INSECTICIDAL ACTIVITY OF PROTEIN EXTRACTS OBTAINED FROM BULBS OF CHILEAN AMARYLLIDACEAE AGAINST TRIALEURODES VAPORARIORUM WESTWOOD AND PSEUDOCOCCUS VIBURNI SIGNORET.

Entomotoxic proteins are produced by plants in defence against insect herbivory. Some carbohydrate-binding proteins exhibit strong insecticidal activity affecting the survival, growth, development and feeding behavior of phytophagous insects. The occurrence of entomotoxic lectins is well documented in the Amaryllidaceae, a plant family spread world-wide. In Chile, this family is represented by numerous species, many of which are also of high ornamental value. Protein extracts were obtained from bulbs of five different species of Chilean Amaryllidaceae. A dose-response assay was carried out with two important pests: the greenhouse whitefly Trialeurodes vaporariorum Westwood and the mealybug Pseudococcus viburni Signoret. The extracts were offered to insects in a liquid artificial diet for three days and the mortality was scored. The Phycella australis Ravenna extract caused the highest insecticidal activity (T. vaporariorum LC50: 7200 µg/mL; P. viburni LC50: 9500 µg/mL). Applied at 1000 µg/mL in the diet the P. australis extract did not repel feeding of these pests. A mannose-binding lectin isolated from the bulbs of P. australis proved to be moderately toxic for these pests (T. vaporariorum LC50: 1127 µg/mL; P. viburni LC50: 2320 µg/mL).

Quantitation and localization of pospiviroids in aphids.

In this paper, the potential role of aphids in viroid transmission was explored. Apterous aphids were fed on pospiviroid-infected plants and viroid targets in the aphids were consequently quantified through RT-qPCR and localized within the aphid body using fluorescence in situ hybridization (FISH). Based on the analytical sensitivity test, the limit of detection (LOD) was estimated at 1.69×10(6) viroid copies per individual aphid body. To localize the viroids in the aphids, a pospiviroid-generic Cy5-labelled probe was used and the fluorescent signal was determined by confocal microscopy. Viroids were clearly observed in the aphid's stylet and stomach, but not in the embryos. Viroids were detected in 29% of the aphids after a 24h feeding period, which suggests only a partial and low concentration viroid uptake by the aphid population including viroid concentrations under the LOD. However, these results show that viroids can be ingested by aphids while feeding on infected plants, thus potentially increasing the transmission risk. The combination of FISH and RT-qPCR provides reliable and fast localization and quantitation of viroid targets in individual aphids and thus constitutes a valuable tool in future epidemiological research.

Transcriptional profiling of the lectin ArathEULS3 from Arabidopsis thaliana toward abiotic stresses.

The family of EUL-related lectins groups all proteins with an Euonymus lectin (EUL) domain, a protein motif which is highly conserved throughout the plant kingdom and occurs as part of many chimeric proteins with different domain architectures. The S3 type EUL lectin from Arabidopsis thaliana (ArathEULS3) has become the model protein within this EUL family. Based on sequence homology to an ABA/NaCl inducible gene from rice and some publicly available high-throughput micro-array data, it was hypothesized that ArathEULS3 is transcriptionally regulated by osmotic stress responses. Here we present a detailed expression analysis of the ArathEULS3 lectin gene. Under normal growth conditions, ArathEULS3 is stably expressed throughout plant development. After ABA, NaCl and methyl jasmonate (MeJA) treatments transcription is upregulated. Furthermore, in silico promoter and co-expression analyses suggested the A. thaliana Homeobox 7 (ATHB-7) as a candidate transcription factor that may regulate ArathEULS3 expression. Taken together, our data confirm that the ArathEULS3 lectin gene indeed shows a stress-inducible expression pattern. We speculate on a role for ArathEULS3 in the plant stress response.

Entomotoxic effects of fungal lectin from Rhizoctonia solani towards Spodoptera littoralis.

