Physiological and ecological warnings that dodders pose an exigent threat to farmlands in Eastern Africa

Invasive holoparasitic plants of the genus Cuscuta (dodder) threaten African ecosystems due to their rapid spread and attack on various host plant species. Most Cuscuta species cannot photosynthesize and hence rely on host plants for nourishment. After attachment through a peg-like organ called a haustorium, the parasites deprive hosts of water and nutrients, which negatively affects host growth and development. Despite their rapid spread in Africa, dodders have attracted limited research attention, although data on their taxonomy, host range, and epidemiology are critical for their management. Here, we combine taxonomy and phylogenetics to reveal the presence of field dodder (Cuscuta campestris) and C. kilimanjari (both either naturalized or endemic to East Africa), in addition to the introduction of the giant dodder (C. reflexa), a south Asian species, in continental Africa. These parasites have a wide host range, parasitizing species across 13 angiosperm orders. We evaluated the possibility of C. reflexa to expand this host range to tea (Camelia sinensis), coffee (Coffea arabica), and mango (Mangifera indica), crops of economic importance to Africa, for which haustorial formation and vascular-bundle connections in all three crops revealed successful parasitism. However, only mango mounted a successful postattachment resistance response. Furthermore, species distribution models predicted high habitat suitability for Cuscuta spp. across major tea- and coffee-growing regions of Eastern Africa, suggesting an imminent risk to these crops. Our findings provide relevant insights into a poorly understood threat to biodiversity and economic wellbeing in Eastern Africa, and provide critical information to guide development of management strategies to avert Cuscuta spp. spread.

Microscopy and habitat suitability modeling provide an early warning that dodder’s invasion in Eastern Africa poses a threat to important cash crops.     

Metabolite changes with induction of Cuscuta haustorium and translocation from host plants

Cuscuta is a stem holoparasitic plant without leaves or roots, parasitizing various types of host plants and causing major problems for certain crops. Cuscuta is known as a generalist and, thus, must have unique parasite strategies to cope with different host plants. For elucidating metabolic responses and mechanisms of parasitization, metabolomic approaches using GC/MS were applied. We compared five stages of Cuscuta japonica: early stage seedlings, with far red light (FR) cue, with contact signal, haustorium induced seedlings by both signals and adult plant parasites on host plants. Sugars, amino acids, organic acids, nucleic acids, and polyols were identified from the polar phase fraction. The apical part contained metabolite profiles different from the haustorium induced part or the basal part. Amino acid and some organic acids were up-regulated for haustorium induction but decreased after parasitization. After attachment to different host plants, metabolite profiles of Cuscuta japonica changed dramatically due to the absorption of specific host plant metabolites such as pinitol. Cuscuta seedlings attached to pinitol rich host plants contained more pinitol and showed different profiles from those attached to plants having less or lacking pinitol.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> ETO1 specifically interacts with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases

Background  

In Arabidopsis, ETO1 (ETHYLENE-OVERPRODUCER1) is a negative regulator of ethylene evolution by interacting with AtACS5, an isoform of the rate-limiting enzyme, 1-aminocyclopropane-1-carboxylate synthases (ACC synthase or ACS), in ethylene biosynthetic pathway. ETO1 directly inhibits the enzymatic activity of AtACS5. In addition, a specific interaction between ETO1 and AtCUL3, a constituent of a new type of E3 ubiquitin ligase complex, suggests the molecular mechanism in promoting AtACS5 degradation by the proteasome-dependent pathway. Because orthologous sequences to ETO1 are found in many plant species including tomato, we transformed tomato with Arabidopsis ETO1 to evaluate its ability to suppress ethylene production in tomato fruits.     

Factors restraining parasitism of the invasive vine Mikania micrantha by the holoparasitic plant Cuscuta campestris

