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Resistance and susceptibility of plants to fungal pathogens.

Plants are under continuous threat of infection by pathogens endowed with diverse strategies to colonize their host. Comprehensive biochemical and genetic approaches are now starting to reveal the complex signaling pathways that mediate plant disease resistance. Initiation of defense signaling often involves specific recognition of invading pathogens by the products of specialized host resistance (R) genes. Potential resistance signaling components have been identified by mutational analyses to be required for specific resistance in the model Arabidopsis and some crop species. Strikingly, many of the components share similarity to that of innate immune systems in animals. Evidence is also accumulating that plant pathogens have a number of ways to evade host defenses during the early stages of infection, similar to animal pathogens. These strategies are becoming much better understood in a number of plant-pathogen interactions. In this review, we focus on the current knowledge of host factors that control plant resistance and susceptibility to fungal pathogens. The knowledge accumulated in these studies will serve a fundamental basis for combating diseases in strategic molecular agriculture.

Fungal Proteins↗

Pathogenicity of Metarhizium anisopliae (Metsch.) Sorokin and Beauveria bassiana (Balsamo) Vuillemin, to three adult fruit fly species: Ceratitis capitata (Weidemann), C. rosa var. fasciventris Karsch and C. cosyra (Walker) (Diptera :Tephritidae).

The pathogenicity of two isolates of Beauveria bassiana and 12 of Metarhizium anisopliae towards adult fruit flies, Ceratitis capitata and Ceratitis rosa var. fasciventris was tested in the laboratory. Fruit flies were exposed to dry conidia evenly spread on velvet material covering the inner side of a cylindrical plastic tube. All isolates tested were pathogenic to both species of fruit flies. Mortality ranged from 7 to 100% in C. capitata and from 11.4 to 100% in C. rosa var. fasciventris at 4 days post-inoculation. Six isolates, M. anisopliae ICIPE 18, 20, 32, 40, 41 and 62, were highly pathogenic to both C. capitata and C. rosa var. fasciventris. The LT90 values of the most pathogenic isolates ranged between 3-4 days in both insects. Because of the difficulties in rearing C. cosyra, only the isolates that were highly pathogenic to both C. rosa var. fasciventris and C. capitata were tested against adult C. cosyra. They caused mortality of between 72-78% at 4 days post-inoculation. The LT90 values in all the isolates did not exceed 4 days. One of the most pathogenic isolates, M. anisopliae ICIPE 20, was evaluated against C. capitata and C. rosa var. fasciventris in cage experiments using three autoinoculators (maize cob, cheesecloth and Petri dish) in an autoinoculative device consisting of plastic mineral bottle. Mortality of between 70-93% was observed in flies of both species that were captured from the cages and held under laboratory conditions. These results indicate the possibility of fruit fly suppression with entomopathogenic fungi using an autoinoculative device.

Animals↗

The combined effects of pathogens and predators on insect outbreaks.

The economic damage caused by episodic outbreaks of forest-defoliating insects has spurred much research, yet why such outbreaks occur remains unclear. Theoretical biologists argue that outbreaks are driven by specialist pathogens or parasitoids, because host-pathogen and host-parasitoid models show large-amplitude, long-period cycles resembling time series of outbreaks. Field biologists counter that outbreaks occur when generalist predators fail, because predation in low-density defoliator populations is usually high enough to prevent outbreaks. Neither explanation is sufficient, however, because the time between outbreaks in the data is far more variable than in host-pathogen and host-parasitoid models, and far shorter than in generalist-predator models. Here we show that insect outbreaks can be explained by a model that includes both a generalist predator and a specialist pathogen. In this host-pathogen-predator model, stochasticity causes defoliator densities to fluctuate erratically between an equilibrium maintained by the predator, and cycles driven by the pathogen. Outbreaks in this model occur at long but irregular intervals, matching the data. Our results suggest that explanations of insect outbreaks must go beyond classical models to consider interactions among multiple species.

Animals↗

Molecular constraints to interspecies transmission of viral pathogens.

The successful replication of a viral pathogen in a host is a complex process involving many interactions. These interactions develop from the coevolution of pathogen and host and often lead to a species specificity of the virus that can make interspecies transmissions difficult. Nevertheless, viruses do sporadically cross species barriers into other host populations, including humans. In zoonotic infections, many of these interspecies transfer events are dead end, where transmission is confined only to the animal-to-human route but sometimes viruses adapt to enable spread from human to human. A pathogen must overcome many hurdles to replicate successfully in a foreign host. The viral pathogen must enter the host cell, replicate with the assistance of host factors, evade inhibitory host products, exit the first cell and move on to the next, and possibly leave the initial host and transmit to another. Each of these stages may require adaptive changes in the pathogen. Although the factors that influence each stage of the replication and transmission of most agents have not been resolved, the genomics of both hosts and pathogens are now at hand and we have begun to understand some of the molecular changes that enable some viruses to adapt to a new host.

