Gene silencing in broomrapes and other parasitic plants of the Orobanchaceae family: mechanisms, considerations, and future directions.

Holoparasites of the Orobanchaceae family are devastating pests causing severe damage to many crop species and are nearly impossible to control with conventional methods. During past decades RNA interference (RNAi) has been seen as a promising approach to control various crop pests. The exchange of small RNAs (sRNAs) between crops and parasitic plants has been documented indicating a potential for the development of methods to protect them via the delivery of the sRNAs to parasites, called host-induced gene silencing (HIGS). Here we describe various approaches used for gene silencing in plants and suggest solutions to improve the long-distance movement of the silencing triggers to elevate the HIGS efficiency in parasitic plants. We also investigate the important biological processes during parasites life cycle with a focus on broomrape species, providing several appropriate target genes that can be used in, especially, multiplex gene silencing experiments. We also touch on how the application of nanoparticles can improve the stability and delivery of the silencing triggers, highlighting its potential for parasitic plants control. Finally, suggestions for further research and possible directions for RNAi in parasitic plants are provided.

The Physcomitrium (Physcomitrella) patens PpKAI2L receptors for strigolactones and related compounds function via MAX2-dependent and -independent pathways.

In angiosperms, the α/ß hydrolase DWARF14 (D14), along with the F-box protein MORE AXILLARY GROWTH2 (MAX2), perceives strigolactones (SL) to regulate developmental processes. The key SL biosynthetic enzyme CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8) is present in the moss Physcomitrium patens, and PpCCD8-derived compounds regulate moss extension. The PpMAX2 homolog is not involved in the SL response, but 13 PpKAI2LIKE (PpKAI2L) genes homologous to the D14 ancestral paralog KARRIKIN INSENSITIVE2 (KAI2) encode candidate SL receptors. In Arabidopsis thaliana, AtKAI2 perceives karrikins and the elusive endogenous KAI2-Ligand (KL). Here, germination assays of the parasitic plant Phelipanche ramosa suggested that PpCCD8-derived compounds are likely noncanonical SLs. (+)-GR24 SL analog is a good mimic for PpCCD8-derived compounds in P. patens, while the effects of its enantiomer (-)-GR24, a KL mimic in angiosperms, are minimal. Interaction and binding assays of seven PpKAI2L proteins pointed to the stereoselectivity toward (-)-GR24 for a single clade of PpKAI2L (eu-KAI2). Enzyme assays highlighted the peculiar behavior of PpKAI2L-H. Phenotypic characterization of Ppkai2l mutants showed that eu-KAI2 genes are not involved in the perception of PpCCD8-derived compounds but act in a PpMAX2-dependent pathway. In contrast, mutations in PpKAI2L-G, and -J genes abolished the response to the (+)-GR24 enantiomer, suggesting that PpKAI2L-G, and -J proteins are receptors for moss SLs.

CYP707As are effectors of karrikin and strigolactone signalling pathways in Arabidopsis thaliana and parasitic plants.

Karrikins stimulate Arabidopsis thaliana germination, whereas parasitic weeds of the Orobanchaceae family have evolved to respond to host-exuded compounds such as strigolactones, dehydrocostus lactone, and 2-phenylethyl isothiocyanate. In Phelipanche ramosa, strigolactone-induced germination was shown to require one of the CYP707A proteins involved in abscisic acid catabolism. Here, germination and gene expression were analysed to investigate the role of CYP707As in germination of both parasitic plants and Arabidopsis upon perception of germination stimulants, after using pharmacological inhibitors and Arabidopsis mutants disrupting germination signals. CYP707A genes were up-regulated upon treatment with effective germination stimulants in both parasitic plants and Arabidopsis. Obligate parasitic plants exhibited both intensified up-regulation of CYP707A genes and increased sensitivity to the CYP707A inhibitor abscinazole-E2B, whereas Arabidopsis cyp707a mutants still positively responded to germination stimulation. In Arabidopsis, CYP707A regulation required the canonical karrikin signalling pathway KAI2/MAX2/SMAX1 and the transcription factor WRKY33. Finally, CYP707As and WRKY33 also modulated Arabidopsis root architecture in response to the synthetic strigolactone rac-GR24, and wrky33-1 exhibited a shoot hyperbranched phenotype. This study suggests that the lack of host-independent germination in obligate parasites is associated with an exacerbated CYP707A induction and that CYP707As and WRKY33 are new players involved in a variety of strigolactone/karrikin responses.

Seed germination in parasitic plants: what insights can we expect from strigolactone research?

