Protein interaction mapping on a functional shotgun sequence of Rickettsia sibirica

Protein interaction maps can reveal novel pathways and functional complexes, allowing ‘guilt by association’ annotation of uncharacterized proteins. To address the need for large-scale protein interaction analyses, a bacterial two-hybrid system was coupled with a whole genome shotgun sequencing approach for microbial genome analysis. We report the first large-scale proteomics study using this system, integrating de novo genome sequencing with functional interaction mapping and annotation in a high-throughput format. We apply the approach by shotgun sequencing and annotating the genome of Rickettsia sibirica strain 246, an obligate intracellular human pathogen among the Spotted Fever Group rickettsiae. The bacteria invade endothelial cells and cause lysis after large amounts of progeny have accumulated. Little is known about specific Rickettsial virulence factors and their mode of pathogenicity. Analysis of the combined genomic sequence and protein–protein interaction data for a set of virulence related Type IV secretion system (T4SS) proteins revealed over 250 interactions and will provide insight into the mechanism of Rickettsial pathogenicity.     

Biosynthesis of fungal melanins and their importance for human pathogenic fungi

For more than 40 years fungi have been known to produce pigments known as melanins. Predominantly these have been dihydroxyphenylalanine (DOPA)-melanin and dihydroxynaphthalene (DHN)-melanin. The biochemical and genetical analysis of the biosynthesis pathways have led to the identification of the genes and corresponding enzymes of the pathways. Only recently have both these types of melanin been linked to virulence in some human pathogenic and phytopathogenic fungi. The absence of melanin in human pathogenic and phytopathogenic fungi often leads to a decrease in virulence. In phytopathogenic fungi such as Magnaporthe grisea and Colletotrichum lagenarium, besides other possible functions in pathogenicity, DHN-melanin plays an essential role in generating turgor for plant appressoria to penetrate plant leaves. While the function of melanin in human pathogenic fungi such as Cryptococcus neoformans, Wangiella dermatitidis, Sporothrix schenckii, and Aspergillus fumigatus is less well defined, its role in protecting fungal cells has clearly been shown. Specifically, the ability of both DOPA- and DHN-melanins to quench free radicals is thought to be an important factor in virulence. In addition, in several fungi the production of fungal virulence factors, such as melanin, has been linked to a cAMP-dependent signaling pathway. Many of the components involved in the signaling pathway have been identified.     

Ecological fitness, genomic islands and bacterial pathogenicity. A Darwinian view of the evolution of microbes

The compositions of bacterial genomes can be changed rapidly and dramatically through a variety of processes including horizontal gene transfer. This form of change is key to bacterial evolution, as it leads to ‘evolution in quantum leaps’. Horizontal gene transfer entails the incorporation of genetic elements transferred from another organism—perhaps in an earlier generation—directly into the genome, where they form ‘genomic islands’, i.e. blocks of DNA with signatures of mobile genetic elements. Genomic islands whose functions increase bacterial fitness, either directly or indirectly, have most likely been positively selected and can be termed ‘fitness islands’. Fitness islands can be divided into several subtypes: ‘ecological islands’ in environmental bacteria and ‘saprophytic islands’, ‘symbiosis islands’ or ‘pathogenicity islands’ (PAIs) in microorganisms that interact with living hosts. Here we discuss ways in which PAIs contribute to the pathogenic potency of bacteria, and the idea that genetic entities similar to genomic islands may also be present in the genomes of eukaryotes.     

Antisense expression of the Arabidopsis thaliana AtPGIP1 gene reduces polygalacturonase-inhibiting protein accumulation and enhances susceptibility to Botrytis cinerea

Polygalacturonases (PGs) hydrolyze the homogalacturonan of plant cell-wall pectin and are important virulence factors of several phytopathogenic fungi. In response to abiotic and biotic stress, plants accumulate PG-inhibiting proteins (PGIPs) that reduce the activity of fungal PGs. In Arabidopsis thaliana, PGIPs with comparable activity against BcPG1, an important pathogenicity factor of the necrotrophic fungus Botrytis cinerea, are encoded by two genes, AtPGIP1 and AtPGIP2. Both genes are induced by fungal infection through different signaling pathways. We show here that transgenic Arabidopsis plants expressing an antisense AtPGIP1 gene have reduced AtPGIP1 inhibitory activity and are more susceptible to B. cinerea infection. These results indicate that PGIP contributes to basal resistance to this pathogen and strongly support the vision that this protein plays a role in Arabidopsis innate immunity.     

