CULTURING OF CA. LIBERIBACTER ASIATICUS BY SELECTED GENE ADDITIONS AND FUNCTIONAL ANALYSES OF GENES USING AN L. CRESCENS MODEL.

Huanglongbing (HLB) is arguably the most destructive disease of citrus and both zebra chip and psyllid yellows have severe economic consequences for potato and tomato, respectively. Despite intense efforts, Ca. Liberibacter asiaticus (Las), the bacterium likely causing HLB and Ca. L. solanacearum (Lso), likely causing zebra chip potato and tomato diseases, have not been axenically cultured and neither have been proved to be causal agents of disease by completion of Koch's postulates; not one strain of these pathogenic species has even been consistently maintained by serial transfer, genetically manipulated or even reintroduced to their psyllid vectors. By contrast, Liberibacter crescens (Lcr) was readily cultured many years ago using standard bacterial techniques and Lcr is genetically tractable, yet not evidently pathogenic or parasitic to any host, plant or insect. The primary goal of Objective I is culturing of Las or Lso by adding multiple Lcr genes. The secondary goal is to demonstrate Koch's Postulates for the first time in plants and/or psyllids for either Las or Lso, using marked strains to follow the infected cells through the acquisition, systemic infection, propagation and transmission stages using confocal imaging.The goal of Objectives 2 and 3 is to add Las genes to Lcr and feed them to psyllids to understand the processes involved in psyllid acquisition of Liberibacters, beginning with Liberibacter entry into psyllid midgut cells, and possibly extending to elucidating the mechanisms of biofilm formation, disease lesions in the psyllid mid-gut and hemocoel entry.

In preliminary work, the GFP marker gene was chromosomally integrated into transformed Lcr BT-1 cells (Zhang et al. 2009; Naranjo et al. 2019). Las OmpA and five additional Las genes already demonstrated to interact with psyllid gut proteins will be amplified from genomic DNA of their respective Las- infected psyllids by PCR and directionally cloned together with an mCherry gene fused with the (strong) tryptophan promoter (demonstrated functional in Lcr into a colE1 vector. The PCR amplified fragments and mCherry will be subcloned into the expression vector pUFR071 (RepW; De Feyter and Gabriel, 1991) previously transformed into Lcr BT-1 (high frequency), and maintained stably in Lcr (Jain et al., 2019).The Lcr colonies are transparent and luminous initially, requiring two weeks for maturation. The BT-1 cells are highly amenable to co-transformation, or sequential transformation with combinations of compatible plasmids, with selective antibiotics. Las bacterial exconjugant colonies will be verified by mCherry fluorescence, and PCR amplification and DNA sequencing.In order to determine if the modified Lcr is capable of penetrating the ACP gut and/or SGs. Asian citrus psyllids, 4th instar nymphs will be fed a 0.2 microliter droplet of media containing Lcr cells expressing different combinations of Las genes. Psyllids will be analyzed for Lcr establishment/presence by light microscopy based on mCherry fluorescence using laser confocal microscopy. Quantitative real-time PCR (qPCR) amplication may also be used to detect Lcr.A subset of psyllids from the feeding/acquisition assays will transferred to one-year old Murraya paniculata cuttings maintained in otherwise insect-proof cages in a BSL-3P containment facility and reared to the adult stage. Teneral adults will be separated into head, midgut, and body to quantify Lcr colonization by qPCR.