Biochemical Aspects of Parasitism by the Angiosperm Parasites: Host-parasite Interrelationship in Phosphatase Activity

Infection by Cuscuta and Orobanche causes significant losses in dry solids and protein content in host plant or plant part. Changes occur in phosphatase activity towards fructose-1,6-diphosphate at alkaline pH and β-glycerophosphate at acid pH, expressed per mg protein or g fresh tissue. The leaves of all hosts infected by Orobanche show an increase in the alkaline fructosediphosphatase activity, whereas as far as the infection by Cuscuta is concerned the general response is a decrease in the enzyme in the shoots. The alterations in the phosphatase activity towards β-glycerophosphate at acid pH in the shoots are not consistent. However, there is a marked increase in the acid phosphatase activity against β-glycerophosphatase in the roots of the infected hosts. The significance of these findings has been discussed in the light of host-parasite interrelationship.     

The carbohydrate and water balance of beans (Vicia faba) attacked by broomrape (Orobanche crenata)

Orobanche crenata parasitizing beans maintained a slightly higher osmotic pressure than the bean roots, largely because of the higher concentration of sugars in the broomrape tissues. The sugar was withdrawn from the bean mainly as sucrose, which was hydrolysed to glucose and fructose by the Orobanche. These sugars were then rapidly translocated to the developing flower spike. As well as maintaining a high osmotic pressure this hydrolysis ensured a sucrose concentration gradient between host and parasite. In the field, bean plants showed wilt symptoms at about the time that the Orobanche flower spikes emerged. It was found that the higher the level of infection the lower was the water content of the host. This fall in water content was not due to increased water loss by the bean shoots and it seemed unlikely that it was due to water removal by the parasite. It was concluded that the death of the bean was due to desiccation brought about largely by the reduced ability of the carbohydrate-starved roots to extract water from the soil.     

Dehydrocostus lactone is exuded from sunflower roots and stimulates germination of the root parasite Orobanche cumana

The germination of the obligate root parasites of the Orobanchaceae depends on the perception of chemical stimuli from host roots. Several compounds, collectively termed strigolactones, stimulate the germination of the various Orobanche species, but do not significantly elicit germination of Orobanche cumana, a specific parasite of sunflower.Phosphate starvation markedly decreased the stimulatory activity of sunflower root exudates toward O. cumana, and fluridone - an inhibitor of the carotenoid biosynthesis pathway - did not inhibit the production of the germination stimulant in both shoots and roots of young sunflower plants, indicating that the stimulant is not a strigolactone.We identified the natural germination stimulant from sunflower root exudates by bioassay-driven purification. Its chemical structure was elucidated as the guaianolide sesquiterpene lactone dehydrocostus lactone (DCL). Low DCL concentrations effectively stimulate the germination of O. cumana seeds but not of Phelipanche aegyptiaca (syn. Orobanche aegyptiaca). DCL and other sesquiterpene lactones were found in various plant organs, but were previously not known to be exuded to the rhizosphere where they can interact with other organisms.     

Nitrogen and carbon relationships between the parasitic weed Orobanche foetida and susceptible and tolerant faba bean lines

The parasitic weed Orobanche foetida (Poiret) is an emergent agronomical problem on faba bean in Tunisia. The Tunisian breeding programs for faba bean resistance to O. foetida have produced several tolerant lines including the line XBJ90.03-16-1-1-1, which limits both parasite attachments to the host roots and growth of the attached parasites. The present study aims to provide a better understanding of the nutritional relationships between the parasite and this tolerant line in comparison with the susceptible Bachaar genotype. Phloem saps of faba bean were harvested using phloem exudation experiments. The major organic compounds potentially transferred from both faba bean genotypes to the parasite were identified as sucrose, raffinose, stachyose, citrate, malate, asparagine (ASN), aspartate (ASP), glutamine, glutamate, serine, alanine and GABA. However, the phloem exudates of the tolerant line were highly deficient in nitrogen when compared to that of the susceptible line. When attached to roots of the tolerant line, the parasite displayed limited activities of soluble invertases in tubercles, and especially in shoots, suggesting that the low performance of the broomrapes attached to the tolerant line resulted from a reduced capacity to utilize the host-derived carbohydrates. On the other hand, the mechanisms involved in the osmotic adjustment and primary metabolism of the parasite did not differ significantly according to the host genotype: mineral cations, especially potassium and calcium, predominated as the major osmotically-active compounds in both tubercles and shoots; shoots accumulated preferentially hexoses as organic solutes although tubercles accumulated preferentially starch and soluble amino acids, especially ASP and ASN. This suggests an important role for a glutamine-dependent asparagine synthetase (EC 6.3.5.4) in the N metabolism of the parasite.     

