Figure 5: Suppression of cotton NBS-LRR genes increases
susceptibility to Verticillim dahliae.
A: Disease symptoms of the TRV:00 and TRV:NB-ARCplants following inoculation with isolate V991 of V. dahliae . The
images were obtained at 16 days post-inoculation (dpi).
B: Dark and necrotic vascular bundles of the dissected stems
from TRV:00 and TRV:NB-ARC plants at 16 dpi with V.
dahliae .
C: Disease index statistics of TRV:00 , TRV:NB-ARCplants from 12 d to 15 dpi with V. dahliae .
D: Fungal biomass determined by quantitative PCR inTRV:00 and TRV:NB-ARC cotton plants. The relative biomass
is represented by the DNA quantification levels of VerticilliumInternal Transcribed Spacer (ITS) compared to the of cotton UB7 .
CN = samples from the cotyledonary nodes of cotton stems; FN = samples
from the first internode. Values represent the means ± s. d: from three
biological replicates (** P value ≤ 0.01, Student’s t-test).
E: Suppression of cotton NBS-LRR genes attenuates the
expression of defense-related genes upon V. dahliae infection.
Values represent the means ± s. d. from three biological replicates.
Figure 6: The microRNA miR398b can guide the cleavage
of GhCSD1, GhCSD2 and GhCCS in cotton.
A: Schematic diagram ofGhCSD2 target site for miR398b, validated through degradome
sequencing and RNA ligase-mediated rapid random amplification of cDNA
ends (RLM-RACE). Watson-Crick pairing (vertical dashes) and
non-Watson-Crick pairing (colon) are indicated. The number of miR398b
cleavage products is indicated with bold font and arrow.
B-C: Schematic diagram of GhCSD1 (B) andGhCCS (C) target sites of miR398b determined by RLM-RACE
experiments.
D:Schematic
representation
of
miR398b-resistant GhCSD2 (rCSD2 ) vector constructed by
synonymous mutation. The top strand depicts the original miR398b target
site of GhCSD2 from 421 to 441 nt calculated from the start
codon, and the bottom strand shows the sequence post-mutation; bases in
red indicate the nucleotides that were replaced.
E-F: Reverse transcription-quantitative PCR analysis of the
expression of miR398b, the target genes GhCSD2 , GhCSD1 ,GhCCS , and the non-target genes GhCSD3 and GhCOX-5bin roots of wild type, null, miR398b-overexpressing (O8-15, O8-17,
O8-18, O8-41) and miR398b-resistant GhCSD2 overexpressing (T8-14,
T8-15) lines.
Bars
represent means and standard error of three technical replicates.
Figure 7: Overexpression of miR398b in cotton results
in constitutive reactive oxygen species (ROS) accumulation and defective
ROS elimination in response to Verticillium dahliae.
A: Necrotic lesions on treated cotyledons of wild type and
transgenic plants. Images were obtained 48 h post leaf infiltration of
the necrosis and ethylene-inducing peptide 1-like protein 1 (NLP1)
expressed via Agrobacterium tumefaciens- mediated plant
transformation. The gel picture shows the expression analysis ofNLP1 in cotton cotyledons by RT-PCR (e: empty vector; N: NLP1).
B: ROS quantification in NLP1-treated cotyledons of wild type
and transgenic cotton. The bar represents the standard error from three
biological repeats and the lowercase letter above the respective columns
represents the statistical significance of as determined using the
linear mode method, completely randomized AOV, and all-pairwise
comparisons (LSD, 0.05).