The role of cuscutain-propeptide inhibitor in haustoria parasitism and enhanced resistance to dodder in transgenic alfalfa expressing this propeptide

Cuscutain is a cysteine protease produced by dodder (the most important weeds of alfalfa) that is essential for the development and penetration of the haustoria in host. The propeptide subunit of cuscutain has a specific inhibitory function and inhibits the enzymatic activity of the cuscutain. In this study, we introduced the gene encoding the propeptide segment of the cuscutain (signal peptide-less inhibitor) into alfalfa and investigated its roles in parasitism and the alfalfa resistance to C. reflexa. Results demonstrated that cuscutain is mainly expressed in haustoria and the expression of propeptide in transgenic alfalfa plants effectively inhibited cuscutain enzyme activity and consequently interrupted haustoria development at the pathogenic stage. Digitate cells of haustoria could not differentiate into the xylem and phloem hyphae in dodder grown on transgenic alfalfa. Dodder development on transgenic alfalfa lines showed an overall reduction in fecundity and vigor due to imperfect attachment of haustoria. Morphology, nodule development and biomass of transgenic plants indicate that the inhibitory transgene exhibits exquisite specificity for cuscutain enzyme and by expression of the inhibitor in transgenic plants, there was no obvious adverse effect on them. The increased development and growth of dodder-challenged alfalfa transgenic plants compared to controls, showed the efficacy of propeptide in dodder control.     

Host-synthesized cysteine protease-specific inhibitor disrupts <Emphasis Type="Italic">Cuscuta campestris</Emphasis> parasitism in tomato

Dodder (Cuscuta campestris) is one of the most important pests of tomato causing severe losses in yield. Cuscutain is a pre-pro-protein produced by dodder that has a cysteine proteinase function essential for normal development of the haustoria and parasitism, which involves the secretion and activation of cuscutain cysteine protease in the host plant tissue. The propeptide subunit of this enzyme has an inhibitory function and restricts the enzymatic activity of cuscutain. Here, we transformed the inhibitory propeptide segment of this enzyme into tomato and examined the tomato resistance to C. campestris. We demonstrate the expression of inhibitory propeptide in transgenic plants and find that it effectively interrupted cuscutain enzyme activity and haustoria development at the endophytic stage. Mature haustoria infecting transgenic hosts showed defects in searching hyphae development and these structures were not elongate, and in most cases no functional haustoria were formed due to inhibitor expression in the transgenic plants after prehaustoria contact. Dodder grown on transgenic lines showed an overall reduction in vigor and fecundity due to defective attachment of haustoria. The increased growth of dodder-challenged transgenic plants relative to controls, demonstrates the efficacy of cysteine protease inhibition in parasite plant control.     

Dodder hyphae invade the host: a structural and immunocytochemical characterization

Summary.  Dodder (Cuscuta pentagona) hyphae are unique amongst the parasitic weeds for their ability to apparently grow through the walls of the host plant. Closer examination reveals, however, that the hyphae do not grow through the host but rather induce the host to form a new cell wall (or extend the existing wall) to coat the growing hypha. This chimeric wall composed of walls from two species is even traversed by plasmodesmata that connect the two cytoplasms. Compositionally, the chimeric wall is quite different from the walls of either the host or in other cells of the dodder plant, on the basis of immunocytochemical labeling. The most striking differences were in the pectins, with much stronger labeling present in the chimeric wall than in either the host or other dodder walls. Interestingly, labeling with monoclonal antibodies specific to arabinan side chains of rhamnogalacturonan I pectin fraction was highly enriched in the chimeric wall, but antibodies to galactan side chains revealed no labeling. Arabinogalactan protein antibodies labeled the plasma membrane and vesicles at the tips of the hyphae and the complementary host wall, although the JIM8-reactive epitope, associated with very lipophilic arabinogalactan proteins, was found only in dodder cells and not the host. Callose was found in the plasmodesmata and along the forming hyphal wall but was found at low levels in the host wall. The low level of host wall labeling with anticallose indicates that a typical woundlike response was not induced by the dodder. When dodder infects leaf lamina, which have more abundant intercellular spaces than petioles or shoots, the hyphae grew both intra- and extracellularly. In the latter condition, a host wall did not ensheath the parasite and there was clear degradation of the host middle lamellae by the growing hyphae, allowing the dodder to pass between cells. These data indicate that the chimeric walls formed from the growth of the host cell wall in concert with the developing hyphae are unique in composition and structure and represent an induction of a wall type in the host that is not noted in surrounding walls. Received February 1, 2002; accepted July 8, 2002; published online November 29, 2002     

