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.

Pseudomonas aeruginosa elastase disables proteinase-activated receptor 2 in respiratory epithelial cells

Pseudomonas aeruginosa, a major lung pathogen in cystic fibrosis (CF) patients, secretes an elastolytic metalloproteinase (EPa) contributing to bacterial pathogenicity. Proteinase-activated receptor 2 (PAR2), implicated in the pulmonary innate defense, is activated by the cleavage of its extracellular N-terminal domain, unmasking a new N-terminal sequence starting with SLIGKV, which binds intramolecularly and activates PAR2. We show that EPa cleaves the N-terminal domain of PAR2 from the cell surface without triggering receptor endocytosis as trypsin does. As evaluated by measurements of cytosolic calcium as well as prostaglandin E(2) and interleukin-8 production, this cleavage does not activate PAR2, but rather disarms the receptor for subsequent activation by trypsin, but not by the synthetic receptor-activating peptide, SLIGKV-NH(2). Proteolysis by EPa of synthetic peptides representing the N-terminal cleavage/activation sequences of either human or rat PAR2 indicates that cleavages resulting from EPa activity would not produce receptor-activating tethered ligands, but would disarm PAR2 in regard to any further activating proteolysis by activating proteinases. Our data indicate that a pathogen-derived proteinase like EPa can potentially silence the function of PAR2 in the respiratory tract, thereby altering the host innate defense mechanisms and respiratory functions, and thus contributing to pathogenesis in the setting of a disease like CF.

Modulation of endothelial cell KCa3.1 Channels during endothelium-derived hyperpolarizing factor signaling in mesenteric resistance arteries

Arterial hyperpolarization to acetylcholine (ACh) reflects coactivation of KCa3.1 (IKCa) channels and KCa2.3 (SKCa) channels in the endothelium that transfers through myoendothelial gap junctions and diffusible factor(s) to affect smooth muscle relaxation (endothelium-derived hyperpolarizing factor [EDHF] response). However, ACh can differentially activate KCa3.1 and KCa2.3 channels, and we investigated the mechanisms responsible in rat mesenteric arteries. KCa3.1 channel input to EDHF hyperpolarization was enhanced by reducing external [Ca2+]o but blocked either with forskolin to activate protein kinase A or by limiting smooth muscle [Ca2+]i increases stimulated by phenylephrine depolarization. Imaging [Ca2+]i within the endothelial cell projections forming myoendothelial gap junctions revealed increases in cytoplasmic [Ca2+]i during endothelial stimulation with ACh that were unaffected by simultaneous increases in muscle [Ca2+]i evoked by phenylephrine. If gap junctions were uncoupled, KCa3.1 channels became the predominant input to EDHF hyperpolarization, and relaxation was inhibited with ouabain, implicating a crucial link through Na+/K+-ATPase. There was no evidence for an equivalent link through KCa2.3 channels nor between these channels and the putative EDHF pathway involving natriuretic peptide receptor-C. Reconstruction of confocal z-stack images from pressurized arteries revealed KCa2.3 immunostain at endothelial cell borders, including endothelial cell projections, whereas KCa3.1 channels and Na+/K+-ATPase {alpha}2/{alpha}3 subunits were highly concentrated in endothelial cell projections and adjacent to myoendothelial gap junctions. Thus, extracellular [Ca2+]o appears to modify KCa3.1 channel activity through a protein kinase A-dependent mechanism independent of changes in endothelial [Ca2+]i. The resulting hyperpolarization links to arterial relaxation largely through Na+/K+-ATPase, possibly reflecting K+ acting as an EDHF. In contrast, KCa2.3 hyperpolarization appears mainly to affect relaxation through myoendothelial gap junctions. Overall, these data suggest that K+ and myoendothelial coupling evoke EDHF-mediated relaxation through distinct, definable pathways.

Lifestyle-induced metabolic inflexibility and accelerated ageing syndrome: insulin resistance, friend or foe?

