- CRISPR elements provide a new framework for the genealogy of the citrus canker. - citri strains of world-wide origin, a regulated pathogen causing Asiatic citrus canker in several regions of the world. - citri, which provides a new framework for the genealogy of the citrus canker pathogen.. - Conclusions: CRISPR-based typing will further improve the accuracy of the genetic identification of X. - Pathogenic mem- bers of the genus cause diseases on over 300 host plants [1]. - They cause a variety of symptoms, including necrosis, cankers, spots, and blight, and they affect different parts of the plant, in- cluding leaves, stems, and fruits [3]. - One of the most im- portant diseases caused by Xanthomonas is citrus canker, which results in significant yield losses on sus- ceptible citrus species [4, 5]. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - 1 IRD, Cirad, Université de Montpellier, IPME, Montpellier, France Full list of author information is available at the end of the article. - An accurate understanding of the phylogeny and evolution and proper identification of X.. - Notably, new spacers become almost always introduced at the same side of the locus close to the leader sequence. - Making use of the polymorphisms in the CRISPR locus, a typing method has been developed for mycobac- teria called “spoligotyping” (for spacer oligonucleotide typing) [33, 34]. - 1 Schematic representation of the X. - Genes of the cas gene cluster are schematically represented by green arrows. - It was noted that the CRISPR region of rice-pathogenic Xanthomonas oryzae evolves very rapidly and thus provides one of the most striking records of differentiation among bacterial isolates origin- ating from different geographic areas. - However, the first applications for plant-pathogenic bacteria were reported for Erwinia amylovora, the causal agent of fire blight, which can affect most members of the Rosaceae family [42, 43]. - Results from this work suggested that the two main groups are responsible for the worldwide expansion of the angular leaf spot dis- ease on strawberry plants.. - citri strains for the presence of the cas1 gene. - All 57 strains were then subjected to PCR amplification of the complete CRISPR locus, using leader- and terminator-specific primers. - As expected, PCR products were obtained for all of the X. - On the other hand, no DNA amplification occurred when using DNA of the X. - This result suggested that either NCPPB 3213 does not have a CRISPR/Cas system or that the leader and/or terminator sequences are too distant and do not allow annealing of the used PCR primer(s). - of spacers and repeats, with one contig harboring four to five repeats of the leader-proximal end (spacers Xcc_23 to Xcc_20) and another contig harboring 16 to 20 repeats of the terminator-proximal end (spacers Xcc_20 to Xcc_01) (Additional file 3: Figure S3, Additional file 4: Figure S4 and Additional file 5: Figure S5). - Notably, all spacer/repeat arrays were found at the ends of the contigs, suggesting that genome assembly was not complete due to the repeti- tive character of the sequence or due to other factors. - In- deed, scrutiny of the contig ends allowed to identify a short inverted repeat, as typically found at the extremities of an IS element. - Using the full-length ISRso19 element as a query, we found a single contig in the draft genome of NCPPB 3608 with 72% sequence identity, CCWG encompasing most of the IS element. - Sequencing of the amplicon from strain LG097 revealed the presence of spacers Xcc_23, Xcc_22, Xcc_20, Xcc_19 and Xcc_18 (except for 4 bp at the site of the IS element insertion) between the leader region and the IS element. - To amplify the opposite site of the IS element insertion in LG097, we performed a PCR with primers IS-2_fw and Terminator_rev. - Except for strain CFBP 2911, spacer Xcc_23 was likely the most recently ac- quired spacer, which is conserved in most of the 56 strains (except for LG117 and NCPPB 3615). - Most of the 25 spoligotype patterns likely evolved by the deletion of a single spacer/repeat unit although simultaneous de- letion of adjacent spacer/repeat units probably occurred as well, as suggested by the absence of intermediate CRISPR structures (Fig. - In order to decipher the origin of the 37 spacers, the NCBI GenBank was queried for similar