Root Parasitic Plants Research Articles

Are root parasitic plants like any other plant pathogens?

This article is a Commentary on Su et al. 226: 891–908.

Chemical identification of 18-hydroxycarlactonoic acid as an LjMAX1 product and in planta conversion of its methyl ester to canonical and non-canonical strigolactones in Lotus japonicus

Strigolactones (SLs) are a group of plant apocarotenoids that act as rhizosphere signaling molecules for both arbuscular mycorrhizal fungi and root parasitic plants. They also regulate plant architecture as phytohormones. The model legume Lotus japonicus (synonym of Lotus corniculatus) produces canonical 5-deoxystrigol (5DS) and non-canonical lotuslactone (LL). The biosynthesis pathways of the two SLs remain elusive. In this study, we characterized the L. japonicus MAX1 homolog, LjMAX1, found in the Lotus japonicus genome assembly build 2.5. The L. japonicus max1 LORE1 insertion mutant was deficient in 5DS and LL production. A recombinant LjMAX1 protein expressed in yeast microsomes converted carlactone (CL) to 18-hydroxycarlactonoic acid (18-OH-CLA) via carlactonoic acid (CLA). Identity of 18-OH-CLA was confirmed by comparison of the methyl ester derivative of the MAX1 product with chemically synthesized methyl 18-hydroycarlactonoate (18-OH-MeCLA) using LC-MS/MS. (11R)-CL was detected as an endogenous compound in the root of L. japonicus.13C-labeled CL, CLA, and 18-OH-MeCLA were converted to [13C]-5DS and LL in plant feeding experiments using L. japonicus WT. These results showed that LjMAX1 is the crucial enzyme in the biosynthesis of Lotus SLs and that 18-hydroxylated carlactonoates are possible precursors for SL biosynthesis in L. japonicus.

Genetic and Genomic Tools in Sunflower Breeding for Broomrape Resistance.

Broomrape is a root parasitic plant causing yield losses in sunflower production. Since sunflower is an important oil crop, the development of broomrape-resistant hybrids is the prime breeding objective. Using conventional plant breeding methods, breeders have identified resistant genes and developed a number of hybrids resistant to broomrape, adapted to different growing regions worldwide. However, the spread of broomrape into new countries and the development of new and more virulent races have been noted intensively. Recent advances in sunflower genomics provide additional tools for plant breeders to improve resistance and find durable solutions for broomrape spread and virulence. This review describes the structure and distribution of new, virulent physiological broomrape races, sources of resistance for introduction into susceptible cultivated sunflower, qualitative and quantitative resistance genes along with gene pyramiding and marker assisted selection (MAS) strategies applied in the process of increasing sunflower resistance. In addition, it presents an overview of underutilized biotechnological tools, such as phenotyping, -omics, and genome editing techniques, which need to be introduced in the study of sunflower resistance to broomrape in order to achieve durable resistance.

Molecular basis of strigolactone perception in root-parasitic plants: aiming to control its germination with strigolactone agonists/antagonists.

The genus Striga, also called "witchweed", is a member of the family Orobanchaceae, which is a major family of root-parasitic plants. Striga can lead to the formation of seed stocks in the soil and to explosive expansion with enormous seed production and stability once the crops they parasitize are cultivated. Understanding the molecular mechanism underlying the communication between Striga and their host plants through natural seed germination stimulants, "strigolactones (SLs)", is required to develop the technology for Striga control. This review outlines recent findings on the SL perception mechanism, which have been accumulated in Striga hermonthica by the similarity of the protein components that regulate SL signaling in nonparasitic model plants, including Arabidopsis and rice. HTL/KAI2 homologs were identified as SL receptors in the process of Striga seed germination. Recently, this molecular basis has further promoted the development of various types of SL agonists/antagonists as seed germination stimulants or inhibitors. Such chemical compounds are also useful to elucidate the dynamic behavior of SL receptors and the regulation of SL signaling.

