Identification of new protein-coding genes with a potential role in the virulence of the plant pathogen Xanthomonas euvesicatoria

Abstract

Background: Bacteria of the genus Xanthomonas are economically important plant pathogens. Pathogenicity of Xanthomonas spp. depends on the type III-secretion system and additional virulence determinants. The number of sequenced Xanthomonas genomes increases rapidly, however, accurate annotation of these genomes is difficult, because it relies on gene prediction programs. In this study, we used a mass-spectrometry (MS)-based approach to identify the proteome of Xanthomonas euvesicatoria (Xe) strain 85-10 also known as X. campestris pv. vesicatoria, a well-studied member of plant-pathogenic Xanthomonadaceae.

Results: Using different culture conditions, MS-datasets were searched against a six-frame-translated genome database of Xe. In total, we identified 2588 proteins covering 55% of the Xe genome, including 764 hitherto hypothetical proteins. Our proteogenomic approach identified 30 new protein-coding genes and allowed correction of the N-termini of 50 protein-coding genes. For five novel and two N-terminally corrected genes the corresponding proteins were confirmed by immunoblot. Furthermore, our data indicate that two putative type VI-secretion systems encoded in Xe play no role in bacterial virulence which was experimentally confirmed.

Conclusions: The discovery and re-annotation of numerous genes in the genome of Xe shows that also a well-annotated genome can be improved. Additionally, our proteogenomic analyses validates "hypothetical" proteins and will improve annotation of Xanthomonadaceae genomes, providing a solid basis for further studies.

Keywords: Genome re-annotation; Ortho proteogenomic; Proteogenome; T3SS; T4SS; T6SS; Translational start sites; Xanthomonas.

Fig. 1

Experimental workflow of the proteogenomic analysis of Xe. The Xe strains 85–10, 85* and 85-10ΔsX13 were grown in NYG, Minimal medium A pH 7 and XVM2, respectively, at 30 °C until OD₆₀₀ of either 0.5 (exponential), 0.8 (early stationary) or 1.2 (stationary). Proteins extracted from Xe 85–10, 85-10ΔsX13 and 85* cell lysates were separated by 12% SDS PAGE and Tricine PAGE. Gel fractions and cell lysate were digested by trypsin. Samples were analyzed by LC–MS/MS. A database search against a six-frame translation database of Xe 85–10 was performed. Peptides were mapped to the genome of Xe using a TBlastN-based approach. The dataset from Abendroth et al. is originally a comparative study between Xe strains 85–10 and 85-10ΔsX13 and is based on the original genome annotation. The MS spectra of this dataset were also searched against the six-frame database

Fig. 2

Overview of proteins identified in the proteogenomic analysis of Xe

Fig. 3

Schematic overview of chromosomal regions with detected new and corrected protein-coding genes. a Examples of three new protein-coding genes detected by proteogenomics, XCV_PG10, XCV_PG14 and XCV_PG15. b Examples of three corrected protein-coding genes detected by proteogenomics with a close-up of the raxA/B region. All six reading frames are shown. Grey: annotated CDS; orange dashes represent peptide-data detected by MS/MS. Black circle represents a stop codon; the green hexagon represents the possible translation start codon of raxB

Fig. 4

Gene organization of the dksA and XCV1265 regions. a and c dksA and XCV1265 loci of Xe. All six reading frames are shown. Grey: annotated CDS; orange dashes: peptide-data detected by MS/MS; green hexagons: possible translation start codons of dksA and XCV1265. b and d Analysis of potential translation start codons of DksA and XCV1265. Total protein extracts of Xe 85–10 containing pBRM-P(dksA), pBRM-P(dksA_GTG1), pBRM-P (dksA_GTG2), pBRM-P (dksA_GTG3), pBRM-P (XCV1265), pBRM-P (XCV1265_ATG1), pBRM-P (XCV1265_ATG2) or an empty vector (−) were separated by 12% SDS PAGE and analyzed by immunoblotting using a c-Myc-specific antibody. As loading control, membranes were reacted with a GroEL-specific antibody. Experiments were repeated at least twice with similar results

Fig. 5

Validation of five new protein-coding genes. a Detection of the protein synthesis of new Xe proteins. Total protein extracts of Xe 85–10 containing pBRM-P (XCV_PG13), pBRM-P (XCV_PG17), pBRM-P (XCV_PG07), pBRM-P (XCV_PG02) or pBRM-P (XCV_PG06) grown in NYG were separated by 15% SDS PAGE and analyzed by immunoblotting using a c-Myc-specific antibody. b Gene organization of the XCV_PG02 locus. XCV0209 and XCV_PG02 are highlighted. Arrows represent possible translation start codons of XCV_PG02

Fig. 6

Deletion of conserved T6SS components has no effect on Xe virulence. a Schematic representation of the two genomic T6SS loci in Xe. Gene numbers and commonly used gene names of T6SS components identified in Xe 85–10 are given. Dashed lines mark genes deleted in this study. b Xe strains 85–10, 85-10ΔTssFGH1ΔTssFGH2 and 85-10ΔTssI1ΔTssI2 were inoculated into susceptible pepper plants (ECW), resistant pepper plants (ECW-10R) and susceptible tomato plants with an OD₆₀₀ of 0.1. Phenotypes were documented 7 days post inoculation (7 dpi, ECW), 2 dpi (ECW-10R) and 8 dpi (tomato). c Xe strains 85–10, 85-10ΔTssI1ΔTssI2 and 85-10ΔhrcN (T3SS mutant) were inoculated in ECW plants with an OD₆₀₀ of 4 × 10⁻⁵. Bacterial multiplication in leaves was monitored for 12 days. d Xe strains 85–10, 85-10ΔTssFGH1ΔTssFGH2, 85-10ΔavrBs2 and 85-10ΔTssFGH1ΔTssFGH2ΔavrBs2 were inoculated with an OD₆₀₀ of 0.1 into leaves of pepper plants (ECW, ECW-10R, ECW-20R) and tomato plants. Phenotypes were documented 6 dpi (ECW), 2 dpi (ECW-10R, ECW-20R) and 9 dpi (tomato). Leaves of ECW-10R and ECW-20R plants were bleached in EtOH for better visualization of cell death reactions. Experiments were repeated twice with similar results