Proteomic and Virus-induced Gene Silencing (VIGS) Analyses Reveal That Gossypol, Brassinosteroids, and Jasmonic acid Contribute to the Resistance of Cotton to Verticillium dahliae
2013; Elsevier BV; Volume: 12; Issue: 12 Linguagem: Inglês
10.1074/mcp.m113.031013
ISSN1535-9484
AutoresWei Gao, Lu Long, Longfu Zhu, Li Xu, Wenhui Gao, Longqing Sun, Linlin Liu, Xianlong Zhang,
Tópico(s)Plant-Microbe Interactions and Immunity
ResumoVerticillium wilt causes massive annual losses of cotton yield, but the mechanism of cotton resistance to Verticillium dahliae is complex and poorly understood. In this study, a comparative proteomic analysis was performed in resistant cotton (Gossypium barbadense cv7124) on infection with V. dahliae. A total of 188 differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) analysis and could be classified into 17 biological processes based on Gene Ontology annotation. Most of these proteins were implicated in stimulus response, cellular processes and metabolic processes. Based on the proteomic analysis, several genes involved in secondary metabolism, reactive oxygen burst and phytohormone signaling pathways were identified for further physiological and molecular analysis. The roles of the corresponding genes were further characterized by employing virus-induced gene silencing (VIGS). Based on the results, we suggest that the production of gossypol is sufficient to affect the cotton resistance to V. dahliae. Silencing of GbCAD1, a key enzyme involving in gossypol biosynthesis, compromised cotton resistance to V. dahliae. Reactive oxygen species and salicylic acid signaling may be also implicated as regulators in cotton responsive to V. dahliae according to the analysis of GbSSI2, an important regulator in the crosstalk between salicylic acid and jasmonic acid signal pathways. Moreover, brassinosteroids and jasmonic acid signaling may play essential roles in the cotton disease resistance to V. dahliae. The brassinosteroids signaling was activated in cotton on inoculation with V. dahliae and the disease resistance of cotton was enhanced after exogenous application of brassinolide. Meanwhile, jasmonic acid signaling was also activated in cotton after inoculation with V. dahliae and brassinolide application. These data provide highlights in the molecular basis of cotton resistance to V. dahliae. Verticillium wilt causes massive annual losses of cotton yield, but the mechanism of cotton resistance to Verticillium dahliae is complex and poorly understood. In this study, a comparative proteomic analysis was performed in resistant cotton (Gossypium barbadense cv7124) on infection with V. dahliae. A total of 188 differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) analysis and could be classified into 17 biological processes based on Gene Ontology annotation. Most of these proteins were implicated in stimulus response, cellular processes and metabolic processes. Based on the proteomic analysis, several genes involved in secondary metabolism, reactive oxygen burst and phytohormone signaling pathways were identified for further physiological and molecular analysis. The roles of the corresponding genes were further characterized by employing virus-induced gene silencing (VIGS). Based on the results, we suggest that the production of gossypol is sufficient to affect the cotton resistance to V. dahliae. Silencing of GbCAD1, a key enzyme involving in gossypol biosynthesis, compromised cotton resistance to V. dahliae. Reactive oxygen species and salicylic acid signaling may be also implicated as regulators in cotton responsive to V. dahliae according to the analysis of GbSSI2, an important regulator in the crosstalk between salicylic acid and jasmonic acid signal pathways. Moreover, brassinosteroids and jasmonic acid signaling may play essential roles in the cotton disease