Horizontal gene transfer and mutation: Ngrol genes in the genome of Nicotiana glauca

Ngrol genes (NgrolB, NgrolC, NgORF13, and NgORF14) that are similar in sequence to genes in the left transferred DNA (TL-DNA) of Agrobacterium rhizogenes have been found in the genome of untransformed plants of Nicotiana glauca. It has been suggested that a bacterial infection resulted in transformation of Ngrol genes early in the evolution of the genus Nicotiana. Although the corresponding four rol genes in TL-DNA provoked hairy-root syndrome in plants, present-day N. glauca and plants transformed with Ngrol genes did not exhibit this phenotype. Sequenced complementation analysis revealed that the NgrolB gene did not induce adventitious roots because it contained two point mutations. Single-base site-directed mutagenesis at these two positions restored the capacity for root induction to the NgrolB gene. When the NgrolB, with these two base substitutions, was positioned under the control of the cauliflower mosaic virus 35S promoter (P35S), transgenic tobacco plants exhibited morphological abnormalities that were not observed in P35s-RirolB plants. In contrast, the activity of the NgrolC gene may have been conserved after an ancient infection by bacteria. Discussed is the effect of the horizontal gene transfer of the Ngrol genes and mutations in the NgrolB gene on the phenotype of ancient plants during the evolution of N. glauca.

Arabidopsis thaliana PAD4 encodes a lipase-like gene that is important for salicylic acid signaling

The Arabidopsis PAD4 gene previously was found to be required for expression of multiple defense responses including camalexin synthesis and PR-1 gene expression in response to infection by the bacterial pathogen Pseudomonas syringae pv. maculicola. This report describes the isolation of PAD4. The predicted PAD4 protein sequence displays similarity to triacyl glycerol lipases and other esterases. The PAD4 transcript was found to accumulate after P. syringae infection or treatment with salicylic acid (SA). PAD4 transcript levels were very low in infected pad4 mutants. Treatment with SA induced expression of PAD4 mRNA in pad4–1, pad4–3, and pad4–4 plants but not in pad4–2 plants. Induction of PAD4 expression by P. syringae was independent of the regulatory factor NPR1 but induction by SA was NPR1-dependent. Taken together with the previous observation that pad4 mutants have a defect in accumulation of SA upon pathogen infection, these results suggest that PAD4 participates in a positive regulatory loop that increases SA levels, thereby activating SA-dependent defense responses.

Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens

The endogenous plant hormones salicylic acid (SA) and jasmonic acid (JA), whose levels increase on pathogen infection, activate separate sets of genes encoding antimicrobial proteins in Arabidopsis thaliana. The pathogen-inducible genes PR-1, PR-2, and PR-5 require SA signaling for activation, whereas the plant defensin gene PDF1.2, along with a PR-3 and PR-4 gene, are induced by pathogens via an SA-independent and JA-dependent pathway. An Arabidopsis mutant, coi1, that is affected in the JA-response pathway shows enhanced susceptibility to infection by the fungal pathogens Alternaria brassicicola and Botrytis cinerea but not to Peronospora parasitica, and vice versa for two Arabidopsis genotypes (npr1 and NahG) with a defect in their SA response. Resistance to P. parasitica was boosted by external application of the SA-mimicking compound 2,6-dichloroisonicotinic acid [Delaney, T., et al. (1994) Science 266, 1247–1250] but not by methyl jasmonate (MeJA), whereas treatment with MeJA but not 2,6-dichloroisonicotinic acid elevated resistance to Alternaria brassicicola. The protective effect of MeJA against A. brassicicola was the result of an endogenous defense response activated in planta and not a direct effect of MeJA on the pathogen, as no protection to A. brassicicola was observed in the coi1 mutant treated with MeJA. These data point to the existence of at least two separate hormone-dependent defense pathways in Arabidopsis that contribute to resistance against distinct microbial pathogens.

