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Orobanche coerulescens is a parasitic plant that cannot complete its life cycle without a host and is incapable of photosynthesis. The habitats of O. coerulescens span the coasts of Korea and its volcanic islands, Ulleungdo and Dokdo. Those on the volcanic islands exhibit morphological differences and have distinct hosts compared to those on the peninsula. The family of Orobanchaceae, encompassing both autotrophic and parasitic species, serves as a model for evolutionary studies of parasitic states. However, there are limited genome assemblies for the Orobanche genus. In our study, we produced approximately 100x ONT long reads to construct a chromosome-level genome of O. coerulescens. The resulting assembly has a total size of 3,648 Mb with an N50 value of 195 Mb, and 82.0% of BUSCO genes were identified as complete. Results of the repeat annotation revealed that 86.3% of the genome consisted of repeat elements, and 29,395 protein-coding genes were annotated. This chromosome-level genome will be an important biological resource for conserving biodiversity and further understanding parasitic plants., (© 2024. The Author(s).)

Background: Orobanche is an obligate parasite on faba bean in the Mediterranean region, causes considerable yield losses. Breeding tolerant faba bean genotypes to Orobanche is pivotal to sustain production and ensuring global food security, particularly considering the challenges posed by population growth. In the present study, seven faba bean lines and four testers were used in a line×tester mating design during 2020-2021 and 2021-2022 growing seasons. The eleven parents and their 28 F 1 crosses were evaluated under Orobanche free and naturally infested soils., Results: The results demonstrated considerable variations among the evaluated genotypes, wide diversity among the parental materials, and heterotic effects for all studied agronomic traits under Orobanche-free and infested soils. Orbanche infestation displayed a significant adverse impact on all the studied agronomic traits. The genotypes Line1, Line2, Line3, and Line5 displayed superior performance under Orobanche-infested conditions and recorded the highest values of all studied agronomic traits. Additionally, Line1, Line2, Line3, Line5, and Line7 exhibited desirable significant GCA for most evaluated traits under the two infestation conditions. The obtained crosses displayed significant negative or positive heterosis for studied agronomic characters such as plant height, number of branches per plant, number of pods per plant, number of seeds per plant , and seed weight per plant were observed. Furthermore, specific cross combinations such as Line2×Sakha3, Line3×Nubaria5, Line7 × Nubaria5, Line6×Nubaria1, Line5×Sakha3, Line1×Sakha3, and Line1 × Nubaria5 exhibited superior performance in seed yield and contributing traits under Orobanche-infested conditions. Moreover, these specific crosses showed superior efficacy in reducing dry weight of Orobanche spikes. The results obtained from GGE biplot analysis closely aligned with those from the line×tester procedure, affirming the significance of GGE biplot as a valuable statistical tool for assessing genotype combining ability in line× tester data. Both additive and non-additive gene actions were reported to be predominantly involved in the inheritance of the studied agronomic traits in faba bean., Conclusions: The detected genetic diversity within the evaluated faba bean genotypes and their developed crosses exhibits substantial potential for improving faba bean productivity under Orobanche-infested conditions. The parental genotypes, Line1, Line2, Line3, Line5, and Line7, were identified as effective and promising combiners. Moreover, the developed crosses Line2×Sakha3, Line3×Nubaria5, Line7×Nubaria5, Line6×Nubaria1, Line5×Sakha3, Line1×Sakha3, and Line1×Nubaria5 could be considered valuable candidates for developing high-yielding and tolerant faba bean genotypes to Orobanche., (© 2024. The Author(s).)

Orobanche foetida Poiret is the main constraint facing faba bean crop in Tunisia. Indeed, in heavily infested fields with this parasitic plant, yield losses may reach 90%, and the recent estimation of the infested area is around 80,000 ha. Identifying genes involved in the Vicia faba/O. foetida interaction is crucial for the development of effective faba bean breeding programs. However, there is currently no available information on the transcriptome of faba bean responding to O. foetida parasitism. In this study, we employed RNA sequencing to explore the global gene expression changes associated with compatible and incompatible V. faba/O. foetida interactions. In this perspective, two faba bean varieties (susceptible and resistant) were examined at the root level across three stages of O. foetida development (Before Germination (BG), After Germination (AG) and Tubercule Stage (TS)). Our analyses presented an exploration of the transcriptomic profile, including comprehensive assessments of differential gene expression and Gene Ontology (GO) enrichment analyses. Specifically, we investigated key pathways revealing the complexity of molecular responses to O. foetida attack. In this study, we detected differential gene expression of pathways associated with secondary metabolites: flavonoids, auxin, thiamine, and jasmonic acid. To enhance our understanding of the global changes in V. faba response to O. foetida, we specifically examined WRKY genes known to play a role in plant host-parasitic plant interactions. Furthermore, considering the pivotal role of parasitic plant seed germination in this interaction, we investigated genes involved in the orobanchol biosynthesis pathway. Interestingly, we detected the gene expression of VuCYP722C homolog, coding for a key enzyme involved in orobanchol biosynthesis, exclusively in the susceptible host. Clearly, this study enriches our understanding of the V. faba/O. foetida interaction, shedding light on the main differences between susceptible and resistant faba bean varieties during O. foetida infestation at the gene expression level., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Boukteb et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Key Message: The HaOr5 resistance gene is located in a large genomic insertion containing putative resistance genes and provides resistance to O. cumana, preventing successful connection to the sunflower root vascular system. Orobanche cumana (sunflower broomrape) is a parasitic plant that is part of the Orobanchaceae family and specifically infests sunflower crops. This weed is an obligate parasitic plant that does not carry out photosynthetic activity or develop roots and is fully dependent on its host for its development. It produces thousands of dust-like seeds per plant. It possesses a high spreading ability and has been shown to quickly overcome resistance genes successively introduced by selection in cultivated sunflower varieties. The first part of its life cycle occurs underground. The connection to the sunflower vascular system is essential for parasitic plant survival and development. The HaOr5 gene provides resistance to sunflower broomrape race E by preventing the connection of O. cumana to the root vascular system. We mapped a single position of the HaOr5 gene by quantitative trait locus mapping using two segregating populations. The same location of the HaOr5 gene was identified by genome-wide association. Using a large population of thousands of F2 plants, we restricted the location of the HaOr5 gene to a genomic region of 193 kb. By sequencing the whole genome of the resistant line harboring the major resistance gene HaOr5, we identified a large insertion of a complex genomic region containing a cluster of putative resistance genes., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Little is known about the microbiomes of flower parts, and even less information is available regarding these microorganisms' colonization of specific niches in parasitic plants. We investigate the temporal interspecies dynamics of the parasitic plants microbiome of flower stigmas in two stages of development: immature stigmas in flower buds and mature stigmas in opened flowers. We compared two related holoparasitic Orobanche species from localities approximately 90 km apart and characterize their bacterial and fungal communities using 16S rRNA gene and ITS sequences, respectively. We identified from 127 to over 228 OTUs per sample for fungi, sequences belonging to genera: Aureobasidium, Cladosporium, Malassezia, Mycosphaerella, and Pleosporales, constituting approximately 53% of the community in total. In the bacterial profile, we recorded 40 to over 68 OTUs per sample consisting of Enterobacteriaceae, and genera Cellulosimicrobium, Pantoea, and Pseudomonas spp., with an approximately 75% frequency. In microbial communities, higher numbers of OTUs colonizing mature stigmas were recorded than in immature. This implies that the dynamics and concurrence of microbial communities were different between O. alsatica and O. bartlingii and underwent significant changes during flower development. To the best of our knowledge, is the first study of the interspecies and temporal dynamics of the bacterial and fungal microbiomes of pistil stigmas in flowers., (© 2023. The Author(s).)

