The in planta proteome of wild type strains of the fire blight pathogen, Erwinia amylovora.

UNLABELLED: Erwinia amylovora is a Gram-negative plant pathogen that causes fire blight. This disease affects most members of the Rosaceae family including apple and pear. Here, an infection model is introduced to study proteomic changes in a highly virulent E. amylovora strain upon interaction with its host as compared to a lower virulent strain. For this purpose separate shoots of apple rootstocks were wound-infected and when infection became systemic, bacterial cells were isolated and processed for analysis in a proteomics platform combining 2-D fluorescence difference gel electrophoresis and mass spectrometry. Comparing the proteome of the isolates, significant abundance changes were observed in proteins involved in sorbitol metabolism, amylovoran production as well as in protection against plant defense mechanisms. Furthermore several proteins associated with virulence were more abundant in the higher virulent strain. Changes at the proteome level showed good accordance at the transcript level, as was verified by RT-qPCR. In conclusion, this infection model may be a valuable tool to unravel the complexity of plant-pathogen interactions and to gain insight in the molecular mechanisms associated with virulence of E. amylovora, paving the way for the development of plant-protective interventions against this detrimental disease. SIGNIFICANCE: During this research a first time investigation was performed on the proteome of E. amylovora, grown inside a susceptible host plant. This bacterium is the causal agent of fire blight, which can affect most members of the Rosaceae family including apple and pear. To do so, an artificial infection model on shoots of apple rootstocks was optimized and employed. When infection was systemic, bacterial cells were extracted from the plant tissue followed by extraction of the proteins from the bacteria. Further processing of the proteins was done by using a 2-D fluorescence difference gel electrophoresis analysis followed by mass spectrometry. By the use of two strains differing in their virulent ability, we were able to draw conclusions concerning virulence and behavior of different strains inside the host. This research provides a model to investigate plant-pathogen interactions and more importantly, we identified possible new targets for the development of novel control methods against this devastating disease.

A comparative proteome analysis reveals flagellin, chemotaxis regulated proteins and amylovoran to be involved in virulence differences between Erwinia amylovora strains.

Erwinia amylovora is a Gram-negative bacterium that causes the destructive disease fire blight affecting most members of the Rosaceae family, of which apple and pear are economically the most important hosts. E. amylovora has been considered as a homogeneous species in whole, although significant differences in virulence patterns have been observed. However, the underlying causes of the differences in virulence remain to be discovered. In a first-time comparative proteomic approach using E. amylovora, 2D differential in-gel electrophoresis (DIGE) was used to identify proteins that could explain the gradual difference in virulence between four different strains. Two important proteins were identified, FliC and CheY, both involved in flagella structure, motility and chemotaxis, which were more abundant in the least virulent strain. In the highly virulent strains the protein GalF, involved in amylovoran production, was more abundant, which was consistent with the higher expression of the gene and the higher amylovoran content in this strain in vitro. Together, these results confirm the involvement of amylovoran in virulence, but also imply an indirect role of flagellin in virulence as elicitor of plant defence. BIOLOGICAL SIGNIFICANCE: This research provides new insights into our current understanding of the virulence of Erwinia amylovora. This plant-pathogen is considered a homogeneous species although different strains show differences in virulence. Despite the efforts made on the genomic level which resulted in the discovery of virulence factors, the reason for the different virulence patterns between strains has not yet been identified. In our lab we used a comparative proteomic approach, which has never been published before, to identify proteins involved in these differences between strains and hereby possibly involved in virulence. Our results provide interesting insights in virulence and present us with the opportunity to glance into the proteome of E. amylovora.

Phytoextraction of toxic metals: a central role for glutathione.

Phytoextraction has a promising potential as an environmentally friendly clean-up method for soils contaminated with toxic metals. To improve the development of efficient phytoextraction strategies, better knowledge regarding metal uptake, translocation and detoxification in planta is a prerequisite. This review highlights our current understanding on these mechanisms, and their impact on plant growth and health. Special attention is paid to the central role of glutathione (GSH) in this process. Because of the high affinity of metals to thiols and as a precursor for phytochelatins (PCs), GSH is an essential metal chelator. Being an important antioxidant, a direct link between metal detoxification and the oxidative challenge in plants growing on contaminated soils is observed, where GSH could be a key player. In addition, as redox couple, oxidized and reduced GSH transmits specific information, in this way tuning cellular signalling pathways under environmental stress conditions. Possible improvements of phytoextraction could be achieved by using transgenic plants or plant-associated microorganisms. Joined efforts should be made to cope with the challenges faced with phytoextraction in order to successfully implement this technique in the field.

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants.

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the beta-glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.

A promoter from sugarcane bacilliform badnavirus drives transgene expression in banana and other monocot and dicot plants.

A 1369 bp DNA fragment (Sc) was isolated from a full-length clone of sugarcane bacilliform badnavirus (ScBV) and was shown to have promoter activity in transient expression assays using monocot (banana, maize, millet and sorghum) and dicot plant species (tobacco, sunflower, canola and Nicotiana benthamiana). This promoter was also tested for stable expression in transgenic banana and tobacco plants. These experiments showed that this promoter could drive high-level expression of the beta-glucuronidase (GUS) reporter gene in most plant cells. The expression level was comparable to the maize ubiquitin promoter in standardised transient assays in maize. In transgenic banana plants the expression levels were variable for different transgenic lines but was generally comparable with the activities of both the maize ubiquitin promoter and the enhanced cauliflower mosaic virus (CaMV) 35S promoter. The Sc promoter appears to express in a near-constitutive manner in transgenic banana and tobacco plants. The promoter from sugarcane bacilliform virus represents a useful tool for the high-level expression of foreign genes in both monocot and dicot transgenic plants that could be used similarly to the CaMV 35S or maize polyubiquitin promoter.