DISCUSSION
Previously we identified differentially expressed miRNAs including miR8746, miR482, and miR398b, which were putatively involved in targeting NBS-LRR disease resistance transcripts (Wang et al., 2016). In this current study, we demonstrated that overexpressing miR398b made plants more susceptible to V. dahliae in cotton (Figure 2). In support of these finds, miR398 has been reported to negatively regulate MAMP-triggered immunity (Li et al., 2010) and the expression levels of miR398 were down-regulated upon inoculation of plants with incompatible strains DC3000 (avrRpm1) and DC3000 (avrRpt2), but not the compatible strain DC3000 (Jagadeeswaran et al., 2009). This implicates involvement of miR398 in both MTI and ETI. In our study, we found that miR398b can target two NBS-LRR genes (Gh_D05G3257 and Gh_A04G0380 ) and the target site of the genes is in the internal region, a region not completely conserved in NBS-LRR genes. No identical target sequences were found in the homologous genes in Oryza sativa , Arabidopsis thaliana ,Zea maize , Populus trichocarpa and Theobroma cacao .Gh_D05G3257 and Gh_A04G0380 may be unique target genes of miR398b at the post-transcriptional level and contribute to cotton resistance against V. dahliae (Figure 3, Figure 4, Figure 5, Table S1). To our knowledge, this is the first report that miR398b can target plant NBS-LRR genes to regulate defense responses to a pathogen.
ROS is a general term for oxygen-derived metabolic intermediates or radicals including superoxide (O2∙-), hydrogen peroxide (H2O2), hydroxyl radical (OH∙) and singlet oxygen (1O2) (Apel and Hirt, 2004). ROS-generation and -scavenging pathways play an important role both in pathogen virulence and resistance by the host. The ROS burst is reported to be essential for penetration peg formation byV. dahliae , and therefore is indispensable for virulence and colonization by this fungus (Zhao et al., 2016). Moreover, excess ROS produced during plant-pathogen interactions facilitates plant infection and colonization by necrotrophic pathogens (Chung, 2012; Govrin and Levine, 2000). In the host, rapid ROS accumulation also occurs during the cotton-V. dahliae interaction but it is quickly scavenged to maintain homeostasis. Thioredoxin GbNRX1, an important apoplastic ROS-scavenger, plays a positive role in the cotton response to V. dahliae (Li et al., 2016) and silencing of anthocyanidin synthase (GbANS ) in cotton increased H2O2production and cell death around the invasion sites, which in turn leads to increased V. dahliae infection (Long et al., 2018). These results indicate that ROS elimination after the initial burst may be functionally important in cotton resistance to V. dahliae .
Among other players involved in ROS metabolism, superoxide dismutases (SODs) are found in all kingdoms of life and act as the first line of defense against ROS, dismutating superoxide to H2O2 (Miller, 2012). In this study, we found that overexpression of miR398b boosted more H2O2 production when treated with NLP1 and V. dahliae and therefore accelerates the NLP1-triggered cell necrosis (Figure 7). By contrast, overexpression of GhrCSD2 , which is resistant to miR398b, decreased H2O2 content (Figure 7B). These results were strikingly similar to those of recent reports from analyses of rice upon Magnaporthe oryzae infection (Li et al., 2019). Additionally, expression levels of GhMSD1 , GhFSD2 andGhRbohB (the gene putatively encoding NADPH oxidase to catalyze the production of O2∙- ) were decreased in GhrCSD2- overexpression lines upon NLP treatment, but increased in miR398b-overexpression lines compared to WT plants. Meanwhile, transcripts of GhCSD1 , GhCCS andGh_D05G3257 (the other target genes of miR398b) were increased in GhrCSD2- overexpression lines and decreased in miR398b-overexpression lines (Figure S6). These results suggest that a compensatory regulation mechanism may also exist in cotton ROS homeostasis. However, the phenotypes of miR398- and GhrCSD2-overexpression plants in cotton upon pathogen were differ from those in rice. Overexpression of miR398b in cotton impaired cotton resistance to V. dahliae , while overexpression of miR398b in rice enhanced resistance to M. oryzae (Li et al., 2019). We propose that the excessive ROS accumulation in miR398b-overexpressing plants may damage the cell and facilitate V. dahliae infection (Figure 7), consistent with results of previous studies (Chung, 2012; Li et al., 2016).
Taken together, these results suggest that miR398b may function in one of two different ways to regulate plant immunity. First, the results indicate involvement of miR398b in ROS homeostasis through cleavingGhCSD1 , GhCSD2 and GhCCS1 , and second, miR398b suppresses the translation of NBS-LRR genes to control defense response (Figure S7). An in-depth account of how miR398b expression is regulated during cotton-V. dahliae interactions will be an interesting topic for a future study.