Parasitism is a successful life strategy that has evolved independently in several families of vascular plants. The genera Cuscuta and Orobanche represent examples of the two profoundly different groups of parasites: one parasitizing host shoots and the other infecting host roots. In this study, we sequenced and described the overall repertoire of small RNAs from Cuscuta campestris and Orobanche aegyptiaca. We showed that C. campestris contains a number of novel microRNAs (miRNAs) in addition to a conspicuous retention of miRNAs that are typically lacking in other Solanales, while several typically conserved miRNAs seem to have become obsolete in the parasite. One new miRNA appears to be derived from a horizontal gene transfer event. The exploratory analysis of the miRNA population (exploratory due to the absence of a full genomic sequence for reference) from the root parasitic O. aegyptiaca also revealed a loss of a number of miRNAs compared to photosynthetic species from the same order. In summary, our study shows partly similar evolutionary signatures in the RNA silencing machinery in both parasites. Our data bear proof for the dynamism of this regulatory mechanism in parasitic plants., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Key Message: Or Deb2 confers post-attachment resistance to Orobanche cumana and is located in a 1.38 Mbp genomic interval containing a cluster of receptor-like kinase and receptor-like protein genes with nine high-confidence candidates. Sunflower broomrape is a holoparasitic angiosperm that parasitizes on sunflower roots, severely constraining crop yield. Breeding for resistance is the most effective method of control. Or Deb2 is a dominant resistance gene introgressed into cultivated sunflower from a wild-related species that confers resistance to highly virulent broomrape races. The objectives of this study were as follows: (i) locate Or Deb2 into the sunflower genome and determine putative candidate genes and (ii) characterize its underlying resistance mechanism. A segregating population from a cross between the sunflower resistant line DEB2, carrying Or Deb2 , and a susceptible line was phenotyped for broomrape resistance in four experiments, including different environments and two broomrape races (F GV and G TK ). This population was also densely genotyped with microsatellite and SNP markers, which allowed locating Or Deb2 within a 0.9 cM interval in the upper half of Chromosome 4. This interval corresponded to a 1.38 Mbp genomic region of the sunflower reference genome that contained a cluster of genes encoding LRR (leucine-rich repeat) receptor-like proteins lacking a cytoplasmic kinase domain and receptor-like kinases with one or two kinase domains and lacking an extracellular LRR region, which were valuable candidates for Or Deb2 . Rhizotron and histological studies showed that Or Deb2 determines a post-attachment resistance response that blocks O. cumana development mainly at the cortex before the establishment of host-parasite vascular connections. This study will contribute to understand the interaction between crops and parasitic weeds, to establish durable breeding strategies based on genetic resistance and provide useful tools for marker-assisted selection and Or Deb2 map-based cloning., (© 2021. The Author(s).)

Phelipanche aegyptiaca and Orobanche spp. are obligate plant root-parasitic weeds that cause extensive damage in agricultural crop plants. Their germination requires exposure to strigolactones (SLs) exuded by the host plant roots. Here we studied genes in the host plant tomato involved in SL exudation and their impact on parasitic weeds. We provide evidence that CRISPR/Cas9-mediated targeted mutagenesis of two homologous ATP-binding cassette subfamily G (ABCG) genes, ABCG44 (Solyc08g067610) and ABCG45 (Solyc08g067620), in tomato significantly reduces SLs in the root exudate and abolishes germination of the root-parasitic weed P. aegyptiaca. Based on genome sequence similarity between ABCG44 and ABCG45, a 20-bp target sequence in their exon region was selected to design single guide RNA targeting both genes using CRISPR/Cas9. The plant binary vector constructs harboring the specific Cas9 and single guide RNA were transformed into tomato. Selected T 0 mutated tomato plants showed different types of deletions at both gene loci. Genotype analysis of T 1 plants suggested stable inheritance of the introduced mutations without any potential off-target effects. The phenotype of Cas9-mutated plants included increased shoot branching and growth of axillary buds, and reduced length of primary stems. Interestingly, reduced germination of P. aegyptiaca resulted from a decrease in the SL orobanchol in the root exudate of Cas9-mutated plants; however, orobanchol content in the root extract was unchanged compared to control plants. Moreover, in single and double ABCG mutants, expression of the SL-biosynthesis genes CCD8 and MAX1 decreased. The current study offers insights into CRISPR-mediated mutagenesis of ABCG genes, which could serve as an efficient control method to prevent root-parasitic weed germination.