resistance to V. dahliae. The brassinosteroids signaling was activated in cotton on inoculation with V. dahliae and the disease resistance of cotton was enhanced after exogenous application of brassinolide. Meanwhile, jasmonic acid signaling was also activated in cotton after inoculation with V. dahliae and brassinolide application. These data provide highlights in the molecular basis of cotton resistance to V. dahliae. Cotton (Gossypium spp.) is one of the most important economic crops globally. However, the yield of cotton is restricted by many unfavorable environmental conditions including biotic and abiotic stresses. Among these stresses, Verticillium wilt, a soil-borne vascular disease caused by Verticillium dahliae, is a devastating disease of cotton worldwide (1Sal'kova E.G. Guseva N.N. The role of pectolytic enzymes of the verticillium dahliae fungus in the development of cotton wilt.Dokl. Akad. Nauk SSSR. 1965; 163: 515-522PubMed Google Scholar), reducing the quality and yield of the fiber; up to 30% yield reductions can occur during a severe outbreak of the disease (2Cai Y.F. He X.H. Mo J.C. Sun Q. Yang J.P. Liu J.G. Molecular research and genetic engineering of resistance to Verticillium wilt in cotton: A review.Afr. J. Biotechnol. 2009; 8: 7363-7372Google Scholar). Few germplasms have been found with resistance to V. dahliae in upland cotton (Gossypium hirsutum), which contributes ∼95% of the total cotton yield (3Zhang J. Sanogo S. Flynn R. Baral J. Bajaj S. Hughs S.E. Percy R. Germplasm evaluation and transfer of Verticillium wilt resistance from Pima (Gossypium barbadense) to Upland cotton (G. hirsutum).Euphytica. 2012; 187: 147-160Crossref Scopus (74) Google Scholar), and the complex genetics of resistance to V. dahliae in upland cotton cultivars has prevented the efficient breeding of disease-resistant cotton (4Aguado A. Santos B.D.L. Blanco C. Romero F. Study of gene effects for cotton yield and Verticillium wilt tolerance in cotton plant (Gossypium hirsutum L.).Field Crops Res. 2008; 107: 78-86Crossref Scopus (27) Google Scholar, 5Jiang F. Zhao J. Zhou L. Guo W. Zhang T. Molecular mapping of Verticillium wilt resistance QTL clustered on chromosomes D7 and D9 in upland cotton.Sci. China C Life Sci. 2009; 52: 872-884Crossref PubMed Scopus (53) Google Scholar, 6Zhang J.F. Lu Y. Adragna H. Hughs E. Genetic improvement of New Mexico Acala cotton germplasm and their genetic diversity.Crop Sci. 2005; 45: 2363Crossref Scopus (61) Google Scholar). Moreover, the fungus can survive in soil for many years even in the absence of hosts (2Cai Y.F. He X.H. Mo J.C. Sun Q. Yang J.P. Liu J.G. Molecular research and genetic engineering of resistance to Verticillium wilt in cotton: A review.Afr. J. Biotechnol. 2009; 8: 7363-7372Google Scholar). These issues make it difficult to develop an effective and practical management plan for the control of V. dahliae in cotton production. Plants have evolved a complete, multilayered immune system that includes constitutive and inducible defenses to counteract colonization by pathogens (7Jones J.D. Dangl J.L. The plant immune system.Nature. 2006; 444: 323-329Crossref PubMed Scopus (8281) Google Scholar). Several endogenous signal molecules, such as salicylic acid (SA), 1The abbreviations used are:SAsalicylic acid2-DEtwo-dimensional gel electrophoresisqPCRquantitative real time PCRAtArabidopsis thalianaGhGossypium hirsutumGbGossypium barbadensehpihours past inoculationsspsample spot proteinNCBINational Center for Biotechnology informationVIGSvirus-induced gene silencingROSreactive oxygen speciesJAjasmonic acidBRsbrassinosteroidsBLbrassinolideDIdisease index. 