An improved method for the highly specific detection of transcription start sites.

Precise detection of the transcriptional start site (TSS) is a key for characterizing transcriptional regulation of genes and for annotation of newly sequenced genomes. Here, we describe the development of an improved method, designated 'TSS-seq2.' This method is an iterative improvement of TSS-seq, a previously published enzymatic cap-structure conversion method to detect TSSs in base sequences. By modifying the original procedure, including by introducing split ligation at the key cap-selection step, the yield and the accuracy of the reaction has been substantially improved. For example, TSS-seq2 can be conducted using as little as 5 ng of total RNA with an overall accuracy of 96%; this yield a less-biased and more precise detection of TSS. We then applied TSS-seq2 for TSS analysis of four plant species that had not yet been analyzed by any previous TSS method.

Developing for nutrient uptake: Induced organogenesis in parasitic plants and root nodule symbiosis.

Plants have evolved diverse strategies to meet their nutritional needs. Parasitic plants employ haustoria, specialized structures that facilitate invasion of host plants and nutrient acquisition. Legumes have adapted to nitrogen-limited conditions by developing nodules that accommodate nitrogen-fixing rhizobia. The formation of both haustoria and nodules is induced by signals originating from the interacting organisms, namely host plants and rhizobial bacteria, respectively. Emerging studies showed that both organogenesis crucially involves plant hormones such as auxin, cytokinins, and ethylene and also integrate nutrient availability, particularly nitrogen. In this review, we discuss recent advances on hormonal and environmental control of haustoria and nodules development with side-by-side comparison. These underscore the remarkable plasticity of plant organogenesis.

Balanophora genomes display massively convergent evolution with other extreme holoparasites and provide novel insights into parasite-host interactions.

Parasitic plants have evolved to be subtly or severely dependent on host plants to complete their life cycle. To provide new insights into the biology of parasitic plants in general, we assembled genomes for members of the sandalwood order Santalales, including a stem hemiparasite (Scurrula) and two highly modified root holoparasites (Balanophora) that possess chimaeric host-parasite tubers. Comprehensive genome comparisons reveal that hemiparasitic Scurrula has experienced a relatively minor degree of gene loss compared with autotrophic plants, consistent with its moderate degree of parasitism. Nonetheless, patterns of gene loss appear to be substantially divergent across distantly related lineages of hemiparasites. In contrast, Balanophora has experienced substantial gene loss for the same sets of genes as an independently evolved holoparasite lineage, the endoparasitic Sapria (Malpighiales), and the two holoparasite lineages experienced convergent contraction of large gene families through loss of paralogues. This unprecedented convergence supports the idea that despite their extreme and strikingly divergent life histories and morphology, the evolution of these and other holoparasitic lineages can be shaped by highly predictable modes of genome reduction. We observe substantial evidence of relaxed selection in retained genes for both hemi- and holoparasitic species. Transcriptome data also document unusual and novel interactions between Balanophora and host plants at the host-parasite tuber interface tissues, with evidence of mRNA exchange, substantial and active hormone exchange and immune responses in parasite and host.

Strigolactones in Rhizosphere Communication: Multiple Molecules With Diverse Functions.

Strigolactones (SLs) are root-secreted small molecules that influence organisms living in the rhizosphere. While SLs are known as germination stimulants for root parasitic plants and as hyphal branching factors for arbuscular mycorrhizal fungi, recent studies have also identified them as chemoattractants for parasitic plants, sensors of neighboring plants and key players in shaping the microbiome community. Furthermore, the discovery of structurally diverged SLs, including so-called canonical and non-canonical SLs in various plant species, raises the question of whether the same SLs are responsible for their diverse functions 'in planta' and the rhizosphere or whether different molecules play different roles. Emerging evidence supports the latter, with each SL exhibiting different activities as rhizosphere signals and plant hormones. The evolution of D14/KAI2 receptors has enabled the perception of various SLs or SL-like compounds to control downstream signaling, highlighting the complex interplay between plants and their rhizosphere environment. This review summarizes the recent advances in our understanding of the diverse functions of SLs in the rhizosphere.

Phenolic signals for prehaustorium formation in Striga hermonthica.

