DOI: 10.5061/dryad.v15dv422z
title: JAK/STAT signaling regulated intestinal regeneration defends insect pests against insecticidal proteins produced by Bacillus thuringiensis

description: A variety of coordinated host-cell responses are activated as defense
 mechanisms against pore-forming toxins (PFTs). Bacillus thuringiensis (Bt)
 is a widely used biopesticide whose efficacy and precise application
 methods limit its use to replace synthetic pesticides in agricultural
 settings. Here, we analyzed the intestinal defense mechanisms of two
 lepidopteran insect pests after intoxication with a sublethal dose of Bt
 PFTs to find out potential target genes for reduction of defense responses
 using dsRNA to silence their expression. We show that larval intestinal
 epithelium was initially damaged by the PFTs and that larval
 survival was observed after intestinal epithelium regeneration. Further
 analyses showed that the proliferation and differentiation of intestinal
 stem cells after Bt toxins treatments were regulated through JNK and
 JAK/STAT signaling pathways. Repression of intestinal regeneration by
 treating specific dsRNA led to increased toxicity of Bt PFTs to both Chilo
 suppressalis and Spodoptera frugiperda. Consequently, a nano-biopesticide
 was designed to improve pesticidal efficacy based on the combination of
 dsRNA-nanoparticles with Bt bacterial strains. This formulation
 efficiently controlled insect pests suggesting its potential use to reduce
 the use of synthetic pesticides in agricultural settings for pest control.
description: # JAK/STAT signaling regulated intestinal regeneration defends insect
 pests against insecticidal proteins produced by Bacillus thuringiensis
 Give a brief summary of
 dataset contents, contextualized in experimental procedures and results.
 The original data and experimental methods of insecticidal bioassays to
 insect pests, determination of intestinal thickness and cell number, APN
 release, RNA interference assays, Reverse transcript quantitative PCR
 (RT-qPCR), Immunofluorescence microscopy, and pot experiment were present
 here. ## Description of the data and file structure This is a freeform
 section for you to describe how the data are structured and how a
 potential consumer might use them. Be as descriptive as necessary. Keep in
 mind that users of your data might be new to the field and unfamiliar with
 common terminology, metrics, etc. Describe relationships between data
 files, missing data codes, other abbreviations used. Be as descriptive as
 possible. 1\. Fig 1A: Five gradient concentrations (100, 75, 50, 25 and
 12.5 μg/g) of Cry9Aa protein in one mL were mixed thoroughly with 3-g
 artificial diet and transferred into flat glass tubes. Thirty-five
 2-day-old larvae were placed inside each tube and three replicates were
 performed for each treatment. The mortality was calculated after 7 days.
 2\. Fig 1B: Five gradient concentrations (500, 400, 300, 200 and 100 μg/g)
 of Cry9Aa protein in one mL were mixed thoroughly with 3-g artificial diet
 and transferred into flat glass tubes. Thirty-five 3rd instar larvae were
 placed inside each tube and three replicates were performed for each
 treatment. The mortality was calculated after 7 days. 3\. Fig 1D: The
 midgut tissues from third instar larvae that survived toxin treatment were
 dissected after feeding with 200 μg Cry9Aa protein per gram of diet for
 different times (0 - 120 h) and fixed overnight in 4% paraformaldehyde at
 4℃. These midgut tissues were carefully washed three times with PBS and
 labeled with 1:1000 Phalloidin-iFluor 647 (Abcam, Cambridge, UK) for 1 h
 at room temperature (RT). After washed twice with PBS, the tissues were
 labeled with 10 μg/ml Hoechst33342 (Solarbio Life Sciences, Beijing,
 China) final concentration in PBS for 5 min. The tissues were sealed with
 Prolong Diamond Antifade (Invitrogen, Oregon, USA) on microscopic slides
 after washing three times with PBS. Images were obtained using 20X
 objective in the confocal LSM 980 (Zeiss, Germany). Six guts were used for
 each treatment and six images were taken from each midgut. The intestinal
 thickness measurements were based on the data of the cytoskeleton stained
 with Phalloidin-iFluor 647 by using ImageJ software in a total of 36
 images. Then the ratio of the width in each image to the width of the
 control group was used to make the graph. 4\. Fig 1E: Midgut cell numbers
 were counted based on number of nuclei stained with Hoechst33342 and then
 dividing by the gut length as the number of nuclei per μm area of the same
 images of Fig 1D. Then the ratio of the cell numbers in each image to the
 cell numbers of the control group was used to make the graph. 5\. Fig 1F:
 Third instar larvae were treated with 200 μg/g Cry9Aa for 12 and 24 h.
