Genome-Wide Association Analysis Identifies a Genetic Basis of Infectivity in a Model Bacterial Pathogen

Knowledge of the genetic architecture of pathogen infectivity and host resistance is essential for a mechanistic understanding of coevolutionary processes, yet the genetic basis of these interacting traits remains unknown for most host–pathogen systems. We used a comparative genomic approach to explore the genetic basis of infectivity in Pasteuria ramosa, a Gram-positive bacterial pathogen of planktonic crustaceans that has been established as a model for studies of Red Queen host–pathogen coevolution. We sequenced the genomes of a geographically, phenotypically, and genetically diverse collection of P. ramosa strains and performed a genome-wide association study to identify genetic correlates of infection phenotype. We found multiple polymorphisms within a single gene, Pcl7, that correlate perfectly with one common and widespread infection phenotype. We then confirmed this perfect association via Sanger sequencing in a large and diverse sample set of P. ramosa clones. Pcl7 codes for a collagen-like protein, a class of adhesion proteins known or suspected to be involved in the infection mechanisms of a number of important bacterial pathogens. Consistent with expectations under Red Queen coevolution, sequence variation of Pcl7 shows evidence of balancing selection, including extraordinarily high diversity and absence of geographic structure. Based on structural homology with a collagen-like protein of Bacillus anthracis, we propose a hypothesis for the structure of Pcl7 and the physical location of the phenotype-associated polymorphisms. Our results offer strong evidence for a gene governing infectivity and provide a molecular basis for further study of Red Queen dynamics in this model host–pathogen system.     

THE CELLULAR IMMUNE RESPONSE OF DAPHNIA MAGNA UNDER HOST–PARASITE GENETIC VARIATION AND VARIATION IN INITIAL DOSE

In invertebrate–parasite systems, the likelihood of infection following parasite exposure is often dependent on the specific combination of host and parasite genotypes (termed genetic specificity). Genetic specificity can maintain diversity in host and parasite populations and is a major component of the Red Queen hypothesis. However, invertebrate immune systems are thought to only distinguish between broad classes of parasite. Using a natural host–parasite system with a well‐established pattern of genetic specificity, the crustacean Daphnia magna and its bacterial parasite Pasteuria ramosa, we found that only hosts from susceptible host–parasite genetic combinations mounted a cellular response following exposure to the parasite. These data are compatible with the hypothesis that genetic specificity is attributable to barrier defenses at the site of infection (the gut), and that the systemic immune response is general, reporting the number of parasite spores entering the hemocoel. Further supporting this, we found that larger cellular responses occurred at higher initial parasite doses. By studying the natural infection route, where parasites must pass barrier defenses before interacting with systemic immune responses, these data shed light on which components of invertebrate defense underlie genetic specificity.     

霉草科(Triuridaceae)-中国大陆分布新记录科

报道中国大陆霉草科（Ｔｒｉｕｒｉｄａｃｅａｅ）２种分布新记录,大柱霉草（Ｓｃｉａｐｉｌａ ｍｅｇａｓｔｙｌａ Ｆｕｋｕｙａｍａ ｅｔ Ｓｕｚｕｋｉ）和多枝霉草（Ｓｃｉａｐｈｉｌａ ｒａｍｏｓａ Ｆｕｋｕｙａｍａ ｅｔ Ｓｕｚｕｋｉ）。     

Tomato strigolactones are derived from carotenoids and their biosynthesis is promoted by phosphate starvation

* Strigolactones are rhizosphere signalling compounds that mediate host location in arbuscular mycorrhizal (AM) fungi and parasitic plants. Here, the regulation of the biosynthesis of strigolactones is studied in tomato (Solanum lycopersicum). * Strigolactone production under phosphate starvation, in the presence of the carotenoid biosynthesis inhibitor fluridone and in the abscisic acid (ABA) mutant notabilis were assessed using a germination bioassay with seeds of Orobanche ramosa; a hyphal branching assay with Gigaspora spp; and by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. * The root exudates of tomato cv. MoneyMaker induced O. ramosa seed germination and hyphal branching in AM fungi. Phosphate starvation markedly increased, and fluridone strongly decreased, this activity. Exudates of notabilis induced approx. 40% less germination than the wild-type. The LC-MS/MS analysis confirmed that the biological activity and changes therein were due to the presence of several strigolactones; orobanchol, solanacol and two or three didehydro-orobanchol isomers. * These results show that the AM branching factors and parasitic plant germination stimulants in tomato root exudate are strigolactones and that they are biosynthetically derived from carotenoids. The dual activity of these signalling compounds in attracting beneficial AM fungi and detrimental parasitic plants is further strengthened by environmental conditions such as phosphate availability.     

