FISHing for ciliates: Catalyzed reporter deposition fluorescence in situ hybridization for the detection of planktonic freshwater ciliates.

Planktonic ciliate species form multiple trophic guilds and are central components of freshwater food webs. Progress in molecular analytical tools has opened new insight into ciliate assemblages. However, high and variable 18S rDNA copy numbers, typical for ciliates, make reliable quantification by amplicon sequencing extremely difficult. For an exact determination of abundances, the classical morphology-based quantitative protargol staining is still the method of choice. Morphotype analyses, however, are time consuming and need specific taxonomic expertise. Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) may represent a promising tool for the analysis of planktonic ciliates by combining molecular identification with microscopic quantification. We tested the applicability of CARD-FISH using nine cultured ciliate species. Eight species- and three genus-specific oligonucleotide probes were designed based on their 18S rRNA genes. The CARD-FISH protocol was adapted and the specificity of probes was established. We subsequently examined the precision of quantitation by CARD-FISH on single cultures and mock assemblages. Successful tests on lake water samples proved that planktonic ciliates could be identified and quantified in field samples by CARD-FISH. Double hybridizations allowed studying interspecific predator prey interactions between two ciliate species. In summary, we demonstrate that CARD-FISH with species-specific probes can facilitate studies on the population dynamics of closely related, small sized or cryptic species at high sampling frequencies.

Identification of an Isoflavonoid Transporter Required for the Nodule Establishment of the Rhizobium-Fabaceae Symbiotic Interaction.

Nitrogen (N) as well as Phosphorus (P) are key nutrients determining crop productivity. Legumes have developed strategies to overcome nutrient limitation by, for example, forming a symbiotic relationship with N-fixing rhizobia and the release of P-mobilizing exudates and are thus able to grow without supply of N or P fertilizers. The legume-rhizobial symbiosis starts with root release of isoflavonoids that act as signaling molecules perceived by compatible bacteria. Subsequently, bacteria release nod factors, which induce signaling cascades allowing the formation of functional N-fixing nodules. We report here the identification and functional characterization of a plasma membrane-localized MATE-type transporter (LaMATE2) involved in the release of genistein from white lupin roots. The LaMATE2 expression in the root is upregulated under N deficiency as well as low phosphate availability, two nutritional deficiencies that induce the release of this isoflavonoid. LaMATE2 silencing reduced genistein efflux and even more the formation of symbiotic nodules, supporting the crucial role of LaMATE2 in isoflavonoid release and nodulation. Furthermore, silencing of LaMATE2 limited the P-solubilization activity of lupin root exudates. Transport assays in yeast vesicles demonstrated that LaMATE2 acts as a proton-driven isoflavonoid transporter.

Arabidopsis ABCG34 contributes to defense against necrotrophic pathogens by mediating the secretion of camalexin.

Plant pathogens cause huge yield losses. Plant defense often depends on toxic secondary metabolites that inhibit pathogen growth. Because most secondary metabolites are also toxic to the plant, specific transporters are needed to deliver them to the pathogens. To identify the transporters that function in plant defense, we screened Arabidopsis thaliana mutants of full-size ABCG transporters for hypersensitivity to sclareol, an antifungal compound. We found that atabcg34 mutants were hypersensitive to sclareol and to the necrotrophic fungi Alternaria brassicicola and Botrytis cinereaAtABCG34 expression was induced by Abrassicicola inoculation as well as by methyl-jasmonate, a defense-related phytohormone, and AtABCG34 was polarly localized at the external face of the plasma membrane of epidermal cells of leaves and roots. atabcg34 mutants secreted less camalexin, a major phytoalexin in Athaliana, whereas plants overexpressing AtABCG34 secreted more camalexin to the leaf surface and were more resistant to the pathogen. When treated with exogenous camalexin, atabcg34 mutants exhibited hypersensitivity, whereas BY2 cells expressing AtABCG34 exhibited improved resistance. Analyses of natural Arabidopsis accessions revealed that AtABCG34 contributes to the disease resistance in naturally occurring genetic variants, albeit to a small extent. Together, our data suggest that AtABCG34 mediates camalexin secretion to the leaf surface and thereby prevents Abrassicicola infection.

An N-acetylglucosamine transporter required for arbuscular mycorrhizal symbioses in rice and maize.

Most terrestrial plants, including crops, engage in beneficial interactions with arbuscular mycorrhizal fungi. Vital to the association is mutual recognition involving the release of diffusible signals into the rhizosphere. Previously, we identified the maize no perception 1 (nope1) mutant to be defective in early signalling. Here, we report cloning of ZmNope1 on the basis of synteny with rice. NOPE1 encodes a functional homologue of the Candida albicans N-acetylglucosamine (GlcNAc) transporter NGT1, and represents the first plasma membrane GlcNAc transporter identified from plants. In C. albicans, exposure to GlcNAc activates cell signalling and virulence. Similarly, in Rhizophagus irregularis treatment with rice wild-type but not nope1 root exudates induced transcriptome changes associated with signalling function, suggesting a requirement of NOPE1 function for presymbiotic fungal reprogramming.

The Arabidopsis tonoplast is almost devoid of glycoproteins with complex N-glycans, unlike the rat lysosomal membrane.

The distribution of the N-glycoproteome in integral membrane proteins of the vacuolar membrane (tonoplast) or the plasma membrane of Arabidopsis thaliana and, for further comparison, of the Rattus norvegicus lysosomal and plasma membranes, was analyzed. In silico analysis showed that potential N-glycosylation sites are much less frequent in tonoplast proteins. Biochemical analysis of Arabidopsis subcellular fractions with the lectin concanavalin A, which recognizes mainly unmodified N-glycans, or with antiserum against Golgi-modified N-glycans confirmed the in silico results and showed that, unlike the plant plasma membrane, the tonoplast is almost or totally devoid of N-glycoproteins with Golgi-modified glycans. Lysosomes share with vacuoles the hydrolytic functions and the position along the secretory pathway; however, our results indicate that their membranes had a divergent evolution. We propose that protection against the luminal hydrolases that are abundant in inner hydrolytic compartments, which seems to have been achieved in many lysosomal membrane proteins by extensive N-glycosylation of the luminal domains, has instead been obtained in the vast majority of tonoplast proteins by limiting the length of such domains.

ABCC1, an ATP binding cassette protein from grape berry, transports anthocyanidin 3-O-Glucosides.

Accumulation of anthocyanins in the exocarp of red grapevine (Vitis vinifera) cultivars is one of several events that characterize the onset of grape berry ripening (véraison). Despite our thorough understanding of anthocyanin biosynthesis and regulation, little is known about the molecular aspects of their transport. The participation of ATP binding cassette (ABC) proteins in vacuolar anthocyanin transport has long been a matter of debate. Here, we present biochemical evidence that an ABC protein, ABCC1, localizes to the tonoplast and is involved in the transport of glucosylated anthocyanidins. ABCC1 is expressed in the exocarp throughout berry development and ripening, with a significant increase at véraison (i.e., the onset of ripening). Transport experiments using microsomes isolated from ABCC1-expressing yeast cells showed that ABCC1 transports malvidin 3-O-glucoside. The transport strictly depends on the presence of GSH, which is cotransported with the anthocyanins and is sensitive to inhibitors of ABC proteins. By exposing anthocyanin-producing grapevine root cultures to buthionine sulphoximine, which reduced GSH levels, a decrease in anthocyanin concentration is observed. In conclusion, we provide evidence that ABCC1 acts as an anthocyanin transporter that depends on GSH without the formation of an anthocyanin-GSH conjugate.