Kin Recognition in the Parasitic Plant Triphysaria versicolor Is Mediated Through Root Exudates.

Triphysaria is a facultative parasitic plant in the Orobanchaceae that parasitizes the roots of a wide range of host plants including Arabidopsis, Medicago, rice and maize. The important exception to this broad host range is that Triphysaria rarely parasitize other Triphysaria. We explored self and kin recognition in Triphysaria versicolor and showed that exudates collected from roots of host species, Arabidopsis thaliana and Medicago truncatula, induced haustorium development when applied to the roots of Triphysaria seedlings in vitro while those collected from Triphysaria did not. In mixed exudate experiments, Triphysaria exudates did not inhibit the haustorium-inducing activity of those from host roots. Interestingly, when roots of Triphysaria seedlings were treated with either horseradish peroxidase or fungal laccase, the extracts showed haustorium-inducing factor (HIF) activity, suggesting that Triphysaria roots contain the proper substrates for producing HIFs. Transgenic Triphysaria roots overexpressing a fungal laccase gene TvLCC1 showed an increased responsiveness to a known HIF, 2,6-dimethoxy benzoquinone (DMBQ), in developing haustoria. Our results indicate kin recognition in Triphysaria is associated with the lack of active HIFs in root exudates. Treatment of Triphysaria roots with enzymatic oxidases activates or releases molecules that are HIFs. This study shows that exogenously applied oxidases can activate HIFs in Triphysaria roots that had no previous HIF activity. Further studies are necessary to determine if differential oxidase activities in host and parasite roots account for the kin recognition in haustorium development.

Risk versus reward: host dependent parasite mortality rates and phenotypes in the facultative generalist Triphysaria versicolor.

BACKGROUND: Parasitic plants engage in a complex molecular dialog with potential host plants to identify a host and overcome host defenses to initiate development of the parasitic feeding organ, the haustorium, invade host tissues, and withdraw water and nutrients. While one of two critical signaling events in the parasitic plant life cycle (germination via stimulant chemicals) has been relatively well-studied, the signaling event that triggers haustorium formation remains elusive. Elucidation of this poorly understood molecular dialogue will shed light on plant-plant communication, parasitic plant physiology, and the evolution of parasitism in plants. RESULTS: Here we present an experimental framework that develops easily quantifiable contrasts for the facultative generalist parasitic plant, Triphysaria, as it feeds across a broad range of diverse flowering plants. The contrasts, including variable parasite growth form and mortality when grown with different hosts, suggest a dynamic and host-dependent molecular dialogue between the parasite and host. Finally, by comparing transcriptome datasets from attached versus unattached parasites we gain insight into some of the physiological processes that are altered during parasitic behavior including shifts in photosynthesis-related and stress response genes. CONCLUSIONS: This work sheds light on Triphysaria's parasitic life habit and is an important step towards understanding the mechanisms of haustorium initiation factor perception, a unique form of plant-plant communication.

Molecular Dialog Between Parasitic Plants and Their Hosts.

Parasitic plants steal sugars, water, and other nutrients from host plants through a haustorial connection. Several species of parasitic plants such as witchweeds (Striga spp.) and broomrapes (Orobanche and Phelipanche spp.) are major biotic constraints to agricultural production. Parasitic plants are understudied compared with other major classes of plant pathogens, but the recent availability of genomic and transcriptomic data has accelerated the rate of discovery of the molecular mechanisms underpinning plant parasitism. Here, we review the current body of knowledge of how parasitic plants sense host plants, germinate, form parasitic haustorial connections, and suppress host plant immune responses. Additionally, we assess whether parasitic plants fit within the current paradigms used to understand the molecular mechanisms of microbial plant-pathogen interactions. Finally, we discuss challenges facing parasitic plant research and propose the most urgent questions that need to be answered to advance our understanding of plant parasitism.

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.

Factors affecting the efficiency of Rhizobium rhizogenes root transformation of the root parasitic plant Triphysaria versicolor and its host Arabidopsis thaliana.

