Pacbio sequencing of copper-tolerant Xanthomonas citri reveals presence of a chimeric plasmid structure and provides insights into reassortment and shuffling of transcription activator-like effectors among X. citri strains

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.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4408-9) contains supplementary material, which is available to authorized users.

Description of copper tolerantXanthomonas citrisubsp.citriand genotypic comparison with sensitive and resistant strains

“… Size defined based on molecular marker and prior sequencing of plasmids (Silva et al ., ; Gochez et al ., ). …”

“…Cu R or Cu S . In addition, Cu S strains have been mistakenly classified as Cu R (Teixeira et al ., ; Hsiao et al ., ), and Cu R strains have been referred to as Cu T (Argüello et al ., ; Gochez et al ., ). One explanation for this misinformation may be that, in contrast to resistance, which may be assessed by the presence of specific cop genes and bacterial growth on medium containing high copper concentrations, tolerance is more difficult to be characterized due to the lack of a strong or evident genetic marker, as discussed by Brauner et al .…”

PthA4AT, a 7.5‐repeats transcription activator‐like (TAL) effector fromXanthomonas citrissp.citri, triggers citrus canker resistance

“…Previous reports have shown a great variability in the size of the pthA genes from different X. citri strains (Al‐Saadi et al ., ; Gochez et al ., ; Lee et al ., ; Ye et al ., ). Given the mode of action of these TALE proteins, it is expected that this variability will contribute to explain differences in pathogenicity among the strains.…”

“…It would be interesting to isolate and sequence those genes, and test if they also can confer avirulence on citrus hosts. Recently, Pacbio sequencing of X. citri strains also revealed a 6.5‐repeat pthA1 variant in the LM180 strain, obtained from infected grapefruit samples (Gochez et al ., ), but the biological relevance of this TALE gene is unknown. The discovery of a TALE conferring canker resistant in C. limon and C. sinensis paves the way to identify citrus R genes involved in this important aspect of citriculture.…”

“…The screening of X. citri diversity in different citrus‐growing regions has identified TALEs ranking from 6.5 to 29 repeats, which likely contain different RVDs. It is suggested that the diversification is driven by recombination, facilitated by the repetitive structure of the central region, leading to variants differing in the number of repeats (Al‐Saadi et al ., ; Gochez et al ., ; Lee et al ., ; Shiotani et al ., ; Ye et al ., ). It has been demonstrated that TALEs shorter than 6.5 repeats are non‐functional in terms of gene activation; these just could be by‐products of recombination events (Boch et al , ).…”