Variations and reduction of plastome are associated with the evolution of parasitism in Convolvulaceae.

Parasitic lifestyle can often relax the constraint on the plastome, leading to gene pseudogenization and loss, and resulting in diverse genomic structures and rampant genome degradation. Although several plastomes of parasitic Cuscuta have  been reported, the evolution of parasitism in the family Convolvulaceae which is linked to structural variations and reduction of plastome has not been well investigated. In this study, we assembled and collected 40 plastid genomes belonging to 23 species representing four subgenera of Cuscuta and ten species of autotrophic Convolvulaceae. Our findings revealed nine types of structural variations and six types of inverted repeat (IR) boundary variations in the plastome of Convolvulaceae spp. These structural variations were associated with the shift of parasitic lifestyle, and IR boundary shift, as well as the abundance of long repeats. Overall, the degradation of Cuscuta plastome proceeded gradually, with one clade exhibiting an accelerated degradation rate. We observed five stages of gene loss in Cuscuta, including NAD(P)H complex → PEP complex → Photosynthesis-related → Ribosomal protein subunits → ATP synthase complex. Based on our results, we speculated that the shift of parasitic lifestyle in early divergent time promoted relaxed selection on plastomes, leading to the accumulation of microvariations, which ultimately resulted in the plastome reduction. This study provides new evidence towards a better understanding of plastomic evolution, variation, and reduction in the genus Cuscuta.

Comparative secretome analysis of Striga and Cuscuta species identifies candidate virulence factors for two evolutionarily independent parasitic plant lineages.

BACKGROUND: Many parasitic plants of the genera Striga and Cuscuta inflict huge agricultural damage worldwide. To form and maintain a connection with a host plant, parasitic plants deploy virulence factors (VFs) that interact with host biology. They possess a secretome that represents the complement of proteins secreted from cells and like other plant parasites such as fungi, bacteria or nematodes, some secreted proteins represent VFs crucial to successful host colonisation. Understanding the genome-wide complement of putative secreted proteins from parasitic plants, and their expression during host invasion, will advance understanding of virulence mechanisms used by parasitic plants to suppress/evade host immune responses and to establish and maintain a parasite-host interaction. RESULTS: We conducted a comparative analysis of the secretomes of root (Striga spp.) and shoot (Cuscuta spp.) parasitic plants, to enable prediction of candidate VFs. Using orthogroup clustering and protein domain analyses we identified gene families/functional annotations common to both Striga and Cuscuta species that were not present in their closest non-parasitic relatives (e.g. strictosidine synthase like enzymes), or specific to either the Striga or Cuscuta secretomes. For example, Striga secretomes were strongly associated with 'PAR1' protein domains. These were rare in the Cuscuta secretomes but an abundance of 'GMC oxidoreductase' domains were found, that were not present in the Striga secretomes. We then conducted transcriptional profiling of genes encoding putatively secreted proteins for the most agriculturally damaging root parasitic weed of cereals, S. hermonthica. A significant portion of the Striga-specific secretome set was differentially expressed during parasitism, which we probed further to identify genes following a 'wave-like' expression pattern peaking in the early penetration stage of infection. We identified 39 genes encoding putative VFs with functions such as cell wall modification, immune suppression, protease, kinase, or peroxidase activities, that are excellent candidates for future functional studies. CONCLUSIONS: Our study represents a comprehensive secretome analysis among parasitic plants and revealed both similarities and differences in candidate VFs between Striga and Cuscuta species. This knowledge is crucial for the development of new management strategies and delaying the evolution of virulence in parasitic weeds.

Cuscuta chinensis flavonoids alleviate ovarian damage in offspring female mice induced by BPA exposure during pregnancy by regulating the central carbon metabolism pathway.

