Strigolactone Biosynthesis Genes of Rice are Required for the Punctual Entry of Arbuscular Mycorrhizal Fungi into the Roots.

Arbuscular mycorrhiza (AM) is a mutualistic association between most plant species and the ancient fungal phylum Glomeromycota in roots, and it plays a key role in a plant's nutrient uptake from the soil. Roots synthesize strigolactones (SLs), derivatives of carotenoids, and exude them to induce energy metabolism and hyphal branching of AM fungi. Despite the well-documented roles of SLs in the pre-symbiotic phase, little is known about the role of SLs in the process of root colonization. Here we show that the expansion of root colonization is suppressed in the mutants of rice (Oryza sativa) SL biosynthesis genes, carotenoid cleavage dioxygenase D10 and more severely in D17. Interestingly, most of the colonization process is normal, i.e. AM fungal hyphae approach the roots and cling around them, and epidermal penetration, arbuscule size, arbuscule number per hyphopodium and metabolic activity of the intraradical mycelium are not affected in d10 and d17 mutants. In contrast, hyphopodium formation is severely attenuated. Our observations establish the requirement for SL biosynthesis genes for efficient hyphopodium formation, suggesting that SLs are required in this process. Efficient hyphopodium formation is required for the punctual internalization of hyphae into roots and maintaining the expansion of colonization.

Phosphate Treatment Strongly Inhibits New Arbuscule Development But Not the Maintenance of Arbuscule in Mycorrhizal Rice Roots.

Phosphorus (P) is a crucial nutrient for plant growth, but its availability to roots is limited in soil. Arbuscular mycorrhizal (AM) symbiosis is a promising strategy for improving plant P acquisition. However, P fertilizer reduces fungal colonization (P inhibition) and compromises mycorrhizal P uptake, warranting studies on the mechanistic basis of P inhibition. In this study, early morphological changes in P inhibition were identified in rice (Oryza sativa) using fungal cell wall staining and live-cell imaging of plant membranes that were associated with arbuscule life cycles. Arbuscule density decreased, and aberrant hyphal branching was observed in roots at 5 h after P treatment. Although new arbuscule development was severely inhibited, preformed arbuscules remained intact and longevity remained constant. P inhibition was accelerated in the rice pt11-1 mutant, which lacks P uptake from arbuscule branches, suggesting that mature arbuscules are stabilized by the symbiotic P transporter under high P condition. Moreover, P treatment led to increases in the number of vesicles, in which lipid droplets accumulated and then decreased within a few days. The development of new arbuscules resumed within by 2 d. Our data established that P strongly and temporarily inhibits new arbuscule development, but not intraradical accommodation of AM fungi.

Evaluation of the Role of the LysM Receptor-Like Kinase, OsNFR5/OsRLK2 for AM Symbiosis in Rice.

In legume-specific rhizobial symbiosis, host plants perceive rhizobial signal molecules, Nod factors, by a pair of LysM receptor-like kinases, NFR1/LYK3 and NFR5/NFP, and activate symbiotic responses through the downstream signaling components also required for arbuscular mycorrhizal (AM) symbiosis. Recently, the rice NFR1/LYK3 ortholog, OsCERK1, was shown to play crucial roles for AM symbiosis. On the other hand, the roles of the NFR5/NFP ortholog in rice have not been elucidated, while it has been shown that NFR5/NFP orthologs, Parasponia PaNFR5 and tomato SlRLK10, engage in AM symbiosis. OsCERK1 also triggers immune responses in combination with a receptor partner, OsCEBiP, against fungal or bacterial infection, thus regulating opposite responses against symbiotic and pathogenic microbes. However, it has not been elucidated how OsCERK1 switches these opposite functions. Here, we analyzed the function of the rice NFR5/NFP ortholog, OsNFR5/OsRLK2, as a possible candidate of the OsCERK1 partner for symbiotic signaling. Inoculation of AM fungi induced the expression of OsNFR5 in the rice root, and the chimeric receptor consisting of the extracellular domain of LjNFR5 and the intracellular domain of OsNFR5 complemented the Ljnfr5 mutant for rhizobial symbiosis, indicating that the intracellular kinase domain of OsNFR5 could activate symbiotic signaling in Lotus japonicus. Although these data suggested the possible involvement of OsNFR5 in AM symbiosis, osnfr5 knockout mutants were colonized by AM fungi similar to the wild-type rice. These observations suggested several possibilities including the presence of functionally redundant genes other than OsNFR5 or involvement of novel ligands, which do not require OsNFR5 for recognition.

