LATERAL ORGAN BOUNDARIES DOMAIN 25 functions as a key regulator of haustorium development in dodders.

Parasitic plants reduce crop yield worldwide. Dodder (Cuscuta campestris) is a stem parasite that attaches to its host, using haustoria to extract nutrients and water. We analyzed the transcriptome of six C. campestris tissues and identified a key gene, LATERAL ORGAN BOUNDARIES DOMAIN 25 (CcLBD25), as highly expressed in prehaustoria and haustoria. Gene coexpression networks from different tissue types and laser-capture microdissection RNA-sequencing data indicated that CcLBD25 could be essential for regulating cell wall loosening and organogenesis. We employed host-induced gene silencing by generating transgenic tomato (Solanum lycopersicum) hosts that express hairpin RNAs to target and down-regulate CcLBD25 in the parasite. Our results showed that C. campestris growing on CcLBD25 RNAi transgenic tomatoes transited to the flowering stage earlier and had reduced biomass compared with C. campestris growing on wild-type (WT) hosts, suggesting that parasites growing on transgenic plants were stressed due to insufficient nutrient acquisition. We developed an in vitro haustorium system to assay the number of prehaustoria produced on strands from C. campestris. Cuscuta campestris grown on CcLBD25 RNAi tomatoes produced fewer prehaustoria than those grown on WT tomatoes, indicating that down-regulating CcLBD25 may affect haustorium initiation. Cuscuta campestris haustoria growing on CcLBD25 RNAi tomatoes exhibited reduced pectin digestion and lacked searching hyphae, which interfered with haustorium penetration and formation of vascular connections. The results of this study elucidate the role of CcLBD25 in haustorium development and might contribute to developing parasite-resistant crops.

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.

A plant parasite uses light cues to detect differences in host-plant proximity and architecture.

Sunlight filtered by green plant tissue becomes diminished in its ratio of red to far-red wavelengths (R:FR). Some parasitic plants exploit this change by growing towards regions of low R:FR to locate host plants. In principle, variation in R:FR can also convey ecologically relevant information about host proximity or architecture. Here, we demonstrate that the parasitic vine Cuscuta epilinum Weihe (Convolvulaceae) can distinguish fine-scale differences in R:FR associated with differences in the proximity and shape of potential host plants. We conducted dual-choice experiments by placing parasite seedlings between targets, including low R:FR fields manipulated via LED lighting and pairs of model plants exhibiting realistic R and FR reflectance but differing in proximity or shape. Seedlings consistently distinguished between low-R:FR fields of differing intensity. Furthermore, they exhibited preferences for nearer plant models versus identical models placed 4 cm further away and between same-sized models exhibiting shape differences. Our results indicate that parasites can discriminate minute differences in R:FR signatures corresponding to host factors (proximity and shape) that impact seedling survival. This keen sensory ability underpins the parasite's sophisticated foraging behaviour and highlights the broader importance of light cues in plant ecology.

Compensatory sequence variation between trans-species small RNAs and their target sites.

Trans-species small regulatory RNAs (sRNAs) are delivered to host plants from diverse pathogens and parasites and can target host mRNAs. How trans-species sRNAs can be effective on diverse hosts has been unclear. Multiple species of the parasitic plant Cuscuta produce trans-species sRNAs that collectively target many host mRNAs. Confirmed target sites are nearly always in highly conserved, protein-coding regions of host mRNAs. Cuscuta trans-species sRNAs can be grouped into superfamilies that have variation in a three-nucleotide period. These variants compensate for synonymous-site variation in host mRNAs. By targeting host mRNAs at highly conserved protein-coding sites, and simultaneously expressing multiple variants to cover synonymous-site variation, Cuscuta trans-species sRNAs may be able to successfully target multiple homologous mRNAs from diverse hosts.

Tobacco plants (Nicotiana benthamiana) were influenced by silicon and were not infected by dodder (Cuscuta europaea).