The haustorium of the root parasite Triphysaria (Scrophulariaceae), with special reference to xylem bridge ultrastructure

Haustoria of Triphysaria pusilla and T. versicolor subsp. faucibarbata from a natural habitat were analyzed by light and electron microscopy. Secretory trichomes (root hairs) participate in securing the haustorium to the surface of the host root. The keel-shaped intrusive part of the secondary haustorium penetrates to the depth of the vascular tissue of the host. Some of the epidermal interface cells differentiate into xylem elements. A significant number of haustoria do not differentiate further, but in most haustoria one to five of the epidermal xylem elements terminate a similar number of xylem strands. The strands mostly consist of vessel members and they connect host xylem or occasionally host parenchyma to the plate xylem adjacent to the stele of the parasite root. Each strand of this xylem bridge is accompanied by highly protoplasmic parenchyma cells with supposed transfer cell function. Increased surface area of the plasmalemma occurs in these cells as it does in interface parenchyma cells. Graniferous tracheary elements are restricted to the haustorium and occur most frequently in the plate xylem. The plate xylem is also accompanied by highly protoplasmic parenchyma cells. Hyphae of mycorrhizal fungi of the host root occasionally penetrate into the distal part of the xylem bridge. We combine structural observations and physiological facts into a hypothesis for translocation of water and nutrients between host and parasite. Some evolutionary aspects related to endogeny/exogeny of haustoria are discussed, and it is argued that the Triphysaria haustorium represents a greatly advanced and/or reduced condition within Scrophulariaceae.     

Una Struttura Comparabile Con I Lomasomi Nelle Cellule Austoriali di Cuscuta Pentagona Engelm

Abstract

A structure comparable with the lomasomes in baustorial cells of « Cuscuta pentagona » Engelm. — Electron microscopic observations of haustorial cells in « Cuscuta pentagona » revealed to the A. the presence of some vesicular structures, outside the plasma membrane, comparable to the lomasomes of Moore and McAlear. The vesicles usually group in a bag of the plasma membrane and show an envelope consisting of unit membrane; their shape and size are variable and some of them are not well distinct while some others show ribosome-like bodies inside .In the examined cells the plasma membrane shows prominences which are in connection with the vesicle membrane, so that the A. thinks it possible the derivation of the vesicles from the peripherical cytoplasm. The function of these structures is not yet explained; they are only found in non photosyntetic cells, such as some Mycophyta, dark grown « Avena » coleoptiles and dodder's haustorial cells. The previously ultrastructural researchs on dodder have not pointed out lomasomes, which seem to be exclusive of the haustoria, so that it is possible to suppose a connection between the lomasome function and that of the haustoria.     

Proteomic analysis of blue light-induced twining response in Cuscuta australis

The parasitic plant Cuscuta australis (dodder) invades a variety of species by entwining the stem and leaves of a host and developing haustoria. The twining response prior to haustoria formation is regarded as the first sign for dodders to parasitize host plants, and thus has been the focus of studies on the host-parasite interaction. However, the molecular mechanism is still poorly understood. In the present work, we have investigated the different effects of blue and white light on the twining response, and identified a set of proteins that were differentially expressed in dodder seedlings using a proteomic approach. Approximately 1,800 protein spots were detected on each 2-D gel, and 47 spots with increased or decreased protein levels were selected and analyzed with MALDI-TOF-MS. Peptide mass fingerprints (PMFs) obtained for these spots were used for protein identification through cross-species database searches. The results suggest that the blue light-induced twining response in dodder seedlings may be mediated by proteins involved in light signal transduction, cell wall degradation, cell structure, and metabolism.     