Animals↗

Invertebrates as a source of emerging human pathogens.

Despite their importance, little is known about the origins of many emerging human pathogens. However, given the age and current predominance of invertebrates, it is likely that bacteria-invertebrate interactions are not only a present source of human pathogens but have also shaped their evolution. Pathogens of invertebrate and unicellular organisms represent an extensive reservoir of bacterial strains equipped with virulence factors that evolved to overcome the innate immune responses of their hosts. This reservoir might represent a source of new human pathogenic strains and might also foster the spread of novel virulence factors into existing human commensal or pathogenic bacteria. This article examines the available evidence for this concept by examining pairs of closely related bacteria, one of which is benign, but insect associated, and one of which is a human pathogen.

Animals↗

Genetic stratification of pathogen-response-related and other variants within a homogeneous Caucasian Irish population.

Selection pressures from pathogens impact on the worldwide geographic distribution of polymorphisms in certain pathogen-response-associated genes. Such gene-specific effects could lead to confounding by geographic disease associations. We wished to determine if such constraints impinge on the genetic structure of a population of Irish patients and whether variants associated with responses to pathogens showed greater stratification. The counties of origin of each subject's grandparents were used as the geographic variable. F(st), proportional to the extent of population structure, was low (mean F(st)=0.004 across 25 SNPs, range 0.001-0.008) and it was not significantly higher for pathogen response SNPs (F(st)=0.004) than for other SNPs (F(st)=0.003, P=0.21). Correspondence analysis revealed weak trends primarily in approximately northeast to southwest and secondarily in northwest to southeast directions. One-dimensional spatial autocorrelation analysis revealed a weak (Moran's I autocorrelation of -0.10) tendency for SNP frequencies to diverge with greater distance. Two-dimensional autocorrelation indicated a northeast to southwest gradient that was similar for both the pathogen response and other SNPs. The southeastern county, Wexford, showed a distinctive pattern, perhaps consistent with Anglo-Norman settlements. In conclusion, these results indicate that pathogen response SNPs do not exhibit significantly more population structure than other SNPs within this Caucasian population. This suggests that the specific population structure of particular genes may not typically be a cause of strong confounding in genetic studies where population structure is controlled.

Arylsulfotransferase↗

Pathogenicity of vibrio splendidus strains associated with turbot larvae, scophthalmus maximus

Turbot larvae were challenged with eight strains of Vibrio splendidus isolated from diseased larvae, plus a ninth strain pathogenic to scallop larvae (A515; Nicolas et al. 1996). Six strains caused heavy mortality but the scallop pathogen and the other two strains did not. All the strains shared a large number of phenotypic traits, and an attempt was made to relate virulence to genotype and phenotype. Five of the six pathogenic strains were very similar, as shown by RAPD fingerprinting and phenotypic characteristics. The relatedness of the other strains was intermediate between the main pathogenic group and V. splendidus ATCC 33125, but the DNA-DNA homology between the pathogenic group and the reference strain was still high (78% of reassociation rate). The non-pathogenic isolates may be a useful tool for determining the possible virulence factors, as all the isolates differed by few characteristics.

Journal Article↗

Concentration of Cryptosporidium, microsporidia and other water-borne pathogens by continuous separation channel centrifugation.

AIMS: The aim of this study was to determine the effectiveness of continuous separation channel centrifugation for concentrating water-borne pathogens of various taxa and sizes. METHODS AND RESULTS: Cryptosporidium parvum oocysts, Giardia lamblia cysts, Encephalitozoon intestinalis spores and Escherichia coli were seeded into different water matrices at densities ranging from 5 to 10 000 organisms l(-1) and recovered using continuous separation channel centrifugation. All pathogens were enumerated on membrane filters using microscopy. Recovery efficiencies were usually > 90%. Oocyst recovery did not vary with source water turbidity or with centrifuge flow rate up to 250 ml min(-1). Based on excystation, this concentration method did not alter oocyst viability. CONCLUSIONS: Continuous separation channel centrifugation is an effective means of concentrating water-borne pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Methods are needed for detecting pathogens in drinking water to ensure public health. The first step for any pathogen detection procedure is concentration. However, this step has been problematic because recovery efficiencies of conventional methods, like filtration, are often low and variable, which may lead to false negatives. Continuous separation channel centrifugation can simultaneously concentrate multiple pathogens as small as 1 microm with high and reproducible efficiency in a variety of water matrices.