Striga hermonthica is a root parasitic plant that causes considerable crop yield losses. To parasitize host plants, parasitic plants develop a specialized organ called the haustorium that functions in host invasion and nutrient absorption. The initiation of a prehaustorium, the primitive haustorium structure before host invasion, requires the perception of host-derived compounds, collectively called haustorium-inducing factors (HIFs). HIFs comprise quinones, phenolics, flavonoids and cytokinins for S. hermonthica; however, the signaling pathways from various HIFs leading to prehaustorium formation remain largely uncharacterized. It has been proposed that quinones serve as direct signaling molecules for prehaustorium induction and phenolic compounds originating from the host cell wall are the oxidative precursors, but the overlap and distinction of their downstream signaling remain unknown. Here we show that quinone and phenolic-triggered prehaustorium induction in S. hermonthica occurs through partially divergent signaling pathways. We found that ASBr, an inhibitor of acetosyringone in virulence gene induction in the soil bacterium Agrobacterium, compromised prehaustorium formation in S. hermonthica. In addition, LGR-991, a competitive inhibitor of cytokinin receptors, inhibited phenolic-triggered but not quinone-triggered prehaustorium formation, demonstrating divergent signaling pathways of phenolics and quinones for prehaustorium formation. Comparisons of genome-wide transcriptional activation in response to either phenolic or quinone-type HIFs revealed markedly distinct gene expression patterns specifically at the early initiation stage. While quinone DMBQ triggered rapid and massive transcriptional changes in genes at early stages, only limited numbers of genes were induced by phenolic syringic acid. The number of genes that are commonly upregulated by DMBQ and syringic acid is gradually increased, and many genes involved in oxidoreduction and cell wall modification are upregulated at the later stages by both HIFs. Our results show kinetic and signaling differences in quinone and phenolic HIFs, providing useful insights for understanding how parasitic plants interpret different host signals for successful parasitism.

Cytokinins Induce Prehaustoria Coordinately with Quinone Signals in the Parasitic Plant Striga hermonthica.

Orobanchaceae parasitic plants are major threats to global food security, causing severe agricultural damage worldwide. Parasitic plants derive water and nutrients from their host plants through multicellular organs called haustoria. The formation of a prehaustorium, a primitive haustorial structure, is provoked by host-derived haustorium-inducing factors (HIFs). Quinones, including 2,6-dimethoxy-p-benzoquinone (DMBQ), are of the most potent HIFs for various species in Orobanchaceae, but except non-photosynthetic holoparasites, Phelipanche and Orobanche spp. Instead, cytokinin (CK) phytohormones were reported to induce prehaustoria in Phelipanche ramosa. However, little is known about whether CKs act as HIFs in the other parasitic species to date. Moreover, the signaling pathways for quinones and CKs in prehaustorium induction are not well understood. This study shows that CKs act as HIFs in the obligate parasite Striga hermonthica but not in the facultative parasite Phtheirospermum japonicum. Using chemical inhibitors and marker gene expression analysis, we demonstrate that CKs activate prehaustorium formation through a CK-specific signaling pathway that overlaps with the quinone HIF pathway at downstream in S. hermonthica. Moreover, host root exudates activated S. hermonthica CK biosynthesis and signaling genes, and DMBQ and CK inhibitors perturbed the prehaustorium-inducing activity of exudates, indicating that host root exudates include CKs. Our study reveals the importance of CKs for prehaustorium formation in obligate parasitic plants.

Strigolactones are chemoattractants for host tropism in Orobanchaceae parasitic plants.

Parasitic plants are worldwide threats that damage major agricultural crops. To initiate infection, parasitic plants have developed the ability to locate hosts and grow towards them. This ability, called host tropism, is critical for parasite survival, but its underlying mechanism remains mostly unresolved. To characterise host tropism, we used the model facultative root parasite Phtheirospermum japonicum, a member of the Orobanchaceae. Here, we show that strigolactones (SLs) function as host-derived chemoattractants. Chemotropism to SLs is also found in Striga hermonthica, a parasitic member of the Orobanchaceae, but not in non-parasites. Intriguingly, chemotropism to SLs in P. japonicum is attenuated in ammonium ion-rich conditions, where SLs are perceived, but the resulting asymmetrical accumulation of the auxin transporter PIN2 is diminished. P. japonicum encodes putative receptors that sense exogenous SLs, whereas expression of a dominant-negative form reduces its chemotropic ability. We propose a function for SLs as navigators for parasite roots.

Nitrogen represses haustoria formation through abscisic acid in the parasitic plant Phtheirospermum japonicum.

