Microarray analysis of expressed sequence tags from haustoria of the rust fungus Uromyces fabae

Rust fungi are plant parasites which colonise host tissue with an intercellular mycelium that forms haustoria within living plant cells. To identify genes expressed during biotrophic growth, EST sequencing was performed with a haustorium-specific cDNA library from Uromyces fabae. One thousand seventeen ESTs were generated, which assembled into 530 contigs. Several of the most frequently represented sequences in the EST database were identical to the in planta induced genes (PIGs) identified previously (Hahn, M., Mendgen, K., 1997. Characterisation of in planta-induced rust genes isolated from a haustorium-specific cDNA library, Mol. Plant-Microbe Interact. 10, 427-437). Virus-encoded sequences were identified, providing evidence for two novel RNA mycoviruses in U. fabae. Microarray hybridisation revealed many cDNAs that were significantly activated in rust-infected leaves compared to germinated uredospores. Very strong in planta expression was found for two PIGs encoding putative metallothioneins. Furthermore, several genes involved in ribosome biogenesis and translation, glycolysis, amino acid metabolism, stress response, and detoxification showed an increased expression in the parasitic mycelium. These data indicate a strong shift in gene expression in rust fungi between germination and the biotrophic stage of development.     

Molecular cloning and characterization of a tobacco leaf cDNA encoding a phosphate transporter

Phosphate acquired by roots is translocated to and utilized by the upper part of the plant, where the phosphate transport in the cell is also important in the phosphate metabolism. In order to study the role of the phosphate transporter in the regulation of the phosphate movement across the membranes in leaf cells, we isolated and characterized a 2,059 bp tobacco leaf cDNA clone, NtPT1. The 537 amino acid sequences, deduced from NtPT1, exhibited 93 and 91% identites to one of the high affinity phosphate transporters constitutively expressed in potato and tomato roots, respectively. The NtPT1 contains 12 membrane-spanning domain with a central hydrophilic region. The expression of NtPT1 in the yeast high affinity phosphate transporter mutant strain, NS219, complemented the mutant and promoted cell growth significantly. These results strongly suggest that NtPT1 encodes a functional phosphate transporter and that one of the high affinity phosphate transporters expressed in roots is also expressed in leaves. Southern analysis indicated that tobacco phosphate transporter genes are low copy number genes and members of a small multi-gene family.     

Altered growth and improved resistance of Arabidopsis against Pseudomonas syringae by overexpression of the basic amino acid transporter AtCAT1

Amino acid transporters in plants are crucial for distributing amino acids between plant organs and cellular compartments. The H⁺‐coupled plasma membrane transporter CAT1 (cationic amino acid transporter 1) facilitates the high‐affinity uptake of basic amino acids. The uptake of lysine (Lys) via the roots was not altered in loss‐of‐function mutants, in accordance with the minor expression of CAT1 in roots, but plants ectopically overexpressing CAT1 incorporated Lys at higher rates. Exogenous Lys inhibited the primary root of Arabidopsis, whereas lateral roots were stimulated. These effects were augmented by the presence or absence of CAT1. Furthermore, the total biomass of soil‐grown plants ectopically overexpressing CAT1 was reduced and the time to flowering was accelerated. These effects were accompanied by only minor changes in the overall amino acid profile. Interestingly, CAT1 belongs to a specific small cluster of nitrogen‐containing metabolite transporter genes that are rapidly up‐regulated upon infection with Pseudomonas syringae and that may participate in the systemic response of plants to pathogen attack. The overexpression of CAT1 indeed enhanced the resistance to the hemibiotrophic bacterial pathogen P. syringae via a constitutively activated salicylic acid (SA) pathway, which is consistent with the developmental defects and the resistance phenotype.     

Molecular and functional characterization of a family of amino acid transporters from Arabidopsis

More than 50 distinct amino acid transporter genes have been identified in the genome of Arabidopsis, indicating that transport of amino acids across membranes is a highly complex feature in plants. Based on sequence similarity, these transporters can be divided into two major superfamilies: the amino acid transporter family and the amino acid polyamine choline transporter family. Currently, mainly transporters of the amino acid transporter family have been characterized. Here, a molecular and functional characterization of amino acid polyamine choline transporters is presented, namely the cationic amino acid transporter (CAT) subfamily. CAT5 functions as a high-affinity, basic amino acid transporter at the plasma membrane. Uptake of toxic amino acid analogs implies that neutral or acidic amino acids are preferentially transported by CAT3, CAT6, and CAT8. The expression profiles suggest that CAT5 may function in reuptake of leaking amino acids at the leaf margin, while CAT8 is expressed in young and rapidly dividing tissues such as young leaves and root apical meristem. CAT2 is localized to the tonoplast in transformed Arabidopsis protoplasts and thus may encode the long-sought vacuolar amino acid transporter.     

