INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION CONCLUSIONS MATERIALS AND METHODS LITERATURE CITED

Parasitic plants in the Scrophulariaceae invade the roots of neighboring plants and rob them of water, minerals, and nutrients (Kuijt, 1969; Press and Graves, 1995). The consequence to the parasitized plants can be debilitating and some of the world's most destructive agricultural pests are parasitic weeds (Parker and Riches, 1993). Parasitic Scrophulariaceae use allelopathic chemicals that are released by host plan roots to trigger developmental programs critical to the parasitic lifestyle. One such program results in the development of haustoria, specialized root structures that attach to and invade host roots. Once a haustorium has invaded the host root, it serves as the physiological conduit between the parasite and host roots through which the parasite robs host plant resources (Riopel and Timko, 1995). Parasitic Scrophulariaceae typically develop haustoria only in the presence of neighboring plants. Haustorium development can be readily monitored in vitro by applying host factors to parasite roots (Atsatt et al., 1978; Riopel and Musselman, 1979).

The first natural haustorium-inducing factor identified from host roots was 2,6-dimethoxybenzoquinone (DMBQ; Chang and Lynn, 1986). DMBQ induces haustorium development in both obligate and facultative parasites within hours of applying it to their roots (Baird and Riopel, 1984; Chang and Lynn, 1986; Albrecht et al., 1999). DMBQ and related quinones are secondary metabolites widespread in diverse plant families (Handa et al., 1983). These molecules are released into the rhizosphere as components of root exudates and as byproducts of fungal-mediated lignin degradation. Quinones are also produced through the oxidation of phenolic acids, another major constituent of root exudates (Thomson, 1987; Siqueira et al., 1991).

Several naturally occurring quinones and phenolics induce haustoria when applied to parasite roots (Steffens et al., 1982; Riopel and Timko, 1995; Albrecht et al., 1999). An important cue as to how structurally different molecules induce haustoria was obtained from the observation that the redox potentials of active factors fall within a relatively narrow window (Smith et al., 1990, 1996). The design of chemical inhibitors that trap radical intermediates established that haustorium induction is associated with redox cycling between quinone and semiquinone states (Zeng et al., 1996). Redox cycling also generates reactive oxygen intermediates that are largely responsible for quinone toxicity (Obrien, 1991). In fact, DMBQ has been investigated as a possible anticancer agent because of its mammalian cytotoxicity (Brambilla et al., 1988). Parasitic plants have recruited these potentially harmful intermediates to signal haustorium organogenesis.

We would like to understand how parasitic plants perceive and process allelopathic quinones. We are using Triphysaria, previously Orthocarpus (Chuang and Heckard, 1991), a small parasitic Scrophulariaceae that grows as a spring-time annual in coastal bluffs and inland grasslands throughout the Pacific coast, for these studies. Triphysaria is a facultative parasite that can be grown to maturity in the absence of host plants but will parasitize a broad spectrum of hosts, including Arabidopsis and maize (Zea mays; Thurman, 1966; Yoder, 1997). Haustorium development is rapid and can be monitored in vitro by applying host root exudates or haustoria inducing factors to Triphysaria roots (Atsatt et al., 1978; Albrecht et al., 1999). Triphysaria is a simple diploid whose perfect flowers are amenable to genetic manipulations (Chuang and Heckard, 1991; Yoder, 1998). Triphysaria is closely related to the parasitic weeds Striga and Orobanche and can serve as a useful model for these pernicious agricultural pests (Nickrent et al., 1998).

To begin the dissection of genetic pathways governing haustorium development, we characterized cDNAs homologous to transcripts up-regulated in Triphysaria versicolor roots exposed to DMBQ. We asked whether these transcripts were similarly regulated by host root factors by northern and reverse northern analyses. These represent plant genes regulated by subterranean signals produced by other plants. By categorizing the putative functions of DMBQ responsive genes, we can generate models that highlight potential pathways associated with quinone signaling and haustorium morphogenesis.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION CONCLUSIONS MATERIALS AND METHODS LITERATURE CITED

