Publications

4381

ABSTRACT “ Candidatus Liberibacter asiaticus” is the causative bacterium associated with citrus greening disease. “ Ca . Liberibacter asiaticus” is transmitted by Diaphorina citri more efficiently when it is acquired by nymphs rather than adults. Why this occurs is not known. We compared midguts of D. citri insects reared on healthy or “ Ca . Liberibacter asiaticus”-infected citrus trees using quantitative PCR, confocal microscopy, and mitochondrial superoxide staining for evidence of oxidative stress. Consistent with its classification as propagative, “ Ca . Liberibacter asiaticus” titers were higher in adults than in nymphs. Our previous work showed that adult D. citri insects have basal levels of karyorrhexis (fragmentation of the nucleus) in midgut epithelial cells, which is increased in severity and frequency in response to “ Ca . Liberibacter asiaticus.” Here, we show that nymphs exhibit lower levels of early-stage karyorrhexis than adults and are refractory to the induction of advanced karyorrhexis by “ Ca . Liberibacter asiaticus” in the midgut epithelium. MitoSox Red staining showed that guts of infected adults, particularly males, experienced oxidative stress in response to “ Ca . Liberibacter asiaticus.” A positive correlation between the titers of “ Ca . Liberibacter asiaticus” and the Wolbachia endosymbiont was observed in adult and nymph midguts, suggesting an interplay between these bacteria during development. We hypothesize that the resistance of the nymph midgut to late-stage karyorrhexis through as yet unknown molecular mechanisms benefits “ Ca . Liberibacter asiaticus” for efficient invasion of midgut epithelial cells, which may be a factor explaining the developmental dependency of “ Ca . Liberibacter asiaticus” acquisition by the vector.

Prophages, the lysogenic form of bacterial phages, are important genetic entities of ‘Candidatus Liberibacter asiaticus’ (CLas), a nonculturable α-proteobacterium associated with citrus Huanglongbing. Two CLas prophages have been described, SC1 (NC_019549.1, Type 1) and SC2 (NC_019550.1, Type 2), which involve the lytic cycle and the lysogenic cycle, respectively. To explore the prophage repertoire, 523 CLas DNA samples extracted from leaf petioles of CLas-infected citrus were collected from southern China and surveyed for Type 1 and Type 2 prophages by specific PCR. Eighteen samples were found lacking both prophages. One sample, JXGC, sequenced using Illumina HiSeq, generated >100 million short sequence reads (150 bp per read). Read mapping to known prophage sequences showed a sequence coverage of 46% to SC1 and 50% to SC2. BLAST search using SC1 and SC2 as queries identified three contigs from the JXGC de novo assembly that form a circular P-JXGC-3 (31,449 bp), designated as a new Type 3 prophage. Chromosomal integration of P-JXGC-3 was detected to occur within a helicase gene, resulting in a duplication of this gene. P-JXGC-3 had 36 open reading frames (ORFs), 10 of which were not found in Type 1 or Type 2 prophages, including four genes that encoded a restriction-modification (R-M) system (hsdR, hsdS, hsdM1, and hsdM2). Typed by prophage-specific PCR, the CLas strains in southern China contained all combinations of the three prophage types with the exception of a Type 2−Type 3 combination, suggesting active ongoing prophage−phage interactions. Based on gene annotation, P-JXGC-3 is not capable of reproduction via the lytic cycle. The R-M system was speculated to play a role against Type 1 prophage−phage invasion.