Pregnancy is a sensitive window period for bisphenol A (BPA) exposure. BPA can pass through the placenta and cause reproductive damage in offspring female mice. Even BPA that is not metabolized during lactation can be passed through milk. Cuscuta chinensis flavonoids (CCFs) can alleviate reproductive damage caused by BPA, but the mechanism of action is unclear. To investigate the potential mitigating impact of CCFs on ovarian damage resulting from BPA exposure during pregnancy, we administered BPA and CCFs to pregnant mice during the gestational period spanning from 0.5 to 17.5 days. Aseptic collection of serum and ovaries from female mice was conducted on postnatal day 21 (PND21). Serum hormone levels and tissue receptor levels were quantified utilizing ELISA and PCR, while ovaries underwent sequencing and analysis through transcriptomics and metabolomics techniques. Additionally, the assessment of ovarian oxidative stress levels was carried out as part of the comprehensive analysis. The results showed that CCFs administration mitigated the adverse effects induced by BPA exposure on ovarian index, hormone levels, receptor expression, and mRNA expression levels in female offspring mice. The joint analysis of transcriptome and metabolome revealed 48 enriched pathways in positive ion mode and 44 enriched pathways in negative ion mode. Among them, the central carbon metabolism pathway is significantly regulated by BPA and CCFs. The screened sequencing results were verified through qPCR and biochemical kits. In this study, CCFs may participate in the central carbon metabolism pathway by reducing the expression of Kit proto-oncogene (Kit), hexokinase 1 gene (Hk1) and pyruvate kinase M (Pkm) mRNA and increasing the expression of h-ras proto-oncogene (Hras), sirtuin 3 (Sirt3), sirtuin 6 (Sirt6) and TP53 induced glycolysis regulatory phosphatase gene (Tigar) mRNA, thereby resisting the effects of BPA on the body. At the same time, the metabolic levels of D-Fructose 1,6-bisphosphate and L-Asparagine tend to be stable. Moreover, CCFs demonstrated a capacity to diminish the BPA-induced escalation in reactive oxygen species (ROS) and malondialdehyde (MDA). Simultaneously, it exhibited the ability to elevate levels of glutathione (GSH) and catalase (CAT), thereby effectively preventing peroxidation. In summary, CCFs alleviate BPA-induced ovarian damage in offspring female mice by regulating the central carbon metabolism pathway. This study will improve the information on BPA reproductive damage antagonist drugs and provide a theoretical basis for protecting animal reproductive health.

Cuscuta campestris fine-tunes gene expression during haustoriogenesis as an adaptation to different hosts.

The Cuscuta genus comprises obligate parasitic plants that have an unusually wide host range. Whether Cuscuta uses different infection strategies for different hosts or whether the infection strategy is mechanistically and enzymatically conserved remains unknown. To address this, we investigated molecular events during the interaction between field dodder (Cuscuta campestris) and two host species of the Solanum genus that are known to react differently to parasitic infection. We found that host gene induction, particularly of cell wall fortifying genes, coincided with a differential induction of genes for cell wall degradation in the parasite in the cultivated tomato (Solanum lycopersicum) but not in a wild relative (Solanum pennellii). This indicates that the parasite can adjust its gene expression in response to its host. This idea was supported by the increased expression of C. campestris genes encoding an endo-ß-1,4-mannanase in response to exposure of the parasite to purified mono- and polysaccharides in a host-independent infection system. Our results suggest multiple key roles of the host cell wall in determining the outcome of an infection attempt.

Complete chloroplast genomes of five Cuscuta species and their evolutionary significance in the Cuscuta genus.

BACKGROUND: Cuscuta, a parasitic plant species in the Convolvulaceae family, grows in many countries and regions. However, the relationship between some species is still unclear. Therefore, more studies are needed to assess the variation of the chloroplast (cp) genome in Cuscuta species and their relationship with subgenera or sections, thus, providing important information on the evolution of Cuscuta species. RESULTS: In the present study, we identified the whole cp genomes of C. epithymum, C. europaea, C. gronovii, C. chinensis and C. japonica, and then constructed a phylogenetic tree of 23 Cuscuta species based on the complete genome sequences and protein-coding genes. The complete cp genome sequences of C. epithymum and C. europaea were 96,292 and 97,661 bp long, respectively, and lacked an inverted repeat region. Most cp genomes of Cuscuta spp. have tetragonal and circular structures except for C. epithymum, C. europaea, C. pedicellata and C. approximata. Based on the number of genes and the structure of cp genome and the patterns of gene reduction, we found that C. epithymum and C. europaea belonged to subgenus Cuscuta. Most of the cp genomes of the 23 Cuscuta species had single nucleotide repeats of A and T. The inverted repeat region boundaries among species were similar in the same subgenera. Several cp genes were lost. In addition, the numbers and types of the lost genes in the same subgenus were similar. Most of the lost genes were related to photosynthesis (ndh, rpo, psa, psb, pet, and rbcL), which could have gradually caused the plants to lose the ability to photosynthesize. CONCLUSION: Our results enrich the data on cp. genomes of genus Cuscuta. This study provides new insights into understanding the phylogenetic relationships and variations in the cp genome of Cuscuta species.

