Complete Genome Sequencing and Targeted Mutagenesis Reveal Virulence Contributions of Tal2 and Tal4b of Xanthomonas translucens pv. undulosa ICMP11055 in Bacterial Leaf Streak of Wheat

Abstract

Bacterial leaf streak caused by Xanthomonas translucens pv. undulosa (Xtu) is an important disease of wheat (Triticum aestivum) and barley (Hordeum vulgare) worldwide. Transcription activator-like effectors (TALEs) play determinative roles in many of the plant diseases caused by the different species and pathovars of Xanthomonas, but their role in this disease has not been characterized. ICMP11055 is a highly virulent Xtu strain from Iran. The aim of this study was to better understand genetic diversity of Xtu and to assess the role of TALEs in bacterial leaf streak of wheat by comparing the genome of this strain to the recently completely sequenced genome of a U.S. Xtu strain, and to several other draft X. translucens genomes, and by carrying out mutational analyses of the TALE (tal) genes the Iranian strain might harbor. The ICMP11055 genome, including its repeat-rich tal genes, was completely sequenced using single molecule, real-time technology (Pacific Biosciences). It consists of a single circular chromosome of 4,561,583 bp, containing 3,953 genes. Whole genome alignment with the genome of the United States Xtu strain XT4699 showed two major re-arrangements, nine genomic regions unique to ICMP11055, and one region unique to XT4699. ICMP110055 harbors 26 non-TALE type III effector genes and seven tal genes, compared to 25 and eight for XT4699. The tal genes occur singly or in pairs across five scattered loci. Four are identical to tal genes in XT4699. In addition to common repeat-variable diresidues (RVDs), the tal genes of ICMP11055, like those of XT4699, encode several RVDs rarely observed in Xanthomonas, including KG, NF, Y∗, YD, and YK. Insertion and deletion mutagenesis of ICMP11055 tal genes followed by genetic complementation analysis in wheat cv. Chinese Spring revealed that Tal2 and Tal4b of ICMP11055 each contribute individually to the extent of disease caused by this strain. A largely conserved ortholog of tal2 is present in XT4699, but for tal4b, only a gene with partial, fragmented RVD sequence similarity can be found. Our results lay the foundation for identification of important host genes activated by Xtu TALEs as targets for the development of disease resistant varieties.

The translucens group of Xanthomonas translucens: Complicated and important pathogens causing bacterial leaf streak on cereals

“…TALE genes have been identified from three X . translucens strains with a complete genome sequence as well as a few strains with a draft genome sequence (Pesce et al , ; Jaenicke et al ., ; Peng et al , ; Charkhabi et al , ). X .…”

“…Sequence comparison revealed that the two strains had several unique RVDs, including KG, QD, Y*, YK and YD, which have not been identified from other Xanthomas spp. (Peng et al , ; Charkhabi et al , ). It was also found that some TALEs that are widely distributed in other xanthomonads are less frequently present in X .…”

“…It was also found that some TALEs that are widely distributed in other xanthomonads are less frequently present in X . translucens (Charkhabi et al , ).…”

“…translucens pv. translucens strain (DSM 18974 from the United States) (Jaenicke et al ., ; Peng et al , ; Charkhabi et al , ). The main features for the three complete genomes are listed in Table .…”

“…The remaining genome sequences were fragmented containing variable numbers of scaffolds or contigs (see the above web link) and the genome size of these strains ranged from 4.1 to 4.8 Mb. Size difference was observed among different pathovars and also among different strains within a pathovar (Charkhabi et al , ; Hersemann et al , ). The annotated protein‐encoding genes in published X. translucens genomes ranged from 3,160 to 4,413, with pv.…”