Early Morphological Responses of T. versicolor Roots to DMBQ

Morphological changes associated with early haustorium development were observed in T. versicolor root tips shortly after exposure to either 10 µM DMBQ or root exudates from hydroponically grown maize. Haustorium ontogeny in T. versicolor was similar to that described for other secondary haustoria (Atsatt et al., 1978; Riopel and Baird, 1987). The first change observed was a termination of root elongation within 30 min of DMBQ treatment (Fig. 1; data not shown). In Striga ariatica, this period is also hallmarked by a cessation of DNA synthesis (O'Malley and Lynn, 2000). Within 5 h of treatment, epidermal hairs could be seen to elongate and proliferate in the region just behind the root tip (Fig. 1). These hairs function to attach the developing haustorium to the host tissue (Baird and Riopel, 1985; Heide-Jorgensen and Kuijt, 1995). At about this same time, cortical cells underlying the proliferating epidermal hairs began to swell. This resulted in a swelling near the root tip noticeable within 8 h of treatment. The swelling and proliferation of haustorial hairs continued for about 24 h during which time the haustorium was competent to attach to a host root (J.I. Yoder, unpublished data). Under our induction conditions, approximately 70% to 80% of the root tips formed haustoria after DMBQ treatment, whereas virtually no haustoria developed when treated with water.

View larger version (19K): [in this window] [in a new window]   Figure 1.   Time course of haustorium development. T. versicolor were grown on agar media in vertically oriented petri dishes so that their roots grew along the surface of the agar. The roots were then exposed to 10 µM DMBQ and their tips photographed at different times under 2× magnification with a dissecting microscope. The pictures in the figure are a time lapse of the same root. The arrowhead to the right in each picture represents the position of the root tip when the treatment was applied.

Haustorium development in T. versicolor roots was transitory and after about 12 h the root tips reverted to their typical developmental program. This resulted in a normal appearing root distal to the haustorium. The reversion to normal root growth occurred even in the presence of active inducing factors because fresh T. versicolor sown onto these plates formed haustoria. Also, the same root made multiple haustoria if the root came out of contact with the media for a short period of time and then retouched the surface. Similar observations have been made for Striga root cultures (Wolf and Timko, 1992).

Early Transcriptional Responses to DMBQ and Host Exudate Exposure

We made a normalized, subtracted cDNA library enriched for transcripts up-regulated in T. versicolor root tips 2 to 5 h after treatment with DMBQ. Following two rounds of colony hybridization with forward- and reverse-subtracted cDNA probes (see "Materials and Methods"), several hundred clones were selected for further characterization. Inserts from 30 cDNAs were PCR amplified and used as probes on northern blots containing RNA from T. versicolor roots exposed to either DMBQ or water. As seen in Figure 2 and summarized in Table I, five transcripts that were in low abundance in water- treated roots, were strongly up-regulated by DMBQ treatment. Ten cDNAs hybridized to transcripts with basal levels in untreated roots that increased upon DMBQ treatment; these are labeled as "moderate" in Table I. The steady-state levels of transcripts hybridizing to six cDNA clones were unchanged by DMBQ. These may be false positives that were carried through the screening. Hybridization to nine cDNA probes was very weak and it was not possible to conclude whether these transcripts were regulated. From these results we predict that between 50% and 80% of the clones in the subtractive library were up-regulated by DMBQ, about 10% of these strongly.

View larger version (52K): [in this window] [in a new window]   Figure 2.   Northern analysis using early DMBQ-induced transcript (EDIT) probes. Northern filters were prepared from mRNA extracted from T. versicolor root tips exposed to either water (left lane []) or DMBQ (right lane [+]) for 5 h. Inserts of 30 cDNAs were PCR amplified, labeled with 32P, and used as hybridization probes. The putative functions of these clones as predicted from virtual translations are given in Table I.

Host root exudates were made by growing maize seedlings in culture for 2 weeks and recovering the supernatant after centrifuging the agar. Maize root exudates induced haustoria in T. versicolor roots with the same frequency and time course as DMBQ. RNA was extracted from exudate-treated roots and analyzed by northern hybridizations. Three cDNAs, 3K19, 1H09, and 1L17, hybridized to transcripts strongly up-regulated by maize root exudate; these were similarly up-regulated in T. versicolor by DMBQ (Fig. 2). A fourth cDNA (3F08) was not up-regulated by either treatment (Fig. 3; data not shown).