Haustoria in action: Case studies of nitrogen acquisition by woody xylem-tapping hemiparasites from their hosts

Summary This review discusses studies conducted by the author and his colleagues on mistletoes and root hemiparasites native to Western Australia. Morphological characteristics of haustoria are described and their anatomical features are discussed in relation to uptake, transfer, and metabolism of xylem-borne nitrogenous solutes derived from a host. Experimental approaches used include comparisons of xylem sap composition of parasite and host(s), solute pool analyses and enzymatic properties of haustoria, host xylem feeding of¹⁵N-labelled solutes to follow the fate of label in haustoria and body of the parasite, and studies using species-specific nonprotein amino acids to validate successful uptake from hosts or occasional backflow of xylem-borne solutes to a host. Field studies on promiscuous root hemiparasites assess frequencies of exploitation of different hosts.¹⁵N natural abundance assays of host and parasite dry matter demonstrate marked preference bySantalum acuminatum for N₂-fixing as opposed to nonfixing hosts. The ability ofOlax phyllanthi to continue to exploit deep-rooted hosts ranks of importance when xylem water potentials of other hosts go out of sucking range during periods of water stress. Comparisons of xylem sap composition of parasites feeding on different hosts indicate remarkable versatility by haustoria in uptake and utilization of the different major nitrogenous solutes received from these hosts. Solute pools in parasites partly reflect metabolic transformations accentuated by haustoria while also indicating direct throughput from xylem of a host. The review concludes by showing how empirically based modelling techniques can be used to estimate proportional gains of N by parasites from single hosts and repercussions on host growth which accompany such exploitation.Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Phytochelatins govern zinc/copper homeostasis and cadmium detoxification in Cuscuta campestris parasitizing Daucus carota

Cuscuta sp., known with the common name of “dodder”, is an obligate parasite capable of invading stems and leaves of a wide range of host plants. Dodder stem usually coils counterclockwise around the host and, within a few days, develops haustorial structures at each point of contact. As soon as dodder haustoria reach host vascular bundles, they start tapping water, photosynthates and minerals. Metal ions such as zinc (Zn) and copper (Cu) are essential for dodder growth and metabolism, although an exceedingly high (over-homeostatic) supply of these micronutrients can result in growth inhibition and cellular toxicity. Even more so, non-essential metals such as cadmium (Cd), if transferred from the host to the parasite, need to be neutralized by timely detoxification mechanisms. In this work, we showed that Cuscuta campestris Yuncker establishes effective haustorial connections with leaf petioles and blades of Daucus carota L. (carrot), with the consequent transfer of Cd and essential metals (such as Zn and Cu) from the host vascular bundles to the parasite. Following up to this point, we detected the presence in the parasite of significant amounts of glutathione and phytochelatins, even in the absence of Cd exposure. This suggests that thiol peptides in dodder might be particularly important for Zn and Cu homeostasis as well as for Cd detoxification. Finally, we demonstrated that dodder is capable of synthesizing phytochelatins on its own, rather than massively importing them from the host, and also provided evidence for the existence of an endogenous, constitutively expressed, dodder's phytochelatin synthase.     