Envelope-binding domain in the cationic amino acid transporter determines the host range of ecotropic murine retroviruses.

Infection of rodent cells by ecotropic type C retroviruses requires the expression of a cationic amino acid transporter composed of multiple membrane-spanning domains. By exchanging portions of cDNAs encoding the permissive mouse and nonpermissive human transporters and examining their abilities to specify virus infection upon expression in human 293 cells, we have identified the amino acid residues in the extracellular loop connecting the fifth and sixth membrane-spanning segments of the mouse transporter that are required for both envelope gp70 binding and infection. These findings strongly suggest that the role of the mouse transporter in determining infection is to provide an envelope-binding site. This role is analogous to those of host membrane proteins composed of a single membrane-spanning domain that serve as binding proteins or receptors for other enveloped viruses such as human immunodeficiency virus, Epstein-Barr virus, and murine and human coronaviruses.     

Transporters in Arabidopsis roots mediating uptake of amino acids at naturally occurring concentrations

Recent studies of Arabidopsis have identified several transporters as being important for amino acid uptake. We used Arabidopsis plants with altered expression of lysine histidine transporter 1 (LHT1), amino acid permease 1 (AAP1) and amino acid permease 5 (AAP5) with the aim of disentangling the roles of each transporter in the uptake of different amino acids at naturally occurring concentrations (2-50 μM). LHT1 mutants displayed reduced uptake rates of L-Gln, L-Ala, L-Glu and L-Asp but not of L-Arg or L-Lys, while AAP5 mutants were affected in the uptake of L-Arg and L-Lys only. Double mutants (lht1aap5) exhibited reduced uptake of all tested amino acids. In the concentration range tested, AAP1 mutants did not display altered uptake rates for any of the studied amino acids. Expression analysis of amino acid transporter genes with important root functions revealed no major differences in the individual mutants other than for genes targeted for mutation. We conclude that LHT1 and AAP5, but not AAP1, are crucial for amino acid uptake at concentrations typically found in soils. LHT1 and AAP5 displayed complementary affinity spectra, and no redundancy with respect to gene expression was found between the two transporters, suggesting these two transporters have separate roles in amino acid uptake.     

Ten rice peroxidases redundantly respond to multiple stresses including infection with rice blast fungus

Class III plant peroxidases are believed to function in diverse physiological processes including disease resistance and wound response, but predicted low substrate specificities and the presence of 70 or more isoforms have made it difficult to define a specific physiological function(s) for each gene. To select pathogen-responsive POX genes, we analyzed the expression profiles of 22 rice POX genes after infection with rice blast fungus. The expression of 10 POX genes among the 22 genes was induced after fungal inoculation in both compatible and incompatible hosts. Seven of the 10 POX genes were expressed at higher levels in the incompatible host than in the compatible host 6-24 h after inoculation by which time no fungus-induced lesions have appeared. Organ-specific expression and stress-induced expression by wounding and treatment with probenazole, an agrichemical against blast fungus, jasmonic acid, salicylic acid and 1-aminocyclopropane-1-carboxylate, a precursor of ethylene, indicated that rice POXs have individual characteristics and can be classified into several types. A comparison of the amino acid sequences of POXs showed that multiple isoforms with a high sequence similarity respond to stress in different or similar ways. Such redundant responses of POX genes may guarantee POX activities that are necessary for self-defense in plant tissues against environmental stresses including pathogen infection.     

Cloning of a Picea abies monosaccharide transporter gene and expression – analysis in plant tissues and ectomycorrhizas

