Regulatory Modules Involved in the Degradation and Modification of Host Cell Walls During Cuscuta campestris Invasion.

Haustoria of parasitic plants have evolved sophisticated traits to successfully infect host plants. The degradation and modification of host cell walls enable the haustorium to effectively invade host tissues. This study focused on two APETALA2/ETHYLENE RESPONSE FACTOR (ERF) genes and a set of the cell wall enzyme genes principally expressed during the haustorial invasion of Cuscuta campestris Yuncker. The orthogroups of the TF and cell wall enzyme genes have been implicated in the cell wall degradation and modification activities in the abscission of tomatoes, which are currently the phylogenetically closest non-parasitic model species of Cuscuta species. Although haustoria are generally thought to originate from root tissues, our results suggest that haustoria have further optimized invasion potential by recruiting regulatory modules from other biological processes.

Trans-species small RNAs move long distances in a parasitic plant complex.

Parasitic plants exchange various types of RNAs with their host plants, including mRNA, and small non-coding RNA. Among small non-coding RNAs, miRNA production is known to be induced at the haustorial interface. The induced miRNAs transfer to the host plant and activate secondary siRNA production to silence target genes in the host. In addition to interfacial transfer, long-distance movement of the small RNAs has also been known to mediate signaling and regulate biological processes. In this study, we tested the long-distance movement of trans-species small RNAs in a parasitic-plant complex. Small RNA-Seq was performed using a complex of a stem parasitic plant, Cuscuta campestris, and a host, Arabidopsis thaliana. In the host plant's parasitized stem, genes involved in the production of secondary siRNA, AtSGS3 and AtRDR6, were upregulated, and 22-nt small RNA was enriched concomitantly, suggesting the activation of secondary siRNA production. Stem-loop RT-PCR and subsequent sequencing experimentally confirmed the mobility of the small RNAs. Trans-species mobile small RNAs were detected in the parasitic interface and also in distant organs. To clarify the mode of long-distance translocation, we examined whether C. campestris-derived small RNA moves long distances in A. thaliana sgs3 and rdr6 mutants or not. Mobility of C. campestris-derived small RNA in sgs3 and rdr6 mutants suggested the occurrence of direct long-distance transport without secondary siRNA production in the recipient plant.

Epidermal cell-patterning genes of the stem parasitic plant Cuscuta campestris are involved in the development of holdfasts.

Cuscuta campestris, a stem parasitic plant, commences its parasitic behavior by forming a specialized disk-like adhesive structure called a holdfast, which facilitates tight adhesion to the stem surface of the host plant. The morphology of epidermal cells in the holdfast is similar to that of the leaf trichome and root hairs of dicotyledonous plants. However, the regulatory network underlying the development of the holdfast has not been elucidated to date. In this study, we assessed the roles of epidermal cell-patterning genes in the development of a holdfast. Epidermal cell-patterning genes of C. campestris, including CcWER, CcGL3, CcTTG1, CcGL2, and CcJKD, were expressed slightly before the initiation of the outgrowth of stem epidermal cells. CcJKD-silencing repressed CcJKD, CcWER, CcGL3, CcTTG1, CcGL2; therefore, CcJKD is an upstream regulator of other epidermal cell-patterning genes. Unlike other genes, CcCPC was not upregulated after attachment to the host, and was not repressed by CcJKD-silencing. Protein interaction assays demonstrated that CcJKD interacted with CcTTG1 and CcCPC. Furthermore, CcJKD-silencing repressed the outgrowth of holdfast epidermal cells. Therefore, C. campestris invokes epidermal cell-patterning genes for the outgrowth of holdfast epidermal cells, and their regulatory mechanism is different from those for leaf trichome or root hairs.

MassBase: A large-scaled depository of mass spectrometry datasets for metabolome analysis.

