Comparative Metabolomics of Early Development of the Parasitic Plants Phelipanche aegyptiaca and Triphysaria versicolor.

Parasitic weeds of the family Orobanchaceae attach to the roots of host plants via haustoria capable of drawing nutrients from host vascular tissue. The connection of the haustorium to the host marks a shift in parasite metabolism from autotrophy to at least partial heterotrophy, depending on the level of parasite dependence. Species within the family Orobanchaceae span the spectrum of host nutrient dependency, yet the diversity of parasitic plant metabolism remains poorly understood, particularly during the key metabolic shift surrounding haustorial attachment. Comparative profiling of major metabolites in the obligate holoparasite Phelipanche aegyptiaca and the facultative hemiparasite Triphysaria versicolor before and after attachment to the hosts revealed several metabolic shifts implicating remodeling of energy and amino acid metabolism. After attachment, both parasites showed metabolite profiles that were different from their respective hosts. In P. aegyptiaca, prominent changes in metabolite profiles were also associated with transitioning between different tissue types before and after attachment, with aspartate levels increasing significantly after the attachment. Based on the results from 15N labeling experiments, asparagine and/or aspartate-rich proteins were enriched in host-derived nitrogen in T. versicolor. These results point to the importance of aspartate and/or asparagine in the early stages of attachment in these plant parasites and provide a rationale for targeting aspartate-family amino acid biosynthesis for disrupting the growth of parasitic weeds

CRISPR/Cas9-mediated mutagenesis of CAROTENOID CLEAVAGE DIOXYGENASE 8 in tomato provides resistance against the parasitic weed Phelipanche aegyptiaca.

Broomrapes (Phelipanche aegyptiaca and Orobanche spp.) are obligate plant parasites that cause extreme damage to crop plants. The parasite seeds have strict requirements for germination, involving preconditioning and exposure to specific chemicals strigolactones [SLs] exuded by the host roots. SLs are plant hormones derived from plant carotenoids via a pathway involving the Carotenoid Cleavage Dioxygenase 8 (CCD8). Having no effective means to control parasitic weeds in most crops, and with CRISPR/Cas9 being an effective gene-editing tool, here we demonstrate that CRISPR/Cas9-mediated mutagenesis of the CCD8 gene can be used to develop host resistance to the parasitic weed P. aegyptiaca. Cas9/single guide (sg) RNA constructs were targeted to the second exon of CCD8 in tomato (Solanum lycopersicum L.) plants. Several CCD8Cas9 mutated tomato lines with variable insertions or deletions in CCD8 were obtained with no identified off-targets. Genotype analysis of T1 plants showed that the introduced CCD8 mutations are inherited. Compared to control tomato plants, the CCD8Cas9 mutant had morphological changes that included dwarfing, excessive shoot branching and adventitious root formation. In addition, SL-deficient CCD8Cas9 mutants showed a significant reduction in parasite infestation compared to non-mutated tomato plants. In the CCD8Cas9 mutated lines, orobanchol (SL) content was significantly reduced but total carotenoids level and expression of genes related to carotenoid biosynthesis were increased, as compared to control plants. Taking into account, the impact of plant parasitic weeds on agriculture and difficulty to constitute efficient control methods, the current study offers insights into the development of a new, efficient method that could be combined with various collections of resistant tomato rootstocks

Heterogeneity in Ty1-copia group of retroelements in chickpea (Cicer arietinum) genome

Retrotransposons constitute a major fraction of plant genomes and these elements may have played a significant role in evolution and sequence organization of genomes. In order to access the diversity of Ty1-copia group of retroelements, reverse transcriptase (RT) sequences were amplified from chickpea genome, using the primers derived from two conserved domains of RT region. Thirty-six RT regions from independent amplicons were cloned and sequenced. On the basis of homology of deduced amino acids, the RT sequences could be grouped into three major families. The intra-family divergence at amino acid level ranges from 2 to 19%. Though intra-family RT sequences were conserved but no two sequences were identical. The results indicate a high degree of heterogeneity among the Ty1-copia group of retroelements from chickpea. It was possible to isolate RT specific sequences from RNA isolated from stressed seedlings, indicating that some of the retroelements may be functional under certain stress conditions

Phylogeny and historical biogeography analysis support Caucasian and Mediterranean centres of origin of key holoparasitic Orobancheae (Orobanchaceae) lineages

