Isolation and characterization of root-specific phosphate transporter promoters from Medicago trunatula

Promoters of phosphate transporter genes MtPT1 and MtPT2 of Medicago truncatula were isolated by utilizing the gene-space sequence information and by screening of a genomic library, respectively. Two reporter genes, beta-glucuronidase (GUS) and green fluorescent protein (GFP) were placed under the control of the MtPT1 and MtPT2 promoters. These chimeric transgenes were introduced into Arabidopsis thaliana and transgenic roots of M. truncatula, and expression patterns of the reporter genes were assayed in plants grown under different phosphate (Pi) concentrations. The expression of GUS and GFP was only observed in root tissues, and the levels of expression decreased with increasing concentrations of Pi. GUS activities in roots of transgenic plants decreased 10-fold when the plants were transferred from 10 microM to 2 mM Pi conditions, however, when the plants were transferred back to 10 microM Pi conditions, GUS expression reversed back to the original level. The two promoters lead to different expression patterns inside root tissues. The MtPT1 promoter leads to preferential expression in root epidermal and cortex cells, while MtPT2 promoter results in strong expression in the vascular cylinder in the center of roots. Promoter deletion analyses revealed possible sequences involved in root specificity and Pi responsiveness. The promoters are valuable tools for defined engineering of plants, particularly for root-specific expression of transgenes.     

Cytological expression of early response to infection by Heterodera glycines Ichinohe in resistant PI 437654 soybean.

The soybean PI 437654 is resistant to all known races of the soybean cyst nematode (SCN) in the U.S.A. and became a new source of resistance genes in cultivar development. Race 3, a wide-ranging nematode pathotype, was used to examine root cells of PI 437654 and susceptible 'Essex', 2, 3, and 5 days after inoculation (DAI). In initial response to SCN, both genotypes formed syncytia by cell wall dissolutions. Hypertrophy of syncytium component cells and hyperplasia of cells near syncytia were observed. At 2 DAI, incompatible response of PI 437654 to SCN was exhibited: limited cell hypertrophy, inhibition of syncytium growth, initiation of necrosis, and wall appositions. At 3 DAI, cellular events appeared to be a sum of the operative mechanisms for SCN resistance: irregular wall thickening, pronounced wall appositions, necrosis, and nuclear breakdown followed by cytoplasmic collapse. The cells surrounding the syncytia showed necrosis, wall apposition, and accumulation of electron-dense bodies. By 5 DAI, syncytia and neighboring cells were totally devoid of ground plasma and the degeneration process was completed. The normal route for early syncytium development in 'Essex' (increased number of organelles, intense vacuolization, accumulation of dense deposits in vacuoles, and wall ingrowths) suggests the involvement of portions of the developmental pathway of differentiating tissues in organogenesis. Early onset of SCN resistance 2 DAI in PI 437654 suggests rapid activation of genes in a cascade reaction leading to cell death. Key words : soybean, nematode, syncytium, cell death.     

Spatiotemporal deep imaging of syncytium induced by the soybean cyst nematode <Emphasis Type="Italic">Heterodera glycines</Emphasis>

Parasite infections cause dramatic anatomical and ultrastructural changes in host plants. Cyst nematodes are parasites that invade host roots and induce a specific feeding structure called a syncytium. A syncytium is a large multinucleate cell formed by cell wall dissolution-mediated cell fusion. The soybean cyst nematode (SCN), Heterodera glycines, is a major soybean pathogen. To investigate SCN infection and the syncytium structure, we established an in planta deep imaging system using a clearing solution ClearSee and two-photon excitation microscopy (2PEM). Using this system, we found that several cells were incorporated into the syncytium; the nuclei increased in size and the cell wall openings began to be visible at 2 days after inoculation (DAI). Moreover, at 14 DAI, in the syncytium developed in the cortex, there were thickened concave cell wall pillars that resembled “Parthenon pillars.” In contrast, there were many thick board-like cell walls and rarely Parthenon pillars in the syncytium developed in the stele. We revealed that the syncytia were classified into two types based on the pattern of the cell wall structures, which appeared to be determined by the position of the syncytium inside roots. Our results provide new insights into the developmental process of syncytium induced by cyst nematode and a better understanding of the three-dimensional structure of the syncytium in host roots.     

