Comprehensive Genomic Insights Into Unique TALEs of a Xanthomonas translucens pv. undulosa Strain From China

A Xanthomonas translucens pv. undulosa isolate from wheat was characterised; two transcription-activator like effectors with distinct repeat-variable di-residue sequences and one uncommon Xanthomonas outer protein were found. Bacterial leaf streak (BLS) caused by the gram-negative bacterium Xanthomonas translucens includes several pathovars such as undulosa (Xtu), translucens (Xtt) and cerealis (Xtc). Recent genomic studies have reclassified the X. translucens complex into three distinct species: X. translucens sensu stricto (including Xtu and Xtt), X. cerealis sp. nov. (including Xtc) and X. graminis sp. nov. (Tambong et al. 2024). BLS is considered one of the most important bacterial diseases affecting the production of small-grain cereals across the globe (Khojasteh et al. 2019; Osdaghi et al. 2023). It is on the list of quarantine pathogens with a high-risk classification (A2 category) according to the European and Mediterranean Plant Protection Organisation (EPPO) and is therefore subject to strict quarantine policies in various countries (EPPO 2022). According to the cultivars investigated, 50% of diseased leaf area can result in yield losses ranging from 11% to 29% (Duveiller and Maraite 1993). It is estimated that highly susceptible cultivars could lose up to 40% of their yield under wet or sprinkler irrigation conditions (Forster et al. 1986). Because the pathogen has been frequently found on seed surfaces, it is believed to spread over long distances through contaminated seed lots. While there is currently no conclusive proof of seed transmission, this spread may be able to establish the disease even in areas where it has never been previously observed (Sapkota et al. 2020). In China, BLS was first reported in 1956 (Yu and Fang 1956) and has frequently been observed in wheat planting regions of Xinjiang province, China, in recent years (Shah et al. 2019, 2023). Notably, Xtu is frequently identified as the BLS pathogen and has a host range that includes both wheat and barley, posing a serious threat to cereal production (Duveiller et al. 1997; Langlois et al. 2017; Sapkota et al. 2020). Although an earlier study identified BLS-associated strains in China as Xtu based on biochemical characterisation (Chen and Ding 1981), no molecular evidence or genomic data have been reported to confirm this classification or to elucidate the genetic features of Chinese Xtu isolates. Type III effectors (T3SEs) secreted by the type III secretion system (T3SS) are crucial for the virulence of Xanthomonas, especially transcription-activator-like effectors (TALEs) (Boch and Bonas 2010). TALEs feature amino acid sequences characterised by tandem repeats of 33–35 amino acids, with polymorphic residues at positions 12 and 13, known as repeat-variable di-residues (RVDs) (Moscou and Bogdanove 2009). Within the plant cell nucleus, the RVD array enables each repeat to interact with a specific base in the effector-binding elements (EBEs) of a promoter (Boch et al. 2009). This interaction activates the expression of host genes—either susceptibility or resistance genes—thereby facilitating disease progression (Timilsina et al. 2020). Although T3SEs are highly conserved among Xtu strains, recent comparative genomic analyses have revealed notable variation in TALEs, which may contribute to differences in virulence and host adaptation (Gutierrez-Castillo et al. 2024). Several TALEs, often referred to as major TALEs, play critical roles during bacterial infection. For instance, X. cerealis pv. cerealis NXtc01 (previously classified as X. translucens) carries two tal genes, with tal1 demonstrating significant contributions to virulence (Shah et al. 2019). Xtu strain ICMP11055 relies on Tal2 and Tal4b for pathogenicity (Charkhabi et al. 2017). Tal8 from Xtu strain 4699 targets the gene TaNCED_5BS to manipulate the abscisic acid (ABA) biosynthetic pathway in wheat (Peng et al. 2019). On the other hand, non-TALEs, also known as Xanthomonas outer proteins (Xops) function to suppress the immune responses of host plants, contributing to disease development. For instance, several Xops in X. oryzae pv. oryzae have been shown to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) in rice (Sinha et al. 2013; Xu et al. 2022). However, specific Xops, as well as certain TALEs, can also be recognised by the plant as immunity elicitors, triggering defence responses (Schultink et al. 2017; Strauß et al. 2012). Genome sequencing has long been an essential tool for understanding the genetic relationships among strains and mining potential virulence factors in plant