Efficient carbon recycling and modulation of antioxidants involved in elongation of the parasitic plant dodder (Cuscuta spp.) in vitro.

Dodder is a holoparasitic flowering plant that re-establishes parasitism with the host when broken off from the host. However, how in vitro dodder shoots recycle stored nutrients to maintain growth for reparasitizing hosts is not well characterized. Here, the spatial and temporal distribution characteristics of carbohydrates and reactive oxygen species (ROS) were analysed to explore the mechanism of recycling stored nutrients in dodder shoots in vitro. Our results showed that in vitro dodder shoots grew actively for more than 10 d, while dry mass decreased continuously. During this process, the transcript levels and activities of amylases gradually increased until 2 d and then declined in basal stems, which induced starch degradation at the tissue, cellular and subcellular levels. Additionally, the distribution characteristics of H2O2 and the activities and transcript levels of antioxidant enzymes indicated that shoot tips exhibited more robust ROS-scavenging capacity, and basal stems maintained higher ROS accumulation. Comparative proteomics analysis revealed that starch in basal stems acted as an energy source, and the glycolysis, TCA cycle and pentose phosphate pathway represented the energy supply for shoot tip elongation with time. These results indicated that efficient nutrient recycling and ROS modulation facilitated the parasitism of dodder grown in vitro by promoting shoot elongation growth to reach the host.

Cuscuta Species Identification Based on the Morphology of Reproductive Organs and Complete Chloroplast Genome Sequences.

The genus Cuscuta (Convolvulaceae) comprises well-known parasitic plants. Cuscuta species are scientifically valuable, as their life style causes extensive crop damage. Furthermore, dried seeds of C. chinensis are used as a Korean traditional herbal medicine. Despite the importance of Cuscuta species, it is difficult to distinguish these plants by the naked eye. Moreover, plastid sequence information available for Cuscuta species is limited. In this study, we distinguished between C. chinensis and C. japonica using morphological characterisation of reproductive organs and molecular characterisation of chloroplast genomes. The differences in morphological characteristics of reproductive organs such as style, stigma, infrastaminal scale, seed shape and testa ornamentation were useful for distinguishing between C. japonica and C. chinensis. Analysis of chloroplast genomes revealed drastic differences in chloroplast genome length and gene order between the two species. Although both species showed numerous gene losses and genomic rearrangements, chloroplast genomes showed highly similar structure within subgenera. Phylogenetic analysis of Cuscuta chloroplast genomes revealed paraphyletic groups within subgenera Monogynella and Grammica, which is consistent with the APG IV system of classification. Our results provide useful information for the taxonomic, phylogenetic and evolutionary analysis of Cuscuta and accurate identification of herbal medicine.

Waterfowl endozoochory: An overlooked long-distance dispersal mode for Cuscuta (dodder).

PREMISE OF THE STUDY: Dispersal of parasitic Cuscuta species (dodders) worldwide has been assumed to be largely anthropomorphic because their seeds do not match any previously known dispersal syndrome and no natural dispersal vectors have been reliably documented. However, the genus has a subcosmopolitan distribution and recent phylogeographic results have indicated that at least18 historical cases of long-distance dispersal (LDD) have occurred during its evolution. The objective of this study is to report the first LDD biological vector for Cuscuta seeds. METHODS: Twelve northern pintails (Anas acuta) were collected from Suisun Marsh, California and the contents of their lowest part of the large intestine (rectum) were extracted and analyzed. Seed identification was done both morphologically and using a molecular approach. Extracted seeds were tested for germination and compared to seeds not subjected to gut passage to determine the extent of structural changes caused to the seed coat by passing through the digestive tract. KEY RESULTS: Four hundred and twenty dodder seeds were found in the rectum of four northern pintails. From these, 411 seeds were identified as Cuscuta campestris and nine as most likely C. pacifica. The germination rate of C. campestris seeds after gut passage was 55%. Structural changes caused by the gut passage in both species were similar to those caused by an acid scarification. CONCLUSIONS: Endozoochory by waterbirds may explain the historical LDD cases in the evolution of Cuscuta. This also suggests that current border quarantine measures may be insufficient to stopping spreading of dodder pests along migratory flyways.

