Characterization of two tomato aquaporins and expression during the incompatible interaction of tomato with the plant parasite Cuscuta reflexa

A subtractive suppression hybridization technique was used to identify genes that were induced during early phases of the interaction between Cuscuta reflexa, a phanerogamic plant parasite and the incompatible host tomato (Lycopersicon esculentum Mill.). One of the identified genes encodes a new aquaporin (LeAqp2) from tomato. Its function was concluded from the swelling kinetics of LeAqp2-expressing Xenopus laevis oocytes under hypo-osmotic conditions. It was shown that, 6 h after attachment of the plant parasite, the corresponding mRNA accumulated in cells at and adjacent to the attachment site of Cuscuta, while artificial wounding did not modify steady-state LeAqp2- RNA levels. Expression of a close homologue named TRAMP (tomato-ripening-associated protein) was not affected by the plant-plant interaction. Levels of indole-3-acetic acid (IAA) in tomato tissue after infection by Cuscuta have been found to increase at a similar stage of infection. In contrast to the different behavior with respect to infection, IAA induced both LeAqp2 and TRAMP expression. The observed pattern of LeAqp2 expression during the interaction at a stage where cell elongation occurs together with the water-channel activity in the heterologous expression system suggest a function for LeAqp2 during the tomato-Cuscuta interaction.     

The cell wall-modifying xyloglucan endotransglycosylase/hydrolase LeXTH1 is expressed during the defence reaction of tomato against the plant parasite Cuscuta reflexa

A suppressive subtractive hybridization technique was used to identify genes, which were induced during the early phases of the interaction between dodder (Cuscuta reflexa), a phanerogamic parasite, and its incompatible host plant tomato. One of the identified genes encodes a tomato xyloglucan endotransglycosylase/hydrolase (XTH)--an enzyme involved in cell wall elongation and restructuring. The corresponding LeXTH1 mRNA accumulated 6 h after attachment of the parasite. In contrast, wounding did not influence the expression level. Subsequent to LeXTH1 mRNA accumulation, an increase in XTH activity at the infection sites as well as in adjacent tissues was observed. The effect of IAA on LeXTH1 expression was analyzed because the concentration of this phytohormone is known to increase in the tomato tissue during the interaction with the parasite. LeXTH1 mRNA accumulation was in fact induced by external application of auxin. However, in the auxin-insensitive tomato mutant diageotropica, Cuscuta induced LeXTH1-mRNA accumulated with a time course similar to wild type tomato. Thus, auxin appears not to be an essential signal for infection-induced LeXTH1 activation. Our data suggest a role for xyloglucan transglycosylation in defence reactions associated with the incompatible tomato- Cuscuta interaction.     

An attack of the plant parasite Cuscuta reflexa induces the expression of attAGP, an attachment protein of the host tomato

Dodder or Cuscutaceae are holoparasitic plants subsisting on other dicotyledonous plants. The infection process is initiated by adherence of Cuscuta prehaustoria to the host surface, followed by penetration attempts by hyphae. In the case of a successful infection, these organs connect the parasite's vascular tissue to that of the host. Here we show that contact of Cuscuta reflexa prehaustoria to tomato induces the expression of a new arabinogalactan protein (AGP), attAGP, in the tomato precisely at the site of dodder attack. We show that attAGP is a plasma membrane-bound cell wall-localized protein. Using the RNAi technique and attAGP-targeted virus-induced gene silencing, we observed a correlation between attAGP expression level and force of attachment of the parasite to host tomatoes. If the expression level of attAGP was reduced, the C. reflexa attachment capability was significantly reduced, too. We conclude that C. reflexa infection induced a signal in the host leading to expression of tomato attAGP, which promotes the parasite's adherence.     