Orobanche cumana is a holoparasitic plant that attaches to host-plant roots and seriously reduces the yield of sunflower (Helianthus annuus L.). Effective control methods are lacking with only a few known sources of genetic resistance. In this study, a seed-soak agroinoculation (SSA) method was established, and recombinant tobacco rattle virus vectors were constructed to express RNA interference (RNAi) inducers to cause virus-induced gene silencing (VIGS) in sunflower. A host target gene HaTubulin was systemically silenced in both leaf and root tissues by the SSA-VIGS approach. Trans-species silencing of O. cumana genes were confirmed for 10 out of 11 target genes with silencing efficiency of 23.43%-92.67%. Knockdown of target OcQR1, OcCKX5, and OcWRI1 genes reduced the haustoria number, and silencing of OcEXPA6 caused further phenotypic abnormalities such as shorter tubercles and necrosis. Overexpression of OcEXPA6 caused retarded root growth in alfalfa (Medicago sativa). The results demonstrate that these genes play an important role in the processes of O. cumana parasitism. High-throughput small RNA (sRNA) sequencing and bioinformatics analyses unveiled the distinct features of target gene-derived siRNAs in O. cumana such as siRNA transitivity, strand polarity, hotspot region, and 21/22-nt siRNA predominance, the latter of which was confirmed by Northern blot experiments. The possible RNAi mechanism is also discussed by analyzing RNAi machinery genes in O. cumana. Taken together, we established an efficient host-induced gene silencing technology for both functional genetics studies and potential control of O. cumana. The ease and effectiveness of this strategy could potentially be useful for other species provided they are amenable to SSA., (© American Society of Plant Biologists 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Parasitic plants have a unique heterotrophic lifestyle based on the extraction of water and nutrients from host plants. Some parasitic plant species, particularly those of the family Orobanchaceae, attack crops and cause substantial yield losses. The breeding of resistant crop varieties is an inexpensive way to control parasitic weeds, but often does not provide a long-lasting solution because the parasites rapidly evolve to overcome resistance. Understanding mechanisms underlying naturally occurring parasitic plant resistance is of great interest and could help to develop methods to control parasitic plants. In this review, we describe the virulence mechanisms of parasitic plants and resistance mechanisms in their hosts, focusing on obligate root parasites of the genera Orobanche and Striga . We noticed that the resistance (R) genes in the host genome often encode proteins with nucleotide-binding and leucine-rich repeat domains ( NLR proteins), hence we proposed a mechanism by which host plants use NLR proteins to activate downstream resistance gene expression. We speculated how parasitic plants and their hosts co-evolved and discussed what drives the evolution of virulence effectors in parasitic plants by considering concepts from similar studies of plant-microbe interaction. Most previous studies have focused on the host rather than the parasite, so we also provided an updated summary of genomic resources for parasitic plants and parasitic genes for further research to test our hypotheses. Finally, we discussed new approaches such as CRISPR/Cas9-mediated genome editing and RNAi silencing that can provide deeper insight into the intriguing life cycle of parasitic plants and could potentially contribute to the development of novel strategies for controlling parasitic weeds, thereby enhancing crop productivity and food security globally.

Broomrape is an obligate root-parasitic weed that acts as a competitive sink for host photoassimilates. Disruption of essential processes for growth of broomrape using host plant-mediated systemic signals can help to implement more specific and effective management plans of this parasite. Accordingly, we tested the possibility of transient silencing three involved genes (PaM6PR, PaCWI, and PaSUS1) in osmoregulation process of broomrape using syringe agroinfiltration of dsRNA constructs in tomato. The highest decrease in mRNA levels, enzyme activity, and amount of total reducing sugars was observed in Phelipanche aegyptiaca when grown on agroinfiltrated tomato plants by PaM6PR dsRNA construct than control. In addition, PaSUS1 dsRNA construct showed high reduction in mRNA abundance (32-fold fewer than control). The lowest decrease in mRNA levels was observed after infiltration of PaCWI dsRNA construct (eightfold fewer than control). While the highest reduction in PaM6PR and PaSUS1 expression levels was detected in the parasite at 3 days post-infiltration (dpi), the maximum reduction in both of the total reducing sugars amount and M6PR and SUS1 activities was observed at 8 dpi. On the contrary, CWI activity, PaCWI expression level, and amount of total reducing sugars in broomrape shoots simultaneously decreased at the day 3 after the dsRNA construct infiltration against PaCWI. On the whole, our results indicated that the three studied genes especially PaM6PR may constitute appropriate targets for the development of transgenic resistance in host plants using silencing strategy.

A better understanding of broomrape physiological features opens up new perspectives for developing specific management strategies. For this purpose, activities of key enzymes involved in osmoregulation (SAI1, CWI, M6PR, and SUS1) were considered at developmental stages of two important broomrape species (Egyptian and branched broomrape) on tomato. While Egyptian broomrape tubercles had high activities of invertases, branched broomrape shoots revealed high activities of M6PR and SUS1 during both pre- and post-emergence stages except for M6PR at post-emergence stages of P. aegyptiaca. Interestingly, the main accumulation of total reducing sugars was detected in tubercle during pre- and in shoot during post-emergence. Unlike low levels of genes expression (except for CWI) before parasite emergence, significantly higher expression levels of SAI1, SUS1 and M6PR were detected after parasite emergence. Matching the expression levels of SAI1 and SUS1 genes with their corresponding enzymes activities makes them as the suitable candidates for gene silencing strategies., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

