Systematic identification of novel, essential host genes affecting bromovirus RNA replication

Positive-strand RNA virus replication involves viral proteins and cellular proteins at nearly every replication step. Brome mosaic virus (BMV) is a well-established model for dissecting virus-host interactions and is one of very few viruses whose RNA replication, gene expression and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. Previously, our laboratory identified ～100 non-essential host genes whose loss inhibited or enhanced BMV replication at least 3-fold. However, our isolation of additional BMV-modulating host genes by classical genetics and other results underscore that genes essential for cell growth also contribute to BMV RNA replication at a frequency that may be greater than that of non-essential genes. To systematically identify novel, essential host genes affecting BMV RNA replication, we tested a collection of ～900 yeast strains, each with a single essential gene promoter replaced by a doxycycline-repressible promoter, allowing repression of gene expression by adding doxycycline to the growth medium. Using this strain array of ～81% of essential yeast genes, we identified 24 essential host genes whose depleted expression reproducibly inhibited or enhanced BMV RNA replication. Relevant host genes are involved in ribosome biosynthesis, cell cycle regulation and protein homeostasis, among other cellular processes. BMV 2a(Pol) levels were significantly increased in strains depleted for a heat shock protein (HSF1) or proteasome components (PRE1 and RPT6), suggesting these genes may affect BMV RNA replication by directly or indirectly modulating 2a(Pol) localization, post-translational modification or interacting partners. Investigating the diverse functions of these newly identified essential host genes should advance our understanding of BMV-host interactions and normal cellular pathways, and suggest new modes of virus control.     

Chromosomal location and function of genes affecting Pseudomonas aeruginosa nitrate assimilation

Seven known genes control Pseudomonas aeruginosa nitrate assimilation. Three of the genes, designated nas, are required for the synthesis of assimilatory nitrate reductase: nasC encodes a structural component of the enzyme; nasA and nasB encode products that participate in the biosynthesis of the molybdenum cofactor of the enzyme. A fourth gene (nis) is required for the synthesis of assimilatory nitrite reductase. The remaining three genes (ntmA, ntmB, and ntmC) control the assimilation of a number of nitrogen sources. The nas genes and two ntm genes have been located on the chromosome and are well separated from the known nar genes which encode synthesis of dissimilatory nitrate reductase. Our data support the previous conclusion that P. aeruginosa has two distinct nitrate reductase systems, one for the assimilation of nitrate and one for its dissimilation.     

Identification of Yersinia enterocolitica genes affecting survival in an animal host using signature-tagged transposon mutagenesis

Pathogenic Yersinia species are associated with both localized and systemic infections in mammalian hosts. In this study, signature-tagged transposon mutagenesis was used to identify Yersinia enterocolitica genes required for survival in a mouse model of infection. Approximately 2000 transposon insertion mutants were screened for attenuation. This led to the identification of 55 mutants defective for survival in the animal host, as judged by their ability to compete with the wild-type strain in mixed infections. A total of 28 mutants had transposon insertions in the virulence plasmid, validating the screen. Two of the plasmid mutants with severe virulence defects had insertions in an uncharacterized region. Several of the chromosomal insertions were in a gene cluster involved in O-antigen biosynthesis. Other chromosomal insertions identified genes not previously demonstrated as being required for in vivo survival of Y. enterocolitica. These include genes involved in the synthesis of outer membrane components, stress response and nutrient acquisition. One severely attenuated mutant had an insertion in a homologue of the pspC gene (phage shock protein C) of Escherichia coli. The phage shock protein operon has no known biochemical or physiological function in E. coli, but is apparently essential for the survival of Y. enterocolitica during infection.     

Mapping of Agrobacterium tumefaciens chromosomal genes affecting cellulose synthesis and bacterial attachment to host cells.

Six chromosomal transposon mutations in Agrobacterium tumefaciens which result in an inability to synthesize cellulose fibrils were mapped to the vicinity of trp-2 and met-6. Mutations which result in an inability to attach to plant cells, attC43 and attC69, also mapped in this region, as did one Tn5 mutation which caused overproduction of cellulose. Another cellulose overproduction mutation mapped at a distance and was closely linked to ilv-13. The results suggest that there is a region of the A. tumefaciens chromosome located near met-6 which is concerned with the interaction of the bacterium with the host cell surface.     

