Insects and bacteria
Bean bugs were reared in petri dishes (90 mm in diameter and 20-mm high) at 25 °C under a long-day regimen (16-h light, 8-h dark) and fed with soybean seeds and distilled water containing 0.05% ascorbic acid (DWA). Burkholderia symbiont strain SFA119, a MEP-degrading strain conferring MEP resistant in the bean bug, and its GFP-(green fluorescent protein) labeled derivative, strain SJ586, were used in this study. The symbiont was cultured at 30 °C on YG medium (0.5% yeast extract, 0.4% glucose, and 0.1% NaCl). The GFP-labeled strain was constructed by the Tn7 mini-transposon system, as previously described31.
Genome sequencing
DNA was extracted from cultured cells of strain SFA1 by the phenol–chloroform extraction as previously described32. The DNA library for Illumina short reads (the mean insert size: 500 bp) was constructed by using the Covaris S2 ultrasonicator (Covaris) and the KAPA HyperPrep Kit (Kapa Biosystems). For the library construction for Nanopore long reads, Native Barcoding Expansion (EXP-NBD104, Oxford Nanopore Technologies) and the Ligation Sequencing Kit (SQK-LSK109, Oxford Nanopore Technologies) were used. The genome sequencing was performed with NextSeq using the 2 × 151-bp protocol (Illumina) and GridION using an R9.4.1 flow cell (Oxford Nanopore Technologies). The Illumina short reads were processed by using Sickle Ver 1.33 (available at https://github.com/najoshi/sickle) for removing the low-quality and shorter reads. After processing the Nanopore long-reads with Porechop Ver 0.2.3 (available at https://github.com/rrwick/Porechop) and Filtlong Ver 0.2.0 (available at https://github.com/rrwick/Filtlong), error correction was performed by using Canu Ver 1.833. These processed short- and long reads were assembled by using Unicycler Ver 0.4.734, resulting in the eight circular replicons (Supplementary Fig. 1). The assembled genome was annotated by DFAST Ver 1.1.035. After the homology searches of the protein sequences by blastp 2.5.0 + 36 against the COG database (PMID: 25428365), circular replicons were visualized with circos v 0.69-837. The chromosomes and plasmids were assigned according to the genome of Caballeronia (Burkholderia) cordobensis strain YI2338.
Phylogenetic analysis
Nucleotide sequences of 16 S rRNA gene of representative Burkholderia spp. and outgroup species were aligned by using SINA v1.2.1139. Protein sequences of MEP-degrading genes (mpd, pnpB, and mhqA) and a plasmid-transfer gene (traH) on plasmid 2 were subjected to the blastp search against the nr database (downloaded in Jul. 2019) and top ~30 hit sequences were retrieved for each gene. Multiple sequencing alignments of each gene were constructed with L-INS-I of mafft v7.40740. Gap-including and ambiguous sites in the alignments were then removed. Unrooted maximum-likelihood (ML) phylogenetic trees were reconstructed with RAxML v8.2.341 using the GTR + Γ model (for 16 S rRNA gene) or the LG + Γ model42 (for other genes). The bootstrap values of 1000 replicates for all internal branches were calculated with a rapid bootstrapping algorithm43.
Preparation of SFA1 cultures for RNA-seq
Burkholderia symbiont SFA1 was precultured in minimal medium (20 mM phosphate buffer [pH 7.0], 0.01% yeast, 0.1% (NH4)2SO4, 0.02% NaCl, 0.01% MgSO4⋅7H2O, 0.005% CaCl2⋅2H2O, 0.00025% FeSO4⋅7H2O, and 0.00033% EDTA⋅2Na) containing 1.0 mM of MEP on a gyratory shaker (210 rpm) at 30 °C overnight, and subcultured in newly prepared MEP-containing minimal medium under the same conditions for 5 h. As a control, SFA1 was precultured in minimal medium containing 0.1% citrate overnight, and then the overnighter was subcultured in a newly prepared citrate-containing minimal medium under the same conditions for 10 h. The culture was mixed with an equal amount of RNAprotect Bacteria Regent (Qiagen, Valencia, CA, USA), then centrifuged to harvest the cells for the RNA-seq analysis.