View larger version (44K): [in this window] [in a new window]   Figure 3.   Transcription regulation by maize root exudate. A northern blot was prepared containing RNA isolated from T. versicolor roots mock treated with water () or with maize root exudate (+) for 4 h. The blot was sequentially probed with clones 3K19, 1H09, 1L17, and 3F08.

We used reverse northerns to obtain an independent estimate of the proportion of DMBQ regulated clones also up-regulated by host root exudates. Inserts from 141 clones were PCR amplified and arrayed in duplicate on a nylon membrane. The filter was sequentially hybridized with radioactive cDNA probes made from mRNA isolated from DMBQ-treated and untreated roots. Most of the inserts did not hybridize with these probes at levels over significantly over background. This is consistent with previous observations that ³²P-labeled probes synthesized from complex cDNA pools generally detect only highly abundant transcripts (Wang and Brown, 1991). Even when hybridization was detected there were relatively few differences between induced and noninduced conditions (Fig. 4C). This can result if the cDNA probes cross hybridize to nonregulated members of a gene family.

View larger version (114K): [in this window] [in a new window]   Figure 4.   Probe interrogation of EDIT arrays. Inserts from 141 cDNA clones were spotted onto nylon membranes in duplicate. The same filter was sequentially hybridized with forward- and reverse-subtracted probes (A and B), and with non-subtracted probes made from complex root mRNA (C). In A and C, the probes were made from T. versicolor root tips exposed to DMBQ for 2 to 5 h. In B, the probes were made from T. versicolor root tips exposed to maize root exudate. The cDNAs spotted at each position is found in supplemental data online at www.plantphysiol.org.

To reduce the proportion of highly abundant transcripts in the probe populations, we normalized the cDNA probes by forward and reverse hybridizations (Gurskaya et al., 1996). Because this procedure employed restriction digests prior to subtractive hybridization, the probes were further enriched for low abundant gene domains (Yang et al., 1999). Forward-subtracted probes, enriched for transcripts more abundant after DMBQ treatment, and reverse-subtracted probes, enriched for sequences less abundant after DMBQ treatment, were hybridized to the filters (Fig. 4A). In this case, most of the probes hybridized to inserts on the filter and there was considerable variation between induced and noninduced treatments. We standardized the autoradiograph exposures using clones analyzed by northern blots. A visual examination of the standardized autoradiographs led to the estimate that about 80% of the cDNAs hybridized stronger to forward-subtracted than reverse-subtracted probes, a result consistent with the northern analyses. Because hybridization intensities cannot be quantified using normalized probes, we did not attempt to refine this estimate further.

We then made forward- and reverse-subtracted probes from RNA isolated from T. versicolor roots treated with maize exudates. When these probes were hybridized to the cDNA arrays, the hybridization pattern was very similar to that obtained following DMBQ treatments (Fig. 4B). Combining these results from those of the northern blots described above, we conclude that most of the transcripts up-regulated in T. versicolor by DMBQ are similarly up-regulated by maize root exudates.

Putative Functions of DMBQ-Responsive Transcripts

Based on the relative strength of signals in colony hybridizations, we selected 220 of the most differentially regulated cDNAs to sequence. The sequences clustered into 191 nonoverlapping assemblies using The Institute for Genomic Research Assembler program (Sutton et al., 1995). The virtual translations of the assembled sequences were analyzed by BLASTX.

Transcripts predicted to encode distinct proteins were assembled into a unigene set of 137 members with each assembly assigned its own EDIT number. Putative functions could be assigned to 117 EDIT proteins by homology searches of public data banks. An additional 12 EDIT translations had significant homology to proteins predicted from genome projects, typically Arabidopsis, but for which there were no well-annotated functions. Eight EDITs did not share significant homology with any protein sequences in the databases. The proteins predicted to be encoded by the EDITs were assigned to the functional classes shown in Table II. GenBank accession numbers and putative functions of these sequences and assemblies can be found in the supplemental data online at www.plantphysiol.org. Links of each sequence to FASTA files and BLAST reports can be found at http://veghome.ucdavis.edu/Faculty/Yoder/lab/index.html. The potential relevance of some of these clones to allelochemical signaling is discussed below.