Inflorescences of Cuscuta (Convolvulaceae): Diversity, evolution and relationships with breeding systems and fruit dehiscence modes.

Cuscuta (dodder) includes ca. 200 species of plant obligate stem parasites with enormous ecological and economical significance. Inflorescences have been historically used in Cuscuta for species descriptions and identification keys, but no comprehensive study exists to date. The main objectives of this study were to survey the diversity and evolution of inflorescences and to uncover their possible form-function relationships. The inflorescence architecture of 132 Cuscuta taxa was analysed using herbarium specimens and eight species were grown to study their inflorescence development. Inflorescence traits were mapped into a genus phylogeny obtained from a combined analysis of nuclear ITS and plastid trnL-F sequences. To test the hypothesis that inflorescence architecture is connected to sexual reproduction, correlations between inflorescence traits (using Principal Components), sexual reproductive traits (pollen/ovule ratios, corolla length and diameter), fruit charaters (fruit length and width), and the modes of dehiscence were analyzed. Based on their development, three major types of inflorescences were observed: "Cuscuta type", a simple, monochasial scorpioid cyme; "Monogynella type", a compound monochasial scorpioid cymes with the longest primary axes having prolonged vegetative growth and giving the appearance of thyrses; and "Grammica type", a compound monochasial scorpiod cymes with up to five orders of axes. Maximum likelihood analyses suggested Monogynella as the ancestral type, while Cuscuta and Grammica were derived. Overall, the total length of axes exhibited a reduction trend throughout the genus evolution, but it was not correlated with the pedicels length. Inflorescences with similar architectures may exhibit contrasting pollen-ovule ratios. Positive significant correlations were noted between the size of the flower traits and pollen-ovule ratios. Several modes of dehiscence had statistically significant different total axes lengths, suggesting that the infructescence architecture is connected to the modes of dehiscence in Cuscuta and therefore seed dispersal.

Trans-species microRNA loci in the parasitic plant Cuscuta campestris have a U6-like snRNA promoter.

Small regulatory RNAs can move between organisms and regulate gene expression in the recipient. Whether the trans-species small RNAs being exported are distinguished from the normal endogenous small RNAs of the source organism is not known. The parasitic plant Cuscuta campestris (dodder) produces many microRNAs that specifically accumulate at the host-parasite interface, several of which have trans-species activity. We found that induction of C. campestris interface-induced microRNAs is similar regardless of host species and occurs in C. campestris haustoria produced in the absence of any host. The loci-encoding C. campestris interface-induced microRNAs are distinguished by a common cis-regulatory element. This element is identical to a conserved upstream sequence element (USE) used by plant small nuclear RNA loci. The properties of the interface-induced microRNA primary transcripts strongly suggest that they are produced via U6-like transcription by RNA polymerase III. The USE promotes accumulation of interface-induced miRNAs (IIMs) in a heterologous system. This promoter element distinguishes C. campestris IIM loci from other plant small RNAs. Our data suggest that C. campestris IIMs are produced in a manner distinct from canonical miRNAs. All confirmed C. campestris microRNAs with documented trans-species activity are interface-induced and possess these features. We speculate that RNA polymerase III transcription of IIMs may allow these miRNAs to be exported to hosts.

An RNA-seq transcriptome analysis for investigating the anti-lung cancer activity of medicinal Cuscuta chinensis Lam plant.

Cuscuta chinensis Lam. is a traditional medicinal herb used to treat female sterility and male reproductive system disorders. However, the anti-lung cancer properties of Cuscuta chinensis Lam. and possible molecular mechanisms have yet to be explored. Thus, the study's main purpose was to evaluate in vitro and in vivo anti-lung cancer properties of C. chinensis water extract (CLW) in human lung adenocarcinomas and the underlying molecular mechanism involved. Our results demonstrated that CLW caused a significant inhibition of cell viability and induced G1 cycle arrest in lung cancer cells. Furthermore, RNA-seq transcriptome analysis revealed 602 common genes with a significant expression in A549 and H1650 cells under CLW treatment. Functional enrichment analysis suggested that these common genes regulated by CLW mainly involve lung cancer cell proliferation, metastases and apoptosis processes. In addition, forty-six common genes (> 2-fold change) regulated by CLW in A549 and H1650 cells were selected for further validation. In vitro quantitative real-time PCR results confirmed that twelve genes were up-regulated, and four genes were down-regulated in A549 and H1650 cells. The in vivo experiment demonstrated CLW could significantly decrease tumour volume and tumour weight of mice compared with the control group. Moreover, in vivo quantitative real-time PCR results revealed that C11orf96, FGFBP1, FOSB and NPTX1 genes were up-regulated and EGR1, GBP4 and MAP2K6 genes were down-regulated in tumour tissues compared with the control group. These data strongly suggest that CLW could be developed as an efficacious drug for lung cancer treatment.