Animals↗

Using event trees to quantify pathogen levels on root crops from land application of treated sewage sludge.

AIMS: To quantify the incremental exposure of root crops, at point of harvest, to enteric pathogens from sewage sludge applied to agricultural land according to current regulations and guidance (Safe Sludge Matrix). METHODS AND RESULTS: A quantitative risk assessment based on the Source-Pathway-Receptor approach is developed for Cryptosporidium and salmonellas. Event trees are constructed to model the partitioning of pathogens present in raw sewage into sludge at the sewage treatment works and to model to the pathways by which root crops may be exposed to those pathogens after treatment and land application of the sludge. The main barriers are sewage sludge treatment, and decay and dilution of the pathogens in the soil. The exposures are expressed in terms of the arithmetic mean. This represents the total loading and accommodates fluctuations not only in the levels of pathogens present in sewage but also in the removal efficiencies by the various barriers. One source of uncertainty is the degree of by-pass of sludge treatment at operational scale. CONCLUSIONS: The models predict that land application of sewage sludge treated by conventional processes (achieving 2-log removal) increases the exposures of root crops to salmonellas and Cryptosporidium oocysts by counts of 0.070 and 0.033 kg(-1), respectively. These predictions are based on decay periods in the soil of 5 and 12 weeks, respectively, and are therefore worst case in not allowing for the full extent of no harvesting periods. A Monte Carlo simulation predicts that 0.01% of 1-kg batches contained > 50 salmonellas and demonstrates that, for risk assessment, it is acceptable to use the arithmetic mean exposure directly in the dose-response curve. SIGNIFICANCE AND IMPACT OF THE STUDY: The predicted numbers of pathogens on root crops at point of harvest provide a basis for modelling the excess risks to humans consuming such crops. The approach underpins scientifically the Safe Sludge Matrix.

Agriculture↗

A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K-12.

Attaching and effacing (AE) bacteria are a diverse group of gastrointestinal pathogens, comprising members of four genera, that cause the intestinal epithelial microvilli to be replaced with raised clusters of filamentous actin that conform to the surface of attached bacteria. We have cloned a 35.4 kb 'pathogenicity island' from the prototype AE bacterium, enteropathogenic Escherichia coli, containing all previously described AE genes. Transfer of this pathogenicity island to avirulent E. coli converts the recipients into strains that secrete virulence proteins, induce host signal-transduction pathways, and cause AE lesions on cultured epithelial cells. These results demonstrate that this pathogenicity island contains all pathogen-specific genes necessary for inducing AE lesions, and that the defining feature of this class of pathogens can be acquired by an avirulent bacterium in a single genetic step.

Bacterial Adhesion↗

Investigation of mycobacterial recA function: protein introns in the RecA of pathogenic mycobacteria do not affect competency for homologous recombination.

The recA locus of pathogenic mycobacteria differs from that of non-pathogenic species in that it contains large intervening sequences termed protein introns or inteins that are excised by an unusual protein-splicing reaction. In addition, a high degree of illegitimate recombination has been observed in the pathogenic Mycobacterium tuberculosis complex. Homologous recombination is the main mechanism of integration of exogenous nucleic acids in M. smegmatis, a non-pathogenic mycobacterium species that carries an inteinless RecA and is amenable to genetic manipulations. To investigate the function of recA in mycobacteria, recA- strains of M. smegmatis were generated by allelic exchange techniques. These strains are characterized (i) by increased sensitivity towards DNA-damaging agents [ethylmethylsulphonate (EMS), mitomycin C, UV irradiation] and (ii) by the inability to integrate nucleic acids by homologous recombination. Transformation efficiencies using integrative or replicative vectors were not affected in recA- mutants, indicating that in mycobacteria RecA does not affect plasmid uptake or replication. Complementation of the recA- mutants with the recA from M. tuberculosis restored resistance towards EMS, mitomycin C and UV irradiation. Transformation of the complemented strains with suicide vectors targeting the pyrF gene resulted in numerous allelic exchange mutants. From these data, we conclude that the intein apparently does not interfere with RecA function, i.e. with respect to competency for homologous recombination, the RecAs from pathogenic and non-pathogenic mycobacteria are indistinguishable.

Alleles↗

The aerobactin iron transport system genes in Shigella flexneri are present within a pathogenicity island.

