Dynamic integration and excision of filamentous phage XacF1 in Xanthomonas citri pv. citri, the causative agent of citrus canker disease

Abstract

Inovirus XacF1 (7325 nucleotides) is integrated into the genome of Xanthomonas citri pv. citri (Xcc) strains at the host dif site (attB) by the host XerC/D recombination system. The XacF1 attP sequence is located within the coding region of ORF12, a possible phage regulator. After integration, this open reading frame (ORF) is split into two pieces on the host genome. We examined dynamic integration/excision of XacF1 in Xcc strain MAFF 301080 and found that the integration started at 4 h postinfection (p.i.) and peaked at 12 h p.i. Thereafter, the ratio of integrated to free forms remained constant, suggesting equilibrium of integration and excision of XacF1 in the host genome. However, the integrated state became very unstable following a 5'-deletion of ORF12 in XacF1, suggesting that ORF12 plays a key role in the integration cycle of XacF1 in Xcc strains.

Keywords: XerC/D; biocontrol; citrus canker; filamentous phage; integration mechanism.

Figure 1

Detection of integrated forms of XacF1 after infection in Xcc MAFF 301080 as a host. Cells were infected with wild‐type XacF1 (A) or with a ∆XacF1′ mutant that lacked a 5′ major portion of ORF12, but retained attP (B). Arrows indicate bands corresponding to integrated forms. Lanes: M, molecular marker (lambda‐StyI fragments); 1, 2 h p.i.; 2, 4 h p.i.; 3, 6 h p.i.; 4, 12 h p.i.; 5, 24 h p.i.; 6, 48 h p.i.; 7, 72 h p.i.

Figure 2

Stability of integrated forms of XacF1 in Xcc MAFF 301080 cells. (A) A single colony containing a XacF1 prophage was picked and cultivated in NB for 48 h at 28 °C. The cells were then spread onto NA plates, and colonies were formed after three days. Ten single colonies were picked and subjected to PCR for the detection of XacF1‐integrated forms. (B) The same experiments were performed with ∆XacF1′‐integrated cells. Lanes: M, molecular size marker (100‐bp ladder), 1–10, independent single colonies. For comparison, 16S rDNA fragments were also amplified in the same way (Materials and methods). There are two identical rRNA operons in the Xcc genome 18.

Figure 3

Twitching motility of XacF1‐infected bacterial cells. Two microlitre of the cell suspension (Xcc MAFF 301080) was spotted on MM plates. Plates were incubated at 28 °C, and the morphology of the colony edge was observed under a culture microscope (4–10× magnification). (A) Uninfected cells, (B) XacF1‐infected cells, and (C) ∆XacF1′‐infected cells. Bar represents 0.5 mm.

Figure 4

Canker symptoms developed on lemon leaves 1 week p.i by the needle‐pricking method. Leaves were inoculated with uninfected cells (A), with XacF1‐infected cells (B), and with ∆XacF1′‐infected cells (C). Each leaf was inoculated with cells of 10 independent single colonies.