Localized hormone fluxes and early haustorium development in the hemiparasitic plant Triphysaria versicolor

Perhaps the most obvious phenotypes associated with chemical signaling between plants are manifested by parasitic species of Orobanchaceae. The development of haustoria, invasive root structures that allow hemiparasitic plants to transition from autotrophic to heterotrophic growth, is rapid, highly synchronous, and readily observed in vitro. Haustorium development is initiated in aseptic roots of the facultative parasite Triphysaria versicolor when exposed to phenolic molecules associated with host root exudates and rhizosphere bioactivity. Morphological features of early haustorium ontogeny include rapid cessation of root elongation, expansion, and differentiation of epidermal cells into haustorial hairs, and cortical cell expansion. These developmental processes were stimulated in aseptic T. versicolor seedlings by the application of exogenous phytohormones and inhibited by the application of hormone antagonists. Surgically dissected root tips formed haustoria if the root was exposed to haustorial-inducing factors prior to dissection. In contrast, root tips that were dissected prior to inducing-factor treatment were unable to form haustoria unless supplemented with indole-3-acetic acid. A transient transformation assay demonstrated that auxin and ethylene-responsive promoters were up-regulated when T. versicolor was exposed to either exogenous hormones or purified haustoria-inducing factors. These experiments demonstrate that localized auxin and ethylene accumulation are early events in haustorium development and that parasitic plants recruit established plant developmental mechanisms to realize parasite-specific functions.     

An Improved Method for Long-Distance Shipping the Mosquito Parasite Romanomermis culicivorax

To prevent losses during long-distance shipment, a new delivery system was developed for the mosquito parasite Romanomermis culicivorax. A specially designed shipping container, two types of culture media (sand and a mixture of sand, vermiculite, and charcoal [SVC]), and two types of insert (polystyrene and polyester urethane foam [PUF]) were tested. Cultures shipped in SVC/PUF insert combination produced significantly higher yields of preparasites than did the other three combinations tested, and the sand/polystyrene combination produced significantly lower yields than did either sand or SVC media shipped with the PUF insert. Yields from cultures shipped in sand/PUF and SVC/PUF combinations were significantly lower than from unshipped controls. Maximum losses (52%) occurred in cultures shipped when 13-16-wk old. Also, yields of preparasites were significantly lower from cultures shipped singly when compared to similar cultures shipped in groups of eight. Techniques and procedures developed in this study did not completely solve the loss associated with delivery of R. culicivorax. For the first time, however, the system can guarantee the delivery of a quality product. The study defines the importance of the carrier medium, age of cultures, shipment size, and the restriction of the movement of the culture medium.     

Host Cell Autophagy Is Induced by Toxoplasma gondii and Contributes to Parasite Growth

Autophagy has been shown to contribute to defense against intracellular bacteria and parasites. In comparison, the ability of such pathogens to manipulate host cell autophagy to their advantage has not been examined. Here we present evidence that infection by Toxoplasma gondii, an intracellular protozoan parasite, induces host cell autophagy in both HeLa cells and primary fibroblasts, via a mechanism dependent on host Atg5 but independent of host mammalian target of rapamycin suppression. Infection led to the conversion of LC3 to the autophagosome-associated form LC3-II, to the accumulation of LC3-containing vesicles near the parasitophorous vacuole, and to the relocalization toward the vacuole of structures labeled by the phosphatidylinositol 3-phosphate indicator YFP-2×FYVE. The autophagy regulator beclin 1 was concentrated in the vicinity of the parasitophorous vacuole in infected cells. Inhibitor studies indicated that parasite-induced autophagy is dependent on calcium signaling and on abscisic acid. At physiologically relevant amino acid levels, parasite growth became defective in Atg5-deficient cells, indicating a role for host cell autophagy in parasite recovery of host cell nutrients. A flow cytometric analysis of cell size as a function of parasite content revealed that autophagy-dependent parasite growth correlates with autophagy-dependent consumption of host cell mass that is dependent on parasite progression. These findings indicate a new role for autophagy as a pathway by which parasites may effectively compete with the host cell for limiting anabolic resources.Macroautophagy (hereafter referred to as autophagy) is a major catabolic process in which cytosolic constituents are sequestered within double-membraned vesicles (autophagosomes) and subsequently delivered to lysosomes for degradation. Current evidence indicates at least two distinct functions for this process. On the one hand, autophagy can be up-regulated under nutrient-limiting conditions to increase nutrient supply via recycling of the products of autophagic degradation, which may be exported from the lysosome (1). The up-regulation of autophagy upon starvation is thought to be mediated by the suppression of signaling through the mTOR pathway (2). On the other hand, autophagy can serve to maintain cellular homeostasis by facilitating the removal of damaged or deleterious elements, such as misfolded protein aggregates (3). An important example of the latter function is the role of autophagy in restricting the growth of intracellular pathogens, including both free bacteria that have escaped into host cytosol, such as group A Streptococcus, and pathogens, such as Mycobacterium tuberculosis, that reside in parasitophorous vacuoles in macrophages (4, 5). In macrophages infected with Toxoplasma gondii, fusion of the parasitophorous vacuole with lysosomes can be induced in an autophagy-dependent manner when host cell anti-parasitic function is activated via CD40 (6). Autophagy as a component of host defense may be up-regulated by inflammatory agents such as lipopolysaccharide (7) and interferon-γ (8).Although the clearance function of autophagy may enhance pathogen killing in host cells that have been activated to generate antimicrobial or antiparasitic function, in permissive host cells, in which the pathogen is less susceptible to sequestration by the autophagosome, autophagy may conceivably play a quite different role. Modulation of the balance between anabolic and catabolic processes may affect the outcome of competition between pathogen and host cell for limiting nutrients. In particular, the nutritive function of autophagy could favor pathogen expansion by providing greater access to host cell biomass. The intracellular apicomplexan parasite, T. gondii, is a suitable agent for the investigation of this hypothesis, because it has been shown to be highly dependent on its host cell for the supply of several nutrients, including amino acids (9), lipids (10), and purines (11). T. gondii replicates within a parasitophorous vacuole that, in permissive host cells, is protected from lysosomal fusion. Recent evidence indicates that in such permissive cells, in which the parasite can differentiate into bradyzoites associated with chronic infection, the pathogen is able to actively sequester host cell lysosome-derived vesicles, thereby potentially gaining access to their contents (12).The ability of intracellular parasites to regulate host cell autophagy has been little examined, and there is also little information with respect to the impact of these pathogens on host cell signals that potentially affect the autophagic pathway. In addition to mTOR, these include calcium ions, which have been implicated in autophagy induced by endoplasmic reticulum stress (13). In this study, we provide evidence that T. gondii induces host cell autophagy by a mechanism dependent on calcium but independent of mTOR and that it exploits the nutritive function of host autophagy to enhance its proliferation.     