Sequestration of polyacetylenes by the parasite Orobanche hederae (Orobanchaceae) from its host Hedera helix (Araliaceae)

Orobanche hederae Duby and its host Hedera helix L. were collected in the North Italian Trentino-Südtirol region and analyzed for their content of polyacetylenes. Both, the host plant (Hedera) and the parasite (Orobanche) contained the polyacetylene falcarinol and two of its dehydroderivatives. The contents of polyacetylenes in Hedera decrease from roots via stems to leaves and contents in Orobanche are only about one-tenth of the contents in the Hedera roots it parasites on. Moreover, relative contents of the more polar polyacetylenes are higher in Orobanche than in Hedera, implying a bias toward the sequestration of more polar compounds.     

Host plant resistance against broomrapes (Orobanche spp.): defence reactions and mechanisms of resistance

Over 4000 species of angiosperms are able to directly invade and parasitise other plants, but only very few are weedy and parasitise cultivated plants. Together with the witchweeds (Striga spp.) and dodders (Cuscuta spp.), the broomrapes (Orobanche spp.) affect important crops causing complete yield loses with severe infestations. Genetic resistance to parasitisation remains as one of the most desirable components in an integrated control strategy. However, breeding for resistance is a difficult task and many aspects of the host/parasite interaction remain unknown. In the present work, we review the cytological and cytochemical studies investigating the mechanisms of resistance involved in the process leading to an incompatible host–parasite interaction. We have attempted to identify the main gaps and problems related to such studies with parasitic plants and present an in‐depth review of histochemical techniques used to assess mechanisms of resistance against these parasitic plants. Furthermore, we discuss future research directions and novel techniques and finally, how such techniques can be applied and incorporated within a breeding programme.     

Host-specific races in the holoparasitic angiosperm Orobanche minor: implications for speciation in parasitic plants

Background and Aims

Orobanche minor is a root-holoparasitic angiosperm that attacks a wide range of host species, including a number of commonly cultivated crops. The extent to which genetic divergence among natural populations of O. minor is influenced by host specificity has not been determined previously. Here, the host specificity of natural populations of O. minor is quantified for the first time, and evidence that this species may comprise distinct physiological races is provided.

Methods

A tripartite approach was used to examine the physiological basis for the divergence of populations occurring on different hosts: (1) host–parasite interactions were cultivated in rhizotron bioassays in order to quantify the early stages of the infection and establishment processes; (2) using reciprocal-infection experiments, parasite races were cultivated on their natural and alien hosts, and their fitness determined in terms of biomass; and (3) the anatomy of the host–parasite interface was investigated using histochemical techniques, with a view to comparing the infection process on different hosts.

Key Results

Races occurring naturally on red clover (Trifolium pratense) and sea carrot (Daucus carota ssp. gummifer) showed distinct patterns of host specificity: parasites cultivated in cross-infection studies showed a higher fitness on their natural hosts, suggesting that races show local adaptation to specific hosts. In addition, histological evidence suggests that clover and carrot roots vary in their responses to infection. Different root anatomy and responses to infection may underpin a physiological basis for host specificity.