The tobacco Cel7 gene promoter is auxin-responsive and locally induced in nematode feeding sites of heterologous plants

Emerging evidence suggests that plant cell-wall-modifying enzymes induced by root-parasitic nematodes play important roles in feeding cell formation. We previously identified a tobacco endo-β-1,4-glucanase (cellulase) gene, NtCel7 , that was strongly induced in both root-knot and cyst nematode feeding cells. To characterize further the developmental and nematode-responsive regulation of NtCel7 , we isolated the NtCel7 promoter and analysed its expression over a time course of nematode infection and in response to auxin, gibberellin, ethylene and sucrose in soybean and tomato hairy roots and in Arabidopsis containing the NtCel7 promoter fused to the β-glucuronidase (GUS) reporter gene. Histochemical analyses of transgenic plant materials revealed that the NtCel7 promoter exhibited a unique organ-specific expression pattern during plant development suggestive of important roles for NtCel7 in both vegetative and reproductive growth. In all plant species tested, strong GUS expression was observed in root tips and lateral root primordia of uninfected roots with weaker expression in the root vasculature. Further analyses of transgenic Arabidopsis plants revealed expression in shoot and root meristems and the vasculature of most organs during plant development. We also determined that the NtCel7 promoter was induced by auxin, but not gibberellin, ethylene or sucrose. Moreover, strong GUS activity was observed in both cyst and root-knot nematode-induced feeding sites in transgenic roots of soybean, tomato and Arabidopsis. The conserved developmental and nematode-responsive expression of the NtCel7 promoter in heterologous plants indicates that motifs of this regulatory element play a fundamental role in regulating NtCel7 gene expression within nematode feeding sites and that this regulation may be mediated by auxin.     

Evaluation of constitutive viral promoters in transgenic soybean roots and nodules

The efficiency of beta-glucuronidase (GUS) expression was evaluated with five viral promoters to identify the most suitable promoter or promoters for use in soybean hairy roots, including applications to study the symbiotic interaction with Bradyrhizobium japonicum. Levels of GUS activity were fluorimetrically and histochemically assayed when the GUS (uidA) gene was driven by the Cauliflower mosaic virus (CaMV) 35S promoter and enhanced 35S (E35S) promoter, the Cassava vein mosaic virus (CsVMV) promoter, the Figwort mosaic virus (FMV) promoter, and the Strawberry vein banding virus (SVBV2) promoter. We demonstrate that GUS activity was highest when driven by the FMV promoter and that the promoter activity of 35S and SVBV2 was significantly lower than that of the CsVMV and E35S promoters when tested in soybean hairy roots. In mature soybean root nodules, strong GUS activity was evident when the FMV, 35S, and CsVMV promoters were used. These results indicate that the FMV promoter facilitates the strong expression of target genes in soybean hairy roots and root nodules.     

A gene expression analysis of syncytia laser microdissected from the roots of the <Emphasis Type="Italic">Glycine max</Emphasis> (soybean) genotype PI 548402 (Peking) undergoing a resistant reaction after infection by <Emphasis Type="Italic">Heterodera glycines</Emphasis> (soybean cyst nematode)

The syncytium is a nurse cell formed within the roots of Glycine max by the plant parasitic nematode Heterodera glycines. Its development and maintenance are essential for nematode survival. The syncytium appears to undergo two developmental phases during its maturation into a functional nurse cell. The first phase is a parasitism phase where the nematode establishes the molecular circuitry that during the second phase ensures a compatible interaction with the plant cell. The cytological features of syncytia undergoing susceptible or resistant reactions appear the same during the parasitism phase. Depending on the outcome of any defense response, the second phase is a period of syncytium maintenance (susceptible reaction) or failure (resistant reaction). In the analyses presented here, the localized gene expression occurring at the syncytium during the resistant reaction was studied. This was accomplished by isolating syncytial cells from Glycine max genotype Peking (PI 548402) by laser capture microdissection. Microarray analyses using the Affymetrix^® soybean GeneChip^® directly compared Peking syncytia undergoing a resistant reaction to those undergoing a susceptible reaction during the parasitism phase of the resistant reaction. Those analyses revealed lipoxygenase-9 and lipoxygenase-4 as the most highly induced genes in the resistant reaction. The analysis also identified induced levels of components of the phenylpropanoid pathway. These genes included phenylalanine ammonia lyase, chalcone isomerase, isoflavone reductase, cinnamoyl-CoA reductase and caffeic acid O-methyltransferase. The presence of induced levels of these genes implies the importance of jasmonic acid and phenylpropanoid signaling pathways locally at the site of the syncytium during the resistance phase of the resistant reaction. The analysis also identified highly induced levels of four S-adenosylmethionine synthetase genes, the EARLY-RESPONSIVE TO DEHYDRATION 2 gene and the 14-3-3 gene known as GENERAL REGULATORY FACTOR 2. Subsequent analyses studied microdissected syncytial cells at 3, 6 and 9 days post infection (dpi) during the course of the resistant reaction, resulting in the identification of signature gene expression profiles at each time point in a single G. max genotype, Peking.     

