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. 2016 Feb 15:6:21077.
doi: 10.1038/srep21077.

AnnoTALE: bioinformatics tools for identification, annotation, and nomenclature of TALEs from Xanthomonas genomic sequences

Jan Grau  1 , Maik Reschke  2   3 , Annett Erkes  1 , Jana Streubel  2   3 , Richard D Morgan  4 , Geoffrey G Wilson  4 , Ralf Koebnik  5 , Jens Boch  2   3
Affiliations

AnnoTALE: bioinformatics tools for identification, annotation, and nomenclature of TALEs from Xanthomonas genomic sequences

Jan Grau et al. Sci Rep. .

Abstract

Transcription activator-like effectors (TALEs) are virulence factors, produced by the bacterial plant-pathogen Xanthomonas, that function as gene activators inside plant cells. Although the contribution of individual TALEs to infectivity has been shown, the specific roles of most TALEs, and the overall TALE diversity in Xanthomonas spp. is not known. TALEs possess a highly repetitive DNA-binding domain, which is notoriously difficult to sequence. Here, we describe an improved method for characterizing TALE genes by the use of PacBio sequencing. We present 'AnnoTALE', a suite of applications for the analysis and annotation of TALE genes from Xanthomonas genomes, and for grouping similar TALEs into classes. Based on these classes, we propose a unified nomenclature for Xanthomonas TALEs that reveals similarities pointing to related functionalities. This new classification enables us to compare related TALEs and to identify base substitutions responsible for the evolution of TALE specificities.

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Conflict of interest statement

G.G.W. and R.D.M. are employees of New England Biolabs Inc., Ipswich, USA. J.B. is a part owner of a patent governing the use of TALEs.

Figures

Figure 1
Figure 1. Phenotypes and race-specific TALE gene pattern of Philippine Xoo strains.
(a) Leaves of 3- to 4-week old rice cultivar Nipponbare plants were infiltrated with 10 mM MgCl2 or Philippine Xoo strains PXO35 (race 1), PXO132 (race 1), PXO83 (race 2), PXO142 (race 3), PXO143 (race 3), PXO183 (race 5) and PXO99 (race 6), respectively. Plant reactions were documented five days post inoculation. (b) Southern blot analysis. Genomic DNA of the Philippine Xoo strains PXO35 (race 1), PXO132 (race 1), PXO83 (race 2), PXO142 (race 3), PXO143 (race 3), PXO183 (race 5) and the reference strain PXO99 (race 6) was digested with BamHI, separated on an agarose gel, and transferred to a nylon membrane. TALE-containing fragments were detected using a DIG-labelled probe corresponding to 500 bp of the 3′ part of talC. Known repeat numbers of TALEs and their corresponding fragments in the sequenced Xoo strain PXO99 are indicated at the right side. The approximate sizes of marker fragments estimated from the ethidium bromide-stained gel are indicated at the left side. The large fragments in lanes 3 and 5 (PXO83 and PXO143) are due to an absence of the typically conserved BamHI sites in certain TALEs.
Figure 2
Figure 2. Comparison of Xanthomonas oryzae pv. oryzae genomic regions.
ProgressiveMauve alignment of four fully sequenced genomes of Xanthomonas oryzae pv. oryzae (Xoo) strains. Similarly coloured areas represent genomic regions with significant synteny. The genomes of Xoo KACC10331 and Xoo MAFF311018 are shown in reverse complement to simplify the view, because of extensive genomic rearrangements. To indicate this, the origin and the orientation of dnaA is indicated by a black dot with horizontal arrow. Black arrowheads point to the positions of the genomic TALE clusters. The numbering is according to Fig. 5.
Figure 3
Figure 3. Workflow for AnnoTALE.
(a) Diagram of input parameters, workflow, and program parts. The newly assigned names of TALEs consist of Tal (black), TALE class (XY, light blue), and allele number of TALE within a class (123, purple). Yellow boxes represent input parameter, orange boxes show generated data, and green boxes indicate the program parts that perform the individual job. To assign TALEs to existing classes the current class definition can be downloaded and imported into the program. Besides genomic sequences, individual TALE DNA sequences can optionally be loaded into the program. N, N-terminal region; C, C-terminal region; RVD, repeat variable di-residue. (b) Examples of the TALE Class Assignment tool. Two representative TALE classes, AD and AP, are displayed. RVD changes between TALEs are marked in black.
Figure 4
Figure 4. Changes in RVD codons.
(a) Network of codon pairs of RVDs connected to the RVD NS by single-nucleotide or double-nucleotide substitutions. RVDs are boxed in colors according to their DNA base specificities (green: A, orange: G, red: T) and list the observed codon pairs. Frequency in known TALEs is shown below each codon pair. Lines represent possible single-nucleotide (solid line) or double-nucleotide (dotted line) exchanges. Substitutions observed within classes are in red with labels indicating the number of occurrences. (b) Histogram of the number of observed nucleotide substitutions in aligned RVDs of TALEs in a common family.
Figure 5
Figure 5. Distribution of TALE genes in Xoo strains PXO83, PXO99A, MAFF311018 and KACC10331.
Individual TALE genes are represented by arrows, which indicate the orientation of the gene. TALEs are labelled with their new name inside the arrow. Below the arrows, former names or gene numbers are shown in brackets. TALEs with prominent TALEs in their class are labelled in bold above the arrows. Pseudogenes are indicated with the Greek letter phi (Φ). Novel assigned TALE clusters (T-I to T-IX) are depicted above the scheme in roman numbers. Similar colors indicate which cluster the TALEs belong to. Colored bars between the genomes represent the regions on which basis the clusters are defined. TALE clusters T-IV and T-VII are unique for their respective genome. Grey bars represent chosen similar genomic genes in different genomes that were not used to discriminate TALE clusters.
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