BACKGROUND: Rhizobium rhizogenes transformation is commonly used to generate transgenic roots traditionally called hairy roots, for both investigative and commercial applications. While fertile plants can be regenerated from transgenic roots, the transgenic roots are more typically used directly, either to investigate root biology or to produce valuable secondary metabolites. Hairy roots have been particularly useful for genetic studies of rhizosphere interactions; including the recognition of host plant roots by the roots of parasitic angiosperms. RESULTS: In this manuscript we analyzed various environmental, nutritional and procedural conditions for their effects on transformation of the model hemi-parasitic plant Triphysaria versicolor and Arabidopsis thaliana, one of its hosts. We first examined the effects of media, gelling agents and co-incubation times on Triphysaria root transformation and determined that while all three affected transformation rates, the media were the most significant. Once those primary conditions were fixed, we examined the roles of seedling age, explant type, acetosyringone, temperature and illumination on Triphysaria hairy root transformation rates. Using the optimized procedure approximately 70% of Triphysaria seedlings developed transgenic roots as judged by expression of YFP. These conditions were then used to transform Arabidopsis and similar transformation rates were obtained. CONCLUSIONS: Analyses of root transformation factors provides a method recovering transgenic roots from both parasitic plants and their hosts at high frequency. In addition to providing an effective in vitro approach for genetic investigations of parasitic plant-host plant interactions, these results are applicable to genetic studies of non-parasitic plants as well.

Chemical induction of hairpin RNAi molecules to silence vital genes in plant roots.

Understanding the functions encoded by plant genes can be facilitated by reducing transcript levels by hairpin RNA (hpRNA) mediated silencing. A bottleneck to this technology occurs when a gene encodes a phenotype that is necessary for cell viability and silencing the gene inhibits transformation. Here we compared the use of two chemically inducible plant promoter systems to drive hpRNA mediated gene silencing in transgenic, hairy roots. We cloned the gene encoding the Yellow Fluorescence Protein (YFP) into the dexamethasone inducible vector pOpOff2 and into the estradiol induced vector pER8. We then cloned a hpRNA targeting YFP under the regulation of the inducible promoters, transformed Medicago truncatula roots, and quantified YFP fluorescence and mRNA levels. YFP fluorescence was normal in pOpOff2 transformed roots without dexamethasone but was reduced with dexamethasone treatment. Interestingly, dexamethasone removal did not reverse YFP inhibition. YFP expression in roots transformed with pER8 was low even in the absence of inducer. We used the dexamethasone system to silence acetyl-CoA carboxylase gene and observed prolific root growth when this construct was transformed into Medicago until dexamethasone was applied. Our study shows that dexamethasone inducibility can be useful to silence vital genes in transgenic roots.

Horizontal gene transfer is more frequent with increased heterotrophy and contributes to parasite adaptation.

Horizontal gene transfer (HGT) is the transfer of genetic material across species boundaries and has been a driving force in prokaryotic evolution. HGT involving eukaryotes appears to be much less frequent, and the functional implications of HGT in eukaryotes are poorly understood. We test the hypothesis that parasitic plants, because of their intimate feeding contacts with host plant tissues, are especially prone to horizontal gene acquisition. We sought evidence of HGTs in transcriptomes of three parasitic members of Orobanchaceae, a plant family containing species spanning the full spectrum of parasitic capabilities, plus the free-living Lindenbergia Following initial phylogenetic detection and an extensive validation procedure, 52 high-confidence horizontal transfer events were detected, often from lineages of known host plants and with an increasing number of HGT events in species with the greatest parasitic dependence. Analyses of intron sequences in putative donor and recipient lineages provide evidence for integration of genomic fragments far more often than retro-processed RNA sequences. Purifying selection predominates in functionally transferred sequences, with a small fraction of adaptively evolving sites. HGT-acquired genes are preferentially expressed in the haustorium-the organ of parasitic plants-and are strongly biased in predicted gene functions, suggesting that expression products of horizontally acquired genes are contributing to the unique adaptive feeding structure of parasitic plants.

Comparative transcriptome analyses reveal core parasitism genes and suggest gene duplication and repurposing as sources of structural novelty.