Ectomycorrhizas are formed between certain soil fungi and fine roots of woody plants. An important feature of this symbiosis is the supply of photoassimilates to the fungus. Hexoses, formed from sucrose in the common apoplast at the root/fungus interface, can be taken up by both plant and fungal monosaccharide transporters. Recently we characterised a monosaccharide transporter from the ectomycorrhizal fungus Amanita muscaria. This transporter was up-regulated in mycorrhizas, thus increasing the hexose uptake capacity of the fungal partner in symbiosis. In order to characterise host (Picea abies) root monosaccharide transporters, degenerate oligonucleotide primers, designed to match conserved regions from known plant hexose transporters, were used to isolate a cDNA fragment of a transporter by PCR. This fragment was used to identify a presumably full length clone (PaMST1) in a P. abies/A. muscaria mycorrhizal cDNA library. The entire cDNA code for an open reading frame of 513 amino acids, revealing best homology to H⁺/monosaccharide transporters from Ara- bidopsis, Saccharum and Ricinus. PaMST1 was highly expressed in the hypocotyl and in roots of P. abies seedlings, but not in needles. Mycorrhiza formation led to a slight reduction of PaMST1 expression. The results are discussed with special reference to carbon allocation in ectomycorrhizas. Received: 9 October 1999 / Accepted: 22 December 1999     

Induction of amino acid transporters expression by endurance exercise in rat skeletal muscle

We here investigated whether an acute bout of endurance exercise would induce the expression of amino acid transporters that regulate leucine transport across plasma and lysosomal membranes in rat skeletal muscle. Rats ran on a motor-driven treadmill at a speed of 28 m/min for 90 min. Immediately after the exercise, we observed that expression of mRNAs encoding l-type amino acid transporter 1 (LAT1) and CD98 was induced in the gastrocnemius, soleus, and extensor digitorum longus (EDL) muscles. Sodium-coupled neutral amino acid transporter 2 (SNAT2) mRNA was also induced by the exercise in those three muscles. Expression of proton-assisted amino acid transporter 1 (PAT1) mRNA was slightly but not significantly induced by a single bout of exercise in soleus and EDL muscles. Exercise-induced mRNA expression of these amino acid transporters appeared to be attenuated by repeated bouts of the exercise. These results suggested that the expression of amino acid transporters for leucine may be induced in response to an increase in the requirement for this amino acid in the cells of working skeletal muscles.     

Changes in carbon allocation and expression of carbon transporter genes in Betula pendula Roth. colonized by the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr.

Comparative analyses of aspects of the carbon (C) physiology and the expression of C transporter genes in birch (Betula pendula Roth.) colonized by the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. were performed using mycorrhizal (M) and non‐mycorrhizal (NM) plants of similar foliar nutrient status. After six months of growth, the biomass of M plants was significantly lower than that of NM plants. Diurnal C budgets of both sets of plants revealed that M plants exhibited higher rates of photosynthesis and root respiration expressed per unit dry weight. However, the diurnal net C gain of M and NM plants remained similar. Ectomycorrhizal roots contained higher soluble carbohydrate pools and increased activity of cell wall invertase, suggesting that additional C was allocated to these roots and their ectomycorrhizal fungi consistent with an increased sink demand for C due to the presence of the mycobiont. In M roots, the expression of two hexose and one sucrose transporter genes of birch were reduced to less than one‐third of the expression level observed in NM roots. Analysis using a probe against the birch ribosomal internal transcribed spacer region revealed that M roots contained 22% less plant RNA than NM roots. As the expression of birch hexose and sucrose transporter genes was reduced to a much greater extent, this suggests that these specific genes were down‐regulated in response to alterations in C metabolism within M roots.     

Expression of amino acid transporter genes in developmental stages and adult tissues of Antarctic echinoderms

Epithelial cells in the body wall of adult and developmental stages of marine invertebrates absorb dissolved organic material directly from seawater. Despite over a century of study, little is known about the molecular biological mechanisms responsible for this transport process. Previous studies on embryonic and larval Antarctic echinoderms show that amino acid uptake could provide an important supplement of metabolic substrates. In the present study, partial cDNA sequences of 11 putative amino acid transporter genes were isolated from six species of Antarctic echinoderms including the Antarctic sea stars Acodontaster hodgsoni, Diplasterias brucei, Odontaster meridionalis, Odontaster validus, and Perknaster fuscus, and the Antarctic sea urchin Sterechinus neumayeri. Conserved domains of cDNA-deduced amino acid sequences characterized these genes as being members of a family of amino acid transporters (solute carrier family 6). Expression of these genes was detected throughout embryonic and larval development of two species that have contrasting developmental modes (A. hodgsoni: lecithotrophic; O. meridionalis: planktotrophic). In all six species studied, the expression of amino acid transporter genes was detected in tube feet and digestive organs of adult animals, demonstrating that members of a single amino acid transporter gene family are expressed during the entire life history of a marine invertebrate. The identification of these genes is an important step toward developing a mechanistic understanding of amino acid transport capacities in Antarctic marine invertebrates.