We sequenced genomes of five parasite species in family Orobanchaceae to explore the evolutionary role of horizontal gene transfer in plants. Orobanche minor and Aeginetia indica are obligate parasites with no photosynthetic activity, whereas the other three (Pedicularis keiskei, Phtheirospermum japonicum, and Melampyrum roseum) are facultative parasites. By using reference genome sequences and/or transcriptomes of 14 species from Fabaceae and Poaceae, their major host families, we detected 106 horizontally transferred genes (HGT genes), only in the genomes of the two obligate parasites (22 and 84 for Oro. minor and Ae. indica, respectively), whereas none in the three facultative parasites. The HGT genes, respectively, account for roughly 0.1% and 0.2% of the coding genes in the two species. We found that almost all HGT genes retained introns at the same locations as their homologs in potential host species, indicating a crucial role of DNA-mediated gene transfer, rather than mRNA mediated retro transfer. Furthermore, some of the HGT genes might have transferred simultaneously because they located very closely in the host reference genome, indicating that the length of transferred DNA could exceed 100 kb. We confirmed that almost all introns are spliced in the current genome of the parasite species, and that about half HGT genes do not have any missense mutations or frameshift-causing indels, suggesting that some HGT genes may be still functional. Evolutionary analyses revealed that the nonsynonymous-synonymous substitution ratio is on average elevated on the lineage leading to HGT genes, due to either relaxation of selection or positive selection.

Background: Orobanche crenata represents a major biotic constraint to production of faba bean and lentil in Morocco. While this parasitic plant attacks both of these crops, the extent to which Orobanche biotypes specialise in parasitising specific crops is unknown. To address this question, we studied O. crenata that grew on different hosts and quantified their host specificity to faba bean and lentil. The virulence of O. crenata populations on each host was investigated through field trials, pot and Petri dishes assays. Genetic diversity of the parasite populations was also assessed through molecular analyses., Results: The two legume species showed distinct patterns of specificity. Faba bean was more susceptible to both O. crenata populations, while the specificity for lentil by lentil-grown O. crenata was evident at the final stage of the parasite life cycle as shown by correspondence factorial analyses. Considerable internal variation (81%) within O. crenata populations parasitising both legume species was observed by molecular analyses, but significant divergence (19%; Ø = 0.189; P = 0.010) among the populations was detected., Conclusion: These results indicate that O. crenata can adapt to specific host species, which is important knowledge when developing integrated pest management practices for parasitic weed control. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Background and Aims: The broomrapes, Orobanche sensu lato (Orobanchaceae), are common root parasites found across Eurasia, Africa and the Americas. All species native to the western hemisphere, recognized as Orobanche sections Gymnocaulis and Nothaphyllon, form a clade that has a centre of diversity in western North America, but also includes four disjunct species in central and southern South America. The wide ecological distribution coupled with moderate taxonomic diversity make this clade a valuable model system for studying the role, if any, of host-switching in driving the diversification of plant parasites., Methods: Two spacer regions of ribosomal nuclear DNA (ITS + ETS), three plastid regions and one low-copy nuclear gene were sampled from 163 exemplars of Orobanche from across the native geographic range in order to infer a detailed phylogeny. Together with comprehensive data on the parasites' native host ranges, associations between phylogenetic lineages and host specificity are tested., Key Results: Within the two currently recognized species of O. sect. Gymnocaulis, seven strongly supported clades were found. While commonly sympatric, members of these clades each had unique host associations. Strong support for cryptic host-specific diversity was also found in sect. Nothaphyllon, while other taxonomic species were well supported. We also find strong evidence for multiple amphitropical dispersals from central North America into South America., Conclusions: Host-switching is an important driver of diversification in western hemisphere broomrapes, where host specificity has been grossly underestimated. More broadly, host specificity and host-switching probably play fundamental roles in the speciation of parasitic plants., (© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Previous studies in Arabidopsis reported that the MAX2 (more axillary growth 2) gene is a component of the strigolactone (SL) signaling pathway, which regulates a wide range of biological processes, from plant growth and development to environmental stress responses. Orobanche aegyptiaca is a harmful parasitic plant for many economically important crops. Seed germination of O. aegyptiaca is very sensitive to SLs, suggesting that O. aegyptiaca may contain components of the SL signaling pathway. To investigate this hypothesis, we identified and cloned a MAX2 ortholog from O. aegyptiaca for complementation analyses using the Arabidopsis Atmax2 mutant. The so-called OaMAX2 gene could rescue phenotypes of the Atmax2 mutant in various tested developmental aspects, including seed germination, shoot branching, leaf senescence and growth and development of hypocotyl, root hair, primary root and lateral root. More importantly, OaMAX2 could enhance the drought tolerance of Atmax2 mutant, suggesting its ability to restore the drought-tolerant phenotype of mutant plants defected in AtMAX2 function. Thus, this study provides genetic evidence that the functions of the MAX2 orthologs, and perhaps the MAX2 signaling pathways, are conserved in parasitic and non-parasitic plants. Furthermore, the results of our study enable us to develop a strategy to fight against parasitic plants by suppressing the MAX signaling, which ultimately leads to enhanced productivity of crop plants., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A growing body of evidence is pointing to an important role of horizontal gene transfer (HGT) in the evolution of higher plants. However, reports of HGTs of transposable elements (TEs) in plants are still scarce, and only one case is known of a class II transposon horizontally transferred between grasses. To investigate possible TE transfers in dicots, we performed transcriptome screening in the obligate root parasite Phelipanche aegyptiaca (Orobanchaceae), data-mining in the draft genome assemblies of four other Orobanchaceae, gene cloning, gene annotation in species with genomic information, and a molecular phylogenetic analysis. We discovered that the broomrape genera Phelipanche and Orobanche acquired two related nuclear genes (christened BO transposase genes), a new group of the hAT superfamily of class II transposons, from Asian Sisymbrieae or a closely related tribe of Brassicaceae, by HGT. The collinearity of the flanking genes, lack of a classic border structure, and low expression levels suggest that BO transposase genes cannot transpose in Brassicaceae, whereas they are highly expressed in P. aegyptiaca.