pathogens. As of July 2025, the NCBI database includes 35 complete genome sequences of X. translucens isolates, none of which originate from China (Shah et al. 2019). This highlights the need to broaden the geographical scope of pathogen isolation and identification to enhance our understanding of this pathogen. Sequencing the complete genomes of BLS agents could yield valuable resources for investigating their virulence profiles and population genomics, potentially paving the way for breeding small-grain cereals with improved disease resistance. In this study, we present the first complete genome sequence of Xtu isolated from China and provide an analysis focused on its virulence effectors. The bacterial strain used in this study was isolated recently from leaf tissues of infected wheat cultivar Xindong 18 collected from Emin County, Tacheng District, Xinjiang Province, China, in July 2023. The affected plants exhibited parallel yellow-brown streaks on their leaves, originating from water-soaked lesions (Figure 1A). To isolate the causal agent, infected leaf pieces from 10 diseased samples were surface-disinfected in 75% ethanol for 30 s, rinsed three times with sterile water, and homogenised. Serial dilutions of the suspension were plated on nutrient agar (NA) (Khojasteh et al. 2019). After 72 h of incubation at 28°C, bacterial isolates were assessed using Gram staining and colony morphology on NA and yeast extract-dextrose-calcium carbonate (YDC) media. Among the strains displaying similar Gram reactions and colony characteristics on both media, a single representative xanthomonad-like strain, designated XtXJ-1, was selected for further study. The strain was gram-negative and exhibited a round shape with a smooth, glossy surface on NA and formed pale-yellow colonies on YDC (Figure 1B). Subsequently, the strain was identified by a diagnostic PCR test using a pathovar-specific primer pair (Alvandi et al. 2023). The specific primer pair successfully amplified a 229-bp DNA fragment (Figure 1C), confirming the strain's classification as pv. undulosa. Following this, pathogenicity tests were conducted using a spray inoculation method on leaves of 16-day-old wheat (cv. Yangmai 158) under conditions of 75% RH and 28°C (Shah et al. 2019). At 6 days post-inoculation (dpi), water-soaked lesions had expanded on wheat seedling leaves and progressively developed into necrosis until the leaves died, whereas control plants sprayed with sterile water remained asymptomatic (Figure 1D). The same isolate was successfully reisolated from inoculated wheat leaf tissues and confirmed through colony morphology, pathovar-specific PCR and Sanger sequencing of housekeeping genes (fyuA, gyrB, dnaK and rpoD; GenBank accessions PV945493–PV945496) with subsequent BLAST verification, thus fulfilling Koch's postulates. Moreover, the hypersensitive reaction (HR) was assessed on tobacco leaves by applying a bacterial suspension derived from a 48-h culture on NA (Khojasteh et al. 2019) at a concentration of 108 CFU/mL. To assess the virulence of XtXJ-1, we syringe infiltrated leaves of wheat (cv. Yangmai 158) and barley (Hua 30) with a bacterial suspension (OD600 = 0.2) using a needleless syringe. XtXJ-1 caused water-soaking lesions on both wheat and barley, confirming its virulence, albeit at a lower level compared to the highly virulent reference strain XtLr8 (Figure 1E,F). Furthermore, bacterial population dynamics revealed that virulence levels were not directly correlated with in planta bacterial multiplication (Figure 1G), suggesting that other factors beyond bacterial load contribute to disease severity. To investigate the presence of TALEs of strain XtXJ-1 and to assess the potential existence of unique TALEs, Southern blotting was performed following an established method (Khojasteh et al. 2020). The results revealed that XtXJ-1 harbours seven tal genes of different sizes, showing a distinct TALE pattern compared to the X. translucens strains previously reported (Khojasteh et al. 2019; Shah et al. 2019, 2023) (Figure 1H). To uncover further insights into this strain, particularly regarding any unique TALEs, we sent the strain XtXJ-1 for whole genome sequencing. To prepare for whole genome sequencing, the strain was incubated in nutrient broth (NB) overnight at 28°C on a shaker at 220 rpm. Then, 2 mL of this pre-inoculum was transferred to 100 mL of NB and incubated under the same conditions for 6 h to achieve an OD600 of 0.9–1.0. Total genomic DNA of the strain was isolated using the AxyPrep Bacterial Genomic DNA Miniprep Kit (Axygen) from harvested bacterial culture according to the manufacturer's recommendations. The quantity and quality of the isolated DNA were