Bacterial pathogenesis of plants: Future challenges from a microbial perspective

Plant infection is a complicated process. Upon encountering a plant, pathogenic microorganisms must first adapt to life on the epiphytic surface, and survive long enough to initiate an infection. Responsiveness to the environment is critical throughout infection, with intracellular and community-level signal transduction pathways integrating environmental signals and triggering appropriate responses in the bacterial population. Ultimately, phytopathogens must migrate from the epiphytic surface into the plant tissue using motility and chemotaxis pathways. This migration is coupled to overcoming the physical and chemical barriers to entry into the plant apoplast. Once inside the plant, bacteria use an array of secretion systems to release phytotoxins and protein effectors that fulfil diverse pathogenic functions (Fig. 1)(Phan Tran et al., 2011, Melotto & Kunkel, 2013)

Elicitation and evasion of plant innate immunity by beneficial and pathogenic bacteria

Plasma membrane-localized pattern recognition receptors (PRRs) are central components of the plant innate immune system. PRRs perceive characteristic microbial features, termed pathogen-associated molecular patterns (PAMPs), leading to pattern-triggered immunity (PTI). As PAMPs from both pathogenic and beneficial bacteria are potentially recognized, both must employ strategies to evade and/or suppress PTI. Here, I show that exopolysaccharides (EPS) and flagella-driven motility, both of which are regulated by the secondary messenger cyclic-di-GMP, are important virulence factors at different stages during Pseudomonas syringae pv. tomato (Pto) DC3000 infection of Arabidopsis thaliana. High levels of cyclic-di-GMP impaired flagellin accumulation in different Pseudomonas species, and helped bacteria to evade recognition by the PRR FLAGELLIN SENSING 2. In this case, immune evasion was fully explained by the effect of cyclic-di-GMP on flagellin synthesis rather than on EPS production. Nevertheless, an EPS-deficient Pto DC3000 mutant, Δalg/psl/wss, showed compromised virulence, and a combination of two types of EPS appeared to be required for optimal in planta proliferation. In a complementary project, I tested whether PAMP recognition affects the interaction between the legume Medicago truncatula and its symbiotic partner Sinorhizobium meliloti. I transferred the PRR EF-TU RECEPTOR (EFR) from A. thaliana to M. truncatula, conferring recognition of the bacterial EF-Tu protein. Expression of EFR protected M. truncatula against the root pathogen Ralstonia solanacearum without compromising the overall symbiotic interaction with nitrogen-fixing S. meliloti. My results indicate that rhizobium either avoids PAMP recognition, or actively suppresses immune signalling during the infection process. Finally, I engineered a PAMP in S. meliloti by replacing the eliciting inactive flg22 epitope (derived from flagellin) with an eliciting epitope. My results suggest that legumes can be engineered with novel PRRs, as a biotechnological approach for broad-spectrum disease resistance, without perturbing the nitrogen-fixing symbiosis. Overall, my work contributes to our understanding of the molecular interaction between plants and bacteria

Verstehen, Urteilen, Entscheiden: Vergleich zweier Bewertungsmodelle zur Grünen Gentechnik und ihr ethisches Verständnis

Sebastian Pfeilmeier, gegenwärtig Post Graduate Student der Molekularbiologie im Sainsbury Laboratory in Norwich, widmete sich in seiner Masterarbeit dem Vergleich zweier Bewertungsmodelle zur Grünen Gentechnik. Diskutiert wurden zwei Konzeptionen des Instituts TTN: die Studien "Grüne Gentechnik. Ein Bewertungsmodell" aus dem Jahr 2002 sowie das Webportal "Pflanzen.Forschung.Ethik". Die Masterarbeit unter dem Titel "Verstehen, Urteilen, Entscheiden" erscheint nun in gekürzter und überarbeiteter Form in der digitalen Publikationsreihe "TTN Edition". Sie entstand unter der Betreuung von PD Jörg Wernecke am Lehrstuhl für Philosophie und Wissenschaftstheorie der Technischen Universität München

High levels of cyclic-di-GMP in plant-associated Pseudomonas correlate with evasion of plant immunity