Phelipanche aegyptiaca is one of the most destructive root parasitic plants of Orobanchaceae. This plant has significant impacts on crop yields worldwide. Conditioned and host root stimulants, in particular, strigolactones, are needed for unique seed germination. However, no extensive study on this phenomenon has been conducted because of insufficient genomic information. Deep RNA sequencing, including de novo assembly and functional annotation was performed on P. aegyptiaca germinating seeds. The assembled transcriptome was used to analyze transcriptional dynamics during seed germination. Key gene categories involved were identified. A total of 274,964 transcripts were determined, and 53,921 unigenes were annotated according to the NR, GO, COG, KOG, and KEGG databases. Overall, 5324 differentially expressed genes among dormant, conditioned, and GR24-treated seeds were identified. GO and KEGG enrichment analyses demonstrated numerous DEGs related to DNA, RNA, and protein repair and biosynthesis, as well as carbohydrate and energy metabolism. Moreover, ABA and ethylene were found to play important roles in this process. GR24 application resulted in dramatic changes in ABA and ethylene-associated genes. Fluridone, a carotenoid biosynthesis inhibitor, alone could induce P. aegyptiaca seed germination. In addition, conditioning was probably not the indispensable stage for P. aegyptiaca, because the transcript level variation of MAX2 and KAI2 genes (relate to strigolactone signaling) was not up-regulated by conditioning treatment.

Background: Besides gene duplication and de novo gene generation, horizontal gene transfer (HGT) is another important way of acquiring new genes. HGT may endow the recipients with novel phenotypic traits that are important for species evolution and adaption to new ecological niches. Parasitic systems expectedly allow the occurrence of HGT at relatively high frequencies due to their long-term physical contact. In plants, a number of HGT events have been reported between the organelles of parasites and the hosts, but HGT between host and parasite nuclear genomes has rarely been found., Results: A thorough transcriptome screening revealed that a strictosidine synthase-like (SSL) gene in the root parasitic plant Orobanche aegyptiaca and the shoot parasitic plant Cuscuta australis showed much higher sequence similarities with those in Brassicaceae than with those in their close relatives, suggesting independent gene horizontal transfer events from Brassicaceae to these parasites. These findings were strongly supported by phylogenetic analysis and their identical unique amino acid residues and deletions. Intriguingly, the nucleus-located SSL genes in Brassicaceae belonged to a new member of SSL gene family, which were originated from gene duplication. The presence of introns indicated that the transfer occurred directly by DNA integration in both parasites. Furthermore, positive selection was detected in the foreign SSL gene in O. aegyptiaca but not in C. australis. The expression of the foreign SSL genes in these two parasitic plants was detected in multiple development stages and tissues, and the foreign SSL gene was induced after wounding treatment in C. australis stems. These data imply that the foreign genes may still retain certain functions in the recipient species., Conclusions: Our study strongly supports that parasitic plants can gain novel nuclear genes from distantly related host species by HGT and the foreign genes may execute certain functions in the new hosts.

Phelipanche ramosa is a parasitic plant that infects numerous crops worldwide. In Western Europe it recently expanded to a new host crop, oilseed rape, in which it can cause severe yield losses. We developed 13 microsatellite markers for P. ramosa using next-generation 454 sequencing data. The polymorphism at each locus was assessed in a sample of 96 individuals collected in France within 6 fields cultivated with tobacco, hemp or oilseed rape. Two loci were monomorphic. At the other 11 loci, the number of alleles and the expected heterozygosity ranged from 3 to 6 and from 0.31 to 0.60, respectively. Genetic diversity within each cultivated field was very low. The host crop from which individuals were collected was the key factor structuring genetic variation. Individuals collected on oilseed rape were strongly differentiated from individuals collected on hemp or tobacco, which suggests that P. ramosa infecting oilseed rape forms a genetically diverged race. The microsatellites we developed will be useful for population genetics studies and for elucidating host-associated genetic divergence in P. ramosa.

Strigolactones are phytohormones that stimulate seed germination of parasitic plants including Phelipanche aegyptiaca. Strigolactones are derived from carotenoids via a pathway involving the carotenoid cleavage dioxygenases CCD7 and CCD8. We report here identification of PaCCD7 and PaCCD8 orthologous genes from P. aegyptiaca. Expression analysis of PaCCD7 and PaCCD8 genes showed significant variation in their transcript levels in seeds and tubercles of P. aegyptiaca at different developmental stages. These two parasitic PaCCD7 and PaCCD8 genes were silenced in P. aegyptiaca using a trans-silencing approach in Nicotiana benthamiana. The transient knock-down of PaCCD7 and PaCCD8 inhibited tubercle development and the infestation process in host plants. Our results suggest an important role of the strigolactone associated genes (PaCCD7 and PaCCD8) in the parasite life cycle.

Strigolactones (SLs) function as plant hormones that mediate a myriad of developmental responses in higher plants. SLs also act as an environmental signal to stimulate seed germination of parasitic plant species of genera Striga and Orobanche. In contrast to their hormonal roles, genetic mechanisms of how SLs stimulate parasitic seed germination are largely not known. Recently, we established a method to monitor the germination-stimulating activity of SLs in Arabidopsis using temperature as environmental constraint (thermoinhibition). Here, we show that SLs require HY5, a key transcription factor for light signal transduction, to stimulate Arabidopsis seed germination during thermoinhibiton. Genetic analysis suggests the HY5 dependent signaling pathway is independent of other known SL signaling pathways. Thermoinhibibed seeds expressed low level of HY5 while GR24 increase the level at both mRNA and protein level. A role of SLs on activating crucial light signaling components such as HY5 may hint the evolution of parasitism associated with SL usage.

