Molecular characterization of CD40 signaling intermediates

Signal transduction through the CD40 receptor is initiated by binding of its trimeric ligand and propagated by interactions of tumor necrosis factor receptor-associated factor (TRAF) proteins with the multimerized CD40 cytoplasmic domain. Using defined multimeric constructs of the CD40 cytoplasmic domain expressed as either soluble or myristoylated proteins, we have addressed the extent of receptor multimerization needed to initiate signal transduction and identified components of CD40 signaling complexes. Signal transduction in human embryonic kidney 293 cells, measured by nuclear factor kappaB activation, was observed in cells expressing soluble trimeric CD40 cytoplasmic domain and to a lesser extent in cells expressing dimeric CD40 cytoplasmic domain. Nuclear factor kappaB activation was strongest in cells expressing myristoylated trimeric CD40 cytoplasmic domain. Signal transduction through trimeric CD40 cytoplasmic domains with various point mutations in the TRAF binding sites was similar to signal transduction through analogous full-length receptors. Transiently expressed soluble trimeric CD40 cytoplasmic domain was isolated as complexes that contained TRAF2, TRAF3, TRAF5, TRAF6, and the inhibitor of apoptosis protein (c-IAP1). Association of c-IAP1 with the CD40 cytoplasmic domain complex was indirect and dependent on the presence of an intact TRAF1/2/3 binding site. These results suggest that extracellular ligation of CD40 can be bypassed and that soluble trimerized CD40 complexes can be isolated and used to identify components that link CD40 with signaling pathways.     

RPP13 is a simple locus in Arabidopsis thaliana for alleles that specify downy mildew resistance to different avirulence determinants in Peronospora parasitica

Disease resistance (R) genes are found in plants as either simple (single allelic series) loci, or more frequently as complex loci of tandemly repeated genes. These different loci are likely to be under similar evolutionary forces from pathogens, but the contrast between them suggests important differences in mechanisms associated with DNA structure and recombination that generate and maintain R gene diversity. The RPP13 locus in Arabidopsis represents an important paradigm for studying the evolution of an R gene at a simple locus. The RPP13 allele from the accession Nd-1, designated RPP13-Nd, confers resistance to five different isolates of the biotrophic oomycete, Peronospora parasitica (causal agent of downy mildew), and encodes an NBS-LRR type R protein with a putative amino-terminal leucine zipper. The RPP13-Rld allele, cloned from the accession Rld-2, encodes a different specificity. Comparison of three RPP13 alleles revealed a high rate of amino acid divergence within the LRR domain, less than 80% identity overall, compared to the remainder of the protein (> 95% identity). We also found evidence for positive selection in the LRR domain for amino acid diversification outside the core conserved beta-strand/beta-turn motif, suggesting that more of the LRR structure is available for interaction with target molecules than has previously been reported for other R gene products. Furthermore, an amino acid sequence (LLRVLDL) identical in an LRR among RPP13 alleles is conserved in other LZ NBS-LRR type R proteins, suggesting functional significance.     

Basic compatibility of Albugo candida in Arabidopsis thaliana and Brassica juncea causes broad-spectrum suppression of innate immunity

A biotrophic parasite often depends on an intrinsic ability to suppress host defenses in a manner that will enable it to infect and successfully colonize a susceptible host. If the suppressed defenses otherwise would have been effective against alternative pathogens, it follows that primary infection by the "suppressive" biotroph potentially could enhance susceptibility of the host to secondary infection by avirulent pathogens. This phenomenon previously has been attributed to true fungi such as rust (basidiomycete) and powdery mildew (ascomycete) pathogens. In our study, we observed broad-spectrum suppression of host defense by the oomycete Albugo candida (white blister rust) in the wild crucifer Arabidopsis thaliana and a domesticated relative, Brassica juncea. A. candida subsp. arabidopsis suppressed the "runaway cell death" phenotype of the lesion mimic mutant lsd1 in Arabidopsis thaliana in a sustained manner even after subsequent inoculation with avirulent Hyaloperonospora arabidopsis (Arabidopsis thaliana downy mildew). In sequential inoculation experiments, we show that preinfection by virulent Albugo candida can suppress disease resistance in cotyledons to several downy mildew pathogens, including contrasting examples of genotype resistance to H. arabidopsis in Arabidopsis thaliana that differ in the R protein and modes of defense signaling used to confer the resistance; genotype specific resistance in B. juncea to H. parasitica (Brassica downy mildew; isolates derived from B. juncea); species level (nonhost) resistance in both crucifers to Bremia lactucae (lettuce downy mildew) and an isolate of the H. parasitica race derived from Brassica oleracea; and nonhost resistance in B. juncea to H. arabidopsis. Broad-spectrum powdery mildew resistance conferred by RPW8 also was suppressed in Arabidopsis thaliana to two morphotypes of Erysiphe spp. following pre-infection with A. candida subsp. arabidopsis.     