Anatomy and Phenylpropanoid Metabolism in the Incompatible Interaction of Lycopersicon esculentum and Cuscuta reflexa

A time-dependent correlation of anatomical and chemical defence reactions was shown during the incompatible reaction of tomato against the phanerogamic parasite Cuscuta reflexa. Microscopical analysis of the infection sites at the tomato stem revealed the elongation of epidermal, hypodermal and collenchymatic cells beneath the parasitic prehaustorium. After 9–11 days of infection the elongated cells had collapsed forming a visible brownish plaque at the tomato stem followed by a scalariform tissue with lignified and suberized cell walls. Concomitantly, an enhanced accumulation of soluble phenolic compounds (chlorogenic acid and an unidentified hydroxycinnamic acid derivative), as well as a stimulation of peroxidases, was observed. In contrast, PAL activity was not increased. Whereas the stimulation of phenylpropanoid metabolism could also be induced by artificial wounding, the described anatomical changes were only observed during attack of Cuscuta.     

A role for IAA in the infection of Arabidopsis thaliana by Orobanche aegyptiaca

BACKGROUND: Vascular continuity is established between a host plant and the root parasite broomrape. It is generally accepted that the direction of vascular continuity results from polar flow of auxin. Our hypothesis was that chemical disruptions of auxin transport and activity could influence the infection of the host by the parasite. METHODS: A sterile system for the routine infection of Arabidopsis thaliana seedlings in Nunc cell culture plates by germinated seeds of Orobanche aegyptiaca was developed. This method permitted a quantitative assay of the rate of host infection. The three-dimensional structure of the vascular contacts was followed in cleared tissue. IAA (indole acetic acid) or substances that influence its activity and transport were applied locally to the host root. RESULTS: The orientation of the xylem contacts showed that broomrape grafts itself upon the host by acting hormonally as a root rather than a shoot. Local applications of IAA, PCIB (p-chlorophenoxyisobutyric acid) or NPA (naphthylphthalamic acid) all resulted in drastic reductions of Orobanche infection CONCLUSIONS: Broomrape manipulates the host by acting as a sink for auxin. Disruption of auxin action or auxin flow at the contact site could be a novel basis for controlling infection by Orobanche.     

Morphological and anatomical features of Cuscuta pedicellata and C. campestris

Observations on morphological and anatomical features of Cuscuta pedicellata and, to a lesser extent, C. campestris have been made at different stages of their life-cycles. The seedling is filiform with a bulbous radicular end which collapses early. The simple shoot apex is green and photosentitive. Initial "nutation" is followed by "creeping" over the substrate. Host plants less than about three weeks old seem not to be attacked by Cuscuta seedlings. When haustorial contact is established the shoot apex differentiates into buds and scale leaves. The chlorophyll disappears and stomata, which are lacking in the seedling, develop. The apical growth rate increases strongly by frequent cell division and - elongation. This is reflected anatomically by the formation of "cell families" in rows. Weakening and flowering of the host plants are followed by flowering of the parasite. Greening of stem segments of Cuscuta occurs during formation of haustoria and after death of the host. Stem anatomy is characterized by a tripartite cortex in which the middle layer consists of thin-walled angular cells. Characteristic are also the voluminuous intercellular spaces, the cortical and perivascular laticifers, and the central position of the small vascular bundles. Observations on self-parasitism were made. The epidermis of the haustorial cushion elongates and the cytoplasm is mainly positioned towards the host. Pseudo-intercellular spaces are formed between the epidermal cells. The haustorium connects the phloem of the two plants, but vascular elements of any kind have not been observed.     

Studies on Cuscuta reflexa ROXB. V. The level of endogenous hormones in the parasite,Cuscuta reflexa,and its host,Vicia faba L., and a suggested role in the transfer of nutrients from host to parasite

Summary The mechanism of parasitism of Cuscuta, especially the absorption of nutrients from its host, is not clear. As it might be connected with the function of plant hormones, the endogenous levels of all hormone groups in the parasite, Cuscuta reflexa, and its host, Vicia faba, were investigated. Since the content of auxins, gibberellins and cytokinins is higher in the host than in the parasite, there is no indication that any of these phytohormones is involved in the absorption of nutrients by Cuscuta. However, the content of growth inhibitors, especially free abscisic acid, is much higher in Cuscuta than in the host. There is a gradient of abscisic acid with a maximum in the basal, haustoria-bearing stem region in which the transfer of nutrients from host to parasite occurs. The high content of abscisic acid within the parasite may be a causal connection with the parasitic absorption of nutrients from host sieve tubes.This publication is respectfully dedicated to the academician Prof. Dr. A. L. Kursanov, Moscow, on the occasion of the 80th anniversary of his birthday.     