Obligate root-parasitic plants belonging to the Orobanchaceae family are deadly pests for major crops all over the world. Because these heterotrophic plants severely damage their hosts even before emerging from the soil, there is an unequivocal need to design early and efficient methods for their control. The germination process of these species has probably undergone numerous selective pressure events in the course of evolution, in that the perception of host-derived molecules is a necessary condition for seeds to germinate. Although most of these molecules belong to the strigolactones, structurally different molecules have been identified. Since strigolactones are also classified as novel plant hormones that regulate several physiological processes other than germination, the use of autotrophic model plant species has allowed the identification of many actors involved in the strigolactone biosynthesis, perception, and signal transduction pathways. Nevertheless, many questions remain to be answered regarding the germination process of parasitic plants. For instance, how did parasitic plants evolve to germinate in response to a wide variety of molecules, while autotrophic plants do not? What particular features are associated with their lack of spontaneous germination? In this review, we attempt to illustrate to what extent conclusions from research into strigolactones could be applied to better understand the biology of parasitic plants.

Haustorium initiation in the obligate parasitic plant Phelipanche ramosa involves a host-exudated cytokinin signal.

The heterotrophic lifestyle of parasitic plants relies on the development of the haustorium, a specific infectious organ required for attachment to host roots. While haustorium development is initiated upon chemodetection of host-derived molecules in hemiparasitic plants, the induction of haustorium formation remains largely unknown in holoparasitic species such as Phelipanche ramosa. This work demonstrates that the root exudates of the host plant Brassica napus contain allelochemicals displaying haustorium-inducing activity on P. ramosa germinating seeds, which increases the parasite aggressiveness. A de novo assembled transcriptome and microarray approach with P. ramosa during early haustorium formation upon treatment with B. napus root exudates allowed the identification of differentially expressed genes involved in hormone signaling. Bioassays using exogenous cytokinins and the specific cytokinin receptor inhibitor PI-55 showed that cytokinins induced haustorium formation and increased parasite aggressiveness. Root exudates triggered the expression of cytokinin-responsive genes during early haustorium development in germinated seeds, and bio-guided UPLC-ESI(+)-/MS/MS analysis showed that these exudates contain a cytokinin with dihydrozeatin characteristics. These results suggest that cytokinins constitutively exudated from host roots play a major role in haustorium formation and aggressiveness in P. ramosa.

Seed response to strigolactone is controlled by abscisic acid-independent DNA methylation in the obligate root parasitic plant, Phelipanche ramosa L. Pomel.

Seed dormancy release of the obligate root parasitic plant, Phelipanche ramosa, requires a minimum 4-day conditioning period followed by stimulation by host-derived germination stimulants, such as strigolactones. Germination is then mediated by germination stimulant-dependent activation of PrCYP707A1, an abscisic acid catabolic gene. The molecular mechanisms occurring during the conditioning period that silence PrCYP707A1 expression and regulate germination stimulant response are almost unknown. Here, global DNA methylation quantification associated with pharmacological approaches and cytosine methylation analysis of the PrCYP707A1 promoter were used to investigate the modulation and possible role of DNA methylation during the conditioning period and in the PrCYP707A1 response to GR24, a synthetic strigolactone analogue. Active global DNA demethylation occurs during the conditioning period and is required for PrCYP707A1 activation by GR24 and for subsequent seed germination. Treatment with 5-azacytidine, a DNA-hypomethylating molecule, reduces the length of the conditioning period. Conversely, hydroxyurea, a hypermethylating agent, inhibits PrCYP707A1 expression and seed germination. Methylated DNA immunoprecipitation followed by PCR experiments and bisulfite sequencing revealed that DNA demethylation particularly impacts a 78-nucleotide sequence in the PrCYP707A1 promoter. The results here demonstrate that the DNA methylation status during the conditioning period plays a crucial role independently of abscisic acid in the regulation of P. ramosa seed germination by controlling the strigolactone-dependent expression of PrCYP707A1.

Robust method for investigating nitrogen metabolism of 15N labeled amino acids using AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry: application to a parasitic plant-plant interaction.

An AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry (AccQ•Tag-UPLC-PDA-ESI-MS) method is presented here for the fast, robust, and sensitive quantification of (15)N isotopologue enrichment of amino acids in biological samples, as for example in the special biotic interaction between the cultivated specie Brassica napus (rapeseed) and the parasitic weed Phelipanche ramosa (broomrape). This method was developed and validated using amino acid standard solutions containing (15)N amino acid isotopologues and/or biological unlabeled extracts. Apparatus optimization, limits of detection and quantification, quantification reproducibility, and calculation method of (15)N isotopologue enrichment are presented. Using this method, we could demonstrate that young parasite tubercles assimilate inorganic nitrogen as (15)N-ammonium when supplied directly through batch incubation but not when supplied by translocation from host root phloem, contrary to (15)N2-glutamine. (15)N2-glutamine mobility from host roots to parasite tubercles followed by its low metabolism in tubercles suggests that the host-derived glutamine acts as an important nitrogen containing storage compound in the young tubercle of Phelipanche ramosa.