Genes encoding the synthesis and transport of aerobactin, a hydroxamate siderophore associated with increased virulence of enteric bacteria, were mapped within a pathogenicity island in Shigella flexneri. The island, designated SHI-2 for Shigella pathogenicity island 2, was located downstream of selC, the site of insertion of pathogenicity islands in several other enteric pathogens. DNA sequence analysis revealed the presence of multiple insertion sequences upstream and downstream of the aerobactin genes and an integrase gene that was nearly identical to an int gene found in Escherichia coli O157:H7. SHI-2 sequences adjacent to selC were similar to sequences at the junction between selC and pathogenicity islands found in E. coli O157:H7 and in enteropathogenic E. coli, but the junctions between the island and downstream yic genes were variable. SHI-2 also encoded immunity to the normally plasmid-encoded colicins I and V, suggesting a common origin for the aerobactin genes in both S. flexneri and E. coli pColV. Polymerase chain reaction and Southern hybridization data indicate that SHI-2 is present in the same location in Shigella sonnei, but the aerobactin genes are not located within SHI-2 in Shigella boydii or enteroinvasive E. coli. Shigella dysenteriae type 1 strains do not produce aerobactin but do contain sequences downstream of selC that are homologous to SHI-2. The presence of the aerobactin genes on plasmids in E. coli pColV and Salmonella, on a pathogenicity island in S. flexneri and S. sonnei and in a different chromosomal location in S. boydii and some E. coli suggests that these virulence-enhancing genes are mobile, and they may constitute an island within an island in S. flexneri.

Bacterial Proteins↗

The selC-associated SHI-2 pathogenicity island of Shigella flexneri.

Pathogenicity islands are chromosomal gene clusters, often located adjacent to tRNA genes, that encode virulence factors present in pathogenic organisms but absent or sporadically found in related non-pathogenic species. The selC tRNA locus is the site of integration of different pathogenicity islands in uropathogenic Escherichia coli, enterohaemorrhagic E. coli and Salmonella enterica. We show here that the selC locus of Shigella flexneri, the aetiological agent of bacterial dysentery, also contains a pathogenicity island. This pathogenicity island, designated SHI-2 (Shigella island 2), occupies 23.8 kb downstream of selC and contains genes encoding the aerobactin iron acquisition siderophore system, colicin V immunity and several novel proteins. Remnants of multiple mobile genetic elements are present in SHI-2. SHI-2-hybridizing sequences were detected in all S. flexneri strains tested and parts of the island were also found in other Shigella species. SHI-2 may allow Shigella survival in stressful environments, such as those encountered during infection.

Bacterial Proteins↗

A Streptococcus pneumoniae pathogenicity island encoding an ABC transporter involved in iron uptake and virulence.

Restricted iron availability is a major obstacle to growth and survival of pathogenic bacteria during infection. In contrast to Gram-negative pathogens, little is known about how Gram-positive pathogens obtain this essential metal. We have identified two Streptococcus pneumoniae genetic loci, pit1 and pit2, encoding homologues of ABC iron transporters that are required for iron uptake by this organism. S. pneumoniae strains containing disrupted copies of either pit1 or pit2 had decreased sensitivity to the iron-dependent antibiotic streptonigrin, and a strain containing disrupted copies of both pit1 and pit2 was unable to use haemoglobin as an iron source and had a reduced rate of iron uptake. The pit2- strain was moderately and the pit1-/pit2- strain strongly attenuated in virulence in mouse models of pulmonary and systemic infection, showing that the pit loci play a critical role during in vivo growth of S. pneumoniae. The pit2 locus is contained within a 27 kb region of chromosomal DNA that has several features of Gram-negative bacterial pathogenicity islands. This probable pathogenicity island (PPI-1) is the first to be described for S. pneumoniae, and its acquisition is likely to have played a significant role in the evolution of this important human pathogen.

ATP-Binding Cassette Transporters↗

Requirement for the induced expression of a cell wall associated receptor kinase for survival during the pathogen response.

Pathogen infection of angiosperms must rely on some interaction between the extracellular matrix (ECM) and the invading agent, and may be accompanied by signaling between the ECM and cytoplasm. An Arabidopsis cell wall associated receptor kinase (Wak1) has an amino-terminal domain that is tightly associated with the ECM, spans the plasma membrane and has a cytoplasmic protein kinase domain. Wak1 expression is induced when Arabidopsis plants are infected with pathogen, or when the pathogen response is stimulated either by exogenous salicylate (SA) or its analog 2,2-dichloroisonicotinic acid (INA). This Wak1 induction requires the positive regulator NPR1/NIM1. Thus Wak1 is a pathogen-related (PR) protein. Expression of an antisense and a dominant negative allele of Wak1 shows that induced expression of Wak1 is needed for a plant to survive if stimulated by INA. Ectopic expression of the entire Wak1, or the kinase domain alone, can provide resistance to otherwise lethal SA levels. These experiments suggest that Wak1 expression and other PR proteins are protecting plants from detrimental effects incurred during the pathogen response. These results provide a direct link between a protein kinase that could mediate signals from the ECM, to the events that are precipitated by a pathogen infection.