The origin of novel traits is recognized as an important process underlying many major evolutionary radiations. We studied the genetic basis for the evolution of haustoria, the novel feeding organs of parasitic flowering plants, using comparative transcriptome sequencing in three species of Orobanchaceae. Around 180 genes are upregulated during haustorial development following host attachment in at least two species, and these are enriched in proteases, cell wall modifying enzymes, and extracellular secretion proteins. Additionally, about 100 shared genes are upregulated in response to haustorium inducing factors prior to host attachment. Collectively, we refer to these newly identified genes as putative "parasitism genes." Most of these parasitism genes are derived from gene duplications in a common ancestor of Orobanchaceae and Mimulus guttatus, a related nonparasitic plant. Additionally, the signature of relaxed purifying selection and/or adaptive evolution at specific sites was detected in many haustorial genes, and may play an important role in parasite evolution. Comparative analysis of gene expression patterns in parasitic and nonparasitic angiosperms suggests that parasitism genes are derived primarily from root and floral tissues, but with some genes co-opted from other tissues. Gene duplication, often taking place in a nonparasitic ancestor of Orobanchaceae, followed by regulatory neofunctionalization, was an important process in the origin of parasitic haustoria.

Trans-specific gene silencing of acetyl-CoA carboxylase in a root-parasitic plant.

Parasitic species of the family Orobanchaceae are devastating agricultural pests in many parts of the world. The control of weedy Orobanchaceae spp. is challenging, particularly due to the highly coordinated life cycles of the parasite and host plants. Although host genetic resistance often provides the foundation of plant pathogen management, few genes that confer resistance to root parasites have been identified and incorporated into crop species. Members of the family Orobanchaceae acquire water, nutrients, macromolecules, and oligonucleotides from host plants through haustoria that connect parasite and host plant roots. We are evaluating a resistance strategy based on using interfering RNA (RNAi) that is made in the host but inhibitory in the parasite as a parasite-derived oligonucleotide toxin. Sequences from the cytosolic acetyl-CoA carboxylase (ACCase) gene from Triphysaria versicolor were cloned in hairpin conformation and introduced into Medicago truncatula roots by Agrobacterium rhizogenes transformation. Transgenic roots were recovered for four of five ACCase constructions and infected with T. versicolor against parasitic weeds. In all cases, Triphysaria root viability was reduced up to 80% when parasitizing a host root bearing the hairpin ACCase. Triphysaria root growth was recovered by exogenous application of malonate. Reverse-transcriptase polymerase chain reaction (RT-PCR) showed that ACCase transcript levels were dramatically decreased in Triphysaria spp. parasitizing transgenic Medicago roots. Northern blot analysis identified a 21-nucleotide, ACCase-specific RNA in transgenic M. truncatula and in T. versicolor attached to them. One hairpin ACCase construction was lethal to Medicago spp. unless grown in media supplemented with malonate. Quantitative RT-PCR showed that the Medicago ACCase was inhibited by the Triphysaria ACCase RNAi. This work shows that ACCase is an effective target for inactivation in parasitic plants by trans-specific gene silencing.

Evolution of a horizontally acquired legume gene, albumin 1, in the parasitic plant Phelipanche aegyptiaca and related species.

BACKGROUND: Parasitic plants, represented by several thousand species of angiosperms, use modified structures known as haustoria to tap into photosynthetic host plants and extract nutrients and water. As a result of their direct plant-plant connections with their host plant, parasitic plants have special opportunities for horizontal gene transfer, the nonsexual transmission of genetic material across species boundaries. There is increasing evidence that parasitic plants have served as recipients and donors of horizontal gene transfer (HGT), but the long-term impacts of eukaryotic HGT in parasitic plants are largely unknown. RESULTS: Here we show that a gene encoding albumin 1 KNOTTIN-like protein, closely related to the albumin 1 genes only known from papilionoid legumes, where they serve dual roles as food storage and insect toxin, was found in Phelipanche aegyptiaca and related parasitic species of family Orobanchaceae, and was likely acquired by a Phelipanche ancestor via HGT from a legume host based on phylogenetic analyses. The KNOTTINs are well known for their unique "disulfide through disulfide knot" structure and have been extensively studied in various contexts, including drug design. Genomic sequences from nine related parasite species were obtained, and 3D protein structure simulation tests and evolutionary constraint analyses were performed. The parasite gene we identified here retains the intron structure, six highly conserved cysteine residues necessary to form a KNOTTIN protein, and displays levels of purifying selection like those seen in legumes. The albumin 1 xenogene has evolved through >150 speciation events over ca. 16 million years, forming a small family of differentially expressed genes that may confer novel functions in the parasites. Moreover, further data show that a distantly related parasitic plant, Cuscuta, obtained two copies of albumin 1 KNOTTIN-like genes from legumes through a separate HGT event, suggesting that legume KNOTTIN structures have been repeatedly co-opted by parasitic plants. CONCLUSIONS: The HGT-derived albumins in Phelipanche represent a novel example of how plants can acquire genes from other plants via HGT that then go on to duplicate, evolve, and retain the specialized features required to perform a unique host-derived function.