Pectin Remodeling and Involvement of AtPME3 in the Parasitic Plant-Plant Interaction, Phelipanche ramosa-Arabidospis thaliana.

Phelipanche ramosa is a root parasitic plant fully dependent on host plants for nutrition and development. Upon germination, the parasitic seedling develops inside the infected roots a specific organ, the haustorium, thanks to the cell wall-degrading enzymes of haustorial intrusive cells, and induces modifications in the host's cell walls. The model plant Arabidopsis thaliana is susceptible to P. ramosa; thus, mutants in cell wall metabolism, particularly those involved in pectin remodeling, like Atpme3-1, are of interest in studying the involvement of cell wall-degrading enzymes in the establishment of plant-plant interactions. Host-parasite co-cultures in mini-rhizotron systems revealed that parasite attachments are twice as numerous and tubercle growth is quicker on Atpme3-1 roots than on WT roots. Compared to WT, the increased susceptibility in AtPME3-1 is associated with reduced PME activity in the roots and a lower degree of pectin methylesterification at the host-parasite interface, as detected immunohistochemically in infected roots. In addition, both WT and Atpme3-1 roots responded to infestation by modulating the expression of PAE- and PME-encoding genes, as well as related global enzyme activities in the roots before and after parasite attachment. However, these modulations differed between WT and Atpme3-1, which may contribute to different pectin remodeling in the roots and contrasting susceptibility to P. ramosa. With this integrative study, we aim to define a model of cell wall response to this specific biotic stress and indicate, for the first time, the role of PME3 in this parasitic plant-plant interaction.

Structure-activity relationship studies of strigolactone-related molecules for branching inhibition in garden pea: molecule design for shoot branching.

Initially known for their role in the rhizosphere in stimulating the seed germination of parasitic weeds such as the Striga and Orobanche species, and later as host recognition signals for arbuscular mycorrhizal fungi, strigolactones (SLs) were recently rediscovered as a new class of plant hormones involved in the control of shoot branching in plants. Herein, we report the synthesis of new SL analogs and, to our knowledge, the first study of SL structure-activity relationships for their hormonal activity in garden pea (Pisum sativum). Comparisons with their action for the germination of broomrape (Phelipanche ramosa) are also presented. The pea rms1 SL-deficient mutant was used in a SL bioassay based on axillary bud length after direct SL application on the bud. This assay was compared with an assay where SLs were fed via the roots using hydroponics and with a molecular assay in which transcript levels of BRANCHED1, the pea homolog of the maize TEOSINTE BRANCHED1 gene were quantified in axillary buds only 6 h after application of SLs. We have demonstrated that the presence of a Michael acceptor and a methylbutenolide or dimethylbutenolide motif in the same molecule is essential. It was established that the more active analog 23 with a dimethylbutenolide as the D-ring could be used to control the plant architecture without strongly favoring the germination of P. ramosa seeds. Bold numerals refer to numbers of compounds.

The ecologically relevant genetics of plant-plant interactions.

Interactions among plants have been long recognized as a major force driving plant community dynamics and crop yield. Surprisingly, our knowledge of the ecological genetics associated with variation of plant-plant interactions remains limited. In this opinion article by scientists from complementary disciplines, the international PLANTCOM network identified four timely questions to foster a better understanding of the mechanisms mediating plant assemblages. We propose that by identifying the key relationships among phenotypic traits involved in plant-plant interactions and the underlying adaptive genetic and molecular pathways, while considering environmental fluctuations at diverse spatial and time scales, we can improve predictions of genotype-by-genotype-by-environment interactions and modeling of productive and stable plant assemblages in wild habitats and crop fields.

Role of the sucrose synthase encoding PrSus1 gene in the development of the parasitic plant Phelipanche ramosa L. (Pomel).