 Fifteen intact midguts were dissected from alive larvae at the indicated
 times and transferred into 100 μl of 50 mmol/L Tris-HCl, pH 7.5. Vortexed
 30 sec, centrifuged at 21,000 *xg* for 5 min at 4℃, and the supernatants
 containing contents in midgut lumen of these samples were used for APN
 activity analysis. Protein concentration in the samples was measured by
 using Bradford kit (Solarbio Life Sciences, Beijing, China). APN activity
 was assayed using 4 mM L-leucyl-p-nitroanilide as substrate in 50 mM
 Tris-HCl (pH 7.5) buffer. The released p-nitroanilide by the hydrolysis of
 LpNA was monitored at 405 nm for 10 min using FlexStation 3 Multi-Mode
 Microplate Reader (Molecular Devices, USA). The higher APN enzymatic
 activity was the result of an increase in intestinal cell shedding that
 represented intestinal damage condition. In these experiments, we used as
 negative control (Ctrl) a non-toxic Cry9Aa-D125R mutant protein which the
 mutation site is located in domain I helix α3. This mutant lost toxicity
 but retains the same ability to bind to the BBMV as the Cry9Aa. Control
 and all the treatments were performed in three repetitions. The difference
 in OD405 between the experimental group and the blank control group
 represents the* p*-NA release. The date that *p*-NA release of
 experimental group (Cry9A) and the control group (Cry9A-D125R) were to
 used make the graph. 6\. Fig 1H: Quantification of *delta* gene expression
 determined by RT-qPCR in intestines isolated from fifteen larvae treated
 with 200 μg/g Cry9Aa (LC20) for 0, 12, 24, 48, 72, 96 and 120 h. Total RNA
 was extracted from the samples by using Total RNA Kit I (OMEGA Bio-tek,
 GA, USA). Five hundred ng RNA was used for reverse transcription by using
 HiScript III RT SuperMix (Vazyme, Nanjing, China). Twenty-fold dilution of
 cDNA was used as template for qPCR by using SYBR qPCR Master Mix (Vazyme,
 Nanjing, China) through QuantStudio 6 (Applied Biosystems, MA, USA).
 Relative gene expression was calculated in relation to a reference gene,
 *elongation factor-1* (*EF-1*) for striped stem borer using the 2-ΔΔ Ct
 method. The fold change, positive error and negative error were used to
 make the graph. 7\. Fig 2A: JAK/STAT pathway genes expression (*socs36E*,
 *Stat*) in the intestine from fifteen 3rd instar larvae of striped stem
 borer after treatment with Cry9Aa (LC20) for different 6 h and 48 h
 determined by RT-qPCR. Control (Ctrl) were larvae treated with non-toxic
 Cry9Aa-D125R. The data were normalized to uninfected control and *EF-1*
 gene was used as reference gene. The experiment was done on three
 biological replicate samples performed in three independent experiments.