Depository of low-molecular-weight compounds or metabolites detected in various organisms in a non-targeted manner is indispensable for metabolomics research. Due to the diverse chemical compounds, various mass spectrometry (MS) setups with state-of-the-art technologies have been used. Over the past two decades, we have analyzed various biological samples by using gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, or capillary electrophoresis-mass spectrometry, and archived the datasets in the depository MassBase (http://webs2.kazusa.or.jp/massbase/). As the format of MS datasets depends on the MS setup used, we converted each raw binary dataset of the mass chromatogram to text file format, and thereafter, information of the chromatograph peak was extracted in the text file from the converted file. In total, the depository comprises 46,493 datasets, of which 38,750 belong to the plant species and 7,743 are authentic or mixed chemicals as well as other sources (microorganisms, animals, and foods), as on August 1, 2020. All files in the depository can be downloaded in bulk from the website. Mass chromatograms of 90 plant species obtained by LC-Fourier transform ion cyclotron resonance MS or Orbitrap MS, which detect the ionized molecules with high accuracy allowing speculation of chemical compositions, were converted to text files by the software PowerGet, and the chemical annotation of each peak was added. The processed datasets were deposited in the annotation database KomicMarket2 (http://webs2.kazusa.or.jp/km2/). The archives provide fundamental resources for comparative metabolomics and functional genomics, which may result in deeper understanding of living organisms.

Tissue adhesion between distant plant species in parasitism and grafting.

Plant grafting is generally performed between closely related species. Recently, we have discovered that Nicotiana species of Solanaceae show the ability to graft with distantly related plant species beyond the family. Graft adhesion with diverse angiosperms by Nicotiana species was probably facilitated by the secretion of a subclade of ß-1,4-glucanases. The capability of interfamily grafting was also found in the model Orobanchaceae hemiparasitic plant, Phtheirospermum japonicum, which naturally invades to the tissues of host plants of different families. Transcriptome analysis indicated that the same clade of ß-1,4-glucanase plays an important role in plant parasitism. Thus, the tissue adhesion between distant plant species occurs both naturally and artificially. Here, we further observed the capability of interfamily grafting in the stem holoparasitic genus, Cuscuta. These findings indicate that the natural process of tissue adhesion is a potential clue to improve plant-grafting techniques.

Identification of a Flavin Monooxygenase-Like Flavonoid 8-Hydroxylase with Gossypetin Synthase Activity from Lotus japonicus.

Lotus japonicus is a model legume that accumulates 8-hydroxyflavonol derivatives, such as gossypetin (8-hydroxyquercetin) 3-O-glycoside, which confer the yellow color to its petals. An enzyme, flavonoid 8-hydroxylase (F8H; LjF8H), is assumed to be involved in the biosynthesis, but the specific gene is yet to be identified. The LjF8H cDNA was isolated as a flavin adenine dinucleotide (FAD)-binding monooxygenase-like protein using flower buds and flower-specific EST data of L. japonicus. LjF8H is a single copy gene on chromosome III consisting of six exons. The conserved FAD- and NAD(P)H-dependent oxidase motifs were found in LjF8H. Phylogenetic analysis suggested that LjF8H is a member of the flavin monooxygenase group but distinctly different from other known flavonoid oxygenases. Analysis of recombinant yeast microsome expressing LjF8H revealed that the enzyme catalyzed the 8-hydroxylation of quercetin. Other flavonoids, such as naringenin, eriodictyol, apigenin, luteolin, taxifolin and kaempferol, also acted as substrates of LjF8H. This broad substrate acceptance was unlike known F8Hs in other plants. Interestingly, flavanone and flavanonol, which have saturated C-C bond at positions 2 and 3 of the flavonoid C-ring, produced 6-hyroxylflavonoids as a by-product of the enzymatic reaction. Furthermore, LjF8H only accepted the 2S-isomer of naringenin, suggesting that the conformational state of the substrates might affect product specificity. The overexpression of LjF8H in Arabidopsis thaliana and Petunia hybrida synthesized gossypetin and 8-hydroxykaempferol, respectively, indicating that LjF8H was functional in plant cells. In conclusion, this study represents the first instance of cloning and identification of F8Hs responsible for gossypetin biosynthesis.

Mobile Host mRNAs Are Translated to Protein in the Associated Parasitic Plant Cuscuta campestris.