The metabolic syndrome may have its origins in thriftiness, insulin resistance and one of the most ancient of all signalling systems, redox. Thriftiness results from an evolutionarily-driven propensity to minimise energy expenditure. This has to be balanced with the need to resist the oxidative stress from cellular signalling and pathogen resistance, giving rise to something we call 'redox-thriftiness'. This is based on the notion that mitochondria may be able to both amplify membrane-derived redox growth signals as well as negatively regulate them, resulting in an increased ATP/ROS ratio. We suggest that 'redox-thriftiness' leads to insulin resistance, which has the effect of both protecting the individual cell from excessive growth/inflammatory stress, while ensuring energy is channelled to the brain, the immune system, and for storage. We also suggest that fine tuning of redox-thriftiness is achieved by hormetic (mild stress) signals that stimulate mitochondrial biogenesis and resistance to oxidative stress, which improves metabolic flexibility. However, in a non-hormetic environment with excessive calories, the protective nature of this system may lead to escalating insulin resistance and rising oxidative stress due to metabolic inflexibility and mitochondrial overload. Thus, the mitochondrially-associated resistance to oxidative stress (and metabolic flexibility) may determine insulin resistance. Genetically and environmentally determined mitochondrial function may define a 'tipping point' where protective insulin resistance tips over to inflammatory insulin resistance. Many hormetic factors may induce mild mitochondrial stress and biogenesis, including exercise, fasting, temperature extremes, unsaturated fats, polyphenols, alcohol, and even metformin and statins. Without hormesis, a proposed redox-thriftiness tipping point might lead to a feed forward insulin resistance cycle in the presence of excess calories. We therefore suggest that as oxidative stress determines functional longevity, a rather more descriptive term for the metabolic syndrome is the 'lifestyle-induced metabolic inflexibility and accelerated ageing syndrome'. Ultimately, thriftiness is good for us as long as we have hormetic stimuli; unfortunately, mankind is attempting to remove all hormetic (stressful) stimuli from his environment.

Effects of corn silage derived from a genetically modified variety containing two Transgenes on feed intake, milk production, and composition, and the absence of detectable Transgenic deoxyribonucleic acid in milk in holstein dairy cows

The objectives were to compare the chemical composition, nutritive value, feed intake, milk production and composition, and presence in milk of transgenic DNA and the encoded protein Cry1Ab when corn silages containing 2 transgenes (2GM: herbicide tolerance: mepsps and insect resistance: cry1Ab) were fed as part of a standard total mixed ration (TMR) compared with a near isogenic corn silage ( C) to 8 multiparous lactating Holstein dairy cows in a single reversal design study. Cows were fed a TMR ration ad libitum and milked twice daily. Diets contained [ dry matter (DM) basis] 45% corn silage, 10% alfalfa hay, and 45% concentrate (1.66 Mcal of net energy for lactation/kg of DM, 15.8% crude protein, 35% neutral detergent fiber, and 4.1% fat). Each period was 28-d long. During the last 4 d of each period, feed intake and milk production data were recorded and milk samples taken for compositional analysis, including the presence of transgenic DNA and Cry1Ab protein. There was no significant difference in the chemical composition between C and 2GM silages, and both were within the expected range (37.6% DM, 1.51 Mcal of net energy for lactation/kg, 8.6% crude protein, 40% neutral detergent fiber, 19.6% acid detergent fiber, pH 3.76, and 62% in vitro DM digestibility). Cows fed the 2GM silage produced milk with slightly higher protein (3.09 vs. 3.00%), lactose ( 4.83 vs. 4.72%) and solids-not-fat (8.60 vs. 8.40%) compared with C. However, the yield (kg/d) of milk (36.5), 3.5% fat-corrected milk (34.4), fat (1.151), protein (1.106), lactose (1.738), and solids-not-fat ( 3.094), somatic cell count (log(10): 2.11), change in body weight (+ 7.8 kg), and condition score (+ 0.09) were not affected by type of silage, indicating no overall production difference. All milk samples were negative for the presence of transgenic DNA from either trait or the Cry1Ab protein. Results indicate that the 2GM silage modified with 2 transgenes did not affect nutrient composition of the silages and had no effect on animal performance and milk composition. No transgenic DNA and Cry1Ab protein were detected in milk.

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.