Integrated metabolomic and transcriptomic analyses of the parasitic plant Cuscuta japonica Choisy on host and non-host plants.

BACKGROUND: Cuscuta japonica Choisy (Japanese dodder) is a parasitic weed that damages many plants and affects agricultural production. The haustorium of C. japonica plays a key role during parasitism in host plants; in contrast, some non-host plants effectively inhibit its formation. However, the metabolic differences between normal dodder in host plants and dodder inhibition in non-host plants are largely unknown. Here, we utilized an integrative analysis of transcriptomes and metabolomes to compare the differential regulatory mechanisms between C. japonica interacting with the host plant Ficus microcarpa and the non-host plant Mangifera indica. RESULTS: After parasitization for 24 h and 72 h, the differentially abundant metabolites between these two treatments were enriched in pathways associated with α-linolenic acid metabolism, linoleic acid metabolism, phenylpropanoid biosynthesis, and pyrimidine metabolism. At the transcriptome level, the flavor biosynthesis pathway was significantly enriched at 24 h, whereas the plant-pathogen interaction, arginine and proline metabolism, and MARK signaling-plant pathways were significantly enriched at 72 h, based on the differentially expressed genes between these two treatments. Subsequent temporal analyses identified multiple genes and metabolites that showed different trends in dodder interactions between the host and non-host plants. In particular, the phenylpropanoid biosynthesis pathway showed significant differential regulation between C. japonica in host and non-host plants. CONCLUSIONS: These results provide insights into the metabolic mechanisms of dodder-host interactions, which will facilitate future plant protection from C. japonica parasitism.

Parasitic plant small RNA analyses unveil parasite-specific signatures of microRNA retention, loss, and gain.

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.

Molecular Processes of Dodder Haustorium Formation on Host Plant under Low Red/Far Red (R/FR) Irradiation.

Low R/FR irradiation can promote dodder haustorium formation on the host plant; however, the mechanisms underlying the process are still unknown. In this study, we compared the transcriptomic data during the formation of haustorium of Cuscuta chinensis on host plant Arabidopsisthaliana under low (R/FR = 0.1) versus high (R/FR = 0.2) R/FR irradiation at 12 h, 24 h and 72 h time points. The results show that low R/FR radiation significantly promoted the entanglement and haustorium formation. Transcriptome analysis showed that during the early stage of haustorium formation, low R/FR radiation significantly up-regulated ARR-A related genes and down-regulated peroxidase related genes compared with high R/FR radiation. Meanwhile, during the middle stage of haustorium formation, low R/FR treatment significantly increased the expression of genes related to pectinesterase (PE), polygalacturonase (PG) and pectin lyase (Pel) production, while, during the late stage of haustorium formation, peroxidase (Prx)-related genes were differentially expressed under different R/FR treatments. Overall, our findings show that a low R/FR ratio promotes the parasitism of C. chinensis through plant hormone signal transduction and cell wall degradation pathways. This study provides a basis for the control of parasitic plants.

An artificial host system enables the obligate parasite Cuscuta campestris to grow and reproduce in vitro.

Cuscuta campestris is an obligate parasitic plant that requires a host to complete its life cycle. Parasite-host connections occur via a haustorium, a unique organ that acts as a bridge for the uptake of water, nutrients, and macromolecules. Research on Cuscuta is often complicated by host influences, but comparable systems for growing the parasite in the absence of a host do not exist. We developed an axenic method to grow C. campestris on an artificial host system (AHS). We evaluated the effects of nutrients and phytohormones on parasite haustoria development and growth. Haustorium morphology and gene expression were also characterized. The AHS consists of an inert, fibrous stick that mimics a host stem, wicking water and nutrients to the parasite. It enables C. campestris to exhibit a parasitic habit and develop through all stages of its life cycle, including production of new shoots and viable seeds. The phytohormones 1-naphthaleneacetic acid and 6-benzylaminopurine affect haustoria morphology and increase parasite fresh weight and biomass. Unigene expression in AHS haustoria reflects processes similar to those in haustoria on living host plants. The AHS is a methodological improvement for studying Cuscuta biology by avoiding specific host effects on the parasite and giving researchers full control of the parasite environment.

