Assembling highly repetitive <i>Xanthomonas</i> TALomes using Oxford Nanopore sequencing

Erkes, Annett; Grove, René P.; Žarković, Milena; Krautwurst, Sebastian; Koebnik, Ralf; Morgan, Richard D.; Wilson, Geoffrey G.; Hölzer, Martin; Marz, Manja; Boch, Jens; Grau, Jan

doi:10.1101/2022.08.17.504259

Cited by 3 publications

(5 citation statements)

References 72 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…With >100x coverage, the final genome assemblies were confirmed with CheckM [34] to be >99% complete. The draft Xtt CO236 and Xtu CO237 genome assemblies from the raw base called reads did not resolve the TALomes due to the formation of homopolymers in the sequence [17]. Therefore, we used Homopolish [32] to perform extra genome polishing after Flye-based genome assembly, and two polishing steps of the raw FastQ reads using Medaka 1.5.0 [15] to resolve the specific TALEs.…”

Section: Resultsmentioning

confidence: 99%

“…In particular, genomic regions high in repetitive sequence can be difficult to assemble strictly via long-read sequencing. Xanthomonas genomes often contain regions of high repetition, and tools using computational corrections have been designed even for specific gene classes such as transcription activator-like effectors (TALEs), which are important virulence factors for many Xanthomonas species [17].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A recently collectedXanthomonas translucensisolate encodes TAL effectors distinct from older, less virulent isolates

Gutierrez-Castillo,

Barrett,

Roberts

2023

Preprint

View full text Add to dashboard Cite

Xanthomonas translucens, the causal agent of bacterial leaf streak disease (BLS) in cereals, is a re-emerging pathogen that is becoming increasingly destructive across the world. While BLS has caused yield losses in the past, there is anecdotal evidence that newer isolates may be more virulent. We observed that twoXanthomonas translucensisolates collected from two sites in Colorado are more aggressive on current wheat and barley varieties compared to older isolates, and we hypothesize that genetic changes between recent and older isolates contribute to the differences in isolate aggressiveness. To test this, we phenotyped and genetically characterized twoX. translucensisolates collected from Colorado in 2018, which we designated CO236 (from barley) and CO237 (from wheat). Using pathovar-specific phenotyping and PCR primers, we determined that CO236 belongs to pathovar translucens and CO237 belongs to pathovar undulosa. We sequenced the full genomes of the isolates using Oxford Nanopore long-read sequencing, and compared their whole genomes against publishedX. translucensgenomes. This analysis confirmed our pathovar designations for Xtt CO236 and Xtu CO237, and showed that, at the whole-genome level, there were no obvious genomic structural changes between Xtt CO236 and Xtu CO237 and other respective published pathovar genomes. Focusing on pathovar undulosa (Xtu CO237), we then compared putative Type III effectors among all available Xtu isolate genomes and found that they were highly conserved. However, there were striking differences in the presence and sequence of various transcription activator-like effectors (TALE) between Xtu CO237 and published undulosa genomes, which correlate with isolate virulence. Here, we explore the potential implications of the differences in these virulence factors, and provide possible explanations for the increased virulence of recently-emerged isolates.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A recently collectedXanthomonas translucensisolate encodes TAL effectors distinct from older, less virulent isolates

Gutierrez-Castillo,

Barrett,

Roberts

2023

Preprint

View full text Add to dashboard Cite

show abstract

“…With >100× coverage, the final genome assemblies were confirmed with CheckM [34] to be >99 % complete. The draft Xtt CO236 and Xtu CO237 genome assemblies from the raw base-called reads did not resolve the TALomes due to the formation of homopolymers in the sequence [17]. Therefore, we used Homopolish [32] to perform extra genome polishing after Flye-based genome assembly, and two polishing steps of the raw Fig.…”

Section: Complete Genome Sequences Of Xtt Co236 and Xtu Co237 Reveal ...mentioning

confidence: 99%

“…In particular, genomic regions rich in repetitive sequence can be difficult to assemble strictly via long-read sequencing. Xanthomonas genomes often contain regions of high repetition, and tools using computational corrections have been designed even for specific gene classes such as transcription activator-like effectors (TALEs), which are important virulence factors for many Xanthomonas species [17].…”

Section: Introductionmentioning

confidence: 99%

A recently collected Xanthomonas translucens isolate encodes TAL effectors distinct from older, less virulent isolates

