Specific Insect-Virus Interactions Are Responsible for Variation in Competency of Different Thrips tabaci Isolines to Transmit Different Tomato Spotted Wilt Virus Isolates

Local adaptation between sympatric host and parasite populations driven by vector genetics appears to be a factor that influences dynamics of disease epidemics and evolution of insect-vectored viruses. Although T. tabaci is the primary vector of Tomato spotted wilt virus (TSWV) in some areas of the world, it is not an important vector of this economically important plant virus in many areas where it occurs. Previous studies suggest that genetic variation of thrips populations, virus isolates, or both are important factors underlying the localized importance of this species as a vector of TSWV. This study was undertaken to quantify variation in transmissibility of TSWV isolates by T. tabaci, in the ability of T. tabaci to transmit isolates of TSWV, and to examine the possibility that genetic interactions and local adaptation contribute to the localized nature of this species as a vector of TSWV. Isofemale lines of Thrips tabaci from multiple locations were tested for their ability to transmit multiple TSWV isolates collected at the same and different locations as the thrips. Results revealed that the probability of an isofemale line transmitting TSWV varied among virus isolates, and the probability of an isolate being transmitted varied among isofemale lines. These results indicate that the interaction of T. tabaci and TSWV isolate genetic determinants underlie successful transmission of TSWV by T. tabaci. Further analysis revealed sympatric vector-virus pairing resulted in higher transmission than allopatric pairing, which suggests that local adaptation is occurring between T. tabaci and TSWV isolates.     

Amblyomma americanum as a Bridging Vector for Human Infection with Francisella tularensis

The γ-proteobacterium Francisella tularensis causes seasonal tick-transmitted tularemia outbreaks in natural rabbit hosts and incidental infections in humans in the south-central United States. Although Dermacentor variabilis is considered a primary vector for F. tularensis, Amblyomma americanum is the most abundant tick species in this endemic region. A systematic study of F. tularensis colonization of A. americanum was undertaken to better understand its potential to serve as an overwintering reservoir for F. tularensis and as a bridging vector for human infections. Colony-reared A. americanum were artificially fed F. tularensis subspecies holarctica strain LVS via glass capillaries and colonization levels determined. Capillary-fed larva and nymph were initially infected with 10⁴ CFU/tick which declined prior to molting for both stages, but rebounded post-molting in nymphs and persisted in 53% at 10³ to 10⁸ CFU/nymph at 168 days post-capillary feeding (longest sampling time in the study). In contrast, only 18% of adults molted from colonized nymphs maintained LVS colonization at 10¹ to 10⁵ CFU/adult at 168 days post-capillary feeding (longest sampling time). For adults, LVS initially colonized the gut and disseminated to salivary glands by 24 h and had an ID₅₀ of <5CFU in mice. Francisella tularensis infected the ovaries of gravid females, but transmission to eggs was infrequent and transovarial transmission to hatched larvae was not observed. The prolonged persistence of F. tularensis in A. americanum nymphs supports A. americanum as an overwintering reservoir for F. tularensis from which seasonal epizootics may originate; however, although the rapid dissemination of F. tularensis from gut to salivary glands in adults A. americanum is compatible with intermittent feeding adult males acting as bridging vectors for incidental F. tularensis infections of humans, acquisition of F. tularensis by adults may be unlikely based on adult feeding preference for larger mammals which are not involved in maintenance of sylvatic tularemia.     

