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. 2016 Aug 17;7:1225.
doi: 10.3389/fpls.2016.01225. eCollection 2016.

TALE-Like Effectors Are an Ancestral Feature of the Ralstonia Solanacearum Species Complex and Converge in DNA Targeting Specificity

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Free PMC article

TALE-Like Effectors Are an Ancestral Feature of the Ralstonia Solanacearum Species Complex and Converge in DNA Targeting Specificity

Niklas Schandry et al. Front Plant Sci. .
Free PMC article

Abstract

Ralstonia solanacearum, a species complex of bacterial plant pathogens divided into four monophyletic phylotypes, causes plant diseases in tropical climates around the world. Some strains exhibit a broad host range on solanaceous hosts, while others are highly host-specific as for example some banana-pathogenic strains. Previous studies showed that transcription activator-like (TAL) effectors from Ralstonia, termed RipTALs, are capable of activating reporter genes in planta, if these are preceded by a matching effector binding element (EBE). RipTALs target DNA via their central repeat domain (CRD), where one repeat pairs with one DNA-base of the given EBE. The repeat variable diresidue dictates base repeat specificity in a predictable fashion, known as the TALE code. In this work, we analyze RipTALs across all phylotypes of the Ralstonia solanacearum species complex. We find that RipTALs are prevalent in phylotypes I and IV but absent from most phylotype III and II strains (10/12, 8/14, 1/24, and 1/5 strains contained a RipTAL, respectively). RipTALs originating from strains of the same phylotype show high levels of sequence similarity (>98%) in the N-terminal and C-terminal regions, while RipTALs isolated from different phylotypes show 47-91% sequence similarity in those regions, giving rise to four RipTAL classes. We show that, despite sequence divergence, the base preference for guanine, mediated by the N-terminal region, is conserved across RipTALs of all classes. Using the number and order of repeats found in the CRD, we functionally sub-classify RipTALs, introduce a new simple nomenclature, and predict matching EBEs for all seven distinct RipTALs identified. We experimentally study RipTAL EBEs and uncover that some RipTALs are able to target the EBEs of other RipTALs, referred to as cross-reactivity. In particular, RipTALs from strains with a broad host range on solanaceous hosts cross-react on each other's EBEs. Investigation of sequence divergence between RipTAL repeats allows for a reconstruction of repeat array biogenesis, for example through slipped strand mispairing or gene conversion. Using these studies we show how RipTALs of broad host range strains evolved convergently toward a shared target sequence. Finally, we discuss the differences between TALE-likes of plant pathogens in the context of disease ecology.

Keywords: Ralstonia solanacearum; RipTAL; bacterial wilt; crop pathogen; effector adaptation; molecular host–pathogen co-evolution; repetitive sequence; transcription activator like effector (TALE).