Parasitic plants are globally prevalent pathogens that withdraw nutrients from their host plants using an organ known as the haustorium. The external environment including nutrient availability affects the extent of parasitism and to understand this phenomenon, we investigated the role of nutrients and found that nitrogen is sufficient to repress haustoria formation in the root parasite Phtheirospermum japonicum. Nitrogen increases levels of abscisic acid (ABA) in P. japonicum and prevents the activation of hundreds of genes including cell cycle and xylem development genes. Blocking ABA signaling overcomes nitrogen's inhibitory effects indicating that nitrogen represses haustoria formation by increasing ABA. The effect of nitrogen appears more widespread since nitrogen also inhibits haustoria in the obligate root parasite Striga hermonthica. Together, our data show that nitrogen acts as a haustoria repressing factor and suggests a mechanism whereby parasitic plants use nitrogen availability in the external environment to regulate the extent of parasitism.

The Phtheirospermum japonicum isopentenyltransferase PjIPT1a regulates host cytokinin responses in Arabidopsis.

The hemiparasitic plant Phtheirospermum japonicum (Phtheirospermum) is a nutritional specialist that supplements its nutrient requirements by parasitizing other plants through haustoria. During parasitism, the Phtheirospermum haustorium transfers hypertrophy-inducing cytokinins (CKs) to the infected host root. The CK biosynthesis genes required for haustorium-derived CKs and the induction of hypertrophy are still unknown. We searched for haustorium-expressed isopentenyltransferases (IPTs) that catalyze the first step of CK biosynthesis, confirmed the specific expression by in vivo imaging of a promoter-reporter, and further analyzed the subcellular localization, the enzymatic function and contribution to inducing hypertrophy by studying CRISPR-Cas9-induced Phtheirospermum mutants. PjIPT1a was expressed in intrusive cells of the haustorium close to the host vasculature. PjIPT1a and its closest homolog PjIPT1b located to the cytosol and showed IPT activity in vitro with differences in substrate specificity. Mutating PjIPT1a abolished parasite-induced CK responses in the host. A homolog of PjIPT1a also was identified in the related weed Striga hermonthica. With PjIPT1a, we identified the IPT enzyme that induces CK responses in Phtheirospermum japonicum-infected Arabidopsis roots. We propose that PjIPT1a exemplifies how parasitism-related functions evolve through gene duplications and neofunctionalization.

Large-scale sequencing paves the way for genomic and genetic analyses in parasitic plants.

Parasitic plants pose a serious agricultural threat, but are also precious resources for valuable metabolites. The heterotrophic nature of these plants has resulted in the development of several morphological and physiological features that are of evolutionary significance. Recent advances in large-scale sequencing technology have provided insights into the evolutionary and molecular mechanisms of plant parasitism. Genome sequencing has revealed gene losses and horizontal gene transfers in parasitic plants. Mobile signals traveling between the parasite and host may have contributed to the increased fitness of parasitic life styles. Transcriptome analyses implicate shared processes among various parasitic species and the establishment of functional analysis is beginning to reveal molecular mechanisms during host and parasite interactions.

Subtilase activity in intrusive cells mediates haustorium maturation in parasitic plants.

Parasitic plants that infect crops are devastating to agriculture throughout the world. These parasites develop a unique inducible organ called the haustorium that connects the vascular systems of the parasite and host to establish a flow of water and nutrients. Upon contact with the host, the haustorial epidermal cells at the interface with the host differentiate into specific cells called intrusive cells that grow endophytically toward the host vasculature. Following this, some of the intrusive cells re-differentiate to form a xylem bridge (XB) that connects the vasculatures of the parasite and host. Despite the prominent role of intrusive cells in host infection, the molecular mechanisms mediating parasitism in the intrusive cells remain poorly understood. In this study, we investigated differential gene expression in the intrusive cells of the facultative parasite Phtheirospermum japonicum in the family Orobanchaceae by RNA-sequencing of laser-microdissected haustoria. We then used promoter analyses to identify genes that are specifically induced in intrusive cells, and promoter fusions with genes encoding fluorescent proteins to develop intrusive cell-specific markers. Four of the identified intrusive cell-specific genes encode subtilisin-like serine proteases (SBTs), whose biological functions in parasitic plants are unknown. Expression of SBT inhibitors in intrusive cells inhibited both intrusive cell and XB development and reduced auxin response levels adjacent to the area of XB development. Therefore, we propose that subtilase activity plays an important role in haustorium development in P. japonicum.

Three-dimensional reconstructions of haustoria in two parasitic plant species in the Orobanchaceae.