Mikania micrantha (Asteraceae) is one of the 10 most invasive weeds in the world and has caused tremendous economic and environmental losses in southern China. The dodder Cuscuta campestris (Convolvulaceae) is a native holoparasite that can parasitize and suppress M. micrantha, and thus is recommended as an effective control agent. However, the natural growth of dodder lags behind that of M. micrantha and fails to exert direct year-round suppression. To verify the effective parasitic distance, we placed a dodder seedling at a designated distance from the stem of M. micrantha and monitored coiling, haustorium formation, and survival. To verify suitable host stems for the parasite, we grew M. micrantha for more than 6 months to form stems of different sizes, taped dodder seedlings to the stems, and monitored. We used various temperatures to determine the effect on dodder seed germination and M. micrantha sprouting. The results showed that a dodder–host distance of 4 cm decreased the probability of successful parasitism to 0; dodder seedlings cannot parasitize M. micrantha stem diameters ≥0.3 cm; and the temperatures for the highest dodder seed germination and M. micrantha sprouting are 26 and 30 °C, respectively. We conclude that the lack of suitable M. micrantha parts within the dodder’s effective parasitism distance is the major cause of restricted dodder parasitism and that the lower temperature for the highest dodder germination compared to that for the highest M. micrantha sprouting may decrease the possibility of parasitism. To acquire aggressive parasitism, dodder should be manually assisted by dispersing its vegetative form.     

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     

Das Phloem der Haustorien vonCuscuta

Summary Haustoria ofCuscuta odorata R. & P. andC. grandiflora H.B.K. show continuous traces of sieve elements, connecting the phloem of the host with that of theCuscuta shoot. The continuity of this haustorial phloem is discernible by callose fluorescence after staining with aniline blue. The fine structural criteria for sieve tubes are analyzed electronmicroscopically, with special respect to sieve pores, P-protein, and a distinct wall-standing smooth surfaced ER. Within the central part of the haustorium sieve tubes are elongated, while the elements abutting the phloem of theCuscuta shoot are nearly isodiametric in shape. Both elements are associated with rather large companion cells, derived from an unequal division.

     

Trans-species small RNAs move long distances in a parasitic plant complex

Parasitic plants exchange various types of RNAs with their host plants, including mRNA, and small non-coding RNA. Among small non-coding RNAs, miRNA production is known to be induced at the haustorial interface. The induced miRNAs transfer to the host plant and activate secondary siRNA production to silence target genes in the host. In addition to interfacial transfer, long-distance movement of the small RNAs has also been known to mediate signaling and regulate biological processes. In this study, we tested the long-distance movement of trans-species small RNAs in a parasitic-plant complex. Small RNA-Seq was performed using a complex of a stem parasitic plant, Cuscuta campestris, and a host, Arabidopsis thaliana. In the host plant’s parasitized stem, genes involved in the production of secondary siRNA, AtSGS3 and AtRDR6, were upregulated, and 22-nt small RNA was enriched concomitantly, suggesting the activation of secondary siRNA production. Stem-loop RT-PCR and subsequent sequencing experimentally confirmed the mobility of the small RNAs. Trans-species mobile small RNAs were detected in the parasitic interface and also in distant organs. To clarify the mode of long-distance translocation, we examined whether C. campestris-derived small RNA moves long distances in A. thaliana sgs3 and rdr6 mutants or not. Mobility of C. campestris-derived small RNA in sgs3 and rdr6 mutants suggested the occurrence of direct long-distance transport without secondary siRNA production in the recipient plant.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> is a susceptible host plant for the holoparasite <Emphasis Type="Italic">Cuscuta </Emphasis>spec

Arabidopsis thaliana and Cuscuta spec. represent a compatible host–parasite combination. Cuscuta produces a haustorium that penetrates the host tissue. In early stages of development the searching hyphae on the tip of the haustorial cone are connected to the host tissue by interspecific plasmodesmata. Ten days after infection, translocation of the fluorescent dyes, Texas Red (TR) and 5,6-carboxyfluorescein (CF), demonstrates the existence of a continuous connection between xylem and phloem of the host and parasite. Cuscuta becomes the dominant sink in this host–parasite system. Transgenic Arabidopsis plants expressing genes encoding the green fluorescent protein (GFP; 27 kDa) or a GFP–ubiquitin fusion (36 kDa), respectively, under the companion cell (CC)-specific AtSUC2 promoter were used to monitor the transfer of these proteins from the host sieve elements to those of Cuscuta. Although GFP is transferred unimpedly to the parasite, the GFP–ubiquitin fusion could not be detected in Cuscuta. A translocation of the GFP–ubiquitin fusion protein was found to be restricted to the phloem of the host, although a functional symplastic pathway exists between the host and parasite, as demonstrated by the transport of CF. These results indicate a peripheral size exclusion limit (SEL) between 27 and 36 kDa for the symplastic connections between host and Cuscuta sieve elements. Forty-six accessions of A. thaliana covering the entire range of its genetic diversity, as well as Arabidopsis halleri, were found to be susceptible towards Cuscuta reflexa.     

