An Improved Method for TAL Effectors DNA-Binding Sites Prediction Reveals Functional Convergence in TAL Repertoires of <i>Xanthomonas oryzae</i> Strains

<div><p>Transcription Activators-Like Effectors (TALEs) belong to a family of virulence proteins from the <i>Xanthomonas</i> genus of bacterial plant pathogens that are translocated into the plant cell. In the nucleus, TALEs act as transcription factors inducing the expression of susceptibility genes. A code for TALE-DNA binding specificity and high-resolution three-dimensional structures of TALE-DNA complexes were recently reported. Accurate prediction of TAL Effector Binding Elements (EBEs) is essential to elucidate the biological functions of the many sequenced TALEs as well as for robust design of artificial TALE DNA-binding domains in biotechnological applications. In this work a program with improved EBE prediction performances was developed using an updated specificity matrix and a position weight correction function to account for the matching pattern observed in a validation set of TALE-DNA interactions. To gain a systems perspective on the large TALE repertoires from <i>X. oryzae</i> strains, this program was used to predict rice gene targets for 99 sequenced family members. Integrating predictions and available expression data in a TALE-gene network revealed multiple candidate transcriptional targets for many TALEs as well as several possible instances of functional convergence among TALEs.</p></div

Distribution of perfect matches (PM) in the TALE-EBE validation set.

<p>(<b>A</b>) Box plot of the distribution of the number of perfect RVD-nucleotide matches computed for individual negative and positive control TALE-EBE pairs. (<b>B</b>) Distribution of perfect match frequency of individual control TALE-EBE pairs. The frequency corresponds to the ratio of the number of perfect RVD-nucleotide matches to TALE length expressed in number of RVD. (<b>C</b>) Frequency of perfect matches across TALE-DNA positions. The frequency corresponds to the ratio of the number of perfect RVD-nucleotide at the considered position to the total number of RVD-nucleotide pairs at this position in TALE-EBE pairs of the positive or negative control set. (<b>D</b>) Frequency of perfect RVD-nucleotide match between positions 1 and 15 ( = number of PM/15). (<b>E</b>) Frequency of perfect match for TALE-DNA positions beyond 15 ( = number of PM/(length-15)). The p-value of the corresponding two-tailed Wilcoxon test in this comparison is 0.371. ** significant differences, one-tailed Wilcoxon test p-value<0.001; *** significant differences one-tailed Wilcoxon p-value<1e-7.</p

Performances of the EBE prediction software in the TALE-EBE validation set.

<p>(<b>A</b>) Boxplot showing the median (thick line), the lower and upper quartiles (box) and the minimum and maximum (whiskers) of the prediction scores for the set of positive (+) and negative (−) control TALE-DNA interactions using three programs for EBE prediction. Scores were scaled down according to the maximum score on the set to facilitate comparison. Talent scores were scaled x⁻¹ since they follow an inverse scale relative to the other programs, this transformation maintains data structure. ** Indicates significant positive vs. negative differences (one-tailed t-test p-value<0.001). (<b>B</b>) ROC graph showing the true positive and false positive rate of the three EBE predictors based on validation set screenings. Dashed line indicates the theoretical performance of a random classifying program where true positive rate = false positive rate. The inset in the upper right corner shows the rates for Talvez and Storyteller at a higher scale to highlight the differences between the two programs.</p

Comparison of the TALE-candidate target gene network with random networks obtained with shuffled TALEs.

<p>Properties of the TALE-gene network are compared to average values from 100 randomized controls (error bars indicate standard deviation): (<b>A</b>) percent frequency distribution of Talvez prediction ranks of TALE-gene pairs, the percentage of top (#1) ranking TALE-gene pairs is indicated for the TALE-gene network. (<b>B</b>) Number of genes and TALEs in the TALE-gene network compared to control random networks.</p

Possible functional convergence on specific rice TALE targets between <i>X. oryzae</i> pathovars.

<p>Panels (<b>A</b>) and (<b>B</b>) summarize Talvez predictions and expression data respectively for <i>OsHen1</i> and <i>F3H</i> (LOC_Os03g03034). See legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068464#pone-0068464-g006" target="_blank">Figure 6</a> for details.</p

TALEs from multiple <i>Xoo</i> strains may converge onto three distinct MtN3 gene family members.

<p>Panels (<b>A</b>), (<b>B</b>) and (<b>C</b>) summarize Talvez predictions and expression data respectively for <i>Os11N3</i>, <i>Os12N3 (Xa25)</i> and <i>Os8N3</i> (<i>Xa13</i>). From top to bottom: data in bar plots derive from our analysis of microarray data from different rice genotypes and 24 hours after infection time points (hpi). Relevant treatments comparisons are indicated above the graphs. logFC values correspond to log2-transformed fold-change ratios. In the Talvez prediction network snapshots, the rank and score values along the edges represent Talvez prediction output for the connected gene (EBE) in target searches for the corresponding TALE. The bottom part of each panel contains a manual alignment of the RVD sequences from TALEs that are predicted to target the gene under consideration in the panel. Individual residues highlighted in bold deviate from the consensus at that position. The locations of the predicted EBEs on the upstream sequences of the rice gene are marked by lines colored following the same pattern as on the RVD alignment. Numbers on the left indicate the distance in base pair between the most upstream nucleotide of the reported sequence and the ATG. TalC from the African Xoo strain BAI3 which has been reported to target <i>Os11N3</i> <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068464#pone.0068464-Yu1" target="_blank">[18]</a> was included in panel A to illustrate the notion of convergence on gene susceptibility targets at the level of distinct EBEs.</p

Overlap of candidate rice target gene sets for TALEs from various <i>X. oryzae</i> strains.