Phelipanche ramosa L. (Pomel) is a major root-parasitic weed attacking many important crops. Success in controlling this parasite is rare and a better understanding of its unique biology is needed to develop new specific control strategies. In the present study, quantitative polymerase chain reaction experiments showed that sucrose synthase encoding PrSus1 transcripts accumulate at their highest level once the parasite is connected to the host (tomato) vascular system, mainly in the parasite tubercles, which bear numerous adventitious roots. In situ hybridization experiments revealed strong PrSus1 expression in both shoot and root apices, especially in shoot apical meristems and in the vascular tissues of scale leaves and stems, and in the apical meristems and developing xylem in roots. In addition, immunolocalization experiments showed that a sucrose synthase protein co-localized with cell-wall thickening in xylem elements. These findings highlight the role of PrSus1 in the utilization of host-derived sucrose in meristematic areas and in cellulose biosynthesis in differentiating vascular elements. We also demonstrate that PrSus1 is downregulated in response to 2,3,5-triiodobenzoic acid-induced inhibition of polar auxin transport in the host stem, suggesting that PrSus1 activity in xylem maturation is controlled by host-derived auxin.

Germination stimulants of Phelipanche ramosa in the rhizosphere of Brassica napus are derived from the glucosinolate pathway.

Phelipanche ramosa is a major parasitic weed of Brassica napus. The first step in a host-parasitic plant interaction is stimulation of parasite seed germination by compounds released from host roots. However, germination stimulants produced by B. napus have not been identified yet. In this study, we characterized the germination stimulants that accumulate in B. napus roots and are released into the rhizosphere. Eight glucosinolate-breakdown products were identified and quantified in B. napus roots by gas chromatography-mass spectrometry. Two (3-phenylpropanenitrile and 2-phenylethyl isothiocyanate [2-PEITC]) were identified in the B. napus rhizosphere. Among glucosinolate-breakdown products, P. ramosa germination was strongly and specifically triggered by isothiocyanates, indicating that 2-PEITC, in particular, plays a key role in the B. napus-P. ramosa interaction. Known strigolactones were not detected by ultraperformance liquid chromatography-tandem mass spectrometry, and seed of Phelipanche and Orobanche spp. that respond to strigolactones but not to isothiocyanates did not germinate in the rhizosphere of B. napus. Furthermore, both wild-type and strigolactone biosynthesis mutants of Arabidopsis thaliana Atccd7 and Atccd8 induced similar levels of P. ramosa seed germination, suggesting that compounds other than strigolactone function as germination stimulants for P. ramosa in other Brassicaceae spp. Our results open perspectives on the high adaptation potential of root-parasitic plants under host-driven selection pressures.

PrCYP707A1, an ABA catabolic gene, is a key component of Phelipanche ramosa seed germination in response to the strigolactone analogue GR24.

After a conditioning period, seed dormancy in obligate root parasitic plants is released by a chemical stimulus secreted by the roots of host plants. Using Phelipanche ramosa as the model, experiments conducted in this study showed that seeds require a conditioning period of at least 4 d to be receptive to the synthetic germination stimulant GR24. A cDNA-AFLP procedure on seeds revealed 58 transcript-derived fragments (TDFs) whose expression pattern changed upon GR24 treatment. Among the isolated TDFs, two up-regulated sequences corresponded to an abscisic acid (ABA) catabolic gene, PrCYP707A1, encoding an ABA 8'-hydroxylase. Using the rapid amplification of cDNA ends method, two full-length cDNAs, PrCYP707A1 and PrCYP707A2, were isolated from seeds. Both genes were always expressed at low levels during conditioning during which an initial decline in ABA levels was recorded. GR24 application after conditioning triggered a strong up-regulation of PrCYP707A1 during the first 18 h, followed by an 8-fold decrease in ABA levels detectable 3 d after treatment. In situ hybridization experiments on GR24-treated seeds revealed a specific PrCYP707A1 mRNA accumulation in the cells located between the embryo and the micropyle. Abz-E2B, a specific inhibitor of CYP707A enzymes, significantly impeded seed germination, proving to be a non-competitive antagonist of GR24 with reversible inhibitory activity. These results demonstrate that P. ramosa seed dormancy release relies on ABA catabolism mediated by the GR24-dependent activation of PrCYP707A1. In addition, in situ hybridization corroborates the putative location of cells receptive to the germination stimulants in seeds.

Wild Helianthus species: A reservoir of resistance genes for sustainable pyramidal resistance to broomrape in sunflower.

Orobanche cumana Wall., sunflower broomrape, is one of the major pests for the sunflower crop. Breeding for resistant varieties in sunflower has been the most efficient method to control this parasitic weed. However, more virulent broomrape populations continuously emerge by overcoming genetic resistance. It is thus essential to identify new broomrape resistances acting at various stages of the interaction and combine them to improve resistance durability. In this study, 71 wild sunflowers and wild relatives accessions from 16 Helianthus species were screened in pots for their resistance to broomrape at the late emergence stage. From this initial screen, 18 accessions from 9 species showing resistance, were phenotyped at early stages of the interaction: the induction of broomrape seed germination by sunflower root exudates, the attachment to the host root and the development of tubercles in rhizotron assays. We showed that wild Helianthus accessions are an important source of resistance to the most virulent broomrape races, affecting various stages of the interaction: the inability to induce broomrape seed germination, the development of incompatible attachments or necrotic tubercles, and the arrest of emerged structure growth. Cytological studies of incompatible attachments showed that several cellular mechanisms were shared among resistant Helianthus species.

