miR398b targets GhCSD genes and regulates ROS homeostasis
From our previous degradome sequencing data (Wang et al., 2016) and the findings reported in the literature (Beauclair et al., 2010; Sunkar et al., 2006), Cu/Zn SOD family members CSD1 , CSD2 andCCS were also identified as the target genes of miR398b. To determine whether these family members were similarly regulated in cotton, 5’-RNA ligase-mediated rapid RACE (5’-RLM-RACE) experiments were performed, revealing thatGhCSD1 , GhCSD2 and GhCCS are also targets of miR398b and that their target sites are at the 5’UTR region ofGhCSD1 , the fourth exon of GhCSD2 , and the last exon ofGhCCS , respectively (Figure 6A, B, C).
To further understand the function of miR398b and the GhCSDs in cotton upon V. dahliae infection, the constructs of a miR398b-resistant GhCSD2 (GhrCSD2 )-overexpression vector, with six synonymous codon mutations at the miR398b-target sites were prepared (35S:GhrCSD2 ) and transformed into cotton (Figure 6D). Two GhrCSD2 overexpression lines (T8-14 and T8-15) (Figure S1A and 1B) were obtained. The results of the expression analysis revealed that transcript levels of GhCSD1 , GhCSD2 and GhCCSwere decreased with no difference of GhCSD3 and GhCOX-5bexpression levels in miR398b- overexpressing lines (Figure 6E-F). The transcripts of GhCSD2 were highly increased inGhrCSD2- overexpressing lines (Figure 6E). Expression levels ofGhCSD1 , GhCSD2 and GhCCS , the target genes of miR398b, were up-regulated during pathogen infection (Figure S4A). However, transcript levels of GhCSD3 , the non-target gene of miR398b, were decreased after V. dahliae inoculation (Figure S4A). Furthermore, down-regulation of GhCSD1 , GhCSD2 , andGhCCS through virus-induced gene silencing (VIGS) rendered the plants more susceptible to V. dahliae (Figure S4B, C, D).These results indicate that the members of CSD family, GhCSD1 ,GhCSD2 and GhCCS , as targets of miR398b, are required to mount appropriate defense responses in cotton following challenge withV. dahliae .
To investigate the functions of miR398b and GhCSD family genes in cotton resistance, we first examined the subcellular location of the CSD family proteins. The results show that GhCSD2, GhCSD3, and GhCCS were localized in the chloroplast, while GhCSD1 exhibited a similar localization pattern as free GFP protein in the cell (Figure S5). Our results showed some differences from the previous study inArabidopsis , whereby AtCSD3 was found to be localized in peroxisomes in Arabidopsis (Huang et al., 2011). These results demonstrate that the CSD family members localize in different cellular compartments to regulate ROS homeostasis.
To examine whether redox homeostasis was controlled by miR398b and itsGhCSD targets in cotton in response to V. dahliae , we measured the hydrogen peroxide (H2O2) content in cotton roots after Necrosis and ethylene-inducing peptide 1 (Nep1)-like protein 1 (NLP1) treatment. NLP1 are considered as conserved virulence effectors in V. dahliae that function as cytolytic toxins. This protein induces plasma membrane leakage, leading to cytotoxicity and necrosis (Ottmann et al., 2009; Qutob et al., 2006). Furthermore, the production of ROS triggered by NLPs induced the onset of lesion formation (Wang et al., 2004). To validate the role of ROS in cotton response to V. dahliae , VdNLP1 was transiently expressed in the cotyledons of miR398b- and GhrCSD2- transgenic plants. Necrosis could be observed at the infiltration position 48 h post infiltration (Figure 7A). The results show that overexpression ofGhrCSD2 attenuated necrosis occurs in the presence of NLP1, while overexpression of miR398b resulted in accelerated and increased necrosis. Overexpression of miR398b produced more ROS after treatment with NLP1 (Figure 7B). Together, these results demonstrate that miR398b plays an essential role in regulating ROS homeostasis by targeting CSD family genes, and overexpression of miR398b impairs the ability of ROS scavenging and benefits the invasion of V. dahliae .