Contalactone, a contaminant formed during chemical synthesis of the strigolactone reference GR24 is also a strigolactone mimic

Strigolactone (SL) plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. GR24, a synthetic SL analog, is the worldwide reference compound used in all bioassays for investigating the role of SLs in plant development and in rhizospheric interactions. In 2012, the first characterization of the SL receptor reported the detection of an unknown compound after incubation of GR24 samples with the SL receptor. We reveal here the origin of this compound (P270), which comes from a by-product formed during GR24 chemical synthesis. We present the identification of this by-product, named contalactone. A proposed chemical pathway for its formation is provided as well as an evaluation of its bioactivity on pea, Arabidopsis, root parasitic plant seeds and AM fungi, characterizing it as a SL mimic. Quality of GR24 samples can be easily checked by carrying out microscale hydrolysis in a basic aqueous medium to easily detect P270 as indicator of the presence of the contalactone impurity. In all cases, before being used for bioassays, GR24 must be careful purified by preparative HPLC.

Intraspecific seasonal variation of dormancy and mortality of Phelipanche ramosa seeds

SummaryPhelipanche ramosa (Branched broomrape) is an obligate root parasitic plant that is a major pest of oilseed rape in France. Knowledge on seed viability and dormancy under field conditions is crucial to understand how to control P. ramosa, but is as yet unknown. Our study aimed to quantify these processes with a 2‐year seed burial experiment. Two genetically distinct populations of P. ramosa were studied, collected on winter oilseed rape (population O) and hemp (population H). Seed mortality was very low in both populations (4–7% per year). Although obligate parasitic seeds are assumed to germinate only after exposure to germination stimulants from host root exudates, a high proportion of population H seeds germinated spontaneously (up to 90%). Seeds of both populations displayed seasonal dormancy, with timing and magnitude depending on the population. Dormancy was low at the time each native host crop is usually sown. Populations differed in germination dynamics, with seeds of population H germinating faster. The difference in behaviour that we observed between populations is consistent with reported adaptations of pathovars to their preferred hosts. The results indicate that the parasitic plant management requires targeting at the populations concerned. For example, delayed sowing is more promising against population O than against population H.

EDNA-based monitoring of parasitic plant (Sapria himalayana)

Sapria himalayana Griffith., is a root parasitic plant that is exceptionally beautiful and odd-looking and found in Southeast Asia. Now these plants are at risk of extinction as they face a large number of different threats. Appropriate measures and conservation plans are needed and one crucial key for successful conservation is species monitoring. The flower is the only part of S. himalayana that is visible during a short period of time of the year. Thus, conducting a visual survey in the field at the other times of the year would be difficult. DNA from living organisms could be found accumulating in environment and so-called environmental DNA (eDNA). Here, an eDNA-based method was developed to specifically monitor S. himalayana in nature. Detecting the specifically generated amplicons allowed us to monitor the presence of S. himalayana at any time of the year. This developed method would increase the conservation success of the S. himalayana.

Development of phloem connection between the parasitic plant Orobanche cumana and its host sunflower

Orobanche cumana is a root parasitic plant causing considerable yield losses in sunflower cultivation. The holoparasite fulfills its entire demand for water, minerals, and organic nutrients from the host's vascular system. In this study, the ultrastructure of the phloem connection between the haustorium of young O. cumana tubercles and the sunflower root has been examined for the first time. Parasite and host tissues were intermingled at the contact site and difficult to distinguish, but sieve-tube elements of O. cumana and sunflower could be differentiated according to their plastid ultrastructure. While O. cumana sieve-element plastids were larger, often irregular in shape and contained few, small starch inclusions, sieve-element plastids of the host were significantly smaller, always roundish with more and larger starch inclusions. This made it possible to trace the exact contact site of host and parasite sieve elements to show a direct symplastic phloem connection between the two species. The interspecific sieve plate showed more callose on the host side. This allowed detection of newly formed plasmodesmata between host sieve-tube elements and parenchymatic parasite cells, thus showing that undifferentiated cells of the parasite could connect to fully differentiated sieve elements of sunflower. Furthermore, the arrangement of phloem within the O. cumana tubercle as well as differences in sieve-element plastid ultrastructure during shoot development in O. cumana were investigated and are discussed in this paper.