Conclusions

It is speculated that host specificity may isolate races of Orobanche on different hosts, accelerating divergence and ultimately speciation in this genus. The rapid life cycle and broad host range of O. minor make this species an ideal model with which to study the interactions of parasitic plants with their host associates.Key words: Parasitic plant, Orobanche, speciation, divergence, host-specificity, host-specific races     

Pectolytic Activity by the Haustorium of the Parasitic PlantOrobancheL. (Orobanchaceae) in Host Roots

The possible involvement of enzymes in the penetration of intrusivecells of the parasitic angiospermOrobancheinto host root tissueswas studied using cytochemical and immunocytochemical methods.Pectin methyl esterase (PME) was detected, with specific antibodies,in the cytoplasm and cell walls ofOrobancheintrusive cells andin adjacent host apoplast. Depletion and chemical changes ofpectins in host cell walls were shown by histochemical stainingwith PATAg, which detects carbohydrates that are sensitive toperiodic acid, especially pectins, and with the monoclonal antibodiesJIM 5 and JIM 7 that label pectins with low and high rates ofesterification, respectively. Galacturonic sequences with lowrates of esterification were more abundant in host cell wallsadjacent to the parasite, which is consistent with pectin de-methylationby PME release from the parasite. Pectins were absent in middlelamellae and in host cell walls neighbouring mature intrusivecells of the parasite, consistent with further degradation ofpectins by other enzymes. These results provide the first directevidence for the presence and activity of a pectolytic enzymein the infection zone of the haustorium of a parasitic angiosperminsitu.Copyright 1998 Annals of Botany Company Broomrape;Orobanche; parasitic weed; haustorium; pectin methyl esterase; pectin; cell wall.     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> transformed calli of the holoparasitic plant <Emphasis Type="Italic">Phelipanche ramosa</Emphasis> maintain parasitic competence

Phelipanche and Orobanche spp. (broomrapes) are economically important parasitic weeds, causing severe damage to many agricultural crops. However, conventional methods to control these parasitic weeds are often not effective. Targeting molecular and biochemical processes involved in the establishment of the connection between the parasite and the host may offer a new perspective for control. However, progress in the understanding of these processes is hampered by the fact that genetic transformation and regeneration of these parasites is difficult if not impossible due to their specific lifecycle. Phelipanche and Orobanche spp. are holoparasites that need to attach to the roots of a host plant to get their assimilates, nutrients and water to develop and reproduce. The present study describes a highly efficient genetic transformation and regeneration protocol for the root holoparasitic Phelipanche ramosa. We present a new transformation system for P. ramosa using Agrobacterium rhizogenes MSU440 carrying a non-destructive selection marker gene coding for a red fluorescent protein (DsRed1). Using this protocol up to 90% transformation efficiency was obtained. We transformed 4 weeks old P. ramosa calli and transgenic calli expressing DsRed1 were then cultured on host plants. For the first time, we present shoot and flower development of the transgenic parasitic plant P. ramosa after successful connection of transgenic calli with the host plant roots. Moreover, we also present, for the first time, growth and development of P. ramosa shoots and flowers in vitro in the absence of a host plant.     

Schistosoma mansoni: Lysozyme activity in Biomphalaria glabrata during infection with two strains

Levels of lysozyme activity were determined in the hemolymph, digestive gland, and headfoot extracts of M-line stock of snails, Biomphalaria glabrata, during infection with the PR-1 and Lc-1 strains of the trematode, Schistosoma mansoni. At 3 hr postexposure there was a 10-fold increase in the levels of enzyme activity in the hemolymph of snails infected with the Lc-1 strain to which the snail is resistant. This increase was considerably higher when compared to the threefold increase in the PR-1-infected snails. The infection also induced a gradual depletion of lysozyme activity in the headfoot muscles of the two groups of infected snails. There were no changes in the levels of enzyme activity in the digestive gland extracts of the control and the two groups of infected snails. Similar changes in the levels of enzyme activity in the hemolymph and headfoot extracts of infected snails suggest a nonspecific response to a parasite infection and do not indicate that lysozyme is primarily responsible for the destruction of schistosome parasite in a resistant snail host.     

