Abstract
BackgroundXanthomonas citri, a causal agent of citrus canker, has been a well-studied model system due to recent availability of whole genome sequences of multiple strains from different geographical regions. Major limitations in our understanding of the evolution of pathogenicity factors in X. citri strains sequenced by short-read sequencing methods have been tracking plasmid reshuffling among strains due to inability to accurately assign reads to plasmids, and analyzing repeat regions among strains. X. citri harbors major pathogenicity determinants, including variable DNA-binding repeat region containing Transcription Activator-like Effectors (TALEs) on plasmids. The long-read sequencing method, PacBio, has allowed the ability to obtain complete and accurate sequences of TALEs in xanthomonads. We recently sequenced Xanthomonas citri str. Xc-03-1638-1-1, a copper tolerant A group strain isolated from grapefruit in 2003 from Argentina using PacBio RS II chemistry. We analyzed plasmid profiles, copy number and location of TALEs in complete genome sequences of X. citri strains.ResultsWe utilized the power of long reads obtained by PacBio sequencing to enable assembly of a complete genome sequence of strain Xc-03-1638-1-1, including sequences of two plasmids, 249 kb (plasmid harboring copper resistance genes) and 99 kb (pathogenicity plasmid containing TALEs). The pathogenicity plasmid in this strain is a hybrid plasmid containing four TALEs. Due to the intriguing nature of this pathogenicity plasmid with Tn3-like transposon association, repetitive elements and multiple putative sites for origins of replication, we might expect alternative structures of this plasmid in nature, illustrating the strong adaptive potential of X. citri strains. Analysis of the pathogenicity plasmid among completely sequenced X. citri strains, coupled with Southern hybridization of the pathogenicity plasmids, revealed clues to rearrangements of plasmids and resulting reshuffling of TALEs among strains.ConclusionsWe demonstrate in this study the importance of long-read sequencing for obtaining intact sequences of TALEs and plasmids, as well as for identifying rearrangement events including plasmid reshuffling. Rearrangement events, such as the hybrid plasmid in this case, could be a frequent phenomenon in the evolution of X. citri strains, although so far it is undetected due to the inability to obtain complete plasmid sequences with short-read sequencing methods.
Highlights
Xanthomonas citri, a causal agent of citrus canker, has been a well-studied model system due to recent availability of whole genome sequences of multiple strains from different geographical regions
We demonstrate in this study the importance of long-read sequencing for obtaining intact sequences of Transcription Activator-Like Effectors (TALE) and plasmids, as well as for identifying rearrangement events including plasmid reshuffling
Plasmid profile coupled with Southern hybridization indicates the presence of two larger size plasmids in Xc03-1638-1-1 compared to reference Xc-A306 strain The plasmid profile of Xc-03-1638-1-1 was compared with other X. citri strains, XcA2090, XcA1660, XcAEtrog, XcA100 and XcA109 including Xc-A306 (Table 1), a complete reference genome used in many studies (Fig. 1a)
Summary
Xanthomonas citri, a causal agent of citrus canker, has been a well-studied model system due to recent availability of whole genome sequences of multiple strains from different geographical regions. X. citri harbors major pathogenicity determinants, including variable DNA-binding repeat region containing Transcription Activator-like Effectors (TALEs) on plasmids. Xanthomonas citri, the causal agent of citrus canker, represents a well-studied pathosystem due to the detailed understanding of pathogen biology using a combination of approaches including mutational analysis and, more recently, by sequencing hundreds of strains of this pathogen collected around the world. Recent structural studies have determined that the number of repeats and the repeat variable di-residues (RVDs) (12th and 13th amino acid) can be used for predicting the target nucleotide sequence that TALEs bind [3, 4]. The correct determination of the repeat regions of TALEs is important for predicting target sites within the host as well as applications as a tool in genome editing
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