Preparation of midgut symbiont cells for RNA-seq
The oral administration of the symbiont strain SFA1 was performed as described19,44. The symbiont was inoculated to 2nd instar nymphs, and three days after molting to the 3rd instar, nymphs were transdermally administered with 1 µl of 0.2 µM or 20 µM of MEP (dissolved in acetone). One- or three days after the treatment, insects were dissected and the crypt-bearing symbiotic gut region was subjected to the RNA extraction and RNA-seq analysis. As a control, untreated insects were analyzed.
RNA-seq analysis
Total RNA was extracted from triplicate samples from cultures by the hot-phenol method as previously described45 or from the midgut symbiont cells by using RNAiso Plus (Takara Bi, Kusatsu, Shiga, Japan) and the RNeasy mini kit (Qiagen). The extracted total RNA was purified by phenol–chloroform extraction and digestion by DNase (RQ1 RNase-Free DNase, Promega, Fitchburg, WI, USA) and repurified by using a RNeasy Mini Kit. The mRNA in the samples was further enriched by the RiboMinus Transcriptome Isolation Kit bacteria (Thermo Fisher Scientific, Waltham, MA, USA) and the RiboMinus Eukaryote Kit for RNA-Seq (Thermo Fisher Scientific), and purified by using an AMPure XP kit (Beckman Coulter, Brea, CA, USA). The cDNA libraries were constructed from approximately 100 ng of rRNA-depleted RNA samples by the use of a NextUltraRNA library prep kit (New England Biolabs, Ipswich, MA, USA). Size selection of cDNA (200–300 bp) and determination of the size distribution and concentration of the purified cDNA samples were performed as described previously46. In total, 21 cDNA libraries were constructed and sequenced by MiSeq (Illumina, Inc., San Diego, CA, USA). To ensure high sequence quality, the remaining sequencing adapters and the reads with a cutoff Phred score of 15 (for leading and tailing sequences, Phred score of >20) and a length of less than 80 bp in the obtained RNA-seq data were removed by the program Trimmomatic v0.30 using Illumina TruSeq3 adapter sequences for the clipping47. The remaining paired reads were analyzed by FastQC version 0.11.9 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) for quality control, and Bowtie2 ver. 2.2.248 for mapping on the symbiont genome (DDBJ/EMBL/GenBank accession: AP022305–AP022312). After the conversion of the output BAM files to BED files using the bamtobed program in BEDTools ver. 2.14.349, gene expression levels were calculated in TPM (transcripts per kilobase million) values by using in-house scripts46.
Gene deletion and complementation
MEP-degrading genes (mpd, pnpA1, and pnpA2) were deleted by the homologous-recombination-based deletion method using pK18mobsacB or pUC18, as previously described50,51. Primers used for the mutagenesis are listed in Supplementary Table 1. For mpd gene deletion, pK18mobsacB was used to construct a markerless mutant. For single deletion of pnpA1 and pnpA2 genes, pUC18 was used to substitute each gene locus with a kanamycin-resistance gene cassette. The double deletion of pnpA1 and pnpA2 genes was performed by substituting pnpA2 gene locus with a tetracycline-resistance gene cassette in the pnpA1-deletion mutant. Gene complementation of mpd was also performed by homologous recombination using plasmid pUC18 with primers listed in Supplementary Table 1. To investigate growth profiles of the wild-type SFA1, the gene-deletion mutants (Δmpd, ΔpnpA1, ΔpnpA2, and ΔpnpA1/ΔpnpA2), and the mph-complement mutant (Δmpd/mpd+) in the MEP-containing minimal medium, the strains were precultured in minimal medium containing 1.0 mM MEP on a gyratory shaker (210 rpm) at 30 °C overnight, and then cultured in newly prepared MEP-containing minimal medium under the same condition. The growth of cultures was estimated by OD600 measurements. To confirm the basic growth abilities of the mutants, these bacterial strains were pre- and subcultured in minimal medium containing 0.1% glucose under the same conditions. These symbiont strains and mutants were inoculated to the bean bug as described above.
Quantitative PCR
Symbiont titers in the midgut crypts were evaluated by quantitative PCR (qPCR) of bacterial dnaA gene copies. The qPCR was performed by using a KAPA SYBR Fast qPCR Master Mix (Kapa Biosystems) and the LightCycler 96 System (Roche Applied Science) with the following primers: BSdnaA–F (5′-AGC GCG AGA TCA GAC GGT CGT CGA T-3′) and BSdnaA–R (5′-TCC GGC AAG TCG CGC ACG CA-3′).