 The fold change, positive error and negative error were used to make the
 graph. 8\. Fig 2B: The DNA templates for dsRNA were cloned from cDNA of
 larvae midguts. The DNA was ligated into pEasy Blunt Zero vector (TransGen
 Biotech, Beijing, China). A 428-bp fragment of enhanced green fluorescent
 protein gene (*egfp*) was separately constructed. The different dsRNAs
 were synthesized from plasmid DNA using the T7 RiboMAX Express RNAi System
 (Promega, Madison, USA) according to the manufacturer’s instructions. The
 concentration of synthetic dsRNA was determined by using NanoDrop
 (ThermoFisher Scientific, Oregon, USA). Mixed 75 μg ds*socs36E* and
 ds*Stat* with Star Polycation (SPc) complex at 1:1 mass ratio in 1 mL
 nuclease-free water. After incubated for 15 min at RT, fifty striped stem
 borer neonates were treated with 3 g diet mixed with the SPc-dsRNA
 formulation for 48 h. SPc-ds*egfp* was used as negative control. Fifteen
 larvae were collected for reverse transcript quantitative PCR (qRT-PCR)
 and the rest of the treated larvae were transferred to artificial diet
 containing 20 μg of Cry9Aa per g of diet for striped stem borer. The
 mortality was calculated after 7 days. 9\. Fig 2C: Diluted the STAT
 inhibitor (Nifuroxazide) to 200 μmol/L in 1mL buffer, fifty striped stem
 borer neonates were treated with 3 g diet mixed with the STAT inhibitor
 formulation for 48 h. Fifteen larvae were collected for reverse transcript
 quantitative PCR (qRT-PCR) and the rest of the treated larvae were
 transferred to artificial diet containing 20 μg of Cry9Aa per g of diet
 for striped stem borer. The mortality was calculated after 7 days. 10\.
 Fig 2E: The midgut tissues from third instar larvae that survived toxin
 treatment were dissected after feeding with 200 μg Cry9Aa protein per g of
 diet for different times (48, 72, 96 h) after treating with ds*Stat* or
 STAT inhibitor for 48 h and fixed overnight in 4% paraformaldehyde at 4℃.
 These midgut tissues were carefully washed three times with PBS and
 labeled with 1:1000 Phalloidin-iFluor 647 (Abcam, Cambridge, UK) for 1 h
 at room temperature (RT). After washed twice with PBS, the tissues were
 labeled with 10 μg/ml Hoechst33342 (Solarbio Life Sciences, Beijing,
 China) final concentration in PBS for 5 min. The tissues were sealed with
 Prolong Diamond Antifade (Invitrogen, Oregon, USA) on microscopic slides
 after washing three times with PBS. Images were obtained using 20X
 objective in the confocal LSM 980 (Zeiss, Germany). Six guts were used for
 each treatment and six images were taken from each midgut. The intestinal
 thickness measurements were based on the data of the cytoskeleton stained
 with Phalloidin-iFluor 647 by using ImageJ software in a total of 36
 images. Then the ratio of the width in each image to the width of the
 control group was used to make the graph. 11\. Fig 2F: The midgut tissues
 from third instar larvae that survived toxin treatment were dissected
 after feeding with 200 μg Cry9Aa protein per g of diet for different times
 (48, 72, 96 h) afrer treating with ds*Stat* or STAT inhibitor for 48 h and
 fixed overnight in 4% paraformaldehyde at 4℃. These midgut tissues were
 carefully washed three times with PBS and labeled with 1:1000
 Phalloidin-iFluor 647 (Abcam, Cambridge, UK) for 1 h at room temperature
 (RT). After washed twice with PBS, the tissues were labeled with 10 μg/ml
 Hoechst33342 (Solarbio Life Sciences, Beijing, China) final concentration
 in PBS for 5 min. The tissues were sealed with Prolong Diamond Antifade
 (Invitrogen, Oregon, USA) on microscopic slides after washing three times
 with PBS. Images were obtained using 20X objective in the confocal LSM 980
 (Zeiss, Germany). Six guts were used for each treatment and six images
 were taken from each midgut. Midgut cell numbers were counted based on
 number of nuclei stained with Hoechst33342 and then dividing by the gut
 length as the number of nuclei per μm area. Then the ratio of the cell
 numbers in each image to the cell numbers of the control group was used to
 make the graph. 12\. Fig 3A: Quantification of *Jnk*, *puc*, *Egfr *genes
 expression determined by RT-qPCR in intestines isolated from fifteen
 larvae treated with 200 μg/g Cry9Aa (LC20) for 6 and 48 h. Total RNA was
 extracted from the samples by using Total RNA Kit I (OMEGA Bio-tek, GA,
 USA). Five hundred ng RNA was used for reverse transcription by using
 HiScript III RT SuperMix (Vazyme, Nanjing, China). Twenty-fold dilution of
 cDNA was used as template for qPCR by using SYBR qPCR Master Mix (Vazyme,
 Nanjing, China) through QuantStudio 6 (Applied Biosystems, MA, USA).