The extensive diversity of the tribe Orobancheae, the most species-rich lineage of holoparasitic Orobanchaceae, is concentrated in the Caucasus and Mediterranean regions of the Old World. This extant diversity has inspired hypotheses that these regions are also centres of origin of its key lineages, however the ability to test hypotheses has been limited by a lack of sampling and phylogenetic information about the species, especially in the Caucasus region. First, we assessed the phylogenetic relationships of several poorly known, problematic, or newly described species and host-races of four genera of Orobancheae occurring in the Caucasus region–Cistanche, Phelypaea, Phelipanche and Orobanche–using nuclear ribosomal (ITS) and plastid (trnL–trnF) sequence data. Then we applied a probablistic dispersal-extinction-cladogenesis model of historical biogeography across a more inclusive clade of holoparasites, to explicitly test hypotheses of Orobancheae diversification and historical biogeography shifts. In sum, we sampled 548 sequences (including 196 newly generated) from 13 genera, 140 species, and 175 taxa across 44 countries. We find that the Western Asia (particularly the Caucasus) and the Mediterranean are the centre of origin for large clades of holoparasitic Orobancheae within the last 6 million years. In the Caucasus, the centres of diversity are composed both of long-branch taxa and shallow, recently diversified clades, while Orobancheae diversity in the Mediterranean appears to represent mainly recent diversification

Effects of some cereal root exudates on germination of broomrapes (Orobanche spp. and Phelipanche spp.)

Species of broomrape (Orobanche spp. and Phelipanche spp.) are among the most damaging parasitic weed species worldwide. These species reproduce through abundant seed production. Their seeds are protected by complex dormancy mechanisms, in particular a need for host-specific chemical germination cues. Broomrape seeds have been shown to remain viable in the soil for many years. While the depletion of the soil seed bank, e.g. using trap crops that induce suicide germination of broomrape seeds, could potentially be a way to control these weeds, the practical uptake of this approach has remained very limited. To explore the potential of an array of cereal species to serve as trap crop, laboratory experiments were conducted to qualitatively check for the existence of cereal-broomrape interactions and to quantify possible effects on Orobanche/Phelipanche seed banks. For this purpose, seeds of the following cereals were used: wheat, rye, barley, oats, maize, rice, sorghum and pearl millet. Several accessions of O. crenata, O. cumana and P. ramosa were used as parasite species. As host species, pea, sunflower and tomato were grown. Cereal and parasite species were crosswise-combined to assess interactions. Maize was found to be most effective in stimulating the germination of the broomrapes. Among the parasite species, P. ramosa proved most ready to germinate in the presence of cereal root exudates. The interaction was observed in various combinations of maize cultivars and P. ramosa accessions. As a result, strong evidence of germination induction in P. ramosa seeds by maize was collected.Canavar otu türleri (Orobanche spp. and Phelipanche spp.) tüm dünyada en zararlı parazit yabancı ot türleri içerisinde yer almaktadır. Bu türler çok sayıda tohum oluşturarak çoğalırlar. Tohumları, çimlenmenin gerçekleşebilmesi için konukçuya özgü kimyasal bir maddeye ihtiyaç duyan karmaşık bir dormansi mekanizmasıyla korunmaktadır. Canavar otu tohumlarının toprakta uzun yıllar canlı kalabildiği görülmektedir. Tuzak bitki kullanımı ile canavar otu tohumlarının çimlenmesi teşvik edilerek topraktaki tohum bankasının azaltılması bu tür yabancı otların mücadelesinde potansiyel bir yöntem olmakla birlikte, bu yaklaşımın pratikte kullanımı oldukça sınırlı kalmıştır. Bazı tahıl türlerinin tuzak bitki olarak potansiyelini araştırmak amacıyla bir seri laboratuar denemeleri kurulmuş ve tahıl-canavar otu arasındaki etkileşim ile birlikte, Orobanche/Phelipanche tohum bankasına muhtemel etkisi test edilmiştir. Bu amaçla tahıllardan; buğday, çavdar, arpa, yulaf, mısır, çeltik, darı ve hint darısı, parazit türlerden ise O. crenata, O. cumana ve P. ramosa ‘ya ait çeşitli populasyonlar kullanılmıştır. Bezelye, ayçiçeği ve domates konukçu bitki türü olarak yetiştirilmiştir. Aradaki etkileşimin belirlenebilmesi için tahıllar ve parazit türler çaprazlama olarak eşleştirilmiştir. Canavar otlarının çimlenmesini en fazla mısır bitkisinin teşvik ettiği bulunmuştur. Parazit türler içerisinde ise tahıl kök salgılarının çimlenmeyi en fazla teşvik ettiği tür P. ramosa olarak belirlenmiştir. Mısır çeşitleri ve P. ramosa populasyonları arasındaki interaksiyonlar gözlemlenmiştir. Sonuç olarak, mısır bitkisinin P. ramosa tohumlarının çimlenmesini teşvik ettiğine dair kuvvetli kanıtlar elde edilmiştir

Transformation and regeneration of the holoparasitic plant Phelipanche aegyptiaca