MEP treatment of insects
MEP treatment of R. pedestris was performed as previously described19. Soybean seeds were dipped in 0.2 mM MEP for 5 s and dried at room temperature. In each clean plastic container, 15 individuals of 3rd-instar nymphs were reared on three seeds of the MEP-treated soybean and DWA at 25 °C under the long-day regime, and the number of dead insects was counted 24 h after the treatments. The survival rate of the insects was analyzed under Fisher’s exact test by use of the program R ver. 3.6.3 (available at https://www.R-project.org/). Multiple comparisons were corrected by the Bonferroni method.
Bactericidal activities of MEP and its degradation product 3M4N
To measure bactericidal activities of MEP and 3M4N on cultured cells of SFA1, 104 cells of log-phase growing bacteria were mixed with a defined concentration of MEP or 3M4N, and spotted on a YG agar plate. To measure the bactericidal activity against midgut crypt-colonizing cells, the symbiotic organs infected with SFA1 were dissected from 3rd-instar insects, homogenized in PBS, and purified by a 5-µm-size pore Syringe filter to harvest colonizing symbiont cells50. MEP or 3M4N was added to approximately 104 cells of the harvested cells and spotted on a YG agar plate. Bactericidal activities of the chemical compounds were then checked in 24 h after incubation at 30 °C.
HPLC detection of in vitro and in vivo MEP-degrading activities of the symbiont
To determine in vitro MEP-degradation activity, cultured cells of SFA1 were prepared as above, and 106 cells were incubated at 25 °C in 200 µl of MEP solution (2 mM MEP in Tris-Hcl [pH 8.5] with 0.1% Triton X-100) in a 1.5-ml microtube. To determine in vivo MEP-degradation activity, the midgut of a 5th-instar insect infected with SFA1 was dissected, the posterior and anterior parts of the crypt-bearing symbiotic region were closed with 0.2-mm polyethylene fishline (Supplementary Fig. 6a), and incubated at 25 °C in 200 µl of the MEP solution. For the in vivo determination, 250 mM of trehalose, known as a major sugar source of insects’ hemolymph52, was added to the MEP solution to keep the tissue fresh. After incubation for different times, the reaction was stopped by adding 400 µl of methanol. After centrifugation, supernatants were subjected to high-performance liquid chromatography (HPLC) analyses to detect MEP and 3M4N, as previously reported21, and precipitated cells and tissues were subjected to DNA extraction and qPCR to estimate symbiont-cell numbers of each reaction.
LC–ESI–MS detection of 3M4N in feces from 3M4N-fed insects
An insect-rearing system for feeding 3M4N and collecting feces is shown in Supplementary Fig. 7. Insects were fed with DW or DW containing 10 mM 3M4N in a plastic container, in which the solution supplier was covered by 0.5-mm mesh, so that insects were able to drink the solution by probing with their proboscis, but did not directly touch the solution by their legs or body. Twenty insects were reared per container and their feces were accumulated on the bottom of the container for five days. The collected feces (DW- or 3M4N-treated) were suspended in 1 ml of MilliQ water, and the water-soluble fractions were extracted by thorough vortexing. Solids and insoluble fractions were removed from the suspension by centrifugation and subsequent filtration using a cellulose-acetate membrane (Φ, 0.20 μm, ADVANTEC, Tokyo, Japan). The resultant fraction was diluted 10-fold by MilliQ water and analyzed by liquid chromatography–electrospray-ionization mass spectrometry (LC–ESI–MS) according to a previous report53,54,55. HPLC was performed using the Nexera X2 system (Shimadzu, Kyoto, Japan) composed of LC-30AD pump, SPD-M30A photodiode-array detector, and SIL-30AC autosampler. Develosil HB ODS-UG column (ID 2.0 mm × L 75 mm, Nomura Chemical Co., Ltd, Aichi, Japan) was employed with a flow rate of 0.2 mL/min. The following gradient system was used for analysis of metabolites: MilliQ water (solvent A) and methanol (solvent B), 90% A and 10% B at 0–5 min, linear gradient from 90% A and 10% B to 20% A and 80% B at 5–15 min, 20% A and 80% B at 15–20 min, and 90% A and 10% B at 20–25 min. Retention time of 3M4N standard reagent was 14.2 min. Electrospray-ionization mass spectrometry (ESI–MS) in positive and negative ion modes was simultaneously performed using amaZon SL (Bruker, Billerica, MA, USA). 3M4N (MW = 153.14) standard showed a clear peak in negative mode at m/z of 151.53.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Source: Ecology - nature.com