Development and Fecundity of Reesimermis nielseni,a Nematode Parasite of Mosquitoes

Maturation of the mermithid nematode Reesimermis nielseni to the adult stage began by the tenth day after emergence of the nematodes from their hosts at ambient temperatures (24-27 C). Most postparasitic males and females reached the adult stage after 50 and 70 days, respectively. The first females exhibiting egg development and oviposition were observed 25-30 days after emergence, but some oviposition was still taking place 150 days later. Reesimermis nielseni laid an average 2,480 eggs per female over an 18-day oviposition period. A majority of the mature eggs hatched within 7 h after the cultures were flooded. The preparasites are short-lived, and only a few were able to infect exposed hosts after 72 h.     

Chloroplast Differentiation in Tomato Root Galls Induced by the Root Knot Nematode Meloidogyne incognita

Primary roots of tomato, Lycopersicon esculentum cv. Marglobe, were cultured aseptically on agar containing a standard nutrient formulation with or without kinetin. When secondary roots developed, cultures were inoculated with the root-knot nematode, Meloidogyne incognita. Following inoculation, the cultures were divided into two groups which were incubated either in total darkness or in 16-h light-8-h dark cycles. At 24 h, 1, 2, 3, and 4 wk after incubation, roots from all cultures were processed for transmission electron microscopy. Fine structural observation of the parenchyma tissue in galls from the inoculated cultures indicated that starch containing plastids or amyloplasts, which are usually present and remain undifferentiated in these root cells, developed into chloroplasts. These chloroplasts contained a membrane system indistinguishable from those found in leaves of intact plants. Although plastid development was not affected when uninoculated cultures were incubated in the light, differentiation of the amyloplast was induced when roots were cultured on the medium containing kinetin. These results suggest that plastid differentiation in the inoculated tissue may be influenced by an accumulation of kinetin in the gall, which is induced by the nematode and serves as the nutrient sink for its feeding.     

In Vitro Haustorium Development in Roots and Root Cultures of the Hemiparasitic Plant Triphysaria Versicolor

Parasitic plants in the Orobanchaceae invade host plant roots through root organs called haustoria. Parasite roots initiate haustorium development when exposed to specific secondary metabolites that are released into the rhizosphere by host plant roots. While molecular approaches are increasingly being taken to understand the genetic mechanism underlying these events, a limitation has been the lack of a transformation system for parasitic plants. Since the haustorium development occurs in roots of Orobanchaceae, root cultures may be suitable material for transient or stable transformation experiments. To this end, root cultures were obtained from explants, and subsequently calluses, from the hemiparasitic plant Triphysaria versicolor. The cultured roots retained their competence to form haustoria when exposed to host roots, host root exudates, or purified haustorium-inducing factors. The root culture haustoria invaded host roots and initiated a vascular continuity between the parasite and host roots. The ontogeny of haustoria development on root cultures was indistinguishable from that on seedlings roots. Root cultures should provide useful material for molecular studies of haustorium development.     