Cuscuta spp. are obligate parasites that connect to host vascular tissue using a haustorium. In addition to water, nutrients, and metabolites, a large number of mRNAs are bidirectionally exchanged between Cuscuta spp. and their hosts. This trans-specific movement of mRNAs raises questions about whether these molecules function in the recipient species. To address the possibility that mobile mRNAs are ultimately translated, we built upon recent studies that demonstrate a role for transfer RNA (tRNA)-like structures (TLSs) in enhancing mRNA systemic movement. C. campestris was grown on Arabidopsis that expressed a ß-glucuronidase (GUS) reporter transgene either alone or in GUS-tRNA fusions. Histochemical staining revealed localization in tissue of C. campestris grown on Arabidopsis with GUS-tRNA fusions, but not in C. campestris grown on Arabidopsis with GUS alone. This corresponded with detection of GUS transcripts in Cuscuta on Arabidopsis with GUS-tRNA, but not in C. campestris on Arabidopsis with GUS alone. Similar results were obtained with Arabidopsis host plants expressing the same constructs containing an endoplasmic reticulum localization signal. In C. campestris, GUS activity was localized in the companion cells or phloem parenchyma cells adjacent to sieve tubes. We conclude that host-derived GUS mRNAs are translated in C. campestris and that the TLS fusion enhances RNA mobility in the host-parasite interactions.

Development of Parasitic Organs of a Stem Holoparasitic Plant in Genus Cuscuta.

Parasitic plants infect a broad range of plant species including economically important crops. They survive by absorbing water, minerals, and photosynthates from their hosts. To support their way of life, parasitic plants generally establish parasitic organs that allow them to attach to their hosts and to efficiently absorb substances from the vascular system of the host. Here, we summarize the recent progress in understanding the mechanisms underlying the formation of these parasitic organs, focusing on the process depicted in the stem holoparasitic genus, Cuscuta. An attachment structure called "holdfast" on the stem surface is induced by the light and contact stimuli. Concomitantly with holdfast formation, development of an intrusive structure called haustorium initiates in the inner cortex of the Cuscuta stem, and it elongates through apoplastic space of the host tissue. When haustoria reaches to host vascular tissues, they begin to form vascular conductive elements to connect vascular tissue of Cuscuta stem to those of host. Recent studies have shown parasite-host interaction in the interfacial cell wall, and regulation of development of these parasitic structures in molecular level. We also briefly summarize the role of host receptor in the control of compatibility between Cuscuta and hosts, on which occurrence of attachment structure depends, and the role of plant-to-plant transfer of long-distance signals after the establishment of conductive structure.

Low Infection of Phelipanche aegyptiaca in Micro-Tom Mutants Deficient in CAROTENOIDCLEAVAGE DIOXYGENASE 8.

Strigolactones (SLs), a group of plant hormones, induce germination of root-parasitic plants and inhibit shoot branching in many plants. Shoot branching is an important trait that affects the number and quality of flowers and fruits. Root-parasitic plants, such as Phelipanche spp., infect tomato roots and cause economic damage in Europe and North Africa-hence why resistant tomato cultivars are needed. In this study, we found carotenoid cleavage dioxygenase 8-defective mutants of Micro-Tom tomato (slccd8) by the "targeting induced local lesions in genomes" (TILLING) method. The mutants showed excess branching, which was suppressed by exogenously applied SL. Grafting shoot scions of the slccd8 mutants onto wild-type (WT) rootstocks restored normal branching in the scions. The levels of endogenous orobanchol and solanacol in WT were enough detectable, whereas that in the slccd8 mutants were below the detection limit of quantification analysis. Accordingly, root exudates of the slccd8 mutants hardly stimulated seed germination of root parasitic plants. In addition, SL deficiency did not critically affect the fruit traits of Micro-Tom. Using a rhizotron system, we also found that Phelipanche aegyptiaca infection was lower in the slccd8 mutants than in wild-type Micro-Tom because of the low germination. We propose that the slccd8 mutants might be useful as new tomato lines resistant to P. aegyptiaca.

Genome-wide analysis of ATP binding cassette (ABC) transporters in tomato.

ATP binding cassette (ABC) transporters are proteins that actively mediate the transport of a wide range of molecules, such as organic acids, metal ions, phytohormones and secondary metabolites. Therefore, ABC transporters must play indispensable roles in growth and development of tomato, including fruit development. Most ABC transporters have transmembrane domains (TMDs) and belong to the ABC protein family, which includes not only ABC transporters but also soluble ABC proteins lacking TMDs. In this study, we performed a genome-wide identification and expression analysis of genes encoding ABC proteins in tomato (Solanum lycopersicum), which is a valuable horticultural crop and a model plant for studying fleshy fruits. In the tomato genome, a total of 154 genes putatively encoding ABC transporters, including 9 ABCAs, 29 ABCBs, 26 ABCCs, 2 ABCDs, 2 ABCEs, 6 ABCFs, 70 ABCGs and 10 ABCIs, were identified. Gene expression data from the eFP Browser and reverse transcription-semi-quantitative PCR analysis revealed their tissue-specific and development-specific expression profiles. This work suggests physiological roles of ABC transporters in tomato and provides fundamental information for future studies of ABC transporters not only in tomato but also in other Solanaceae species.