The effect of silicon (Si) on tobacco (Nicotiana benthamiana) development and dodder (Cuscuta europaea) - tobacco interaction were studied. Three Si application approaches were tested: tobacco seed priming (2.5 mM Si and 5 mM Si; 2.5S, 5S), watering tobacco plants with Si solution (2.5 mM Si and 5 mM Si; 2.5W, 5W) and foliar application (1 mM Si and 2.5 mM Si; 1F, 2.5F). Dodder was not able to infect the host plant in almost all Si treatments. Only in the control and 2.5W treatments was dodder able to infect its host. A significant increase in all observed antioxidant enzymes activities (POX, CAT and SOD) occurred in the plants of 2.5W treatment after infection in comparison with the uninfected 2.5W treatment and control plants, which indicated the importance of antioxidant enzymes activities in the plant parasite - host interaction. Resistance of Si treated plants to dodder could have been due to the changes in the cell wall properties of the epidermis and cortex where activity of POX was confirmed histochemically. The growth and development of tobacco shoots were evaluated after four and eight weeks of cultivation in the individual Si treatments. The development of shoots was enhanced after eight weeks of cultivation in the 2.5S, 5S, 2.5W and 5W treatments in comparison with the control treatment. However, a negative effect of Si was observed in 1F and 2.5F treatments. In the majority of cases, the plants treated with Si had decreased chlorophyll content when compared to control, except for chl a in 5W plants after 8 weeks of cultivation. Contrary to this, carotenoids increased in all Si treated plants after eight weeks cultivation in comparison with the control. The secondary xylem formation in tobacco was enhanced after 4 and 8 weeks cultivation in shoots of plants receiving the 2.5S, 5S, 2.5W and 5W treartments. The cambium was the most active in producing secondary xylem in the 2.5S treatment. Protein profile and antioxidant enzymes activities (POX, CAT and SOD) were altered by Si treatment. After 8 weeks of cultivation, activities of POX were significantly decreased in 2.5S, 5S, 2.5W and 5W in comparison with control. Catalase was decreased in 2.5S, 5S and 5W in comparison with the control, however, 1F and 2.5F treatments had significantly increased CAT and SOD activities. The specific activity of POX was confirmed histochemically in Si treated plants in the cell walls of several stem tissues like the epidermis, cortex and pith. A small amount of H2O2 was detected in leaves in the control and Si treated plants. The amount of O2- decreased in all treatments with time. The highest Si concentration in the plants (almost 800 mg . kg-1 d. w.) was detected in the 2.5W, 5W treatments.

Salinity effect on Cuscuta campestris Yunck. Parasitism on Arabidopsis thaliana L.

Stem holoparasitic flowering plants of the genus Cuscuta are globally distributed invasive species and agricultural pests. The present research represents the combined effect of salt stress (e.g. abiotic stress) and Cuscuta campestris infection (e.g. biotic stress) on the model host plant Arabidopsis thaliana and the response of the parasite to salinity. The response of these parasites to abiotic stress conditions including salinity is poorly studied. Arabidopsis plants were continuously irrigated with 0, 50 and 150 mM NaCl and subjected to C. campestris infection. The influence of both abiotic and biotic stresses on the major osmoprotectant L-proline and three antioxidant enzymes - catalase, superoxide dismutase and guaiacol peroxidase, was assessed in both the parasite and the host plant. All four biochemical markers were differentially affected by stress, showing that the influence of C. campestris parasitism and its interaction with salinity is mostly in the site of infection (direct response) and also in roots (indirect vertical response) rather than on non-infected leaves of infected plants (indirect horizontal response). Despite its absence of soil contact, C. campestris was also significantly affected by salinity (indirect response). The mutual adaptation of the parasite-host pair to salinity slightly altered the regular response to abiotic stress of A. thaliana, but no detrimental additive effect of biotic and abiotic stress was observed.

MicroRNAs from the parasitic plant Cuscuta campestris target host messenger RNAs.

