A nuclear-targeted effector of Rhizophagus irregularis interferes with histone 2B mono-ubiquitination to promote arbuscular mycorrhisation

Abstract

Arguably, symbiotic arbuscular mycorrhizal (AM) fungi have the broadest host range of all fungi, being able to intracellularly colonise root cells in the vast majority of all land plants. This raises the question how AM fungi effectively deal with the immune systems of such a widely diverse range of plants. Here, we studied the role of a nuclear-localisation signal-containing effector from Rhizophagus irregularis, called Nuclear Localised Effector1 (RiNLE1), that is highly and specifically expressed in arbuscules. We showed that RiNLE1 is able to translocate to the host nucleus where it interacts with the plant core nucleosome protein histone 2B (H2B). RiNLE1 is able to impair the mono-ubiquitination of H2B, which results in the suppression of defence-related gene expression and enhanced colonisation levels. This study highlights a novel mechanism by which AM fungi can effectively control plant epigenetic modifications through direct interaction with a core nucleosome component. Homologues of RiNLE1 are found in a range of fungi that establish intimate interactions with plants, suggesting that this type of effector may be more widely recruited to manipulate host defence responses.

Keywords: Rhizophagus irregularis; H2B mono-ubiquitination; arbuscular mycorrhiza (AM); effector; plant defence; symbiosis.

Fig. 1

RiNLE1 is specifically expressed in arbuscules and localises in the plant nucleus. (a) RNA in situ hybridisations of the arbuscule‐containing cell‐specific MtPT4 gene in mycorrhized Medicago truncatula roots. Arrow indicates in situ signal. (b) RNA in situ hybridisations of RiNLE1 in mycorrhized M. truncatula roots. Arrow indicates in situ signal. (c) Negative control of RNA in situ hybridisation in which RiNLE1 sense probe sets are used. (d) Negative control of RNA in situ hybridisation in which probe sets are omitted to reveal the background signal. (a–d) Bars, 25 μm. (e–h) PT4p::GFP‐RiNLE1ΔSP expressed in M. truncatula mycorrhizal roots. (e) GFP‐RiNLE1 accumulates in the nucleolus and other nuclear bodies of the arbuscule‐containing cells, as indicated by arrowheads. (f) Corresponding red fluorescence resulting from co‐expression of the DsRed1 protein under the control of the Arabidopsis thaliana Ubiquitin10 promoter, used as marker for cytoplasm and nucleus. (g) Corresponding bright field image. (h) Corresponding overlay of GFP, DsRed and bright field image. (d–g) Bars, 10 μm.

Fig. 2

RiNLE1 is secreted and translocates to the plant nucleus. (a) Yeast signal sequence trap showing that RiNLE1 is a secreted protein. RiNLE1, representing a fusion of full‐length RiNLE1 with an invertase, and the empty pYST0 vector (EV) were transformed into Saccharomyces cerevisiae Y02321, and grown on SD/−Leu and sucrose selection medium at different dilutions for 3 d at 30°C. (b) RiNLE1 can translocate to the plant nucleus. Expression of UBp::BCP1sp‐GFP (upper row) and UBp::MtBCP1sp‐GFP‐RiNLE1 (lower row), using the signal peptide of Medicago BLUE COPPER PROTEIN 1 (BCP1) to secrete GFP or GFP‐tagged RiNLE1 to the apoplast, in Nicotiana benthamiana leaves. By contrast with the GFP control, the RiNLE1 fusion protein can be observed inside the plant nucleus indicating that NLE1 can translocate into the plant cell after been secreted to the apoplast. Bars, 10 μm.

Fig. 3

Overexpression of RiNLE1 enhances mycorrhizal root colonisation in Medicago truncatula. (a) Quantification of the level of mycorrhization in UBp::RiNLE1 transgenic roots and empty vector control roots, 3 wk post inoculation. Indicated are the percentage of intraradical hyphae (IntrHyphae), arbuscules and overall level of colonisation (Total). Data are represented as mean ± SD of eight independently transformed plants per construct. Student’s t‐test. *, P < 0.05; (b) qPCR analyses showing increased RiEF expression level as the marker for fungal colonisation in RiNLE1 overexpression samples. No significant difference between EV and RiNLE1 samples is observed for arbuscular‐specific MtPT4 gene expression. Expression levels were normalised using MtEF1 as reference. Data are represented as mean ± SD of eight independently transformed plants per construct. Student’s t‐test. *, P < 0.05; **, P < 0.01.