Chlamydia trachomatis Relies on Autonomous Phospholipid Synthesis for Membrane Biogenesis

The obligate intracellular parasite Chlamydia trachomatis has a reduced genome and is thought to rely on its mammalian host cell for nutrients. Although several lines of evidence suggest C. trachomatis utilizes host phospholipids, the bacterium encodes all the genes necessary for fatty acid and phospholipid synthesis found in free living Gram-negative bacteria. Bacterially derived phospholipids significantly increased in infected HeLa cell cultures. These new phospholipids had a distinct molecular species composition consisting of saturated and branched-chain fatty acids. Biochemical analysis established the role of C. trachomatis-encoded acyltransferases in producing the new disaturated molecular species. There was no evidence for the remodeling of host phospholipids and no change in the size or molecular species composition of the phosphatidylcholine pool in infected HeLa cells. Host sphingomyelin was associated with C. trachomatis isolated by detergent extraction, but it may represent contamination with detergent-insoluble host lipids rather than being an integral bacterial membrane component. C. trachomatis assembles its membrane systems from the unique phospholipid molecular species produced by its own fatty acid and phospholipid biosynthetic machinery utilizing glucose, isoleucine, and serine.     

Morphological and plant hormonal changes during parasitization by Cuscuta japonica on Momordica charantia

The holostemparasitic plant Cuscuta parasitizes various plants and sucks nutrients from the host stem. We used Cuscuta japonica as the parasite and Momordica charantia as the host plant, and described their interaction. The parasitized Momordica stems started swelling as a hypertrophic response within 3 days after parasitization. Concurrently, the Cuscuta stem grew rapidly and developed bigger scale leaves than usual. Parasitized Momordica stems reduced photosynthetic activity. Histological observation revealed no programmed cell death but an increased number of vascular bundles in the Momordica stem, especially near the Cuscuta hyphae. The defensive response of Momordica mainly involved the SA pathway. Drastic increase of tZ- and DZ-type cytokinins in Momordica stems would play an important role for hypertrophy. Cuscuta had higher cZ endogenously and our results imply that each subtype of CK might play different roles during parasitization process. Comprehensive plant hormone analysis provides new insights into plant interaction studies.     

Comparison of phospholipid effects on insulin-sensitive low Km cyclic AMP phosphodiesterase in adipocyte plasma membranes and microsomes

Both adipocyte plasma membranes and microsomes possess insulin-sensitive low K_m cyclic AMP phosphodiesterase activity. The activity of the enzyme from both sources was susceptible to activation by several anionic phospholipids. Activators of the plasma membrane enzyme were lysophosphatidylglycerol > lysophosphatidylcholine > lysophosphatidylserine > phosphatidylserine > phosphatidylglycerol. These same phospholipids activated the microsomal enzyme but the extent of activation by each phospholipid was reversed. Neutral phospholipids and other anionic phospholipids were without effect. The phospholipids had no effect on high K_m cAMP phosphodiesterase in either membrane. The results suggest that the phospholipid headgroup was an important determinant for enzyme activation by phospholipid. The increased susceptibility of the plasma membrane enzyme to lysophospholipid may be attributed to a difference in the plasma membrane enzyme compared to the microsomal membrane enzyme or to differences in plasma membrane and microsomal membrane phospholipid composition and their ability to regulate low K_m cAMP phosphodiesterase activity.     

