP2
Genetic reagent (D. melanogaster)wMel wmkThis paper; Perlmutter et al., 2019; PMID:31504075Expresses codon-
optimized transgene;
UAS promoter
Genetic reagent (D. melanogaster)wBol1b wmkThis paperExpresses codon-
optimized transgene;
UAS promoter
Genetic reagent (D. melanogaster)wBif wmkThis paperExpresses codon-
optimized transgene;
UAS promoter
Genetic reagent (D. melanogaster)wCaub wmkThis paperExpresses codon-
optimized transgene;
UAS promoter
Genetic reagent (D. melanogaster)wInn/wBor wmkThis paperExpresses codon-
optimized transgene;
UAS promoter; wInn
and wBor wmk have
same exact sequence
Genetic reagent (D. melanogaster)wSuzi wmkThis paperExpresses codon-
optimized transgene;
UAS promoter
Genetic reagent (D. melanogaster)wRec wmkThis paperExpresses codon-
optimized transgene;
UAS promoter
Genetic reagent (D. melanogaster)HA-wmkThis paperExpresses codon-
optimized transgene;
UAS promoter; 3 X HA
tag epitope in linker
between HTH domains
of wMel wmk
Genetic reagent (D. melanogaster)5’ wRec wmkThis paperExpresses codon-optimized transgene; UAS promoter;
Sequence has additional nine amino acids
starting at upstream alternative start codon
Genetic reagent (D. melanogaster)5’ wSuzi wmkThis paperExpresses codon-optimized transgene; UAS promoter;
Sequence has
additional nine amino acids starting at
upstream alternative
start codon
Genetic reagent (D. melanogaster)wMel wmk (new)This paperExpresses codon-optimized transgene; UAS promoter;
Same exact sequence
as wMel wmk, newly transformed strain
Genetic reagent (D. melanogaster)wSuzi codonThis paperExpresses codon-optimized transgene; UAS promoter; Same as wMel wmk, but with 16th amino acid position using TCC Serine codon from wSuzi wmk strain
Genetic reagent (D. melanogaster)wRec codonThis paperExpresses codon-optimized transgene; UAS promoter; Same as wMel wmk, but with 16th amino acid position using AGC Serine codon from wRec wmk strain
Recombinant DNA reagentpTIGER (plasmid)Ferguson et al., 2012; PMID:22328499Modified pUASp
plasmid for enhanced germline expression under Gal4/UAS control
Sequence-based reagentRp49_FThis paperPCR primersCGGTTACGGAT
CGAACAAGC
Sequence-based reagentRp49_RThis paperPCR primersCTTGCGCTTCT
TGGAGGAGA
Sequence-based reagentwmk_homologs_opt_FThis paperPCR primersCTGTATGCCATTG
CCGAGACCCT
Sequence-based reagentwmk_homologs_opt_RThis paperPCR primersTCACCAGATCCTTG
GCGATCTTCATC
Sequence-based reagentMsl-2_FThis paperPCR primersGGATTAACGCGGT
CTAAGCATGTGTAACTG
Sequence-based reagentMsl-2_RThis paperPCR primersGTATGCCGTCTG
GGCCATGATG
Commercial assay or kitDirect-zol RNA MiniPrep KitZymoR2051
Commercial assay or kitSuperscript VILO cDNA Synthesis KitThermoFisher11754050
Chemical compound, drugDNase, RNase-freeAmbion, Life TechnologiesAM2222
Chemical compound, drugiTaq Universal SYBR Green MixBio-Rad1725120
Software, algorithmGraphPad Prism 8GraphPad Prism 8RRID:SCR_002798
Software, algorithmGeneious Pro v.2019.2; Geneious Pro v.2020.2.4GeneiousRRID:SCR_010519
Software, algorithmjModelTestjModelTestRRID:SCR_015244
Software, algorithmRNAfold WebServerUniversity of Vienna,
Gruber et al., 2008,
Lorenz et al., 2011
PMID:18424795; PMID:22115189http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi
  1. **Reagents source from this paper may be obtained from Bordenstein lab.

Additional files

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