Characterization of Synthetic-Lethal Mutants Reveals a Role for the Saccharomyces Cerevisiae Guanine-Nucleotide Exchange Factor Cdc24p in Vacuole Function and Na(+) Tolerance

Cdc24p is the guanine-nucleotide exchange factor for the Cdc42p GTPase, which controls cell polarity in Saccharomyces cerevisiae. To identify new genes that may affect cell polarity, we characterized six UV-induced csl (CDC24 synthetic-lethal) mutants that exhibited synthetic-lethality with cdc24-4(ts) at 23°. Five mutants were not complemented by plasmid-borne CDC42, RSR1, BUD5, BEM1, BEM2, BEM3 or CLA4 genes, which are known to play a role in cell polarity. The csl3 mutant displayed phenotypes similar to those observed with calcium-sensitive, Pet(-) vma mutants defective in vacuole function. CSL5 was allelic to VMA5, the vacuolar H(+)-ATPase subunit C, and one third of csl5 cdc24-4(ts) cells were elongated or had misshapen buds. A cdc24-4(ts) Δvma5::LEU2 double mutant did not exhibit synthetic lethality, suggesting that the csl5/vma5 cdc24-4(ts) synthetic-lethality was not simply due to altered vacuole function. The cdc24-4(ts) mutant, like Δvma5::LEU2 and csl3 mutants, was sensitive to high levels of Ca(2+) as well as Na(+) in the growth media, which did not appear to be a result of a fragile cell wall because the phenotypes were not remedied by 1 M sorbitol. Our results indicated that Cdc24p was required in one V-ATPase mutant and another mutant affecting vacuole morphology, and also implicated Cdc24p in Na(+) tolerance.     

Phosphoenolpyruvate Carboxylase in Arabidopsis Leaves Plays a Crucial Role in Carbon and Nitrogen Metabolism

