key: cord-0818748-s6uyh2n6
authors: Nazir, Amina; Dong, Zhaoxia; Liu, Jin; Zhang, Xianglilan; Tahir, Rana Adnan; Ashraf, Neelma; Qing, Hong; Peng, Donghai; Tong, Yigang
title: Sequence Analysis of a Jumbo Bacteriophage, Xoo-sp14, That Infects Xanthomonas oryzae pv. oryzae
date: 2020-11-25
journal: Microbiol Resour Announc
DOI: 10.1128/mra.01072-20
sha: 8087a70f6bcb16808bb0d4aabcf436dffa09d928
doc_id: 818748
cord_uid: s6uyh2n6

A jumbo bacteriophage, Xoo-sp14, infecting Xanthomonas oryzae pv. oryzae was isolated from rice fields in China. Here, we report the complete genome sequence of this phage, revealing that it had a linear double-stranded DNA (dsDNA) molecule 232,104 bp long, with a G+C content of 58%. It has 251 annotated protein-coding sequences.

X anthomonas oryzae pv. oryzae is considered one of the most important agents of bacterial blight (BB) on rice (1) . This disease creates serious threats to this major crop, leading to destruction and an alarming situation for global food security (2) . Bacteriophages are one of the most abundant entities that exist everywhere in the environment and are potent for controlling bacterial diseases (3) . Previously, we isolated multiple bacteriophages as potential biocontrol agents against X. oryzae pv. oryzae (4) .

In this study, we isolated and characterized a novel Myoviridae bacteriophage, Xoo-sp14, which infects X. oryzae pv. oryzae strain PXO99A. Xoo-sp14 was isolated from an agricultural soil (118°359E, 35°199N) in Shandong, east China, through the sample enrichment method (4). The soil sample was mixed in sterile water and shaken in order to release the phage particles. The bacteriophage was extracted through a 0.22-mm filter from the mixture. The presence of phage was then confirmed through a spot testing assay with double-layer plates, followed by three single plaque purification steps (4). For phage replication, a filtered medium was incubated with PXO99A in nutrient broth at 28°C with shaking at 220 rpm for 24 h.

Genomic DNA was extracted using a phenol-chloroform protocol (5) . The sequencing library of phage was prepared using the New England BioLabs (NEB) Ultra II kit v3. Whole-phage-genome sequencing was performed by Berry Genomics Biotechnology Co., Ltd. (Beijing, China), using Illumina HiSeq 2500 paired-end sequencing technology, with an average read length of 150 bp. The low-quality sequences and short reads were filtered by Trimmomatic software v0.36 (6) . SOAP de novo version 2.0 was used to assemble the sequences (7) . The complete genome of Xoo-sp14 was annotated by Rapid Annotation using Subsystem Technology (RAST; http://rast.nmpdr.org) (8) . All predicted open reading frames (ORFs) were manually double checked by searching against the nonredundant National Center for Biotechnology Information (NCBI) database by using PSI-BLAST (E value = 0.0001). A tRNA scanner (http://lowelab.ucsc.edu/ tRNAscan-SE/index.html) was used to predict tRNA genes (9) . After the high-throughput sequencing, we got 3,687,722 filtered paired-end reads. All the reads were assembled into a single contig, and the depth of coverage was approximately 2,234Â.

Xoo-sp14 had a linear double-stranded DNA (dsDNA) molecule of 232,104 bp, with a G1C content of 58%. A total of 251 ORFs and no tRNA were found in the genome of Xoo-sp14. A total of 13 genes are unknown, and 190 were annotated as hypothetical proteins. However, 48 ORFs were identified as corresponding to putative functional proteins, highlighting the novelty of the phage. Based on putative functions of the ORFs, the Xoo-sp14 genome was divided into nucleotide metabolism (kinase, dihydrofolate reductase, dCTP deaminase, thymidylate synthase, RNA polymerase beta subunit, and phosphatase), replication (DNA helicase and DNA polymerase), structural components (major capsid protein, major tail protein, minor tail protein, and large terminase subunit), and lysis (cell wall hydrolase, lysozyme, and protease) modules.

Detailed genomic analysis of Xoo-sp14 will contribute to future insights into the unique structural attributes of jumbo phages and their potential as biocontrol agents.

Default parameters were used for all software used in this study. Data availability. The complete genome sequence of Xoo-sp14 was submitted to GenBank under the accession number MT939492. The raw sequence reads were deposited to NCBI under the SRA accession number SRR12606159.

Novel insights into rice innate immunity against bacterial and fungal pathogens

The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice

The characteristics and genome analysis of vB_AviM_AVP, the first phage infecting Aerococcus viridans

Isolation and characterization of a novel phage Xoo-sp2 that infects Xanthomonas oryzae pv. oryzae

Molecular cloning: a laboratory manual

Trimmomatic: a flexible trimmer for Illumina sequence data

SOAPdenovo-Trans: de novo transcriptome assembly with short RNA-Seq reads

The RAST Server: rapid annotations using subsystems technology

The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs

This research was supported by a grant from The National Key Research and Development Program of China (2018YFA0903000), the National Natural Science Foundation of China (81672001), and the China MoST Emergency Project on COVID-19 (2020YFC0840800).We declare no conflicts of interest.