README: JAK/STAT signaling regulated intestinal regeneration defends insect pests against insecticidal proteins produced by Bacillus thuringiensis

https://doi.org/10.5061/dryad.v15dv422z

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.

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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.