assessed using an ND-100 spectrophotometer (NanoDrop Technologies) as stated previously (Shah et al. 2021). Genomic DNA of XtXJ-1 was sequenced by long-read Nanopore sequencing technology and Illumina Hiseq 6000 (OE Biotech Co. Ltd). The library preparation was carried out using the Nextera DNA Library Prep kit, following the manufacturer's protocols. Quantification of the prepared library was verified using a Qubit 3.0 fluorometer (Invitrogen). The raw reads were filtered and then assembled with Unicycler v. 0.4.9 (Wick et al. 2017) using the combination of single-molecule real-time (SMRT) and the second-generation sequencing data. Nanopore sequencing data were assembled using Canu v. 1.8 (Chin et al. 2016; Hunt et al. 2015) for preliminary genome assembly. Finally, the de novo assembly was further polished with Pilon v. 1.23 (Bankevich et al. 2012) to correct the base level errors. GeneMarkS-2+ v. 6.1 (Lomsadze et al. 2021) and prodigal v. 2.6.3 (Hyatt et al. 2010) were used to predict genes in the assembled genome. The tRNA and rRNA families of the genome were predicted using tRNAscan-SE v. 2.0 (Chan et al. 2021) and Barrnap v. 0.7 software (Seemann 2013), respectively. Subsequently, genome annotation was carried out using DIAMOND software v. 0.9.29.130 (Buchfink et al. 2015) and visualised using Circos software v. 0.69 to gather gene function information. T3SEs were identified by EffectiveT3 v. 2.0.2 software with default parameters (Arnold et al. 2009). The genome of strain XtXJ-1 spans 4,722,928 bp (Figure 2A) and exhibits a G + C content of 67.7%, achieved with a sequencing coverage of 219×. The N50 value is 4,722,928, while the L50 is 1. Through gene element detection, we identified 56 tRNA genes, 4 rRNA genes, and a total of 4001 coding DNA sequences (CDS) (Table 1). Subsequent functional annotation analysis categorised 84 of these predicted genes mapped to the Clusters of Orthologous Groups (COGs) database, and 2203 genes were classified within KEGG pathways. Notably, the EffectiveT3 analysis revealed that 398 genes encode T3SEs (Table S1). Using the Average Nucleotide Identity (ANI)-m calculator JSpeciesWS with an all-vs.-all approach (Richter et al. 2015), we conducted a detailed analysis of our strain against the genome sequences of all completely assembled Xtt and Xtu strains available in the NCBI database. An unweighted pair-group method with arithmetic means (UPGMA) distance clustering plot was created based on ANI-m values and visualised in R software. Comparative genomic analysis revealed that strain XtXJ-1 is closely related to other X. translucens strains and is classified as pv. undulosa. Within the Xtu clade, the Chinese strain XtXJ-1 forms a distinct branch, clearly separated from Xtu strains isolated from Argentina, Canada, Iran, Mexico, the United States and Uruguay. The closest relative to XtXJ-1 was the Iranian Xtu strain ICMP11055, sharing 99.46% estimated nucleotide identity (Figure 2B). Furthermore, TALE genes were annotated and categorised using AnnoTALE v. 1.5 (Grau et al. 2016). Our sequence analysis confirmed the findings from Southern blotting, revealing that strain XtXJ-1 harbours seven TALE genes located on its chromosome (Figure 2C). Of these seven TALEs, five are shared with other X. translucens strains and belong to the classes TalDA, TalDD, TalCT, TalDF and TalCZ. The remaining two TALEs, classified as TalDB and TalKR, exhibit distinct RVD sequences that differ by several amino acids from previously characterised TALE classes in X. translucens strains (Table 2). Such subtle variations may reflect adaptive evolution in response to promoter differences among host wheat cultivars, potentially conferring selective advantages under local environmental pressures (Xu et al. 2019). To identify Xop effectors, we conducted BLASTp searches using amino acid sequences retrieved from EuroXanth DokuWiki (Costa et al. 2024), applying cut-off criteria of E-value ≤ 1e−5, ≥ 50% query coverage, and ≥ 35% identity (Pearson 2013). Using the same approach, we extended our analysis to all currently sequenced and completely assembled Xtu genomes available in NCBI and visualised the distribution of identified effectors using R software (Figure 2D). For strain XtXJ-1, we identified 30 Xop genes on the chromosome, with XopP, XopX, XopAV1 and XopAZ appearing in multiple copies. Alongside the conserved core set of Xop, this strain also contained XopAO, which has been reported less frequently in previous studies. The presence of a less common Xop may signify specialised functions or adaptations that allow XtXJ-1 to develop in specific environmental conditions or host interactions. In recent years, identification, genome sequencing