Phosphoenolpyruvate carboxylase (PEPC) is a crucial enzyme that catalyzes an irreversible primary metabolic reaction in plants. Previous studies have used transgenic plants expressing ectopic PEPC forms with diminished feedback inhibition to examine the role of PEPC in carbon and nitrogen metabolism. To date, the in vivo role of PEPC in carbon and nitrogen metabolism has not been analyzed in plants. In this study, we examined the role of PEPC in plants, demonstrating that PPC1 and PPC2 were highly expressed genes encoding PEPC in Arabidopsis (Arabidopsis thaliana) leaves and that PPC1 and PPC2 accounted for approximately 93% of total PEPC activity in the leaves. A double mutant, ppc1/ppc2, was constructed that exhibited a severe growth-arrest phenotype. The ppc1/ppc2 mutant accumulated more starch and sucrose than wild-type plants when seedlings were grown under normal conditions. Physiological and metabolic analysis revealed that decreased PEPC activity in the ppc1/ppc2 mutant greatly reduced the synthesis of malate and citrate and severely suppressed ammonium assimilation. Furthermore, nitrate levels in the ppc1/ppc2 mutant were significantly lower than those in wild-type plants due to the suppression of ammonium assimilation. Interestingly, starch and sucrose accumulation could be prevented and nitrate levels could be maintained by supplying the ppc1/ppc2 mutant with exogenous malate and glutamate, suggesting that low nitrogen status resulted in the alteration of carbon metabolism and prompted the accumulation of starch and sucrose in the ppc1/ppc2 mutant. Our results demonstrate that PEPC in leaves plays a crucial role in modulating the balance of carbon and nitrogen metabolism in Arabidopsis.Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is a crucial enzyme that functions in primary metabolism by irreversibly catalyzing the conversion of phosphoenolpyruvate (PEP) and HCO₃⁻ to oxaloacetate (OAA) and inorganic phosphate. PEPC is found in all plants, green algae, and cyanobacteria, and in most archaea and nonphotosynthetic bacteria, but not in animals or fungi (Chollet et al., 1996; O’Leary et al., 2011a). Several isoforms of PEPC are present in plants, including plant-type PEPCs and one bacterium-type PEPC (Sánchez and Cejudo, 2003; Sullivan et al., 2004; Mamedov et al., 2005; Gennidakis et al., 2007; Igawa et al., 2010). Arabidopsis (Arabidopsis thaliana) possesses three plant-type PEPC genes, AtPPC1, AtPPC2, and AtPPC3, and one bacterium-type PEPC gene, AtPPC4. Unlike plant-type PEPCs, bacterium-type PEPCs lack a seryl-phosphorylation domain near the N terminus, a typical domain conserved in plant-type PEPCs (Sánchez and Cejudo, 2003). Plant-type PEPCs form class 1 PEPCs, which exist as homotetramers. Recently, bacterium-type PEPCs have been reported to interact with plant-type PEPCs to form heterooctameric class 2 PEPCs in several species, including unicellular green algae (Selenastrum minutum), lily (Lilium longiflorum), and castor bean (Ricinus communis; O’Leary et al., 2011a).Because of the irreversible nature of the enzymatic reactions catalyzed by PEPC isoforms, they are strictly regulated by a variety of mechanisms. PEPC is an allosteric enzyme and is activated by its positive effector, Glc-6-P, and inhibited by its negative effectors, malate, Asp, and Glu (O’Leary et al., 2011a). Control by reversible phosphorylation is another important mechanism that regulates the activity of PEPC. In this reaction, phosphorylation catalyzed by PEPC kinase changes the sensitivity of PEPC to its allosteric effectors (Nimmo, 2003). In addition, monoubiquitination may also regulate plant-type PEPC activity (Uhrig et al., 2008). Recent research in castor oil seeds suggests that bacterium-type PEPC is a catalytic and regulatory subunit of class 2 PEPCs, as class 1 and class 2 PEPCs show significant differences in their sensitivity to allosteric inhibitors (O’Leary et al., 2009, 2011b).A number of studies on PEPC function have been performed in a variety of organisms (O’Leary et al., 2011a). The best described function of PEPC is in fixing photosynthetic CO₂ during C4 and Crassulacean acid metabolism photosynthesis. However, in most nonphotosynthetic tissues and the photosynthetic tissues of C3 plants, the fundamental function of PEPC is to anaplerotically replenish tricarboxylic acid cycle intermediates (Chollet et al., 1996). PEPC also functions in malate production in guard cells and legume root nodules (Chollet et al., 1996). A chloroplast-located PEPC isoform in rice (Oryza sativa) was recently found to be crucial for ammonium assimilation (Masumoto et al., 2010). In addition, previous work in Arabidopsis suggested that AtPPC4 might play a role in drought tolerance (Sánchez et al., 2006).Transgenic plants expressing ectopic PEPC forms with diminished feedback inhibition showed an increase in overall organic nitrogen content at the expense of starch and soluble sugars (Rademacher et al., 2002; Chen et al., 2004; Rolletschek et al., 2004). However, the in vivo function of PEPC in carbon and nitrogen metabolism has not been reported previously.To further investigate the function of PEPC in higher plants, we isolated and characterized mutants of Arabidopsis deficient in the expression of the PEPC-encoding genes PPC1 and PPC2. We demonstrated that PPC1 and PPC2 were the most highly expressed PEPC genes in the leaves. To further define their role, we produced a double mutant (ppc1/ppc2) deficient in the expression of the PPC1 and PPC2 genes. We then conducted a detailed molecular, biochemical, and physiological characterization of this double mutant.     

Topographic Heterogeneity Plays a Crucial Role for Grasshopper Diversity in a Southern African Megabiodiversity Hotspot

Topographic heterogeneity as a determinant of insect diversity pattern has been little studied. Responses of grasshopper assemblages to three hill sizes were assessed in the arid Succulent Karoo, South Africa. This area is one of the world’s 25 hotspots for conservation priorities. Small hills overall were more speciose than medium or large hills. There were also significantly higher densities of small-sized grasshoppers on small hills than on medium or large ones. The slopes of the three hill sizes did not differ significantly either in their species richness or abundance, and there was no significant difference in species richness between summits only of the three hill sizes. Patterns of grasshopper species dominance were markedly variable among sites, but with clear differences between small and larger hills, associated with vegetation characteristics. Vegetation cover and grass cover was less on the small hills. Grasshopper taxonomic groups varied among the three hill sizes, with small hills being taxonomically more diverse, supporting species from four families and nine subfamilies, while medium and large hills only supported Acrididae. It is concluded that topography has a remarkably strong effect on various aspects of grasshopper spatial heterogeneity and that small hills in particular are a major factor to consider in spatial conservation planning.     