Summary: The plant innate immune system employs plasma membrane‐localized receptors that specifically perceive pathogen/microbe‐associated molecular patterns (PAMPs/MAMPs). This induces a defence response called pattern‐triggered immunity (PTI) to fend off pathogen attack. Commensal bacteria are also exposed to potential immune recognition and must employ strategies to evade and/or suppress PTI to successfully colonize the plant. During plant infection, the flagellum has an ambiguous role, acting as both a virulence factor and also as a potent immunogen as a result of the recognition of its main building block, flagellin, by the plant pattern recognition receptors (PRRs), including FLAGELLIN SENSING2 (FLS2). Therefore, strict control of flagella synthesis is especially important for plant‐associated bacteria. Here, we show that cyclic‐di‐GMP [bis‐(3′‐5′)‐cyclic di‐guanosine monophosphate], a central regulator of bacterial lifestyle, is involved in the evasion of PTI. Elevated cyclic‐di‐GMP levels in the pathogen Pseudomonas syringae pv. tomato (Pto) DC3000, the opportunist P. aeruginosa PAO1 and the commensal P. protegens Pf‐5 inhibit flagellin synthesis and help the bacteria to evade FLS2‐mediated signalling in Nicotiana benthamiana and Arabidopsis thaliana. Despite this, high cellular cyclic‐di‐GMP concentrations were shown to drastically reduce the virulence of Pto DC3000 during plant infection. We propose that this is a result of reduced flagellar motility and/or additional pleiotropic effects of cyclic‐di‐GMP signalling on bacterial behaviour

Adaptive remodeling of the bacterial proteome by specific ribosomal modification regulates Pseudomonas infection and niche colonisation

Post-transcriptional control of protein abundance is a highly important, underexplored regulatory process by which organisms respond to their environments. Here we describe an important and previously unidentified regulatory pathway involving the ribosomal modification protein RimK, its regulator proteins RimA and RimB, and the widespread bacterial second messenger cyclic-di-GMP (cdG). Disruption of rimK affects motility and surface attachment in pathogenic and commensal Pseudomonas species, with rimK deletion significantly compromising rhizosphere colonisation by the commensal soil bacterium P. fluorescens, and plant infection by the pathogens P. syringae and P. aeruginosa. RimK functions as an ATP-dependent glutamyl ligase, adding glutamate residues to the C-terminus of ribosomal protein RpsF and inducing specific effects on both ribosome protein complement and function. Deletion of rimK in P. fluorescens leads to markedly reduced levels of multiple ribosomal proteins, and also of the key translational regulator Hfq. In turn, reduced Hfq levels induce specific downstream proteomic changes, with significant increases in multiple ABC transporters, stress response proteins and non-ribosomal peptide synthetases seen for both ΔrimK and Δhfq mutants. The activity of RimK is itself controlled by interactions with RimA, RimB and cdG. We propose that control of RimK activity represents a novel regulatory mechanism that dynamically influences interactions between bacteria and their hosts; translating environmental pressures into dynamic ribosomal changes, and consequently to an adaptive remodeling of the bacterial proteome

Plant microbiota feedbacks through dose-responsive expression of general non-self response genes

The ability of plants to perceive and react to biotic and abiotic stresses is critical for their health. We recently identified a core set of genes consistently induced by members of the leaf microbiota, termed general non-self response (GNSR) genes. Here we show that GNSR components conversely impact leaf microbiota composition. Specific strains that benefited from this altered assembly triggered strong plant responses, suggesting that the GNSR is a dynamic system that modulates colonization by certain strains. Examination of the GNSR to live and inactivated bacteria revealed that bacterial abundance, cellular composition and exposure time collectively determine the extent of the host response. We link the GNSR to pattern-triggered immunity, as diverse microbe- or danger-associated molecular patterns cause dynamic GNSR gene expression. Our findings suggest that the GNSR is the result of a dose-responsive perception and signalling system that feeds back to the leaf microbiota and contributes to the intricate balance of plant–microbiome interactions

Pseudomonas syringae addresses distinct environmental challenges during plant infection through the coordinated deployment of polysaccharides

Prior to infection, phytopathogenic bacteria face a challenging environment on the plant surface, where they are exposed to nutrient starvation and abiotic stresses. Pathways enabling surface adhesion, stress tolerance, and epi phytic survival are important for successful plant pathogenesis. Understanding the roles and regulation of these path ways is therefore crucial to fully understand bacterial plant infections. The phytopathogen Pseudomonas syringae pv. tomato (Pst) encodes multiple polysaccharides that are implicated in biofilm formation, stress survival, and virulence in other microbes. To examine how these polysaccharides impact Pst epiphytic survival and pathogenesis, we ana lysed mutants in multiple polysaccharide loci to determine their intersecting contributions to epiphytic survival and infection. In parallel, we used qRT–PCR to analyse the regulation of each pathway. Pst polysaccharides are tightly coordinated by multiple environmental signals. Nutrient availability, temperature, and surface association strongly af fect the expression of different polysaccharides under the control of the signalling protein genes ladS and cbrB and the second messenger cyclic-di-GMP. Furthermore, functionally redundant, combinatorial phenotypes were observed for several polysaccharides. Exopolysaccharides play a role in mediating leaf adhesion, while α-glucan and alginate together confer desiccation tolerance. Our results suggest that polysaccharides play important roles in overcoming environmental challenges to Pst during plant infection