Functional genomics of a generalist parasitic plant: laser microdissection of host-parasite interface reveals host-specific patterns of parasite gene expression.

BACKGROUND: Orobanchaceae is the only plant family with members representing the full range of parasitic lifestyles plus a free-living lineage sister to all parasitic lineages, Lindenbergia. A generalist member of this family, and an important parasitic plant model, Triphysaria versicolor regularly feeds upon a wide range of host plants. Here, we compare de novo assembled transcriptomes generated from laser micro-dissected tissues at the host-parasite interface to uncover details of the largely uncharacterized interaction between parasitic plants and their hosts. RESULTS: The interaction of Triphysaria with the distantly related hosts Zea mays and Medicago truncatula reveals dramatic host-specific gene expression patterns. Relative to above ground tissues, gene families are disproportionally represented at the interface including enrichment for transcription factors and genes of unknown function. Quantitative Real-Time PCR of a T. versicolor ß-expansin shows strong differential (120x) upregulation in response to the monocot host Z. mays; a result that is concordant with our read count estimates. Pathogenesis-related proteins, other cell wall modifying enzymes, and orthologs of genes with unknown function (annotated as such in sequenced plant genomes) are among the parasite genes highly expressed by T. versicolor at the parasite-host interface. CONCLUSIONS: Laser capture microdissection makes it possible to sample the small region of cells at the epicenter of parasite host interactions. The results of our analysis suggest that T. versicolor's generalist strategy involves a reliance on overlapping but distinct gene sets, depending upon the host plant it is parasitizing. The massive upregulation of a T. versicolor ß-expansin is suggestive of a mechanism for parasite success on grass hosts. In this preliminary study of the interface transcriptomes, we have shown that T. versicolor, and the Orobanchaceae in general, provide excellent opportunities for the characterization of plant genes with unknown functions.

The TvPirin gene is necessary for haustorium development in the parasitic plant Triphysaria versicolor.

The rhizosphere is teemed with organisms that coordinate their symbioses using chemical signals traversing between the host root and symbionts. Chemical signals also mediate interactions between roots of different plants, perhaps the most obvious being those between parasitic Orobanchaceae and their plant hosts. Parasitic plants use specific molecules provided by host roots to initiate the development of haustoria, invasive structures critical for plant parasitism. We took a transcriptomics approach to identify parasitic plant genes associated with host factor recognition and haustorium signaling and previously identified a gene, TvPirin, which is transcriptionally up-regulated in roots of the parasitic plant Triphysaria versicolor after being exposed to the haustorium-inducing molecule 2,6-dimethoxybenzoquinone (DMBQ). Because TvPirin shares homology with proteins associated with environmental signaling in some plants, we hypothesized that TvPirin may function in host factor recognition in parasitic plants. We tested the function of TvPirin in T. versicolor roots using hairpin-mediated RNA interference. Reducing TvPirin transcripts in T. versicolor roots resulted in significantly less haustoria development in response to DMBQ exposure. We determined the transcript levels of other root expressed transcripts and found that several had reduced basal levels of gene expression but were similarly regulated by quinone exposure. Phylogenic investigations showed that TvPirin homologs are present in most flowering plants, and we found no evidence of parasite-specific gene duplication or expansion. We propose that TvPirin is a generalized transcription factor associated with the expression of a number of genes, some of which are involved in haustorium development.

Transformation and regeneration of the holoparasitic plant Phelipanche aegyptiaca.

BACKGROUND: 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. RESULTS: Here we demonstrate that transformation of Phelipanche aegyptiaca is achieved by infection of 3 month-old in vitro grown P. aegyptiaca calli with Agrobacterium rhizogenes 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. CONCLUSIONS: 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.

Host plant resistance to parasitic weeds; recent progress and bottlenecks.