PEX基因在过氧化物酶体形成及真菌致病性中的作用

过氧化物酶体(peroxisomes)是一类真核生物中普遍存在的细胞器,参与β-氧化、乙醛酸循环等多种重要的生化代谢。研究表明,过氧化物酶体在植物病原真菌侵染寄主过程中具有着举足轻重的作用。参与过氧化物酶体形成与增殖的基因,通常称为PEX基因。近年来,越来越多的PEX基因在植物病原真菌中得到鉴定,真菌过氧化物酶体的形成机制及其在植物病原真菌生长发育和致病过程中的作用越来越受到研究者的关注。本文围绕PEX 基因在过氧化物酶体形成中的作用、对过氧化物酶体相关生化代谢的影响,以及与植物病原真菌生长发育和致病性的关系进行了综述,以期为植物病原真菌致病机理研究和病害防控提供借鉴和参考。     

Gene disruption to evaluate the role of fungal candidate virulence genes

Gene disruption is a powerful genetic tool that can define pathogenic or virulence factors. In the past two years gene disruption approaches have been used to identify fungal virulence genes. The capsule genes, an alpha subunit of G protein and certain kinases of Cryptococcus neoformans have clearly been demonstrated to be associated with pathogenicity. In Candida albicans at least four genes involved in hyphal formation have been disrupted and tested for virulence. In other fungi, such as Histoplasma capsulatum, however, more efficient gene disruption methods need to be developed before such approaches can be regularly used for identifying virulence genes.     

In search of decoy/guardee to R genes: Deciphering the role of sugars in defense against Fusarium wilt in chickpea

Plant responses are coordinately controlled by both external and internal signals. Apt perception of pathogen attack and its appropriate conversion to internal signals ultimately determine the outcome of innate immunity. The present review predicts the involvement of unconventional ‘guard/decoy model’ in chickpea-Fusarium encounter. Rapid alkalinization factor is predicted to act as initial ‘Gatekeeper decoy’ counteracting fungal entry. Phospholipases and cystatins probably function as ‘Guardees’ being shielded by R gene(s). Serine Threonine Kinases decodes external pathogenic signals to in planta defense alarms. 14.3.3 provides clues to the wilt mechanism. The versatile sugars serve as signal generators and transmitters maintaining intra and inter cellular connectivity during stress.Key words: R gene, decoy, guardee, RALF, ROS, STK, 14.3.3, sugar, defense‘Survival for existence’ is the dictum followed by the entire living world. Similarly ‘survival of the fittest’ is nature''s preference. Owing to the extensive surveillance system of higher organisms resistance becomes the natural rule while susceptibility the exception.¹ All living entities are being exposed to a plethora of interactions ranging from mutualism to antagonism.² However the adaptive strategies opted by the plants are unique, versatile and still grossly unknown which have attracted the researchers since decades towards looking into the varied responses and diversification of plant adaptation.Plants are hosts to a large number of organisms such as symbiotic/pathogenic bacteria, phytopathogenic fungi, harmful viruses and nematodes. All have their own stratagem to gain over their host.³ However only the plant-fungal interaction with Chickpea-Fusarium case study in particular, shall be the focal area of the present review. Fungi are classified as necrotrophic and biotrophic according to their nutritional requirements.⁴ Necrotrophs apply ‘brute force’ by killing host cells and thriving on their dead remains while biotrophs prefer subtler ‘modus operandi’—the stealth mechanism used to derive nutrients from live host cells.⁵ Irrespective of the pathogen type and their mode of nutrition procurement, perception of attack lies central to effective induction of innate immunity in plants.     