Alleles↗

Constitutive expression of a celery mannitol dehydrogenase in tobacco enhances resistance to the mannitol-secreting fungal pathogen Alternaria alternata.

Our previous observation that host plant extracts induce production and secretion of mannitol in the tobacco pathogen Alternaria alternata suggested that, like their animal counterparts, plant pathogenic fungi might produce the reactive oxygen quencher mannitol as a means of suppressing reactive oxygen-mediated plant defenses. The concurrent discovery that pathogen attack induced mannitol dehydrogenase (MTD) expression in the non-mannitol-containing host tobacco suggested that plants, unlike animals, might be able to counter this fungal suppressive mechanism by catabolizing mannitol of fungal origin. To test this hypothesis, transgenic tobacco plants constitutively expressing a celery Mtd cDNA were produced and evaluated for potential changes in resistance to both mannitol- and non-mannitol-secreting pathogens. Constitutive expression of the MTD transgene was found to confer significantly enhanced resistance to A. alternata, but not to the non-mannitol-secreting fungal pathogen Cercospora nicotianae. These results are consistent with the hypothesis that MTD plays a role in resistance to mannitol-secreting fungal plant pathogens.

Alternaria↗

Prevalence of enteric pathogens among community based asymptomatic individuals.

BACKGROUND AND AIMS: The objective of this study was to describe the prevalence of pathogenic microorganisms in asymptomatic individuals in a community study in Melbourne, Australia. METHODS: The study population was a subset of 2803 individuals participating in the Water Quality Study; a community based randomized trial. Faecal specimens (1091) were collected over a 3-month period from asymptomatic individuals. Specimens were tested for a range of bacteria including Salmonella, Shigella and Campylobacter species. Rotavirus and adenovirus were detected using a Rota-Adeno latex kit, and protozoa were detected using a permanent stain (modified iron-haemotoxylin). RESULTS: Twenty-eight known pathogens were identified from the 1091 faecal specimens, a total carriage rate of 2.6%. Giardia species were present in 18 specimens (1.6%), Salmonella in four (0.4%), Campylobacter in one (0.1%), Cryptosporidium in four (0.4%) and adenovirus in one (0.1%). Blastocystis hominis was found in 65 specimens. The median age of those without a pathogen was 12.5 years compared with 6.6 years for those with a pathogen (P=0.02). CONCLUSIONS: Except for Giardia, pathogens were rarely found in asymptomatic individuals in the community. The prevalence of pathogens was higher in children than adults.

Adolescent↗

Pathogenicity of MSH2 missense mutations is typically associated with impaired repair capability of the mutated protein.

BACKGROUND & AIMS: Inherited deleterious mutations in mismatch repair genes MLH1, MSH2, and MSH6 predispose to hereditary nonpolyposis colorectal cancer. A major diagnostic challenge is the difficulty in evaluating the pathogenicity of missense mutations. Previously we showed that most missense variants in MSH6 do not impair MMR capability and are associated with no or low cancer susceptibility, whereas in MLH1, functional studies distinguished nontruncating mutations with severe defects from those not or slightly impaired in protein expression or function. The present study was undertaken to evaluate the pathogenicity of inherited missense mutations in MSH2. METHODS: Fifteen mutated MSH2 proteins including 14 amino acid substitutions and one in-frame deletion were tested for expression/stability, MSH2/MSH6 interaction, and repair efficiency. The genetic and biochemical data were correlated with the clinical data. Comparative sequence analysis was performed to assess the value of sequence homology as a tool for predicting functional results. RESULTS: None of the studied MSH2 mutations destroyed the protein or abolished MSH2/MSH6 interaction, whereas 12 mutations impaired the repair capability of the protein. Comparative sequence analysis correctly predicted functional studies for 13 of 14 amino acid substitutions. CONCLUSIONS: Interpretation was pathogenic for 12, nonpathogenic for 2, and contradictory for 1 mutation. The pathogenicity could not be distinguished unambiguously by phenotypic characteristics, although correlation between the absence of staining for MSH2 and pathogenicity of the missense mutation was notable. Unlike in MSH6 and MLH1, the pathogenicity of missense mutations in MSH2 was always associated with impaired repair capability of the mutated protein.

Adaptor Proteins, Signal Transducing↗