Parasiticidal activity of a novel synthetic peptide from the core α-helical region of NK-lysin.

NK-lysin is an anti-microbial peptide that plays a critical role in innate immunity against infectious pathogens through its selective membrane disruptive property. We previously expressed and purified a full-length chicken NK-lysin (cNKL) recombinant protein, and demonstrated its in vitro anti-parasitic activity against the apicomplexan protozoan, Eimeria, the etiologic agent of avian coccidiosis. This study evaluated the in vitro and in vivo anti-parasitic properties of a synthetic peptide (cNK-2) incorporating a predicted membrane-permeating, amphipathic α-helix of the full-length cNKL protein. The cNK-2 peptide exhibited dose- and time-dependent in vitro cytotoxic activity against E. acervulina and E. tenella sporozoites. The cytotoxic activity of 1.5 µM of cNK-2 peptide against E. acervulina following 6h incubation was equal to that of 2.5 µM of melittin, the principal active component of apitoxin (bee venom) that also exhibits anti-microbial activity. Even greater activity was detected against E. tenella, where 0.3 µM of cNK-2 peptide was equivalent to 2.5 µM of melittin. Against Neospora caninum tacyzoites, however, the cytotoxic activity of cNK-2 peptide was inferior to that of melittin. Transmission electron microscopy of peptide-treated E. tenella sporozoites revealed disruption of the outer plasma membrane and loss of intracellular contents. In vivo administration of 1.5 µM of cNK-2 peptide increased protection against experimental E. acervulina infection, as measured by greater body weight gain and reduced fecal oocyst shedding, compared with saline controls. These results suggest that the cNK-2 synthetic peptide is a novel anti-infective peptide that can be used for protection against avian coccidiosis during commercial poultry production.

Gene expression profiling and shared promoter motif for cell wall-modifying proteins expressed in soybean cyst nematode-infected roots.

We hypothesized that soybean cyst nematode (SCN; Heterodera glycines) co-opts part or all of one or more innate developmental process in soybean (Glycine max) to establish its feeding structure, syncytium, in soybean roots. The syncytium is formed within the vascular bundle by partial degradation of cell walls and membranes between adjacent parenchyma cells. A mature syncytium incorporates as many as 200 cells into one large multinucleated cell. Gene expression patterns for several cell wall-modifying proteins were compared in multiple tissues undergoing major shifts in cell wall integrity. These included SCN-colonized roots, root tips where vascular differentiation occurs, flooded roots (aerenchyma), adventitious rooting in hypocotyls, and leaf abscission zones. A search in the 5' upstream promoters of these genes identified a motif (SCNbox1: WGCATGTG) common to several genes that were up-regulated in several different tissues. The polygalacturonase 11 promoters (GmPG11a/b) include the SCNbox1 motif. The expression pattern for GmPG11a was examined further in transgenic soybean containing a PG11a promoter fused to a ß-glucuronidase (GUS) reporter gene. GUS expression was highest in cells undergoing radial expansion in the stele and/or cell wall dissolution. GUS staining was not observed in cortical cells where a lateral root tip or a growing nematode emerged through the root cortex.

Gene expression profiles for cell wall-modifying proteins associated with soybean cyst nematode infection, petiole abscission, root tips, flowers, apical buds, and leaves.

Changes in transcript accumulation for cell wall-modifying proteins were examined in excised soybean root pieces colonized by soybean cyst nematodes (SCN), Heterodera glycines, using RT-PCR and soybean Affymetrix GeneChips. Sequence-specific PCR primer pairs were prepared from sequence data for core sequences in the GenBank soybean database and consensus sequences derived from the assembly of soybean ESTs. In addition, to identify previously uncharacterized soybean transcripts, degenerate primers were prepared for conserved motifs in cellulases (endo-1,4-beta-glucanases, EGases) and polygalacturonases (PGs) and these were used to amplify segments of transcripts that were then extended with 3' and 5' RACE. Several novel EGase and PG transcripts were identified. Gene expression patterns were determined by real-time RT-PCR for 11 EGases, three expansins (EXPs), 14 PGs, two pectate lyases (PLs), and two xyloglucan endotransglucosylase/hydrolases (XTHs) in soybean roots inoculated with SCN, non-inoculated roots, serial dissections of root tips, leaf abscission zones, flowers, apical buds, and expanding leaves. A large number of genes associated with cell wall modifications are strongly up-regulated in root pieces colonized by SCN. However, in contrast to most of the transcripts for cell wall proteins, two XTH transcripts were specifically down-regulated in the colonized root pieces. Gene expression in serial dissections of root tips (0-2 mm, and 2-7 mm) and whole roots indicate that the SCN up-regulated genes are associated with a wide range of developmental processes in roots. Also of interest, many of the cDNAs examined were up-regulated in petiole abscission zones induced to abscise with ethylene.

Simultaneous hyperaccumulation of nickel, manganese, and calcium in Alyssum leaf trichomes.

We have developed commercially viable phytoremediation/phytomining technologies employing Alyssum Ni-hyperaccumulator species to quantitatively extract Ni from soils. The majority of Ni is stored either in Alyssum leaf epidermal cell vacuoles or in the basal portions only of the numerous stellate trichomes. Here, we report simultaneous and region-specific localization of high levels of Ni, Mn, and Ca within Alyssum trichomes as determined by scanning electron microscopy/energy-dispersive X-ray analysis (SEM/EDX). Plants were grown in high Ni soil, achieving up to 48 400 microg g(-1) Ni in total leaf concentration; however, Ca and Mn were not enriched in the experimental soils. The region-specific localization of hyperaccumulated Ca, Mn, and Ni occurred in three soil types, five Alyssum species/ecotypes, and over a wide range of soil Ni concentrations. The metal concentration in the trichome basal compartment was approximately 15-20% dry weight, the highest ever reported for healthy vascular plant tissue.