Strigolactone-Like Bioactivity via Parasitic Plant Germination Bioassay.

Strigolactones are a class of plant hormones involved in shoot branching, growth of symbiotic arbuscular mycorrhizal fungi, and germination of parasitic plant seeds. Assaying new molecules or compound exhibiting strigolactone-like activities is therefore important but unfortunately time-consuming and hard to implement because of the extremely low concentrations at which they are active. Seeds of parasite plants are natural integrator of these hormones since they can perceive molecule concentrations in the picomolar to nanomolar range stimulating their germination. Here we describe a simple and inexpensive method to evaluate the activity of these molecules by scoring the germination of parasitic plant seeds upon treatment with these molecules. Up to four molecules can be assayed from a single 96-well plate by this method. A comparison of SL-like bioactivities between molecules is done by determining the EC50 and the maximum percentage of germination.

A Phelipanche ramosa KAI2 protein perceives strigolactones and isothiocyanates enzymatically.

Phelipanche ramosa is an obligate root-parasitic weed that threatens major crops in central Europe. In order to germinate, it must perceive various structurally divergent host-exuded signals, including isothiocyanates (ITCs) and strigolactones (SLs). However, the receptors involved are still uncharacterized. Here, we identify five putative SL receptors in P. ramosa and show that PrKAI2d3 is involved in the stimulation of seed germination. We demonstrate the high plasticity of PrKAI2d3, which allows it to interact with different chemicals, including ITCs. The SL perception mechanism of PrKAI2d3 is similar to that of endogenous SLs in non-parasitic plants. We provide evidence that PrKAI2d3 enzymatic activity confers hypersensitivity to SLs. Additionally, we demonstrate that methylbutenolide-OH binds PrKAI2d3 and stimulates P. ramosa germination with bioactivity comparable to that of ITCs. This study demonstrates that P. ramosa has extended its signal perception system during evolution, a fact that should be considered for the development of specific and efficient biocontrol methods.

Purification, characterization, and mode of action of a rhamnogalacturonan hydrolase from Irpex lacteus, tolerant to an acetylated substrate.

A novel rhamnogalacturonase (RGase) acting on an acetylated substrate was detected in the commercial preparation Driselase, an enzymatic mixture derived from the basidiomycete Irpex lacteus. The activity was isolated by hydrophobic interaction chromatography, gel filtration, and preparative isoelectric focusing, resulting in the isolation of five different rhamnogalacturonan hydrolases exhibiting various isoelectric points from 6.2 to 7.7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry analyses after trypsin cleavage of the five fractions revealed that the five rhamnogalacturonases have a molar mass of 55 kDa without any divergences in the identified peptides. The RGase with a pI of 7.2 exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40 degrees C and 50 degrees C. Its mode of action was analyzed by mass spectrometry of the oligosaccharides produced after hydrolysis of acetylated and nonacetylated rhamnogalacturonan. Oligomers esterified by an acetyl group on the reducing galacturonic acid residue or fully acetylated were detected in the hydrolysate showing that the novel enzyme is able to bind acetylated galacturonic acid in its active site.

Management of Infection by Parasitic Weeds: A Review.

Parasitic plants rely on neighboring host plants to complete their life cycle, forming vascular connections through which they withdraw needed nutritive resources. In natural ecosystems, parasitic plants form one component of the plant community and parasitism contributes to overall community balance. In contrast, when parasitic plants become established in low biodiversified agroecosystems, their persistence causes tremendous yield losses rendering agricultural lands uncultivable. The control of parasitic weeds is challenging because there are few sources of crop resistance and it is difficult to apply controlling methods selective enough to kill the weeds without damaging the crop to which they are physically and biochemically attached. The management of parasitic weeds is also hindered by their high fecundity, dispersal efficiency, persistent seedbank, and rapid responses to changes in agricultural practices, which allow them to adapt to new hosts and manifest increased aggressiveness against new resistant cultivars. New understanding of the physiological and molecular mechanisms behind the processes of germination and haustorium development, and behind the crop resistant response, in addition to the discovery of new targets for herbicides and bioherbicides will guide researchers on the design of modern agricultural strategies for more effective, durable, and health compatible parasitic weed control.