sequences using the BLASTN algorithm. - citri, reflecting their high conservation in this patho- var of the species X. - Using stringent thresholds (E-value smaller than 0.1 and at least 90% coverage of the query sequence), we found significant matches between eight spacers and sequences from Xanthomonas-specific bacteriophages, which were however restricted to the 14 unique spacers of strain CFBP 2911 (Table 1. - All five bacteriophages belong to the order of Causovirales, with CP1 being a member of the Siphoviridae and the others being members of the Podoviridae. - Spacer Xcc_31 was also similar to a sequence in the genome of the Ralstonia-related blood disease bacter- ium R229 (GenBank accession number FR854082), which likely belongs to an integrated prophage and encodes a DNA polymerase A (GenBank accession number CCA83269.1) (Additional file 7: Table S1).. - White boxes indicate the absence of the corresponding spacer. - Spacers Xcc_22, Xcc_20 and Xcc_01 were similar to sequences in the X. - However, BLASTP search of the coding sequence revealed 80% sequence identity with protein I of the Xanthomonas campestris filamentous bacteriophage ΦLf (GenBank accession number AAB88261) [49]. - A simi- lar region with 74% sequence identity over the whole length is present in the genomes of the X. - The 25 spoligotypes of the 56 X.. - Comparison of the discriminatory power of CRISPR typing with other genotyping methods. - Presence of CRISPR loci in citrus-infecting xanthomonads In the present study, we analyzed 57 strains of X. - citri among citrus-infecting xanthomonads and our results demonstrate that the cas1 gene is a useful diagnostic marker for the presence or absence of the CRISPR/Cas system and could be used to differentiate citrus pathogens of the genus Xanthomonas.. - oryzae [40, 41], the CRISPR locus of X. - Consequently, the small size of the X. - This means that these strains only differ due to loss of one or more of the 23 spacers. - beginning of the twentieth century and to analyse their repertoire of spacers [52].. - CRISPR arrays represent a signature of the long history of interactions between bacteria and bacteriophages or other extrachromosomal genetic elements. - In addition to the hits in the CRISPR loci of completely sequenced X. - In addition, a sequence in the genomic contig of the Ralstonia-related blood disease bac- terium R229 (GenBank accession number FR854082) was related to spacer Xcc_31. - this sequence encodes a DNA- dependent DNA polymerase with homology to DNA poly- merases of the Xanthomonas-specific bacteriophages phiL7, OP1 and Xp10 [58–60]. - Possibly, the genomic sequence of the blood disease bacterium R229 corresponds to a pro- phage with similarity to Xanthomonas-specific bacterio- phages. - Xanthomonas bacteriophages f20-Xaj and f30-Xaj also matched with several spacers of the 14 unique spacers of strain CFBP 2911 (Additional file 7: Table S1).. - Only four of the 23 older spacers matched to se- quences in GenBank that did not correspond to the CRISPR arrays of X. - It was surpris- ing that none of the older and conserved 23 spacers matched to a sequence from a bacteriophage genome whereas all the observed hits of the CFBP 2911-specific spacers corresponded to sequences from bacteriophages that have been isolated over the last 50 years. - It is not clear whether this observation is merely due to sampling effects or if it reflects the fact that the sources for the 23 old spacers got extinct and only a few of the homolo- gous sequences were vertically inherited and thus pre- served in the form of prophages or remnants thereof.. - Multiple genetic events have contributed to the CRISPR array diversity within X. - Second, it is possible but unlikely as well that none of the 56 strains except for CFBP 2911 was challenged by alien DNA elements, such as bacteriophages or plasmids, since they had acquired spacer Xcc_23. - Given the important role of the Cas proteins for spacer acquisition in the CRISPR/Cas system, we com- pared the sequences of the cas gene cluster of strain CFBP 2911 with those of other strains. - cas8c genes of the majority of strains suffer from a frame-shift mutation due to a short tandem repeat of two base pairs (AG). - occurred in the repeat between spacers Xcc_20 and Xcc_21 (LB302, LB305, LG115 and NCPPB 3608,) and another