Methylation at the C-3′ in D-Ring of Strigolactone Analogs Reduces Biological Activity in Root Parasitic Plants and Rice

Strigolactones (SLs) regulate plant development and induce seed germination in obligate root parasitic weeds, e.g. Striga spp. Because organic synthesis of natural SLs is laborious, there is a large need for easy-to-synthesize and efficient analogs. Here, we investigated the effect of a structural modification of the D-ring, a conserved structural element in SLs. We synthesized and investigated the activity of two analogs, MP13 and MP26, which differ from previously published AR8 and AR36 only in the absence of methylation at C-3′. The de-methylated MP13 and MP26 were much more efficient in regulating plant development and inducing Striga seed germination, compared with AR8. Hydrolysis assays performed with purified Striga SL receptor and docking of AR8 and MP13 to the corresponding active site confirmed and explained the higher activity. Field trials performed in a naturally Striga-infested African farmer’s field unraveled MP13 as a promising candidate for combating Striga by inducing germination in host’s absence. Our findings demonstrate that methylation of the C-3′ in D-ring in SL analogs has a negative impact on their activity and identify MP13 and, particularly, MP26 as potent SL analogs with simple structures, which can be employed to control Striga, a major threat to global food security.

Suicidal germination as a control strategy for Striga hermonthica (Benth.) in smallholder farms of sub‐Saharan Africa

Societal Impact StatementInfestation by the parasitic plant Striga hermonthica is a severe threat to food security in sub‐Saharan Africa, impacting the production of the major staple crops pearl millet and sorghum, equating to 7–10 billion $ losses. Using Striga seed dependency on host‐released germination stimulants, we have developed and validated a method for addressing the problem of accumulated parasite seedbanks—the major obstacle in combating Striga infestation in African rain‐fed fields. Application of our method promises to alleviate the problem posed by this pernicious weed by increasing crop production for smallholder farmers. Summary The root parasitic plant Striga hermonthica is a major threat to global food security, causing enormous losses in yields of the main staple crops in sub‐Saharan Africa, which include pearl millet, sorghum, maize and rice. Sustainable Striga control should ideally lead to the depletion of the vast, long‐lived Striga seedbank, and this can be achieved by inducing suicidal seed germination through application of strigolactone (SL) analogs in the absence of host plants. However, this “suicidal germination” strategy has not been evaluated under the natural rain‐fed conditions that prevail in Striga‐prone regions. We have developed and validated a protocol for suicidal germination in laboratory and natural conditions in Striga‐infested rain‐fed African fields. Three SL analogs were tested and these resulted to between 65% and 55% reduction in Striga emergence in pearl millet and sorghum fields, respectively. We conclude that suicidal germination is an effective method for reducing the Striga seedbank. Moreover, the minimal demands of our protocol, in terms of water consumption and amount of selected SL analogs, make it affordable and applicable at a large scale in African rain‐fed agriculture, holding promise for sustainable cleaning of heavily Striga‐infested fields in sub‐Saharan Africa.

Chemical screening of novel strigolactone agonists that specifically interact with DWARF14 protein

Strigolactones (SLs) are a class of plant hormones that regulate shoot branching as well as being known as root-derived signals for parasitic and symbiotic interactions. The physical interaction between SLs and the DWARF14 (D14) receptor family can be examined by differential scanning fluorimetry (DSF) that monitors the changes in protein melting temperature (Tm). The Tm of D14 is lowered by bioactive SLs in DSF analysis. In this report, we screened the compounds that lower the Tm of Arabidopsis D14 (AtD14) as potential candidates for SL agonists using DSF analysis. Subsequent physiological analyzes revealed that 113D10 acts as a novel SL agonist in a D14-dependent manner. Intriguingly, 113D10 has a chemical structure different from natural SLs in that it does not possess an enol ether bond that connects to a methylbutenolide moiety. Moreover, 113D10 does not stimulate seed germination of root parasitic plants. Accordingly, 113D10 can be a useful tool for SL studies and agricultural applications.