Parasitic witchweeds (Striga spp.) and broomrapes (Orobanche and Phelipanche spp.) directly invade the roots of crop plants connecting to the vascular system and abstracting nutrients and water. As a consequence they cause devastating losses in crop yield. Genetic resistance to parasitic weeds is a highly desirable component of any control strategy. Resistance to parasitic plants can occur at different stages of the parasite lifecycle: before attachment to the host, during penetration of the root or after establishment of vascular connections. New studies are beginning to shed light on the molecular mechanisms and signaling pathways involved in plant-plant resistance. The first resistance gene to Striga, encoding a CC-NBS-LRR Resistance protein (R) has been identified and cloned suggesting that host plants resist attack from parasitic plants using similar surveillance mechanisms as those used against fungal and bacterial pathogens. It is becoming clear that the salicylic acid (SA) signaling pathway plays an important role in resistance to parasitic plants and genes encoding pathogenesis-related (PR) proteins are upregulated in a number of the resistant interactions. New strategies for engineering resistance to parasitic plants are also being explored, including the expression of parasite-specific toxins in host roots and RNAi to silence parasite genes crucial for development.

A single-electron reducing quinone oxidoreductase is necessary to induce haustorium development in the root parasitic plant Triphysaria.

Parasitic plants in the Orobanchaceae develop haustoria in response to contact with host roots or chemical haustoria-inducing factors. Experiments in this manuscript test the hypothesis that quinolic-inducing factors activate haustorium development via a signal mechanism initiated by redox cycling between quinone and hydroquinone states. Two cDNAs were previously isolated from roots of the parasitic plant Triphysaria versicolor that encode distinct quinone oxidoreductases. QR1 encodes a single-electron reducing NADPH quinone oxidoreductase similar to zeta-crystallin. The QR2 enzyme catalyzes two electron reductions typical of xenobiotic detoxification. QR1 and QR2 transcripts are upregulated in a primary response to chemical-inducing factors, but only QR1 was upregulated in response to host roots. RNA interference technology was used to reduce QR1 and QR2 transcripts in Triphysaria roots that were evaluated for their ability to form haustoria. There was a significant decrease in haustorium development in roots silenced for QR1 but not in roots silenced for QR2. The infrequent QR1 transgenic roots that did develop haustoria had levels of QR1 similar to those of nontransgenic roots. These experiments implicate QR1 as one of the earliest genes on the haustorium signal transduction pathway, encoding a quinone oxidoreductase necessary for the redox bioactivation of haustorial inducing factors.

The evolution of parasitism in plants.

The multiple independent origins of plant parasitism suggest that numerous ancestral plant lineages possessed the developmental flexibility to meet the requirements of a parasitic life style, including such adaptations as the ability to recognize host plants, form an invasive haustorium, and regulate the transfer of nutrients and other molecules between two different plants. In this review, we focus on the Orobanchaceae, which are unique among the parasitic plants in that extant member species include the full range of host dependence from facultative to obligate parasites. The recent emergence of genomic resources for these plants should provide new insights into parasitic plant evolution and enable the development of novel genetic strategies for controlling parasitic weeds.

Engineering host resistance against parasitic weeds with RNA interference.

Host genetic resistance is a key component of integrated pest management. The present authors and others are investigating the use of RNA interference (RNAi) as a genetic tool for engineering host resistance against parasitic weeds. The general approach is to transform a host plant with a plasmid encoding a double stranded hairpin RNA (hpRNA) targeted against one or more vital parasite genes. When the hpRNAs are specifically designed against parasite gene sequences, the hpRNA should have no phenotypic effect on the host. They will, however, have a dramatic effect on the parasites that have taken up the parasite-specific RNAi from the host via the haustorium. The current status of using RNAi technology for controlling parasitic weeds is reviewed. A key component to success with RNAi technology is identifying the best parasite genes to silence. Some of the criteria for RNAi targets are discussed, the existing status of parasitic plant sequence databases is described and internet access points to the parasite genome data are highlighted. Sequence information obtained from different parasite species can be used to clone the homologous gene from a particular pest or can be directly transformed into crop plants.

Trans-specific gene silencing between host and parasitic plants.

