Characterization and manipulation of the prophages/phages of ‘Candidatus Liberibacter asiaticus’ for the control of citrus huanglongbing

Characterization and manipulation of the prophages/phages of 'Candidatus Liberibacter asiaticus' for the control of citrus huanglongbing

Report Date: 01/17/2017
Project: 803   Year: 2016
Category: CLas Bacteria
Author: Yongping Duan
Sponsor: Citrus Research and Development Foundation

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 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. In order to define the mechanisms that phage are employing to overcome abiotic stress, we designed and optimized specific primer sets for quantitative reverse transcription PCR (qRT-PCR) for genes within the phage region that were likely regulated by heat treatment and other stress. Because this analyses are based on mRNA transcript level and not on genomic DNA, the upregulation of phage genes reflects the relative level of transcript of active living cells. In order to ensure adequate generation of cDNA from Las, we found it necessary to use individual primers specific for the targeted region instead of generalized primers such as random hexamers. The cDNA that was generated in this fashion was then used as the template for qRT-PCR. The present analysis included three biological samples for each condition and three technical replicates for each sample for statistical purposes. Particular genes that were found to be upregulated included: CLIBASIA_5590 encoding an unknown protein, CLIBASIA_5610 encoding a putative phage terminase (large subunit), CLIBASIA_5665 encoding an unknown protein, CLIBASIA_5390, which has the conserved sodium: dicarboxilate symporter family domain, CLIBASIA_5525 encoding a guanylate kinase that catalyzes the reaction ATP + GMP <->ADP + GDP. Phage genes found to be downregulated included: CLIBASIA_00005 encoding an unknown protein, CLIBASIA_00010, which has an NTPase domain of typical DNA-packaging enzyme, CLIBASIA_00030 encoding a putative DNA polymerase of bacteriophage origin, CLIBASIA_5565, which has the conserved domain TolA protein and is thought to be required for the translocation of the phage DNA. This data correlated well with what was seen via our previous RNA-Seq analysis and helps reveal the transcriptional response of the phage to abiotic stress factors. Given that previous studies on thermotherapy showed an overall reduction in Las titer in citrus affected by HLB post heat treatment, harnessing the ability to control these particular genes may allow us to lower the bacterium�s ability to handle stress. Based on the variations of Las prophages/phages, we recognized certain molecular mechanisms behind the symptom variations and their association with “mild strains” of Las bacteria and host tolerance/resistance. The titration dynamics between 16S DNA-based and phage gene-based results revealed the association of host tolerance with the dynamics. 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 thermo-therapy. To investigate the effects of stress on the genes involved in the phage lytic cycle, we identified several phage genes that were over-expressed in citrus plant after heat treatment. These results indicated that thermotherapy has a direct effect on Las bacteria by actively regulating specific phage genes. To further evaluate genes related to stages of the phage lytic cycle, we further compared Las genes expression profile from two distinct insect vectors, psyllids, and mealybugs. Mealybugs were found to contain much higher titers of Type D when compared with psyllids, indicating that the phage may be more active in the mealybugs than in the psyllids. Using an enrichment method to acquire RNAseq data, we compared the Las transcriptomes between the two insect vectors, and revealed that psyllids samples contained more than four times reads of the Las 16s rRNA than those of mealybugs samples, indicating much higher bacterial titers in psyllids than in mealybugs. However, mealybug�s transcriptome profiling showed much higher expression level of prophage genes than those in psyllids, where expression level of prophage genes was completely absent or extremely low. Interestingly, more than 2/3 of the highly expressed genes in mealybugs were identified as prophage/phage genes. Interestingly, eight genes with the highest level of expression in mealybugs were identified as the highly expressed ones in Las-infected citrus after heat treatment. These results indicates that both stresses caused by thermotherapy and in mealybugs environment triggered similar signaling pathways, and result in the expression of prophage genes that may induce lytic cycle and eventually reduce Las titer in citrus plant treated with heat stress, and maintain low titer in mealybugs.

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