and comparative genomics of X. translucens have advanced rapidly on a global scale (Fu et al. 2024; Goettelmann et al. 2021; Heiden et al. 2023; Ledman et al. 2023; Shah et al. 2023). However, molecular studies of X. translucens in China—particularly those focusing on virulence effectors—remain relatively limited. In this study, we have presented the isolation of a novel strain of Xtu, designated XtXJ-1, from infected wheat leaves in Xinjiang, China, which is the first whole genome annotation of an Xtu strain from China. To establish the novelty of this strain, we conducted a Southern blot analysis, which revealed that XtXJ-1 harbours seven TALEs with a distinct hybridisation pattern compared to those reported for previously characterised X. translucens strains (Khojasteh et al. 2020; Peng et al. 2016), indicating differences in tal gene composition or organisation. Following this, the genome of this strain was sequenced using both Illumina and Oxford Nanopore PromethION long-read direct DNA sequencing technologies. Interestingly, phylogenetic analysis showed that Chinese strain XtXJ-1 is placed in a different branch from other Xtu strains. This finding suggests that XtXJ-1 may have diverged significantly from other strains, potentially due to geographic isolation, host specialisation or distinct environmental pressures in Xinjiang, China. However, additional genome sequences of Xtu strains, particularly from China or other under-represented regions, are needed to confirm this observation. Furthermore, our analysis focused on TALEs and Xops, revealing two novel TALEs and an uncommon Xop (Gutierrez-Castillo et al. 2024; Shah et al. 2021). The current findings supported our earlier observation that the distribution of TALEs within the genus is uneven, and this variability extends to species and pathovars as well (Khojasteh et al. 2020). The conservation and variability of TALEs suggest adaptive evolution among strains; the uncommon TALEs and Xops possessed by XtXJ-1 may serve as significant virulence factors, potentially overcoming existing plant defence mechanisms and posing a severe threat to wheat production. In conclusion, the genome profile of the Chinese Xtu strain XtXJ-1 provides valuable insights into the genetic makeup of Xtu and serves as a foundation for future studies of the BLS pathosystem in cereals. This genomic data serves as a valuable resource for investigating the roles of bacterial effectors in pathogenicity and identifying effector targets in small-grain cereals, which is crucial for developing crop resistance breeding strategies. The whole genome sequence of XtXJ-1 not only facilitates the prediction of pathogenicity-associated genes but also enables the development of advanced genome-based detection techniques for more efficient and cost-effective monitoring of seed infections. As the incidence and geographical range of BLS continue to expand, particularly amid climate change, it is critical to recognise emerging threats. Therefore, ongoing research into X. translucens is essential for safeguarding agricultural productivity against this evolving pathogen. Qi Wang: writing – original draft, software, visualization, methodology, investigation, validation, formal analysis, data curation. Ying Li: validation. Linlin Liu: validation. Yong Wang: validation. Jiali Yan: validation. Syed Mashab Ali Shah: methodology, formal analysis. Zhengyin Xu: conceptualization. Xiameng Xu: conceptualization. Baishi Hu: resources, investigation. Haifeng Gao: resources, investigation. Moein Khojasteh: funding acquisition, writing – review and editing, conceptualization, supervision. Gongyou Chen: supervision, writing – review and editing, project administration, conceptualization. This study was supported by the National Natural Science Foundation of China (Grant 32350410395), the National Foreign Expert Program (Grant QN2023134006) of the Ministry of Science and Technology of the People's Republic of China, the Morning Star Postdoctoral Incentive Program by Shanghai Jiao Tong University, and the Shanghai Postdoctoral Excellence Program (Grant 2023339). The authors declare no conflicts of interest. The complete genome assembly of Chinese Xtu strain XtXJ-1 has been deposited at GenBank under the accession number CP156747, with the nucleotide sequences of housekeeping genes fyuA, gyrB, dnaK and rpoD deposited under accession numbers PV945493–PV945496. Table S1:List of type III secretion system effectors (T3SEs) in Xanthomonas translucens pv. undulosa strain XtXJ-1 identified using EffectiveT3, AnnoTALE and BLASTp. The table includes gene IDs, predicted effector names and genomic location. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.