一株来源于土壤的横梗霉菌及其产羧甲基纤维素酶性质

从广州徐闻农垦丰收农场土壤样品中分离到一株产羧甲基纤维素酶菌株,结合菌株形态特征和ITS基因序列同源性分析结果,表明该菌株为Lichtheimia sp.的未定种,其系统分类学关系与横梗霉菌Lichtheimia ramosa isolate K12(MN190291.1)最近,故命名为:横梗霉菌XWNR1(Lichtheimia ramosa XWNR1),该菌种保藏号为CCTCC NO:M2018230。采用刚果红染色法发现该菌粗酶液能够降解羧甲基纤维素钠产生透明圈,初步确定该菌具有产羧甲基纤维素酶的能力。通过优化该菌发酵培养条件,结果表明:该菌株以2.0×10^6接种浓度、1%接种量,28℃180 r/min恒温振荡培养60 h时,菌株分泌的羧甲基纤维素酶具有最好的活力。酶学性质初步研究结果表明,该羧甲基纤维素酶在最适pH 7.0、最适反应温度40℃条件下具有较好的pH稳定性和热稳定性。     

Parasitism drives host genome evolution: Insights from the Pasteuria ramosa–Daphnia magna system

Because parasitism is thought to play a major role in shaping host genomes, it has been predicted that genomic regions associated with resistance to parasites should stand out in genome scans, revealing signals of selection above the genomic background. To test whether parasitism is indeed such a major factor in host evolution and to better understand host–parasite interaction at the molecular level, we studied genome‐wide polymorphisms in 97 genotypes of the planktonic crustacean Daphnia magna originating from three localities across Europe. Daphnia magna is known to coevolve with the bacterial pathogen Pasteuria ramosa for which host genotypes (clonal lines) are either resistant or susceptible. Using association mapping, we identified two genomic regions involved in resistance to P. ramosa, one of which was already known from a previous QTL analysis. We then performed a naïve genome scan to test for signatures of positive selection and found that the two regions identified with the association mapping further stood out as outliers. Several other regions with evidence for selection were also found, but no link between these regions and phenotypic variation could be established. Our results are consistent with the hypothesis that parasitism is driving host genome evolution.     

Temperature-dependent costs of parasitism and maintenance of polymorphism under genotype-by-environment interactions

The maintenance of genetic variation for infection-related traits is often attributed to coevolution between hosts and parasites, but it can also be maintained by environmental variation if the relative fitness of different genotypes changes with environmental variation. To gain insight into how infection-related traits are sensitive to environmental variation, we exposed a single host genotype of the freshwater crustacean Daphnia magna to four parasite isolates (which we assume to represent different genotypes) of its naturally co-occurring parasite Pasteuria ramosa at 15, 20 and 25 degrees C. We found that the cost to the host of becoming infected varied with temperature, but the magnitude of this cost did not depend on the parasite isolate. Temperature influenced parasite fitness traits; we found parasite genotype-by-environment (G x E) interactions for parasite transmission stage production, suggesting the potential for temperature variation to maintain genetic variation in this trait. Finally, we tested for temperature-dependent relationships between host and parasite fitness traits that form a key component of models of virulence evolution, and we found them to be stable across temperatures.     

Cloning of the unculturable parasite Pasteuria ramosa and its Daphnia host reveals extreme genotype-genotype interactions

The degree of specificity in host-parasite interactions has important implications for ecology and evolution. Unfortunately, specificity can be difficult to determine when parasites cannot be cultured. In such cases, studies often use isolates of unknown genetic composition, which may lead to an underestimation of specificity. We obtained the first clones of the unculturable bacterium Pasteuria ramosa, a parasite of Daphnia magna. Clonal genotypes of the parasite exhibited much more specific interactions with host genotypes than previous studies using isolates. Clones of P. ramosa infected fewer D. magna genotypes than isolates and host clones were either fully susceptible or fully resistant to the parasite. Our finding enhances our understanding of the evolution of virulence and coevolutionary dynamics in this system. We recommend caution when using P. ramosa isolates as the presence of multiple genotypes may influence the outcome and interpretation of some experiments.     

Exploitation of immunofluorescence for the quantification and characterization of small numbers of Pasteuria endospores

The Pasteuria group of endospore-forming bacteria has been studied as a biocontrol agent of plant-parasitic nematodes. Techniques have been developed for its detection and quantification in soil samples, and these mainly focus on observations of endospore attachment to nematodes. Characterization of Pasteuria populations has recently been performed with DNA-based techniques, which usually require the extraction of large numbers of spores. We describe a simple immunological method for the quantification and characterization of Pasteuria populations. Bayesian statistics were used to determine an extraction efficiency of 43% and a threshold of detection of 210 endospores g(-1) sand. This provided a robust means of estimating numbers of endospores in small-volume samples from a natural system. Based on visual assessment of endospore fluorescence, a quantitative method was developed to characterize endospore populations, which were shown to vary according to their host.