<p>Venn diagram of rice genes from the network assigned to distinct sets according to the strain of origin of their cognate TALE(s).</p

Comparison of EBE predictors rankings of known TALE targets in genomic searches.

<p>Comparison of EBE predictors rankings of known TALE targets in genomic searches.</p

Image_6_Functional and Genome Sequence-Driven Characterization of tal Effector Gene Repertoires Reveals Novel Variants With Altered Specificities in Closely Related Malian Xanthomonas oryzae pv. oryzae Strains.PDF

<p>Rice bacterial leaf blight (BLB) is caused by Xanthomonas oryzae pv. oryzae (Xoo) which injects Transcription Activator-Like Effectors (TALEs) into the host cell to modulate the expression of target disease susceptibility genes. Xoo major-virulence TALEs universally target susceptibility genes of the SWEET sugar transporter family. TALE-unresponsive alleles of OsSWEET genes have been identified in the rice germplasm or created by genome editing and confer resistance to BLB. In recent years, BLB has become one of the major biotic constraints to rice cultivation in Mali. To inform the deployment of alternative sources of resistance in this country, rice lines carrying alleles of OsSWEET14 unresponsive to either TalF (formerly Tal5) or TalC, two important TALEs previously identified in West African Xoo, were challenged with a panel of strains recently isolated in Mali and were found to remain susceptible to these isolates. The characterization of TALE repertoires revealed that talF and talC specific molecular markers were simultaneously present in all surveyed Malian strains, suggesting that the corresponding TALEs are broadly deployed by Malian Xoo to redundantly target the OsSWEET14 gene promoter. Consistent with this, the capacity of most Malian Xoo to induce OsSWEET14 was unaffected by either talC- or talF-unresponsive alleles of this gene. Long-read sequencing and assembly of eight Malian Xoo genomes confirmed the widespread occurrence of active TalF and TalC variants and provided a detailed insight into the diversity of TALE repertoires. All sequenced strains shared nine evolutionary related tal effector genes. Notably, a new TalF variant that is unable to induce OsSWEET14 was identified. Furthermore, two distinct TalB variants were shown to have lost the ability to simultaneously induce two susceptibility genes as previously reported for the founding members of this group from strains MAI1 and BAI3. Yet, both new TalB variants retained the ability to induce one or the other of the two susceptibility genes. These results reveal molecular and functional differences in tal repertoires and will be important for the sustainable deployment of broad-spectrum and durable resistance to BLB in West Africa.</p

Image_4_Functional and Genome Sequence-Driven Characterization of tal Effector Gene Repertoires Reveals Novel Variants With Altered Specificities in Closely Related Malian Xanthomonas oryzae pv. oryzae Strains.TIFF

<p>Rice bacterial leaf blight (BLB) is caused by Xanthomonas oryzae pv. oryzae (Xoo) which injects Transcription Activator-Like Effectors (TALEs) into the host cell to modulate the expression of target disease susceptibility genes. Xoo major-virulence TALEs universally target susceptibility genes of the SWEET sugar transporter family. TALE-unresponsive alleles of OsSWEET genes have been identified in the rice germplasm or created by genome editing and confer resistance to BLB. In recent years, BLB has become one of the major biotic constraints to rice cultivation in Mali. To inform the deployment of alternative sources of resistance in this country, rice lines carrying alleles of OsSWEET14 unresponsive to either TalF (formerly Tal5) or TalC, two important TALEs previously identified in West African Xoo, were challenged with a panel of strains recently isolated in Mali and were found to remain susceptible to these isolates. The characterization of TALE repertoires revealed that talF and talC specific molecular markers were simultaneously present in all surveyed Malian strains, suggesting that the corresponding TALEs are broadly deployed by Malian Xoo to redundantly target the OsSWEET14 gene promoter. Consistent with this, the capacity of most Malian Xoo to induce OsSWEET14 was unaffected by either talC- or talF-unresponsive alleles of this gene. Long-read sequencing and assembly of eight Malian Xoo genomes confirmed the widespread occurrence of active TalF and TalC variants and provided a detailed insight into the diversity of TALE repertoires. All sequenced strains shared nine evolutionary related tal effector genes. Notably, a new TalF variant that is unable to induce OsSWEET14 was identified. Furthermore, two distinct TalB variants were shown to have lost the ability to simultaneously induce two susceptibility genes as previously reported for the founding members of this group from strains MAI1 and BAI3. Yet, both new TalB variants retained the ability to induce one or the other of the two susceptibility genes. These results reveal molecular and functional differences in tal repertoires and will be important for the sustainable deployment of broad-spectrum and durable resistance to BLB in West Africa.</p