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).