NBS-LRR genes are targets for miR398b and involved in cotton resistance to V. dahliae.
In our previous degradome sequencing data (Wang et al., 2016), twoNBS-LRRgenes (Gh_A04G0380 and Gh_D05G3257 ) were identified as putative targets of miR398b.Ghir_D05G34520and Ghir_A04G004640 (the corresponding genes ofGh_D05G3257 and Gh_A04G0380 in 3rd generation genome data of cotton) contain a CC-NBS-ARC domain and two internal repeat regions. The miR398b target site is localized in the first internal repeat region of Ghir_D05G34520 (Figure 3A, Figure S2A, B). The expression level of Gh_D05G3257 was examined, revealing that the transcripts of Gh_D05G3257 were decreased in miR398b-overexpression lines (Figure 3B). The transcripts ofGh_D05G3257 were up-regulated in both WT and transgenic plants upon inoculation of pathogen, while the expression levels ofGh_D05G3257 in transgenic plants were significantly reduced as compared with WT plants (Figure 3B). Also, Gh_D05G3257transcripts were highly expressed in roots, hypocotyls, and stems, tissues of which are heavily colonized by V. dahliae . The expression levels of Gh_D05G3257 were significantly up-regulated upon V. dahliae infection (Figure S3A, B). Two reads containing the cleavage site were detect in our normalized degradome data, which may suggested that the expression level of Gh_D05G3257 andGh_A04G0380 may not be suppressed through cleavage (Figure 3A). A plant small RNA target analysis server psRNATarget (Dai et al., 2018) was used to identify the interaction between miR398b andGh_D05G3257 , Gh_A04G0380 genes. Analyses of these results showed that miR398b repressed the expression of NBS-LRRgenes by translational inhibition (Table S1) and this may be the reason that it is difficult to verify cleavage by 5’random amplification of cDNA ends (RACE).
To confirm the repression activity of miR398b on NBS-LRR genes, a luciferase reporter system was employed with about 100 bp-length target sites inserted into the 5’ UTR region of the Luciferase (LUC) encoding gene, and a miRNA precursor sequence was cloned into another vector driven by the CaMV35s promoter (Figure 3C). Both constructs were transformed into Agrobacterium and infiltrated intoNicotiana benthamiana leaves for transient expression assays, where miR157 was used as the negative control and the target site ofGhCSD2 as a positive control. Analyses of these results revealed that the luminescence intensity of 4NB-LUC, 5NB-LUC and CSD2-LUC were decreased when co-expressed with miR398b than when expressed alone or co-expressed with miR157 (Figure 3D). The ratios of LUC activity to control renilla luciferase (RLUC) activity were also detected and showed consistent results in the extracted total protein from the infiltrated sites of the leaf (Figure 3E).
Additionally, the 21 bp target sites of 5NB (5NBts ) and the mutated target sites (mts) of 5NB (5NBmts ) were fused at the 5’-terminus of GFP , respectively (Figure 4A). The35S:5NBts-GFP and 35S:5NBmts-GFP constructs were co-expressed with either the 35S:miR398b or the 35S:miR157vector by Agrobacterium -mediated transient transformation in tobacco leaves. Analyses of the results revealed that the fluorescence intensity and protein content of 5NBts-GFP were clearly decreased when co-expressed with miR398b than when expressed alone or co-expressed with miR157. However, 5NBmts-GFP intensity and protein levels were not influenced when co-expressed with miR398b or miR157 (Figure 4B, C). These results demonstrate that these two NBS-LRR genes could be inhibited at the post-transcription level by miR398b.
To further understand the function of the two NBS-LRR genes in cotton response to V. dahliae , VIGS was employed to knock down the expression levels of both genes. The results revealed that knock-down of NBS-LRR genes made the plants more susceptible toV. dahliae (Figure 5A). The TRV:NB-ARC plants showed severe necrotic vascular bundles with a higher disease indices compared to the TRV:00 plants (Figure 5B, C). Also, more fungal biomass could be detected in TRV:NB-ARC plants inoculated with V. dahliae - (Figure 5D). We also examined the expression pattern of some defense-related genes in TRV:NB-ARC plants upon infection byV. dahliae . The results indicate that the expression of genesGhEDS1 , GhPAD4 , GhWRKY28 (genes related to the salicylic acid synthesis and signaling pathway) (Miao et al., 2019) andGhAOS (jasmonate acid synthesis-related gene) (Hu et al., 2019) were reduced in TRV:NB-ARC plants compared with TRV:00plants during V. dahliae infection (Figure 5E). These results indicate that knock-down of NB-ARC genes reduces cotton resistance to V. dahliae by attenuating defense response genes related to SA and JA signal pathways.