'Conserved hypothetical' proteins: prioritization of targets for experimental study

Comparative genomics shows that a substantial fraction of the genes in sequenced genomes encodes ‘conserved hypothetical’ proteins, i.e. those that are found in organisms from several phylogenetic lineages but have not been functionally characterized. Here, we briefly discuss recent progress in functional characterization of prokaryotic ‘conserved hypothetical’ proteins and the possible criteria for prioritizing targets for experimental study. Based on these criteria, the chief one being wide phyletic spread, we offer two ‘top 10’ lists of highly attractive targets. The first list consists of proteins for which biochemical activity could be predicted with reasonable confidence but the biological function was predicted only in general terms, if at all (‘known unknowns’). The second list includes proteins for which there is no prediction of biochemical activity, even if, for some, general biological clues exist (‘unknown unknowns’). The experimental characterization of these and other ‘conserved hypothetical’ proteins is expected to reveal new, crucial aspects of microbial biology and could also lead to better functional prediction for medically relevant human homologs.     

Exploring Candidate Genes for Pericarp Russet Pigmentation of Sand Pear (Pyrus pyrifolia) via RNA-Seq Data in Two Genotypes Contrasting for Pericarp Color

Sand pear (Pyrus pyrifolia) russet pericarp is an important trait affecting both the quality and stress tolerance of fruits. This trait is controlled by a relative complex genetic process, with some fundamental biological questions such as how many and which genes are involved in the process remaining elusive. In this study, we explored differentially expressed genes between the russet- and green-pericarp offspring from the sand pear (Pyrus pyrifolia) cv. ‘Qingxiang’ × ‘Cuiguan’ F1 group by RNA-seq-based bulked segregant analysis (BSA). A total of 29,100 unigenes were identified and 206 of which showed significant differences in expression level (log₂fold values>1) between the two types of pericarp pools. Gene Ontology (GO) analyses detected 123 unigenes in GO terms related to ‘cellular_component’ and ‘biological_process’, suggesting developmental and growth differentiations between the two types. GO categories associated with various aspects of ‘lipid metabolic processes’, ‘transport’, ‘response to stress’, ‘oxidation-reduction process’ and more were enriched with genes with divergent expressions between the two libraries. Detailed examination of a selected set of these categories revealed repressed expressions of candidate genes for suberin, cutin and wax biosynthesis in the russet pericarps.Genes encoding putative cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) that are involved in the lignin biosynthesis were suggested to be candidates for pigmentation of sand pear russet pericarps. Nine differentially expressed genes were analyzed for their expressions using qRT-PCR and the results were consistent with those obtained from Illumina RNA-sequencing. This study provides a comprehensive molecular biology insight into the sand pear pericarp pigmentation and appearance quality formation.     

Prioritizing Disease Candidate Proteins in Cardiomyopathy-Specific Protein-Protein Interaction Networks Based on “Guilt by Association” Analysis

The cardiomyopathies are a group of heart muscle diseases which can be inherited (familial). Identifying potential disease-related proteins is important to understand mechanisms of cardiomyopathies. Experimental identification of cardiomyophthies is costly and labour-intensive. In contrast, bioinformatics approach has a competitive advantage over experimental method. Based on “guilt by association” analysis, we prioritized candidate proteins involving in human cardiomyopathies. We first built weighted human cardiomyopathy-specific protein-protein interaction networks for three subtypes of cardiomyopathies using the known disease proteins from Online Mendelian Inheritance in Man as seeds. We then developed a method in prioritizing disease candidate proteins to rank candidate proteins in the network based on “guilt by association” analysis. It was found that most candidate proteins with high scores shared disease-related pathways with disease seed proteins. These top ranked candidate proteins were related with the corresponding disease subtypes, and were potential disease-related proteins. Cross-validation and comparison with other methods indicated that our approach could be used for the identification of potentially novel disease proteins, which may provide insights into cardiomyopathy-related mechanisms in a more comprehensive and integrated way.     