Causes of root growth retardation induced by ultraviolet-B irradiation of shoots in Barley seedlings

In barley seedlings (Hordeum vulgare L.) during two days after irradiation of shoots with UV-B (0.5 W/m², 6 h), the rate of elongation of primary roots decreased 2–3 times compared to that in control plants. The modulus of elasticity of roots (ε) increased at most twofold in 12 h after the onset of irradiation; the hydraulic conductivity (L _p) diminished by a factor of two in 12 h, and the root osmotic pressure gradually decreased by 0.08 MPa in 24 h. Changes in ε and L _p were shown to be related to oxidative stress in growing roots, which was evidenced from the increase in H₂O₂ level up to 15-fold increase in 6 h and in activity of guaiacol peroxidase (3.5-fold in 12 h). After 48 h, the characteristics of oxidative metabolism and root characteristics ε and L _p became identical in untreated and treated plants. On the third day, the rate of root growth in treated plants reached its initial value. It is concluded that the main causes of retardation of root growth under these conditions were as follows: the increase in cell wall rigidity related to formation of oxidative cross-links in the apoplast and the decrease in root osmotic pressure due to limited transport of assimilates from irradiated leaves. After the intensity of UV-B irradiation applied to shoots was enhanced (1.6 W/m², 4 h), another physiological status of roots was observed on the 2nd day characterized by twofold increase in L _p, tenfold decreased root elongation rate, and by a progressing increase of root diameter in growing roots. The comparison of root responses induced by irradiation of shoots with the root responses to sodium salicylate and ABA suggests that both agents might participate in the transmission of signals from irradiated leaves to roots.     

Sink-stimulated photosynthesis and sink-dependent increase in nitrate uptake: nitrogen and carbon relations of the parasitic association Cuscuta reflexa–Ricinus communis

Ricinus communis L. was grown under limiting N supply in quartz sand culture, fed with 0.2, 1 or 5 mol m^?3 NO₃^?, or in liquid culture with 0.022, 0.05 or 0.5 mol m^?3 NO₃^?. Some of the plants were infected with Cuscuta reflexa Roxb. As occurred for the host, dry matter production and growth of C. reflexa were severely depressed with decreasing N supply to the host. When parasitized by C. reflexa, the shoot and root dry weight of Ricinus was diminished at all levels of N nutrition, but the total dry weight of host plus parasite was almost the same as that of uninfected Ricinus. In contrast to the situation in Lupinus albus (Jeschke et al. 1994b), infection by Cuscuta resulted in increased tissue N levels in the host and the N content of the system Ricinus plus C. reflexa was the same or even somewhat larger than that of uninfected plants. This indicated a sink-dependent stimulation of nitrate uptake. As a result of decreased root weights, nitrate uptake g^?1 FW was stimulated by 80, 60 or only 40% at 0.2, 1 or 5 mol m^?3 nitrate supply. Increased nitrate uptake was reflected, particularly at low N supply, in xylem transport; xylem sap nitrate concentrations were substantially elevated, while those of amino acids were decreased in parasitized plants. This indicated an inhibition of nitrate assimilation in roots of parasitized plants under limiting N supply. Besides these effects on N relations, C. reflexa induced a substantial sink-dependent stimulation of net photosynthesis in host leaves and a concomitant increase in stomatal opening and transpiration. This stimulation depended on the relative sink size induced by Cuscuta, on nitrogen nutrition and on leaf age, indicating that delayed senescence of leaves contributes to the overall effects of Cuscuta on its host. The Cuscuta-induced inhibition of nitrate assimilation in the roots and the increase in nitrate uptake suggest that nitrate reduction was shifted towards the leaves in the presence of C. reflexa. The stimulating effects of C. reflexa in the Ricinus-Cuscuta association are compared with the strongly inhibitory effects occurring in the tripartite association L. albus–Rhizobium–Cuscuta reflexa.     