Fig. 4

Dexamethasone (DEX)‐induced expression of RiNLE1 suppresses defence gene expression in Medicago truncatula roots. (a) Induction of RiNLE1 expression suppresses host defence gene expression based on RNA‐seq data. Heatmap of DEGs that show significant (>2‐fold, P < 0.05) downregulation upon 20 h of DEX treatment in RiNLE1‐expressing roots compared with empty vector controls. Expression values reflect log₂ transformed transcripts per million (TPM). Columns were clustered using Euclidean distance, rows were ranged by fold change from high to low. (b) qRT‐PCR analysis of selected defence‐related genes (see also Supporting Information Table S6) based on RNA‐seq analyses in independent DEX‐induced RiNLE1‐expressing and EV control roots. MtEF1 was used as reference. Data are represented as mean ± SD of three biological replicates. Student’s t‐test was used. *, P < 0.05. (c) RiNLE1 inhibited Cladosporium fulvum AVR4 effector and Solanum lycopersicum receptor‐like protein Cf‐4 induced HR. Nicotiana benthamiana leaves were infiltrated with the following combinations of Agrobacterium strains and grown for 4 d: 1, 0.04 OD₆₀₀ AVR4 + 0.04 OD₆₀₀ Cf‐4 (positive control); 2, 0.04 OD₆₀₀ AVR4 + 0.04 OD₆₀₀ Cf‐4 + 1 OD₆₀₀ GFP (negative control); 3, 0.04 OD₆₀₀ AVR4 + 0.04 OD₆₀₀ Cf‐4 + 1 OD₆₀₀ RiNLE1; 4, 1 OD₆₀₀ RiNLE1 (negative control). (d) Trypan blue staining of the leaves in (c) to visualise accumulation of HR.

Fig. 5

RiNLE1 interacts with H2B. (a) Co‐IP assay in transiently transformed Nicotiana benthamiana leaves confirmed the interaction between RiNLE1 and MtH2B.1 (Medtr4g064020). Free GFP and a FLAG‐tagged nuclear‐localising protein MtSPX3 (Medtr0262s0060) were included as controls. For the input blots, 0.2% input extract was loaded to detect FLAG‐ and GFP‐tagged proteins using anti‐FLAG or anti‐GFP antibodies, respectively. After co‐immunoprecipitation, 20% of the eluate was loaded for detection. Total protein levels were detected using Coomassie brilliant blue staining as the loading control. (b) Y2H assay showing the interaction between RiNLE1 and MtH2B.1, using RiNLE1 as bait (pGBKT7) and H2B.1 as prey (pGADT7). Serial dilutions of overnight mated yeast cultures were plated on selective medium. DDO was used as the control plate to show mating was successful. Selection was carried out on QDO plates containing aureobasidin and LacZ staining as additional selection markers. The positive (pGBKT7_53 + pGADT7_T), negative (pGBKT7_lam + pGADT7_T), and EV (GBKT7_RiNLE1 + pGADT7) controls are shown.

Fig. 6

RiNLE1 inhibits H2B mono‐ubiquitination levels. (a) Western blot analysis of nuclear protein extracts from 20 h DEX‐induced EV and RiNLE1 Medicago truncatula roots. Detection was carried out using anti‐H2Bub (upper panel) or anti‐H3 antibody (lower panel). The expected sizes of H2Bub and H3 are 28 kDa and 17 kDa, respectively. Similar results were obtained in three independent experiments. (b) Quantification of the relative band intensities using imagej software based on the three independent experiments, including the one shown in (a), normalised to H3 levels. Error bars indicate SD from the three replicates. Student’s t‐test was used. *, P < 0.05.

Fig. 7

H2Bub levels influence defence gene expression and arbuscular mycorrhization. (a) Western blot analysis of nuclear protein extracts from 20 h DEX‐induced EV, MtH2BK144A and MtHUB1 expressing Medicago roots. Detection using anti‐H2Bub (upper panel) and anti‐H3 antibody (lower panel). (b) qPCR analysis of selected defence‐related genes in DEX‐induced H2BK144A roots compared with EV induced roots. MtEF1 was used as the reference gene. Medtr8g010320, Defensin related; Medtr2g010600, Catabolite activator protein (CAP); Medtr2g035210, ABA‐responsive protein; Medtr8g096900, pathogenesis‐related thaumatin family protein; Medtr2g029910, peroxidase family protein; Medtr7g110780, Chitinase, Medtr3g108520, gibberellin 2‐beta‐dioxygenase; Medtr8g074335, Chitinase (Class Ib)/Hevein; Medtr7g103390, Myb/SANT‐like DNA‐binding domain protein; Medtr2g089835, wound‐responsive family protein. Error bars indicate SD from three replicates. (c) Quantification of mycorrhization in eight independently transformed M. truncatula roots expressing PT4p::MtHUB1 and eight EV transformed roots as control at 3 wk post inoculation with Rhizophagus irregularis using the intersect method. Student’s t‐test was used. *, P < 0.05. Data are represented as mean ± SD. (d) qPCR analyses of MtHUB1, RiEF and MtPT4 in PT4p::MtHUB1 expressing and EV. MtEF1 was used as reference. Data are represented as mean ± SD of eight biological replicates. Student’s t‐test. *, P < 0.05. (e) qRT‐PCR analysis of defence‐related gene expression from DEX‐induced MtHUB1 samples compared with DEX‐induced EV samples. MtEF1 was used as reference. Data are represented as mean ± SD of three biological replicates. Student’s t‐test was used. *, P < 0.05.