Atomic structure of a thermostable subdomain of HIV-1 gp41

Infection by HIV-1 involves the fusion of viral and cellular membranes with subsequent transfer of viral genetic material into the cell. The HIV-1 envelope glycoprotein that mediates fusion consists of the surface subunit gp120 and the transmembrane subunit gp41. gp120 directs virion attachment to the cell–surface receptors, and gp41 then promotes viral–cell membrane fusion. A soluble, α-helical, trimeric complex within gp41 composed of N-terminal and C-terminal extraviral segments has been proposed to represent the core of the fusion-active conformation of the HIV-1 envelope. A thermostable subdomain denoted N34(L6)C28 can be formed by the N-34 and C-28 peptides connected by a flexible linker in place of the disulfide-bonded loop region. Three-dimensional structure of N34(L6)C28 reveals that three molecules fold into a six-stranded helical bundle. Three N-terminal helices within the bundle form a central, parallel, trimeric coiled coil, whereas three C-terminal helices pack in the reverse direction into three hydrophobic grooves on the surface of the N-terminal trimer. This thermostable subdomain displays the salient features of the core structure of the isolated gp41 subunit and thus provides a possible target for therapeutics designed selectively to block HIV-1 entry.

Role of Dynactin in Endocytic Traffic: Effects of Dynamitin Overexpression and Colocalization with CLIP-170

The flow of material from peripheral, early endosomes to late endosomes requires microtubules and is thought to be facilitated by the minus end-directed motor cytoplasmic dynein and its activator dynactin. The microtubule-binding protein CLIP-170 may also play a role by providing an early link to endosomes. Here, we show that perturbation of dynactin function in vivo affects endosome dynamics and trafficking. Endosome movement, which is normally bidirectional, is completely inhibited. Receptor-mediated uptake and recycling occur normally, but cells are less susceptible to infection by enveloped viruses that require delivery to late endosomes, and they show reduced accumulation of lysosomally targeted probes. Dynactin colocalizes at microtubule plus ends with CLIP-170 in a way that depends on CLIP-170’s putative cargo-binding domain. Overexpression studies using p150Glued, the microtubule-binding subunit of dynactin, and mutant and wild-type forms of CLIP-170 indicate that CLIP-170 recruits dynactin to microtubule ends. These data suggest a new model for the formation of motile complexes of endosomes and microtubules early in the endocytic pathway.

Protective immunity to HIV-1 in SCID/beige mice reconstituted with peripheral blood lymphocytes of exposed but uninfected individuals

Immunodeficiency typically appears many years after initial HIV infection. This long, essentially asymptomatic period contributes to the transmission of HIV in human populations. In rare instances, clearance of HIV-1 infection has been observed, particularly in infants. There are also reports of individuals who have been frequently exposed to HIV-1 but remain seronegative for the virus, and it has been hypothesized that these individuals are resistant to infection by HIV-1. However, little is known about the mechanism of immune clearance or protection against HIV-1 in these high-risk individuals because it is difficult to directly demonstrate in vivo protective immunity. Although most of these high-risk individuals show an HIV-1-specific cell-mediated immune response using in vitro assays, their peripheral blood lymphocytes (PBLs) are still susceptible to HIV infection in tissue culture. To study this further in vivo, we have established a humanized SCID mouse infection model whereby T-, B-, and natural killer-cell defective SCID/beige mice that have been reconstituted with normal human PBLs can be infected with HIV-1. When the SCID/beige mice were reconstituted with PBLs from two different multiply exposed HIV-1 seronegative individuals, the mice showed resistance to infection by two strains of HIV-1 (macrophage tropic and T cell tropic), although the same PBLs were easily infected in vitro. Mice reconstituted with PBLs from non-HIV-exposed controls were readily infected. When the same reconstituted mice were depleted of human CD8 T cells, however, they became susceptible to HIV-1 infection, indicating that the in vivo protection required CD8 T cells. This provides clear experimental evidence that some multiply exposed, HIV-1-negative individuals have in vivo protective immunity that is CD8 T cell-dependent. Understanding the mechanism of such protective immunity is critical to the design and testing of effective prophylactic vaccines and immunotherapeutic regimens.