Up-regulation of genes involved in N-acetylglucosamine uptake and metabolism suggests a recycling mode of chitin in intraradical mycelium of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi colonize roots and form two kinds of mycelium, intraradical mycelium (IRM) and extraradical mycelium (ERM). Arbuscules are characteristic IRM structures that highly branch within host cells in order to mediate resource exchange between the symbionts. They are ephemeral structures and at the end of their life span, arbuscular branches collapse from the tip, fungal cytoplasm withdraws, and the whole arbuscule shrinks into fungal clumps. The exoskeleton of an arbuscule contains structured chitin, which is a polymer of N-acetylglucosamine (GlcNAc), whereas a collapsed arbuscule does not. The molecular mechanisms underlying the turnover of chitin in AM fungi remain unknown. Here, a GlcNAc transporter, RiNGT, was identified from the AM fungus Rhizophagus irregularis. Yeast mutants defective in endogenous GlcNAc uptake and expressing RiNGT took up (14)C-GlcNAc, and the optimum uptake was at acidic pH values (pH 4.0-4.5). The transcript levels of RiNGT in IRM in mycorrhizal Lotus japonicus roots were over 1000 times higher than those in ERM. GlcNAc-6-phosphate deacetylase (DAC1) and glucosamine-6-phosphate isomerase (NAG1) genes, which are related to the GlcNAc catabolism pathway, were also induced in IRM. Altogether, data suggest the existence of an enhanced recycling mode of GlcNAc in IRM of AM fungi.

Earliest colonization events of Rhizophagus irregularis in rice roots occur preferentially in previously uncolonized cells.

Arbuscular mycorrhizal (AM) fungi form a symbiotic association with several plant species. An arbuscule, a finely branched structure of AM fungi, is formed in root cells and plays essential roles in resource exchange. Because arbuscules are ephemeral, host cells containing collapsed arbuscules can be recolonized, and a wide region of roots can be continuously colonized by AM fungi, suggesting that repetitive recolonization in root cells is required for continuous mycorrhization. However, recolonization frequency has not been quantified because of the lack of appropriate markers for visualization of the cellular processes after arbuscule collapse; therefore, the nature of the colonization sequence remains uncertain. Here we observed that a green fluorescent protein (GFP)-tagged secretory carrier membrane protein (SCAMP) of rice was expressed even in cells with collapsed arbuscules, allowing live imaging coupled with GFP-SCAMP to evaluate the colonization and recolonization sequences. The average lifetime of intact arbuscules was 1-2 d. Cells with collapsed arbuscules were rarely recolonized and formed a new arbuscule during the observation period of 5 d, whereas de novo colonization occurred even in close proximity to cells containing collapsed arbuscules and contributed to the expansion of the colonized region. Colonization spread into an uncolonized region of roots but sparsely into a previously colonized region having no metabolically active arbuscule but several intercellular hyphae. Therefore, we propose that a previously colonized region tends to be intolerant to new colonization in rice roots. Our observations highlight the overlooked negative impact of the degeneration stage of arbuscules in the colonization sequence.

Lipid droplets of arbuscular mycorrhizal fungi emerge in concert with arbuscule collapse.

Plants share photosynthetically fixed carbon with arbuscular mycorrhizal (AM) fungi to maintain their growth and nutrition. AM fungi are oleogenic fungi that contain numerous lipid droplets in their syncytial mycelia during most of their life cycle. These lipid droplets are probably used for supporting growth of extraradical mycelia and propagation; however, when and where the lipid droplets are produced remains unclear. To address these issues, we investigated the correlation between intracellular colonization stages and the appearance of fungal lipid droplets in roots by a combination of vital staining of fungal structures, selective staining of lipids and live imaging. We discovered that a surge of lipid droplets coincided with the collapse of arbuscular branches, indicating that arbuscule collapse and the emergence of lipid droplets may be associated processes. This phenomenon was observed in the model AM fungus Rhizophagus irregularis and the ancestral member of AM fungi Paraglomus occultum. Because the collapsing arbuscules were metabolically inactive, the emerged lipid droplets are probably derived from preformed lipids but not de novo synthesized. Our observations highlight a novel mode of lipid release by AM fungi.

The soybean mycorrhiza-inducible phosphate transporter gene, GmPT7, also shows localized expression at the tips of vein endings of senescent leaves.