Background and Aims: A fundamental element in the evolution of obligate root-parasitic angiosperms is their ability to germinate only in response to chemical stimulation by roots, to ensure contact with a nearby nourishing host. The aim of this study was to explore inheritance of the unique germination control in this group of plants., Methods: Analysis was made of the segregation of spontaneous (non-induced) germination that appeared in hybrid progenies derived from crosses between Orobanche cernua and O. cumana, which, like all other Orobanche species, are totally dependent on chemical stimulation for the onset of germination, and show negligible spontaneous germination in their natural seed populations., Key Results and Conclusions: F(1) and F(2) seeds did not germinate in the absence of chemical stimulation, but significant spontaneous germination was found in some F(3) seed families. This indicates that the prevention of non-induced germination in Orobanche seeds, i.e. dependence on an external chemical stimulation for seed germination, is genetically controlled, that this genetic control is expressed in a seed tissue with maternal origin (presumably the perisperm that originates from the nucellus) and that genetic variation for this trait exists in Orobanche species. Similar segregation results were obtained in reciprocal crosses, suggesting that stimulated germination is controlled by nuclear genes.

The dried succulent stems of Cistanche (Cistanche deserticola Y. C. Ma and Cistanche tubulosa Wight.) are one of the most widely used components of traditional Chinese medicines. However, it is often confused and substituted with the roots of Orobanche pycnostachya, Boschniakia rossica (Cham. & Schltdl.) Standl., Cistanche sinensis Beck, and Cistanche salsa (C. A. Mey.) Beck. In this study, we identified psbA-trnH regions from species and tested their suitable for the identification of the above mentioned taxa. The psbA-trnH sequences showed considerable variations between species and thus were revealed as a promising candidate for barcoding of Cistanche species. Additionally, the average genetic distance of psbA-trnH ranging from 0.077% to 0.743%. In contrast, the intra-specific variation among Cistanche species was found to be significantly different from those of other species, with percentages of variation studied ranged from 0% to 0.007%. The sequence difference between the psbA-trnH sequences of Cistanche species and Orobanche pycnostachya ranged from 0.979% to 1.149%. The distance between the Cistanche species and Boschniakia rossica ranged from 1.066% to 1.224%. Our results suggest that the psbA-trnH intergenic spacer region represent a barcode that can be used to identify Cistanche species and other morphologically undistinguishable species.

Orobanche spp. (broomrape) are parasitic plants which subsist on the roots of a wide range of hosts, including tomato, causing severe losses in yield quality and quantity. Large amounts of mannitol accumulate in this parasitic weed during development. Mannose 6-phosphate reductase (M6PR) is a key enzyme in mannitol biosynthesis, and it has been suggested that mannitol accumulation may be very important for Orobanche development. Therefore, the Orobanche M6PR gene is a potential target for efforts to control this parasite. Transgenic tomato plants were produced bearing a gene construct containing a specific 277-bp fragment from Orobanche aegyptiaca M6PR-mRNA, in an inverted-repeat configuration. M6PR-siRNA was detected in three independent transgenic tomato lines in the R1 generation, but was not detected in the parasite. Quantitative RT-PCR analysis showed that the amount of endogenous M6PR mRNA in the tubercles and underground shoots of O. aegyptiaca grown on transgenic host plants was reduced by 60%-80%. Concomitant with M6PR mRNA suppression, there was a significant decrease in mannitol level and a significant increase in the percentage of dead O. aegyptiaca tubercles on the transgenic host plants. The detection of mir390, which is involved with cytoplasmic dsRNA processing, is the first indication of the existence of gene-silencing mechanisms in Orobanche spp. Gene silencing mechanisms are probably involved with the production of decreased levels of M6PR mRNA in the parasites grown on the transformed tomato lines.

Holoparasitic plants such as Orobanche spp. have lost their photosynthetic ability, so photoresponses to optimize photosynthesis are not necessary in these plants. Photoresponses are also involved in the regulation of plant development but the photoresponses of holoparasites have not been characterized in detail. In this study, the phytochrome (phy)-related photoresponse of Orobanche minor was investigated. Its photoreceptor, phytochrome A (OmphyA), was also characterized. Light effects on germination, shoot elongation, anthocyanin biosynthesis, and OmphyA expression and subcellular localization were analyzed. Red light (R):far-red light (FR) reversible inhibition of O. minor seed germination demonstrated that phy-mediated responses are retained in this holoparasite. Shoot elongation was inhibited by FR but not by R. This pattern is unique among known patterns of plant photoresponses. Additionally, molecular analysis showed that OmphyA is able to respond to the light signals. Interestingly, the unique pattern of photoresponses in O. minor seems to have been modified for adaptation to its parasitic life cycle. We hypothesize that this alteration has resulted from the loss or alteration of some phy-signaling components. Elucidation of altered components in phy signaling in this parasite will provide useful information not only about its physiological characteristics but also about general plant photoreception systems.

Many parasitic angiosperms have a broad host range and are therefore considered to be host generalists. Orobanche minor is a nonphotosynthetic root parasite that attacks a range of hosts from taxonomically disparate families. In the present study, we show that O. minor sensu lato may comprise distinct, genetically divergent races isolated by the different ecologies of their hosts. Using a three-pronged approach, we tested the hypothesis that intraspecific taxa O. minor var. minor and O. minor ssp. maritima parasitizing either clover (Trifolium pratense) or sea carrot (Daucus carota ssp.gummifer), respectively, are in allopatric isolation. Morphometric analysis revealed evidence of divergence but this was insufficient to define discrete, host-specific taxa. Intersimple sequence repeat (ISSR) marker-based data provided stronger evidence of divergence, suggesting that populations were isolated from gene flow. Phylogenetic analysis, using sequence-characterized amplified region (SCAR) markers derived from ISSR loci, provided strong evidence for divergence by clearly differentiating sea carrot-specific clades and mixed-host clades. Low levels of intrapopulation SCAR marker sequence variation and floral morphology suggest that populations on different hosts are probably selfing and inbreeding. Morphologically cryptic Orobanche taxa may therefore be isolated from gene flow by host ecology. Together, these data suggest that host specificity may be an important driver of allopatric speciation in parasitic plants.