Effects of Host Size and Parasite Burden on Sex Ratio in the Mosquito Parasite Octomyomermis muspratti

The ratio of Octomyomermis muspratti to the host mosquito at the time of exposure had little effect on the ratio of male to female parasites that resulted. However, the ratio of males to females increased as the number of parasites/host increased. Hosts with a single nematode produced fewer than 1% males in comparison with hosts with 8 parasites which produced about 40% males; hosts with 10 or more nematodes generally produced more male than female nematodes. Males of O. muspratti usually emerged before females because of the earlier death of multiply-infected mosquitoes. The size of the host at the time of invasion bad no significant influence on nematode sex ratios. Since mating is apparently necessary for reproduction in O. muspratti, the low male to female ratios that occur will be important in developing successful mass production techniques.     

Systematic Analysis of Diguanylate Cyclases That Promote Biofilm Formation by Pseudomonas fluorescens Pf0-1

Cyclic di-GMP (c-di-GMP) is a broadly conserved, intracellular second-messenger molecule that regulates biofilm formation by many bacteria. The synthesis of c-di-GMP is catalyzed by diguanylate cyclases (DGCs) containing the GGDEF domain, while its degradation is achieved through the phosphodiesterase activities of EAL and HD-GYP domains. c-di-GMP controls biofilm formation by Pseudomonas fluorescens Pf0-1 by promoting the cell surface localization of a large adhesive protein, LapA. LapA localization is regulated posttranslationally by a c-di-GMP effector system consisting of LapD and LapG, which senses cytoplasmic c-di-GMP and modifies the LapA protein in the outer membrane. Despite the apparent requirement for c-di-GMP for biofilm formation by P. fluorescens Pf0-1, no DGCs from this strain have been characterized to date. In this study, we undertook a systematic mutagenesis of 30 predicted DGCs and found that mutations in just 4 cause reductions in biofilm formation by P. fluorescens Pf0-1 under the conditions tested. These DGCs were characterized genetically and biochemically to corroborate the hypothesis that they function to produce c-di-GMP in vivo. The effects of DGC gene mutations on phenotypes associated with biofilm formation were analyzed. One DGC preferentially affects LapA localization, another DGC mainly controls swimming motility, while a third DGC affects both LapA and motility. Our data support the conclusion that different c-di-GMP-regulated outputs can be specifically controlled by distinct DGCs.     

A Study of 'Phloeotracheids' in Haustoria of Santalaceous Root Parasites using Scanning Electron Microscopy

Tracheary elements within haustoria of six santalaceous rootparasites have been examined by scanning electron microscopywith particular reference to those cells which contain granules.These cells have been called ‘phloeotracheids’ byearly workers. The granules lie free within the lumen of thecell from which a protoplast is absent at maturity. The granulesoccur in short vessel elements and in imperforate cells whichare thought to represent a specialized type of tracheid. Phloeotracheidsare confirmed in Exocarpus bidwilliiand Mida salicifolia andreported for the first time in Buckleya distichophylla, Comandraumbellata, Nestronia umbellataand Pyrularia pubera. The structureof phloeotracheids shown by scanning electron microscopy isdiscussed in relation to previous studies.     

银杏根中黄酮类化合物的研究

采用丙酮浸泡、回流从银杏根中提取得到黄酮,以紫外分光光度法测定提取物中总黄酮含量为6．93％,以HPLC法测定其中槲皮素含量为0．064％;结果表明：银杏根黄酮存在槲皮素甙,不含山奈酚、异鼠李素等物质。     

Correlations between Haustoria Formation and Parasitic Development in Orthocarpus purpurascens (Scrophulariaceae)

Three lines of evidence correlate the parasitic performane ofOrthocarpus purpuruscens Benth. with numbers of haustoria produced:(i) the pattern of variation in numbers of haustoria producedin agar culture with different chemical stimuli correspondsclosely to the variation pattern of parasite vigour producedby a range of host plants; (ii) the progeny of plants demonstratingvigorous growth with hosts produce significantly more haustoriathan progeny from parents exhibiting weak parasitic development;(iii) conversely, seedlings that produce high numbers of haustoriain agar culture grow significantly better when transplantedwith hosts than do seedlings with low numbers of haustoria.Haustoria-forming potential is heritable, but highly influencedby environmental factors. Potential number of haustoria is aproduct of the concentration and/or quality of haustoria inducingstimuli, and the parasite's individual ability to respond. Intra-populationdifferences in parasitic development appear to be largely dueto the quantity rather than the quality of substrates receivedfrom host plants. haustoria, Orthocarpus purpurarcens, parasitic development