GmPT7 was originally identified as an arbuscular mycorrhiza-inducible gene of soybean that encodes a member of subfamily I in the PHOSPHATE TRANSPORTER 1 family. In the present study, we established conditions under which a number of dwarf soybean plants complete their life cycles in a growth chamber. Using this system, we grew transgenic soybean with a GmPT7 promoter-ß-glucuronidase fusion gene and evaluated GmPT7 expression in detail. GmPT7 was highly expressed in mature, but not in collapsed, arbuscule-containing cortical cells, suggesting its importance in the absorption of fungus-derived phosphate and/or arbuscule development. GmPT7 was also expressed in the columella cells of root caps and in the lateral root primordia of non-mycorrhizal roots. The expression of GmPT7 occurred only in the late stage of phosphorus translocation from leaves to seeds, after water evaporation from the leaves ceased, and later than the expression of GmUPS1-2, GmNRT1.7a and GmNRT1.7b, which are possibly involved in nitrogen export. GmPT7 expression was localized in a pair of tracheid elements at the tips of vein endings of senescent leaves. Transmission electron microscopy revealed that the tip tracheid elements in yellow leaves were still viable and had intact plasma membranes. Thus, we think that GmPT7 on the plasma membranes transports phosphate from the apoplast into the tip elements. GmPT7 knockdown resulted in no significant effects, the function of GmPT7 remaining to be clarified. We propose a working model in which phosphate incorporated in vein endings moves to seeds via xylem to phloem transfer.

The bifunctional plant receptor, OsCERK1, regulates both chitin-triggered immunity and arbuscular mycorrhizal symbiosis in rice.

Plants are constantly exposed to threats from pathogenic microbes and thus developed an innate immune system to protect themselves. On the other hand, many plants also have the ability to establish endosymbiosis with beneficial microbes such as arbuscular mycorrhizal (AM) fungi or rhizobial bacteria, which improves the growth of host plants. How plants evolved these systems managing such opposite plant-microbe interactions is unclear. We show here that knockout (KO) mutants of OsCERK1, a rice receptor kinase essential for chitin signaling, were impaired not only for chitin-triggered defense responses but also for AM symbiosis, indicating the bifunctionality of OsCERK1 in defense and symbiosis. On the other hand, a KO mutant of OsCEBiP, which forms a receptor complex with OsCERK1 and is essential for chitin-triggered immunity, established mycorrhizal symbiosis normally. Therefore, OsCERK1 but not chitin-triggered immunity is required for AM symbiosis. Furthermore, experiments with chimeric receptors showed that the kinase domains of OsCERK1 and homologs from non-leguminous, mycorrhizal plants could trigger nodulation signaling in legume-rhizobium interactions as the kinase domain of Nod factor receptor1 (NFR1), which is essential for triggering the nodulation program in leguminous plants, did. Because leguminous plants are believed to have developed the rhizobial symbiosis on the basis of AM symbiosis, our results suggest that the symbiotic function of ancestral CERK1 in AM symbiosis enabled the molecular evolution to leguminous NFR1 and resulted in the establishment of legume-rhizobia symbiosis. These results also suggest that OsCERK1 and homologs serve as a molecular switch that activates defense or symbiotic responses depending on the infecting microbes.

The effects of microbial fertilizers application on growth, yield and some biochemical changes in the leaves and seeds of guar (Cyamopsis tetragonoloba L.).

Guar (Cyamopsis tetragonoloba L.) is a summer legume that is becoming a crucial industrial crop because of its high gum and protein content. Thus far, the combined effects of arbuscular mycorrhizal fungi (AMF) and Bradyrhizobium on the yield and chemical composition of guar plants are not well studied. Therefore, the current investigation was designed to estimate the individual as well as the combined effects of AMF and Bradyrhizobium on plant growth, yield and nutritional quality of seeds and leaves of guar. AMF and/or Bradyrhizobium inoculation improved chemical composition of guar seeds and its morpho-physiological (plant height, fresh weight, dry weight, and yield production) traits. In addition to increased guar growth and yield production, the inoculation of AMF and/or Bradyrhizobium increased guar leaf and seed minerals, fiber, lipids, crude protein and ash contents. At primary metabolites, there were increases in sugar levels including raffinose stachyose, verbascose and galactomannan. These increases in sugar provided a route for organic acids, amino acids and fatty acids production. Interestingly, there was an increase in essential amino acids and unsaturated fatty acids. At the bioactive secondary metabolite levels, biofertilizers improved phenols and flavonoids levels and anthocyanin and polyamines biosynthesis. In line with these increases, precursors of anthocyanin (phenylalanine, p-coumaric acid, and cinnamic acid) and the levels of polyamines (diaminopropane, putrescine, cadaverine, spermidine, spermine, and agmatine) were increased. Overall, for the first time, our study shed the light on how AMF and Bradyrhizobium improved guar yield and metabolism. Our findings suggested that the combined inoculation of AMF and Bradyrhizobium is an innovative approach to improve guar growth, yield production and yield quality.