Fungicide resistance: the dose rate debate

It makes economic sense to use as little fungicide as possible on a crop. In many settings, it is common to apply less than the manufacturer's recommended dose. If sources of disease are scarce, or conditions are unsuitable for it to increase, the reduced control from a low dose may be adequate. In other cases, a big reduction in dose may cause little reduction in control, again permitting savings - especially for growers prepared to run a little risk. But the label recommendations for most fungicides state that to avoid resistance, a full dose must always be used. Are individual cost-savings therefore endangering everyone's access to an exceptionally useful tool? The emergence of fungicide resistance is evolution in action. In all cases, it involves the genetic replacement of the original susceptible population of the pathogen by a new population with genetically distinct biochemistry, which confers resistance. The resistant biochemistry originates in rare genetic mutations, so rare that initially the population is hardly altered. Replacement of susceptible forms by resistant ones happens because, with fungicide present, the resistant form multiplies more rapidly than the susceptible form. The key point to notice is that only the relative rates of multiplication of the resistant and susceptible types are involved in the evolution of resistance. The absolute rates are irrelevant.

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.

A qualitative host-pathogen interaction in the Theobroma cacao-Moniliophthora perniciosa pathosystem

The aim of this study was to test whether resistance of clones of Theobroma cacao ( cocoa) varied between isolates of Moniliophthora (formerly Crinipellis) perniciosa, the cause of witches' broom disease. Developing buds of vegetatively propagated T. cacao grown in greenhouses in the UK were inoculated with 16 000 spores of M. perniciosa per meristem in water, under conditions where water condensed on the inoculated shoot for at least 12 h after inoculation. The proportion of successful inoculations varied between clones and was inversely correlated with time to symptom production or broom formation. A specific interaction was demonstrated among three single-spore isolates of M. perniciosa and the clone Scavina 6 (SCA 6) and a variety of susceptible clones. Isolates Castenhal-I and APC3 were equally likely to infect SCA 6 and the other clones, but isolate Gran Couva A9 never infected SCA 6, although it was as virulent on the other clones. The interaction was maintained when the wetness period was extended to 70 h. Offspring of SCA 6 x Amelonado matings were all susceptible to both Castenhal-I and GC-A5, with no evidence of greater variability in susceptibility to GC-A5 than Castanhal-I. This suggests recessive inheritance of a single homozygous factor conferring resistance to GC-A5, from SCA 6. The progenies were slightly more susceptible to Castanhal-I than GC-A5. The implications for managing the disease are discussed.

The use of bioluminescence for monitoring in planta growth dynamics of a Pseudomonas syringae plant pathogen

The use of bioluminescence was evaluated as a tool to study Pseudomonas syringae population dynamics in susceptible and resistant plant environments. Plasmid pGLITE, containing the luxCDABE genes from Photorhabdus luminescens, was introduced into Pseudomonas syringae pv. phaseolicola race 7 strain 1449B, a Gram-negative pathogen of bean (Phaseolus vulgaris). Bacteria recovered from plant tissue over a five-day period were enumerated by counting numbers of colony forming units and by measurement of bioluminescence. Direct measurement of bioluminescence from leaf disc homogenates consistently reflected bacterial growth as determined by viable counting, but also detected subtle effects of the plant resistance response on bacterial viability. This bioluminescence procedure enables real time measurement of bacterial metabolism and population dynamics in planta, obviates the need to carry out labour intensive and time consuming traditional enumeration techniques and provides a sensitive assay for studying plant effects on bacterial cells.

