A fungal sRNA silences a host plant transcription factor to promote arbuscular mycorrhizal symbiosis

Abstract

Cross-kingdom RNA interference (ckRNAi) is a mechanism of interspecies communication where small RNAs (sRNAs) are transported from one organism to another; these sRNAs silence target genes in trans by loading into host AGO proteins. In this work, we investigated the occurrence of ckRNAi in Arbuscular Mycorrhizal Symbiosis (AMS). We used an in silico prediction analysis to identify a sRNA (Rir2216) from the AM fungus Rhizophagus irregularis and its putative plant gene target, the Medicago truncatula MtWRKY69 transcription factor. Heterologous co-expression assays in Nicotiana benthamiana, 5' RACE reactions and AGO1-immunoprecipitation assays from mycorrhizal roots were used to characterize the Rir2216-MtWRKY69 interaction. We further analyzed MtWRKY69 expression profile and the contribution of constitutive and conditional MtWRKY69 expression to AMS. We show that Rir2216 is loaded into an AGO1 silencing complex from the host plant M. truncatula, leading to cleavage of a host target transcript encoding for the MtWRKY69 transcription factor. MtWRKY69 is specifically downregulated in arbusculated cells in mycorrhizal roots and increased levels of MtWRKY69 expression led to a reduced AM colonization level. Our results indicate that MtWRKY69 silencing, mediated by a fungal sRNA, is relevant for AMS; we thus present the first experimental evidence of fungus to plant ckRNAi in AMS.

Keywords: Medicago truncatula; Rhizophagus irregularis; arbuscular mycorrhizal symbiosis; cross‐kingdom RNA interference; small RNA.

Fig. 1

In silico analysis of the interaction between Rir2216 and MtWRKY69. (a) (Upper panel) Target prediction results of potential targets of Rir2216 in the Medicago truncatula transcriptome with their associated description, expectation, and alignment. (Lower panel) Alignment of the target site within the MtWRKY69 sequence with that of Rir2216 and the associated hybridization energy of the sRNA‐mRNA pair. For the alignment, the following symbols are used: colon (:) denotes standard Watson‐Crick base pairs; dot (.) denotes G‐U wobble pairs. (b) Phylogenetic relationship of MtWRKY69 orthologues. Protein sequences were aligned with mafft v.7.511 (Katoh & Standley, 2013), and their phylogenetic relationship was inferred with IQ‐Tree 2.0.7 (Minh et al., 2020) with the tree rooted on the outlier sequence of Arabidopsis thaliana WRKY1. Sequences, whose corresponding CDSs contain the Rir2216 binding site, are indicated in green. (c) Nucleotide diversity (pi) across the CDS alignment of MtWRKY69 orthologous belonging to Fabaceae family, calculated with a 21‐mer sliding‐windows method (i.e. Pi for all the possible 21‐mer across the alignment in steps of 1 nt) excluding sites having alignment gaps. The most conserved 21‐mers in the alignment overlaps with the binding site for Rir2216. Notably, the predicted binding site for Rir2216 has a higher level of conservation than the sequence encoding the WRKY domain.

Fig. 2

Rir2216 is a bona fide ckRNAi sRNA targeting MtWRKY69. (a) On the left: transversal section of a mycorrhizal root under the laser microdissector before (upper panel) and after (down) the cut. Arbuscule‐containing cells are indicated by a red line. Bar, 50 mm. On the right: Normalized expression values of MtWRKY69, MtPT4, and MtAGO1 transcript abundance in cells collected by laser microdissection: CC‐cortical cells from noncolonized roots; Arb‐cortical cells containing arbuscules. Box plots display the median (horizontal line), the quartiles (boxes) and 1.5 interquartile range (whiskers); each dot corresponds to an independent replicate. Statistical analysis was performed using one‐way analysis of variance (Kruskal–Wallis test; *, P < 0.05). (b) Western blot of co‐expression assays using proteins extracted from Nicotiana benthamiana leaves expressing WRKY‐YFP alone, in combination with Rir2216, or with the plant miR159b. The lower panel corresponds to a Ponceau staining of the gel showing the Rubisco protein. (c) The cleavage site (arrow) and frequencies (indicated by the ratio of the number of clones showing that 5′ end to the total number of sequenced recombinant clones) detected using a 5′ RACE on Medicago truncatula mycorrhizal roots. (d) (Upper panel left) Western blot (WB) of anti‐AGO1 (left lane) and anti‐myc‐tag (central lane) and no‐antibody (right lane) immunoprecipitations with anti‐AGO1 and anti‐myc‐tag antibodies. Full images of blots are shown in Supporting Information Fig. S6; (lower panel left) Gel electrophoresis of stem‐loop reverse transcription polymerase chain reaction assays on Rir2216, Rir434, and Rir196 using RNA extracted from the immunoprecipitated fractions. (Right) Gel electrophoresis of stem‐loop reverse transcription polymerase chain reaction assays on Rir2216, Rir434, Rir196, and Mt‐miR398a‐3p from input RNA from noninoculated (Mock) and mycorrhizal roots (Myc).

Fig. 3

MtWRKY69 transcription factor modulates fungal colonization. (a) Normalized expression value of MtWRKY69 and the AM‐responsive gene MtPT4 in plants expressing Scarlet or MtWRKY69 under the CaMV35S promoter (OE = overexpressing). (b) Frequency of mycorrhizal colonization (F%), intensity of colonization (M%) and arbuscules abundance (A%) in composite plants expressing Scarlet or MtWRKY69 under the CaMV35S promoter at 60 d post inoculation (dpi). (c) Representative images of R. irregularis‐colonized roots from composite plants overexpressing Scarlet or MtWRKY69. Arrows indicate arbuscule‐containing cells. cc, central cylinder; c, cortical cells; ep, epidermal cells. Bars correspond to 50 μm. (d) Frequency of mycorrhizal colonization (F%), intensity of colonization (M%) and arbuscules abundance (A%) in composite plants expressing Scarlet or MtWRKY69 under the MtPT4 promoter at 60 dpi. In a, b and d, box plots display the median (horizontal line), the quartiles (boxes) and 1.5 interquartile range (whiskers); each dot corresponds to an independent replicate. Statistical analysis was performed using one‐way analysis of variance (ANOVA; *, P < 0.05; **, P < 0.01).