Molecular Components of Arabidopsis Intact Vacuoles Clarified with Metabolomic and Proteomic Analyses.

We analyzed the metabolites and proteins contained in pure intact vacuoles isolated from Arabidopsis suspension-cultured cells using capillary electrophoresis-mass spectrometry (CE-MS), Fourier transform-ion cyclotron resonance (FT-ICR)-MS and liquid chromatography (LC)-MS. We identified 21 amino acids and five organic acids as major primary metabolites in the vacuoles with CE-MS. Further, we identified small amounts of 27 substances including well-known vacuolar molecules, but also some unexpected substances (e.g. organic phosphate compounds). Non-target analysis of the vacuolar sample with FT-ICR-MS suggested that there are 1,106 m/z peaks that could predict the 5,090 molecular formulae, and we have annotated 34 compounds in these peaks using the KNapSAck database. By conducting proteomic analysis of vacuolar sap, we found 186 proteins in the same vacuole samples. Since the vacuole is known as a major degradative compartment, many of these were hydrolases, but we also found various oxidoreductases and transferases. The relationships between the proteins and metabolites in the vacuole are discussed.

Differentiation of vascular elements in haustoria of Cuscuta japonica.

Parasitic plants establish vascular-conducting cells in an intrusive organ called haustorium. In haustoria of a stem parasitic plant, Cuscuta japonica, the presence of cells expressing cell-type-specific genes of phloem companion cell, phloem sieve element, procambial cell and xylem vessel has recently been demonstrated. Differentiation of these vascular cells is regulated in a manner similar to that in conventional vascular tissues. However, the initiation of procambial cells occurs concomitantly with the differentiation of vascular-conducting cells. The differentiation process of phloem also differed from that of conventional vascular tissues because enucleation of sieve elements appeared to be impeded. These results collectively imply that the vascular differentiation process in haustoria of parasitic plants may be different from that in conventional vascular tissues.

Organization of Vascular Cells in the Haustorium of the Parasitic Flowering Plant Cuscuta japonica.

The stem parasite dodder, Cuscuta japonica, has evolved a specialized root-like organ, the haustorium, which is differentiated from the stem. In order to take up water and nutrients, C. japonica reprograms haustorial cells to vascular cells, connecting the host's vascular system to its own. However, little is known about vascular differentiation in haustoria. In this study, we first confirmed the temporal and spatial expression profiles of vascular cell type-specific genes, CjAPL, CjSEOR1, CjWOX4 and CjTED7, to examine whether phloem companion cells, developing sieve elements, procambial cells and differentiating xylem cells, respectively, are present in the haustoria. CjAPL and CjSEOR1 decreased, and CjWOX4 showed a transient increase before the onset of xylem vessel formation, and then decreased. CjTED7 increased coincidentally with xylem vessel formation. In situ hybridization demonstrated that CjWOX4-expressing cells and phloem-conducting cells are in close proximity, and occupied a domain distinguishable from xylem vessels, suggesting differentiation of a phloem/procambial domain and a xylem domain in the haustorium. Secondly, expression of regulatory genes that are involved in determination of the fate of procambial cells was investigated. Expression patterns of CjCLE41, CjGSK3 and CjBES1suggested that TDIF-TDR-GSK3-mediated signaling is activated in haustoria. The natural antisense transcript of CjCLE41 was detected in haustoria, implying the sense regulation of CjCLE41. Expression profiles of the regulatory genes, combined with those of cell type-specific marker genes, suggest that reprogramming of haustorial cells to vascular cells is regulated in a way that allows the immediate formation of xylem vessels by alleviating inhibition of xylem differentiation.

Phloem-Conducting Cells in Haustoria of the Root-Parasitic Plant Phelipanche aegyptiaca Retain Nuclei and Are Not Mature Sieve Elements.

