Exploiting the Las and Lam phage for potential control of HLB (Year 2)

Exploiting the Las and Lam phage for potential control of HLB (Year 2)

Report Date: 08/15/2015
Project: 723   Year: 2015
Category: CLas Bacteria
Author: Dean Gabriel
Sponsor: Citrus Research and Development Foundation

One of the primary goals of this work is to identify a small molecule treatment that can be used to activate the phage lytic cycle genes encoded by Las prophage, thus bringing about the death of Las bacteria carrying these prophage. The majority of Candidatus Liberibacter asiaticus (Las) strains carry bacteriophage similar to SC1 and SC2 of Las UF506. Lytic phage particles are formed and have been reported in both periwinkles and in citrus, but not in infected psyllids. We reported that relative mRNA expression levels of prophage late genes SC2-gp095 (“peroxidase”) and SC1-gp110 (�holin�) were much higher in periwinkle than in citrus. We now report that these genes were silent in psyllids. We also reported that both the prophage holin (SC1_gp110) and endolysin (SC1_gp035) were functional, and that strong expression of the holin gene alone in Las cells would be sufficient to kill the cells, whether or not phage particles were formed. This could be part of the explanation as to why Las has to date not been cultured (Fleites et al. 2014). The Las holin promoter was strongly active in Liberibacter crescens (Lcr), a culturable proxy for Las, but nearly silent in E. coli. Further, activity of the holin promoter in Lcr was suppressed by aqueous extracts from psyllids applied outside of the Liberibacter cells, indicating cell penetration. The suppressor activity was sensitive to heat and proteinase treatment, indicating direct repression by a protein, and size fractionation demonstrated the the size was 10-50 kDa. Electrophoretic mobility shift assays (EMSAs) were used to demonstrate specific binding of a protein to the Las holin promoter, and small, overlapping holin promoter fragments were used as competitor DNAs, to further delineate the target binding sites of the protein. The DNA-binding protein was purified by DNA affinity capture, and MALDI-TOF analyses are underway to identify the protein. We also reported that the peroxidase on SC2 is a reactive oxygen species (ROS) scavenger and confirmed that it has peroxidase activity. SC2-gp095 was cloned in a shuttle vector and transformed into L. crescens (Lcr). Transformed Lcr cells showed 20-25% enhanced resistance to hydrogen peroxide on agar plates, 47% higher enzymatic activity and enhanced growth in liquid cultures. A non-classical secretion potential was confirmed by enzymatic and Western blot analyses in Lcr. Transient expression of SC2_gp095 in planta resulted in strong transcriptional down-regulation of RbohB, the key gatekeeper of the H2O2 mediated defense signaling in plants, helping explain the surprisingly long incubation period (years) before HLB symptoms appear in Las-infected citrus. Las peroxidase is therefore a secreted, horizontally acquired effector that suppresses host symptom development, a tactic used by most biotrophic plant pathogens. In psyllids, stringent control of all lytic cycle genes, including SC1 excision and replication (early genes) as well as the lysis genes and the peroxidase (late genes), appears to be critical to prevent Las from being detected by the insect innate immune system. Upon examination of SC1 and SC2 promoter regions, we found strong matches to a predicted Las chromosomally encoded LexA-like repressor, The lytic cycles of many phages are under the control of the bacterial SOS (suicide program) response. In the stable “lysogenic” or “prophage” state, the phage is kept dormant by suppression of early gene activation, often by using a phage C1-like repressor. In Las, the C1-like repressor is chromosomally encoded and has a LexA domain. Externally applied chemicals, as well as heat shocks have been used in other bacterial systems to activate the SOS response and derepress various phage lytic cycle genes by disabling LexA. We have recently developed indirect evidence that Las LexA may repress SC1 early gene activity, making this a potential new molecular target for HLB control.


Your browser does not support pdfs, click here to download the file.