Assembling highly repetitive Xanthomonas TALomes using Oxford Nanopore sequencing.

BACKGROUND: Most plant-pathogenic Xanthomonas bacteria harbor transcription activator-like effector (TALE) genes, which function as transcriptional activators of host plant genes and support infection. The entire repertoire of up to 29 TALE genes of a Xanthomonas strain is also referred to as TALome. The DNA-binding domain of TALEs is comprised of highly conserved repeats and TALE genes often occur in gene clusters, which precludes the assembly of TALE-carrying Xanthomonas genomes based on standard sequencing approaches. RESULTS: Here, we report the successful assembly of the 5 Mbp genomes of five Xanthomonas strains from Oxford Nanopore Technologies (ONT) sequencing data. For one of these strains, Xanthomonas oryzae pv. oryzae (Xoo) PXO35, we illustrate why Illumina short reads and longer PacBio reads are insufficient to fully resolve the genome. While ONT reads are perfectly suited to yield highly contiguous genomes, they suffer from a specific error profile within homopolymers. To still yield complete and correct TALomes from ONT assemblies, we present a computational correction pipeline specifically tailored to TALE genes, which yields at least comparable accuracy as Illumina-based polishing. We further systematically assess the ONT-based pipeline for its multiplexing capacity and find that, combined with computational correction, the complete TALome of Xoo PXO35 could have been reconstructed from less than 20,000 ONT reads. CONCLUSIONS: Our results indicate that multiplexed ONT sequencing combined with a computational correction of TALE genes constitutes a highly capable tool for characterizing the TALomes of huge collections of Xanthomonas strains in the future.

Epigenetic features improve TALE target prediction.

BACKGROUND: The yield of many crop plants can be substantially reduced by plant-pathogenic Xanthomonas bacteria. The infection strategy of many Xanthomonas strains is based on transcription activator-like effectors (TALEs), which are secreted into the host cells and act as transcriptional activators of plant genes that are beneficial for the bacteria.The modular DNA binding domain of TALEs contains tandem repeats, each comprising two hyper-variable amino acids. These repeat-variable diresidues (RVDs) bind to their target box and determine the specificity of a TALE.All available tools for the prediction of TALE targets within the host plant suffer from many false positives. In this paper we propose a strategy to improve prediction accuracy by considering the epigenetic state of the host plant genome in the region of the target box. RESULTS: To this end, we extend our previously published tool PrediTALE by considering two epigenetic features: (i) chromatin accessibility of potentially bound regions and (ii) DNA methylation of cytosines within target boxes. Here, we determine the epigenetic features from publicly available DNase-seq, ATAC-seq, and WGBS data in rice.We benchmark the utility of both epigenetic features separately and in combination, deriving ground-truth from RNA-seq data of infections studies in rice. We find an improvement for each individual epigenetic feature, but especially the combination of both.Having established an advantage in TALE target predicting considering epigenetic features, we use these data for promoterome and genome-wide scans by our new tool EpiTALE, leading to several novel putative virulence targets. CONCLUSIONS: Our results suggest that it would be worthwhile to collect condition-specific chromatin accessibility data and methylation information when studying putative virulence targets of Xanthomonas TALEs.

PrediTALE: A novel model learned from quantitative data allows for new perspectives on TALE targeting.

Plant-pathogenic Xanthomonas bacteria secrete transcription activator-like effectors (TALEs) into host cells, where they act as transcriptional activators on plant target genes to support bacterial virulence. TALEs have a unique modular DNA-binding domain composed of tandem repeats. Two amino acids within each tandem repeat, termed repeat-variable diresidues, bind to contiguous nucleotides on the DNA sequence and determine target specificity. In this paper, we propose a novel approach for TALE target prediction to identify potential virulence targets. Our approach accounts for recent findings concerning TALE targeting, including frame-shift binding by repeats of aberrant lengths, and the flexible strand orientation of target boxes relative to the transcription start of the downstream target gene. The computational model can account for dependencies between adjacent RVD positions. Model parameters are learned from the wealth of quantitative data that have been generated over the last years. We benchmark the novel approach, termed PrediTALE, using RNA-seq data after Xanthomonas infection in rice, and find an overall improvement of prediction performance compared with previous approaches. Using PrediTALE, we are able to predict several novel putative virulence targets. However, we also observe that no target genes are predicted by any prediction tool for several TALEs, which we term orphan TALEs for this reason. We postulate that one explanation for orphan TALEs are incomplete gene annotations and, hence, propose to replace promoterome-wide by genome-wide scans for target boxes. We demonstrate that known targets from promoterome-wide scans may be recovered by genome-wide scans, whereas the latter, combined with RNA-seq data, are able to detect putative targets independent of existing gene annotations.

Transcriptional Reprogramming of Rice Cells by Xanthomonas oryzae TALEs.

Rice-pathogenic Xanthomonas oryzae bacteria cause severe harvest loss and challenge a stable food supply. The pathogen virulence relies strongly on bacterial TALE (transcription activator-like effector) proteins that function as transcriptional activators inside the plant cell. To understand the plant targets of TALEs, we determined the genome sequences of the Indian X. oryzae pv. oryzae (Xoo) type strain ICMP 3125T and the strain PXO142 from the Philippines. Their complete TALE repertoire was analyzed and genome-wide TALE targets in rice were characterized. Integrating computational target predictions and rice transcriptomics data, we were able to verify 12 specifically induced target rice genes. The TALEs of the Xoo strains were reconstructed and expressed in a TALE-free Xoo strain to attribute specific induced genes to individual TALEs. Using reporter assays, we could show that individual TALEs act directly on their target promoters. In particular, we show that TALE classes assigned by AnnoTALE reflect common target genes, and that TALE classes of Xoo and the related pathogen X. oryzae pv. oryzicola share more common target genes than previously believed. Taken together, we establish a detailed picture of TALE-induced plant processes that significantly expands our understanding of X. oryzae virulence strategies and will facilitate the development of novel resistances to overcome this important rice disease.

Evolution of Transcription Activator-Like Effectors in Xanthomonas oryzae.

Transcription activator-like effectors (TALEs) are secreted by plant-pathogenic Xanthomonas bacteria into plant cells where they act as transcriptional activators and, hence, are major drivers in reprogramming the plant for the benefit of the pathogen. TALEs possess a highly repetitive DNA-binding domain of typically 34 amino acid (AA) tandem repeats, where AA 12 and 13, termed repeat variable di-residue (RVD), determine target specificity. Different Xanthomonas strains possess different repertoires of TALEs. Here, we study the evolution of TALEs from the level of RVDs determining target specificity down to the level of DNA sequence with focus on rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) strains. We observe that codon pairs coding for individual RVDs are conserved to a similar degree as the flanking repeat sequence. We find strong indications that TALEs may evolve 1) by base substitutions in codon pairs coding for RVDs, 2) by recombination of N-terminal or C-terminal regions of existing TALEs, or 3) by deletion of individual TALE repeats, and we propose possible mechanisms. We find indications that the reassortment of TALE genes in clusters is mediated by an integron-like mechanism in Xoc. We finally study the effect of the presence/absence and evolutionary modifications of TALEs on transcriptional activation of putative target genes in rice, and find that even single RVD swaps may lead to considerable differences in activation. This correlation allowed a refined prediction of TALE targets, which is the crucial step to decipher their virulence activity.

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

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.