Dodders (Cuscuta spp.) are obligate parasitic plants that obtain water and nutrients from the stems of host plants via specialized feeding structures called haustoria. Dodder haustoria facilitate bidirectional movement of viruses, proteins and mRNAs between host and parasite, but the functional effects of these movements are not known. Here we show that Cuscuta campestris haustoria accumulate high levels of many novel microRNAs (miRNAs) while parasitizing Arabidopsis thaliana. Many of these miRNAs are 22 nucleotides in length. Plant miRNAs of this length are uncommon, and are associated with amplification of target silencing through secondary short interfering RNA (siRNA) production. Several A. thaliana mRNAs are targeted by 22-nucleotide C. campestris miRNAs during parasitism, resulting in mRNA cleavage, secondary siRNA production, and decreased mRNA accumulation. Hosts with mutations in two of the loci that encode target mRNAs supported significantly higher growth of C. campestris. The same miRNAs that are expressed and active when C. campestris parasitizes A. thaliana are also expressed and active when it infects Nicotiana benthamiana. Homologues of target mRNAs from many other plant species also contain the predicted target sites for the induced C. campestris miRNAs. These data show that C. campestris miRNAs act as trans-species regulators of host-gene expression, and suggest that they may act as virulence factors during parasitism.

The parasitic plant Cuscuta australis is highly insensitive to abscisic acid-induced suppression of hypocotyl elongation and seed germination.

Around 1% of angiosperms are parasitic plants. Their growth and development solely or partly depend on host plants from which they extract water, nutrients, and other molecules using a parasitic plant-specific organ, the haustorium. Strong depletion of nutrients can result in serious growth retardation and in some cases, death of the hosts. The genus Cuscuta (dodder) comprises about 200 holoparasitic species occurring on all continents. Their seedlings have no roots and cotyledons but are only string-like hypocotyls. When they contact suitable host plants, haustoria are formed and thereafter seedlings rapidly develop into vigorously growing branches without roots and leaves. This highly specialized lifestyle suggests that Cuscuta plants likely have unique physiology in development and stress responses. Using germination and seedling growth assays, we show that C. australis seeds and seedlings are highly insensitive to abscisic acid (ABA). Transcriptome analysis and protein sequence alignment with Arabidopsis, tomato, and rice homologs revealed that C. australis most likely consists of only four functional ABA receptors. Given that Cuscuta plants are no longer severely challenged by drought stress, we hypothesize that the ABA-mediated drought resistance pathway in Cuscuta spp. might have had degenerated over time during evolution.

Differential effects of ephemeral colonization by arbuscular mycorrhizal fungi in two Cuscuta species with different ecology.

Seedlings of parasitic Cuscuta species are autotrophic but can survive only a short period of time, during which they must locate and attach to a suitable host. They have an ephemeral root-like organ considered not a "true" root by most studies. In the present study, two species with contrasting ecology were examined: Cuscuta gronovii, a North American riparian species, and Cuscuta campestris, an invasive dodder that thrives in disturbed habitats. The morphology, structure, and absorptive capability of their root-like organ were compared, their potential for colonization by two species of arbuscular mycorrhizal fungi (AMF) was assessed, and the effect of the AMF on seedling growth and survival was determined. The root of both species absorbed water and interacted with AMF, but the two species exhibited dissimilar growth and survival patterns depending on the colonization level of their seedlings. The extensively colonized seedlings of C. gronovii grew more and survived longer than non-colonized seedlings. In contrast, the scarce colonization of C. campestris seedlings did not increase their growth or longevity. The differential growth responses of the AMF-colonized and non-colonized Cuscuta species suggest a mycorrhizal relationship and reflect their ecology. While C. gronovii roots have retained a higher ability to interact with AMF and are likely to take advantage of fungal communities in riparian habitats, the invasive C. campestris has largely lost this ability possibly as an adaptation to disturbed ecosystems. These results indicate that dodders have a true root, even if much reduced and ephemeral, that can interact with AMF.

EASTERN DODDER (CUSCUTA MONOGYNA VAHL.) SEED GERMINATION AFFECTED BY SOME HERBACEOUS DISTILLATES.

Eastern dodder (Cuscuta monogyna Vahl.) is one of the noxious parasitic weeds that infected many ornamental trees in green spaces and gardens. Our purpose is to find natural inhibitors for prevention of its seed germination. In order to reach this aim, laboratory studies were conducted by using of herbaceous distillates of Dracocephalum moldavica, Nasturtium officinalis, Malva neglecta, Mentha piperita, Mentha pulegium, Rosa damascene, Ziziphora tenuior, and Urtica dioica on seed germination of C. monogyna. Z. tenuior distillate stimulated C. monogyna seed germination, whereas others reduced this parasitic weed's seed germination. D. moldavica caused maximum inhibition on weed seed germination. Seedling growth of C. monogyna was more affected than its seed germination. All of these herbaceous distillates reduced C. monogyna seedling length so that the latter decreased from 28.2 mm in distilled water to 4.5, 3.97, 3.85, 3.67, 3.1, 2.87, 2.57, 1.9, and 1.17 in M. pulegium, M. piperita, F. officinalis, Z. tenuior, N. officinalis, M. neglecta, R. damascene, U. dioica and D. moldavica, respectively. By using these medicinal plants distillates instead of herbicides, the parasitic weed seedling length and host plant infection will reduce.