Identification and expression analysis of arbuscular mycorrhiza-inducible phosphate transporter genes of soybean.

Soybeans, the world's leading leguminous crop, establish mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi. AM fungi colonize root cortical cells forming arbuscules, highly branched fungal structures. Arbuscules are enveloped by plant-derived periarbuscular membranes through which plants obtain mineral nutrients, particularly phosphate. We searched the soybean genome in silico, and found 14 Pht1 genes encoding phosphate transporters putatively localized on the plasma membranes. Time course analyses involving reverse transcription-PCR indicated that three of these were AM-inducible. GmPT10 and GmPT11 were induced on fungal colonization, while a transcript of GmPT7 appeared in the later stages. The transport activities of GmPT10 and GmPT11 were confirmed by complementation of a yeast mutant. Soybean hairy roots expressing the GmPT10-green fluorescent protein (GFP) or GmPT11-GFP fusion protein under the control of corresponding promoter showed GFP fluorescence on the branch domains of periarbuscular membranes, indicating that active phosphate transport occurred there.

Expression of plant genes for arbuscular mycorrhiza-inducible phosphate transporters and fungal vesicle formation in sorghum, barley, and wheat roots.

Sorghum shows strong growth stimulation on arbuscular mycorrhizal (AM) symbiosis, while barley and wheat show growth depression. We identified the AM-inducible phosphate transporter genes of these cereals. Their protein products play major roles in phosphate absorption from arbuscules, intracellular fungal structures. Unexpectedly, barley and wheat expressed the AM-inducible genes at high levels. Hence the cause of their growth depression appears to be unrelated to the transcription of these genes. Notably, fungal vesicles were formed significantly more in barley and wheat than in sorghum. This study yielded new clues for investigation of the mechanism underlying these various responses.

Localized expression of the Dwarf14-like2a gene in rice roots on infection of arbuscular mycorrhizal fungus and hydrolysis of rac-GR24 by the encoded protein.

Strigolactones (SLs) are plant hormones that control diverse aspects of the shoot and root growth and are exuded into the soil as recruitment signals for arbuscular mycorrhizal (AM) fungi. SL signaling in plants is transduced via the α/ß-hydrolase receptor Dwarf14 (D14). The D14 family consists of D14, Dwarf14-like (D14L), and Dwarf14-like 2 (D14L2) clades in rice. The D14L receptor is known to condition pre-symbiotic perception of AM fungi. In this study, it was found that the Dwarf14-like2a (D14L2a) gene expression was significantly induced by AM fungal colonization. The transcript of D14L2a appeared not only in mature arbuscule-containing cells but also in epidermal/cortical cells at an early colonization stage and near the elongating intercellular hyphae. D14L2a transcript was detected normally in mycorrhizal roots of str1-2 mutant that form stunted arbuscules, suggesting that the gene expression is independent of arbuscule development. Moreover, the recombinant D14L2a protein exhibited hydrolase activity of synthetic SL, rac-GR24. Based on these results, we discussed the role of D14L2 in the establishment of AM symbiosis.

Dynamics of periarbuscular membranes visualized with a fluorescent phosphate transporter in arbuscular mycorrhizal roots of rice.

In arbuscular mycorrhizal (AM) symbiosis, host plants supply photosynthates to AM fungi and, in return, they receive inorganic nutrients such as phosphate from finely branched fungal arbuscules. Plant cortical cells envelope arbuscules with periarbuscular membranes that are continuous with the plant plasma membranes. We prepared transgenic rice (Oryza sativa) plants that express a fusion of green fluorescent protein with rice AM-inducible phosphate transporter, OsPT11-GFP, and grew them with AM fungi. The fluorescence of the fusion transporter was observed in the arbuscule branch domain, where active nutrient exchange seems to occur. In contrast, a signal was not detected around intracellular hyphal coils on colonization by either Glomus mosseae or Gigaspora rosea, making the difference between Arum- and Paris-type mycorrhizae ambiguous. We also invented a simple device involving glass-bottomed Petri dishes for in planta observation of fluorescent proteins in living AM roots with an inverted fluorescence microscope. The plant bodies remain completely intact, avoiding any stressful procedure such as cutting, staining, etc. Since rice roots exhibit a very low level of autofluorescence, the device enabled clear time-lapse imaging to analyze the formation, function and degeneration of arbuscules. In cortical cells, arbuscules seemed to be functional for only 2-3 d. Suddenly, the arbuscular branches became fragile and they shrank. At this stage, however, the periarbuscular membranes appeared intact. Then, the fluorescence of the transporter disappeared within only 2.5-5.5 h. The collapse of arbuscules occurred in the subsequent several days. Thus, our device has a great advantage for investigation of dynamic features of AM symbiosis.

