ABSTRACT
“
Candidatus
Liberibacter asiaticus” is an uncultured alphaproteobacterium that systemically colonizes its insect host both inter- and intracellularly and also causes a severe, crop-destroying disease of citrus called huanglongbing, or citrus “greening.”
In planta
, “
Ca
. Liberibacter asiaticus” is also systemic but phloem limited. “
Ca
. Liberibacter asiaticus” strain UF506 carries two predicted prophages, SC1 and SC2. Bacteriophage particles have been observed in experimentally “
Ca
. Liberibacter asiaticus”-infected periwinkle but not in any other host. Comparative gene expression analysis of predicted SC1 late genes showed a much higher level of late gene expression, including holin transcripts (SC1_gp110), in “
Ca
. Liberibacter asiaticus”-infected periwinkle relative to “
Ca
. Liberibacter asiaticus”-infected citrus. To functionally characterize predicted holin and endolysin activity, SC1_gp110 and two predicted endolysins, one within SC1 (SC1_gp035) and another well outside the predicted prophage region (CLIBASIA_04790), were cloned and expressed in
Escherichia coli
. Both SC1 genes inhibited bacterial growth consistent with holin and endolysin function. The holin (SC1_gp110) promoter region was fused with a
uidA
reporter on pUFR071, a wide bacterial host range (repW) replicon, and used to transform
Liberibacter crescens
strain BT-1 by electroporation. BT-1 is the only liberibacter strain cultured to date and was used as a proxy for “
Ca
. Liberibacter asiaticus.” pUFR071 was >95% stable without selection in BT-1 for over 20 generations. The reporter construct exhibited strong constitutive glucuronidase (GUS) activity in culture-grown BT-1 cells. However, GUS reporter activity in BT-1 was suppressed in a dose-dependent manner by crude aqueous extracts from psyllids. Taken together with plant expression data, these observations indicate that “
Ca
. Liberibacter asiaticus” prophage activation may limit “
Ca
. Liberibacter asiaticus” host range and culturability.