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.

Dodder transmission of phytoplasmas.

Transmission of phytoplasmas from naturally infected plant host species using the parasitic plant Cuscuta spp. (dodder) to Catharanthus roseus (Madagascar periwinkle) is an effective way to maintain a wide range of phytoplasmas for further research. Here, we describe transmission via dodder from an infected medicinal plant Rehmannia glutinosa var. purpurea and from a symptomatic redcurrant plant (Ribes spp.) to C. roseus indicator plants using a "stable bridges" method. In both cases, typical symptoms of phytoplasma disease on periwinkle plants were obtained: virescent flowers with an increased number of axillary shoots and smaller leaves after transmission from R. glutinosa, and greening petals (virescence) after transmission from Ribes spp. Phytoplasmas could be detected in donor and recipient plants by electron microscopy and by polymerase chain reaction (PCR) assays using universal phytoplasma primer pairs. Restriction fragment length polymorphism analyses of PCR fragments can also be used to confirm the identity of the phytoplasmas from donor and recipient plants.

Interspecific RNA interference of SHOOT MERISTEMLESS-like disrupts Cuscuta pentagona plant parasitism.

Infection of crop species by parasitic plants is a major agricultural hindrance resulting in substantial crop losses worldwide. Parasitic plants establish vascular connections with the host plant via structures termed haustoria, which allow acquisition of water and nutrients, often to the detriment of the infected host. Despite the agricultural impact of parasitic plants, the molecular and developmental processes by which host/parasitic interactions are established are not well understood. Here, we examine the development and subsequent establishment of haustorial connections by the parasite dodder (Cuscuta pentagona) on tobacco (Nicotiana tabacum) plants. Formation of haustoria in dodder is accompanied by upregulation of dodder KNOTTED-like homeobox transcription factors, including SHOOT MERISTEMLESS-like (STM). We demonstrate interspecific silencing of a STM gene in dodder driven by a vascular-specific promoter in transgenic host plants and find that this silencing disrupts dodder growth. The reduced efficacy of dodder infection on STM RNA interference transgenics results from defects in haustorial connection, development, and establishment. Identification of transgene-specific small RNAs in the parasite, coupled with reduced parasite fecundity and increased growth of the infected host, demonstrates the efficacy of interspecific small RNA-mediated silencing of parasite genes. This technology has the potential to be an effective method of biological control of plant parasite infection.

Plant defenses against parasitic plants show similarities to those induced by herbivores and pathogens.

Herbivores and pathogens come quickly to mind when one thinks of the biotic challenges faced by plants. Important but less appreciated enemies are parasitic plants, which can have important consequences for the fitness and survival of their hosts. Our knowledge of plant perception, signaling, and response to herbivores and pathogens has expanded rapidly in recent years, but information is generally lacking for parasitic species. In a recent paper we reported that some of the same defense responses induced by herbivores and pathogens--notably increases in jasmonic acid (JA), salicylic acid (SA), and a hypersensitive-like response (HLR)--also occur in tomato plants upon attack by the parasitic plant Cuscuta pentagona (field dodder). Parasitism induced a distinct pattern of JA and SA accumulation, and growth trials using genetically-altered tomato hosts suggested that both JA and SA govern effective defenses against the parasite, though the extent of the response varied with host plant age. Here we discuss similarities between the induced responses we observed in response to Cuscuta parasitism to those previously described for herbivores and pathogens and present new data showing that trichomes should be added to the list of plant defenses that act against multiple enemies and across Kingdoms.

Parasitism by Cuscuta pentagona sequentially induces JA and SA defence pathways in tomato.