The novel cyst nematode effector protein 19C07 interacts with the Arabidopsis auxin influx transporter LAX3 to control feeding site development

Plant-parasitic cyst nematodes penetrate plant roots and transform cells near the vasculature into specialized feeding sites called syncytia. Syncytia form by incorporating neighboring cells into a single fused cell by cell wall dissolution. This process is initiated via injection of esophageal gland cell effector proteins from the nematode stylet into the host cell. Once inside the cell, these proteins may interact with host proteins that regulate the phytohormone auxin, as cellular concentrations of auxin increase in developing syncytia. Soybean cyst nematode (Heterodera glycines) Hg19C07 is a novel effector protein expressed specifically in the dorsal gland cell during nematode parasitism. Here, we describe its ortholog in the beet cyst nematode (Heterodera schachtii), Hs19C07. We demonstrate that Hs19C07 interacts with the Arabidopsis (Arabidopsis thaliana) auxin influx transporter LAX3. LAX3 is expressed in cells overlying lateral root primordia, providing auxin signaling that triggers the expression of cell wall-modifying enzymes, allowing lateral roots to emerge. We found that LAX3 and polygalacturonase, a LAX3-induced cell wall-modifying enzyme, are expressed in the developing syncytium and in cells to be incorporated into the syncytium. We observed no decrease in H. schachtii infectivity in aux1 and lax3 single mutants. However, a decrease was observed in both the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant. In addition, ectopic expression of 19C07 was found to speed up lateral root emergence. We propose that Hs19C07 most likely increases LAX3-mediated auxin influx and may provide a mechanism for cyst nematodes to modulate auxin flow into root cells, stimulating cell wall hydrolysis for syncytium development.     

Activity of the Arabidopsis RD29A and RD29B promoter elements in soybean under water stress

The constitutive and drought-induced activities of the Arabidopsis thaliana RD29A and RD29B promoters were monitored in soybean (Glycine max (L.) Merr.] via fusions with the visual marker gene β-glucuronidase (GUS). Physiological responses of soybean plants were monitored over 9 days of water deprivation under greenhouse conditions. Data were used to select appropriate time points to monitor the activities of the respective promoter elements. Qualitative and quantitative assays for GUS expression were conducted in root and leaf tissues, from plants under well-watered and dry-down conditions. Both RD29A and RD29B promoters were significantly activated in soybean plants subjected to dry-down conditions. However, a low level of constitutive promoter activity was also observed in both root and leaves of plants under well-watered conditions. GUS expression was notably higher in roots than in leaves. These observations suggest that the respective drought-responsive regulatory elements present in the RD29X promoters may be useful in controlling targeted transgenes to mitigate abiotic stress in soybean, provided the transgene under control of these promoters does not invoke agronomic penalties with leaky expression when no abiotic stress is imposed.     

Preferential expression of a plant cystatin at nematode feeding sites confers resistance to Meloidogyne incognita and Globodera pallida

The expression patterns of three promoters preferentially active in the roots of Arabidopsis thaliana have been investigated in transgenic potato plants in response to plant parasitic nematode infection. Promoter regions from the three genes, TUB-1, ARSK1 and RPL16A were linked to the GUS reporter gene and histochemical staining was used to localize expression in potato roots in response to infection with both the potato cyst nematode, Globodera pallida and the root-knot nematode, Meloidogyne incognita. All three promoters directed GUS expression chiefly in root tissue and were strongly up-regulated in the galls induced by feeding M. incognita. Less activity was associated with the syncytial feeding cells of the cyst nematode, although the ARSK1 promoter was highly active in the syncytia of G. pallida infecting soil grown plants. Transgenic potato lines that expressed the cystatin OcIDeltaD86 under the control of the three promoters were evaluated for resistance against Globodera sp. in a field trial and against M. incognita in containment. Resistance to Globodera of 70 +/- 4% was achieved with the best line using the ARSK1 promoter with no associated yield penalty. The highest level of partial resistance achieved against M. incognita was 67 +/- 9% using the TUB-1 promoter. In both cases this was comparable to the level of resistance achieved using the constitutive cauliflower mosaic virus 35S (CaMV35S) promoter. The results establish the potential for limiting transgene expression in crop plants whilst maintaining efficacy of the nematode defence.     

