Bioanalyzer 2100

Infected leaves were collected 7 days after inoculation and immediately frozen in liquid nitrogen. Samples were ground to a fine powder in liquid nitrogen and total RNA was isolated with the RNeasy Plant Mini Kit (Qiagen). After DNase treatment (Promega), RNA was further purified using the RNeasy Plant Mini Kit columns and the quality was assessed using the Bioanalyzer 2100. For library preparation, around 10 μg of total RNA was processed with the mRNA-Seq Sample Preparation kit (Illumina). Libraries were prepared for the pair 9 isolates, including BCL-75 (1 for Cs and 1 for Cs/Ax) and 630550 (1 for Cs and 1 for Cs/Ax). Each library was sequenced using the Illumina HiSeq2500 platform (125 bp paired-end reads).

Amplification of the bacterial 16S rRNA gene’s V3–V4 variable region was achieved through a biphasic PCR method for 25 cycles at 55 °C. In summary, the PCR utilized a set of two primers: the forward primer was 5ʹ-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACG-GGNGGCWGCAG and the reverse primer was 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACA-GGACTACHVGGGTATC-TAATCC-3′. The PCR products underwent evaluation using 2% agarose gel electrophoresis. The 16S rRNA libraries were then purified with magnetic beads (AMPure XP), in line with the specifications given by Beckman Coulter, Wycombe, UK. To verify the purity of the samples, a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) was employed. The second phase of the PCR involved attaching Illumina Nextera barcodes to the products of the initial PCR using i5 forward and i7 reverse primers. These amplified products were then purified following the same procedure as the initial round. DNA quantification was carried out using the QuantiFluor® ONE dsDNA System (Promega), and the Bioanalyzer 2100 was again used for assessing the quality of the samples. The amplified 16S rRNA gene and the prepared library, developed using this two-step PCR method, were sequenced using the MiSeq v3 Reagent Kit from Illumina, Inc.

Infected leaves were collected at 3, 5, and 7 days after inoculation with Pt104 and immediately frozen in liquid nitrogen. Samples were ground to a fine powder in liquid nitrogen and total RNA was isolated with the isolate II RNA Mini Kit (Bioline, NSW, Australia). After DNase treatment (Promega, NSW, Australia), RNA was further purified by on-column DNase treatment, and the quality was assessed using the Bioanalyzer 2100. For library preparation, approximately 10 μg of total RNA was processed with the mRNA-Seq Sample Preparation kit (Illumina), which was then sequenced on the Illumina HiSeq2500 platform (125 bp paired-end reads).