DOI: 10.5061/dryad.73n5tb3bm
title: Diverse PGRPs cooperatively maintain homeostasis of facultative symbiont <em>Arsenophonus</em> to promote reproduction of <em>Nilaparvata lugens</em>

description: Peptidoglycan recognition proteins (PGRPs) play a critical role in insect
 innate immunity in defending against pathogen invasion and regulating the
 homeostasis of endosymbionts. Arsenophonus, an emerging clade of
 intracellular symbionts, has been extensively studied for its roles in
 host reproductive manipulation and environmental adaptation. However, the
 molecular mechanisms by which host insects maintain Arsenophonus
 homeostasis through PGRPs remain largely unexplored. Here, we investigated
 the functional roles of PGRPs in regulating Arsenophonus homeostasis in
 the brown planthopper, Nilaparvata lugens, a devastating pest of rice
 crops. While a previous study reported the existence of only two PGRPs
 (PGRP-LC and PGRP-LB) in N. lugens, we further determined that PGRP-LC
 produced two functional isoforms, PGRP-LCa and PGRP-LCb, respectively.
 PGRP-LCa and PGRP-LCb shared identical intracellular domains but possessed
 different extracellular domains. Simultaneously, Arsenophonus was observed
 to localize to the gut muscle layer, fat bodies and ovarian tissues by
 TEM. Functional assays revealed that PGRP-LCa and PGRP-LCb bound not only
 peptidoglycan but also directly interacted with cultured Arsenophonus.
 Artificially transfected Arsenophonus upregulated the expression of
 PGRP-LCa, PGRP-LCb, and Relish, while suppressing PGRP-LB. PGRP-LCa or
 Relish knockdown increased Arsenophonus density, whereas silencing
 PGRP-LCb reduced the symbiont load and female fecundity by the non-IMD
 pathway. Notably, inhibiting PGRP-LB not only elevated Arsenophonus
 density but also triggered symbiont dispersal from the gut muscle layer
 into epithelium. Our findings indicate that PGRPs cooperatively maintain
 Arsenophonus homeostasis to ensure the fecundity in N. lugens. These
 studies provide insight into the interaction of immunity between host and
 endosymbiont.
description: Nymphs of <em>N. lugens</em> were injected with
 dsRNAs targeting PGRP-LCa, PGRP-LCb, PGRP-LB, Relish, or control dsGFP.
 qRT-PCR was used to quantify transcript levels of PGRP-LCa, PGRP-LCb, and
 PGRP-LB in whole bodies or selected tissues. Relative expression was
 calculated using the 2 <sup>−ΔΔCt</sup> or 2
 <sup>−ΔCt</sup> method with appropriate reference genes.
 Statistical comparisons between treatments were performed using Student’s
 t-test in GraphPad Prism, and the corresponding P values and test
 statistics are included in the tables where applicable. For full
 experimental details and interpretation, please refer to the associated
 article in <em>Insect Molecular Biology</em>.
description: # Diverse PGRPs cooperatively maintain homeostasis of facultative symbiont
 *Arsenophonus* to promote reproduction of *Nilaparvata lugens* Dataset
 DOI: [10.5061/dryad.73n5tb3bm](https://doi.org/10.5061/dryad.73n5tb3bm) ##
 Description of the data and file structure Overview This dataset contains
 the raw experimental data underlying the manuscript: Faliang Qin, Ping Li,
 Xuan He, Sen Zheng, Lihaotian Gao, Shuo Wang, Xingya Wang. Diverse PGRPs
 cooperatively maintain homeostasis of facultative symbiont *Arsenophonus*
 to promote reproduction of *Nilaparvata lugens*. *Insect Molecular
 Biology*, 2025. The experiments investigate the role of PGRPs
 in *Arsenophonus* homeostasis in *N. lugens*. The data include: (1)
 Tissue-specific expression profiles of *PGRP-LB*, *PGRP-LCa*, and
 *PGRP-LCb* in female adults of *N. lugens*; (2) Effects of *Arsenophonus*
 injection on PGRPs expression; (3) Effects of PGRPs RNAi on the relative
 abundance of *Arsenophonus* in whole body or tissues of *N .lugens*;
 (4) Effects of PGRPs RNAi on egg production. All data are provided in one
 Excel workbook: Rawdata.xlsx Dryad datasets are treated as independent
 publications; this README describes the content of each worksheet and the
 main variables, so that the dataset can be understood and reused without
 referring to the article. For full experimental details and
 interpretation, please refer to the associated article in *Insect
 Molecular Biology*. ### Files and variables #### File: Rawdata.xlsx
 **Description:** Sheet: Fig 1. These data were used to generate: **Figure
 1C–E**: The conservative domains and tissue profiles of PGRPs in ***N.