Defense response of resistant host Impatiens balsamina to the parasitic angiosperm Cuscuta japonica

The response of the stem of a resistant host (Impatiens baslamina) to infection by the parasitic flowering plant Cuscuta japonica was studied with light and electron microscopy. The intra- and interfascicular cambial cells in the host stem first reacted to the penetrating upper haustorium by dividing, and the differentiation of the host xylem (vascular) tissues proceeded toward interfascicular areas from vascular bundles. When the host vascular tissue was invaded by the endophyte (haustorial portion in the host stem), the host xylem was displaced, and host vessels became occluded with parenchyma cells, resulting in tyloses. As the parasitism progressed, areas of the host stem penetrated by the endophyte became swollen via secondary growth and cell division in the parenchymatous cortex, pith, and interfascicular areas. During this intrusion by the endophyte, darkly stained necrotic reactions were detected at the interface between the host tissue and the invading endophyte. The results suggested that in the host tissues penetrated by the parasite, the formation of secondary tissue and swellings caused by active cell division of ground tissue and host vessel occlusion by tyloses constitute the host structural defense against the parasite.     

水稻茎伸长生长与植物激素

赤霉素（GA）,生长素（IAA）,脱落酸（ABA）和乙烯影响水稻茎（或节间）的伸长,其中赤霉素与水稻茎伸长生长的关系最密切。GA1是植物体内刺激茎伸长的至关重要的赤霉素,GA3已作为最常用的外源激素诱导水稻的节间伸长。水稻茎秆的伸受激素浓度和敏感性的双重控制,激素浓度或敏感性任一方的改变都有可能导致株高的变异。赤霉素如此显著地促进茎的伸长可能与增加细胞分裂和促使细胞壁松弛有关。而生长素主要促进细胞伸长。植物激素促进水稻茎长的分子机理的研究已有较大的进展,预期这方面的研究和应用在未来几年内将有新的突破。     

水稻茎伸长生长与植物激素

赤霉素（GA）、生长素（IAA）、脱落酸（ABA）和乙烯影响水稻茎（或节间）的伸长,其中赤霉素与水稻茎伸长生长的关系最密切。GA1是植物体内刺激茎伸长的至关重要的赤霉素, GA3已作为最常用的外源激素诱导水稻的节间伸长。水稻茎秆的伸长受激素浓度和敏感性的双重控制,激素浓度或敏感性任一方的改变都有可能导致株高的变异。赤霉素如此显著地促进茎的伸长可能与增加细胞分裂和促使细胞壁松弛有关。而生长素主要促进细胞伸长。植物激素促进水稻茎伸长的分子机理的研究已有较大的进展,预期这方面的研究和应用在未来几年内将有新的突破。     

Structure and Development of the Endophyte in the Parasitic Angiosperm <Emphasis Type="Italic">Cuscuta japonica</Emphasis>