The C-Terminal Portion of an Archaeal Toxin,aRelE, Plays a Crucial Role in Protein Synthesis Inhibition

Mutations of amino acids in the C-terminal region of an archaeal toxin, aRelE, from Pyrococcus horikoshii were characterized with respect to protein synthesis inhibitory activity and 70S ribosome-binding activity. The results suggest that basic residues at the C-terminal region in aRelE play a crucial role both in 70S ribosome binding and in protein synthesis inhibition activities.     

A Crucial Role for CDC42 in Senescence-Associated Inflammation and Atherosclerosis

Risk factors for atherosclerosis accelerate the senescence of vascular endothelial cells and promote atherogenesis by inducing vascular inflammation. A hallmark of endothelial senescence is the persistent up-regulation of pro-inflammatory genes. We identified CDC42 signaling as a mediator of chronic inflammation associated with endothelial senescence. Inhibition of CDC42 or NF-κB signaling attenuated the sustained up-regulation of pro-inflammatory genes in senescent human endothelial cells. Endothelium-specific activation of the p53/p21 pathway, a key mediator of senescence, also resulted in up-regulation of pro-inflammatory molecules in mice, which was reversed by Cdc42 deletion in endothelial cells. Likewise, endothelial-specific deletion of Cdc42 significantly attenuated chronic inflammation and plaque formation in atherosclerotic mice. While inhibition of NF-κB suppressed the pro-inflammatory responses in acute inflammation, the influence of Cdc42 deletion was less marked. Knockdown of cdc-42 significantly down-regulated pro-inflammatory gene expression and restored the shortened lifespan to normal in mutant worms with enhanced inflammation. These findings indicate that the CDC42 pathway is critically involved in senescence-associated inflammation and could be a therapeutic target for chronic inflammation in patients with age-related diseases without compromising host defenses.     

Lpg0393 of Legionella pneumophila Is a Guanine-Nucleotide Exchange Factor for Rab5, Rab21 and Rab22

Legionella pneumophila, a human intracellular pathogen, encodes about 290 effector proteins that are translocated into host cells through a secretion machinery. Some of these proteins have been shown to manipulate or subvert cellular processes during infection, but functional roles of a majority of them remain unknown. Lpg0393 is a newly identified Legionella effector classified as a hypothetical protein. Through X-ray crystallographic analysis, we show that Lpg0393 contains a Vps9-like domain, which is structurally most similar to the catalytic core of human Rabex-5 that activates the endosomal Rab proteins Rab5, Rab21 and Rab22. Consistently, Lpg0393 exhibited a guanine-nucleotide exchange factor activity toward the endosomal Rabs. This work identifies the first example of a bacterial guanine-nucleotide exchange factor that is active towards the Rab5 sub-cluster members, implying that the activation of these Rab proteins might be advantageous for the intracellular survival of Legionella.     

Pyruvate Carboxylase Plays a Crucial Role in Carbon Metabolism of Extra- and Intracellularly Replicating Listeria monocytogenes