 lugens*** (C) Tissue-specific expression profiles of *PGRP-LB* in female
 adults of *N. lugens.* (D)  Tissue-specific expression profiles of
 *PGRP-LCa* in female adults of *N. lugens.* (E) Tissue-specific expression
 profiles of *PGRP-LCb* in female adults of *N. lugens.* The relative
 expression of genes was calculated using the 2^-ΔΔCt^ method with *N.
 lugens* 18S rRNA gene as an internal standard for normalization. FB, Fat
 body; SG, Salivary glands; Ov, Ovary. Data are means ± SEMs. Pairwise
 comparisons were performed using Student’s t- test (**P* &lt; 0.05, ***P*
 &lt; 0.01, ****P* &lt; 0.001). sheet: Fig 4. These data were used to
 generate: **Figure 4A–D**: ***Arsenophonus*** **could induce the innate
 immunity of** ***N. lugens.*** (A) Relative expression level of *PGRP-LCa*
 at 3 h, 6 h, and 12 h after the cultured *Arsenophonus* injection.
 (B) Relative expression level of *PGRP-LCb* at 3 h, 6 h, and 12 h after
 the cultured *Arsenophonus* injection. (C) Relative expression level of
 *Relsh* at 3 h, 6 h and 12 h after the cultured *Arsenophonus* injection.
 (D) Relative expression level of *PGRP-LB* at 3 h, 6 h and 12 h after the
 cultured *Arsenophonus* injection. Each treatment was replicated three
 times with 10 individuals per replication. The relative expression of
 genes was calculated using the 2^-ΔΔCt^ method with *N. lugens* 18S rRNA
 gene as an internal standard for normalization. Data are means ± SEMs.
 Significant differences between treatments are indicated by asterisks
 (**P* &lt; 0.05; ***P* &lt; 0.01; ****P* &lt; 0.001). sheet: Fig 5. These
 data were used to generate: Figure 5A–I: ***Arsenophonus*** density was
 regulated by the Imd pathway of ***N. lugens.*** (A) *Arsenophonus*
 density in whole body of female adults (0-6 h after eclosion) after
 microinjection ds*PGRP-LCa* and ds*GFP* (n = 24). (B) *Arsenophonus*
 density in the gut and carcass of female adults (0-6 h after eclosion)
 after microinjection ds*PGRP-LCa* and ds*GFP* (Each treatment was
 replicated four times with 10 individuals per replication). (C) The number
 eggs in ds*PGRP-LCa* and ds*GFP-injected* female adults within 10 d after
 eclosion (n = 15). (D) *Arsenophonus* density in whole body of female
 adults (0-6 h after eclosion) after ds*PGRP-LCb* and ds*GFP* injection (n
 = 24). (E) *Arsenophonus* density in the gut and carcass of female adults
 (0-6 h after eclosion) after ds*PGRP-LCb* and ds*GFP* injection (Each
 treatment was replicated four times with 10 individuals per replication).
 (F) The number eggs in ds*PGRP-LCa* and ds*GFP-injected* female adults
 within 10 d after eclosion (n = 15). (G)  *Arsenophonus* density in whole
 body of female adults (0-6 h after eclosion) after ds*GFP* and ds*Relish*
 injection (n = 24). (H) *Arsenophonus* density in the gut and carcass of
 female adults (0-6 h after eclosion) after ds*GFP* and ds*Relish*
 injection (Each treatment was replicated four times with 10 individuals
 per replication). (I) The number eggs in ds*Relish* and ds*GFP-injected*
 female adults within 10 d after eclosion (n = 15). The relative symbiont
 density was calculated using the 2^-ΔCt^ method with *N. lugens* 18S rRNA
 gene as an internal standard for normalization. Data are means ± SEMs.
 Significant differences between treatments are indicated by asterisks (*P
 &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001). sheet: Fig 6. These data were
 used to generate: Figure 6A-B, and C: ***PGRP-LB*** **regulates**
 ***Arsenophonus*** density and infection in the gut of ***N.
 lugens***. (A) *Arsenophonus* density in whole body of female adults (0-6
 h after eclosion) after microinjection ds*PGRP-LCa* and ds*GFP* (n = 24).