Plasmodium vivax Population Structure and Transmission Dynamics in Sabah Malaysia

Despite significant progress in the control of malaria in Malaysia, the complex transmission dynamics of P. vivax continue to challenge national efforts to achieve elimination. To assess the impact of ongoing interventions on P. vivax transmission dynamics in Sabah, we genotyped 9 short tandem repeat markers in a total of 97 isolates (8 recurrences) from across Sabah, with a focus on two districts, Kota Marudu (KM, n = 24) and Kota Kinabalu (KK, n = 21), over a 2 year period. STRUCTURE analysis on the Sabah-wide dataset demonstrated multiple sub-populations. Significant differentiation (F _ST  = 0.243) was observed between KM and KK, located just 130 Km apart. Consistent with low endemic transmission, infection complexity was modest in both KM (mean MOI  = 1.38) and KK (mean MOI  = 1.19). However, population diversity remained moderate (H _E  = 0.583 in KM and H _E  = 0.667 in KK). Temporal trends revealed clonal expansions reflecting epidemic transmission dynamics. The haplotypes of these isolates declined in frequency over time, but persisted at low frequency throughout the study duration. A diverse array of low frequency isolates were detected in both KM and KK, some likely reflecting remnants of previous expansions. In accordance with clonal expansions, high levels of Linkage Disequilibrium (I _A ^S >0.5 [P<0.0001] in KK and KM) declined sharply when identical haplotypes were represented once (I _A ^S  = 0.07 [P = 0.0076] in KM, and I _A ^S = -0.003 [P = 0.606] in KK). All 8 recurrences, likely to be relapses, were homologous to the prior infection. These recurrences may promote the persistence of parasite lineages, sustaining local diversity. In summary, Sabah''s shrinking P. vivax population appears to have rendered this low endemic setting vulnerable to epidemic expansions. Migration may play an important role in the introduction of new parasite strains leading to epidemic expansions, with important implications for malaria elimination.     

Genetic and host-associated differentiation within Thrips tabaci Lindeman (Thysanoptera: Thripidae) and its links to Tomato spotted wilt virus-vector competence

Of eight thelytokous populations of onion thrips (Thrips tabaci) collected from potato (three populations), onion (four) or Chrysanthemum (one) hosts from various regions of Australia, only those from potato were capable of transmitting Tomato spotted wilt virus (TSWV) in controlled transmission experiments. Genetic differentiation of seven of these eight populations, and nine others not tested for TSWV vector competence, was examined by comparison of the DNA sequences of mitochondrial cytochrome oxidase subunit 1 (COI) gene. All Australian populations of T. tabaci grouped within the European ‘L2'' clade of Brunner et al. (2004). Within this clade the seven populations from potato, the three from onion, and the four from other hosts (Chrysanthemum, Impatiens, lucerne, blackberry nightshade) clustered as three distinct sub-groupings characterised by source host. Geographical source of thrips populations had no influence on genetic diversity. These results link genetic differentiation of thelytokous T. tabaci to source host and to TSWV vector capacity for the first time.     

CTLA4 Variants and Haplotype Contribute Genetic Susceptibility to Myasthenia Gravis in Northern Chinese Population

Background

Cytotoxic T lymphocyte-associated antigen-4 (CTLA4), a critical negative regulator of the T-cell response, has been considered a candidate for many autoimmune diseases. Evidence from Caucasians supported a genetic predisposition of CTLA4 to myasthenia gravis (MG), but the contribution in East Asians has not been established.

Objectives

To investigate the role of CTLA4 variants in the susceptibility to MG and the contribution to subtypes of MG.

Methods

Six autoimmune disease-related risk alleles of CTLA4 (rs1863800, rs733618, rs4553808, rs5742909, rs231775, and rs3087243) were investigated for MG in northern Chinese. 168 patients with MG (mean age 37.1±20.5 years, 64 men and 104 women) and 233 healthy controls (mean age 53.3±8.7 years, 96 men and 137 women) were screened, and the contribution of CTLA4 to the general risk of MG and each subgroup was explored.

Results

rs1863800*C, rs733618*C, and rs231775*G were significantly associated with the whole cohort of patients with MG after permutation correction for multiple-testing adjustment (P = 0.027, 0.001, and 0.032, respectively). A risk haplotype (CCACG) [odds ratio (OR) = 1.535, range = 1.150–2.059, P = 0.004)] was also identified. The stratified subtype analysis indicated that the positive contribution was possibly derived from early onset MG (EOMG), seropositive MG (SPMG), female patients, and MG without thymoma. No association was observed in juvenile MG/LOMG, and MG coupled with thymoma.