The human pathogen L. monocytogenes is a facultatively intracellular bacterium that survives and replicates in the cytosol of many mammalian cells. The listerial metabolism, especially under intracellular conditions, is still poorly understood. Recent studies analyzed the carbon metabolism of L. monocytogenes by the ¹³C isotopologue perturbation method in a defined minimal medium containing [U-¹³C₆]glucose. It was shown that these bacteria produce oxaloacetate mainly by carboxylation of pyruvate due to an incomplete tricarboxylic acid cycle. Here, we report that a pycA insertion mutant defective in pyruvate carboxylase (PYC) still grows, albeit at a reduced rate, in brain heart infusion (BHI) medium but is unable to multiply in a defined minimal medium with glucose or glycerol as a carbon source. Aspartate and glutamate of the pycA mutant, in contrast to the wild-type strain, remain unlabeled when [U-¹³C₆]glucose is added to BHI, indicating that the PYC-catalyzed carboxylation of pyruvate is the predominant reaction leading to oxaloacetate in L. monocytogenes. The pycA mutant is also unable to replicate in mammalian cells and exhibits high virulence attenuation in the mouse sepsis model.Listeria monocytogenes is a human pathogen that can cause systemic infections, especially in immunocompromised people, with symptoms such as septicemia, (encephalo)meningitis, placentitis, and stillbirth. These Gram-positive bacteria are able to enter the cytosol of many mammalian cells after being taken up via normal or induced phagocytosis by professional phagocytes, mainly macrophages and dendritic cells, and nonphagocytic cells, such as epithelial cells, fibroblasts, and endothelial cells (1, 8, 13). While the virulence genes and their regulation (4, 21), as well as the encoded virulence factors (20, 22), necessary for the various steps of the intracellular replication cycle of L. monocytogenes have been extensively studied in the past few decades, there is still little information concerning the metabolic capacities and the metabolic adaptation processes (10) that enable these bacteria to efficiently replicate in the cytosol of their host cells.The information on listerial metabolism obtained from the genome sequence (7) suggests that these heterotrophic bacteria are capable of utilizing a variety of carbohydrates as carbon sources, since a large number of genes encoding phosphoenolpyruvate (PEP)-phosphotransferase systems (PTS) were identified. Furthermore, all genes encoding the enzymes necessary for the catabolism of glycerol and dihydroxyacetone are present in the L. monocytogenes genome (7, 11). This genomic information is in accord with data from previous and more recent physiological studies (11, 17, 24).Most genes encoding the enzymes for the major catabolic pathways, namely, glycolysis, the citrate cycle, and the pentose phosphate cycle, are present in L. monocytogenes. The citrate cycle, however, seems to be interrupted, since the genes encoding 2-oxoglutarate dehydrogenase have not been identified in all L. monocytogenes strains sequenced so far, including EGD-e (7), or in Listeria innocua strain Clip 11262. This enzymatic gap in the citrate cycle was recently confirmed by ¹³C isotopologue perturbation studies using uniformly ¹³C-labeled glucose. The results showed that two C₄ amino acids, aspartate and threonine, are generated in L. monocytogenes, predominantly from building blocks comprising one or three ¹³C atoms, respectively (2). These data suggested that oxaloacetate, the direct or indirect precursor of both amino acids, is generated by an anaplerotic reaction assembling precursors composed of one and three carbon atoms, respectively. This can be afforded by the carboxylation of pyruvate catalyzed by the ATP-dependent pyruvate carboxylase (PYC) encoded by pycA.The genes encoding the enzymes for most anabolic pathways, but not those for the biosynthesis of thiamine (vitamin B₁), riboflavin (vitamin B₂), biotin, and thiotic acid (lipoate), were also identified in L. monocytogenes. However, these bacteria grow efficiently in a mineral salt medium containing a suitable carbon source (e.g., glucose) and these four cofactors only when the amino acids cysteine, methionine, glutamine, arginine, valine, isoleucine, and leucine are also added (17). According to Tsai and Hodgson, strain 10403S requires only methionine and cysteine (24). The missing sulfate reductase in L. monocytogenes readily explains the strict requirement for cysteine/methionine as a sulfur source, while the missing nitrate reductase may be the reason for the stimulatory growth effect of glutamine and arginine as reduced nitrogen sources. However, the need for the three branched-chain amino acids (BCAA) valine, isoleucine, and leucine for efficient growth of L. monocytogenes EGD-e (references 17 and 24 and our unpublished results) is less obvious, since L. monocytogenes has the complete genetic set for synthesis of the BCAA, indicating the role of metabolic intermediates in listerial growth.The central precursor for the biosynthesis of the BCAA is pyruvate, which is channeled into their biosynthetic pathways either directly, via oxidative decarboxylation of pyruvate to acetyl-coenzyme A (CoA), or more indirectly via oxaloacetate (generated by pyruvate carboxylation) to aspartate and further to threonine. Thus, biosynthesis of the BCAA may compete with the PYC-mediated generation of oxaloacetate for the common substrate pyruvate. These data suggest that PYC may play an important role in the carbon metabolism of L. monocytogenes.To more precisely determine the significance of this anaplerotic enzyme for listerial metabolism and pathogenesis, we generated a mutant of L. monocytogenes EGD-e defective in pycA, the gene encoding PYC, and studied the replication of this mutant under different extra- and intracellular growth conditions. The results show that PYC indeed plays a crucial role in the intracellular replication of L. monocytogenes and hence in the infection process.     