The effects of the Rhizoctonia solani lectin (RSA) on the growth, development and survival of an economically important caterpillar in agriculture and horticulture, the cotton leafworm, Spodoptera littoralis were studied. The high lectin concentration present in the sclerotes of the soil pathogen R. solani allowed the purification of large amounts of the pure lectin for feeding experiments with cotton leafworm. Rearing of insects on a diet containing different concentrations of RSA exerted a strong effect on the larval weight gain. This effect was visible at the lowest concentration of 0.1 % RSA at day 8 and day 11. Interestingly with 1 % RSA, there was a dramatic reduction in larval weight of 89 % at the end of L6 which was followed by a high mortality rate of 82 % in the treated larvae. Furthermore, the other developmental stages of pupation and adult formation were also affected. In addition, the data demonstrated that the combination of RSA with Bt toxin yielded synergistic effects. For instance, 0.03 % RSA+0.005 % Bt toxin caused reduced growth rate and higher mortalities. These findings suggest that RSA is an interesting tool that can be used for bioengineering insect resistance in important agronomical crops.

Carbohydrate-binding agents (CBAs) inhibit HIV-1 infection in human primary monocyte-derived macrophages (MDMs) and efficiently prevent MDM-directed viral capture and subsequent transmission to CD4+ T lymphocytes.

Carbohydrate-binding agents (CBAs) have been proposed as innovative anti-HIV compounds selectively targeting the glycans of the HIV-1 envelope glycoprotein gp120 and preventing DC-SIGN-directed HIV capture by dendritic cells (DCs) and transmission to CD4(+) T-lymphocytes. We now show that CBAs efficiently prevent R5 HIV-1 infection of human primary monocyte-derived macrophage (MDM) cell cultures in the nanomolar range. Both R5 and X4 HIV-1 strains were efficiently captured by the macrophage mannose-binding receptor (MMR) present on MDM. HIV-1 capture by MMR-expressing MDM was inhibited by soluble mannose-binding lectin and MMR antibody. Short pre-exposure of these HIV-1 strains to CBAs is able to prevent virus capture by MDM and subsequent syncytia formation in cocultures of the CBA-exposed HIV-1-captured MDM and uninfected CD4(+) T-lymphocytes. The potential of CBAs to impair MDM in their capacity to capture and to transmit HIV to T-lymphocytes might be an important property to be taken into consideration in the eventual choice to select microbicide candidate drugs for clinical investigation.

Antiviral activity of carbohydrate-binding agents against Nidovirales in cell culture.

Coronaviruses are important human and animal pathogens, the relevance of which increased due to the emergence of new human coronaviruses like SARS-CoV, HKU1 and NL63. Together with toroviruses, arteriviruses, and roniviruses the coronaviruses belong to the order Nidovirales. So far antivirals are hardly available to combat infections with viruses of this order. Therefore, various antiviral strategies to counter nidoviral infections are under evaluation. Lectins, which bind to N-linked oligosaccharide elements of enveloped viruses, can be considered as a conceptionally new class of virus inhibitors. These agents were recently evaluated for their antiviral activity towards a variety of enveloped viruses and were shown in most cases to inhibit virus infection at low concentrations. However, limited knowledge is available for their efficacy towards nidoviruses. In this article the application of the plant lectins Hippeastrum hybrid agglutinin (HHA), Galanthus nivalis agglutinin (GNA), Cymbidium sp. agglutinin (CA) and Urtica dioica agglutinin (UDA) as well as non-plant derived pradimicin-A (PRM-A) and cyanovirin-N (CV-N) as potential antiviral agents was evaluated. Three antiviral tests were compared based on different evaluation principles: cell viability (MTT-based colorimetric assay), number of infected cells (immunoperoxidase assay) and amount of viral protein expression (luciferase-based assay). The presence of carbohydrate-binding agents strongly inhibited coronaviruses (transmissible gastroenteritis virus, infectious bronchitis virus, feline coronaviruses serotypes I and II, mouse hepatitis virus), arteriviruses (equine arteritis virus and porcine respiratory and reproductive syndrome virus) and torovirus (equine Berne virus). Remarkably, serotype II feline coronaviruses and arteriviruses were not inhibited by PRM-A, in contrast to the other viruses tested.