Conclusion

A predisposing effect of rs1863800*C, rs733618*C, and rs231775*G of CTLA4 gene to general risk of MG in Chinese was demonstrated for the first time, which was likely derived from EOMG, SPMG, MG without thymoma and the female patients.     

Nematode Control Related to Fusarium Wilt in Soybean and Root Rot and Zinc Deficiency in Corn

Nematode and disease problems of irrigated, double-cropped soybean and corn, and zinc deficiency of corn were investigated. Ethylene dibromide, phenamiphos, and aldicarb were equally effective for controlling nematodes and increasing yields of corn planted minimum-till and soybean planted in a moldboard plow prepared seedbed. The residual effects on yields of nematicides applied to the preceeding crop occurred during 3 years for soybean and 1 year for corn. Fusarium wilt symptoms of soybean that developed during 2 years of the study were less severe in all nematicide-treated plots than in control plots. Typical zinc deficiency symptoms on 30-day-old corn plants were observed during 1 year of the study in certain plots. Symptoms were not evident on plants grown on plots treated with ethylene dibromide, and only occasional plants had symptoms on plots treated with phenamiphos and aldicarb. The amount of yield response directly related to nematode control could not be determined because of the apparent interaction of nematodes on the expression of Fusarium wilt of soybean. Our study strongly indicates that the expression of Fusarium wilt of soybean and zinc deficiency in corn are influenced by nematodes and that nematicides will reduce their severity.     

Meloidogyne incognita and Tomato Response to Thiamine,Ascorbic Acid, L-arginine,and L-glutamic Acid

The influence of solutions of ascorbic acid, thiamine, L-arginine, and L-gtutamic acid on egg hatch, juvenile survival, and development and reproduction of Meloidogyne incognita in susceptible and resistant tomatoes was studied. Maximum inhibition of egg hatch occurred at 2,000, 4,000, and 2,000 ppm for ascorbic acid, L-arginine, and L-glutamic acid, respectively. Larval survival was significantly reduced by concentrations of 2,000 ppm ascorbic acid and 1,000 ppm of L-arginine. Maximum inhibition of egg hatch and mortality of juveniles was achieved at a concentration of 4,000 ppm of ascorbic acid and L-arginine. L-glutamic acid and thiamine had respective moderate and minimal toxic effects. Foliar sprays of ascorbic acid, L-arginine, or L-glutamic acid suppressed the numbers of root galls, females, and egg masses on the susceptible tomato cultivar Tropic. Ascorbic acid and L-arginine had highly significant effects when applied to foliage before inoculation with nematodes. Thiamine had little effect. All sprays suppressed the numbers of root galls and females in roots of the resistant cultivar VFN8 when treatments were applied before inoculation. They were not, however, effective as post-inoculation treatments. Growth of a susceptible cultivar was improved by post-inoculation and pre-inoculation treatments when compared with the control plants which had neither nematode infection nor chemical treatment. No positive growth response to chemical treatment was seen in resistant control plants.     

Wolbachia Infection in a Natural Parasitoid Wasp Population

The maternally transmitted bacterium Wolbachia pipientis is well known for spreading and persisting in insect populations through manipulation of the fitness of its host. Here, we identify three new Wolbachia pipientis strains, wHho, wHho2 and wHho3, infecting Hyposoter horticola, a specialist wasp parasitoid of the Glanville fritillary butterfly. The wHho strain (ST435) infects about 50% of the individuals in the Åland islands in Finland, with a different infection rate in the two mitochondrial (COI) haplotypes of the wasp. The vertical transmission rate of Wolbachia is imperfect, and lower in the haplotype with lower infection rate, suggesting a fitness trade-off. We found no association of the wHho infection with fecundity, longevity or dispersal ability of the parasitoid host. However, preliminary results convey spatial associations between Wolbachia infection, host mitochondrial haplotype and parasitism of H. horticola by its hyperparasitoid, Mesochorus cf. stigmaticus. We discuss the possibility that Wolbachia infection protects H. horticola against hyperparasitism.     