1The abbreviations used are:SAsalicylic acid2-DEtwo-dimensional gel electrophoresisqPCRquantitative real time PCRAtArabidopsis thalianaGhGossypium hirsutumGbGossypium barbadensehpihours past inoculationsspsample spot proteinNCBINational Center for Biotechnology informationVIGSvirus-induced gene silencingROSreactive oxygen speciesJAjasmonic acidBRsbrassinosteroidsBLbrassinolideDIdisease index. ethylene (ET), and jasmonic acid (JA), are synthesized and activate distinct defense pathways involved in complex defense signaling networks (8Bari R. Jones J.D. Role of plant hormones in plant defence responses.Plant Mol. Biol. 2009; 69: 473-488Crossref PubMed Scopus (1675) Google Scholar). Among these molecules, JA usually acts with ethylene to induce resistance against necrotrophic pathogens, whereas SA-mediated defense responses are effective against hemi-biotrophs and biotrophs and are critical for systemic acquired resistance (9Dempsey D.A. Klessig D.F. SOS - too many signals for systemic acquired resistance?.Trends Plant Sci. 2012; 17: 538-545Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). In addition, defense signaling pathways mediated by SA and JA frequently act antagonistically to mediate defense against specific types of pathogens (10Brooks D.M. Bender C.L. Kunkel B.N. The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis thaliana.Mol. Plant Pathol. 2005; 6: 629-639Crossref PubMed Scopus (261) Google Scholar, 11Spoel S.H. Johnson J.S. Dong X. Regulation of tradeoffs between plant defenses against pathogens with different lifestyles.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 18842-18847Crossref PubMed Scopus (469) Google Scholar, 12Spoel S.H. Koornneef A. Claessens S.M. Korzelius J.P. Van Pelt J.A. Mueller M.J. Buchala A.J. Métraux J.P. Brown R. Kazan K. Van Loon L.C. Dong X. Pieterse C.M. NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol.Plant Cell. 2003; 15: 760-770Crossref PubMed Scopus (849) Google Scholar). For example, SA accumulation and SA-derived signaling are induced by virulent Pseudomonas syringae infection, which enhances susceptibility to Alternaria brassicicola by inhibiting JA-mediated defense responses in Arabidopsis (11Spoel S.H. Johnson J.S. Dong X. Regulation of tradeoffs between plant defenses against pathogens with different lifestyles.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 18842-18847Crossref PubMed Scopus (469) Google Scholar). Nevertheless, the phytotoxin coronatine, a structural analog of JA produced by Pseudomonas syringae, can suppress SA-derived responses in the host (10Brooks D.M. Bender C.L. Kunkel B.N. The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis thaliana.Mol. Plant Pathol. 2005; 6: 629-639Crossref PubMed Scopus (261) Google Scholar). Other endogenous signal molecules, such reactive oxygen species (ROS), auxin and brassinosteroids (BRs), can also influence defense signaling and resistance (13Grant M.R. Jones J.D. Hormone (dis)harmony moulds plant health and disease.Science. 2009; 324: 750-752Crossref PubMed Scopus (328) Google Scholar, 14Li A. Zhang R. Pan L. Tang L. Zhao G. Zhu M. Chu J. Sun X. Wei B. Zhang X. Jia J. Mao L. Transcriptome analysis of H2O2-treated wheat seedlings reveals a H2O2-responsive fatty acid desaturase gene participating in powdery mildew resistance.PLoS ONE. 2011; 6: e28810Crossref PubMed Scopus (37) Google Scholar, 15Lamb C. Dixon R.A. The oxidative burst in plant disease resistance.Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 251-275Crossref PubMed Scopus (2538) Google Scholar, 16Pieterse C.M. Leon-Reyes A. Van der Ent S. Van Wees S.C. Networking by small-molecule hormones in plant immunity.Nat. Chem. Biol. 2009; 5: 308-316Crossref PubMed Scopus (1551) Google Scholar, 17Fradin E.F. Zhang Z. Juarez Ayala J.C. Castroverde C.D. Nazar R.N. Robb J. Liu C.M. Thomma B.P. Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1.Plant Physiol. 