Herpes simplex virus helicase-primase inhibitors are active in animal models of human disease

Herpes simplex virus infections are the cause of significant morbidity, and currently used therapeutics are largely based on modified nucleoside analogs that inhibit viral DNA polymerase function. To target this disease in a new way, we have identified and optimized selective thiazolylphenyl-containing inhibitors of the herpes simplex virus (HSV) helicase-primase enzyme. The most potent compounds inhibited the helicase, the primase and the DNA-dependent ATPase activities of the enzyme with IC50 (50% inhibitory concentration) values less than 100 nM. Inhibition of the enzymatic activities was through stabilization of the interaction between the helicase-primase and DNA substrates, preventing the progression through helicase or primase catalytic cycles. Helicase-primase inhibitors also prevented viral replication as demonstrated in viral growth assays. One compound, BILS 179 BS, displayed an EC50 (effective concentration inhibiting viral growth by 50%) of 27 nM against viral growth with a selectivity index greater than 2,000. Antiviral activity was also demonstrated for multiple strains of HSV, including strains resistant to nucleoside-based therapies. Most importantly, BILS 179 BS was orally active against HSV infections in murine models of HSV-1 and HSV-2 disease and more effective than acyclovir when the treatment frequency per day was reduced or when initiation of treatment was delayed up to 65 hours after infection. These studies validate the use of helicase-primase inhibitors for the treatment of acute herpesvirus infections and provide new lead compounds for optimization and design of superior anti-HSV agents.     

Overexpression and assembly of the herpes simplex virus type 1 helicase-primase in insect cells

Herpes simplex virus type 1 (HSV-1) encodes a helicase-primase that consists of three polypeptides encoded by the UL5, UL8, and UL52 genes (Crute, J.J., Tsurumi, T., Zhu, L., Weller, S.K., Olivo, P.D., Challberg, M.D., Mocarski, E.S., and Lehman, I.R. (1989) Proc. Natl. Acad, Sci, U.S.A. 86, 2186-2189). To obtain sufficient quantities of the enzyme for study, we have overexpressed the three genes using the baculovirus expression system. We find that the fully active enzyme can be assembled in vivo by triply infecting Spodoptera frugiperda SF9 cells with a baculovirus recombinant for each gene. The recombinant enzyme which we have purified to near homogeneity from the insect cells has a molecular weight of 270,000 and is composed of the three polypeptides encoded by the UL5, UL8, and UL52 genes. The enzyme possesses DNA-dependent ATPase, DNA-dependent GTPase, DNA helicase, and DNA primase activities that are essentially identical to the enzyme isolated from HSV-1-infected cells.     

Sequence specificity of the core-binding factor.

The core-binding factor (CBF) binds the conserved core motif in mammalian type C retrovirus enhancers. We analyzed the phosphate contacts made by CBF on the Moloney murine leukemia virus enhancer by ethylation interference assay. The phosphate contacts span 9 bp centered around the consensus core site. To examine the sequence preferences for CBF binding, we employed the technique of selected and amplified binding sequence footprinting (T. K. Blackwell and H. Weintraub, Science 250:1104-1110, 1990). The consensus binding site for CBF defined by selected and amplified binding sequence footprinting is PyGPyG GTPy.     