 Relative gene expression was calculated in relation to a reference gene,
 *elongation factor-1* (*EF-1*) for striped stem borer using the 2-ΔΔ Ct
 method. The fold change, positive error and negative error were used to
 make the graph. 13\. Fig 3B: Mixed 75 μg ds*Jnk* or ds*Egfr* with Star
 Polycation (SPc) complex at 1:1 mass ratio in 1 mL nuclease-free water.
 After incubated for 15 min at RT, fifty striped stem borer neonates were
 treated with 3 g diet mixed with the SPc-dsRNA formulation for 48 h.
 SPc-ds*egfp* was used as negative control. Fifteen larvae were collected
 for reverse transcript quantitative PCR (qRT-PCR) and the rest of the
 treated larvae were transferred to artificial diet containing 20 μg of
 Cry9Aa per g of diet for striped stem borer. The mortality was calculated
 after 7 days. 14\. Fig 3C: Diluted the JNK inhibitor (SP600125) to 200
 μmol/L in 1mL buffer, fifty striped stem borer neonates were treated with
 3 g diet mixed with the STAT inhibitor formulation for 48 h. Fifteen
 larvae were collected for reverse transcript quantitative PCR (qRT-PCR)
 and the rest of the treated larvae were transferred to artificial diet
 containing 20 μg of Cry9Aa per g of diet for striped stem borer. The
 mortality was calculated after 7 days. 15\. Fig 3E: The midgut tissues
 from third instar larvae that survived toxin treatment were dissected
 after feeding with 200 μg Cry9Aa protein per g of diet for different times
 (48, 72, 96 h) after treating with ds*Jnk* or JNK inhibitor for 48 h and
 fixed overnight in 4% paraformaldehyde at 4℃. These midgut tissues were
 carefully washed three times with PBS and labeled with 1:1000
 Phalloidin-iFluor 647 (Abcam, Cambridge, UK) for 1 h at room temperature
 (RT). After washed twice with PBS, the tissues were labeled with 10 μg/ml
 Hoechst33342 (Solarbio Life Sciences, Beijing, China) final concentration
 in PBS for 5 min. The tissues were sealed with Prolong Diamond Antifade
 (Invitrogen, Oregon, USA) on microscopic slides after washing three times
 with PBS. Images were obtained using 20X objective in the confocal LSM 980
 (Zeiss, Germany). Six guts were used for each treatment and six images
 were taken from each midgut. The intestinal thickness measurements were
 based on the data of the cytoskeleton stained with Phalloidin-iFluor 647
 by using ImageJ software in a total of 36 images. Then the ratio of the
 width in each image to the width of the control group was used to make the
 graph. 16\. Fig 3F: The midgut tissues from third instar larvae that
 survived toxin treatment were dissected after feeding with 200 μg Cry9Aa
 protein per g of diet for different times (48, 72, 96 h) after treating
 with ds*Jnk* or JNK inhibitor for 48 h and fixed overnight in 4%