Peripheral Blood Mononuclear Cell Traffic Plays a Crucial Role in Mother-to-Infant Transmission of Hepatitis B Virus

The role of peripheral blood mononuclear cells (PBMCs) in HBV intrauterine infection is not fully defined. Particularly the origin of PBMCs in HBV-infected neonates remains to be addressed. We carried out a population-based nested case-control study by enrolling 312 HBsAg-positive mothers and their babies. PBMC HBV DNA as well as serum HBsAg and HBV DNA was tested in cohort entry samples. Totally, 45.5% (142/312) of the newborns were found to be infected with HBV in perinatal transmission. 119 mother-infant pairs were identified to be different in the genetic profile of maternal and fetal PBMCs by AS-PCR and hemi-nested PCR. Among them, 57.1% (68/119) of the maternal PBMCs in index cases were positive for HBV DNA while 83.8% (57/68) of the HBV DNA positive maternal PBMCs passed the placental barrier and entered the fetus. Furthermore, maternal PBMC HBV infection was significantly associated with newborn infants HBV infection. PBMC traffic from mother to fetus resulted in a 9.5-fold increased risk of HBV infection in PBMC HBV DNA positive newborn infants. These data indicate that maternal PBMCs infected with HBV contribute to HBV intrauterine infection of newborn infants via PBMC traffic from mother to fetus.     

Crucial Role of Hyaluronan in Neointimal Formation after Vascular Injury

Background

Hyaluronan (HA) is a primary component of the extracellular matrix of cells, and it is involved in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the role of HA in neointimal formation after vascular injury and determine its tissue-specific role in vascular smooth muscle cells (VSMCs) by using a cre-lox conditional transgenic (cTg) strategy.

Methods and Results

HA was found to be expressed in neointimal lesions in humans with atherosclerosis and after wire-mediated vascular injury in mice. Inhibition of HA synthesis using 4-methylumbelliferone markedly inhibited neointimal formation after injury. In vitro experiments revealed that low-molecular-weight HA (LMW-HA) induced VSMC activation, including migration, proliferation, and production of inflammatory cytokines, and reactive oxygen species (ROS). The migration and proliferation of VSMCs were mediated by the CD44/RhoA and CD44/ERK1/2 pathways, respectively. Because HA synthase 2 (HAS2) is predominantly expressed in injured arteries, we generated cTg mice that overexpress the murine HAS2 gene specifically in VSMCs (cHAS2/CreSM22α mice) and showed that HA overexpression markedly enhanced neointimal formation after cuff-mediated vascular injury. Further, HA-overexpressing VSMCs isolated from cHAS2/CreSM22α mice showed augmented migration, proliferation, and production of inflammatory cytokines and ROS.

Conclusion

VSMC-derived HA promotes neointimal formation after vascular injury, and HA may be a potential therapeutic target for cardiovascular disease.     

Distal C Terminus of CaV1.2 Channels Plays a Crucial Role in the Neural Differentiation of Dental Pulp Stem Cells

L-type voltage-dependent Ca_V1.2 channels play an important role in the maintenance of intracellular calcium homeostasis, and influence multiple cellular processes. C-terminal cleavage of Ca_V1.2 channels was reported in several types of excitable cells, but its expression and possible roles in non-excitable cells is still not clear. The aim of this study was to determine whether distal C-terminal fragment of Ca_V1.2 channels is present in rat dental pulp stem cells and its possible role in the neural differentiation of rat dental pulp stem cells. We generated stable Ca_V1.2 knockdown cells via short hairpin RNA (shRNA). Rat dental pulp stem cells with deleted distal C-terminal of Ca_V1.2 channels lost the potential of differentiation to neural cells. Re-expression of distal C-terminal of Ca_V1.2 rescued the effect of knocking down the endogenous Ca_V1.2 on the neural differentiation of rat dental pulp stem cells, indicating that the distal C-terminal of Ca_V1.2 is required for neural differentiation of rat dental pulp stem cells. These results provide new insights into the role of voltage-gated Ca²⁺ channels in stem cells during differentiation.     

The Polyploid State Plays a Tumor-Suppressive Role in the Liver

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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.