in different tissues and in defense responses.
A: Reverse transcription-quantitative PCR analysis for
expression levels of miR398b in different tissues. R: root; S: stem; L:
leaf; P: petal; A: anther; 0 d: 0 d ovule; 5 d: 5 d fiber after
flowering. The expression levels of miR398b were normalized toGhUB7 . The bar represents the standard error from three
biological repeats and the lowercase letter above the column represents
significant difference by statistical analysis using the linear mode
method, completely randomized AOV, and all-pairwise comparisons (LSD,
0.05).
B: Histochemical β-glucuronidase (GUS) staining analysis of the
expression patterns of pmiR398b::GUS in two independent lines of
transgenic Arabidopsis leaves and roots after subjected to 12 h
for flg22, nlp20, and Verticillium dahliae treatments. Bars = 200
μm.
Figure 2: Overexpression of miR398b attenuates cotton
resistance to Verticillium dahliae.
A:Verticillium wilt disease symptoms of null cotton plants (ON and TN) and
miR398b overexpressing lines (O8-17 and O8-18) at 16 days
post-inoculation with V. dahliae.
B: Dark, necrotic vascular bundles of the dissected stems of
each line were photographed 16 days after V. dahliae inoculation.
Fluorescence imaging shows V592-GFP hyphae in the vascular bundles of
miR398b overexpression lines.
C: Disease index statistics of null plants (ON and TN) and
miR398b overexpressing lines (O8-17 and O8-18) at 16 d after
inoculation. The bar represents the standard error from three biological
repeats and the lowercase above the column represent the significance by
statistical analysis using the linear mode method, completely randomized
AOV, and all-pairwise comparisons (LSD, 0.05).
D: PCR quantification of fungal biomass. The biomass represents
the levels of amplification of the Verticillium internal
transcribed spacer (ITS) region compared to the levels of cottonGhUB7 amplification. Values represent the means ± s. d. from
three biological replicates (** P<0.01, Student’s t-test).
Figure
3: The cotton microRNA miR398b inhibits the expression ofNBS-LRR defense signaling associated genes.
A: Schematic diagram of 4NB and 5NB target sites
of miR398b validated through degradome sequencing.
B: RT-qPCR analysis of expression levels of NBS-LRRgenes in root samples of miR398b overexpressing lines and in root
samples of wild type plants and miR398b overexpressing lines inoculated
with Verticillium dahliae . The bar represents the standard error
from three biological repeats and the lowercase above the column
represent the significance by statistical analysis using the linear mode
method, completely randomized AOV, and all-pairwise comparisons (LSD,
0.05).
C: Schematics of dual-luciferase system containing 4NB ,5NB target sites in the 5’UTR region of the luciferase (LUC)
gene, respectively.
D: Inhibition of the expression of NBS-LRR genes by
miR398b. Agrobacterium-mediated transient expression of the 4NB-LUC and
5NB-LUC fusion proteins with miR398b and miR157 in tobacco leaf cells
with Agrobacterium tumefaciens cultures grown to the same
optical density. After 48 h, LUC luminescence was examined using a
cryogenically cooled CCD camera (Lumazome PyLoN 2048B)
E: Quantification of LUC/RLUC intensity and miR398b mRNA
levels. Values represent the means ± s. d. from three biological
replicates (* P value ≤ 0.05, ** P value ≤ 0.01, Student’s t-test).
Figure 4: MicroRNA miR398b suppresses the expression ofNBS-LRR defense signaling gene in cotton.
A: Schematics of 5NBts (5NB target sites)-green fluorescent
protein (GFP) and 5NBmts (5NB-mutated target sites)-GFP fusion proteins
and alignment sequences of 5NBts and 5NBmts with miR398b and miR157,
respectively. Bases in red indicate the mutated nucleotides.
B: GFP fluorescence shows that miR398b represses the expression
of 5NB. Agrobacterium tumefaciens mediated transient expression
of 5NBts-GFP and 5NBmts-GFP fusion proteins with miR398b and miR157 in
tobacco leaf cells at the indicated optical densities (O.D.). Laser
confocal microscopy was used to observe the green fluorescence 60 h
after infiltration by A. tumefaciens . * indicated the injection
site, Bars=100 μM.
C: Western blot analysis with an anti-GFP antibody reveals the
expression levels of 5NBts-GFP and 5NBmts-GFP proteins following each
treatment. The total protein was stained with Coomassie brilliant blue
as a loading control.