Parasitic plants infect other plants by forming haustoria, specialized multicellular organs consisting of several cell types, each of which has unique morphological features and physiological roles associated with parasitism. Understanding the spatial organization of cell types is, therefore, of great importance in elucidating the functions of haustoria. Here, we report a three-dimensional (3-D) reconstruction of haustoria from two Orobanchaceae species, the obligate parasite Striga hermonthica infecting rice (Oryza sativa) and the facultative parasite Phtheirospermum japonicum infecting Arabidopsis (Arabidopsis thaliana). In addition, field-emission scanning electron microscopy observation revealed the presence of various cell types in haustoria. Our images reveal the spatial arrangements of multiple cell types inside haustoria and their interaction with host roots. The 3-D internal structures of haustoria highlight differences between the two parasites, particularly at the xylem connection site with the host. Our study provides cellular and structural insights into haustoria of S. hermonthica and P. japonicum and lays the foundation for understanding haustorium function.

Orobanchaceae parasite-host interactions.

Parasitic plants in the family Orobanchaceae, such as Striga, Orobanche and Phelipanche, often cause significant damage to agricultural crops. The Orobanchaceae family comprises more than 2000 species in about 100 genera, providing an excellent system for studying the molecular basis of parasitism and its evolution. Notably, the establishment of model Orobanchaceae parasites, such as Triphysaria versicolor and Phtheirospermum japonicum, that can infect the model host Arabidopsis, has greatly facilitated transgenic analyses of genes important for parasitism. In addition, recent genomic and transcriptomic analyses of several Orobanchaceae parasites have revealed fascinating molecular insights into the evolution of parasitism and strategies for adaptation in this family. This review highlights recent progress in understanding how Orobanchaceae parasites attack their hosts and how the hosts mount a defense against the threats.

Ethylene signaling mediates host invasion by parasitic plants.

Parasitic plants form a specialized organ, a haustorium, to invade host tissues and acquire water and nutrients. To understand the molecular mechanism of haustorium development, we performed a forward genetics screening to isolate mutants exhibiting haustorial defects in the model parasitic plant Phtheirospermum japonicum. We isolated two mutants that show prolonged and sometimes aberrant meristematic activity in the haustorium apex, resulting in severe defects on host invasion. Whole-genome sequencing revealed that the two mutants respectively have point mutations in homologs of ETHYLENE RESPONSE 1 (ETR1) and ETHYLENE INSENSITIVE 2 (EIN2), signaling components in response to the gaseous phytohormone ethylene. Application of the ethylene signaling inhibitors also caused similar haustorial defects, indicating that ethylene signaling regulates cell proliferation and differentiation of parasite cells. Genetic disruption of host ethylene production also perturbs parasite invasion. We propose that parasitic plants use ethylene as a signal to invade host roots.

An auxin transport network underlies xylem bridge formation between the hemi-parasitic plant Phtheirospermum japonicum and host Arabidopsis.

Parasitic plants form vascular connections with host plants for efficient material transport. The haustorium is the responsible organ for host invasion and subsequent vascular connection. After invasion of host tissues, vascular meristem-like cells emerge in the central region of the haustorium, differentiate into tracheary elements and establish a connection, known as a xylem bridge, between parasite and host xylem systems. Despite the importance of this parasitic connection, the regulatory mechanisms of xylem bridge formation are unknown. Here, we show the role of auxin and auxin transporters during the process of xylem bridge formation using an Orobanchaceae hemiparasitic plant, Phtheirospermum japonicum The auxin response marker DR5 has a similar expression pattern to tracheary element differentiation genes in haustoria. Auxin transport inhibitors alter tracheary element differentiation in haustoria, but biosynthesis inhibitors do not, demonstrating the importance of auxin transport during xylem bridge formation. The expression patterns and subcellular localization of PIN family auxin efflux carriers and AUX1/LAX influx carriers correlate with DR5 expression patterns. The cooperative action of auxin transporters is therefore responsible for controlling xylem vessel connections between parasite and host.

Genome Sequence of Striga asiatica Provides Insight into the Evolution of Plant Parasitism.

Parasitic plants in the genus Striga, commonly known as witchweeds, cause major crop losses in sub-Saharan Africa and pose a threat to agriculture worldwide. An understanding of Striga parasite biology, which could lead to agricultural solutions, has been hampered by the lack of genome information. Here, we report the draft genome sequence of Striga asiatica with 34,577 predicted protein-coding genes, which reflects gene family contractions and expansions that are consistent with a three-phase model of parasitic plant genome evolution. Striga seeds germinate in response to host-derived strigolactones (SLs) and then develop a specialized penetration structure, the haustorium, to invade the host root. A family of SL receptors has undergone a striking expansion, suggesting a molecular basis for the evolution of broad host range among Striga spp. We found that genes involved in lateral root development in non-parasitic model species are coordinately induced during haustorium development in Striga, suggesting a pathway that was partly co-opted during the evolution of the haustorium. In addition, we found evidence for horizontal transfer of host genes as well as retrotransposons, indicating gene flow to S. asiatica from hosts. Our results provide valuable insights into the evolution of parasitism and a key resource for the future development of Striga control strategies.

