In planta proteomics and proteogenomics of the biotrophic barley fungal pathogen blumeria graminis f. sp hordei

To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity. Molecular & Cellular Proteomics 8: 2368-2381, 2009.

Plant pathogens as suppressors of host defense

This chapter reviews our current knowledge about mechanisms of suppression developed by pathogens to avoid host defense responses. In general, plants perceive pathogens by diverse pathogen- or microbe- or even damage-associated molecular patterns (PAMPs, MAMPs, DAMPs) and induce a variety of defense mechanisms referred to as horizontal or basal resistance, nowadays designated PAMP-triggered immunity (PTI). In addition, plants can also recognize specific pathogen-derived effectors and have derived a highly specific defense response termed effector-triggered immunity (ETI), classically called R gene-mediated, specific or vertical resistance. Both PTI and ETI are responses to potential dangers and have common components. Fungal, oomycete, and bacterial pathogens have evolved various effector-based mechanisms of suppression that interfere with such components. Plants strongly depend on RNA gene silencing to interfere with viral pathogens. Plant viruses counteract this response by encoding suppressor proteins of RNA silencing.

Phytohormones and plant-herbivore-pathogen interactions: integrating the molecular with the ecological

Current research into indirect phytopathogen–herbivore interactions (i.e., interactions mediated by the host plant) is carried out in two largely independent directions: ecological/mechanistic and molecular. We investigate the origin of these approaches and their strengths and weaknesses. Ecological studies have determined the effect of herbivores and phytopathogens on their host plants and are often correlative: the need for long-term manipulative experiments is pressing. Molecular/cellular studies have concentrated on the role of signaling pathways for systemic induced resistance, mainly involving salicylic acid and jasmonic acid, and more recently the cross-talk between these pathways. This cross-talk demonstrates how interactions between signaling mechanisms and phytohormones could mediate plant–herbivore–pathogen interactions. A bridge between these approaches may be provided by field studies using chemical induction of defense, or investigating whole-organism mechanisms of interactions among the three species. To determine the role of phytohormones in induced resistance in the field, researchers must combine ecological and molecular methods. We discuss how these methods can be integrated and present the concept of “kaleidoscopic defense.” Our recent molecular-level investigations of interactions between the herbivore Gastrophysa viridula and the rust fungus Uromyces rumicis on Rumex obtusifolius, which were well studied at the mechanistic and ecological levels, illustrate the difficulty in combining these different approaches. We suggest that the choice of the right study system (possibly wild relatives of model species) is important, and that molecular studies must consider the environmental conditions under which experiments are performed. The generalization of molecular predictions to ecologically realistic settings will be facilitated by “middle-ground studies” concentrating on the outcomes of the interactions.

In planta proteomics and proteogenomics of the biotrophic barley fungal pathogen blumeria graminis f. sp hordei

To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity. Molecular & Cellular Proteomics 8: 2368-2381, 2009.

Microbial impacts on plant-herbivore interactions: the indirect effects of a birch pathogen on a birch aphid

The role of indirect interactions in structuring communities is becoming increasingly recognised. Plant fungi can bring about changes in plant chemistry which may affect insect herbivores that share the same plant, and hence the two may interact indirectly. This study investigated the indirect effects of a fungal pathogen (Marssonina betulae) of silver birch (Betula pendula) on an aphid (Euceraphis betulae), and the processes underpinning the interaction. There was a strong positive association between natural populations of the aphid and leaves bearing high fungal infection. In choice tests, significantly more aphids settled on leaves inoculated with the fungus than on asymptomatic leaves. Individual aphids reared on inoculated leaves were heavier, possessed longer hind tibiae and displayed enhanced embryo development compared with aphids reared on asymptomatic leaves; population growth rate was also positively correlated with fungal infection when groups of aphids were reared on inoculated branches. Changes in leaf chemistry were associated with fungal infection with inoculated leaves containing higher concentrations of free-amino acids. This may reflect a plant-initiated response to fungal attack in which free amino acids from the degradation of mesophyll cells are translocated out of infected leaves via the phloem. These changes in plant chemistry are similar to those occurring during leaf senescence, and are proposed as the mechanistic basis for the positive interaction between the fungus and aphid.

Evolutionary bi-stability in pathogen transmission mode

Many pathogens transmit to new hosts by both infection (horizontal transmission) and transfer to the infected host's offspring (vertical transmission). These two transmission modes require speci®c adap- tations of the pathogen that can be mutually exclusive, resulting in a trade-off between horizontal and vertical transmission. We show that in mathematical models such trade-offs can lead to the simultaneous existence of two evolutionary stable states (evolutionary bi-stability) of allocation of resources to the two modes of transmission. We also show that jumping between evolutionary stable states can be induced by gradual environmental changes. Using quantitative PCR-based estimates of abundance in seed and vege- tative parts, we show that the pathogen of wheat, Phaeosphaeria nodorum, has jumped between two distinct states of transmission mode twice in the past 160 years, which, based on published evidence, we interpret as adaptation to environmental change. The ®nding of evolutionary bi-stability has impli- cations for human, animal and other plant diseases. An ill-judged change in a disease control programme could cause the pathogen to evolve a new, and possibly more damaging, combination of transmission modes. Similarly, environmental changes can shift the balance between transmission modes, with adverse effects on human, animal and plant health.