Horizontal Gene Transfer and Assortative Recombination within the Acinetobacter baumannii Clinical Population Provide Genetic Diversity at the Single carO Gene, Encoding a Major Outer Membrane Protein Channel

We described previously the presence in Acinetobacter baumannii of a novel outer membrane (OM) protein, CarO, which functions as an l-ornithine OM channel and whose loss was concomitant with increased carbapenem resistance among clonally related nosocomial isolates of this opportunistic pathogen. Here, we describe the existence of extensive genetic diversity at the carO gene within the A. baumannii clinical population. The systematic analysis of carO sequences from A. baumannii isolates obtained from public hospitals in Argentina revealed the existence of four highly polymorphic carO variants among them. Sequence polymorphism between the different A. baumannii CarO variants was concentrated in three well-defined protein regions that superimposed mostly to predicted surface-exposed loops. Polymorphism among A. baumannii CarO variants was manifested in differential electrophoretic mobilities, antigenic properties, abilities to form stable oligomeric structures, and l-ornithine influx abilities through the A. baumannii OM under in vivo conditions. Incongruence between the phylogenies of the clinical A. baumannii isolates analyzed and those of the carO variants they harbor suggests the existence of assortative (entire-gene) carO recombinational exchange within the A. baumannii population. Exchange of carO variants possessing differential characteristics mediated by horizontal gene transfer may constitute an A. baumannii population strategy to survive radically changing environmental conditions, such as the leap from inanimate sources to human hosts and vice versa, persistence in a compromised host, and/or survival in health care facilities.     

Infestation of leek by Thrips tabaci as related to spatial and temporal patterns of undersowing

Full field undersowing of leeks with clovers suppresses populations of adult and larval Thrips tabaci. To explore the conditions for application of this approach of IPM in commercial practice, variations in the spatial and temporal pattern of clover undersowing were studied. Effects on thrips populations, crop growth and the development of thrips feeding symptoms were recorded. Assessment of the yield in quantity and quality was made. Sowing clover (Trifolium fragiferum) between rows resulted in significant and sufficient thrips population suppression and good yields. Similar results were achieved by full field sowing of clover simultaneous with leek crop planting. Reduced thrips feeding symptoms were found in intercropped leeks.     

Genetic Code-guided Protein Synthesis and Folding in Escherichia coli

Universal genetic codes are degenerated with 61 codons specifying 20 amino acids, thus creating synonymous codons for a single amino acid. Synonymous codons have been shown to affect protein properties in a given organism. To address this issue and explore how Escherichia coli selects its “codon-preferred” DNA template(s) for synthesis of proteins with required properties, we have designed synonymous codon libraries based on an antibody (scFv) sequence and carried out bacterial expression and screening for variants with altered properties. As a result, 342 codon variants have been identified, differing significantly in protein solubility and functionality while retaining the identical original amino acid sequence. The soluble expression level varied from completely insoluble aggregates to a soluble yield of ∼2.5 mg/liter, whereas the antigen-binding activity changed from no binding at all to a binding affinity of > 10⁻⁸ m. Not only does our work demonstrate the involvement of genetic codes in regulating protein synthesis and folding but it also provides a novel screening strategy for producing improved proteins without the need to substitute amino acids.     