Paleovirology of ‘syncytins’, retroviral env genes exapted for a role in placentation

The development of the emerging field of ‘paleovirology’ allows biologists to reconstruct the evolutionary history of fossil endogenous retroviral sequences integrated within the genome of living organisms and has led to the retrieval of conserved, ancient retroviral genes ‘exapted’ by ancestral hosts to fulfil essential physiological roles, syncytin genes being undoubtedly among the most remarkable examples of such a phenomenon. Indeed, syncytins are ‘new’ genes encoding proteins derived from the envelope protein of endogenous retroviral elements that have been captured and domesticated on multiple occasions and independently in diverse mammalian species, through a process of convergent evolution. Knockout of syncytin genes in mice provided evidence for their absolute requirement for placenta development and embryo survival, via formation by cell–cell fusion of syncytial cell layers at the fetal–maternal interface. These genes of exogenous origin, acquired ‘by chance’ and yet still ‘necessary’ to carry out a basic function in placental mammals, may have been pivotal in the emergence of mammalian ancestors with a placenta from egg-laying animals via the capture of a founding retroviral env gene, subsequently replaced in the diverse mammalian lineages by new env-derived syncytin genes, each providing its host with a positive selective advantage.     

Hypervariable genes—experimental error or hidden dynamics

In a homogeneous group of samples, not all genes of high variability stem from experimental errors in microarray experiments. These expression variations can be attributed to many factors including natural biological oscillations or metabolic processes. The behavior of these genes can tease out important clues about naturally occurring dynamic processes in the organism or experimental system under study. We developed a statistical procedure for the selection of genes with high variability denoted hypervariable (HV) genes. After the exclusion of low expressed genes and a stabilizing log-transformation, the majority of genes have comparable residual variability. Based on an F-test, HV genes are selected as having a statistically significant difference from the majority of variability stabilized genes measured by the ‘reference group’. A novel F-test clustering technique, further noted as ‘F-means clustering’, groups HV genes with similar variability patterns, presumably from their participation in a common dynamic biological process. F-means clustering establishes, for the first time, groups of co-expressed HV genes and is illustrated with microarray data from patients with juvenile rheumatoid arthritis and healthy controls.     

The Fus3/Kss1 MAP kinase homolog Amk1 regulates the expression of genes encoding hydrolytic enzymes in Alternaria brassicicola

Mitogen-activated protein (MAP) kinases have been shown to be required for virulence in diverse phytopathogenic fungi. To study its role in pathogenicity, we disrupted the Amk1 MAP kinase gene, a homolog of the Fus3/Kss1 MAP kinases in Saccharomyces cerevisiae, in the necrotrophic Brassica pathogen, Alternaria brassicicola. The amk1 disruption mutants showed null pathogenicity on intact host plants. However, amk1 mutants were able to colonize host plants when they were inoculated on a physically damaged host surface, or when they were inoculated along with nutrient supplements. On intact plants, mutants expressed extremely low amounts of several hydrolytic enzyme genes that were induced over 10-fold in the wild-type during infection. These genes were also dramatically induced in the mutants on wounded plants. These results imply a correlation between virulence and the expression level of specific hydrolytic enzyme genes plus the presence of an unidentified pathway controlling these genes in addition to or in conjunction with the Amk1 pathway.     

Delivering the goods: Fungal secretion modulates virulence during host–pathogen interactions

Fungi secrete a variety of compounds that have wide ranging effects on society and govern the outcome of host–pathogen interactions. The secreted products range from powerful toxins and carcinogens, to beneficial compounds such as ethanol used in common commercial practices, and the ‘wonder drug’ penicillin. Much research in the past 50 y has focused on identifying the genes and their functions relating to the fungal secretome. Recent advances into the mechanisms by which phytopathogenic fungal secretion systems function and modulate virulence have broad implications for the agricultural and biotechnological industries. In this review, we focus on secretion mechanisms in phytopathogenic fungi with examples from key plant–pathogen systems. Current progress and knowledge gaps regarding secretion pathways and their regulation are discussed. We highlight possible approaches to using novel molecular techniques to generate alternative control methods to synthetic pesticides.     

植物病原真菌分泌蛋白质组学研究进展

分泌蛋白质组是指在特定时间和特定条件下,由组织或细胞等分泌的全部蛋白质。在病原真菌与植物的相互作用过程中,病原真菌会分泌大量的蛋白质和代谢产物,在病原真菌对植物的侵入、定殖和扩展等致病过程中起着重要作用。本文主要介绍了分泌蛋白质在植物病原真菌致病性中的作用、重要植物病原真菌分泌蛋白质组的研究进展、及植物病原真菌分泌蛋白质组的生物信息学预测分析等,对于全面了解植物病原真菌的致病机理具有重要意义。