Colonisation of Oncidium orchid roots by the endophyte Piriformospora indica restricts Erwinia chrysanthemi infection, stimulates accumulation of NBS-LRR resistance gene transcripts and represses their targeting micro-RNAs in leaves

Abstract

Background: Erwinia chrysanthemi (Ec) is a destructive pathogen which causes soft-rot diseases in diverse plant species including orchids. We investigated whether colonization of Oncidium roots by the endophytic fungus Piriformospora indica (Pi) restricts Ec-induced disease development in leaves, and whether this might be related to the regulation of nucleotide binding site-leucine rich repeat (NBS-LRR) Resistance (R) genes.

Results: Root colonization of Oncidium stackings by Pi restricts progression of Ec-induced disease development in the leaves. Since Pi does not inhibit Ec growth on agar plates, we tested whether NBS-LRR R gene transcripts and the levels of their potential target miRNAs in Oncidium leaves might be regulated by Pi. Using bioinformatic tools, we first identified NBS-LRR R gene sequences from Oncidium, which are predicted to be targets of miRNAs. Among them, the expression of two R genes was repressed and the accumulation of several regulatory miRNA stimulated by Ec in the leaves of Oncidium plants. This correlated with the progression of disease development, jasmonic and salicylic acid accumulation, ethylene synthesis and H₂O₂ production after Ec infection of Oncidium leaves. Interestingly, root colonization by Pi restricted disease development in the leaves, and this was accompanied by higher expression levels of several defense-related R genes and lower expression level of their target miRNA.

Conclusion: Based on these data we propose that Pi controls the levels of NBS-LRR R mRNAs and their target miRNAs in leaves. This regulatory circuit correlates with the protection of Oncidium plants against Ec infection, and molecular and biochemical investigations will demonstrate in the future whether, and if so, to what extent these two observations are related to each other.

Keywords: Erwinia chrysanthemi; Oncidium; Piriformospora indica; Resistance gene; microRNA.

Fig. 1

Phylogenetic analysis of R proteins of Oncidium, Phalaenopsis equestris and Dendrobium officinale orchids. Bootstrap Neighbor-Joint tree was constructed for the R proteins from Oncidium (ONC, green), Phalaenopsis equestris (PEQU, red) and Dendrobium officinale (DEND, blue) using MEGA6.01 and the respective NBARC domains (from P-loop to MHD-like domain) (Fig. 2). The sequences were compared to 15 known R protein sequences: TNL: RPP-1 (AAC72977), RPS4 (BAB11393); NL: Pi9 (ABB88855), Pi2 (ABC94599), Pib (BAA76281); XNL: Prf (U65391); CNL: Rp1 (AAP81262), RXO1 (AY935244), Xa1 (BAA25068), Pita (AAK00132), Cre3 (AAC05834), Lr10 (aaq01784), RPM1 (NP187360), RPP13 (AF209732) and HERO (CAD29728)

Fig. 2

Detection of the pathogen in leaf tissues in Pi-colonized/−uncolonized Oncidium. a E. chrysenthemi (Ec) was locally inoculated on the second leaf of Pi-colonized/−uncolonized cuttings, respectively. Local and distal leaves were collected separately. b Ec DNA levels in leaves were detected by qPCR of 16S rDNA 1, 2 and 3 days after infection, Pi DNA in leaves and roots were detected with EF-hand DNA primer pair 10 days after inoculation, data represent the means ± SE of 3 replicates and were normalized to the plant ACTIN DNA level, values with the same letter were not significantly different (p < 0.05). c Levels of endogenous salicylic acid, jasmonic acid, ethylene and H₂O₂ 24 h after infection of the leaf with Ec. Data represent the means ± SE of 3 replicates, values with the same letter were not significantly different (p < 0.05). PI: qPCR for Pi and Ec DNA in roots/leaves of Pi-colonized cuttings. CK: uncolonized plants. EC1d, EC2d and EC3d indicates the detection of the presence Pi and Ec in Pi-colonized/−uncolonized plants 1, 2, or 3 days after Ec infection, relative values normalized to the plant ACTIN DNA level. CK: control plant. P: Pi-colonized plants; (P)EL: local infected leaf of Pi-uncolonized (EL) or -colonized (PEL) plants inoculated with Ec. (P)ED: distal leaves of Pi-uncolonized (ED) or –colonized (PED) plants inoculated with Ec

Fig. 3

Expression of R genes after Ec infection of leaves of Pi-colonized or -uncolonized Oncidium. Expression levels of R genes 24 h after Ec infection of Pi-colonized (2 weeks) or –uncolonized Oncidium plants. 24 h after infection, the leaves were harvested for qRT-PCR analyses. CK: control plant without Pi colonization and Ec infection. P: Pi-colonized plants; (P)EL: local infected leaf of Pi-uncolonized (EL) or -colonized (PEL) plants. (P)ED: distal leaves of Pi-uncolonized (ED) or –colonized (PED) plants. SA: leaves treated with 1.0 mM salicylic acid for 24 h. MeJA: leaves treated with 0.1 mM methyl jasmonic acid for 24 h. Data represent the means ± SE of 3 replicates and were normalized to the Oncidium ACTIN mRNA level, values with the same letter were not significantly different (p < 0.05)

Fig. 4

Expression of miRNAs after leaf infection of Pi-colonized or –uncolonized Oncidium with Ec. miRNA levels in Pi-colonized (2 weeks) or –uncolonized Oncidium. 24 h after Ec infection, the leaves were harvested for qRT-PCR analyses. CK: control plant without Pi colonization and Ec infection. P: Pi-colonized plants; (P)EL: local infected leaf of Pi-uncolonized (EL) or -colonized (PEL) plants inoculated with Ec for 24 h. (P)ED: distal leaves of Pi-uncolonized (ED) or –colonized (PED) plants inoculated with Ec for 24 h. SA: leaves treated with 1.0 mM salicylic acid for 24 h. MeJA: leaves treated with 0.1 mM methyl jasmonic acid for 24 h. Data represent the means ± SE of 3 replicates and were normalized to the U6 snRNA level, values with the same letter were not significantly different (p < 0.05)

Fig. 5

A model describing the regulation of miRNA and NB-LRR R mRNA levels in Oncidium leaves after Ec infection and root colonization by Pi