The endophyte, that is, the haustorial part within the tissues of the host plant Impatiens balsamina, of the parasitic angiosperm Cuscuta japonica was studied with light and electron microscopy. The endophyte consisted mainly of vacuolated parenchymatous axial cells and elongate, superficial (epidermal) cells. Then the elongate, epidermal cells separated from each other and transformed into filamentous cells, called searching hyphae. The hyphae grew independently either intercellularly or intracellularly in the host parenchyma. The apical end of the hyphal cells was characterized by conspicuous, large nuclei with enlarged nucleoli and very dense cytoplasm with abundant organelles, suggesting that the hyphal cells penetrating host tissue were metabolically very active. Numerous osmiophilic particles and chloroplasts were noted in the hyphae. The osmiophilic particles were assumed to be associated with elongation of the growing hyphe. Plasmodemata connections between the searching hyphal cells of the parasite and the host parenchyma cells were not detected. Hyphal cells that reached the host xylem differentiated into water-conducting xylic hyphae by thickening of the secondary walls. A xylem bridge connecting the parasite and the host was confirmed from serial sections. Some hyphal cells that reached the host phloem differentiated into nutrient-conducting phloic hyphae. Phloic hyphae had a thin layer of peripheral cytoplasm with typical features of sieve-tube members in autotrophic angiosperms, i.e., parallel arrays of smooth endoplasmic reticulum, mitochondria, and plastids with starch granules. Interspecific open connections via the sieve pores of the host sieve elements and plasmodesmata of the parasite phloic hyphae were very rarely observed, indicating that the symplastic translocation of assimilate to the parasite from the host occurred.     

Biochemical Aspects of Parasitism by the Angiosperm Parasites: Starch Accumulation

The investigation dealt with starch accumulation in four species of Cuscuta (Cuscuta campestris, C. indecora, C. planiflora and C. reflexa), a leafy mistletoe (Dendrophthoe falcata) and a chlorophyll-lacking root parasite (Orobanche aegyptiaca). The highest content of starch occurred In O. aegyptiaca, with a maximum of 45 per cent of dry weight Starch in Cuscuta filaments and mistletoe leaves showed a maximum of about 10 per cent of dry weight. The starch content varied along the length of the Cuscuta vine, with a maximum in the apical region. Orobanche had a higher starch content when it was still submerged than it was fully developed. Cuscuta vines did not show any marked diurnal alteration in the starch content. The content of ethanol-soluble carbohydrate was only a tenth of the starch in Orobanche, but was relatively higher in the other parasites. the neutral sugars in Cuscuta filaments were sucrose and glucose, whereas fructose was also present in mistletoe and Orobanche. Raffinose and stachyose were absent or present only ill traces in parasite tissue. Starch granules from Cuscuta and Orobanche bad ADPG/UDPG-starch synthetase activity and homogenates starch phosphorylase activity. The former enzyme appeared to be responsible for synthesis of starch and the latter for utilization. The four different species of Cuscnta, growing on alfalfa, had more or less the same activity of starch synthetase and also of phosphorylase activity. Hosts infected by Cuscuta had significantly less starch per plant than the controls. A characteristic feature of invasion by Cuscuta and Orobanche was increased phosphorylase activity in the host tissues. The protein content of the tissues of Cuscuta and Orobanche was of a lower level than that of the host shoot system or foliage, indicating that the parasite differed from the host in having a higher carbon (of starch) to nitrogen (of protein) ratio.     

Structure and development of the upper haustorium in the parasitic flowering plant Cuscuta japonica (Convolvulaceae)

The anatomical and ultrastructural development of the haustorium of the Cuscuta japonica, a holoparasitic angiosperm, growing on the host plant Impatiens balsamina was studied. After the shoot tips of light-grown parasite seedlings contacted the host, the upper haustorium (external to the host organ) developed through three main successive stages of the haustorial initials, the meristem, and the endophyte primoridium (EP) within the middle layer of the cortex of the parasite stem. The haustorial initial cells were characterized by abundant starch-bearing amyloplasts and mitochondria with an expanded intermembrane space. The meristem cells had numerous large chloroplasts with well-developed thylakoids, reflecting the capability for photosynthesis. Commonly, all three stages of haustorial cells contained conspicuous, large nuclei with enlarged nucleoli and dense cytoplasm including many other organelles, indicating a very active metabolism. In the final stage of upper haustorium development, the meristem cells differentiated into the EP, a host-penetrating tissue. The primordium had smaller file cells at the proximal end and elongate digitate cells at the distal end. The file cells divided actively, while the digitate cells contained abundant chloroplasts, dictyosomes, rough endoplasmic reticulum, and other organelles, suggesting that the EP was cytohistologically well organized for penetration into the host tissue.     