Localized expression of arbuscular mycorrhiza-inducible ammonium transporters in soybean.

The majority of land plants acquire soil nutrients, such as phosphorus and nitrogen, not only through the root surface but also through arbuscular mycorrhizal (AM) fungi. Soybean is the most important leguminous crop in the world. We found 16 ammonium transporter genes in the soybean genome, five of which are AM inducible. Among them, promoter-reporter analysis indicated that the most abundantly transcribed gene, GmAMT4.1, showed specific expression in arbusculated cortical cells. Moreover, the GmAMT4.1-green fluorescent protein fusion was localized on the branch domain of periarbuscular membranes but not on the trunk region, indicating that active ammonium transfer occurs around the arbuscule branches.

A mutant strain Arabidopsis thaliana that lacks vacuolar membrane zinc transporter MTP1 revealed the latent tolerance to excessive zinc.

A mutant line of Arabidopsis thaliana that lacks a vacuolar membrane Zn(2+)/H(+) antiporter MTP1 is sensitive to zinc. We examined the physiological changes in this loss-of-function mutant under high-Zn conditions to gain an understanding of the mechanism of adaptation to Zn stress. When grown in excessive Zn and observed using energy-dispersive X-ray analysis, wild-type roots were found to accumulate Zn in vacuolar-like organelles but mutant roots did not. The Zn content of mutant roots, determined by chemical analysis, was one-third that of wild-type roots grown in high-Zn medium. Severe inhibition of root growth was observed in mtp1-1 seedlings in 500 muM ZnSO(4). Suppression of cell division and elongation by excessive Zn was reversible and the cells resumed growth in normal medium. In mutant roots, a marked formation of reactive oxygen species (ROS) appeared in the meristematic zone, where the MTP1 gene was highly expressed. Zn treatment enhanced the expression of several genes involved in Zn tolerance: namely, the plasma membrane Zn(2+)-export ATPase, HMA4, and plasma and vacuolar membrane proton pumps. CuZn-superoxide dismutases, involved in the detoxification of ROS, were also induced. The expression of plasma membrane Zn-uptake transporter, ZIP1, was suppressed. The up- or down-regulation of these genes might confer the resistance to Zn toxicity. These results indicate an essential role of MTP1 in detoxification of excessive Zn and provide novel information on the latent adaptation mechanism to Zn stress, which is hidden by MTP1.

Isolation of Native Arbuscular Mycorrhizal Fungi within Young Thalli of the Liverwort Marchantia paleacea.

Arbuscular mycorrhizal fungi (AMF) are a group of soil microorganisms that establish symbioses with most land plant species. "Root trap culture" generally has been used for isolating a single regenerated spore in order to establish a monospecific, native AMF line. Roots may be co-colonized with multiple AMF species; however, only a small portion of AMF within roots sporulate, and do so only under certain conditions. In this study, we tested whether young thalli (<2 mm) of the liverwort Marchantia paleacea harbour monospecific AMF, and can be used as a vegetative inoculant line. When M. paleacea gemmae were co-cultivated with roots obtained from the field, the young thalli were infected by AMF via rhizoids and formed arbuscules after 18 days post-sowing. Ribosomal DNA sequencing of the AMF-colonized thalli (mycothalli) revealed that they harboured phylogenetically diverse AMF; however, new gemmae sown around transplanted mycothalli showed evidence of colonization from phylogenetically uniform Rhizophagus species. Of note, mycothalli can also be used as an inoculum. These results suggest that the young thalli of M. paleacea can potentially isolate monospecific AMF from field soil in a spore-independent manner.

Beneficial associations between Brassicaceae plants and fungal endophytes under nutrient-limiting conditions: evolutionary origins and host-symbiont molecular mechanisms.