A new UHPLC-MS/MS method for the direct determination of strigolactones in root exudates and extracts.

Strigolactones (SLs) are the most representative germination stimulants for seeds of root parasitic plants, and they show activity even at concentrations below 10-10 M. The low amounts of stimulants produced by the host and their rapid degradability make it crucial to develop analytical methods with very low limits of quantification. To develop a sensitive and validated analytical method for the simultaneous quantification of seven SLs [7-oxoorobanchyl acetate (1), solanacol (2), orobanchol (4), strigol (5), fabacyl acetate (6), orobanchyl acetate (7), and 5-deoxystrigol (8)]. SLs were analysed using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), with (±)-GR24 (3) employed as internal standard (IS). Validation was based on selectivity, linearity, precision of the peak areas (repeatability and intermediate precision), detection and quantification limits, and stability. A simple, rapid and reliable UHPLC-MS/MS method has been validated for the routine analysis of seven SLs and has been successfully applied to quantify them in exudates and extracts from tomato roots (Solanum lycopersicum). The limits of quantifications range from 0.05μg/L for 5-deoxystrigol to 0.96μg/L for solanacol. The method provides a useful tool for research in all the fields related to SLs, both for studies related to their function as hormones, and signalling molecules in the rhizosphere, without sample preparation required for extracts and root exudates in less than 11minutes.

Low Infection of Phelipanche aegyptiaca in Micro-Tom Mutants Deficient in CAROTENOIDCLEAVAGE DIOXYGENASE 8.

Strigolactones (SLs), a group of plant hormones, induce germination of root-parasitic plants and inhibit shoot branching in many plants. Shoot branching is an important trait that affects the number and quality of flowers and fruits. Root-parasitic plants, such as Phelipanche spp., infect tomato roots and cause economic damage in Europe and North Africa—hence why resistant tomato cultivars are needed. In this study, we found carotenoid cleavage dioxygenase 8-defective mutants of Micro-Tom tomato (slccd8) by the “targeting induced local lesions in genomes” (TILLING) method. The mutants showed excess branching, which was suppressed by exogenously applied SL. Grafting shoot scions of the slccd8 mutants onto wild-type (WT) rootstocks restored normal branching in the scions. The levels of endogenous orobanchol and solanacol in WT were enough detectable, whereas that in the slccd8 mutants were below the detection limit of quantification analysis. Accordingly, root exudates of the slccd8 mutants hardly stimulated seed germination of root parasitic plants. In addition, SL deficiency did not critically affect the fruit traits of Micro-Tom. Using a rhizotron system, we also found that Phelipanche aegyptiaca infection was lower in the slccd8 mutants than in wild-type Micro-Tom because of the low germination. We propose that the slccd8 mutants might be useful as new tomato lines resistant to P. aegyptiaca.

Structural basis for specific inhibition of the highly sensitive ShHTL7 receptor

Striga hermonthica is a root parasitic plant that infests cereals, decimating yields, particularly in sub‐Saharan Africa. For germination, Striga seeds require host‐released strigolactones that are perceived by the family of HYPOSENSITIVE to LIGHT (ShHTL) receptors. Inhibiting seed germination would thus be a promising approach for combating Striga. However, there are currently no strigolactone antagonists that specifically block ShHTLs and do not bind to DWARF14, the homologous strigolactone receptor of the host. Here, we show that the octyl phenol ethoxylate Triton X‐100 inhibits S. hermonthica seed germination without affecting host plants. High‐resolution X‐ray structures reveal that Triton X‐100 specifically plugs the catalytic pocket of ShHTL7. ShHTL7‐specific inhibition by Triton X‐100 demonstrates the dominant role of this particular ShHTL receptor for Striga germination. Our structural analysis provides a rationale for the broad specificity and high sensitivity of ShHTL7, and reveals that strigolactones trigger structural changes in ShHTL7 that are required for downstream signaling. Our findings identify Triton and the related 2‐[4‐(2,4,4‐trimethylpentan‐2‐yl)phenoxy]acetic acid as promising lead compounds for the rational design of efficient Striga‐specific herbicides.