Quinone Detoxification

Signal Transduction and Transcript Regulation

View larger version (31K): [in this window] [in a new window]   Figure 5.   Regulation of TvPirin by DMBQ. A northern blot containing RNA isolated from T. versicolor root tips 0, 0.5, 1, 5, and 24 h after exposure to DMBQ was probed sequentially probed with TvPirin (1L17) and an unsequenced, constitutively expressed cDNA.

Haustorium Ontogeny

Mitochondrial Genes and Maternal Factors in DMBQ Recognition

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION CONCLUSIONS MATERIALS AND METHODS LITERATURE CITED

The transition of parasitic plants from autotrophic to heterotrophic growth is coupled to transcriptional changes in many genes. Some of the proteins predicted to be activated during this period, such as those functioning in quinone detoxification, are probably not associated with parasitism. Comparing the expression of these genes in closely related parasitic and nonparasitic Scrophulariaceae can test this hypothesis (Matvienko et al., 2001). One of the long-standing questions in parasitic plant biology has been whether the genes that encode developmental pathways associated with parasitism are of an endogenous or exogenous origin (Atsatt, 1973). Most of the genes we examined have closely related homologs in other plants, so we can conclude that at least many of the genes responsive during haustorium development also function in autotrophic plants. Our experiments do not, however, rule out the involvement of extrinsic genes introduced into the parasitic lineage of Scrophulariaceae through endosymbiosis or horizontal gene transfer. In either case, the cDNAs described here represent a class of plant genes that are responsive to signal molecules released by other plants. As such they should be useful markers for monitoring plant-plant interactions and their regulatory elements may facilitate the engineering of allelopathy to improve crop performance.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION CONCLUSIONS MATERIALS AND METHODS LITERATURE CITED

Materials

Triphysaria versicolor seeds were harvested from mature capsules on plants growing in a grassland stand near Napa California. Seeds were collected and pooled from hundreds of different plants growing within about 1 ha.

DMBQ was obtained from Pfalz and Bauer Inc. (Waterbury, CT). Maize (Zea mays) root exudates were prepared from in vitro-grown seedlings. Maize seeds (no. 3245IR, Pioneer Hi-Bred International, Johnston, IA) were surface sterilized with bleach and placed into 100 mL of 0.6% (w/v) phytoagar in Magenta culture boxes, four seeds per box (112 plants total). After 2 weeks growth at 25°C, the plants and their roots were removed from the agar, which was centrifuged at 10,000 rpm for 30 min and the supernatant filter sterilized and dried under a vacuum. The resulting powder was resuspended in 100 mL water and added to the parasite roots without further dilution. Approximately 1 mL of exudate corresponds to 1 g fresh root weight.

Haustorium Induction

T. versicolor seeds were surface sterilized and plated at 16°C in petri dishes containing 0.25× Hoagland solution [1.25 mM Ca(NO₃)₂, 1.25 mM KNO₃, 0.25 mM KH₂PO₄, and 0.5 mM MgSO₄], 1% (w/v) Suc, and 0.6% (w/v) phytoagar as previously described (Delavault et al., 1998). Two weeks after germination, seedlings were transplanted along one edge of square petri dishes containing 0.25× Hoagland media in 1% (w/v) agar, 10 seedlings to a dish. The dishes were then incubated for 10 d at 25°C at a near vertical orientation so that the roots grew down along the surface of the agar.

To induce haustoria, 2 mL of either maize root exudate or 10 µM DMBQ (dissolved in water) was applied to T. versicolor roots. An equal number of plants were mock treated with 2 mL water. For optimal induction, the plates needed to be kept horizontal for 2 h after exposure before returning them to the vertical orientation.

Construction of the Subtractive cDNA Library

The roots of 500 T. versicolor seedlings were treated with either 10 µM DMBQ or water for 2 to 5 h at which time 5-mm pieces were cut off the root tips and frozen in liquid nitrogen. The root tips were ground in liquid nitrogen and resuspended in a 1:1 mixture of RNA extraction buffer (0.1 M Tris-HCl, pH 9.0, 0.1 M LiCl, 10 mM EDTA, and 1% [w/v] SDS) and saturated phenol at 70°C. After vortexing and centrifugation (15 min, 6,000g), the water phase was collected and RNA extracted as described by Pawlowski et al. (1994).