Unprecedented organelle genomic variations in morning glories reveal independent evolutionary scenarios of parasitic plants and the diversification of plant mitochondrial complexes.

BACKGROUND: The morning glories (Convolvulaceae) are distributed worldwide and produce economically important crops, medicinal herbs, and ornamentals. Members of this family are diverse in morphological characteristics and trophic modes, including the leafless parasitic Cuscuta (dodders). Organelle genomes were generally used for studying plant phylogeny and genomic variations. Notably, plastomes in parasitic plants always show non-canonical features, such as reduced size and accelerated rates. However, few organelle genomes of this group have been sequenced, hindering our understanding of their evolution, and dodder mitogenome in particular. RESULTS: We assembled 22 new mitogenomes and 12 new plastomes in Convolvulaceae. Alongside previously known ones, we totally analyzed organelle genomes of 23 species in the family. Our sampling includes 16 leafy autotrophic species and 7 leafless parasitic dodders, covering 8 of the 12 tribes. Both the plastid and mitochondrial genomes of these plants have encountered variations that were rarely observed in other angiosperms. All of the plastomes possessed atypical IR boundaries. Besides the gene and IR losses in dodders, some leafy species also showed gene and intron losses, duplications, structural variations, and insertions of foreign DNAs. The phylogeny reconstructed by plastid protein coding sequences confirmed the previous relationship of the tribes. However, the monophyly of 'Merremieae' and the sister group of Cuscuta remained uncertain. The mitogenome was significantly inflated in Cuscuta japonica, which has exceeded over 800 kb and integrated massive DNAs from other species. In other dodders, mitogenomes were maintained in small size, revealing divergent evolutionary strategies. Mutations unique to plants were detected in the mitochondrial gene ccmFc, which has broken into three fragments through gene fission and splicing shift. The unusual changes likely initially happened to the common ancestor of the family and were caused by a foreign insertion from rosids followed by double-strand breaks and imprecise DNA repairs. The coding regions of ccmFc expanded at both sides after the fission, which may have altered the protein structure. CONCLUSIONS: Our family-scale analyses uncovered unusual scenarios for both organelle genomes in Convolvulaceae, especially in parasitic plants. The data provided valuable genetic resources for studying the evolution of Convolvulaceae and plant parasitism.

Bidirectional lncRNA Transfer between Cuscuta Parasites and Their Host Plant.

Dodder species (Cuscuta spp.) are holoparasites that have extensive material exchange with their host plants through vascular connections. Recent studies on cross-species transfer have provided breakthrough insights, but little is known about the interaction mechanisms of the inter-plant mobile substances in parasitic systems. We sequenced the transcriptomes of dodder growing on soybean hosts to characterize the long non-coding RNA (lncRNA) transfer between the two species, and found that lncRNAs can move in high numbers (365 dodder lncRNAs and 14 soybean lncRNAs) in a bidirectional manner. Reverse transcription-polymerase chain reaction further confirmed that individual lncRNAs were trafficked in the dodder-soybean parasitic system. To reveal the potential functions of mobile transcripts, the Gene Ontology terms of mobile lncRNA target genes were predicted, and mobile dodder target genes were found to be mainly enriched in "metabolic process", "catalytic activity", "signaling", and "response to stimulus" categories, whereas mobile soybean target genes were enriched in organelle-related categories, indicating that specific mobile lncRNAs may be important in regulating dodder parasitism. Our findings reveal that lncRNAs are transferred between dodder and its host soybean plants, which may act as critical regulators to coordinate the host-dodder interaction at the whole parasitic level.

Karyotype asymmetry in Cuscuta L. subgenus Pachystigma reflects its repeat DNA composition.

Cuscuta is a cytogenetically diverse genus, with karyotypes varying 18-fold in chromosome number and 127-fold in genome size. Each of its four subgenera also presents particular chromosomal features, such as bimodal karyotypes in Pachystigma. We used low coverage sequencing of the Cuscuta nitida genome (subgenus Pachystigma), as well as chromosome banding and molecular cytogenetics of three subgenus representatives, to understand the origin of bimodal karyotypes. All three species, C. nitida, C. africana (2n = 28) and C. angulata (2n = 30), showed heterochromatic bands mainly in the largest chromosome pairs. Eighteen satellite DNAs were identified in C. nitida genome, two showing similarity to mobile elements. The most abundant were present at the largest pairs, as well as the highly abundant ribosomal DNAs. The most abundant Ty1/Copia and Ty3/Gypsy elements were also highly enriched in the largest pairs, except for the Ty3/Gypsy CRM, which also labelled the pericentromeric regions of the smallest chromosomes. This accumulation of repetitive DNA in the larger pairs indicates that these sequences are largely responsible for the formation of bimodal karyotypes in the subgenus Pachystigma. The repetitive DNA fraction is directly linked to karyotype evolution in Cuscuta.