Brassicaceae plants have lost symbiotic interactions with mutualistic mycorrhizal fungi, but, nonmycorrhizal Brassicaceae associate with diverse taxonomic groups of mutualistic root-endophytic fungi. Distantly related fungal endophytes of Brassicaceae plants transfer phosphorus to the hosts and promote plant growth, thereby suggesting that the beneficial function was independently acquired via convergent evolution. These beneficial interactions appear tightly regulated by the tryptophan-derived secondary metabolite pathway, which specifically developed in Brassicaceae. Importantly, phosphate availability and types of colonizing microbes appear to influence the metabolite pathway. Thus, endophytes of Brassicaceae may have evolved to adapt to the Brassicaceae-specific traits. Future comparative functional analyses among well-defined endophytic fungi and their relatives with distinct life strategies and host plants will help understand the mechanisms that establish and maintain beneficial interactions.

Infection Unit Density as an Index of Infection Potential of Arbuscular Mycorrhizal Fungi.

The effective use of arbuscular mycorrhizal (AM) fungal function to promote host plant phosphate uptake in agricultural practice requires the accurate quantitative evaluation of AM fungal infection potential in field soil or AM fungal inoculation material. The number of infection units (IUs), intraradical fungal structures derived from single root entries formed after a short cultivation period, may reflect the number of propagules in soil when pot soil is completely permeated by the host root. However, the original IU method, in which all AM propagules in a pot are counted, requires the fine tuning of plant growing conditions and is considered to be laborious. The objective of the present study was to test whether IU density, not the total count of IU, but the number of IUs per unit root length, reflects the density of AM fungal propagules in soil. IU density assessed after 12 d of host plant cultivation and 3,3'-diaminobenzidine (DAB) staining showed a stronger linear correlation with propagule density than the mean infection percentage (MIP). In addition, IU density was affected less by the host plant species than MIP. We suggest that IU density provides a more rapid and reliable quantitation of the propagule density of AM fungi than MIP or the original IU method. Thus, IU density may be a more robust index of AM fungal infection potential for research and practical applications.

Arabidopsis immunophilin-like TWD1 functionally interacts with vacuolar ABC transporters.

Previously, the immunophilin-like protein TWD1 from Arabidopsis has been demonstrated to interact with the ABC transporters AtPGP1 and its closest homologue, AtPGP19. Physiological and biochemical investigation of pgp1/pgp19 and of twd1 plants suggested a regulatory role of TWD1 on AtPGP1/AtPGP19 transport activities. To further understand the dramatic pleiotropic phenotype that is caused by loss-of-function mutation of the TWD1 gene, we were interested in other TWD1 interacting proteins. AtMRP1, a multidrug resistance-associated (MRP/ABCC)-like ABC transporter, has been isolated in a yeast two-hybrid screen. We demonstrate molecular interaction between TWD1 and ABC transporters AtMRP1 and its closest homologue, AtMRP2. Unlike AtPGP1, AtMRP1 binds to the C-terminal tetratricopeptide repeat domain of TWD1, which is well known to mediate protein-protein interactions. Domain mapping proved that TWD1 binds to a motif of AtMRP1 that resembles calmodulin-binding motifs; and calmodulin binding to the C-terminus of MRP1 was verified. By membrane fractionation and GFP-tagging, we localized AtMRP1 to the central vacuolar membrane and the TWD1-AtMRP1 complex was verified in vivo by coimmunoprecipitation. We were able to demonstrate that TWD1 binds to isolated vacuoles and has a significant impact on the uptake of metolachlor-GS and estradiol-beta-glucuronide, well-known substrates of vacuolar transporters AtMRP1 and AtMRP2.

Varietal differences in the growth responses of rice to an arbuscular mycorrhizal fungus under natural upland conditions.

Seedlings of three rice (Oryza sativa L.) varieties (one indica, ARC5955; and two japonica, Nipponbare and Koshihikari) with or without pre-colonization by the arbuscular mycorrhizal fungus Funneliformis mosseae were transplanted into an upland field and grown to maturity. Pre-colonization had no effect on the yield of Nipponbare or Koshihikari. However, pre-colonized ARC5955 exhibited a strong tendency toward increased yield, which was accompanied by increases in the percentage of ripened grain and the 1000-grain weight. The rice roots were also colonized by indigenous arbuscular mycorrhizal fungi in the field, but these had only limited effects on shoot biomass and grain yields. We speculate that F. mosseae may have exhibited priority effects, allowing it to dominate the rice roots. There was no significant difference in the contents of most mineral elements in the shoots of pre-colonized ARC5955 at harvest, indicating that some other factor is responsible for the observed yield increase.