Cuscuta europaea plastid apparatus in various developmental stages: localization of THF1 protein.

It was generally accepted that Cuscuta europaea is mostly adapted to a parasitic lifestyle with no detectable levels of chlorophylls. We found out relatively high level of chlorophylls (Chls a+b) in young developmental stages of dodder. Significant lowering of Chls (a+b) content and increase of carotenoid concentration was typical only for ontogenetically more developed stages. Lower content of photosynthesis-related proteins involved in Chls biosynthesis and in photosystem formation as well as low photochemical activity of PSII indicate that photosynthesis is not the main activity of C. europaea plastids. Previously, it has been shown in other species that the Thylakoid Formation Protein 1 (THF1) is involved in thylakoid membrane differentiation, plant-fungal and plant-bacterial interactions and in sugar signaling with its preferential localization to plastids. Our immunofluorescence localization studies and analyses of haustorial plasma membrane fractions revealed that in addition to plastids, the THF1 protein localizes also to the plasma membrane and plasmodesmata in developing C. europaea haustorium, most abundantly in the digitate cells of the endophyte primordium. These results are supported by western blot analysis, documenting the highest levels of the THF1 protein in "get together" tissues of dodder and tobacco. Based on the fact that photosynthesis is not a typical process in the C. europaea haustorium and on the extra-plastidial localization pattern of the THF1, our data support rather other functions of this protein in the complex relationship between C. europaea and its host.

Long-distance transport of mRNA via parenchyma cells and phloem across the host-parasite junction in Cuscuta.

It has been shown that the parasitic plant dodder (Cuscuta pentagona) establishes a continuous vascular system through which water and nutrients are drawn. Along with solutes, viruses and proteins, mRNA transcripts are transported from the host to the parasite. The path of the transcripts and their stability in the parasite have yet to be revealed. To discover the route of mRNA transportation, the in situ reverse transcriptase-polymerase chain reaction (RT-PCR) technique was used to locally amplify host transcript within parasitic tissue. The stability of host mRNA molecules was also checked by monitoring specific transcripts along the growing dodder thread. Four mRNAs, alpha and beta subunits of PYROPHOSPHATE (PPi)-DEPENDENT PHOSPHOFRUCTOKINASE (LePFP), the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and GIBBERELLIC ACID INSENSITIVE (LeGAI), were found to move from host (tomato (Solanum lycopersicum)) to dodder. LePFP mRNA was localized to the dodder parenchyma cells and to the phloem. LePFP transcripts were found in the growing dodder stem up to 30 cm from the tomato-dodder connection. These results suggest that mRNA molecules are transferred from host to parasite via symplastic connections between parenchyma cells, move towards the phloem, and are stable for a long distance in the parasite. This may allow developmental coordination between the parasite and its host.

Development of Cuscuta species on a partially incompatible host: induction of xylem transfer cells.

The growth of dodders, Cuscuta reflexa and Cuscuta japonica, on the partially incompatible host poinsettia ( Euphorbia pulcherrima) is studied. Poinsettia responds by bark growths to the formation of the dodder haustoria and prevents dodder from obtaining normal growth. The growth instead becomes extremely branched, coral-like, and dodder lacks the ability to form haustoria. After a period of coral-like growth, long shoots sprout, resembling the normal growth. These long shoots mark an ending phase for dodder, which dies shortly after without having flowered. During the coral-like growth phase, dodder develops transfer cells in the parenchyma cells bordering the vessels of the xylem in the shoot. The transfer cells have not been observed when dodder is grown on the compatible host Pelargonium zonale. A coral-like growth phase has also been observed at the establishing phase when dodder is grown in vitro on agar; later a more normal growth form takes over. In this coral phase, xylem transfer cells are also developed. The fluorochromes carboxyfluorescein and Texas Red were loaded into the host in the phloem and xylem, respectively, and detection of these fluorochromes in the dodder stem indicated that a functional haustorial contact developed for both vascular systems. The results show that Cuscutaspp. have the genetic ability to develop xylem transfer cells and use this in response to developmental stress.

Dodder hyphae invade the host: a structural and immunocytochemical characterization.

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.

Attachment of the parasitic weed dodder to the host.

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.