A Type 3 prophage of “Candidatus Liberibacter asiaticus” carrying a restriction-modification system

Authors: ZHENG, Z - South China Agricultural University; BAO, M - South China Agricultural University; WU, F - South China Agricultural University; Van Horn, Christopher; Chen, Jianchi; DENG, X - South China Agricultural University

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/29/2017
Publication Date: 3/15/2018
Citation: Zheng, Z., Bao, M., Wu, F., Van Horn, C.R., Chen, J., Deng, X. 2018. A Type 3 prophage of “Candidatus Liberibacter asiaticus” carrying a restriction-modification system. Phytopathology. 108(4):454-461. doi:10.1094/PHYTO-08-17-0282-R.
DOI: https://doi.org/10.1094/PHYTO-08-17-0282-R

Interpretive Summary: “Candidatus Liberibacter asiaticus” is a non-culturable bacterial pathogen of citrus Huanglongbing (HLB, yellow shoot disease, also known as citrus greening disease), a highly destructive disease currently threatening the citrus industry around the world. Recent studies have shown that the HLB pathogen has two phages or bacterial viruses. The phages could destroy the HLB pathogen but also could bring in genes to enhance virulence of the pathogen. Therefore, phages are important issues in HLB research. This study was to search for new phages of HLB pathogen through a next generation sequencing (NGS) approach and analyses of a large collection of HLB pathogen samples. A total of 523 HLB DNA samples collected from southern China during 2013 and 2016 were studied. A new phage, called P-JXGC-3, was found and analyzed. The phage carried a unique bacterial defense system, called restriction-modification system, which could be used to enhance the pathogen survival under field condition. The new phage information facilitates our understanding of phage biology of the HLB pathogen that will help to formulate new HLB management strategies.

Technical Abstract: Prophages, the lysogenic form of bacterial phages, are important genetic entities of “Candidatus Liberibacter asiaticus” (CLas), a non-culturable alfa-proteobacterium associated with citrus Huanglongbing (HLB). Two different CLas prophages are currently known, SC1 (Type 1) that has a lytic cycle and SC2 (Type 2) associated with bacterial virulence. To explore the prophage repertoire, 523 DNA samples isolated from leaf petioles of CLas infected citrus were collected from southern China and surveyed for prophage status using Type-specific PCR. Eighteen samples were found lacking both Type 1 and Type 2 prophages. One sample, JXGC, was selected for sequencing using Illumina HiSeq format that generated >100M short sequence reads (150 bp per read). Read mapping experiments showed sequence coverage of 46% to SC1 and 50% to SC2. BLAST search using SC1 and SC2 as queries identified three circular contigs (31,449 bp) representing new Type 3 prophageP-JXGC-3. The integration site pf P-JXGC-3 occurred within a helicase gene, resulting in a gene duplication in the CLas chromosome. P-JXGC-3 had 36 open reading frames (ORFs), ten were unique to SC1 and SC2, including a restriction-modification (R-M) system (hsdR, hsdS, hsdM1 and hsdM2) predicted for CLas cell defense against foreign DNA invasion. Typed by prophage specific PCR, the CLas population in southern China contained all combinations of the three prophage types, with the exception of Type 2-Type 3, suggesting active prophage/phage interactions involving lytic Type 1 prophage and lysogenic Type 2 and Type 3 prophages.