Real-time PCR has become the method of choice for accurate and in-depth expression studies of candidate genes. To avoid bias, real-time PCR is referred to one or several internal control genes that should not fluctuate among treatments. A need for reference genes in the parasitic plant Orobanche ramosa has emerged, and the studies in this area have not yet been evaluated. In this study, the genes 18S rRNA, Or-act1, Or-tub1, and Or-ubq1 were compared in terms of expression stability using the BestKeeper software program. Among the four common endogenous control genes, Or-act1 and Or-ubq1 were the most stable in O. ramosa samples. In parallel, a study was carried out studying the expression of the transcription factor Or-MYB1 that seemed to be implicated during preinfection stages. The normalization strategy presented here is a prerequisite to accurate real-time PCR expression profiling that, among other things, opens up the possibility of studying messenger RNA levels of low-copy-number-like transcription factors.

The phylogenetic relationships of the non-photosynthetic Orobanche sensu lato (Orobanchaceae), which includes some of the economically most important parasitic weeds, remain insufficiently understood and controversial. This concerns both the phylogenetic relationships within the genus, in particular its monophyly or lack thereof, and the relationships to other holoparasitic genera such as Cistanche or Conopholis. Here we present the first comprehensive phylogenetic study of this group based on a region from the plastid genome (rps2 gene). Although substitution rates appear to be elevated compared to the photosynthetic members of Orobanchaceae, relationships among the major lineages Cistanche, Conopholis plus Epifagus, Boschniakia rossica (Cham. & Schltdl.) B. Fedtsch., B. himalaica Hook. f. & Thomson, B. hookeri Walp. plus B. strobilacea A. Gray, and Orobanche s. l. remain unresolved. Resolution within Orobanche, however, is much better. In agreement with morphological, cytological and other molecular phylogenetic evidence, five lineages, corresponding to the four traditionally recognised sections (Gymnocaulis, Myzorrhiza, Orobanche, Trionychon) and O. latisquama Reut. ex Boiss. (of sect. Orobanche), can be distinguished. A combined analysis of plastid rps2 and nuclear ITS sequences of the holoparasitic genera results in more resolved and better supported trees, although the relationships among Orobanche s. l., Cistanche, and the clade including the remaining genera is unresolved. Therefore, rps2 is a marker from the plastid genome that is well-suited to be used in combination with other already established nuclear markers for resolving generic relationships of Orobanche and related genera.

Plastid sequences are among the most widely used in phylogenetic and phylogeographic studies in flowering plants, where they are usually assumed to evolve like non-recombining, uniparentally transmitted, single-copy genes. Among others, this assumption can be violated by intracellular gene transfer (IGT) within cells or by the exchange of genes across mating barriers (horizontal gene transfer, HGT). We report on HGT of a plastid region including rps2, trnL-F, and rbcL in a group of non-photosynthetic flowering plants. Species of the parasitic broomrape genus Phelipanche harbor two copies of rps2, a plastid ribosomal gene, one corresponding to the phylogenetic position of the respective species, the other being horizontally acquired from the related broomrape genus Orobanche. While the vertically transmitted copies probably reside within the plastid genome, the localization of the horizontally acquired copies is not known. With both donor and recipient being parasitic plants, a possible pathway for the exchange of genetic material is via a commonly attacked host.

We present the first study on the diversity and evolution of Ty1-copia and Ty3-gypsy retroelements in a group of non-photosynthetic flowering plants. To this end partial sequences of the reverse transcriptase (rt) gene were obtained from 20 clones for each retroelement type from seven and six accessions of Orobanche and Phelipanche (Orobanchaceae), respectively. Overall sequence similarity is higher in Ty3-gypsy elements than in Ty1-copia elements in agreement with the results from other angiosperm groups. Higher sequence diversity and stronger phylogenetic structure, especially of Ty1-copia sequences, in Orobanche species compared to Phelipanche species support the previously suggested hypothesis (based on karyological and cytological data) that genomes of Orobanche species are more dynamic than those of Phelipanche species. No evidence was found for intraspecific differences of retroelement diversity nor for differences between pest taxa and their putative wild relatives, e.g., O. crenata and O. owerini. The occurrence of a few sequences from Phelipanche species in clades otherwise comprising sequences from Orobanche species might be due to horizontal gene transfer, but the alternative of vertical transmission cannot be rejected unambiguously.

Broomrapes (Orobanche spp.) are holoparasitic weeds that cause devastating losses in many economically important crops. The molecular mechanisms that control the early stages of host infection in Orobanche are poorly understood. In the present study, the role of peroxidase has been examined during pre-infection growth and development of O. ramosa, using an in vitro model system. Peroxidase activity was histochemically localized at the tips of actively growing radicles and nascent attachment organs. Addition of exogenous catalase resulted in a significant reduction in the apical growth rate of the radicle. The prx1 gene encoding a putative class III peroxidase was cloned from a cDNA library of O. ramosa and was found to be expressed specifically during the early stages of the parasitic life cycle. The exogenous addition of sucrose resulted in significantly reduced prx1 transcript levels and in a dramatic change in radicle development from polarized apical growth to isotropic growth and the formation of tubercle-like structures. The results indicate an important role of peroxidases during the early parasitic stages of Orobanche.

Orobanche minor is a non-photosynthetic root holoparasitic plant. Although it is known that photosynthesis-related genes are inactivated or have been eliminated from the plastid genomes of holoparasites, little is known about the alterations in their genes involved in the signaling networks by which light regulates photosynthesis. Cryptochromes (crys), which are blue-light receptors, appear to control both photosynthesis-related and non-photosynthetic responses to light in higher plants. Because we are interested in to what extent a cry-mediated light signaling network remains in the holoparasites, we cloned CRY homologous cDNA from O. minor (OmCRY1) and used real-time RT-PCR to compare its expression under natural daylight and darkness. We found that the OmCRY1 has a high degree of homology with CRY1 s from photosynthetic plants. Expression of the OmCRY1 gene was higher in plants grown in the dark than that in the plants grown under natural daylight. This is the first report of the gene expression of a blue-light receptor in non-photosynthetic plants.