Sterols and fatty acids of some non-photosynthetic angiosperms

Sterols and fatty acids were extracted and identified from three parasitic angiosperms, Cuscuta campestris, Monotropa uniflora and M. hypopitys. Each plant contained the typical 16 and 18-carbon fatty acids of angiosperms, but the partially-photosynthetic Cuscuta contained much larger quantities of linolenic acid than the non-green Monotropa species which had smaller amounts of linolenic acid characteristic of non-photosynthetic tissue. Sterol quantity was three times higher in Cuscuta than in the Monotropa species. Sitosterol was the major sterol in all species with smaller amounts of campesterol and cholesterol.     

Brassinosteroid induction of AtACS4 encoding an auxin-responsive 1-aminocyclopropane-1-carboxylate synthase 4 in Arabidopsis seedlings

Brassinosteroid (BR), an endogenous steroid growth regulator of higher plants, enhances expansion and division of the cell in a number of plant species. It has been recently reported that a shared auxin–BR signalling pathway is involved in the seedling growth in Arabidopsis . Here, we show that BR specifically enhanced the expression of AtACS4 , which encodes an auxin-responsive ACC synthase 4, by a distinct temporal induction mechanism compared with that of IAA in etiolated Arabidopsis seedlings. This BR induction of AtACS4 was undetectable in the light-grown seedlings. In addition, BR failed to activate the AtACS4 gene in auxin-resistant1 ( axr1-3 ) and auxin-resistant2 ( axr2-1 ), both of which are auxin-resistant mutants. Thus, it appears that there is a possible regulatory link between light, auxin and BR to control ethylene synthesis in Arabidopsis young seedlings. Analysis of transgenic Arabidopsis plants harbouring AtACS4::GUS fusion revealed the AtACS4 promoter-driven GUS activity in the highly elongating zone of the hypocotyls in response to BR treatment. Furthermore, Arabidopsis plants homozygous for the T-DNA insertion in the AtACS4 gene exhibited longer hypocotyls and roots than those of control seedlings. Taken together, these results suggest that the BR-induced ethylene production may participate in the elongation growth response in early seedling development of Arabidopsis .     

The parasitic mechanism of the holostemparasitic plant Cuscuta

Cuscuta is a stem holoparasitic plant without leaves or roots, which develops a haustorium and sucks nutrients from host plants. The genus Cuscuta comprises about 200 species, many of which can cause severe problems for certain crops. The parasitic process in Cuscuta begins in finding and attaching to a host plant and then developing a haustorium. The process does not always require any chemical signal, but does require a light signal. Finding a host involves detecting the lower red light:far-red light ratio near a potential host plant by phytochrome. A contact signal is also necessary for haustorium induction. Apparently, cytokinin increase is downstream of the light and contact signal and is critical for haustorium induction. This pathway, however, appears to be slightly different from a standard pathway. The direct connection between Cuscuta and its host involves both the xylem and phloem, and mRNA and proteins can translocate. Several features indicate that Cuscuta is a useful model plant for parasite plant research as well as plant–plant interaction research. These include the simple anatomical structure and seedling development, no chemical requirement for haustorium induction, and the wide range of host plants.     

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.     

Parasitic plant small RNA analyses unveil parasite-specific signatures of microRNA retention,loss, and gain

Parasitism is a successful life strategy that has evolved independently in several families of vascular plants. The genera Cuscuta and Orobanche represent examples of the two profoundly different groups of parasites: one parasitizing host shoots and the other infecting host roots. In this study, we sequenced and described the overall repertoire of small RNAs from Cuscuta campestris and Orobanche aegyptiaca. We showed that C. campestris contains a number of novel microRNAs (miRNAs) in addition to a conspicuous retention of miRNAs that are typically lacking in other Solanales, while several typically conserved miRNAs seem to have become obsolete in the parasite. One new miRNA appears to be derived from a horizontal gene transfer event. The exploratory analysis of the miRNA population (exploratory due to the absence of a full genomic sequence for reference) from the root parasitic O. aegyptiaca also revealed a loss of a number of miRNAs compared to photosynthetic species from the same order. In summary, our study shows partly similar evolutionary signatures in the RNA silencing machinery in both parasites. Our data bear proof for the dynamism of this regulatory mechanism in parasitic plants.

MicroRNAs in parasitic plants reflect their lifestyle.     