 paraformaldehyde at 4℃. These midgut tissues were carefully washed three
 times with PBS and labeled with 1:1000 Phalloidin-iFluor 647 (Abcam,
 Cambridge, UK) for 1 h at room temperature (RT). After washed twice with
 PBS, the tissues were labeled with 10 μg/ml Hoechst33342 (Solarbio Life
 Sciences, Beijing, China) final concentration in PBS for 5 min. The
 tissues were sealed with Prolong Diamond Antifade (Invitrogen, Oregon,
 USA) on microscopic slides after washing three times with PBS. Images were
 obtained using 20X objective in the confocal LSM 980 (Zeiss, Germany). Six
 guts were used for each treatment and six images were taken from each
 midgut. Midgut cell numbers were counted based on number of nuclei stained
 with Hoechst33342 and then dividing by the gut length as the number of
 nuclei per μm area. Then the ratio of the cell numbers in each image to
 the cell numbers of the control group was used to make the graph. 17\. Fig
 4A: Quantification of *Jnk*, *Stat92E *genes expression determined by
 RT-qPCR in intestines isolated from fifteen larvae treated with 200 μg/g
 Cry9Aa (LC20) for different times (0 - 96 h). Total RNA was extracted from
 the samples by using Total RNA Kit I (OMEGA Bio-tek, GA, USA). Five
 hundred ng RNA was used for reverse transcription by using HiScript III RT
 SuperMix (Vazyme, Nanjing, China). Twenty-fold dilution of cDNA was used
 as template for qPCR by using SYBR qPCR Master Mix (Vazyme, Nanjing,
 China) through QuantStudio 6 (Applied Biosystems, MA, USA). Relative gene
 expression was calculated in relation to a reference gene, *elongation
 factor-1* (*EF-1*) for striped stem borer using the 2-ΔΔ Ct method. The
 fold change, positive error and negative error were used to make the
 graph. 18\. Fig 4B: Quantification of *Jnk*, *Stat, delta genes expression
 determined by RT-qPCR in intestines isolated from fifteen larvae treated
 with 200 μg/g Cry9Aa (LC20) for different times (6 or 48 h) after treating
 with ds*Jnk or ds*Stat* for 48 h. Relative gene expression was calculated
 in relation to a reference gene, *elongation factor-1* (*EF-1*) for
 striped stem borer using the 2-ΔΔ Ct method. The fold change, positive
 error and negative error were used to make the graph. 19\. Fig 4F:
 Quantification of length of seventy-two larvae treated with Cry9Aa (LC20)
 for 120 h after treating with or without silencing *Jnk *or *Stat92E *for
 48 h. 20\. Fig 5A: Mixed 75 μg dsRNA with Star Polycation (SPc) complex at
 1:1 mass ratio in 1 mL nuclease-free water. After incubated for 15 min at
 RT, fifty fall armyworm neonates were treated with 3 g diet mixed with the
 SPc-dsRNA formulation for 48 h. SPc-ds*egfp* was used as negative control.