Figures

FIGURE 1
FIGURE 1
RipTAL abundance differs across Ralstonia solanacearum phylotypes but RipTALs sequences are similar within and different across phylotypes. (A) Abundance of ripTALs in strains from distinct phylotypes. The assessment is based on PCR analysis with primers flanking the repeats and was carried out on a broad collection of Rssc strains covering all phylotypes and different degrees of host adaptation. (B,C) Pairwise NTR and CTR sequence identities of depicted RipTALs from closely related Rssc strains (B) and RipTALs from different Rssc phylotypes or the Xanthomonas TALE AvrBs3 (C) are given in percent. Light and dark gray background indicates identities in the NTR and CTR, respectively.
FIGURE 2
FIGURE 2
Comparison of RVD compositions of novel RipTALs across all four Ralstonia solanacearum phylotypes reveals limited diversity. Cartoon displays RVD compositions of newly identified RipTALs separated by class. Each repeat is depicted as an oval. Capital letters inside the repeats indicate amino acids (single letter code) in position 12 and 13 (RVD) of each repeat. Repeats are color-coded based on the preferred base of repeat residue 13, which is the key base specificity determinant, with a color code given at the bottom. Strains bearing a particular ripTAL are given next to the RipTAL identifier in black text. Text in brackets gives the sequevar of this strain, n.d. indicates that this strain has not been clearly assigned to a sequevar. Underlined strain name indicates that the given ripTAL was studied as a representative in functional assays. RipTALI-2 to RipTALI-6 were described previously (de Lange et al., 2013) and are shown in Supplementary Figure S1.
FIGURE 3
FIGURE 3
In planta expressed RipTALs of all classes show nuclear localization. Confocal laser scanning microscopy images of Arabidopsis thaliana protoplasts expressing depicted YFP-tagged RipTALs and a nuclear-targeted mCherry. Scale bars represent 50 μm.
FIGURE 4
FIGURE 4
All RipTALs activated promoters bearing predicted G0 Effector Binding Elements (EBEs). (A) All RipTALs were tested against pepper Bs3 promoter derivatives, bearing the RipTAL EBE in place of the AvrBs3 binding site, preceded by the given base (indicated by color-coded boxplots) upstream of a uidA CDS. Background levels were determined using the same promoter-reporter in combination with AvrBs3. Experiments were repeated twice and all results are shown. G0EBEs were activated significantly (p < 0.01, Wilcoxon rank-sum test), while the others were not. (B) Boxshade alignment of G0EBEs corresponding to depicted RipTALs.
FIGURE 5
FIGURE 5
RipTALs form functional groups based on cross activation. Increase in GUS-reporter activity for RipTALs on promoters with predicted EBEs. Columns indicate promoter-embedded EBEs tested. The last column provides information on the natural host range of the RipTAL bearing strains identified in this study. All full-length RipTALs were tested against all EBEs. For each RipTAL-EBE combination the median fold activation is given. Underlined values indicate predicted RipTAL-EBE combinations. Blue background is used for RipTAL-EBE combinations that were significantly greater than 1 (p < 0.01, determined by Wilcoxon rank-sum test).
FIGURE 6
FIGURE 6
HD repeat 8 of RipTALs from broad host-range strains is unable to discriminate between adenine and cytosine. White boxplots show fold GUS change over background on the RipTALI-1 EBE reporter. Gray boxplots show fold GUS change over background for the same EBE where base 8 was changed from cytosine to adenine. The number of replicates is given below each plot.
FIGURE 7
FIGURE 7
Comparison of repeats within a TALE a ripTAL uncovers pronounced differences in their inter-repeat identities. Individual repeats of the TALE avrBs3 (upper right) and ripTALI-1 (lower left) were, aligned, ordered as in their native CRD, and pairwise identities were calculated (percentages in cells). Values <97% are displayed in white font, values ≥97 in black font. Color-coding of cells indicates identities between two given repeats, with a color key given upper right. Black-framed cells indicate comparison of a repeat against itself. Numbers above or adjacent to repeats indicate the position of the repeat within the given array.
FIGURE 8
FIGURE 8
Inter-repeat comparisons of RipTALs from closely related Rssc strains uncovers position-dependent conservation (PDC) of repeats. Individual repeats were aligned and pairwise identities were calculated. Repeats are shown in their native order. Cells are color coded by their percentage identities value according to the color code to the right. Identities <97% are displayed in white font, those ≥97% in black font. Numbers indicate the position of the repeat within the CRD. (A) Comparison between all repeats of the CRD of ripTALI-1 (columns) versus all repeats of the CRD of ripTALI-8 (rows). (B) Comparison between all repeats of the CRD of ripTALIII-1 (columns) versus all repeats of the CRD of ripTALII-1 (rows).
FIGURE 9
FIGURE 9
Inspection of ripTAL CRDs indicates the molecular mechanisms shaping ripTAL CRD composition. Closely related repeats are colored in the same color, with exception of gray that does not indicate any relatedness. (A) Specificity altering SNPs. Repeats 10-13 of RipTALI-1 and RipTALI-8 are shown. The RVDs and cognate codons of repeat 12 of each RipTAL are further given in yellow boxes. The two depicted SNPs constitute the only polymorphisms between these two repeats. (B) Repeat duplication by segmental gene conversion. Repeats 7–11 of RipTALI-9 are shown, as well as repeats 7-12 of RipTALI-1 and RipTALIII-1. Green color indicates HD repeats that are highly similar in sequence, within each array. A less related HD repeat is displayed in dark gray. Proposed segmental gene conversion events are indicated by dashed lines with arrowheads. Polymorphic bases between repeats 8, 10, and 11 of the respective ripTAL are displayed to the right of the cartoon display. Next to the base comparison, repeat RVD, as well as the position within the array, colored according to the fill color of that repeat in the cartoon display is given. (C) Duplication of adjacent repeats by slipped-strand mispairing exemplified on ripTALII-1. Repeats 1-6 of a proposed ancestral repeat array are shown to the left. A slipped-strand mispairing DNA intermediate is shown above. Repeats 1–7 of the resulting product are shown to the right. Slipped-strand mispairing leads to a duplication of the repeat colored in orange. (D) A recombination event leads to loss of all repeats except one in ripTALI-7. The remaining repeat is fusion of ripTALI-1 repeats 1 (pink) and 16 (blue). To the right, polymorphic bases of repeats 1 and 16 are shown. The ripTAL designation, repeat RVD and repeat position are given to the right of the sequence of polymorphic bases, colored according to the fill color of that repeat.

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