Evaluation of cacao (Theobroma cacao L.) clones against seven Colombian isolates of Moniliophthora roreri (Cif.) Evans et al., representing four major genetic groupings of the pathogen in cacao

Artificial pod inoculation was used to compare the relative aggressiveness of seven Colombian isolates of Moniliophthora roreri (the causal agent of moniliasis or frosty pod disease), representing four major genetic groupings of the pathogen in cacao (cocoa), when applied to five diverse cacao genotypes (ICS-1, ICS-95, TSH-565, SCC-61 and CAP-34) at La Suiza Experimental Farm, Santander Department, Colombia. The following variables were evaluated 9 weeks after inoculation of 2- to 3-month-old pods with spore suspensions (1.2 x 10(5) spores mL(-1)): (i) disease incidence (DI); (ii) external severity (ES); and (iii) internal severity (IS). IS was found to be of greatest value in classifying the reaction of the host genotype against M. roreri. Genetic variation reported between isolates and cacao genotypes was not matched by similar diversity in their aggressiveness. All isolates were generally highly aggressive against most cacao genotypes, with only two isolates showing reduced IS and ES reactions. There was considerable variation between clones in the IS and ES scores, but one cultivated clone (ICS-95) displayed a significant level of resistance against all seven isolates. This clone may be useful in cacao breeding initiatives for resistance to moniliasis of cacao.

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.

Genotype and temperature influence pea aphid resistance to a fungal entomopathogen

The influence of temperature on life history traits of four Acyrthosiphon pisum clones was investigated, together with their resistance to one genotype of the fungal entomopathogen Erynia neoaphidis . There was no difference among aphid clones in development rate, but they did differ in fecundity. Both development rate and fecundity were influenced by temperature, but all clones showed similar responses to the changes in temperature (i.e. the interaction term was nonsignificant). However, there were significant differences among clones in susceptibility to the pathogen, and this was influenced by temperature. Furthermore, the clones differed in how temperature influenced susceptibility, with susceptibility rankings changing with temperature. Two clones showed changes in susceptibility which mirrored changes in the in vitro vegetative growth rate of E. neoaphidis at different temperatures, whereas two other clones differed considerably from this expected response. Such interactions between genotype and temperature may help maintain heritable variation in aphid susceptibility to fungal pathogen attack and have implications for our understanding of disease dynamics in natural populations. This study also highlights the difficulties of drawing conclusions about the efficacy of a biological control agent when only a restricted range of pest genotypes or environmental conditions are considered.

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.

DNA cleavage and antitumour activity of platinum(II) and copper(II) compounds derived from 4-methyl-2-N-(2-pyridylmethyl)aminophenol: spectroscopic, electrochemical and biological investigation

The reaction of the redox-active ligand, Hpyramol (4-methyl-2-N-(2-pyridylmethyl)aminophenol) with K2PtCl4 yields monofunctional square-planar [Pt(pyrimol)Cl], PtL-Cl, which was structurally characterised by single-crystal X-ray diffraction and NMR spectroscopy. This compound unexpectedly cleaves supercoiled double-stranded DNA stoichiometrically and oxidatively, in a non-specific manner without any external reductant added, under physiological conditions. Spectro-electrochemical investigations of PtL-Cl were carried out in comparison with the analogue CuL-Cl as a reference compound. The results support a phenolate oxidation, generating a phenoxyl radical responsible for the ligand-based DNA cleavage property of the title compounds. Time-dependent in vitro cytotoxicity assays were performed with both PtL-Cl and CuL-Cl in various cancer cell lines. The compound CuL-Cl overcomes cisplatin-resistance in ovarian carcinoma and mouse leukaemia cell lines, with additional activity in some other cells. The platinum analogue, PtL-Cl also inhibits cell-proliferation selectively. Additionally, cellular-uptake studies performed for both compounds in ovarian carcinoma cell lines showed that significant amounts of Pt and Cu were accumulated in the A2780 and A2780R cancer cells. The conformational and structural changes induced by PtL-Cl and CuL-Cl on calf thymus DNA and phi X174 supercoiled phage DNA at ambient conditions were followed by electrophoretic mobility assay and circular dichroism spectroscopy. The compounds induce extensive DNA degradation and unwinding, along with formation of a monoadduct at the DNA minor groove. Thus, hybrid effects of metal-centre variation, multiple DNA-binding modes and ligand-based redox activity towards cancer cell-growth inhibition have been demonstrated. Finally, reactions of PtL-Cl with DNA model bases (9-Ethylguanine and 5'-GMP) followed by NMR and MS showed slow binding at Guanine-N7 and for the double stranded self complimentary oligonucleotide d(GTCGAC)(2) in the minor groove.