Structure and Function of Glycosylated Tandem Repeats from Candida albicans Als Adhesins

Tandem repeat (TR) regions are common in yeast adhesins, but their structures are unknown, and their activities are poorly understood. TR regions in Candida albicans Als proteins are conserved glycosylated 36-residue sequences with cell-cell aggregation activity (J. M. Rauceo, R. De Armond, H. Otoo, P. C. Kahn, S. A. Klotz, N. K. Gaur, and P. N. Lipke, Eukaryot. Cell 5:1664–1673, 2006). Ab initio modeling with either Rosetta or LINUS generated consistent structures of three-stranded antiparallel β-sheet domains, whereas randomly shuffled sequences with the same composition generated various structures with consistently higher energies. O- and N-glycosylation patterns showed that each TR domain had exposed hydrophobic surfaces surrounded by glycosylation sites. These structures are consistent with domain dimensions and stability measurements by atomic force microscopy (D. Alsteen, V. Dupres, S. A. Klotz, N. K. Gaur, P. N. Lipke, and Y. F. Dufrene, ACS Nano 3:1677–1682, 2009) and with circular dichroism determination of secondary structure and thermal stability. Functional assays showed that the hydrophobic surfaces of TR domains supported binding to polystyrene surfaces and other TR domains, leading to nonsaturable homophilic binding. The domain structures are like “classic” subunit interaction surfaces and can explain previously observed patterns of promiscuous interactions between TR domains in any Als proteins or between TR domains and surfaces of other proteins. Together, the modeling techniques and the supporting data lead to an approach that relates structure and function in many kinds of repeat domains in fungal adhesins.Yeast adhesins are a diverse set of cell adhesion proteins that mediate adhesion to host cells, environmental substrates, other fungi, and coinfecting bacteria (6, 8, 20, 21, 23, 29). The adhesins share common features, including compact N-terminal domains similar to Ig or lectin domains, Thr-rich midpieces, often in tandem repeats, and long highly glycosylated Ser/Thr-rich C-terminal regions that extend the functional domains out from the cell surface. No structures for the Thr-rich midpieces are known, but they can mediate aggregation of fungal cells (33, 35, 47). The prevalence and conservation of such repeats argue that they are functionally important, despite limited data on their structure and function.In Candida albicans, the Als adhesins are homologous proteins, products of 8 loci that encode numerous alleles of cell surface adhesins (16). In each mature Als protein, there are, from the N terminus, three tandem Ig-like domains, a β-sheet-rich conserved 127-residue amyloid-forming T region, a variable number of 36-residue tandem repeats (TRs), and a highly glycosylated stalk region that extends the N-terminal domains away from the cell surface (Fig. 1) (16, 33, 41). The C termini of these and other wall-associated adhesins are covalently cross-linked into the cell wall through transglycosylation of a modified glycosylphosphatidylinositol (GPI) anchor (18, 25). This modular design, including tandem repeats, is typical of fungal adhesins (8).Open in a separate windowFig. 1.Schematic diagram of the sequence of Als5p. The regions are named above, and the number of amino acid residues in each region is shown below. The modeled sequences are in the TR region.The Als protein Ig-like region, T region, and TR region all have protein-protein interaction activities (26, 33, 35). The Ig-like regions can interact with diverse mammalian proteins, presumably in a way analogous to antibody-antigen binding, as has been shown in the homologous protein α-agglutinin from Saccharomyces cerevisiae (8, 24, 26, 35). The T regions interact through formation of amyloid-like structures both in vivo and in vitro (33, 34a, 36). An insight into the function of the tandem repeats followed from observations that Als proteins initiate and maintain cell-to-cell aggregations, either spontaneously (“autoaggregation”) or following adhesion to a bead-bound defined ligand (10, 11, 36). Aggregation is more extensive for Als proteins with more tandem repeats (26, 35). This result suggested that the tandem repeats are uniquely structured to facilitate or mediate the aggregative function. Circular dichroism spectroscopy of the TR region of Als5p shows a β-sheet-rich structure in the soluble protein (35).In support of their direct involvement in aggregation, the repeat region of the C. albicans adhesin Als5p mediates cell-cell aggregation in the absence of the Ig-like and T domains (35). Moreover, the repeats can also potentiate binding of Als5p to fibronectin (35). Thus, the TR domains mediate cellular aggregation and increased binding to fibronectin. In addition, TR domains and their amino acid sequences are highly conserved across several Candida species (3). These properties need to be explained by their three-dimensional structure.Because there are no homologous structures known, we modeled by two independent ab initio methods. Rosetta assembles structures by combining short peptide structures extracted from the protein structural database PDB (38), then combines structures in a Monte Carlo approach, and assesses energetics of assembled structures. Rosetta has recently been shown to generate accurate models for protein-sized domains (40). We also predicted structures with LINUS, which generates randomized structures and rapidly estimates energetics to choose low-energy models (45). The models were supported by structural analyses with atomic force microscopy and circular dichroism spectroscopy. Functional assays showed that the TR domains can mediate binding activities predicted from the calculated structures.     