A herpes simplex virus 1 recombinant lacking the glycoprotein G coding sequences is defective in entry through apical surfaces of polarized epithelial cells in culture and in vivo

During infection of a new host, the first surfaces encountered by herpes simplex viruses are the apical membranes of epithelial cells of mucosal surfaces. These cells are highly polarized, and the protein composition of their apical and basolateral membranes are very different, so that different viral entry pathways have evolved for each surface. To determine whether the viral glycoprotein G (gG) is specifically required for efficient infection of a particular surface of polarized cells, apical and basal surfaces were infected with wild-type virus or a gG deletion mutant. After infection of polarized cells in culture, the gG− virus was deficient in infection of apical surfaces but was able to infect cells through basal membranes, replicate, and spread into surrounding cells. The gG-dependent step in apical infection was a stage beyond attachment. After in vivo infection of apical surfaces of epithelial cells of nonscarified mouse corneas, infection by glycoprotein C− or gG− virus was considerably reduced as compared with that observed after infection with wild-type virus. In contrast, when corneas were scarified, allowing virus access to other cell surfaces, the gG and glycoprotein C deletion mutants infected eyes as efficiently as wild-type viruses. A secondary mutation allowing infection of apical surfaces by gG− virus arose readily during passage of the virus in nonpolarized cells, indicating that either the gG-dependent step of apical infection can be bypassed or that another viral protein can acquire the same function.

Isolation and characterization of powdery mildew-resistant Arabidopsis mutants

A compatible interaction between a plant and a pathogen is the result of a complex interplay between many factors of both plant and pathogen origin. Our objective was to identify host factors involved in this interaction. These factors may include susceptibility factors required for pathogen growth, factors manipulated by the pathogen to inactivate or avoid host defenses, or negative regulators of defense responses. To this end, we identified 20 recessive Arabidopsis mutants that do not support normal growth of the powdery mildew pathogen, Erysiphe cichoracearum. Complementation analyses indicated that four loci, designated powdery mildew resistant 1–4 (pmr1–4), are defined by this collection. These mutants do not constitutively accumulate elevated levels of PR1 or PDF1.2 mRNA, indicating that resistance is not simply due to constitutive activation of the salicylic acid- or ethylene- and jasmonic acid-dependent defense pathways. Further Northern blot analyses revealed that some mutants accumulate higher levels of PR1 mRNA than wild type in response to infection by powdery mildew. To test the specificity of the resistance, the pmr mutants were challenged with other pathogens including Pseudomonas syringae, Peronospora parasitica, and Erysiphe orontii. Surprisingly, one mutant, pmr1, was susceptible to E. orontii, a very closely related powdery mildew, suggesting that a very specific resistance mechanism is operating in this case. Another mutant, pmr4, was resistant to P. parasitica, indicating that this resistance is more generalized. Thus, we have identified a novel collection of mutants affecting genes required for a compatible interaction between a plant and a biotrophic pathogen.

Three-dimensional structure of poliovirus receptor bound to poliovirus

Poliovirus initiates infection by binding to its cellular receptor (Pvr). We have studied this interaction by using cryoelectron microscopy to determine the structure, at 21-Å resolution, of poliovirus complexed with a soluble form of its receptor (sPvr). This density map aided construction of a homology-based model of sPvr and, in conjunction with the known crystal structure of the virus, allowed delineation of the binding site. The virion does not change significantly in structure on binding sPvr in short incubations at 4°C. We infer that the binding configuration visualized represents the initial interaction that is followed by structural changes in the virion as infection proceeds. sPvr is segmented into three well-defined Ig-like domains. The two domains closest to the virion (domains 1 and 2) are aligned and rigidly connected, whereas domain 3 diverges at an angle of ≈60°. Two nodules of density on domain 2 are identified as glycosylation sites. Domain 1 penetrates the “canyon” that surrounds the 5-fold protrusion on the capsid surface, and its binding site involves all three major capsid proteins. The inferred pattern of virus–sPvr interactions accounts for most mutations that affect the binding of Pvr to poliovirus.