 (B) *Arsenophonus* density in the gut and carcass of female adults (0-6 h
 after eclosion) after microinjection dsPGRP-LCa ds*GFP* (Each treatment
 was replicated four times with 10 individuals per replication). (C) The
 number eggs in ds*PGRP-LB* and ds*GFP-injected* female adults within 10 d
 after eclosion (n = 15). The relative symbiont density was calculated
 using the 2^-ΔCt^ method with *N. lugens* 18S rRNA gene as an internal
 standard for normalization. Data are means ± SEMs. Significant differences
 between treatments are indicated by asterisks (**P &lt; 0.01; ***P &lt;
 0.001). sheet: Fig S2. These data were used to generate Figure S2: The
 abundance of *Arsenophonus* in the gut of *N.lugens.* The different gut
 parts of female adult (2 d after eclosion) were dissected and used to
 extract the DNA for qPCR. Each treatment was replicated three times with
 10 individuals per replication. The relative symbiont density was
 calculated using the 2^-ΔCt^ method with *N. lugens* 18S rRNA gene as an
 internal standard for normalization. Data are means ± SEMs. n = 3.
 Significant differences between treatments are indicated by asterisks
 (***P* &lt; 0.01). sheet: Fig S5. These data were used to generate Figure
 S5: The binding affinities of recombinant PGRP-LCa and PGRP-LCb to
 different type PGN. The relative band intensities in Western blot analysis
 were quantified using ImageJ software. sheet: Fig S6. These data were used
 to generate: Figure S6: **Heated-*Arsenophonus*** **could induce the
 innate immunity of** ***N. lugens.*** The heat-killed *Arsenophonus* were
 microinjected into fifth-instar nymphs of *N. lugens* to quantify the gene
 responses associated with the IMD pathway. (A) Relative expression level
 of *PGRP-LCa* at 3 h, 6 h, and 12 h after the heat-killed *Arsenophonus*
 injection. (B) Relative expression level of *PGRP-LCb* at 3 h, 6 h, and 12
 h after the heat-killed *Arsenophonus* injection. (C) Relative expression
 level of *Relish* at 3 h, 6 h and 12 h after the heat-killed
 *Arsenophonus* injection. (D) Relative expression level of *PGRP-LB* at 3
 h, 6 h and 12 h after the heat-killed *Arsenophonus* injection. Each
 treatment was replicated three times with 10 individuals per replication.
 The relative expression of genes was calculated using the 2^-ΔΔCt^ method
 with *N. lugens* 18S rRNA gene as an internal standard for normalization.
 Data are means ± SEMs. Significant differences between treatments are
 indicated by asterisks (**P* &lt; 0.05; ****P* &lt; 0.001).  sheet: Fig
 S7.These data were used to generate Figure S7A-E: Relative expression of
 genes after dsRNA microinjection. (A) Relative expression of *PGRP-LCa* in
 ds*GFP*-injected and ds*PGRP-LCa*-injected female adult (0-6 h after
 eclosion) of *N.lugens* (n = 3). (B) Relative expression of *PGRP-LCb and
 Relish* in ds*GFP*-injected and ds*PGRP-LCb*-injected female adult (0-6 h
 after eclosion) of *N.lugens* (n = 3). (C) Relative expression of *Relish*
 in ds*GFP*-injected and ds*Relish*-injected female adult (0-6 h after
 eclosion) of *N.lugens* (n = 3). (D) Relative expression of *PGRP-LB and
 Relish* in ds*GFP*-injected and ds*PGRP-LB*-injected female adult (0-6 h
 after eclosion) of *N.lugens* (n = 3). (E) Relative expression of
 *PGRP-LCb and Relish* in ds*GFP*-injected and ds*PGRP-LCb +
 Relish*-injected female adult (0-6 h after eclosion) of *N.lugens* (n =
 3). (F) Relative abundance of Arsenophonus in the gut of *N. lugens* after
 ds*PGRP-LCb*+*Relish* injection. The relative expression of genes was
 calculated using the 2^-ΔΔCt^ method with *N. lugens* 18S rRNA gene as an
 internal standard for normalization. Data are means ± SEMs. Significant
 differences between treatments are indicated by asterisks (**P* &lt; 0.05;
 ****P* &lt; 0.001).