Nanodrop spectrophotometer

Firstly, dried starter samples were powdered with the help of sterile mortar and pestle and 1g of powdered sample were taken and homogenized in 9 ml of 0.85% physiological saline and subsequently filtered through 4 layers of sterile cheese-cloth. The resulting filtered solutions were centrifuged at 14000 g for 10 min at 4°C (Lv et al., 2013 (link); Sha et al., 2018 (link)) and then the pellets were subjected to total community DNA extraction using the ProMega DNA extraction kit (ProMega, United States) according to the manufacturer’s instructions. Subsequently, the RNA was eliminated from the cellular lysate by administering the RNase solution after incubation at 35°C for 15 min. The residual proteins were removed by adding protein precipitation solution and then centrifuging at maximum speed. Finally, the DNA was precipitated by adding isopropanol, and purified with two washes of 70% ethanol. Quality of DNA was checked on 0.8% agarose gel by measuring the concentration using Nano-Drop spectrophotometer (AG-6135, Eppendorf, Germany). The DNA was kept at -20°C until further processing.

Each testis tissue was thawed, removed from RNAlater and pad-dried. Thereafter, RNA was extracted from each tissue using Innu Prep RNA mini kit (Analytik Jena, Jena, Germany), following the manufacturer’s protocol. The concentration and purity of extracted RNA was determined using a Nanodrop spectrophotometer (Eppendorf Nanodrop BioPhotometer plus, Hamburg, Germany. To determine RNA quality, gel electrophoresis using 1% agarose (w/v) in 1 × LB buffer, was performed and viewed using a UV transilluminator (ChemiDoc XRS, Bio-Rad Laboratories, Hercules, CA, USA). RNA samples which had OD_260/280 of 1.8–2.0 as determined by a Nanodrop spectrophotometer, and showed distinct 28S and 18S ribosomal RNA bands after gel electrophoresis, were selected for the downstream experiments.

This was done according to our published protocol (Chowdhury et al., 2017a (link),b (link)). Total RNA was isolated from leaf samples by Trizol (Invitrogen) method, followed by quantification with a nanodrop spectrophotometer (Eppendorf). Two micrograms of RNA were used to synthesize cDNA using random primers. Real time PCR (RT-qPCR) was carried out in 96-well blocks in 20μl reaction volume (Applied Biosystems 7500 Real-Time PCR System, USA) using Power SYBR Green master mix (Applied Biosystems, USA). Transcript sequences were amplified by gene-specific primers. Relative fold changes in gene expression were determined by a standard method, particularly by calculating ΔΔCt value. The specificity of the PCR reactions was adjusted based on a melting curve analysis of the amplified products utilizing the standard method installed in the system. The pathogen gene 18s rDNA was used as an internal control for the pathogen (Sayler and Yang, 2007 (link)). Tomato actin gene was used as internal control according to our protocol (Ray et al., 2015 ). The list of primers both for the pathogen and the tomato plants used in this study is given below.
List of primers used for quantitative real-time PCR:

Genomic DNA samples were extracted using the CTAB method⁵⁷ on five individual gut homogenates from each biological replicate and treatment. The quantity and quality of extracted gDNA were determined using a NanoDrop Spectrophotometer (Eppendorf AG, Germany) and stored at −20 °C prior to the analysis. The pyrosequencing of V2-V3 hyper-variable regions of the 16S rRNA gene amplicons was carried out on a 454 GS FLX Titanium system (Roche, Penzberg, Germany)^{60 (link),62} . Universal barcoded primers Ba27F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and Ba519R (5′-TAT TAC CGC GGC KGC TG-3′) containing A and B sequencing adaptors (454 Life Sciences), key sequences and multiplex identifiers were used to prepare the pyrosequencing amplicons^{63 (link)}. Before pyrosequencing, amplicons were purified from gel using AxyPrep DNA gel extraction kit (Axygen Scientific, Inc.), quantified using Quant-iT Pico Green dsDNA assay (Invitrogen, Germany) and pooled in an equimolar ratio before being subjected to emulsion PCR and breaking. GS FLX Titanium chemistry was used to perform pyrosequencing from A-end according to the supplier protocol.

Bacteria were collected and washed with ice-cold PBS before mixing with pre-prepared red blood cell membranes as described in Preparation of CMCB. Both EcN and CMCB were diluted to reach an optical density (OD) value of ~0.15 in LB, and incubated at 37 °C with gently shaking. The OD value of cultures was recorded at 600 nm at various time points by nanodrop spectrophotometer (Eppendorf, German).

Total genomic DNA of Xss-V₂–18 was isolated using the Bacterial Genomic DNA Extraction Kit (TaKaRa, China). The isolated gDNA pellet was re-suspended in double-distilled water. Bacterial plasmid DNA was isolated using the Plasmid Miniprep Kit (Omega, USA). The quality and quantity of genomic DNA and plasmid DNA were checked with NanoDrop spectrophotometer (Eppendorf). Routine plasmids isolation from E. coli was carried out by using the plasmid DNA Mini Kit (GBS Biotechnology, China).