The V domain of herpesvirus Ig-like receptor (HIgR) contains a major functional region in herpes simplex virus-1 entry into cells and interacts physically with the viral glycoprotein D

The herpesvirus entry mediator C (HveC), previously known as poliovirus receptor-related protein 1 (PRR1), and the herpesvirus Ig-like receptor (HIgR) are the bona fide receptors employed by herpes simplex virus-1 and -2 (HSV-1 and -2) for entry into the human cell lines most frequently used in HSV studies. They share an identical ectodomain made of one V and two C2 domains and differ in transmembrane and cytoplasmic regions. Expression of their mRNA in the human nervous system suggests possible usage of these receptors in humans in the path of neuron infection by HSV. Glycoprotein D (gD) is the virion component that mediates HSV-1 entry into cells by interaction with cellular receptors. We report on the identification of the V domain of HIgR/PRR1 as a major functional region in HSV-1 entry by several approaches. First, the epitope recognized by mAb R1.302 to HIgR/PRR1, capable of inhibiting infection, was mapped to the V domain. Second, a soluble form of HIgR/PRR1 consisting of the single V domain competed with cell-bound full-length receptor and blocked virion infectivity. Third, the V domain was sufficient to mediate HSV entry, as an engineered form of PRR1 in which the two C2 domains were deleted and the V domain was retained and fused to its transmembrane and cytoplasmic regions was still able to confer susceptibility, although at reduced efficiency relative to full-length receptor. Consistently, transfer of the V domain of HIgR/PRR1 to a functionally inactive structural homologue generated a chimeric receptor with virus-entry activity. Finally, the single V domain was sufficient for in vitro physical interaction with gD. The in vitro binding was specific as it was competed both by antibodies to the receptor and by a mAb to gD with potent neutralizing activity for HSV-1 infectivity.

Gene transfer of Fas ligand induces tumor regression in vivo

The Fas–Fas ligand (FasL) system plays an important role in the induction of lymphoid apoptosis and has been implicated in the suppression of immune responses. Herein, we report that gene transfer of FasL inhibits tumor cell growth in vivo. Although such inhibition is expected in Fas+ tumor cell lines, marked regression was unexpectedly observed after FasL gene transfer into the CT26 colon carcinoma that does not express Fas. Infection by an adenoviral vector encoding FasL rapidly eliminated tumor masses in the Fas+ Renca tumor by inducing cell death, whereas the elimination of Fas− CT26 cells was mediated by inflammatory cells. Analysis of human malignancies revealed Fas, but not FasL, expression in a majority of tumors and susceptibility to FasL in most Fas+ cell lines. These findings suggest that gene transfer of FasL generates apoptotic responses and induces potent inflammatory reactions that can be used to induce the regression of malignancies.

Isolation of Ethylene-Insensitive Soybean Mutants That Are Altered in Pathogen Susceptibility and Gene-for-Gene Disease Resistance1

Plants commonly respond to pathogen infection by increasing ethylene production, but it is not clear if this ethylene does more to promote disease susceptibility or disease resistance. Ethylene production and/or responsiveness can be altered by genetic manipulation. The present study used mutagenesis to identify soybean (Glycine max L. Merr.) lines with reduced sensitivity to ethylene. Two new genetic loci were identified, Etr1 and Etr2. Mutants were compared with isogenic wild-type parents for their response to different soybean pathogens. Plant lines with reduced ethylene sensitivity developed similar or less-severe disease symptoms in response to virulent Pseudomonas syringae pv glycinea and Phytophthora sojae, but some of the mutants developed similar or more-severe symptoms in response to Septoria glycines and Rhizoctonia solani. Gene-for-gene resistance against P. syringae expressing avrRpt2 remained effective, but Rps1-k-mediated resistance against P. sojae races 4 and 7 was disrupted in the strong ethylene-insensitive etr1-1 mutant. Rps1-k-mediated resistance against P. sojae race 1 remained effective, suggesting that the Rps1-k locus may encode more than one gene for disease resistance. Overall, our results suggest that reduced ethylene sensitivity can be beneficial against some pathogens but deleterious to resistance against other pathogens.