Baculovirus infection influences host protein expression in two established insect cell lines

We identified host proteins that changed in response to host cell susceptibility to baculovirus infection. We used three baculovirus-host cell systems utilizing two cell lines derived from pupal ovaries, Hz-AM1 (from Helicoverpa zea) and Hv-AM1 (from Heliothis virescens). Hv-AM1 cells are permissive to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and semi-permissive to H. zea single nucleopolyhedrovirus (HzSNPV). Hz-AM1 cells are non-permissive to AcMNPV. We challenged each cell line with baculovirus infection and after 24 h determined protein identities by MALDI TOF/TOF mass spectrometry. For Hv-AM1 cells, 21 proteins were identified, and for Hz-AM1 cells, 19 proteins were newly identified (with 8 others having been previously identified). In the permissive relationship, 18 of the proteins changed in expression by 70% or more in AcMNPV infected Hv-AM1 cells as compared with non-infected controls; 12 were significantly decreased and 6 cellular proteins were significantly increased. We also identified 3 virus-specific proteins. In the semi-permissive infections, eight proteins decreased by 2-fold or more. Non-permissive interactions did not lead to substantial changes in host cell protein expression. We hypothesize that some of these proteins act in determining host cell specificity for baculoviruses.     

A genome-wide screen for genes affecting eisosomes reveals Nce102 function in sphingolipid signaling

The protein and lipid composition of eukaryotic plasma membranes is highly dynamic and regulated according to need. The sphingolipid-responsive Pkh kinases are candidates for mediating parts of this regulation, as they affect a diverse set of plasma membrane functions, such as cortical actin patch organization, efficient endocytosis, and eisosome assembly. Eisosomes are large protein complexes underlying the plasma membrane and help to sort a group of membrane proteins into distinct domains. In this study, we identify Nce102 in a genome-wide screen for genes involved in eisosome organization and Pkh kinase signaling. Nce102 accumulates in membrane domains at eisosomes where Pkh kinases also localize. The relative abundance of Nce102 in these domains compared with the rest of the plasma membrane is dynamically regulated by sphingolipids. Furthermore, Nce102 inhibits Pkh kinase signaling and is required for plasma membrane organization. Therefore, Nce102 might act as a sensor of sphingolipids that regulates plasma membrane function.     

Mutant genes affecting higher plant meiosis

Summary That meiosis is conditioned by a large number of genes majority of which are present in a dominant state, is evidenced by the detection of numerous monogenic recessive mutant genes which affect the premeiotic, meiotic and post-meiotic course of events. These genes are site- and stage-specific, and a few are sex specific. Of these, the most prevalent are the mutant genes affecting male meiosis and causing male sterility (ms genes) and those inhibiting synapsis and chiasma formation (synaptic genes) and leading to gametic sterility. Majority of the mutant genes affect the entire chromosomal complement but a few influence only specific chromosomes of a complement so that the chromosomes behave differentially within a genome of the same species. Some mutant genes alter chromosome form and function, others modify integrity, degree of spiralization, movement and migration of chromosomes. Their cytogenetic behaviour, genetic significance and breeding utility are described and discussed.Dedicated to Professor Dr. Werner Gottschalk, Director, Institute of Genetics, University of Bonn, Federal Republic of Germany, on his 65th birthday for his excellent scientific contribution and humane nature     

Low and homogeneous copy number of plasmid-borne symbiont genes affecting host nutrition in Buchnera aphidicola of the aphid Uroleucon ambrosiae

The bacterial endosymbiont of aphids, Buchnera aphidicola, often provides amino acids to its hosts. Plasmid amplification of leucine (leuABCD) and tryptophan (trpEG) biosynthesis genes may be a mechanism by which some Buchnera over-produce these nutrients. We used quantitative polymerase chain reaction to assess the leuABCD/trpEG copy variability within Uroleucon ambrosiae, an aphid with a wide diet breadth and range. Both leuABCD and trpEG abundances are: (i) similar for aphids across 15 populations, and (ii) low compared to Buchnera from other aphid species (particularly trpEG). Consequently, the plasmid location of trpEG combined with Buchnera's chromosomal polyploidy may functionally limit, rather than increase, tryptophan production within Uroleucon ambrosiae.     

