The objective of this research will 1) characterize Pr-D (FP3) and its role and disease suppression; 2) investigate the dynamics of the prophages/phages in Las bacteria by revealing the variations in gene expression and recombination; and 3) identify critical elements, such as heat and chemical stress that facilitates lytic activities of the prophages. In addition, we will demonstrate whether or not if the ‘cross protection’ using mild strains of Las bacteria will work for the HLB pathosystem along with quantitative detection protocols for prophage-based strain differentiation. We harvested various Las-infected citrus and periwinkle samples showing symptoms ranging from mild to severe, and used for isolation and enrichment of prophage/phage apart from plant and bacterial host materials by differential centrifugation, PEG precipitation, and CsCl density fractionation. Absolute and relative amounts of prophage/phage and Las bacteria existed in total DNA and in various fractions have been evaluated by using specific LJ900 primers targeting the repeat sequence in the LasAI gene located within a prophage region of the Las genome and 16S rDNA primers for detecting Las genomic DNA. We developed a protocol that could isolate and enrich lytic phages from fresh plant tissues. The Las phages were enriched in certain fractions of the purification process, particularly in PEG precipitated pellet and certain ClCs fractions, consistent with the biochemical and biophysical property of free phages. For further confirmation, these fractions were directly examined by electron microscopy, and we were able to find some particles consisting of head-tail fiber structure typical of small bacteriophages, with diameter of the heads varies from 50-70 nm and the length and the width of the tails varies from 140-190 nm and 10-20 nm respectively. We have establish a digital PCR (dPCR) system for accurate quantification of HLB and Las prophage/phage. After optimization, we are able to detect as low as 1-2 copy numbers of targeted DNA molecule/.L sample. To further improve the quantification accuracy of absolute and relative amounts of phages and the bacterium, we designed new sets of primers and probes targeting only single copy genes. Based on Las genome analysis, we targeted gene 05560 in the prophage region, .-operon gene in the Las genome, and a CitLGT gene (limonoid UDP-glucosyltransferase-like) in the citrus genome as an internal measurement reference. The specificity and sensitivity of the new primers is currently being tested and we are optimizing an dPCR-based assay for accurate measurement of lytic phage activities in Las-infected materials. Duan Lab to detect bacterial transcripts in mixed eukaryotic/ prokaryotic samples at set time points throughout a typical course of thermotherapy treatment. Overall, the analysis revealed that, depending upon the time at which the samples was taken, between 4% and 9% of the total predicted genes for Las appear to be differentially regulated during the thermotherapy process compared to a sample taken at time zero. These genes provide initial evidence of how the bacteria itself is modifying its transcriptional activity in response to the increase in temperature. Although a majority of the regulated genes found are defined as hypothetical, several do have a predicted function and their contributions to the effects of heat therapy are now under investigation. Their regulation has now been confirmed and transcripts are being quantified via real-time PCR. Additional plants have also been stressed with heat for verification purposes and to ensure the accuracy of the RNA-seq results. Identification of these genes is leading the way towards deciphering the molecular mechanisms behind thermotherapy in an effort to find alternative methods of achieving the same reduction in Las titer as seen with thermotherapy that will work in the roots as well as the foliage. Construction of a transcriptional reporter system is also currently in progress for the final verification of the genes identified as being involved in stress response to heat in plants subjected to thermotherapy. This system will also allow future experimentation to rapidly identify other catalysts that can produce the same reduction in bacterial numbers as thermotherapy. Purification of the ~10Kb FP3 region has been achieved from both periwinkle and citrus, though the amount purified from citrus appears to be less than that from periwinkle (as would be expected from the lower bacterial titer found in citrus). This should allow the region to be sequenced in its entirety and comparisons made to help characterize the role of FP3 found in citrus vs periwinkle.