Carotenoid inhibitors reduce strigolactone production and Striga hermonthica infection in rice

The strigolactones are internal and rhizosphere signalling molecules in plants that are biosynthesised through carotenoid cleavage. They are secreted by host roots into the rhizosphere where they signal host-presence to the symbiotic arbuscular mycrorrhizal (AM) fungi and the parasitic plants of the Orobanche, Phelipanche and Striga genera. The seeds of these parasitic plants germinate after perceiving these signalling molecules. After attachment to the host root, the parasite negatively affects the host plant by withdrawing water, nutrients and assimilates through a direct connection with the host xylem. In many areas of the world these parasites are a threat to agriculture but so far very limited success has been achieved to minimize losses due to these parasitic weeds. Considering the carotenoid origin of the strigolactones, in the present study we investigated the possibilities to reduce strigolactone production in the roots of plants by blocking carotenoid biosynthesis using carotenoid inhibitors. Hereto the carotenoid inhibitors fluridone, norflurazon, clomazone and amitrole were applied to rice either through irrigation or through foliar spray. Irrigation application of all carotenoid inhibitors and spray application of amitrole significantly decreased strigolactone production, Striga hermonthica germination and Striga infection, also in concentrations too low to affect growth and development of the host plant. Hence, we demonstrate that the application of carotenoid inhibitors to plants can affect S. hermonthica germination and attachment indirectly by reducing the strigolactone concentration in the rhizosphere. This finding is useful for further studies on the relevance of the strigolactones in rhizosphere signalling. Since these inhibitors are available and accessible, they may represent an efficient technology for farmers, including poor subsistence farmers in the African continent, to control these harmful parasitic weeds.     

A peptide from insects protects transgenic tobacco from a parasitic weed

Parasitic plants present some of the most intractable weed problems for agriculture in much of the world. Species of root parasites such as Orobanche can cause enormous yield losses, yet few control measures are effective and affordable. An ideal solution to this problem is the development of parasite-resistant crops, but this goal has been elusive for most susceptible crops. Here we report a mechanism for resistance to the parasitic angiosperm Orobanche based on expression of sarcotoxin IA in transgenic tobacco. Sarcotoxin IA is a 40-residue peptide with antibiotic activity, originally isolated from the fly, Sarcophaga peregrina. The sarcotoxin IA gene was fused to an Orobanche-inducible promoter, HMG2, which is induced locally in the host root at the point of contact with the parasite, and used to transform tobacco. The resulting transgenic plants accumulated more biomass than non-transformed plants in the presence of parasites. Furthermore, plants expressing sarcotoxin IA showed enhanced resistance to O. aegyptiaca as evidenced by abnormal parasite development and higher parasite mortality after attachment as compared to non-transformed plants. The transgenic plants were similar in appearance to non-transformed plants suggesting that sarcotoxin IA is not detrimental to the host.     

Cell Wall Degrading Enzymes in Cuscuta reflexa and Its Hosts

Pectin degrading enzymes, hemicellulose degrading enzyme andcellulose degrading enzymes were studied in Cuscuta reflexaRoxb., its susceptible hosts, Brassica campestris L., Cocciniaindica W. & A. Datura innoxia Mill, Helianthus annuus L.,Holoptelea indica Planch, Lantana camara L., Medicago sativaL., Manihot utilissima Pohl, Petunia hybrida X Hort exvilm,Pisum sativum L., Phaseolus vulgaris L. and Solanum nigrum L.and non-susceptible plants Ipomoea batata Lam. and Solanum tuberosumL. Pectin esterase and polygalacturonase were present in higheramounts in Cuscuta parasitic on P. vulgaris and S. nigrum, whichneeded more time for haustorial establishment. Exo-l, 4-ß-D-glucosidaseactivity was found in Cuscuta but could not be detected in itshosts. Xylanase and cellulase activity of host plants increasedwhile cellobiase activity decreased as a result of infectionby the parasite. Higher pectin esterase, polygalacturonase,xylanase and exo-l, 4-ß-D-glucosidase activities inthe haustorial region of the parasite is likely to bring aboutthe lysis of the cell wall of the host plant and thus facilitatethe penetration of the parasite haustoria into the host sieveelement, which is necessary for the transport of nutrients betweenthe host and the parasite. Key words: Cell wall degrading enzymes, Cuscuta reflexa     

<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.