Phelipanche aegyptiaca parasitizes a wide range of plants, including important crops, and causes serious damage to their production. P. aegyptiaca develops a specialized intrusive organ called a haustorium that establishes connections to the host's xylem and phloem. In parallel with the development of xylem vessels, the differentiation of phloem-conducting cells has been demonstrated by the translocation of symplasmic tracers from the host to the parasite. However, it is unclear yet whether haustorial phloem-conducting cells are sieve elements. In this study, we identified phloem-conducting cells in haustoria by the host-to-parasite translocation of green fluorescent protein (GFP) from AtSUC2pro::GFP tomato sieve tubes. Haustorial GFP-conducting cells contained nuclei but not callose-rich sieve plates, indicating that phloem-conducting cells in haustoria differ from conventional sieve elements. To ascertain why the nuclei were not degenerated, expression of the P. aegyptiaca homologs NAC-domain containing transcription factor (NAC45), NAC45/86-dependent exonuclease-domain protein 1 (NEN1), and NEN4 was examined. However, these genes were more highly expressed in the haustorium than in tubercle protrusion, implying that nuclear degradation in haustoria may not be exclusively controlled by the NAC45/86-NEN regulatory pathway. Our results also suggest that the formation of plasmodesmata with large size exclusion limits is independent of nuclear degradation and callose deposition.

Arabinogalactan Proteins Accumulate in the Cell Walls of Searching Hyphae of the Stem Parasitic Plants, Cuscuta campestris and Cuscuta japonica.

Stem parasitic plants (Cuscuta spp.) develop a specialized organ called a haustorium to penetrate their hosts' stem tissues. To reach the vascular tissues of the host plant, the haustorium needs to overcome the physical barrier of the cell wall, and the parasite-host interaction via the cell wall is a critical process. However, the cell wall components responsible for the establishment of parasitic connections have not yet been identified. In this study, we investigated the spatial distribution patterns of cell wall components at a parasitic interface using parasite-host complexes of Cuscuta campestris-Arabidopsis thaliana and Cuscuta japonica-Glycine max. We focused on arabinogalactan proteins (AGPs), because AGPs accumulate in the cell walls of searching hyphae of both C. campestris and C. japonica. We found more AGPs in elongated haustoria than in pre haustoria, indicating that AGP accumulation is developmentally regulated. Using in situ hybridization, we identified five genes in C. campestris that encode hyphal-expressed AGPs that belong to the fasciclin-like AGP (FLA) family, which were named CcFLA genes. Three of the five CcFLA genes were expressed in the holdfast, which develops on the Cuscuta stem epidermis at the attachment site for the host's stem epidermis. Our results suggest that AGPs are involved in hyphal elongation and adhesion to host cells, and in the adhesion between the epidermal tissues of Cuscuta and its host.

TOMATOMICS: A Web Database for Integrated Omics Information in Tomato.

Solanum lycopersicum (tomato) is an important agronomic crop and a major model fruit-producing plant. To facilitate basic and applied research, comprehensive experimental resources and omics information on tomato are available following their development. Mutant lines and cDNA clones from a dwarf cultivar, Micro-Tom, are two of these genetic resources. Large-scale sequencing data for ESTs and full-length cDNAs from Micro-Tom continue to be gathered. In conjunction with information on the reference genome sequence of another cultivar, Heinz 1706, the Micro-Tom experimental resources have facilitated comprehensive functional analyses. To enhance the efficiency of acquiring omics information for tomato biology, we have integrated the information on the Micro-Tom experimental resources and the Heinz 1706 genome sequence. We have also inferred gene structure by comparison of sequences between the genome of Heinz 1706 and the transcriptome, which are comprised of Micro-Tom full-length cDNAs and Heinz 1706 RNA-seq data stored in the KaFTom and Sequence Read Archive databases. In order to provide large-scale omics information with streamlined connectivity we have developed and maintain a web database TOMATOMICS (http://bioinf.mind.meiji.ac.jp/tomatomics/). In TOMATOMICS, access to the information on the cDNA clone resources, full-length mRNA sequences, gene structures, expression profiles and functional annotations of genes is available through search functions and the genome browser, which has an intuitive graphical interface.

Rapid identification of causal mutations in tomato EMS populations via mapping-by-sequencing.