A model for the evolution of pathogen-induced leaf shedding

A size-structured plant population model is developed to study the evolution of pathogen-induced leaf shedding under various environmental conditions. The evolutionary stable strategy (ESS) of the leaf shedding rate is determined for two scenarios: i) a constant leaf shedding strategy and ii) an infection load driven leaf shedding strategy. The model predicts that ESS leaf shedding rates increase with nutrient availability. No effect of plant density on the ESS leaf shedding rate is found even though disease severity increases with plant density. When auto-infection, that is increased infection due to spores produced on the plant itself, plays a key role in further disease increase on the plant, shedding leaves removes disease that would otherwise contribute to disease increase on the plant itself. Consequently leaf shedding responses to infections may evolve. When external infection, that is infection due to immigrant spores, is the key determinant, shedding a leaf does not reduce the force of infection on the leaf shedding plant. In this case leaf shedding will not evolve. Under a low external disease pressure adopting an infection driven leaf shedding strategy is more efficient than adopting a constant leaf shedding strategy, since a plant adopting an infection driven leaf shedding strategy does not shed any leaves in the absence of infection, even when leaf shedding rates are high. A plant adopting a constant leaf shedding rate sheds the same amount of leaves regardless of the presence of infection. Based on the results we develop two hypotheses that can be tested if the appropriate plant material is available.

Spatio-temporal analysis of an invasive plant pathogen (Phytophthora ramorum) in England and Wales

Phytophthora ramorum is a damaging invasive plant pathogen and was first discovered in the UK in 2002. Spatial point analyses were applied to the occurrence of this disease in England and Wales during the period of 2003-2006 in order to assess its spatio-temporal spread. Out of the 4301 garden centres and nurseries (GCN) surveyed, there were 164, 105, 123 and 41 sites with P. ramorum in 2003, 2004, 2005 and 2006, respectively. Spatial analysis of the observed point patterns of GCN outbreaks suggested that these sites were significantly clumped within a radius of ca 60 km in 2003, but not in later years. Further analyses were conducted to determine the relationship of GCN outbreak sites over two consecutive years and thus to infer possible disease spread over time. This analysis suggested that disease spread among GCN sites was most likely to have occurred within a distance of 60 km for 2003-2004, but not for the later years. There were 35, 63, 81 and 58 sites with P. ramorum in the semi-natural environment (SNE). Analyses were carried out to assess whether infected GCN sites could act as an inoculum source of infected SNE plants or vice versa. In all years, there was a significant spatial closeness among GCN and SNE outbreak sites within a distance of 1 km. But a significant relationship over a longer distance (within 60 km) was only observed between cases in 2003 and 2004. These analyses suggest that statutory actions taken so far appear to have reduced the extent of long-distance spread of P. ramorum among garden centres and nurseries, but not the disease spread at a shorter distance between GCN and SNE sites.

Apical leaf necrosis as a defence mechanism against pathogen attack: effects of high nutrient availability on onset and rate of necrosis

An outdoor experiment was conducted to increase understanding of apical leaf necrosis in the presence of pathogen infection. Holcus lanatus seeds and Puccinia coronata spores were collected from two adjacent and otherwise similar habitats with differing long-term N fertilization levels. After inoculation, disease and necrosis dynamics were observed during the plant growing seasons of 2003 and 2006. In both years high nutrient availability resulted in earlier disease onset, a higher pathogen population growth rate, earlier physiological apical leaf necrosis onset and a reduced time between disease onset and apical leaf necrosis onset. Necrosis rate was shown to be independent of nutrient availability. The results showed that in these nutrient-rich habitats H. lanatus plants adopted necrosis mechanisms which wasted more nutrients. There was some indication that these necrosis mechanisms were subject to local selection pressures, but these results were not conclusive. The findings of this study are consistent with apical leaf necrosis being an evolved defence mechanism.

The staphylococcus aureus surface protein IsdA mediates resistance to the innate defenses of human skin

Resistance to human skin innate defenses is crucial for survival and carriage of Staphylococcus aureus, a common cutaneous pathogen and nasal colonizer. Free fatty acids extracted from human skin sebum possess potent antimicrobial activity against S. aureus. The mechanisms by which S. aureus overcomes this host defense during colonization remain unknown. Here, we show that S. aureus IsdA, a surface protein produced in response to the host, decreases bacterial cellular hydrophobicity rendering them resistant to bactericidal human skin fatty acids and peptides. IsdA is required for survival of S. aureus on live human skin. Reciprocally, skin fatty acids prevent the production of virulence determinants and the induction of antibiotic resistance in S. aureus and other Gram-positive pathogens. A purified human skin fatty acid was effective in treating systemic and topical infections of S. aureus suggesting that our natural defense mechanisms can be exploited to combat drug-resistant pathogens.