Whole Genome Sequencing And Analysis Of "C Andidatus Liberibacter Asiaticus",the Associated Pathogen Of Citrus Huanglongbing

Citrus Huanglongbing?HLB?,one of the most destructive citrus diseases,caused the large economic losses in citrus industry worldwide.HLB was caused by the phloem-limited fastidious?-Proteobacteriacea Gram-negative bacteria,"Candidatus Liberibacter spp.",including three species,"Ca.L.asiaticus"?CLas?,"Ca.L.africanus"?CLaf?and"Ca.L.americanus"?CLam?.Of three species,"Ca.L.asiaticus"is most widely distributed in the world.Due to the current uncultivable of"Ca.L.asiaticus",the traditional research methods,e.g.morphology,biology,and etiology,were difficult to conduct.With the development of DNA sequencing technology,genomic analysis became an important and useful methods on microbial research.Especially on the current status that most microorganisms cannot be cultured,the genome sequencing can provide the possibility to get insight into the gene function of uncultured microorganisms.In this study,the genomic sequencing of"Ca.L.asiaticus"strain A4?Guangdong,China?,"Ca.L.asiaticus"strain HHCA?California,USA?and FL17?Florida,USA?were conducted by a combination protocol,named Enrichment-Enlarge-Next generation sequencing?EEN?.The variation of prophage sequence in the CLas genome was analyzed by whole genome sequence comparison.The population diversity and genetic variation of CLas strains collected from different geographical region in southern China were analyzed based on the prophage types.The CRISPR?clustered regularly interspaced short palindromic repeats?/cas?CRISPR associated protein genes?system was also identified and used for analysis of the relationship between different CLas strains and phages.In addition,a five-copy genes wereidentified in the CLas genome and used for design primers to increase the sensitivity of"Ca.L.asiaticus"detection.The main conclusion of this study were as follows,1.The EEN method was raised and first used in this study to enhance the proportion of CLas DNA in total plant DNA.The high quality of CLas strain A4 genome was obtained.A total of 3.28×10⁷ reads were obtained from the Illumina Miseq sequencing platform.636,810 reads were extracted as the CLas-reads by using Psy62 strain,SC1 phage and SC2phage sequence as references.Assembly was performed by both Velvet 1.2.10 and CLC genomic workbench 7.5.The CLas strain A4 genome consisted of 1,233,514 bp with the average GC content of 36.4%.Sequence comparison revealed the"Ca.L.asiaticus"strain A4 only harbored the Type 2?SC2-like?prophage?named CGdP2?and didn't harbor the Type 1?SC1-like?prophage.Annotation result showed the prophage CGdP2 have 43 genes,and only 24 genes showed highly similarity?>99%?with SC2 phage sequence,FP2prophage sequence and gxpsy genome.Comparing with Psy62 genome,three main large insertions?1,890 bp,2,542 bp and 2,107 bp?were identified in A4 genome.The 1,890 bp insertion was newly confirmed in A4 genome,and not found on Psy62 strain,gxpsy strain and Ishi-1 strain.Two putative protein genes were identified on the 1,890 bp insertion.2.The EEN method was used to sequence the HLB samples collected from California?HHCA?and Florida?FL17?.The HHCA sample was the first reported HLB in California.Sequence analysis revealed the CLas strain HHCA only harbored the Type 2prophage,while the CLasstrain FL17 only harbored the Type 1 prophage.Whole genome sequence comparison among all published CLas genomes revealed the chromosomal region of CLaswas conserved,while the prophage region was highly variable.The CLasstrains from different geographical location had different types of prophages.However,a combination analysis?including genome analysis,reads mapping and specific loci?revealed two current"Ca.L.asiaticus"Californian strains,HHCA and SGCA5,harbored different prophages.3.The prophage type-specific primers were designed based on specific region by comparing two types of prophage sequences.A total of 566 CLas strains collected from southern China were used for prophage type analysis by type-specific primers PCR.Based on the prophage types,the diversity of CLas population and the genetic variation of CLaswere analyzed.The result revealed that more than 90%of total CLasstrains only harbored a single prophage,6.36%of CLasstrains harbored both two prophages,and 3.18%of CLas strains didn't harbor any of two prophages.The genetic structure analysis revealed three major groups of nine CLaspopulations in southern China.The CLaspopulations from Guangdong,Fujian,Hunan,Jiangxi and Zhejiang were clustered in one?group I?with a relatively low genetic distance?<0.03?.Further,CLaspopulations from two neighbouring provinces,the Guangxi and Guizhou province,were clustered together?group II?with the genetic distance of 0.0114.The Group III including CLaspopulation from two non-adjacent provinces,Hainan and Yunnan province,with a genetic distance of 0.1366.In addition,two specific loci of Type 2 prophage,T2-6 and T2-8 locus,showed highly variable among different Type 2 CLas strains.4.A CRISPR array was identified in A4 genome and a Cas4 protein with a relatively conserved domain was also identified in the adjacent region of CRISPR array.In addition,several genes adjacent to CRSIPR array were found to be associated with DNA/RNA processing function motifs,indicating the CLas CRISPR/cas system possesses all key components to be fully function.Sequence comparison of CRISPR array among different CLasstains revealed that different prophage type strains can have different spacer sequence.Considering of the predominant of single prophage in CLaspopulation in southern China,it could be interpreted as the two prophages were in competition in a CLas cell.It also can be hypothesized that one pre-established"Ca.L.asiaticus"prophage in a CLas cell could use its CRISPR/cas system to defeat the invasion of the other phage DNA.5.Sequence analysis revealed a total of tenrepeat loci in A4 genome.Five out of ten loci contained a common of 390-bp sequence.Annotation result showed five copies sequence harbored a same gene,ribonucleotide reductase?-subunit gene?nrdB?.Three nrdBgenes were in long form?nrdB^L,1,059 bp?and others two were in short form?nrdB^S,378 bp?.The nrdB^L encoded all active sites of the enzyme while nrdB^S didn't contain any active site.The nrdB^S,shared>99%identity to 3'end of nrdB^L.The common sequence shared by fivenrdB genes were conserved among different"Ca.L.asiaticus"strains.Phylogenetic analyses based on nrdB nucleotide and amino acid sequences showed a distinct monophyletic lineage of CLas,similar as based on 16S rRNA gene sequences.A nrdB-based primer set RNRf/RNRr was designed and evaluated in real-time PCR against262 HLB samples collected from China and U.S.A.Compared to the current standard primer set HLBas/HLBr derived from the 16S rDNA sequence,RNRf/RNRr had tripling the detection sensitivity.In addition,RNRf/RNRr was more than twice the stability of primer set LJ900f/LJ900r derived from multi-copy prophage.