 Fifteen larvae were collected for reverse transcript quantitative PCR
 (qRT-PCR). 15 g artificial diet were incorporated with 7.5 μg Cry1Fa (0.5
 μg/g) or 15 μg Vip3Aa (1 μg/g) and evenly packed into 24-well culture
 plates. One 2-day-old larva was placed in each well. Three repetitions
 were performed for each treatment. The mortality was calculated after 7
 days. 21\. Fig 5C: The midgut tissues from third instar larvae that
 survived toxin treatment were dissected after feeding with 30 μg Cry1F
 protein per g of diet for 72 h after treating with or without ds*Stat* for
 48 h and fixed overnight in 4% paraformaldehyde at 4℃. These midgut
 tissues were carefully washed three times with PBS and labeled with 1:1000
 Phalloidin-iFluor 647 (Abcam, Cambridge, UK) for 1 h at room temperature
 (RT). After washed twice with PBS, the tissues were labeled with 10 μg/ml
 Hoechst33342 (Solarbio Life Sciences, Beijing, China) final concentration
 in PBS for 5 min. The tissues were sealed with Prolong Diamond Antifade
 (Invitrogen, Oregon, USA) on microscopic slides after washing three times
 with PBS. Images were obtained using 20X objective in the confocal LSM 980
 (Zeiss, Germany). Six guts were used for each treatment and six images
 were taken from each midgut. The intestinal thickness measurements were
 based on the data of the cytoskeleton stained with Phalloidin-iFluor 647
 by using ImageJ software in a total of 36 images. Then the ratio of the
 width in each image to the width of the control group was used to make the
 graph. 22\. Fig 5D: The midgut tissues from third instar larvae that
 survived toxin treatment were dissected after feeding with 30 μg Cry1F
 protein per g of diet for 72 h after treating with or without ds*Stat* for
 48 h and fixed overnight in 4% paraformaldehyde at 4℃. These midgut
 tissues were carefully washed three times with PBS and labeled with 1:1000
 Phalloidin-iFluor 647 (Abcam, Cambridge, UK) for 1 h at room temperature
 (RT). After washed twice with PBS, the tissues were labeled with 10 μg/ml
 Hoechst33342 (Solarbio Life Sciences, Beijing, China) final concentration
 in PBS for 5 min. The tissues were sealed with Prolong Diamond Antifade
 (Invitrogen, Oregon, USA) on microscopic slides after washing three times
 with PBS. Images were obtained using 20X objective in the confocal LSM 980
 (Zeiss, Germany). Six guts were used for each treatment and six images
 were taken from each midgut. Midgut cell numbers were counted based on
 number of nuclei stained with Hoechst33342 and then dividing by the gut
 length as the number of nuclei per μm area. Then the ratio of the cell
 numbers in each image to the cell numbers of the control group was used to
 make the graph. 23\. Fig 5G: *delta gene expression determined by RT-qPCR
 in intestines isolated from fifteen 4th instar fall armyworm larvae
 treated with Cry1Fa or Vip3Aa (LC20) for 36 h after treating with or
 without ds*Stat for 48 h. The data were normalized to uninfected control
 intestines and ecdysoneless (*ECD*) gene was used as reference gene on
 biological replicate samples in three independent experiments. 24\. Fig
 5I: Quantification of length from seventy-two 4th instar larvae treated
 with Cry1Fa (LC20) for 72 h with or without silencing *Stat92E *for 48 h.
 25\. Fig 7A: Two rice seedlings (Oryza sativa L.) at four-leaf stage per
 pot were utilized for spraying assay. Five mL formulation containing
 1.8ⅹ109 cfu Bt serovar *fukuokaensis *strain with cry9Aa gene preserved in
 our laboratory mixed with 60 μg ds*Stat samples embedded in SPc at 1:1
 mass ratio as previously reported were sprayed on the seedlings. After 1
 h, six second-instar striped stem borer larvae were placed on each plant.
 Two pots were used for each treatment. Three replicates were performed.
 Mortality of the first three days of nuclease-free water, SPc-dsStat
 *complex,* *SPc-ds*egfp *complex with Bt, SPc-ds*Stat *complex with Bt
 were calculated. 26\. Fig 7D: Two rice seedlings (Oryza sativa L.) at
 four-leaf stage per pot were utilized for spraying assay. Five mL
 formulation containing 1.8ⅹ109 cfu Bt serovar *fukuokaensis *strain with
 cry9Aa gene preserved in our laboratory mixed with 60 μg ds*Stat samples
 embedded in SPc at 1:1 mass ratio as previously reported were sprayed on
 the seedlings. After 1 h, six second-instar striped stem borer larvae were
 placed on each plant. Two pots were used for each treatment. Three
 replicates were performed. The plant height of rice seedlings of
 nuclease-free water without larvae, nuclease-free water with larvae,
 SPc-dsStat *complex with larvae, SPc-ds*egfp *complex with Bt and larvae,*
 *SPc-ds*Stat *complex with Bt and larvae were calculated after 7 days.