Factors affecting the efficiency of Rhizobium rhizogenes root transformation of the root parasitic plant Triphysaria versicolor and its host Arabidopsis thaliana

BackgroundRhizobium rhizogenes transformation is commonly used to generate transgenic roots traditionally called hairy roots, for both investigative and commercial applications. While fertile plants can be regenerated from transgenic roots, the transgenic roots are more typically used directly, either to investigate root biology or to produce valuable secondary metabolites. Hairy roots have been particularly useful for genetic studies of rhizosphere interactions; including the recognition of host plant roots by the roots of parasitic angiosperms.ResultsIn this manuscript we analyzed various environmental, nutritional and procedural conditions for their effects on transformation of the model hemi-parasitic plant Triphysaria versicolor and Arabidopsis thaliana, one of its hosts. We first examined the effects of media, gelling agents and co-incubation times on Triphysaria root transformation and determined that while all three affected transformation rates, the media were the most significant. Once those primary conditions were fixed, we examined the roles of seedling age, explant type, acetosyringone, temperature and illumination on Triphysaria hairy root transformation rates. Using the optimized procedure approximately 70% of Triphysaria seedlings developed transgenic roots as judged by expression of YFP. These conditions were then used to transform Arabidopsis and similar transformation rates were obtained.ConclusionsAnalyses of root transformation factors provides a method recovering transgenic roots from both parasitic plants and their hosts at high frequency. In addition to providing an effective in vitro approach for genetic investigations of parasitic plant-host plant interactions, these results are applicable to genetic studies of non-parasitic plants as well.

Secondary Metabolite Accumulation Associates with Ecological Succession of Endophytic Fungi in Cynomorium songaricum Rupr.

Cynomorium songaricum Rupr. is a rare root-parasitic plant distributed in the desert ecosystem. Little is known about the role of endophytes in accumulation of metabolites in C. songaricum. Here, the correlations between the seven active components (total sugars, flavonoids, protocatechuic acid, catechins, tannins, gallic acid, and ursolic acid) and the endophytic fungi of C. songaricum were investigated, and their causal relationships are discussed further. The results showed that the accumulation of these components and the assembly of endophytic fungi changed with different plant developmental stages. Diverse relationships including positive and negative correlation were found among chemicals and endophytic fungal operational taxonomic units based on correlation coefficient matrices, which demonstrated that the accumulation of secondary metabolites in C. songaricum is closely related to the endophytic fungal community composition. These results present new opportunities to deeply understand plant-fungal symbioses and secondary metabolite productions.

Target-based selectivity of strigolactone agonists and antagonists in plants and their potential use in agriculture.

Strigolactones (SLs) are small carotenoid-derived molecules that possess a wide spectrum of functions, including plant hormonal activities and chemical mediation of rhizosphere communication with both root parasitic plants and symbiotic arbuscular mycorrhizal fungi. Chemicals that regulate the functions of SLs may therefore have the potential to become widely used in agricultural applications. For example, various SL analogs and mimics have been developed to reduce the seed banks of root parasites in the field. Other analogs and mimics act selectively to suppress branching, with weak, or no stimulation, of germination in root parasites. In addition, some antagonists for SL receptors have been developed based on the mechanisms of SL perception. A better understanding of the modes of action of SL perception by various receptors will help to support the design of SL analogs, mimics, and antagonists with high activity and selectivity. Here, we review the compounds reported so far from the viewpoint of their selectivity to their targets, and the possibilities for their use in agriculture.

Partitioning of Fungal Endophyte Assemblages in Root-Parasitic Plant Cynomorium songaricum and Its Host Nitraria tangutorum.