2009; 150: 320-332Crossref PubMed Scopus (341) Google Scholar). Plants have evolved regulatory defense mechanisms to adapt efficiently to changes in their complex environment during pathogen invasion. Crosstalk among signal molecules provides plants with a powerful capacity to finely regulate the immune response. The molecular mechanisms involved in plant immunity are complex. salicylic acid two-dimensional gel electrophoresis quantitative real time PCR Arabidopsis thaliana Gossypium hirsutum Gossypium barbadense hours past inoculation sample spot protein National Center for Biotechnology information virus-induced gene silencing reactive oxygen species jasmonic acid brassinosteroids brassinolide disease index. salicylic acid two-dimensional gel electrophoresis quantitative real time PCR Arabidopsis thaliana Gossypium hirsutum Gossypium barbadense hours past inoculation sample spot protein National Center for Biotechnology information virus-induced gene silencing reactive oxygen species jasmonic acid brassinosteroids brassinolide disease index. During the past two decades, extensive studies have enriched our understanding of the molecular mechanism of cotton resistance to V. dahliae. Production of phytoalexins, including terpenoids, and phenylpropanoid substances, is induced quickly in cotton on infection by V. dahliae (18Liu C.J. Heinstein P. Chen X.Y. Expression pattern of genes encoding farnesyl diphosphate synthase and sesquiterpene cyclase in cotton suspension-cultured cells treated with fungal elicitors.Mol. Plant. Microbe Interact. 1999; 12: 1095-1104Crossref PubMed Scopus (50) Google Scholar, 19Xu L. Zhu L. Tu L. Liu L. Yuan D. Jin L. Long L. Zhang X. Lignin metabolism has a central role in the resistance of cotton to the wilt fungus Verticillium dahliae as revealed by RNA-Seq-dependent transcriptional analysis and histochemistry.J. Exp. Bot. 2011; 62: 5607-5621Crossref PubMed Scopus (322) Google Scholar). Gossypol is one of the most important sesquiterpene phytoalexin, which exists specifically in cotton and plays a crucial role in the defense against the invasion of pathogens and insects (20Luo P. Wang Y.H. Wang G.D. Essenberg M. Chen X.Y. Molecular cloning and functional identification of (+)-delta-cadinene-8-hydroxylase, a cytochrome P450 mono-oxygenase (CYP706B1) of cotton sesquiterpene biosynthesis.Plant J. 2001; 28: 95-104Crossref PubMed Scopus (105) Google Scholar). Though many phytoalexin-related genes have been shown to be important in mediating cotton defense, the molecular mechanism is unknown (21Townsend B.J. Poole A. Blake C.J. Llewellyn D.J. Antisense suppression of a (+)-delta-cadinene synthase gene in cotton prevents the induction of this defense response gene during bacterial blight infection but not its constitutive expression.Plant Physiol. 2005; 138: 516-528Crossref PubMed Scopus (72) Google Scholar, 22Xu Y.H. Wang J.W. Wang S. Wang J.Y. Chen X.Y. Characterization of GaWRKY1, a cotton transcription factor that regulates the sesquiterpene synthase gene (+)-delta-cadinene synthase-A.Plant Physiol. 2004; 135: 507-515Crossref PubMed Scopus (350) Google Scholar). As sequencing technology develops, a number of genes related to disease resistance (e.g. aerobic metabolism enzymes, pathogen-related proteins, ethylene biosynthesis and response genes, etc.) have been identified from resistant cotton cultivars (Gossypium barbadense cv7124 or Pima 90) through suppression subtractive hybridization (23Zuo K. Wang J. Wu W. Chai Y. Sun X. Tang K. Identification and characterization of differentially expressed ESTs of Gossypium barbadense infected by Verticillium dahliae with suppression subtractive hybridization.Mol. Biol. 2005; 39: 191-199Crossref Scopus (23) Google Scholar, 24Xu L. Zhu L.F. Tu L.L. Guo X.P. Long L. Sun L.Q. Gao W. Zhang X.L. Differential gene expression in cotton defence response to Verticillium dahliae by SSH.J. Phytopathol. 