Evidence for a race-specific resistance factor in some lettuce (Lactuca sativa L.) cultivars previously considered to be universally susceptible to Bremia lactucae regel

Summary Previously undetected race-specific resistance to Bremia lactucae (downy mildew) was located in many lettuce cultivars hitherto considered to be universally susceptible to this disease. This resistance factor(s) may also be widely distributed in other cultivars known to carry combinations of already recognised factors R1 to R11. Specific virulence to match this resistance is almost invariably present in pathogen collections. This situation may be either a relic of the evolutionary history of the B. lactucae — L. sativa asssociation or may reflect a rare mutation in B. lactucae for avirulence on all but a few specialised L. sativa genotypes.     

Herpes simplex-1 helicase-primase. Identification of two nucleoside triphosphatase sites that promote DNA helicase action.

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase comprised of the products of the UL5, UL8, and UL52 genes (Crute, J. J., and Lehman, I. R. (1991) J. Biol. Chem. 266, 4484-4488). A steady state kinetic analysis of the enzyme isolated from HSV-1-infected CV-1 cells or insect cells expressing the enzyme after infection with recombinant baculoviruses has shown it to possess two sites capable of hydrolyzing nucleoside triphosphates in a DNA-dependent manner. One site (Site I) hydrolyzes both ATP and GTP; the second (Site II) hydrolyzes only ATP. These two sites are contained within a subassembly of the helicase-primase formed by coexpression of the UL5 and UL52 genes in insect cells. Sites I and II are activated by separate DNA effector sites, both of which support DNA helicase action. These findings are likely to be of importance in understanding how helicases in general catalyze the unwinding of duplex DNA and, in particular, how the helicase-primase functions at the HSV-1 replication fork.     

An Association Between High Peroxidase Activity in Lettuce (Lactuca sativa) and Field Resistance to Downy Mildew (Bremia lactucae)

The association between variation for pre-infection peroxidase activity and levels of field resistance-susceptibility to downy mildew (Bremia lactucae) was investigated in lettuce (Lactuca sativa) cultivars, accessions of L. serriola (prickly lettuce), segregating F₂ populations and selected F₃ families from a cross between field resistant and susceptible lettuce cultivars. A trend was apparent in this series of experiments indicating that one component of field resistance could be related to a high level of peroxidase activity prior to infection. The data suggest that in breeding programmes there could be merit in imposing primary selection for high peroxidase activity prior to field selection for resistance.     

Trichoderma spp. and Bacillus spp. as growth promoters in maize (Zea mays L.)

Microbes that are beneficial to plants are used to enhance the crop growth, yield and are alternatives to chemical fertilizers. Trichoderma and Bacillus are the predominant plant growth-promoting fungi and bacteria. The objective of this study was select, characterize, and evaluate isolates of Trichoderma spp. and Bacillus spp. native from the northern region of Sinaloa, Mexico, and assess their effect on growth promotion in maize (Zea mays L.). In greenhouse conditions, four Trichoderma isolates and twenty Bacillus isolates, as well as two controls, were tested in a completely randomized design with three replicates. We selected the two best strains of Trichoderma and Bacillus: TB = Trichoderma asperellum, TF = Trichoderma virens, B14 = Bacillus cereus sensu lato and B17 = Bacillus cereus, which were evaluated in the field in a completely randomized blocks in factorial arrangement design with three replicates applying different rates of nitrogen fertilizer (0, 150 kg N/ha, and 300 kg N/ha). Treatments 5 (B17 = B. cereus) and 11 (TF = T. virens) both fertilized with 150 kg N/ha showed similar yields and they did not reveal significant differences from the treatments fertilized with 300 kg N/ha. This indicated that treatment 5 (B17= B. cereus with 150 kg N/ha) and treatment 11 (TF= T. virens with 150 kg N/ha) were efficient as growth promoters, by not showing significant differences in root volume and dry weight of foliage. The results indicated a reduction of 50% in the rate of nitrogen to fertilizer required for maize (Zea mays L.) crops. These microorganisms Trichoderma and Bacillus could be an alternative to reduce the use of chemical fertilizers in maize.