Genetic diversity of dodder (<Emphasis Type="Italic">Cuscuta</Emphasis> spp.) collected from commercial cranberry production as revealed in the<Emphasis Type="Italic">trn</Emphasis>L (UAA) intron

Swamp dodder (Cuscuta gronovii) is a parasitic plant detrimental to cranberries. Observation of emergence of dodder seeds collected from a cultivated cranberry bog in Massachusetts revealed 2 or more peak emergence times during 4 consecutive growing seasons. Molecular methods were used to investigate genetic variation among the emerging dodder seedlings. On emergence, dodder seedlings were collected and analyzed for DNA sequence diversity in thetrnL (UAA) intron, a noncoding region of chloroplast DNA. DNA sequence analysis of 87 seedlings collected during the 1999 and 2000 growing seasons revealed the presence of 2 dodder ecotypes, designated A and B. Comparative DNA sequence analysis indicated that in thetrnL (UAA) intron, the sequence of ecotype A is identical to that ofCuscuta gronovii, whereas the sequence of ecotype B is closest to that ofCuscuta attenuata (99.3% sequence identity; 293 bases considered). ABg/II restriction enzyme cut site was identified that distinguished between thetrnL (UAA) introns of ecotypes A and B. Restriction fragment length polymorphism (RFLP) was used to analyze the sequences of 100 seedlings collected during the growing seasons of 2001 and 2002. Only 10 of the 187 samples were ecotype A, all of which emerged on or before May 7 in the growing seasons. Therefore, the predominant dodder haplotype found in this study may be a close relative ofC. attenuata and notC. gronovii, the common species found in cranberry bogs.     

Mineral composition ofCuscuta nevadensis Johnston (dodder) in relationship to its hosts

Summary The mineral composition of 16 samples of dodder (Cuscuta nevadensis Johnston) and leaves of eight species of its host plants collected from the northern Mojave Desert were examined to determine relationships between parasitism and transport of mineral elements from the host to the parasite. The K concentration of dodder was fairly constant for a wide range of host plants. It was sometimes less than and sometimes greater than that of the host. The concentrations of Ca, Mg, B, Mn, Mo, and Sr in dodder were always much lower than those of leaves of host plants. Those of Fe, P, and Al were sometimes higher. Sodium was generally excluded from dodder regardless of host concentration. Positive and significant correlation coefficients were obtained for concentration in doddervs concentration in leaves of host plants for P, Na, Ca, Mg, Cu, Mn, Mo, Sr, Ba, and Li. The coefficient of variation was less in the dodder than in leaves of the different host plants for 7 of 17 metals.     

Development and Structure of the Haustorium of the Parasite Rhamphicarpa fistulosa (Scrophulariaceae)

Rhamphicarpa fistulosa (Hochst.) Benth. (Scrophulariaceae), a parasite of African cereals, develops secondary haustoria which penetrate the roots of the host plant. Light and electron microscopy have been used to study the structure and development of haustoria in this species, which, until now, have not been well characterized. Haustoria are initiated in the hypodermis of the parasite roots. A meristematic strand is developed between the parasite root stele and the host-parasite interface. From this strand, cells differentiate into xylem elements after penetration of the host root. Xylem differentiation follows an acropetal pattern. Mature haustoria are characterized by a continuous xylem bridge between water conducting elements of parasite and host. A detailed study of the hostparasite interface revealed the presence of collapsed and compressed host cells at the lateral interface (between parasite cells and host cortex), whereas the central interface between parasite cells and the host stele is almost devoid of host cell remnants. Implications of these observations for the penetration mechanisms are discussed.     

Assunzione Di Metaboliti Da Parte Di « Cuscuta Epithymum » Parassita Di « Trifolium Repens »

Abstract

Uptake of metabolites by CUSCUTA. — The distribution of the radioactivity in the filaments of Cuscuta epithymum attached to the host (Trifolium repens) growing in an atmosphere of C¹⁴O₂ indicates that the dodder uptakes from the host solely, or mainly, sucrose. The other radioactive compounds present in the extracts of Cuscuta appear to be derived from the sucrose rather than uptaken from the host.     

First report of parasite dodder (Cuscuta campestris) on jujube trees (Ziziphus jujuba) from Iran

Jujube (Zizyphus jujuba Mill.) as a naturally host for dodder (Cuscuta campestris Yunck.) is newly recorded from Southern Khorasan province of Iran. This is the first report of C. campestris, a stem parasite plant, naturally infecting jujube trees worldwide.     