insertion had occurred in spacer Xcc_18 (LG097) (Fig. - Our results thus further confirm an Indian origin of the A w strains from Florida, in agreement with outbreak in- vestigation and previously produced genotyping data . - In such cases, binary data of the spoligotype might be unable to pro- vide sufficient information to accurately establish geno- typic relationships among bacterial isolates. - Here, we took advantage of the availability of genome sequence data for 42 out of the 56 X. - Five of the seven pathotype A w strains have been se- quenced and allow as well their phylogenetic reconstruction [18]. - Nevertheless, we conclude that CRISPR elements provide a new and useful framework for the genealogy of the citrus canker pathogen X. - citri strains used in this study is representative of the worldwide genetic and. - Among them, we used the draft ge- nomes of 42 strains out of the 56 strains tested in this study to confirm our PCR amplification data (Add- itional file 10: Table S3) [18].. - oryzae), resulting in an amplicon of 221 bp, was de- signed and used to evaluate the presence of the CRISPR/. - PCR primers corresponding to the leader and terminator re- gions of the CRISPR locus were designed based on five. - In cases where we could not amplify and/or sequence the full-length CRISPR array, we designed PCR primers corresponding to internal regions of the CRISPR array. - Specifically, we designed two forward primers targeting spacers Xcc_21 and Xcc_19 and two re- verse primers targeting spacers Xcc_18 and Xcc_02, counting from the terminator of the CRISPR locus (Table 4. - Based on genome se- quence data, we designed specific primers corresponding to conserved regions of the IS element (Table 4). - Several pri- mer combinations were used to determine the position of the IS element and to elucidate the presence and order of CRISPR spacers, e.g., combinations Leader_fw and IS-1_. - of the CRISPR array. - the CRISPR array next to an IS element. - To compare the CRISPR loci of the 56 X.. - In order to produce a distance tree for 56 strains, a presence/absence matrix was produced based on the distribution of the polymorph- ism of fragment for AFLP and the distribution of CRISPR spacers for CRISPR typing, respectively.. - Structure of the CRISPR array of X. - Spacer Xcc_18*: 4 bp, indicated by dashes, are deleted due to the IS element insertion.. - PCR amplification of spacer/repeat units next to the IS element of five X. - Multiple sequence alignments of the seven cas gene open reading frames, which were retrieved from the corresponding GenBank files (Table 3).. - Primer design for PCR amplification of the CRISPR array from X. - A, amplification of the full-length CRISPR arrays using primers Leader_fw and Terminator_rev. - B, amplifica- tion to internal regions of the CRISPR arrays using spacer-specific primers.. - The host range of the genus Xanthomonas. - Geo- referenced spatiotemporal analysis of the urban citrus canker epidemic in Florida. - dieffenbachiae leads to a taxonomic revision of the X.. - The leucine-responsive regulatory protein (lrp) gene for characterization of the relationship among Xanthomonas species. - A MLVA genotyping scheme for global surveillance of the citrus pathogen Xanthomonas citri pv. - Highly polymorphic markers reveal the establishment of an invasive lineage of the citrus bacterial pathogen Xanthomonas citri pv. - A putative RNA- interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. - Genome sequence and rapid evolution of the rice pathogen Xanthomonas oryzae pv. - Genome-based population structure analysis of the strawberry plant pathogen Xanthomonas fragariae reveals two distinct groups that evolved independently before its species description. - Distribution of Xanthomonas citri strains in relation to the sensitivity to phages CP1 and CP2. - A comparative study of the strains of Xanthomonas campestris pv. - oryzae, changes its host range by duplication and deletion of the small domain in the deduced tail fiber gene. - Genomic characterization of the intron- containing T7-like phage phiL7 of Xanthomonas campestris. - Microevolution of the direct repeat region of. - Numerical index of the discriminatory ability of typing systems: an application of Simpson ’ s index of diversity
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