The PCR-Select Subtraction Kit (CLONTECH, Palo Alto, CA) was used to make a cDNA library enriched for transcripts differentially abundant in T. versicolor root tips after exposure to DMBQ. This protocol uses suppression PCR to enrich for transcripts differentially expressed in the tester material (Diatchenko et al., 1996). The procedure also contains a hybridization step that equalizes transcript abundance (Gurskaya et al., 1996). We performed the subtractive hybridization using 2 µg poly(A⁺) RNA isolated from DMBQ-treated root tips as tester, and 2 µg poly(A⁺) RNA from water-treated root tips as driver (CLONTECH, 2000). Following PCR amplification, subtracted cDNAs were cloned into pCR2.1 using the TA Cloning System (Invitrogen, Carlsbad, CA). Recombinant clones were identified on X-Gal-containing plates. The average insert size was 650 bp.

Approximately 2,500 single colonies were picked into individual wells of 384-well trays (Nalge Nunc International, Rochester, NY) containing 0.2 mL of Luria broth. Replica trays containing 4.4% (w/v) glycerol were frozen at 80°C. Colonies were then replica plated from the multiwell trays onto seven, 8- × 12-cm nylon filters using a 384-pin replicator (V & P Scientific, Inc., San Diego). Each clone was spotted twice for total of 768 colonies per filter. The colonies were grown overnight on the filters, lysed, DNA fixed to the membrane, and prehybridized in 6.7% (w/v) SDS and 6.7× SSPE at 68°C (Sambrook et al., 1989).

The filters were hybridized sequentially with forward- and reverse-subtracted probes to identify false positives (CLONTECH, 1999). Forward-subtracted probes were made using mRNA obtained from DMBQ-induced root tips as tester and water-treated roots as driver. These probes were enriched for transcripts induced in DMBQ treated roots. Reverse-subtracted probes were similarly prepared except that the tester mRNA was derived from water-treated root tips, whereas the driver mRNA from DMBQ-treated roots. Reverse-subtracted probes were enriched for transcripts less abundant after DMBQ treatment.

About 2% of the 2,500 clones strongly hybridized to both forward- and reverse-subtracted probes. These clones were picked, the inserts amplified by PCR, pooled, and added as cold blocking agent in subsequent hybridizations. Clones giving the greatest differential in hybridization signal strength between the forward- and reverse-subtracted clones were selected for sequencing.

Sequence Analysis

Selected recombinant clones were picked into 0.2 mL of Luria broth in individual wells of a 384-well tray. Insert cDNA was PCR amplified from the intact clones using the primers 1 and 2R (CLONTECH, 2000). One side of the PCR-amplified inserts were sequenced using an Applied Biosystems 377 DNA sequencer (PE Applied Biosystems, Foster City, CA) and the insert sequences trimmed of vector sequences using Sequencher software (Gene Codes Co., Ann Arbor, MI). To identify redundancies, each sequence was searched against the entire database of sequences using a local BLAST. Some cDNAs were also sequenced from the other end. The sequences were clustered into 191 nonoverlapping assemblies using The Institute for Genomic Research Assembler program (Sutton et al., 1995). The virtual translations of the assembled sequences were analyzed by BLASTX to identify homologous sequences in the GenBank database (Benson et al., 1998).

cDNA Array Preparation and Probing

PCR-amplified insert DNA (100 ng µL) from 141 clones was put into individual wells of a 384-well tray. The cDNA inserts were then arrayed in duplicate onto an 8- × 12-cm nylon filter using a 384-pin replicator (V & P Scientific, Inc.) and the DNA fixed to the membrane. The filters were sequentially hybridized to radioactive cDNA probes and visualized by autoradiography. Filters were washed of probe between hybridizations by immersing into boiling 0.5% (w/v) SDS.

Northern Hybridizations

Total RNA was denatured in dimethyl sulfoxide/glyoxal, separated on 1.4% (w/v) agarose gels (Sambrook et al., 1989), and blotted onto Hybond N⁺ nylon membrane according to the recommendations of the manufacturer (Amersham, Arlington Heights, IL). The blots were hybridized in 6.7% (w/v) SDS ande 6.7× SSPE at 65°C (Sambrook et al., 1989). The cDNA inserts used as probes were radiolabeled by random priming using (-³²P) dCTP (3,000 Ci mmol¹; ICN Pharmaceuticals, Costa Mesa, CA).