In this study, the isolation and characterization of a phytochrome A (PHYA) homologous cDNA (OmPHYA) in the non-photosynthetic holoparasitic plant Orobanche minor are described. The present findings provide the first report of the presence of a PHYA homolog in the holoparasite. This study found that OmPHYA is of similar size to the other PHYAs of green plants and shows 72, 77, and 77% amino acid sequence identity with PHYA in Arabidopsis, potato, and tobacco respectively. The OmPHYA contains a conserved chromophore attachment cysteine at position 323. Although OmPHYA shows high sequence identity with other PHYAs in green plants, 13 amino acid substitutions located in both the N and C-terminal domains are observed (a total of 26 amino acids). OmPHYA is encoded by a single gene within the O. minor genome. The abundance of the OmPHYA transcript as well as nuclear translocation of OmphyA occurs in a light-dependent manner.

The rbcL sequences of 106 specimens representing 28 species of the four recognized sections of Orobanche were analyzed and compared. Most sequences represent pseudogenes with premature stop codons. This study confirms that the American lineage (sects. Gymnocaulis and Myzorrhiza) contains potentially functional rbcL-copies with intact open reading frames and low rates of non-synonymous substitutions. For the first time, this is also shown for a member of the Eurasian lineage, O. coerulescens of sect. Orobanche, while all other investigated species of sects. Orobanche and Trionychon contain pseudogenes with distorted reading frames and significantly higher rates of non-synonymous substitutions. Phylogenetic analyses of the rbcL sequences give equivocal results concerning the monophyly of Orobanche, and the American lineage might be more closely related to Boschniakia and Cistanche than to the other sections of Orobanche. Additionally, species of sect. Trionychon phylogenetically nest in sect. Orobanche. This is in concordance with results from other plastid markers (rps2 and matK), but in disagreement with other molecular (nuclear ITS), morphological, and karyological data. This might indicate that the ancestor of sect. Trionychon has captured the plastid genome, or parts of it, of a member of sect. Orobanche. Apart from the phylogenetically problematic position of sect. Trionychon, the phylogenetic relationships within sect. Orobanche are similar to those inferred from nuclear ITS data and are close to the traditional groupings traditionally recognized based on morphology. The intraspecific variation of rbcL is low and is neither correlated with intraspecific morphological variability nor with host range. Ancestral character reconstruction using parsimony suggests that the ancestor of O. sect. Orobanche had a narrow host range.

Orobanche is the largest genus among the holoparasitic members of Orobanchaceae. We present the first molecular phylogenetic analysis (using nuclear ITS sequences) that includes members of all sections of Orobanche, Gymnocaulis, Myzorrhiza, Trionychon, and Orobanche. Orobanche is not monophyletic, but falls into two lineages: (1) the Orobanche group comprises Orobanche sect. Orobanche and the small Near Asian genus Diphelypaea and is characterized by a chromosome base number of x=19 and (2) the Phelipanche group contains Orobanche sects. Gymnocaulis, Myzorrhiza, and Trionychon and possesses a chromosome base number of x=12. The relationships between these two groups and to other genera such as Boschniakia or Cistanche remain unresolved. Within the Orobanche group, Orobanche macrolepis and Orobanche anatolica (including Orobanche colorata) constitute two phylogenetically distinct lineages. Intrasectional structurings proposed by some authors for O. sect. Orobanche are not confirmed by the molecular data. In most cases, intraspecific sequence divergence between accessions, if present, is negligible and not correlated with morphological or ecological traits. In a few cases, however, there is evidence for the presence of cryptic taxa.

RAPD markers were used to study variation among 20 taxa in the genus OROBANCHE: O. alba, O. amethystea, O. arenaria, O. ballotae, O. cernua, O. clausonis, O. cumana, O. crenata, O. densiflora, O. foetida, O. foetida var. broteri, O. gracilis, O. haenseleri, O. hederae, O. latisquama, O. mutelii, O. nana, O. ramosa, O. rapum-genistae and O. santolinae. A total of 202 amplification products generated with five arbitrary RAPD primers was obtained and species-specific markers were identified. The estimated Jaccard's differences between the species varied between 0 and 0.864. The pattern of interspecific variation obtained is in general agreement with previous taxonomic studies based on morphology, and the partition into two different sections (Trionychon and Orobanche) is generally clear. However, the position in the dendrogram of O. clausonis did not fit this classification since it clustered with members of section TRIONYCHON: Within this section, O. arenaria was relatively isolated from the other members of the section: O. mutelii, O. nana and O. ramosa. Within section Orobanche, all O. ramosa populations showed a similar amplification pattern, whereas differences among O. crenata populations growing on different hosts were found. Orobanche foetida and O. densiflora clustered together, supporting the morphological and cytological similarities and the host preferences of these species.

Orobanche species characterization using plastid sequences as molecular markers revealed that O. cumana contains at least two distinct rbcL sequences: one similar in size to the truncated rbcL pseudogene from O. cernua, a closely related species, and another with a size comparable to that of rbcL plastid genes from autotrophic plants. In this work, the nucleotide sequences of these two copies are reported and analysed. The organization of the O. cumana plastid genome was investigated using a long-distance PCR strategy in order to determine their localization. Because of the non-plastid localization of the rbcL larger copy, Southern blot and PCR chromosome-walking experiments were carried out to better characterize this transferred sequence and to identify its localization. Then the mode of multiple transfer of genetic information from plastid to nucleus and the concomitant plastid sequence disorganization and migration during parasitic plant evolution are discussed.