The noncanonical type III secretion system of Xanthomonas translucens pv. graminis is essential for forage grass infection

Abstract

Xanthomonas translucens pv. graminis (Xtg) is a gammaproteobacterium that causes bacterial wilt on a wide range of forage grasses. To gain insight into the host-pathogen interaction and to identify the virulence factors of Xtg, we compared a draft genome sequence of one isolate (Xtg29) with other Xanthomonas spp. with sequenced genomes. The type III secretion system (T3SS) encoding a protein transport system for type III effector (T3E) proteins represents one of the most important virulence factors of Xanthomonas spp. In contrast with other Xanthomonas spp. assigned to clade 1 on the basis of phylogenetic analyses, we identified an hrp (hypersensitive response and pathogenicity) gene cluster encoding T3SS components and a representative set of 35 genes encoding putative T3Es in the genome of Xtg29. The T3SS was shown to be divergent from the hrp gene clusters of other sequenced Xanthomonas spp. Xtg mutants deficient in T3SS regulating and structural genes were constructed to clarify the role of the T3SS in forage grass colonization. Italian ryegrass infection with these mutants led to significantly reduced symptoms (P < 0.05) relative to plants infected with the wild-type strain. This showed that the T3SS is required for symptom evocation. In planta multiplication of the T3SS mutants was not impaired significantly relative to the wild-type, indicating that the T3SS is not required for survival until 14 days post-infection. This study represents the first major step to understanding the bacterial colonization strategies deployed by Xtg and may assist in the identification of resistance (R) genes in forage grasses.

Comparative genomics of host adaptive traits in Xanthomonas translucens pv. graminis

“…Genome data based on sequencing of single paired-end libraries only, revealed 284 to 296 scaffolds, while the combination with mate-pair sequencing data, as applied for Xtg29, reduced the number of scaffolds to three. In contrast, a previous Xtg29 assembly had 788 contigs (> 500 bp) and 12 scaffolds upon sequencing with the 454 FLX System [12]. De novo annotation of the 454 assembly by using the same strategy as applied in the present study, revealed 3619 predicted coding sequences (CDS) while 3543 CDS were identified for the Illumina MiSeq-based assembly of the re-sequenced Xtg29 genome (Additional file 2: Table S2).…”

“…In view of the sequencing costs, homopolymer resolution, and observed GC bias in library preparations [22, 23], we decided to apply the Illumina MiSeq System by sequencing (2 × 250 bp) paired-end libraries constructed with the TrueSeq DNA LT Sample Prep Kit (Illumina Inc., San Diego, United States). The Swiss strain Xtg29, which has previously been sequenced by applying the Roche 454 Genome Sequencer FLX System [12], was re-sequenced in order to prevent bias in comparative genome analysis due to deviating sequencing strategies. Genome assembly of Xtg29 was moreover facilitated by (2 × 250 bp) sequencing of an additional 8 kb mate-pair library on an Illumina MiSeq System (Illumina Inc., San Diego, United States).…”

“…Moreover, plant-inducible promoter (PIP) boxes [41] were identified by regular expression search as described recently [12]. Class III signal peptides in type IV pilin sequences were identified based on the core motif [GAS]-[ACFGILMNPQSTVWY]4-[DE] as reported by Imam et al [42].…”

“…graminis strain Xtg29 revealed the presence of a non-canonical type III secretion system with deviations in gene order and sequence homology in comparison to the classical T3SS, which is well conserved among other Xanthomonas species [12]. Site-directed knockout mutagenesis of the main regulator HrpG and the structural components HrpE and HrcR in Xtg29 impaired bacterial virulence on Italian ryegrass, but the tested mutants were not affected in in planta colonization [12]. Therefore, it is likely that Xtg possesses further virulence factors, which crucially influence its pathogenicity on Italian ryegrass.…”

Complete Genome Sequence of the Barley Pathogen Xanthomonas translucens pv. translucens DSM 18974 T (ATCC 19319 T )

“…The Illumina reads were mapped with the Burrows–Wheeler aligner (6) onto the contig to obtain the consensus sequence of a circular chromosome of 4,715,357 bp with an average coverage of 223× and a G+C content of 67.7%. Automated genome annotation carried out by means of the GenDB platform (7) with the Prodigal gene prediction software (8) revealed 3,736 protein-coding genes, 54 tRNAs, and two rRNA operons.…”