The Thioredoxin GbNRX1 Plays a Crucial Role in Homeostasis of Apoplastic Reactive Oxygen Species in Response to Verticillium dahliae Infection in Cotton

Examining the proteins that plants secrete into the apoplast in response to pathogen attack provides crucial information for understanding the molecular mechanisms underlying plant innate immunity. In this study, we analyzed the changes in the root apoplast secretome of the Verticillium wilt-resistant island cotton cv Hai 7124 (Gossypium barbadense) upon infection with Verticillium dahliae. Two-dimensional differential gel electrophoresis and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis identified 68 significantly altered spots, corresponding to 49 different proteins. Gene ontology annotation indicated that most of these proteins function in reactive oxygen species (ROS) metabolism and defense response. Of the ROS-related proteins identified, we further characterized a thioredoxin, GbNRX1, which increased in abundance in response to V. dahliae challenge, finding that GbNRX1 functions in apoplastic ROS scavenging after the ROS burst that occurs upon recognition of V. dahliae. Silencing of GbNRX1 resulted in defective dissipation of apoplastic ROS, which led to higher ROS accumulation in protoplasts. As a result, the GbNRX1-silenced plants showed reduced wilt resistance, indicating that the initial defense response in the root apoplast requires the antioxidant activity of GbNRX1. Together, our results demonstrate that apoplastic ROS generation and scavenging occur in tandem in response to pathogen attack; also, the rapid balancing of redox to maintain homeostasis after the ROS burst, which involves GbNRX1, is critical for the apoplastic immune response.Cotton (Gossypium spp.) is one of the most economically important crops worldwide and a number of pathogens affect the growth and development of cotton plants. The soil-borne pathogen Verticillium dahliae (V. dahliae) causes the destructive vascular disease Verticillium wilt, which results in devastating reductions in plant mass, lint yield, and fiber quality (Bolek et al., 2005; Cai et al., 2009). To date, Verticillium wilt has not been effectively controlled in the most common cultivated cotton species, upland cotton (Gossypium hirsutum), and cultivars with stably inherited resistance to this disease are currently unavailable (Aguado et al., 2008; Jiang et al., 2009; Zhang et al., 2012a). Unlike upland cotton, sea-island cotton (Gossypium barbadense), which is only cultivated on a small scale, possesses Verticillium wilt resistance. Exploring the molecular mechanisms involved in the defense responses against V. dahliae invasion in G. barbadense can provide useful information for generating wilt-resistant G. hirsutum species through molecular breeding.During the past decades, progress has been made in studying the defense responses against V. dahliae infection in cotton. Global analyses have demonstrated that several signaling pathways, including those mediated by salicylic acid, ethylene, jasmonic acid, and brassinosteroids, activate distinct processes involved in V. dahliae defense (Bari and Jones, 2009; Grant and Jones, 2009; Gao et al., 2013a). Accumulating evidence indicates that many V. dahliae-responsive genes, such as GbWARKY1, GhSSN, GbERF, GhMLP28, GhNDR1, GhMKK2, and GhBAK1 (Qin et al., 2004; Gao et al., 2011, 2013b; Li et al., 2014a; Sun et al., 2014; Yang et al., 2015), play crucial roles in defense against Verticillium wilt. In addition, the biosynthesis of terpenoids, lignin, and gossypol also makes important contributions to V. dahliae resistance in cotton (Tan et al., 2000; Luo et al., 2001; Xu et al., 2011; Gao et al., 2013a). Together, these studies have greatly improved our understanding of the complex innate defense systems against V. dahliae infection in cotton.The initial interaction between plants and pathogens takes place in the apoplast, the compartment of the plant cell outside the cell membrane, including the cell wall and intercellular space (Dietz, 1997). In response to pathogen colonization, the attacked plant cells undergo significant cellular and molecular changes, such as reinforcement of the cell wall and secretion of antimicrobial molecules into the apoplastic space (Bednarek et al., 2010). Thus, the apoplast serves as the first line of defense against microbe invasion, and apoplast immunity can be considered an important component of the plant immune response to pathogens.Upon recognition of pathogen infection, rapid production of reactive oxygen species [the reactive oxygen species (ROS) burst] occurs in the apoplast (Lamb and Dixon, 1997; Torres et al., 2006; Torres, 2010). This ROS burst is regarded as a core component of the early plant immune response (Daudi et al., 2012; Doehlemann and Hemetsberger, 2013). During defense responses, apoplastic ROS can diffuse into the cytoplasm and serve as signals, interacting with other signaling processes such as phosphorylation cascades, calcium signaling, and hormone-mediated pathways (Kovtun et al., 2000; Mou et al., 2003). Apoplastic ROS can also directly strengthen the host cell walls by oxidative cross linking of glycoproteins (Bradley et al., 1992; Lamb and Dixon, 1997) or the precursors of lignin and suberin polymers (Hückelhoven, 2007). Moreover, apoplastic ROS can directly affect pathogens by degrading nucleic acids and peptides from microbes or causing lipid peroxidation and membrane damage in the microbe (Mehdy, 1994; Lamb and Dixon, 1997; Apel and Hirt, 2004; Montillet et al., 2005).ROS levels in the apoplast increase rapidly in response to a variety of pathogens, but subsequently return to basal levels. The rapid production and dissipation of apoplastic ROS indicate that this process is finely regulated. Two classes of enzymes, NADPH oxidases and class III peroxidases, account for the rapid ROS burst in the apoplast (Bolwell et al., 1995; O’Brien et al., 2012). NADPH oxidases are directly phosphorylated by the receptor-like kinase BIK1 to enhance ROS generation (Li et al., 2014b). Also, due to the toxicity of high levels of ROS, plants have evolved enzymatic and nonenzymatic mechanisms to eliminate ROS, thereby preventing or reducing oxidative damage (Rahal et al., 2014; Torres et al., 2006). However, the molecular system responsible for the regulation of apoplastic ROS homeostasis during the immune response is not well understood.In this study, we performed a comparative analysis of the apoplastic proteomes in control roots compared with V. dahliae-inoculated roots of Gossypium barbadense (wilt-resistant sea-island cotton) using the two-dimensional differential gel electrophoresis (2D-DIGE) technique. Among the differentially expressed apoplastic proteins, ROS-related proteins were found to be major components, including a thioredoxin, GbNRX1, which functions as an ROS scavenger in response to V. dahliae infection. Knock-down of GbNRX1 expression in cotton by virus-induced gene silencing (VIGS) resulted in reduced resistance to V. dahliae. Our results demonstrate that maintaining apoplastic ROS homeostasis is a crucial component of the apoplastic immune response and that GbNRX1 is an important regulator of this process.