Jm109

The Escherichia coli strain JM109 (Takara) was used for cloning purposes. The compatible haploid T type strains UeT14 (a1b1 CCTCC AF 2015016) and UeT55 (a2b2 CCTCC AF 2015015) and their derivatives (listed in Supplementary Table S1) were used in this study. The strains of U. maydis used in this study are also listed in Supplementary Table S1. The growth conditions and media for E. coli (Russell and Sambrook, 2001 ), U. maydis (Holliday, 1974 ), and U. esculenta (Zhang et al., 2019 (link)) have previously been described. The growth conditions of Zea mays (the early golden bantam) and the wild Z. latifolia used for pathogenic development assays of tested strains before and after stem injection have previously been described (Flor-Parra et al., 2007 (link); Zhang et al., 2019 (link)).

In this study, R. microplus ticks were collected from Yongjing County, Gansu Province, and maintained under laboratory conditions. These ticks were identified using morphology by the Animal Research Institute (Lanzhou Veterinary Research Institute). Approximately 0.5 mg of eggs and 1 g of larvae were used for miRNA extraction, and another 1 g of larvae was placed in a cloth bag attached to cattle for 25 ± 3 days to produce engorged adult female ticks. To analyze the expression profile of miRNAs in R. microplus, unfed larvae, adult female ticks and eggs were ground in liquid nitrogen to extract total RNA using TRIzol reagent (Invitrogen, Cat No. 15596-026). The concentration of RNA was 400 ng/mL (260/280 = 2.00). Some RNAs were used to construct libraries for small RNA sequencing, and some were used for miRNA validation. The synthesis of first-strand cDNAs was performed according to the protocol for transcriptase XL (Avian Myeloblastosis Virus, AMV) (TaKaRa, Shiga, Japan) with a loop primer of miRNAs and oligo dT18. PCR was performed to obtain the miRNAs sequence. The PCR product was ligated to the T-Easy vector (TaKaRa, Japan), which was transformed into JM109 (TaKaRa, Japan). The sequence was obtained by GenScript (Nanjing, China).

E. coli JM109 (Takara, Shiga, Japan) was used for sequencing analysis, and E. coli BL21(DE3) (Takara), pColdTF (Takara), and pColdI (Takara) were used to express BmeTC^X genes. NMR spectra were recorded using a Bruker DPX 400 spectrometer (Billerica, MA, USA) at 400 MHz for protons (¹H) and 100 MHz for carbon (¹³C). GC–MS was performed on a JMS-T100GCV spectrometer (JEOL, Tokyo, Japan) equipped with a DB-1 capillary column (30 m × 0.25 mm × 0.25 µm; J&W Scientific. Inc., Folsom, CA, USA), using the EI mode operated at 70 eV. GC analyses were performed using a Shimadzu GC-2014 chromatograph equipped with a flame ionization detector and using a DB-1 capillary column (30 m × 0.25 mm × 0.25 µm; J&W Scientific, Inc.). GC and GC–MS conditions for the BmeTC^X products were as follows: injection temperature = 300 °C, column temperature = 220–300 °C (1 °C min⁻¹). GC and GC–MS conditions for the volatile compounds were as follows: injection temperature = 200 °C, column temperature = 40–300 °C (5 °C min⁻¹) for GC and 30–300 °C (5 °C min^-1) for GC–MS. Compound 6 was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Two ambergris samples (NSMT M55020 and NSMT M55019; Supplementary Fig. 7) stored in the National Museum of Nature and Science (Japan) for more than 30 years were used for the analysis of volatile components.