Long read and single molecule DNA sequencing simplifies genome assembly and TAL effector gene analysis of Xanthomonas translucens

Peng et al. BMC Genomics (2016) 17:21 DOI 10.1186/s12864-015-2348-9 RESEARCH ARTICLE Open Access Long read and single molecule DNA sequencing simplifies genome assembly and TAL effector gene analysis of Xanthomonas translucens Zhao Peng1, Ying Hu2, Jingzhong Xie1, Neha Potnis3, Alina Akhunova1, Jeffrey Jones3, Zhaohui Liu4, Frank F. White1,3* and Sanzhen Liu1* Abstract Background: The species Xanthomonas translucens encompasses a complex of bacterial strains that cause diseases and yield loss on grass species including important cereal crops. Three pathovars, X. translucens pv. undulosa, X. translucens pv. translucens and X. translucens pv.cerealis, have been described as pathogens of wheat, barley, and oats. However, no complete genome sequence for a strain of this complex is currently available. Results: A complete genome sequence of X. translucens pv. undulosa strain XT4699 was obtained by using PacBio long read, single molecule, real time (SMRT) DNA sequences and Illumina sequences. Draft genome sequences of nineteen additional X. translucens strains, which were collected from wheat or barley in different regions and at different times, were generated by Illumina sequencing. Phylogenetic relationships among different Xanthomonas strains indicates that X. translucens are members of a distinct clade from so-called group 2 xanthomonads and three pathovars of this species, undulosa, translucens and cerealis, represent distinct subclades in the group 1 clade. Knockout mutation of type III secretion system of XT4699 eliminated the ability to cause water-soaking symptoms on wheat and barley and resulted in a reduction in populations on wheat in comparison to the wild type strain. Sequence comparison of X. translucens strains revealed the genetic variation on type III effector repertories among different pathovars or within one pathovar. The full genome sequence of XT4699 reveals the presence of eight members of the Transcription-Activator Like (TAL) effector genes, which are phylogenetically distant from previous known TAL effector genes of group 2 xanthomonads. Microarray and qRT-PCR analyses revealed TAL effectorspecific wheat gene expression modulation. Conclusions: PacBio long read sequencing facilitates the assembly of Xanthomonas genomes and the multiple TAL effector genes, which are difficult to assemble from short read platforms. The complete genome sequence of X. translucens pv. undulosa strain XT4699 and draft genome sequences of nineteen additional X. translucens strains provides a resource for further genetic analyses of pathogenic diversity and host range of the X. translucens species complex. TAL effectors of XT4699 strain play roles in modulating wheat host gene expressions. Keywords: Bacterial leaf streak, X. translucens, PacBio, TAL effectors * Correspondence: ; 1 Department of Plant Pathology, Kansas State University, Manhattan, KS, USA Full list of author information is available at the end of the article © 2015 Peng et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Peng et al. BMC Genomics (2016) 17:21 Background Bacterial pathogens of the genus Xanthomonas cause disease symptoms in a wide range of plant species, including many economically important cereal crops [1]. The species X. translucens represents a complex of strains that are pathogenic on various members of the Poaceae, including wheat, barley, oat, rye and other grass species. Bacterial leaf streak (BLS) and black chaff symptoms in the grain spikes on wheat are caused by X. translucens pv. undulosa strains. Outbreaks of BLS occur sporadically in central Great Plains and are associated with relatively warm and humid conditions, although the disease has been prevalent in recent recurrent years in the northern Great Plains [2]. X. translucens strains have been classified by pathogenicity types and DNA fingerprinting technologies [3]. Strains causing disease symptoms on barley and wheat are named as X. translucens pv. undulosa, while strains only pathogenic on barley are called X. translucens pv. translucens [4]. Although some strains of X. translucens pv. cerealis behave similarly as X. translucens pv. undulosa in pathogenicity types, they are distinguishable by DNA fingerprinting [3]. Phylogenic analyses of various X. translucens, do not align with the pathovar designations, and clarifications await genomic analyses on larger strain collections. In addition, many strains that were isolated from other species, often have been reported to cause disease symptoms on wheat [3]. For example, thirtythree bacterial strains isolated from diseased ornamental asparagus were identified as X. translucens pv. undulosa using DNA fingerprinting and cross inoculation [5]. Next-generation sequencing technologies have made transformational changes over the Sanger sequencing by improving throughput and reducing cost [6, 7]. The draft genome sequences provide valuable information on major genome contents and enable genome comparison among strains of interest [8, 9]. Currently, draft genome sequences are available for four strains from the X. translucens group. Draft genomic data for X. translucens pv. undulosa strain DAR61454, X. translucens pv. transluencs strain DSM18974, X. translucens pv. cerealis CFBP2541, and X. translucens pv. graminis ART-Xtg29 were generated by using Illumina or Roche 454 short read sequencing platforms [8–10]. At the same time, genome assemblies based on Illumina and Roche 454 sequencing are fragmented, and most assemblies failed to assemble complex repetitive sequences, including Transcription Activator-Like (TAL) effector genes, which occur in multiple gene copies and contain multiple simple near-perfect repeats within each gene. TAL effector genes typically have highly conserved N- and C-terminal sequences, and harbor 12.5–28.5 units of 102 or 105 bp repeats in the central regions [1]. Recently, a singlemolecule real-time (SMRT) sequencing technology was Page 2 of 19 developed by Pacific Bioscience (PacBio) and produces long sequence reads with no obvious sequencing biases and may allow better resolution of long repetitive DNA features in a genome. Due to a high error rate of PacBio reads, a high sequencing depth (e.g., 50× or higher) is usually required for a high-quality de novo assembly [11–13]. In this study, the complete genome sequence of X. translucens pv. undulosa strain XT4699 was generated by using high-depth PacBio and Illumina (...truncated)