The alfalfa (Medicago sativa) TDY1 gene encodes a mitogen-activated protein kinase homolog.

Development of root nodules, specifically induction of cortical cell division for nodule initiation, requires expression of specific genes in the host and microsymbiont. A full-length cDNA clone and the corresponding genomic clone encoding a MAP (mitogen-activated protein) kinase homolog were isolated from alfalfa (Medicago sativa). The genomic clone, TDY1, encodes a 68.9-kDa protein with 47.7% identity to MMK4, a previously characterized MAP kinase homolog from alfalfa. TDY1 is unique among the known plant MAP kinases, primarily due to a 230 amino acid C-terminal domain. The putative activation motif, Thr-Asp-Tyr (TDY), also differs from the previously reported Thr-Glu-Tyr (TEY) motif in plant MAP kinases. TDY1 messages were found predominantly in root nodules, roots, and root tips. Transgenic alfalfa and Medicago truncatula containing a chimeric gene consisting of 1.8 kbp of 5' flanking sequence of the TDY1 gene fused to the beta-glucuronidase (GUS) coding sequence exhibited GUS expression primarily in the nodule parenchyma, meristem, and vascular bundles, root tips, and root vascular bundles. Stem internodes stained intensely in cortical parenchyma, cambial cells, and primary xylem. GUS activity was observed in leaf mesophyll surrounding areas of mechanical wounding and pathogen invasion. The promoter was also active in root tips and apical meristems of transgenic tobacco. Expression patterns suggest a possible role for TDY1 in initiation and development of nodules and roots, and in localized responses to wounding.     

A soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) polyubiquitin promoter gives strong constitutive expression in transgenic soybean

The success of plant genetic transformation relies greatly on the strength and specificity of the promoters used to drive genes of interest. In this study, we analyzed gfp gene expression mediated by a polyubiquitin promoter (Gmubi) from soybean (Glycine max) in stably transformed soybean tissues. Strong GFP expression was observed in stably transformed proliferative embryogenic tissues. In whole transgenic plants, GFP expression was observed in root tips, main and lateral roots, cotyledons and plumules in young plants as well as in leaf veins, petioles, flower petals, pollen, pods and developing seeds in mature plants. GFP expression was localized mainly in epidermal cells, leaf mesophyll, procambium and vascular tissues. Introduction of an intron-less version of the Gmubi promoter (Gmupri) displayed almost the same GFP expression pattern albeit at lower intensities. The Gmubi promoter showed high levels of constitutive expression and represents an alternative to viral promoters for driving gene expression in soybean.     

Global functional analyses of rice promoters by genomics approaches

Promoters play key roles in conferring temporal, spatial, chemical, developmental, or environmental regulation of gene expression. Promoters that are subject to specific regulations are useful for manipulating foreign gene expression in plant cells, tissues, or organs with desirable patterns and under controlled conditions, and have been important for both basic research and applications in agriculture biotechnology. Recent advances in genomics technologies have greatly facilitated identification and study of promoters in a genome scale with high efficiency. Previously we have generated a large T-DNA tagged rice mutant library (TRIM), in which the T-DNA was designed with a gene/promoter trap system, by placing a promoter-less GUS gene next to the right border of T-DNA. GUS activity screens of this library offer in situ and in planta identifications and analyses of promoter activities in their native configurations in the rice genome. In the present study, we systematically performed GUS activity screens of the rice mutant library for genes/promoters constitutively, differentially, or specifically active in vegetative and reproductive tissues. More than 8,200 lines have been screened, and 11% and 22% of them displayed GUS staining in vegetative tissues and in flowers, respectively. Among the vegetative tissue active promoters, the ratio of leaf active versus root active is about 1.6. Interestingly, all the flower active promoters are anther active, but with varied activities in different flower tissues. To identify tissue specific ABA/stress up-regulated promoters, we compared microarray data of ABA/stress induced genes with those of tissue-specific expression determined by promoter trap GUS staining. Following this approach, we showed that the peroxidase 1 gene promoter was ABA up-regulated by 4 fold within 1 day of exposure to ABA and its expression is lateral root specific. We suggest that this be an easy bioinformatics approach in identifying tissue/cell type specific promoters that are up-regulated by hormones or other factors. Su-May Yu and Swee-Suak Ko equally contributed to this work.