Analysis of lectin concentrations in different Rhizoctonia solani strains.

Lectins are carbohydrate-binding proteins that contain at least one carbohydrate binding domain which can bind to a specific mono- or oligosaccharide. These proteins are widely distributed in plants. However, over the last decade evidence is accumulating that lectins occur also in numerous fungi belonging to both the Ascomycota and Basiodiomycota. Rhizoctonia solani is known to be an important pathogen to a wide range of host plants. In this study, isolates of R. solani from different anastomosis groups have been screened for the presence of lectin using agglutination assays to detect and quantitate lectin activity. The evaluation included determination of the lectin content in mycelium as well as in sclerotia. The amount of lectin in the sclerotia was higher than in the mycelium of the same strains. The R. solani strains with the highest amounts of lectin have been selected for cultivation, extraction and purification of the lectin.

Lectin histochemical investigations of the distal gut of chicks with special emphasis on the follicle-associated epithelium.

Carbohydrates on epithelial cell surfaces play an important role as attachment sites for different microorganisms like bacteria, viruses and protozoa. To obtain more information about the distribution of carbohydrates on the luminal surface along the intestine, lectin histochemical studies on different gut segments of chicks of different age groups were carried out using a panel of 13 lectins with specificities for Man, Glc, Gal, GalNAc, GlcNAc or GlcNAc oligosaccharides and Sia. Furthermore, we tried to find out whether previously reported specificities of certain lectins for M cells (membranous or multifold cells) in the bursa of Fabricius (BF) can be observed also on M cells of the intestine. As a result we were able to demonstrate binding of all lectins employed in these studies in all investigated gut segments. In some cases, the application of the same lectin led to varying staining intensities of the same histological structures in different age-groups (e.g. staining of the brush border with WGA, LEA, MAA or Conarva) or different gut segments (e.g. staining of goblet cells with CMA II, LEA and MPA). Hence, terminal carbohydrate residues of glycoconjugates on the intestinal epithelium vary depending on age and organ site. As glycoconjugates can act as attachment sites for microorganisms, these differences in the distribution of sugar residues may be one explanation for the site-specificity of certain pathogens. Furthermore, the binding of lectins to the follicle-associated epithelium (FAE) of the BF differs from that to the FAE of the intestine again stressing the site specificity of lectin binding. Thus, up to now no universal M-cell marker along the chicken intestine exists.

Structure of an RNase-related protein from Calystegia sepium.

The structure of a catalytically inactive RNase-related protein from Calystegia sepium (CalsepRRP) has been resolved by protein crystallography at a resolution of 2.05 A and an R factor of 20.74%. Although the protein is completely devoid of ribonuclease activity, it adopts the typical alpha + beta structure of non-base-specific RNases. Analysis of the structure revealed that two amino-acid substitutions in the 'active' P1 site, in combination with the less hydrophobic/aromatic character of the B1 base-recognition site and a completely disrupted B2 base-recognition site, might account for this complete lack of activity.

Ee-CBP, a hevein-type antimicrobial peptide from bark of the spindle tree (Euonymus europaeus L.).

Ee-CBP, a hevein-type antimicrobial peptide was isolated from the bark of the spindle tree (Euonymus europaeus L.). This 4992.5 Da protein exhibited a very strong antifungal activity against five different fytopathogenic fungi that were tested. Concentrations required to inhibit the growth of Botrytis cinerea in agar diffusion assays and microtiterplate assays were 5 micrograms/ml and 1 microgram/ml, respectively. Comparative tests further indicated that Ee-CBP is a more potent antifungal protein than Ac-AMP2, an antimicrobial peptide from seeds of Amaranthus caudatus L. when tested with the same fungus.