Macromolecular trafficking between Nicotiana tabacum and the holoparasite Cuscuta reflexa

Transgenic tobacco plants expressing green fluorescent protein (GFP) under the control of the companion cell-specific promoter, AtSUC2, were parasitized by the holoparasite Cuscuta reflexa (dodder). GFP, moving in the translocation stream of the host, was transferred to the Cuscuta phloem via the absorbing hyphae of the parasite. An identical pattern of transfer was observed for the phloem-mobile probe, carboxyfluorescein. Following uptake by the parasite, GFP was translocated and unloaded from the Cuscuta phloem in meristematic sink tissues. Contrary to published data, these observations suggest the presence of a functional symplastic pathway between Cuscuta and its hosts, and demonstrate a considerable capacity for macromolecular exchange between plant species.     

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.     

Influence of Shading on the Gross Composition of Cuscuta Species on Medicago sativa

A study was made of the alterations in growth characteristics of Cuscuta Campestris, Cuscuta indecora and Cuscuta reflexa on Medicago sativa as a consequence of a limitation in light intensity. There was a decrease in the content of dry solids, protein and total phosphorus in the filament mass. A decrease in these parameters occurred also in the tissues of the host, but was less pronounced that in the parasite. The response by the host to parasite growth was not always the same in the shade as in light. Phosphorus showed a distinct tendency toward firm retention in the infection complex in the dark.     

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     

Interaction of Gibberellic Acid and Indole-3-acetic Acid in the Growth of Excised Cuscuta Shoot Tips in Vitro

Gibberellic acid (GA₃) induced a marked elongation of 2.5-centimeter shoot tips of Cuscuta chinensis Lamk. cultured in vitro. In terms of the absolute amount of elongation, this growth may be the largest reported for an isolated plant system. The response to hormone was dependent on an exogenous carbohydrate supply. The hormone-stimulated growth was due to both cell division and cell elongation. The growth response progressively decreased if GA₃ was given at increasingly later times after culturing, but the decreased growth response could be restored by the application of indole-3-acetic acid (IAA) to the apex. Explants deprived of GA₃ gradually lost their ability to transport IAA basipetally, but this ability was also restored by auxin application. The observations are explained on the basis that: (a) the growth of Cuscuta shoot tip in vitro requires, at least, both an auxin and a gibberellin; and (b) in the absence of gibberellin the cultured shoot tip explants lose the ability to produce and/or transport auxin.     

Resistance and tolerance in a host plant-holoparasitic plant interaction: genetic variation and costs

Host organisms are believed to evolve defense mechanisms (i.e., resistance and/or tolerance) under selective pressures exerted by natural enemies. A prerequisite for the evolution of resistance and tolerance is the existence of genetic variation in these traits for natural selection to act. However, selection for resistance and/or tolerance may be constrained by negative genetic correlations with other traits that affect host fitness. We studied genetic variation in resistance and tolerance against parasitic infection and the potential fitness costs associated with these traits using a novel study system, namely the interaction between a flowering plant and a parasitic plant. In this system, parasitic infection has significant negative effects on host growth and reproduction and may thus act as a selective agent. We conducted a greenhouse experiment in which we grew host plants, Urtica dioica, that originated from a single natural population and represented 20 maternal families either uninfected or infected with the holoparasitic dodder, Cuscuta europaea. that originated from the same site. We calculated correlations among resistance, tolerance, and host performance to test for costs of resistance and tolerance. We measured resistance as parasite performance (quantitative resistance) and tolerance as the slopes of regressions relating the vegetative and reproductive biomass of host plants to damage level (measured as parasite biomass). We observed significant differences among host families in parasite resistance and in parasite tolerance in terms of reproductive biomass, a result that suggests genetic variation in these traits. Furthermore, we found differences in resistance and tolerance between female and male host plants. In addition, the correlations indicate costs of resistance in terms of host growth and reproduction and costs of tolerance in terms of host reproduction. Our results thus indicate that host tolerance and resistance can evolve as a response to infection by a parasitic plant and that costs of resistance and tolerance may be one factor maintaining genetic variation in these traits.