Genetic characterization of the interaction between Italian ryegrass (Lolium multiflorum Lam.) and Xanthomonas translucens pv. graminis

Abstract

Grasslands are among the largest ecosystems in the world and represent the main source of natural low-cost feed for ruminants. Ryegrasses are major components of such grassland systems and Italian ryegrass (Lolium multiflorum Lam.) is a very widely used plant species for intensive hay and silage production. Xanthomonas translucens pv. graminis (Xtg) is a γ-proteobacterial plant pathogen and causes bacterial wilt on a number of forage grass species including the genera Agrostis, Alopecurus, Bromus, Dactylis, Festuca, Lolium, Phleum, Poa and Trisetum. The pathogen spreads through wounds and breaches caused when mowing with contaminated equipment. Compared to all plant species included in the host range of Xtg, L. multiflorum is particularly susceptible and the disease can lead to complete yield losses in pure and mixed stands depending on cultivar susceptibility. Breeding for resistant cultivars by means of phenotypic recurrent selection is the most practicable means of disease control and has lead to the development of cultivars with improved resistance to bacterial wilt. Nevertheless, due to the out-breeding reproduction mode of L. multiflorum and the population-based breeding schemes used for breeding forage grasses, further progress is difficult to obtain and highly susceptible individuals still occur in advanced breeding material. Since resistance mechanisms of L. multiflorum and virulence mechanisms of Xtg are largely unknown, the main objective of this thesis was to understand in more detail the interaction of Xtg with L. multiflorum. The application of genomic approaches combined with transcriptome analyses enables the identification of genes and genomic regions that affect host resistance and pathogen virulence. The first aim of this thesis was to elucidate the existence of race-specificity in the L. multiflorum-Xtg interaction and to identify genomic regions that contribute to Xtg resistance using marker-trait associations (chapter 2). No major race-specific interactions were found in 62 L. multiflorum genotypes infected with six Xtg isolates. Molecular marker-resistance associations revealed one simple sequence repeat (SSR) marker on linkage group (LG) 5 to be significantly associated with bacterial wilt resistance using the same plant genotypes across all six bacterial isolates. The identified SSR marker explained up to 37.4% of the total variance of area under the disease progress curve (AUDPC) values. The second aim of this thesis was to identify candidate genes in L. multiflorum for Xtg resistance by means of cross-species hybridization to a cDNA microarray of perennial ryegrass (L. perenne) using a resistant and a susceptible L. multiflorum genotype (chapter 3). Major transcriptomic differences were observed between the susceptible and the resistant L. multiflorum genotype especially after Xtg infection. Transcriptome analyses of solely the resistant genotype after Xtg infection revealed a number of promising candidate genes for bacterial wilt resistance such as the low silicon transporter (Lsi1) and the germin-like protein 6 (GLP6). To investigate bacterial factors which might be the target of plant resistance mechanisms, we screened for known virulence genes in Xtg. Phytopathogenic Xanthomonas spp. have many virulence associated characteristics in common; nevertheless, the symptoms caused and the significance of these virulence factors for the interaction with their hosts may differ greatly. However, the type III secretion system (T3SS) represents one of the major virulence factors found in most Xanthomonas spp. and mediates the delivery of disease promoting effector proteins and avirulence products into their host. Screening Xtg for genes encoding T3SS components was performed using conserved primers based on publicly available sequences of Xanthomonas spp. Despite high conservation among the sequences encoding T3SS components of Xanthomonas spp., this approach was only successful for the hrpG gene of Xtg. After amplification and sequencing of the hrpG gene, a mutant deficient of the hrpG gene was obtained by means of double homologous recombination (chapter 4). Virulence of the ΔhrpG mutant was significantly reduced when infecting L. multiflorum plants. However, in planta analyses revealed that the ΔhrpG mutant was still able to survive and multiply inside its host. Due to distant phylogenetic relationships between Xtg and the other sequenced Xanthomonas spp. and in order to elucidate the existence of a T3SS and other virulence mechanisms of Xtg, the whole genome of the Xtg29 isolate was sequenced by means of 454 sequencing. This shotgun sequencing approach enabled a preliminary analysis of the gene cluster encoding components of the T3SS, genes encoding effectors and other bacterial virulence factors (chapter 5) and has revealed an entire T3SS gene cluster and genes encoding 22 putative effector proteins. Additional functional characterization of the genes encoding virulence factors and effector proteins may enable the identification of resistance mechanisms inside the host plants. Overall, the data presented in this thesis provides a more comprehensive knowledge of the interaction between Xtg and L. multiflorum and represent a valuable basis for the development of tools for marker-assisted selection (MAS) in the future.