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

Abstract

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.

Figure 1. Mutation of the orthologous genes Os04g01520 and GRMZM2G176737 disrupted coloniztion by R. irregularis in rice and maize, respectively

A, Percentage root length colonization in rice plants (n = 3) segregating Osnope1 (+/+, wild type, −/− homozygous Osnope1, C complemented line C4) at 6 weeks post infection (wpi). Means groups assigned for each fungal structure indicated by letters (adj. P < 0.05). B–G, WGA-staining of fungal structures and propidium-iodide counterstained plant cell walls of rice roots inoculated with R. irregularis at 6 wpi as examined by laser scanning confocal microscopy. B, Hyphopodium (HP) and arbuscule differentiation in a wild type (WT) root C, Misshapen and highly septate (arrows) hyphopodial hypha on the surface of the root of a Osnope1 homozygote. D, Detail of a hyphopodia on a Osnope1 homozygous plant, showing several aborted penetration attempts (arrowheads). Morphologically equivalent arbuscules formed in the roots of wild type (E) and Osnope1 homozygous (F) plants. G, Arbuscules formed in root cortical cells of the complemented line C4. H, Percentage R. irregularis root length colonization of maize plants segregating Zmnope1 (+/+, wild type, +/−, heterozygote, −/− homozygous mutant) at 6 wpi. Represented as A. I and J, WGA-staining of fungal structures and propidium-iodide counterstained plant cell walls of maize roots inoculated with R. irregularis at 6 wpi, as examined by laser scanning confocal microscopy. On wild type (I) roots the fungus develops normal hyphopodia and extensively colonizes the root forming frequent arbuscules. J, Misshapen hyphopodium on the roots of a nope1-1 homozygous mutant showing mutiple septa (arrows) and absence of root pentration. HP, hyphopodium; A, arbuscule. Scale bar = 50µm.

Figure 2. Rice NOPE1 mediates GlcNAc transport in C. albicans

A, Ten-fold cell dilution series of C. albicans strains spotted onto plates with indicated sugar. B, C. albicans strains grown overnight in glucose containing medium and resuspended in fresh medium containing either 50 mM glucose or 50 mM GlcNAc. C, [H³]GlcNAc uptake in C. albicans strains at 20 mM and 200 mM GlcNAc. GlcNAc, N-acetylglucosamine. Boxes show 1st quartile, median and 3rd quartile. Whiskers extend to the most extreme points within 1.5× box length; outlying values beyond this range are shown as unfilled circles. Means groups were calculated post hoc independently for the two GlcNAc treatments and are indicated by letters (p < 0.05). For description of strains see Supplemental Information Table S5.

Figure 3. NOPE1 mediates GlcNAc transport in rice and Arabidopsis

A, Laser scanning confocal microscopy of A. thaliana roots expressing Ubq_prom::YPF::AtNOPE1. Three independent lines were analyzed to determine reproducible localization patterns, here shown for line At4731y-4 with the YFP-AtNOPE1 signal shown in green (left). Corresponding cells stained with Propidium Iodide (PI) shown in red (centre). Overlay (yellow, right). Scale bar: 5 µm. B, Time course of [³H]GlcNAc uptake in roots of Osnope1, wild type and genetically complemented mutant line C4. Means and SEs of three biologically independent experiments are shown (*P ≤ 0.05). Please note that the 0.5min value corresponds to unspecific adsorption of medium to the protoplasts. C, Time course of [³H]GlcNAc export activity of wild type rice roots at 0 and 5mM (50×) GlcNAc external concentration. Means and SEs of three biological replicates are shown. (**p ≤ 0.01).

Figure 4. R. irregularis transcriptional response to root exudates from rice wild type and Osnope1 mutant plants

A, Venn diagrams indicating number of significantly induced fungal genes (P ≤ 0.05, one way ANOVA) in response to treatment with exudates from wild type relative to Osnope (top) and Osnope relative to wild type (bottom). B, Time-resolved Gene Ontology analysis for Biological Process terms (p ≤0.01) for fungal genes induced when treated with root exudates from wild type (top) or from Osnope1 (bottom). The colour code indicates the significance of gene enrichment (p-value).