Haustorium Inducing Factors for Parasitic Orobanchaceae.

Parasitic plants in the Orobanchaceae family include devastating weed species, such as Striga, Orobanche, and Phelipanche, which infest important crops and cause economic losses of over a billion US dollars worldwide, yet the molecular and cellular processes responsible for such parasitic relationships remain largely unknown. Parasitic species of the Orobanchaceae family form specialized invasion organs called haustoria on their roots to enable the invasion of host root tissues. The process of forming haustoria can be divided into two steps, prehaustorium formation and haustorium maturation, the processes occurring before and after host attachment, respectively. Prehaustorium formation is provoked by host-derived signal molecules, collectively called haustorium-inducing factors (HIFs). Cell wall-related quinones and phenolics have been known for a long time to induce haustoria in many Orobanchaceae species. Although such phenolics are widely produced in plants, structural specificities exist among these molecules that modulate their competency to induce haustoria in different parasitic plant species. In addition, the plant hormone cytokinins, structurally distinct from phenolic compounds, also trigger prehaustorium formation in Orobanchaceae. Recent findings demonstrate their involvement as rhizopsheric HIFs for Orobanche and Phelipanche species and thus address new activities for cytokinins in haustorium formation in Orobanchaceae, as well as in rhizospheric signaling. This review highlights haustorium-inducing signals in the Orobanchaceae family in the context of their host origin, action mechanisms, and species specificity.

Reactive Oxygen Species (ROS) Generation Is Indispensable for Haustorium Formation of the Root Parasitic Plant Striga hermonthica.

The parasitic witchweed Striga hermonthica causes devastating damage to crops in sub-Saharan Africa, yet the mechanism of its parasitism is not well understood. Parasitic plants form a special organ called a haustorium to obtain water and nutrients from host plants. The haustorium is induced by host-derived small molecules, collectively named haustorium-inducing factors (HIFs). The most active HIF known to date is 2,6-dimethoxy-p-benzoquinone (DMBQ), originally isolated from sorghum root extracts. It has been suggested that DMBQ is produced by oxidation of its precursor, syringic acid, and that reactive oxygen species (ROS) and peroxidases are involved in the process. However, the roles of ROS in haustorium formation after HIF recognition remain to be elucidated. Here, we investigated the effects of various inhibitors of ROS and ROS-regulating enzymes on haustorium formation in S. hermonthica. Inhibitors of NADPH oxidases and peroxidases inhibited haustorium formation during treatment with DMBQ, syringic acid, and host root extracts, suggesting that ROS production and/or regulation via NADPH oxidases and peroxidases are essential for haustorium formation. We observed hydrogen peroxide accumulation in the haustorium upon treatment with various HIFs. Our results suggest that ROS and ROS-regulating enzymes are indispensable in downstream signaling of HIFs for haustorium formation.

The Structural Integrity of Lignin Is Crucial for Resistance against Striga hermonthica Parasitism in Rice.

Striga species are parasitic weeds that seriously constrain the productivity of food staples, including cereals and legumes, in Sub-Saharan Africa and Asia. In eastern and central Africa, Striga spp. infest as much as 40 million hectares of smallholder farmland causing total crop failure during severe infestation. As the molecular mechanisms underlying resistance are yet to be elucidated, we undertook a comparative metabolome study using the Striga-resistant rice (Oryza sativa) cultivar 'Nipponbare' and the susceptible cultivar 'Koshihikari'. We found that a number of metabolites accumulated preferentially in the Striga-resistant cultivar upon Striga hermonthica infection. Most apparent was increased deposition of lignin, a phenylpropanoid polymer mainly composed of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) aromatic units, around the site of interaction in Nipponbare. The increased deposition of lignin was accompanied by induction of the expression of corresponding enzyme-encoding genes in the phenylpropanoid pathway. In addition, perturbing normal lignin composition by knocking down or overexpressing the genes that regulate lignin composition, i.e. p-COUMARATE 3-HYDROXYLASE or FERULATE 5-HYDROXYLASE, enhanced susceptibility of Nipponbare to S hermonthica infection. These results demonstrate that enhanced lignin deposition and maintenance of the structural integrity of lignin polymers deposited at the infection site are crucial for postattachment resistance against S hermonthica.