# Unveiling Prophage Diversity and Host Interactions in Liberibacter: Genomic Insights for Phage Therapy Against Citrus Huanglongbing

## TL;DR

This study explores prophage diversity in Liberibacter bacteria to develop phage therapy for citrus Huanglongbing disease.

## Contribution

The discovery of 13 new prophage types and the identification of active Type 4 prophages for potential phage therapy.

## Key findings

- 191 prophages were identified across 48 Liberibacter genomes, including 13 new types.
- Active Type 4 prophages were found to be promising for phage therapy against CLas.
- Prophages impact host processes like gene expression and stress response.

## Abstract

Huanglongbing (HLB) is a destructive disease in citrus plants, putatively caused by the bacterium Candidatus Liberibacter asiaticus (CLas), which currently has no cure. In this study, we examined the genomes of 48 Liberibacter strains and identified 191 prophages, which are virus-like entities found within the bacterial genomes. These prophages were classified into 17 types, 13 of which were newly discovered. Our analysis showed that the number of prophages varied across genomes, and we identified six key biological processes impacted by these prophages, such as changes in gene expression and stress responses. Some prophages contained genes linked to virulence factors, potentially aiding in CLas’s global spread. Notably, 19 of the identified Type 4 prophages were active, suggesting their potential for developing phage therapy as a treatment for HLB. This research contributes valuable insights into the diversity of Liberibacter and how prophages interact with their host.

HLB is a severe and devastating disease affecting citrus plants, for which no cure is currently available. The pathogen CLas is an unculturable, phloem-limited α-proteobacterium associated with HLB. Phages, known for their ability to directly lyse pathogenic bacteria, have been widely recognized as effective control agents in both medical and agricultural fields. In this study, we identified 191 prophages across 48 Liberibacter genomes using comparative genomics and phylogenetic methods. These prophages were classified into 17 distinct types, with 13 novel types, named NF1 to NF13. Diversity analysis revealed that the number of prophages per genome ranged from one to eight, with an average of four. Annotation showed that the genome sizes of NF1–NF13 ranged from 10,160 bp to 72,736 bp, with an average size of 27,616 bp, containing between 7 and 66 genes (average: 27 genes). Further functional enrichment of the sequences within 5 kb upstream and downstream of the integration site identified six main aspects, involving four core biological processes: genome structure modification, gene expression regulation, stress response activation, and metabolic pathway modulation. It is speculated that the insertion of the prophage significantly impacts the host. Six virulence factors, including carB, clpC, flhA, algW, rffG, and IlpA, were identified in the prophages. Interestingly, prophages containing carB or clpC are predominantly found in CLas, which may contribute to the global colonization of CLas. Notably, among the 35 identified Type 4 prophages, 19 were found to be active. Type 4 is the only active prophage type, making it a promising candidate for developing phage therapy against CLas. This study is significant for a deeper understanding of the diversity of Liberibacter and the interactions between prophages and their hosts.