The GUS reporter-aided analysis of the promoter activities of Arabidopsis ACC synthase genes AtACS4, AtACS5, and AtACS7 induced by hormones and stresses

Ethylene biosynthesis in higher plants is regulated developmentallyand environmentally. To investigate the regulation of ACC synthasegene expression, the promoters of Arabidopsis ACS genes, AtACS4,AtACS5, and AtACS7, were fused to a GUS reporter gene, and therecombinant transgenes were introduced into Arabidopsis to producethree groups of AtACS::GUS transgenic plants. Histochemic andfluorometric study of these transgenic plants revealed thatpromoters of AtACS4, AtACS, and AtACS7 are all active in dark-germinatedseedlings. AtACS5 has the highest promoter activity in leavesof 2-week-old light-grown seedlings among the three AtACS genesstudied. In the mature leaves, AtACS4 and AtACS7 genes are expressedin both veins and areoles, whereas AtACS5 is expressed at ahigher level in the areoles and epidermal cells surroundingtrichomes. The promoter activities of all these AtACS genesare found in the reproductive organs. AtACS5 and AtACS7 arehighly expressed in petals, sepals, carpels, stamens, caulineleaves, inflorescence stems, and siliques, while AtACS4 expressionis undetectable in the petals of open flowers. All three AtACSgenes are expressed in root tissue. In the 2-week-old light-grownArabidopsis, the AtACS4 promoter is responsive to the planthormones IAA, ethylene, and ABA, and to darkness and wounding;the AtACS5 promoter to IAA, ABA, salt, high temperature, andwounding; and the AtACS7 promoter to GA₃, ethylene, and ABA,and to darkness and salt. Low-temperature treatment abolishesthe darkness-induced AtACS7 gene expression, but not that ofAtACS4. Each AtACS gene has a unique expression profile duringgrowth and development. It appears that at any developmentalstage or any growth period of Arabidopsis, there is always amember of AtACS multigene family that is actively expressed. Key words: ACC synthase, Arabidopsis, ethylene, gene expression, GUS histochemical staining, reporter, stress treatments     

Studies on Mature Seeds of Cuscuta pedicellata and C. campestris by Electron Microscopy

The seeds of Cuscuta pedicellata have been investigated by transmissionand scanning electron microscopy. Additional observations havebeen made on seeds of C. campestris by SEM only. The seed coatconsists of an outer single epidermis, two different palisadelayers, and an inner multiparenchyma layer. The outer epidermalwall in C. pedicellata has a thick cuticle and zones rich inpectic substances. The thicker ‘U-shaped’ cell wallsin the outer palisade layer are strengthened by a wall layerof hemicellulose. The inner palisade layer has thick walledcells with a ‘light line’. The inner cell wall ofthe compressed multiparenchyma layer has a thin cuticle. A fairlythick cuticle is positioned directly on the endosperm surface.The aleurone cell walls are different from the remaining endospermwalls. The latter are thick and believed to be of galactomannans.There is a ‘clear’ zone between the plasmalemmaand the cell wall in the aleurone cells. The embryo cells arepacked with lipids and proteins. In Cuscuta campestris mostendosperm has been absorbed during the seed development. Theembryo apex has two minute leaf primordia. The features of theCuscuta seeds are discussed in relation to functional and environmentalconditions. Cuscuta pedicellata, Cuscuta campestris, seed, seed coat, cuticle, cell walls, endosperm, aleurone cells, galactomannan, embryo, TEM, SEM     

Transfer of phloem-mobile substances from the host plants to the holoparasite Cuscuta sp

During the development of the haustorium, searching hyphae of the parasite and the host parenchyma cells are connected by plasmodesmata. Using transgenic tobacco plants expressing a GFP-labelled movement protein of the tobacco mosaic virus, it was demonstrated that the interspecific plasmodesmata are open. The transfer of substances in the phloem from host to the parasite is not selective. After simultaneous application of (3)H-sucrose and (14)C-labelled phloem-mobile amino acids, phytohormones, and xenobiotica to the host, corresponding percentages of the translocated compounds are found in the parasite. An open continuity between the host phloem and the Cuscuta phloem via the haustorium was demonstrated in CLSM pictures after application of the phloem-mobile fluorescent probes, carboxyfluorescein (CF) and hydroxypyrene trisulphonic acid (HPTS), to the host. Using a Cuscuta bridge (14)C-sucrose and the virus PVY(N) were transferred from one host plant to the another. The results of translocation experiments with labelled compounds, phloem-mobile dyes and the virus should be considered as unequivocal evidence for a symplastic transfer of phloem solutes between Cuscuta species and their compatible hosts.