Epidermal derivatives as xylem elements and transfer cells: a study of the host-parasite interface in two species of Triphysaria (Scrophulariaceae)

Summary Haustoria ofTriphysaria pusilla andT. versicolor subsp.faucibarbata from a natural habitat were analysed by light and electron microscopy. The keel-shaped edge of the secondary haustorium generally splits the epidermis and cortex of the host root parallel to the root axis, and penetrates to the host vascular tissue. Anticlinally elongated epidermal cells of the haustorium constitute most of the host/parasite interface. Some of these epidermal cells are divided by oblique cell walls. Some of their oblique daughter cells as well as some undivided epidermal cells differentiate into xylem elements. Single epidermal cells occasionally intrude into the vascular tissue of the host and individual host cells can be invaded. The surface area of the plasmalemma in parasitic parenchymatous interface cells is increased by the differentiation of wall labyrinths characteristic of transfer cells and by the development of membrane-lined cytoplasmic tubules or flattened sacs which become embedded in the partly lignified interface cell-wall. Mycorrhizal fungal hyphae enter the xylem bridge in some haustoria. Implications of these observations for the function of the haustorium are discussed.     

Attachment of the parasitic weed dodder to the host

Summary.  The parasitic weed dodder (Cuscuta pentagona L.) invades a number of potential host species, but the mechanisms responsible for ensuring tight adhesion to the wide variety of host surfaces have yet to be identified. In this study, a battery of microscopy protocols is used to examine the host–parasite interface in an effort to deduce these mechanisms. As the dodder shoot approaches the host tissue, epidermal cells in the parasite shoot elongate and differentiate into secretory type trichomes. The trichome cell walls are malleable, allowing them to elongate towards the host and bend their walls to conform to the shape of the host cell surface. The presence of osmiophilic particles (probable cell-wall-loosening complexes) at far greater numbers than found in other species presages the expansion and malleable nature of the epidermal cells. In addition to the changes in cell shape, the dodder trichome cells secrete an electron-opaque cementing substance that covers the host–parasite interface. When probed with antibodies that recognize cell wall components, the cement reacted only with antibodies that recognize chiefly de-esterified pectins but not other common wall constituents. These data indicate that dodder utilizes both a cementing layer of pectin and a radically modified epidermal cell wall to secure the parasite to the perspective host. Received January 29, 2001 Accepted November 28, 2001     

Occurrence of Mycoplasma-like Organisms in Parenchyma Cells of Cuscuta odorata (Ruiz et Pav.)

In shoots of the dodder Cuscuta odorata mycoplasma-like organisms (MLO) were observed to occur in nearly all mature sieve elements. Their frequency within the sieve elements varied from a few organisms to high concentrations. In addition, MLO, were found in other cell types. By identifying these cells and investigating the location in the vascular tissue three different types of cells infected with MLO could be distinguished: (1) phloem parenchyma cells, (2) parenchyma cells separating the five vascular bundles of C. odorata and (3) cells in the outer region of the vascular system next to the cells looking like an endodermis. Within the cells of the types 2 and 3, MLO occurred in large numbers and at different morphological structures., Therefore, we assume that the MLO multiply in these cells.     

Modelling the flow and partitioning of carbon and nitrogen in the holoparasite Cuscuta reflexa Roxb. and its host Lupinus albus L.: I. Methods for estimating net flows

