INTRODUCTION
Cotton (Gossypium spp.) cultivation has a major impact in human society since it is widely used as a source of fiber and seed oil. Verticillium wilt disease caused by the soil-borne vascular diseaseVerticillium dahliae is one of the major threats limiting cotton productivity. Diseased cotton is characterized by wilting, stunting, chlorosis, vascular discoloration, early senescence (Xu et al., 2013). Strains of V. dahliae isolated from cotton have been characterized as causing a defoliating or non-defoliating phenotype, and the genetic basis for the defoliating phenotype was recently established (Zhang et al. 2019). In either case, there are few resistant germplasm sources or efficient management measures available to control this pathogen, especially post-infection. Verticillium dahliae has a very broad host range, infecting over 200 plant species, and survives for years in the soil, precluding crop rotation as a strategy for disease control (Klosterman et al. 2009).
The evolutionary arms race between plants and pathogens has prompted the development of innate immune responses. One of these immune responses is known as MAMP (microbe-associated molecular pattern)-triggered immunity (MTI), and this response serves as the first barrier to guard against the invasion of pathogens (Chisholm et al., 2006). Another immune response is known as effector-triggered immunity (ETI), which is carried out by dominant resistance (R) proteins by recognizing pathogen effector proteins directly or indirectly, to trigger gene-for-gene resistance (Jones and Dangl, 2006). The canonical R proteins contain a nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains. The core nucleotide binding domain in NBS-LRR proteins is known as the NB-ARC domain since it is well studied in APAF-1 (apoptotic protease-activating factor-1), R proteins, and CED-4 (Caenorhabditis elegans death-4 protein) (van der Biezen and Jones, 1998). In plants, NBS-LRR proteins mediate pathogen-specific effector triggered immunity, and the genes that encode them are widely used as markers in plant breeding to generate disease resistance. A drawback of R -mediated resistance is that it occurs at the expense of fitness (Tian et al., 2003), which suggests that the expression of an R gene must be highly regulated, and strictly inactivated in the absence of a pathogen.
Reactive oxygen species (ROS) also play multiple roles in MTI and ETI defense. For example, ROS can function as secondary messengers directly or indirectly to activate the expression of defense-related genes and induce programmed cell death during the hypersensitive response (HR) (Mittler, 2017; Mittler et al., 2011). However, excessive ROS adversely affects many cellular functions by causing oxidative damage to DNA, RNA, proteins and membranes (Apel and Hirt, 2004). Plants have evolved many antioxidative systems to eliminate ROS, including enzymatic and nonenzymatic mechanisms. Enzymatic ROS scavenging mechanisms in plants include superoxide dismutase (SOD), glutathione peroxidase (GPX), ascorbate peroxidase (APX), and catalase (CAT). SODs act as the first line of defense against ROS. There are three types of SODs in plants based on different metal ligands involved copper/zinc SOD (Cu/Zn–SOD, also known as CSD), manganese SOD (Mn–SOD) and iron SOD (Fe–SOD) (Guan et al., 2013). There are three CSD isozymes which are localized in different cellular compartments inArabidopsis : cytosolic CSD1, chloroplastic CSD2 and peroxisomeic CSD3 (Huang et al., 2011). In Arabidopsis , there is a copper chaperone for superoxide dismutase (CCS, which delivers copper to the CSD) to activate all three CSD isozymes activities (Huang et al., 2011). Overexpression of CSD1 and CSD2 enhances the tolerance of transgenic plants to UV and high light treatment, salt and heavy metal stresses (Leng et al., 2017; Sunkar et al., 2006). SODs have also been reported to play roles in the hypersensitive response (HR) during cotton-Xanthomonas campestris interaction and barley-Blumeria graminis interaction (Voloudakis et al., 2006; Xu et al., 2014b).
MicroRNAs (miRNAs) are approximately 21 or 22 nucleotide (nt) long non-coding RNAs that play essential roles in gene silencing by targeting mRNA for cleavage, or by translational repression in both plants and animals (Reinhart et al., 2000; Reinhart et al., 2002). Primary miRNAs (pri-miRNAs) are transcribed by RNA polymerase II but contain an imperfect stem-loop or hairpin structure (Voinnet, 2009; Xie et al., 2015). miRNAs are released from their pri-miRNAs by RNase III-like Dicer-like enzymes in plants (Margis et al., 2006), and then associate with argonaute (AGO) protein (Mallory and Vaucheret, 2010) to inhibit gene expression at transcriptional gene silencing (TGS) or post-transcriptional gene silencing (PTGS) levels (Bologna and Voinnet, 2014). Hundreds of miRNAs have now been discovered through deep-sequencing and genetic approaches (Meyers et al., 2006), and they have been shown to play vital roles in plant development (Couzigou and Combier, 2016; Guo et al., 2017; Huang et al., 2017; Zhang et al., 2017) and responses to biotic and abiotic stresses (Deng et al., 2018; Ding et al., 2017; Kumar, 2014; Li et al., 2017).
Intriguingly, miRNAs are considered as important regulators of Rgene expression. For example, two miRNAs, nta-miR6019 and nta-miR6020, were reported to guide the cleavage of the tobacco mosaic virus (TMV) resistance gene, N , which is a toll and interleukin-1 receptor-NBS-LRR immune receptor (Li et al., 2012). Furthermore, miR482/2118 is another well-known miRNA that targets the NBS-LRRresistance genes (Shivaprasad et al., 2012). Therefore, miRNA-mediated repression of NBS-LRR genes is an efficient mechanism for plants to balance the trade-off between growth and defense. The miRNA known specifically as miR398 has been reported to play a role in responses to various abiotic stresses by modulating the expression of its target genes (Zhu et al., 2011; Wang et al., 2016). miR398b was the first miRNA reported to be down-regulated in response to biotic stress (P. syringae ) in Arabidopsis (Jagadeeswaran et al., 2009; Li et al., 2010). In our previous work, we also found that miR398b plays a role in temperature stress through miRNA and degradome sequencing (Wang et al., 2016). To date, four targets of miR398 have been reported through computational prediction and sequence analysis: CSD1 ,CSD2 , CCS and COX-5b (a subunit of the mitochondrial cytochrome c oxidase) in Arabidopsis (Beauclair et al., 2010; Jones-Rhoades and Bartel, 2004). Some studies also show that miR398 negatively regulates the PTI response and resistance to pathogenic bacteria (Li et al., 2010). However, whether miR398 can participate in the ETI response and resistance to fungal pathogensV. dahliae is not known. Here, we investigated the potential role of miR398b in cotton-V. dahliae interaction. The results indicate that miR398b can target both NBS-LRR genes and CSD family genes to suppress the resistance of cotton to V. dahliae .