<p>Abstract</p> <p>Background</p> <p>Transformation and subsequent regeneration of holoparasitic plants has never been reported, in part due to challenges in developing transformation protocols, but also because regeneration of obligate parasites is difficult since their survival depends completely on successful haustorium penetration of a host and the formation of vascular connections. The recent completion of a massive transcriptome sequencing project (the Parasitic Plant Genome Project) will fuel the use of genomic tools for studies on parasitic plants. A reliable system for holoparasite transformation is needed to realize the full value of this resource for reverse genetics and functional genomics studies.</p> <p>Results</p> <p>Here we demonstrate that transformation of <it>Phelipanche aegyptiaca </it>is achieved by infection of 3 month-old <it>in vitro </it>grown <it>P. aegyptiaca </it>calli with <it>Agrobacterium rhizogenes </it>harboring the yellow fluorescent protein (YFP). Four months later, YFP-positive regenerated calli were inoculated onto tomato plants growing in a minirhizotron system. Eight days after inoculation, transgenic parasite tissue formed lateral haustoria that penetrated the host and could be visualized under UV illumination through intact host root tissue. YFP-positive shoot buds were observed one month after inoculation.</p> <p>Conclusions</p> <p>This work constitutes a breakthrough in holoparasitic plant research methods. The method described here is a robust system for transformation and regeneration of a holoparasitic plant and will facilitate research on unique parasitic plant capabilities such as host plant recognition, haustorial formation, penetration and vascular connection.</p

Phelipanche cernua Pomel (Orobanchaceae), a prioritary name for the western mediterranean species recently redescribed as Ph. inexpectata

The specific parasite of Lactuca (Compositae) described in 2005 from the Iberian Peninsula as Phelipanche inexspectata (Orobanchaceae) and known so far in northeastern Spain and southern France, is shown to also occur in mountain areas of northern Africa, where it had been previously described under the neglected name Phelipanche cernua. Given the recent proposal to consider the aforementioned Lactuca parasite a mere variant of Phelipanche schultzii, we stress the neat differences between both species

Pollen morphology of the Central European broomrapes (Orobanchaceae: Orobanche, Phelipanche and Orobanchella) and its taxonomical implications

Pollen grains of 450 samples of 25 species of the genus Orobanche and Phelipanche occurring in Central Europe were investigated using light and scanning electron microscopy. Palynological data on 18 species are reported here for the first time. The usefulness of micromorphological studies on pollen of Orobanche and Phelipanche is demonstrated. Previous research showed the separation of Phelipanche from Orobanche, a finding which is also supported by pollen aperture type and exine ornamentation. The pollen of Phelipanche is tricolpate, while that of Orobanche is inaperturate. Our research showed that one of the species included so far within Orobanche, namely O. coerulescens, has tricolpate pollen with microreticulate sculpture. As a consequence of the finding of tricolpate pollen and intermediate macromorphological characteristics of this species between Orobanche and Phelipanche, it is proposed to transfer it to a new genus, Orobanchella, described in the paper. A comparison of the main characteristics distinguishing Orobanche, Phelipanche, Boulardia and Orobanchella is also presented and discussed

The first evidence of a host-to-parasite mitochondrial gene transfer in Orobanchaceae

Several parasitic plants are known to have acquired mitochondrial genes via a horizontal transfer from their hosts. However, mitochondrial gene transfer in this direction has not yet been found in the parasite-rich family Orobanchaceae. Based on a phylogenetic analysis of the mitochondrial atp6 gene in selected species of Orobanche s.l., we provide evidence of a host-to-parasite transfer of this gene in O. coerulescens, which is a Eurasiatic species that parasitises Artemisia (Asteraceae). We did not find the original Orobanche atp6 gene in this species, which suggests that it has been replaced by a gene that was acquired from Asteraceae. In addition, our data suggest the occurrence of a second HGT event in the atp6 sequence - from Asteraceae to Phelipanche. Our results support the view that the transfer of genetic material from hosts to parasites influences the mitochondrial genome evolution in the latter

Phylogenetic relationships within Orobanche and Phelipanche (Orobanchaceae) from Central Europe, focused on problematic aggregates, taxonomy, and host ranges

Holoparasitic genera within the family Orobanchaceae are characterized by greatly reduced vegetative organs; therefore, molecular analysis has proved to be a useful tool in solving taxonomic problems in this family. For this purpose, we studied all species of the genera Orobanche and Phelipanche occurring in Central Europe, specifically in Poland, the Czech Republic, Slovakia, and Austria, supplemented by samples mainly from Spain, France, Germany, and Ukraine. They were investigated using nuclear sequences (ITS region) and a plastid trnL- trnF region. The aim of this study was to examine phylogenetic relationships within Orobanche and Phelipanche from Central Europe; we focused on problematic species and aggregates, recent taxonomic changes in these (rank and secondary ranks), and host ranges. The most interesting results concern the exlusion of O. mayeri from O. alsatica aggr. Additionally, following the rules of traditional taxonomy, the correct names and types of some secondary ranks are given and, as a result of this, a new combination below the Phelipanche genus is made ( P . sect. Trionychon ). The host ranges of the investigated species in Central Europe include 102 species from 12 families, most often from Asteraceae. For this purpose, ca. 400 localities were examined in the field. Moreover, data acquired from the literature and European and Asian herbaria were use