Figure 1.
Growth effects of P. indica on Oncidium orchid.
(A) Seedlings inoculated with P. indica for 8 weeks showed significantly enhanced growth and root development. (B–D) Anatomic structures of roots colonized by P. indica for 24 hours. Hyphae (green spots in B, arrow head) and penetration site (red spots in C, arrow head) were overlayed in bright field (D, arrow head). Bar = 50 µm. (E) Microscopic structure of roots colonized by P. indica for 5 days. A large number of hyphae were widespread over the root surface and tip, elongation zone and mature zone. Bar = 200 µm. (F, G) Microscopic structures of transverse sections and longitudinal sections of roots colonized by P.indica for 5 days. Hyphae fully colonized the velamen. Chloroplast autofluorescence (red) was also detected in cortex. Bar = 200 µm. (H) Microscopic structures of transverse section of roots colonized by P. indica for 5 days. P. indica was restricted in the velamen and not detectable in the exodermis of Oncidium roots. Chloroplast autofluorescence (red) was detected in cortex. Bar = 50 µm. (I) Micrograph of root cross sections from seedlings colonized with P. indica for 5 days. Without chlorophyll fluorescence in velamen, the penetration sites (red spot) were clearly detected. Bar = 20 µm. (J) Growth quantification of seedling colonized with P. indica for 8 weeks. Fresh weight, plant height, leaf number, leaf wide, stem diameter, root number and diameter were analyzed. Error bars represent SD for three independent experiments. *, P value<0.05; **, P<0.001. Hyphae were stained with chitin-specific WGA-AF488 (green). Penetrated sites (Ps) were stained with lectin-specific conA-AF633 (red) [88]. Samples were analyzed and photographed with an Olympus IX71 inverted microscope system (Japan). Ve, velamen; EX, exodermis; Cort: cortex; Ch: Chloroplast. Hy, hyphae; Ps, penetration site.
Table 1.
Category distribution of small RNAs in root tissues of Oncidium orchid ± P. indica colonization.
Table 2.
Family members and counts of conserved miRNAs detected in root tissues of Oncidium orchid ± P. indica colonization.
Figure 2.
GO analysis of the conserved miRNA target candidates.
The potential targets of miRNAs were predicted with psRNATarget by alignment to mRNAs from Oncidium, Oryza stativa and A. thaliana. The best hit results were chosen and annotated using Blast 2 GO.
Table 3.
Target genes prediction for miRNAs in P. indica colonized- Oncidium roots.
Figure 3.
Expression analyses of miRNAs in Oncidium orchid ± P. indica by qPCR.
Roots colonized ± P. indica for 8 weeks were sampled. Data represent the mean ± SD of 3 replicates and normalization by 5.8S rRNA. e represents the decimal point. * indicates antisense strand; no P. indica indicates that the novel miRNAs were specifically found in the–P. indica orchid library. P. indica indicates that the novel miRNAs were specifically found in the +P. indica orchid library.
Figure 4.
Expression analyses of the target genes of miRNAs by qPCR.
Roots colonized with P. indica for 0, 1, 3 and 8 weeks were sampled and mRNA expression level was analyzed by qPCR. Data represents the mean ± SD of 3 replicates, and were normalized to the Actin mRNA level.