Luria bertani

Luria Bertani (LB), and nutrient medium (NM) and constituents of growth media were purchased from HI Media. E. coli ATCC 25922 and S. aureus ATCC 25923 are type strains, which were taken from culture collection of department of Microbiology, while B. subtilis AST5-2, P. aeruginosa AL2-14B^{32 (link)} and K. pneumoniae AWD5^{33 (link)}, were isolated in an unrelated work, and identified by 16S rDNA sequence analysis. P. aeruginosa AL2-14b was plant endophyte while B. subtilis AST5-2 and K. pneumoniae AWD5 were isolated from soil. The bacterial strains were grown on LB or NM as per the requirements, and stored on nutrient agar (NA) slants at −20 °C. The cultures were revived on LB broth and sub-cultured after regular intervals.

Restriction enzymes were purchased from New England Biolabs (NEB), USA. Taq polymerase and T4 DNA ligase were purchased from Bangalore Genei, India. Gel extraction kit and plasmid isolation kit were purchased from Qiagen, India. Recombinant yeast strain P. pastoris X-33 harbouring Lip11 gene from Yarrowia lipolytica was taken from the laboratory culture collection. This strain has been submitted to Microbial Type Culture Collection (MTCC) with MTCC number 9517. Zeocine was from Invitrogen. The triacylglycerides, p-np esters used in the experiments were procured from Sigma Aldrich. Luria bertani, tryptone, yeast extract, yeast nitrogen base and methanol were purchased from Hi-Media. Sodium chloride was taken from Sisco Research Laboratories Pvt. Ltd. India (SRL). Glycosylation kit was procured from G Bioscience (USA).

In this study, an Indian strain of M. oryzae, i.e., B157 (international race IC9) isolated from Hyderabad, and its cytoplasmic GFP-expressing transformant (M. oryzaeGFP) were grown on yeast extract glucose (YEG) media (Saha et al., 2020 (link)). The blast-susceptible indica rice cultivar- C0-43, was grown at 27°C under a 16:8 light/dark photoperiod. The binary vector pCAMBIA1302 was streaked onto Luria Bertani (Himedia, Mumbai, India) agar plates supplemented with 50 mg/L Kanamycin (Himedia, Mumbai, India) and incubated overnight at 37°C. Fungal growth assays were performed on a complete medium (CM) supplemented with or without dsRNA, at 28°C for 7 days. For conidiation, the fungi were grown in dark on YEG plates for 8–10 days. The fungal biomass was then scraped on 10 days after inoculation and homogenized into uniform slurry by vortexing. Spores were separated from the mycelial debris by filtering the slurry through sterile MiraCloth (Calbiochem, Darmstadt, Germany), and then centrifuged at 7,000 rpm for 7 min. The conidia were visualized and counted under a microscope using a hemocytometer (Neubauer, Marienfeld, Germany). The length and breadth of conidia were measured keeping a 20-μm scale reference using an built-in microscope camera. Microscopic examination of at least 50 conidia per replicate was done in at least three independent experiments.