Global survey of genetic variation in CCR5, RANTES, and MIP-1α: Impact on the epidemiology of the HIV-1 pandemic

Expression of CC chemokine receptor 5 (CCR5), the major coreceptor for HIV-1 cell entry, and its ligands (e.g., RANTES and MIP-1α) is widely regarded as central to the pathogenesis of HIV-1 infection. By surveying nearly 3,000 HIV+ and HIV− individuals from worldwide populations for polymorphisms in the genes encoding RANTES, MIP-1α, and CCR5, we show that the evolutionary histories of human populations have had a significant impact on the distribution of variation in these genes, and that this may be responsible, in part, for the heterogeneous nature of the epidemiology of the HIV-1 pandemic. The varied distribution of RANTES haplotypes (AC, GC, and AG) associated with population-specific HIV-1 transmission- and disease-modifying effects is a striking example. Homozygosity for the AC haplotype was associated with an increased risk of acquiring HIV-1 as well as accelerated disease progression in European Americans, but not in African Americans. Yet, the prevalence of the ancestral AC haplotype is high in individuals of African origin, but substantially lower in non-Africans. In a Japanese cohort, AG-containing RANTES haplotype pairs were associated with a delay in disease progression; however, we now show that their contribution to HIV-1 pathogenesis and epidemiology in other parts of the world is negligible because the AG haplotype is infrequent in non-Far East Asians. Thus, the varied distribution of RANTES, MIP-1α, and CCR5 haplotype pairs and their population-specific phenotypic effects on HIV-1 susceptibility and disease progression results in a complex pattern of biological determinants of HIV-1 epidemiology. These findings have important implications for the design, assessment, and implementation of effective HIV-1 intervention and prevention strategies.

Tomato Ve disease resistance genes encode cell surface-like receptors

In tomato, Ve is implicated in race-specific resistance to infection by Verticillium species causing crop disease. Characterization of the Ve locus involved positional cloning and isolation of two closely linked inverted genes. Expression of individual Ve genes in susceptible potato plants conferred resistance to an aggressive race 1 isolate of Verticillium albo-atrum. The deduced primary structure of Ve1 and Ve2 included a hydrophobic N-terminal signal peptide, leucine-rich repeats containing 28 or 35 potential glycosylation sites, a hydrophobic membrane-spanning domain, and a C-terminal domain with the mammalian E/DXXXLφ or YXXφ endocytosis signals (φ is an amino acid with a hydrophobic side chain). A leucine zipper-like sequence occurs in the hydrophobic N-terminal signal peptide of Ve1 and a Pro-Glu-Ser-Thr (PEST)-like sequence resides in the C-terminal domain of Ve2. These structures suggest that the Ve genes encode a class of cell-surface glycoproteins with receptor-mediated endocytosis-like signals and leucine zipper or PEST sequences.

An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense

Plants contain RNA-dependent RNA polymerase (RdRP) activities that synthesize short cRNAs by using cellular or viral RNAs as templates. During studies of salicylic acid (SA)-induced resistance to viral pathogens, we recently found that the activity of a tobacco RdRP was increased in virus-infected or SA-treated plants. Biologically active SA analogs capable of activating plant defense response also induced the RdRP activity, whereas biologically inactive analogs did not. A tobacco RdRP gene, NtRDRP1, was isolated and found to be induced both by virus infection and by treatment with SA or its biologically active analogs. Tobacco lines deficient in the inducible RDRP activity were obtained by expressing antisense RNA for the NtRDRP1 gene in transgenic plants. When infected by tobacco mosaic virus, these transgenic plants accumulated significantly higher levels of viral RNA and developed more severe disease symptoms than wild-type plants. After infection by a strain of potato virus X that does not spread in wild-type tobacco plants, the transgenic NtRDRP1 antisense plants accumulated virus and developed symptoms not only locally in inoculated leaves but also systemically in upper uninoculated leaves. These results strongly suggest that inducible RdRP activity plays an important role in plant antiviral defense.