Species of Orobanchaceae parasitize the roots of nearby host plants to rob them of water and other nutrients. Parasitism can be debilitating to the host plant, and some of the world's most pernicious agricultural pests are parasitic weeds. We demonstrate here that interfering hairpin constructs transformed into host plants can silence expression of the targeted genes in the parasite. Transgenic roots of the hemi-parasitic plant Triphysaria versicolor expressing the GUS reporter gene were allowed to parasitize transgenic lettuce roots expressing a hairpin RNA containing a fragment of the GUS gene (hpGUS). When stained for GUS activity, Triphysaria roots attached to non-transgenic lettuce showed full GUS activity, but those parasitizing transgenic hpGUS lettuce lacked activity in root tissues distal to the haustorium. Transcript quantification indicated a reduction in the steady-state level of GUS mRNA in Triphysaria when they were attached to hpGUS lettuce. These results demonstrate that the GUS silencing signal generated by the host roots was translocated across the haustorium interface and was functional in the parasite. Movement across the haustorium was bi-directional, as demonstrated in double-junction experiments in which non-transgenic Triphysaria concomitantly parasitized two hosts, one transgenic for hpGUS and the other transgenic for a functional GUS gene. Observation of GUS silencing in the second host demonstrated that the silencing trigger could be moved from one host to another using the parasite as a physiological bridge. Silencing of parasite genes by generating siRNAs in the host provides a novel strategy for controlling parasitic weeds.

Agrobacterium tumefaciens and Agrobacterium rhizogenes transformed roots of the parasitic plant Triphysaria versicolor retain parasitic competence.

Parasitic plants in the Orobanchaceae invade roots of neighboring plants to rob them of water and nutrients. Triphysaria is facultative parasite that parasitizes a broad range of plant species including maize and Arabidopsis. In this paper we describe transient and stable transformation systems for Triphysaria versicolor Fischer and C. Meyer. Agrobacterium tumefaciens and Agrobacterium rhizogenes were both able to transiently express a GUS reporter in Triphysaria seedlings following vacuum infiltration. There was a correlation between the length of time seedlings were conditioned in the dark prior to infiltration and the tissue type transformed. In optimized experiments, nearly all of the vacuum infiltrated seedlings transiently expressed GUS activity in some tissue. Calluses that developed from transformed tissues were selected using non-destructive GUS staining and after several rounds of in vivo GUS selection, we recovered uniformly staining GUS calluses from which roots were subsequently induced. The presence and expression of the transgene in Triphysaria was verified using genomic PCR, RT PCR and Southern hybridizations. Transgenic roots were also obtained by inoculating A. rhizogenes into wounded Triphysaria seedlings. Stable transformed roots were identified using GUS staining or fluorescent microscopy following transformation with vectors containing GFP, dsRED or EYFP. Transgenic roots derived from both A. tumefaciens and A. rhizogenes transformations were morphologically normal and developed haustoria that attached to and invaded lettuce roots. Transgenic roots also remained competent to form haustoria in response to purified inducing factors. These transformation systems will allow an in planta assessment of genes predicted to function in plant parasitism.

Pscroph, a parasitic plant EST database enriched for parasite associated transcripts.

BACKGROUND: Parasitic plants in the Orobanchaceae develop invasive root haustoria upon contact with host roots or root factors. The development of haustoria can be visually monitored and is rapid, highly synchronous, and strongly dependent on host factor exposure; therefore it provides a tractable system for studying chemical communications between roots of different plants. DESCRIPTION: Triphysaria is a facultative parasitic plant that initiates haustorium development within minutes after contact with host plant roots, root exudates, or purified haustorium-inducing phenolics. In order to identify genes associated with host root identification and early haustorium development, we sequenced suppression subtractive libraries (SSH) enriched for transcripts regulated in Triphysaria roots within five hours of exposure to Arabidopsis roots or the purified haustorium-inducing factor 2,6 dimethoxybenzoquinone. The sequences of over nine thousand ESTs from three SSH libraries and their subsequent assemblies are available at the Pscroph database http://pscroph.ucdavis.edu. The web site also provides BLAST functions and allows keyword searches of functional annotations. CONCLUSION: Libraries prepared from Triphysaria roots treated with host roots or haustorium inducing factors were enriched for transcripts predicted to function in stress responses, electron transport or protein metabolism. In addition to parasitic plant investigations, the Pscroph database provides a useful resource for investigations in rhizosphere interactions, chemical signaling between organisms, and plant development and evolution.