Nodulated Lupinus albus L. was grown on quartz sand in the greenhouseand supplied with a N-free culture solution. Half the plantswere infected with Cuscuta reflexa Roxb. at 33 DAS. An empiricallybased modelling technique was developed to quantitatively depictuptake, flow and utilization of C and N in the host plant andbetween host and parasite over a 12 d period. The modellingincorporated C: N ratios of solutes in phloem and pressure-inducedxylem sap, net increments of C and N and respiratory lossesof C. For assessing the transfer of solutes from host phloemto Cuscuta it was not possible to use the C: N ratio of phloemsap close to the site of parasite attachment, a procedure whichwould have assumed non-specific withdrawal of phloem-borne solutes,since this would have implied unimpeded mass flow from hostto parasite. The relative intake of C and N by the parasiteby specific withdrawal of nitrogenous and carbonaceous solutesfrom the phloem was obtained independently by assuming thatxylem intake occurred non-specifically. Xylem import was thusobtained (a) from transpiration and tissue water increment ofCuscuta and the concentrations of N and C in xylem sap and (b)from the Ca²⁺ increment of Cuscuta and the ratios Ca: N andCa: C in lupin xylem sap, assuming that Ca²⁺ intake occurredsolely via xylem. By subtracting net xylem import from totaluptake of C and N by Cuscuta the methods resulted in comparableratios of C: N intake from the phloem. The average ratio (53.4)was smaller than the C:N ratio in host phloem (85.6) indicatingspecific withdrawal of solutes with a distinct preference forN. Using this ratio, modelling of flows of C and N was possibleand showed that Cuscuta abstracted C and N mainly from the hostphloem, but xylem supply was nutrient-dependent and amountedto 6.4% of the N but only 0.5% of the C demand. The resultsindicated that Cuscuta exerted a very strong sink and competedefficiently with the root, the major sink of L. albus, by attracting81% of the current photosynthate and more N (223%) than wascurrently fixed. The massive demand of the parasite led to lossesparticularly of N from leaves and the root and apart from causingcarbon losses it appeared to induce a sink-dependent stimulationof photosynthesis. In contrast, nitrogen fixation in the Cuscuta-infectedlupin was inhibited to 37% of the control. Key words: Cuscuta reflexa, Lupinus albus, carbon, nitrogen, phloem, xylem, transport, parasites, modelling     

Modelling of the flows and partitioning of carbon and nitrogen in the holoparasite Cuscuta reflexa Roxb. and its host Lupinus albus L.: II. Flows between host and parasite and within the parasitized host

A recently developed empirically based modelling technique wasused to quantify uptake, flow and utilization of C and N inLupinus albus L., uninfected and parasitized by Cuscuta reflexaRoxb. plants over a 12 d period during flowering and early fruitsetting of the host. The modelling combined data on molar C:Nratios in host phloem and pressure-induced xylem sap, net incrementsof C and N in host and parasite plant parts and respiratorylosses of C. The modelling of the solute transfer between hostand Cuscuta was achieved by assuming non-specific intake fromthe xylem. The models predicted that Cuscuta derived 99.5% ofits carbon and 93.6% of its nitrogen demand from the host phloem.The overriding sink strength of the parasite diverted most ofthe basipetally translocated host assimilates and massivelycompeted with the host root and inhibited fruit setting. Carbonincorporation in Cuscuta consumed 56%, respiration 24% and secretionby extrafloral nectaries 1.8% of the current host photosynthate.Root respiration was inhibited by 59% and carbon was mobilizedfrom host root and leaves. Competition by the parasite for Nwas even more severe and Cuscuta incorporated nitrogen equalling223% of current fixation, but N₂ fixation of the host was severelyrestricted to 37%. Withdrawal of N from host phloem led to severelosses of N from leaves and the root and marked decreases inN concentration. It required massive xylem-to-phloem transferof N, because the xylem as the major supply route for N wasnot exploited substantially by Cuscuta. The results are discussedin relation to likely causes for parasite-induced pathogeniceffects, suggesting that Cuscuta affected the host adverselyby depriving it mainly of its nitrogen, but that causal to incipientnitrogen deficiency and restricted N2 fixation was the superiorsink potential of Cuscuta, which prevented adequate supply ofassimilates to the nodulated root. The dominating sink potentialof Cuscuta is compared with the similarly strong sink competitionexerted by fruits at the stage of seed filling in annual plants. Key words: Cuscuta reflexa, Lupinus albus, parasitism, carbon, nitrogen, phloem, xylem, transport