Differential Expression of Tomato Spotted Wilt Virus-Derived Viral Small RNAs in Infected Commercial and Experimental Host Plants

Background

Viral small RNAs (vsiRNAs) in the infected host can be generated from viral double-stranded RNA replicative intermediates, self-complementary regions of the viral genome or from the action of host RNA-dependent RNA polymerases on viral templates. The vsiRNA abundance and profile as well as the endogenous small RNA population can vary between different hosts infected by the same virus influencing viral pathogenicity and host response. There are no reports on the analysis of vsiRNAs of Tomato spotted wilt virus (TSWV), a segmented negative stranded RNA virus in the family Bunyaviridae, with two of its gene segments showing ambisense gene arrangement. The virus causes significant economic losses to numerous field and horticultural crops worldwide.

Principal Findings

Tomato spotted wilt virus (TSWV)-specific vsiRNAs were characterized by deep sequencing in virus-infected experimental host Nicotiana benthamiana and a commercial, susceptible host tomato. The total small (s) RNA reads in TSWV-infected tomato sample showed relatively equal distribution of 21, 22 and 24 nt, whereas N. benthamiana sample was dominated by 24 nt total sRNAs. The number of vsiRNA reads detected in tomato was many a magnitude (~350:1) higher than those found in N. benthamiana, however the profile of vsiRNAs in terms of relative abundance 21, 22 and 24 nt class size was similar in both the hosts. Maximum vsiRNA reads were obtained for the M RNA segment of TSWV while the largest L RNA segment had the least number of vsiRNAs in both tomato and N. benthamiana. Only the silencing suppressor, NSs, of TSWV recorded higher antisense vsiRNA with respect to the coding frame among all the genes of TSWV.

Significance

Details of the origin, distribution and abundance of TSWV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. It also has major implications toward our understanding of the differential processing of vsiRNAs in antiviral defense and viral pathogenicity.     

Interrelationship of Heterodera schachtii and Meloidogyne hapla on Tomato

Invasion of tomato (Lycopersicon esculentum L.) roots by combined and sequential inoculations of Meloidogyne hapla and a tomato population of Heterodera schachtii was affected more by soil temperature than by nematode competition. Maximum invasion of tomato roots, by M. hapla and H. schachtii occurred at 30 and 26 C, respectively. Female development and nematode reproduction (eggs per plant) of M. hapla was adversely affected by H. schachtii in combined inoculations of the two nematode species. Inhibition of M. hapla development and reproduction on tomato roots from combined nematode inoculations was more pronounced as soil temperature was increased over a range of 18-30 C and with prior inoculation of tomato with H. schachtii. M. hapla minimally affected H. schachtii female development, but there was significant reduction in the buildup of H. schachtii when M. hapla inoculation preceded that of H. schachtii by 20 days.     

Cultivation of the Pine Wilt Nematode, Bursaphelenchus xylophilus,in Axenic Culture Media

The pine wilt nematode, Bursaphelenchus xylophilus, has been cultured axenically in vitro on soy peptone/yeast extract or modified Caenorhabditis medium supplemented with cholesterol and hemoglobin. Although growth, development and reproduction were best in soy peptone/yeast extract medium, satisfactory population size increases were observed in the chemically defined Caenorhaditis medium.