A host-free transcriptome for haustoriogenesis in Cuscuta campestris: Signature gene expression identifies markers of successive development stages.

The development of the infection organ of the parasitic angiosperm genus Cuscuta is a dynamic process that is normally obscured from view as it happens endophytically in its host. We artificially induced haustoriogenesis in Cuscuta campestris by far-red light to define specific morphologically different stages and analyze their transcriptional patterns. This information enabled us to extract sets of high-confidence housekeeping and marker genes for the different stages, validated in a natural infection setting on a compatible host. This study provides a framework for more reproducible investigations of haustoriogenesis and the processes governing host-parasite interactions in shoot parasites, with C. campestris as a model species.

Mitochondrial genomes of two parasitic Cuscuta species lack clear evidence of horizontal gene transfer and retain unusually fragmented ccmFC genes.

BACKGROUND: The intimate association between parasitic plants and their hosts favours the exchange of genetic material, potentially leading to horizontal gene transfer (HGT) between plants. With the recent publication of several parasitic plant nuclear genomes, there has been considerable focus on such non-sexual exchange of genes. To enhance the picture on HGT events in a widely distributed parasitic genus, Cuscuta (dodders), we assembled and analyzed the organellar genomes of two recently sequenced species, C. australis and C. campestris, making this the first account of complete mitochondrial genomes (mitogenomes) for this genus. RESULTS: The mitogenomes are 265,696 and 275,898 bp in length and contain a typical set of mitochondrial genes, with 10 missing or pseudogenized genes often lost from angiosperm mitogenomes. Each mitogenome also possesses a structurally unusual ccmFC gene, which exhibits splitting of one exon and a shift to trans-splicing of its intron. Based on phylogenetic analysis of mitochondrial genes from across angiosperms and similarity-based searches, there is little to no indication of HGT into the Cuscuta mitogenomes. A few candidate regions for plastome-to-mitogenome transfer were identified, with one suggestive of possible HGT. CONCLUSIONS: The lack of HGT is surprising given examples from the nuclear genomes, and may be due in part to the relatively small size of the Cuscuta mitogenomes, limiting the capacity to integrate foreign sequences.

LATERAL ORGAN BOUNDARIES DOMAIN 25 functions as a key regulator of haustorium development in dodders.

Parasitic plants reduce crop yield worldwide. Dodder (Cuscuta campestris) is a stem parasite that attaches to its host, using haustoria to extract nutrients and water. We analyzed the transcriptome of six C. campestris tissues and identified a key gene, LATERAL ORGAN BOUNDARIES DOMAIN 25 (CcLBD25), as highly expressed in prehaustoria and haustoria. Gene coexpression networks from different tissue types and laser-capture microdissection RNA-sequencing data indicated that CcLBD25 could be essential for regulating cell wall loosening and organogenesis. We employed host-induced gene silencing by generating transgenic tomato (Solanum lycopersicum) hosts that express hairpin RNAs to target and down-regulate CcLBD25 in the parasite. Our results showed that C. campestris growing on CcLBD25 RNAi transgenic tomatoes transited to the flowering stage earlier and had reduced biomass compared with C. campestris growing on wild-type (WT) hosts, suggesting that parasites growing on transgenic plants were stressed due to insufficient nutrient acquisition. We developed an in vitro haustorium system to assay the number of prehaustoria produced on strands from C. campestris. Cuscuta campestris grown on CcLBD25 RNAi tomatoes produced fewer prehaustoria than those grown on WT tomatoes, indicating that down-regulating CcLBD25 may affect haustorium initiation. Cuscuta campestris haustoria growing on CcLBD25 RNAi tomatoes exhibited reduced pectin digestion and lacked searching hyphae, which interfered with haustorium penetration and formation of vascular connections. The results of this study elucidate the role of CcLBD25 in haustorium development and might contribute to developing parasite-resistant crops.