Ecosystem screening approach for pathogen-associated microorganisms affecting host disease

The microbial community in which a pathogen evolves is fundamental to disease outcome. Species interacting with a pathogen on the host surface shape the distribution, density, and genetic diversity of the inoculum, but the role of these species is rarely determined. The screening method developed here can be used to characterize pathogen-associated species affecting disease. This strategy involves three steps: (i) constitution of the microbial community, using the pathogen as a trap; (ii) community selection, using extracts from the pathogen as the sole nutrient source; and (iii) molecular identification and the screening of isolates focusing on their effects on the growth of the pathogen in vitro and host disease. This approach was applied to a soilborne plant pathogen, Phytophthora parasitica, structured in a biofilm, for screening the microbial community from the rhizosphere of Nicotiana tabacum (the host). Two of the characterized eukaryotes interfered with the oomycete cycle and may affect the host disease. A Vorticella species acted through a mutualistic interaction with P. parasitica, disseminating pathogenic material by leaving the biofilm. A Phoma species established an amensal interaction with P. parasitica, strongly suppressing disease by inhibiting P. parasitica germination. This screening method is appropriate for all nonobligate pathogens. It allows the definition of microbial species as promoters or suppressors of a disease for a given biotope. It should also help to identify important microbial relationships for ecology and evolution of pathogens.     

Mutations in coliphage p1 affecting host cell lysis

A total of 103 amber mutants of coliphage P1 were tested for lysis of nonpermissive cells. Of these, 83 caused cell lysis at the normal lysis time and have defects in particle morphogenesis. Five amber mutants, with mutations in the same gene (gene 2), caused premature lysis and may have a defect in a lysis regulator. Fifteen amber mutants were unable to cause cell lysis. Artificially lysed cells infected with five of these mutants produced viable phage particles, and phage particles were seen in thin sections of unlysed, infected cells. However, phage production by these mutants was not continued after the normal lysis time. We conclude that the defect of these five mutants is in a lysis function. The five mutations were found to be in the same gene (designated gene 17). The remaining 10 amber mutants, whose mutations were found to be in the same gene (gene 10), were also unable to cause cell lysis. They differed from those in gene 17 in that no viable phage particles were produced from artificially lysed cells, and no phage particles were seen in thin sections of unlysed, infected cells. We conclude that the gene 10 mutants cannot synthesize late proteins, and it is possible that gene 10 may code for a regulator of late gene expression for P1.     

Baculovirus host-range

Baculoviruses are used as microbial insecticides, protein expression vectors, epitope display platforms, and most recently as vectors for gene therapy. Understanding the mechanisms that control baculovirus host-range and tissue tropisms are important for assessing their safety and for improving their properties for these biotechnology applications. In the past two decades some progress has been made and several baculovirus genes that influence host-range have been identified. Despite this progress, our understanding of the underlying mechanisms that restrict baculovirus host-range is still limited. Here we review what is currently known about baculovirus genes that influence virus host-range.     

Baculovirus Host-Range

Baculoviruses are used as microbial insecticides, protein expression vectors, epitope display platforms, and most recently as vectors for gene therapy. Understanding the mechanisms that control baculovirus host-range and tissue tropisms are important for assessing their safety and for improving their properties for these biotechnology applications. In the past two decades some progress has been made and several baculovirus genes that influence host-range have been identified. Despite this progress, our understanding of the underlying mechanisms that restrict baculovirus host-range is still limited. Here we review what is currently known about baculovirus genes that influence virus host-range     

Willow leaf traits affecting host use by the leaf-gall-forming sawfly

Gall-forming insects usually have very restricted host ranges, but plant traits affecting patterns of host use have rarely been examined. The sawfly Phyllocolpa sp. (Hymenoptera: Tenthredinidae) forms leaf-roll galls on three of seven Salix (Salicaceae) species that occur together on riverbanks in central Japan. We have attempted to explain this host-use pattern by invoking three plant traits: synchrony of leaf flush with the oviposition period of the sawfly, intrinsic leaf quality as a potential larval food, and leaf morphology. Two Salix species frequently used by the sawfly, Salix eriocarpa and Salix pierotii, had similar leaf traits suitable for larval survival. The third species, Salix serissaefolia, was used relatively less often and the sawfly frequently stopped laying eggs on the plant during oviposition, suggesting ovipositional selection. S. serissaefolia had the smallest leaves, and survival of sawfly larvae was lower on S. serissaefolia than on S. eriocarpa and S. pierotii, because of gall destruction, by other herbivorous insects, and leaf-size restrictions. Among the four unused species, Salix chaenomeloides had a late leaf-flush phenology, Salix gracilistyla had inferior leaf quality, and Salix gilgiana had linear leaves; these seemed to be critical factors for non-use. Salix subfragilis was also unused, but the reason for this could not be explained by the three leaf traits studied.