While plant responses to herbivores and pathogens are well characterized, responses to attack by other plants remain largely unexplored. We measured phytohormones and C(18) fatty acids in tomato attacked by the parasitic plant Cuscuta pentagona, and used transgenic and mutant plants to explore the roles of the defence-related phytohormones salicylic acid (SA) and jasmonic acid (JA). Parasite attachment to 10-day-old tomato plants elicited few biochemical changes, but a second attachment 10 d later elicited a 60-fold increase in JA, a 30-fold increase in SA and a hypersensitive-like response (HLR). Host age also influenced the response: neither Cuscuta seedlings nor established vines elicited a HLR in 10-day-old hosts, but both did in 20-day-old hosts. Parasites grew larger on hosts deficient in SA (NahG) or insensitive to JA [jasmonic acid-insensitive1 (jai1)], suggesting that both phytohormones mediate effective defences. Moreover, amounts of JA peaked 12 h before SA, indicating that defences may be coordinated via sequential induction of these hormones. Parasitism also induced increases in free linolenic and linoleic acids and abscisic acid. These findings provide the first documentation of plant hormonal signalling induced by a parasitic plant and show that tomato responses to C. pentagona display characteristics similar to both herbivore- and pathogen-induced responses.

Are herbicide-resistant crops the answer to controlling Cuscuta?

BACKGROUND: Herbicide-resistant crop technology could provide new management strategies for the control of parasitic plants. Three herbicide-resistant oilseed rape (Brassica napus L.) genotypes were used to examine the response of attached Cuscuta campestris Yuncker to glyphosate, imazamox and glufosinate. Cuscata campestris was allowed to establish on all oilseed rape genotypes before herbicides were applied. RESULTS: Unattached seedlings of C. campestris, C. subinclusa Durand & Hilg. and C. gronovii Willd. were resistant to imazamox and glyphosate and sensitive to glufosinate, indicating that resistance initially discovered in C. campestris is universal to all Cuscuta species. Glufosinate applied to C. campestris attached to glufosinate-resistant oilseed rape had little impact on the parasite, while imazamox completely inhibited C. campestris growth on the imidazolinone-resistant host. The growth of C. campestris on glyphosate-resistant host was initially inhibited by glyphosate, but the parasite recovered and resumed growth within 3-4 weeks. CONCLUSION: The ability of C. campestris to recover was related to the quality of interaction between the host and parasite and to the resistance mechanism of the host. The parasite was less likely to recover when it had low compatibility with the host, indicating that parasite-resistant crops coupled with herbicide resistance could be highly effective in controlling Cuscuta.

[Study of quality control on Cuscuta chinensis and C. australia].

OBJECTIVE: To study the estimate method of C. chinensis and C. australia. METHOD: HPLC was used to determine the contents of four kinds of flavones of C. chinensis and C. australia growing on different hosts. RESULT: C. chinensis and C. australia growing on different hosts both had hyperoside, quercetin, kaempferol and isorhamnetin. The content range of hyperoside was 2.790-6.502 mg/g and was higher than other flavones. The content ranges of quercetin, kaempferol and isorhamnetin were 0.025-0.176 mg/g, 0.001-0.213 mg/g and 0.001-0.077 mg/g, respectively. CONCLUSION: The contents of hyperoside and quercetin are higher in C. chineasis than in C. australia. The contents of kaempferol and isorhamnetin are lower in C. chinensis than in C. australia. The hosts influence flavones content of C. chinensis and C. australia.

Volatile chemical cues guide host location and host selection by parasitic plants.

The importance of plant volatiles in mediating interactions between plant species is much debated. Here, we demonstrate that the parasitic plant Cuscuta pentagona (dodder) uses volatile cues for host location. Cuscuta pentagona seedlings exhibit directed growth toward nearby tomato plants (Lycopersicon esculentum) and toward extracted tomato-plant volatiles presented in the absence of other cues. Impatiens (Impatiens wallerana) and wheat plants (Triticum aestivum) also elicit directed growth. Moreover, seedlings can distinguish tomato and wheat volatiles and preferentially grow toward the former. Several individual compounds from tomato and wheat elicit directed growth by C. pentagona, whereas one compound from wheat is repellent. These findings provide compelling evidence that volatiles mediate important ecological interactions among plant species.