The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in plants, leading to improved breeding stocks and superior tomato varieties. However, until recently, the identification of causal mutations that underlie particular phenotypes has been a very lengthy task that many laboratories could not afford because of spatial and technical limitations. Here, we describe a simple protocol for identifying causal mutations in tomato using a mapping-by-sequencing strategy. Plants displaying phenotypes of interest are first isolated by screening an EMS mutant collection generated in the miniature cultivar Micro-Tom. A recombinant F2 population is then produced by crossing the mutant with a wild-type (WT; non-mutagenized) genotype, and F2 segregants displaying the same phenotype are subsequently pooled. Finally, whole-genome sequencing and analysis of allele distributions in the pools allow for the identification of the causal mutation. The whole process, from the isolation of the tomato mutant to the identification of the causal mutation, takes 6-12 months. This strategy overcomes many previous limitations, is simple to use and can be applied in most laboratories with limited facilities for plant culture and genotyping.

Circadian Oscillation of the Lettuce Transcriptome under Constant Light and Light-Dark Conditions.

Although, the circadian clock is a universal biological system in plants and it orchestrates important role of plant production such as photosynthesis, floral induction and growth, there are few such studies on cultivated species. Lettuce is one major cultivated species for both open culture and plant factories and there is little information concerning its circadian clock system. In addition, most of the relevant genes have not been identified. In this study, we detected circadian oscillation in the lettuce transcriptome using time-course RNA sequencing (RNA-Seq) data. Constant light (LL) and light-dark (LD) conditions were used to detect circadian oscillation because the circadian clock has some basic properties: one is self-sustaining oscillation under constant light and another is entrainment to environmental cycles such as light and temperature. In the results, 215 contigs were detected as common oscillating contigs under both LL and LD conditions. The 215 common oscillating contigs included clock gene-like contigs CCA1 (CIRCADIAN CLOCK ASSOCIATED 1)-like, TOC1 (TIMING OF CAB EXPRESSION 1)-like and LHY (LATE ELONGATED HYPOCOTYL)-like, and their expression patterns were similar to those of Arabidopsis. Functional enrichment analysis by GO (gene ontology) Slim and GO Fat showed that the GO terms of response to light stimulus, response to stress, photosynthesis and circadian rhythms were enriched in the 215 common oscillating contigs and these terms were actually regulated by circadian clocks in plants. The 215 common oscillating contigs can be used to evaluate whether the gene expression pattern related to photosynthesis and optical response performs normally in lettuce.

A bioinformatics approach to distinguish plant parasite and host transcriptomes in interface tissue by classifying RNA-Seq reads.

BACKGROUND: The genus Cuscuta is a group of parasitic plants that are distributed world-wide. The process of parasitization starts with a Cuscuta plant coiling around the host stem. The parasite's haustorial organs then establish a vascular connection allowing for access to the phloem content. The host and the parasite form new cellular connections, suggesting coordination of developmental and biochemical processes. Simultaneous monitoring of gene expression in the parasite's and host's tissues may shed light on the complex events occurring between the parasitic and host cells and may help to overcome experimental limitations (i.e. how to separate host tissue from Cuscuta tissue at the haustorial connection). A novel approach is to use bioinformatic analysis to classify sequencing reads as either belonging to the host or to the parasite and to characterize the expression patterns. Owing to the lack of a comprehensive genomic dataset from Cuscuta spp., such a classification has not been performed previously. RESULTS: We first classified RNA-Seq reads from an interface region between the non-model parasitic plant Cuscuta japonica and the non-model host plant Impatiens balsamina. Without established reference sequences, we classified reads as originating from either of the plants by stepwise similarity search against de novo assembled transcript sets of C. japonica and I. balsamina, unigene sets of the same genus, and cDNA sequences of the same family. We then assembled de novo transcriptomes from the classified read sets. We assessed the quality of the classification by mapping reads to contigs of both plants, achieving a misclassification rate low enough (0.22-0.39%) to be used reliably for differential gene expression analysis. Finally, we applied our read classification method to RNA-Seq data from the interface between the non-model parasitic plant C. japonica and the model host plant Glycine max. Analysis of gene expression profiles at 5 parasitizing stages revealed differentially expressed genes from both C. japonica and G. max, and uncovered the coordination of cellular processes between the two plants. CONCLUSIONS: We demonstrated that reliable identification of differentially expressed transcripts in undissected interface region of the parasite-host association is feasible and informative with respect to differential-expression patterns.