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.

BACKGROUND: 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. 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. RESULTS: We 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. CONCLUSIONS: We 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.

Recent advances in the understanding of Xanthomonas citri ssp. citri pathogenesis and citrus canker disease management.

Taxonomic status: Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; Species Xanthomonas citri ssp. citri (Xcc). Host range: Compatible hosts vary in their susceptibility to citrus canker (CC), with grapefruit, lime and lemon being the most susceptible, sweet orange being moderately susceptible, and kumquat and calamondin being amongst the least susceptible. Microbiological properties: Xcc is a rod-shaped (1.5-2.0 × 0.5-0.75 µm), Gram-negative, aerobic bacterium with a single polar flagellum. The bacterium forms yellow colonies on culture media as a result of the production of xanthomonadin. Distribution: Present in South America, the British Virgin Islands, Africa, the Middle East, India, Asia and the South Pacific islands. Localized incidence in the USA, Argentina, Brazil, Bolivia, Uruguay, Senegal, Mali, Burkina Faso, Tanzania, Iran, Saudi Arabia, Yemen and Bangladesh. Widespread throughout Paraguay, Comoros, China, Japan, Malaysia and Vietnam. Eradicated from South Africa, Australia and New Zealand. Absent from Europe.

Molecular characterization of XopAG effector AvrGf2 from Xanthomonas fuscans ssp. aurantifolii in grapefruit.

Xanthomonas fuscans ssp. aurantifolii group C strains exhibit host specificity on different citrus species. The strains possess a type III effector, AvrGf2, belonging to the XopAG effector gene family, which restricts host range on citrus. We dissected the modular nature and mode of action of AvrGf2 in grapefruit resistance. XopAG effectors possess characteristic features, such as a chloroplast localization signal, a cyclophilin-binding domain characteristic amino acid sequence motif (GPLL) and a C-terminal domain-containing CLNAxYD. Mutation of GPLL to AASL in AvrGf2 abolished the elicitation of the hypersensitive response (HR), whereas mutation of only the first amino acid to SPLL delayed the HR in grapefruit. Yeast two-hybrid experiments showed strong interaction of AvrGf2 with grapefruit cyclophilin (GfCyp), whereas AvrGf2-SPLL and AvrGf2-AASL mutants showed weak and no interaction, respectively. Molecular modelling and in silico docking studies for the cyclophilin-AvrGf2 interaction predicted the binding of citrus cyclophilins (CsCyp, GfCyp) to hexameric peptides spanning the cyclophilin-binding domain of AvrGf2 and AvrGf2 mutants (VAGPLL, VASPLL and VAAASL) with affinities equivalent to or better than a positive control peptide (YSPSA) previously demonstrated to bind CsCyp. In addition, the C-terminal domain of XopAG family effectors contains a highly conserved motif, CLNAxYD, which was identified to be crucial for the induction of HR based on site-directed mutagenesis (CLNAxYD to CASAxYD). Our results suggest a model in which grapefruit cyclophilin promotes a conformational change in AvrGf2, thereby triggering the resistance response.