Endophytic fungi are an integral part and even seen as host organs of plant, influencing physiology, ecology, and development of host plants. However, little is known about micro-ecosystems and functional interactions of endophytic fungi in root-parasitic interactions of Cynomorium songaricum and its host Nitraria tangutorum. Here, distribution and dynamics of endophytic fungi were objectively investigated in their associations with C. songaricum and N. tangutorum based on mycobiome studies using high-throughput sequencing. Results suggest that endophytic fungi may be exchanged between C. songaricum and its host N. tangutorum probably through haustorium, connection of xylem and phloem in the vascular system. The similarity of endophytic fungal composition between C. songaricum and parasitized N. tangutorum was 3.88% which was significantly higher than the fungal similarity of 0.10% observed between C. songaricum and non-parasitized N. tangutorum. The similarities of fungal community in parasitized N. tangutorum were much closer to C. songaricum than to the non-parasitized N. tangutorum. The composition of endophytic fungi in these associations increased in progressive developmental stages of C. songaricum from sprouting to above ground emergence, and decreased subsequently probably due to host recognition and response by fungi. However, the shared fungal operational taxonomic units (OTUs) increased among interactions of C. songaricum with parasitized and non-parasitized N. tangutorum. Studies of bioactivity on culturable endophytic fungi showed that isolates such as Fusarium spp. possess the ability to promote seed germination of C. songaricum. Our study reports for the first time the special ecological system of endophytic fungi in C. songaricum and its host N. tangutorum. Overall, we hypothesize that a deeper understanding of the sharing, movement, and role of endophytic fungi between root-parasitic plant and its host may lead to finding alternative approaches to help increase the output of ethno-pharmacologically important medicinal plants.

Structural diversity in the strigolactones.

Strigolactones (SLs) are a class of signalling molecules secreted by the roots of plants into the rhizosphere. On the one hand, they serve as the signal for recruiting arbuscular mycorrhizal fungi which have a symbiotic relationship with plants. On the other hand, they are also host detection signals for the non-symbiotic, pathogenic, root parasitic plants, which use the SLs as germination stimulants. Finally, recently the SLs were discovered to be a new class of plant hormones that regulate processes such as branching/tillering and root architecture. Intriguingly, >25 different SLs have already been discovered that all have the so-called D-ring but otherwise display many differences in structure and functional groups. In this review, we will critically discuss the structural diversity in the SLs. How are they synthesized in plants; how has this structural diversity possibly evolved; what is the biological relevance of this diversity; and what does this imply for the perception of the SLs by receptors in the plant itself and in other organisms? Finally, we conclude that only little is known about the biological significance of this structural diversity, and we will give an outlook on how to elucidate their importance further.

In vitro conservation of Alectra chitrakutensis: a critically endangered root parasitic plant of high medicinal importance

The present study provides an effective in vitro propagation method for critically endangered parasitic plant Alectra chitrakutensis (M.A. Rau) R. Prasad & R.D. Dixit (Scrophulariaceae). The plant comprises tremendous medicinal properties to treat many diseases such as leprosy, leucoderma, tuberculosis and paralysis. The protocol has been developed using rhizome explant on the modified Murashige and Skoog medium containing various phytohormones to raise complete plantlets without any host interaction. 0.5 mg/l kinetin and 1.0 mg/l NAA was found to be the most excellent medium combination to obtain plantlets with an average shoot length of 11 ± 1.04 cm; and 100% response for rooting. The same medium combination also supported flowering in vitro. Further, comparative molecular and chemical characterization of in vitro and in vivo grown plants were carried out by performing RAPD and HPTLC analysis, respectively, to make sure the genetic as well as chemical stability among the plants. RAPD analysis with MAP (01-20) and OPJ primers (01-20) exhibited a monomorphic band pattern which revealed the genetic similarities among the tested plants. Similarly, presence of azafrin, the most important pharmaceutically active compound in in vitro generated plants indicates the efficacy of protocol to fulfill the objective of in vitro propagation of this critically endangered plant species. Moreover, the present study is the first report towards in vitro conservation of parasitic plant A. chitrakutensis without host from the rhizome explant.