Gutierrez-Castillo,

Barrett,

Roberts

2024

Microbial Genomics

View full text Add to dashboard Cite

Xanthomonas translucens, the causal agent of bacterial leaf streak disease (BLS) in cereals, is a re-emerging pathogen that is becoming increasingly destructive across the world. While BLS has caused yield losses in the past, there is anecdotal evidence that newer isolates may be more virulent. We observed that two X. translucens isolates collected from two sites in Colorado, USA, are more aggressive on current wheat and barley varieties compared to older isolates, and we hypothesize that genetic changes between recent and older isolates contribute to the differences in isolate aggressiveness. To test this, we phenotyped and genetically characterized two X. translucens isolates collected from Colorado in 2018, which we designated CO236 (from barley) and CO237 (from wheat). Using pathovar-specific phenotyping and PCR primers, we determined that CO236 belongs to pathovar translucens (Xtt) and CO237 belongs to pathovar undulosa (Xtu). We sequenced the full genomes of the isolates using Oxford Nanopore long-read sequencing, and compared their whole genomes against published X. translucens genomes. This analysis confirmed our pathovar designations for Xtt CO236 and Xtu CO237, and showed that, at the whole-genome level, there were no obvious genomic structural changes between Xtt CO236 and Xtu CO237 and other respective published pathovar genomes. Focusing on pathovar undulosa (Xtu CO237), we then compared putative type III effectors among all available Xtu isolate genomes and found that they were highly conserved. However, there were striking differences in the presence and sequence of various transcription activator-like effectors between Xtu CO237 and published undulosa genomes, which correlate with isolate virulence. Here, we explore the potential implications of the differences in these virulence factors, and provide possible explanations for the increased virulence of recently emerged isolates.

show abstract

“…True T3E orthogroups were further manually annotated and curated by integrating BLASTP, paperblast (81) and the EUROXANTH database (42). Because genes encoding TALEs are often poorly assembled due to their repetitive sequences (82), only TALE orthologs for which the complete N-terminal domain upstream of the repeat region was present were considered.…”

Section: Regulation Of Type Three Effector Gene Expressionmentioning

confidence: 99%

Comparative transcriptomics reveal a highly polymorphicXanthomonasHrpG virulence regulon

Quiroz Monnens,

Roux,

Cunnac

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

Bacteria of the genusXanthomonascause economically significant diseases in various crops. Their virulence is dependent on the translocation of type III effectors (T3Es) into plant cells by the type III secretion system (T3SS), a process regulated by the master response regulator HrpG. Although HrpG has been studied for over two decades, its regulon across diverseXanthomonasspecies, particularly beyond type III secretion, remains understudied. In this study, we conducted transcriptome sequencing to explore the HrpG regulons of 17Xanthomonasstrains, encompassing six species and nine pathovars, each exhibiting distinct host and tissue specificities. We employed constitutive expression of plasmid-bornehrpG*, which encodes a constitutively active form of HrpG, to induce the regulon. Our findings reveal substantial inter- and intra-specific diversity in the HrpG* regulons across the strains. Besides 21 genes directly involved in the biosynthesis of the T3SS, the core HrpG* regulon is limited to only five additional genes encoding the transcriptional activator HrpX, the two T3E proteins XopR and XopL, a major facility superfamily (MFS) transporter, and the phosphatase PhoC. Interestingly, genes involved in chemotaxis and genes encoding enzymes with carbohydrate-active and proteolytic activities are variably regulated by HrpG*. The diversity in the HrpG* regulon suggests that HrpG-dependent virulence inXanthomonasmight be achieved through several distinct strain-specific strategies, potentially reflecting adaptation to diverse ecological niches. These findings enhance our understanding of the complex role of HrpG in regulating various virulence and adaptive pathways, extending beyond T3Es and the T3SS.

show abstract

Assembling highly repetitive Xanthomonas TALomes using Oxford Nanopore sequencing

Cited by 3 publications

References 72 publications

A recently collectedXanthomonas translucensisolate encodes TAL effectors distinct from older, less virulent isolates

A recently collectedXanthomonas translucensisolate encodes TAL effectors distinct from older, less virulent isolates

A recently collected Xanthomonas translucens isolate encodes TAL effectors distinct from older, less virulent isolates

Comparative transcriptomics reveal a highly polymorphicXanthomonasHrpG virulence regulon

Contact Info

Product

Resources

About