2011; 159: 606-615Crossref Scopus (45) Google Scholar). Using RNA-Seq-dependent transcriptional analysis, a subset of genes participating in lignin metabolism was demonstrated to be very important in the resistance of cotton to V. dahliae (19Xu L. Zhu L. Tu L. Liu L. Yuan D. Jin L. Long L. Zhang X. Lignin metabolism has a central role in the resistance of cotton to the wilt fungus Verticillium dahliae as revealed by RNA-Seq-dependent transcriptional analysis and histochemistry.J. Exp. Bot. 2011; 62: 5607-5621Crossref PubMed Scopus (322) Google Scholar). Additionally, defense- and stress-related proteins, such as pathogenesis-related proteins and proteins likely to be involved in the oxidative burst, sugars, ethylene signaling, and isoprenoid synthesis, have recently been suggested to be involved in cotton response to V. dahliae (25Wang F.X. Ma Y.P. Yang C.L. Zhao P.M. Yao Y. Jian G.L. Luo Y.M. Xia G.X. Proteomic analysis of the sea-island cotton roots infected by wilt pathogen Verticillium dahliae.Proteomics. 2011; 11: 4296-4309Crossref PubMed Scopus (70) Google Scholar, 26Zhao F.A. Fang W. Xie D. Zhao Y. Tang Z. Li W. Nie L. Lv S. Proteomic identification of differentially expressed proteins in Gossypium thurberi inoculated with cotton Verticillium dahliae.Plant Sci. 2012; 185–186: 176-184Crossref PubMed Scopus (27) Google Scholar). Most of the candidate genes involving in disease resistance are isolated from transcriptomic analysis, whereas only few genes have been functionally characterized (27Munis M.F. Tu L. Deng F. Tan J. Xu L. Xu S. Long L. Zhang X. A thaumatin-like protein gene involved in cotton fiber secondary cell wall development enhances resistance against Verticillium dahliae and other stresses in transgenic tobacco.Biochem. Biophys. Res. Commun. 2010; 393: 38-44Crossref PubMed Scopus (104) Google Scholar, 28Shi J. An H.L. Zhang L. Gao Z. Guo X.Q. GhMPK7, a novel multiple stress-responsive cotton group C MAPK gene, has a role in broad spectrum disease resistance and plant development.Plant Mol. Biol. 2010; 74: 1-17Crossref PubMed Scopus (75) Google Scholar). Ve1 is the only R gene isolated using map-based cloning from tomato (Solanum lycopersicum) and has been shown to provide race-specific resistance to race 1 strains of V. dahliae and V. albo-atrum in tomato and Arabidopsis (17Fradin E.F. Zhang Z. Juarez Ayala J.C. Castroverde C.D. Nazar R.N. Robb J. Liu C.M. Thomma B.P. Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1.Plant Physiol. 2009; 150: 320-332Crossref PubMed Scopus (341) Google Scholar, 29Fradin E.F. Abd-El-Haliem A. Masini L. van den Berg G.C. Joosten M.H. Thomma B.P. Interfamily transfer of tomato Ve1 mediates Verticillium resistance in Arabidopsis.Plant Physiol. 2011; 156: 2255-2265Crossref PubMed Scopus (194) Google Scholar). Although several genes homologous to Ve1 have been cloned from cotton (30Zhang B. Yang Y. Chen T. Yu W. Liu T. Li H. Fan X. Ren Y. Shen D. Liu L. Dou D. Chang Y. Island cotton Gbve1 gene encoding a receptor-like protein confers resistance to both defoliating and non-defoliating isolates of Verticillium dahliae.PLoS ONE. 2012; 7: e51091Crossref PubMed Scopus (93) Google Scholar, 31Gao X. Wheeler T. Li Z. Kenerley C.M. He P. Shan L. Silencing GhNDR1 and GhMKK2 compromises cotton resistance to Verticillium wilt.Plant J. 2011; 66: 293-305Crossref PubMed Scopus (185) Google Scholar), it is unclear whether Ve1-mediated resistance signaling also exists in cotton. In this study, we performed a comparative proteomic analysis on Mock and V. dahliae inoculated roots of G. barbadense cv7124, which shows high resistance to V. dahliae, at different time points. A number of differentially expressed proteins were identified. Furthermore, three classes of genes, involved in gossypol metabolism, BR signaling, and JA signaling were characterized using virus-induced gene silencing (VIGS). Our data suggest that gossypol, BRs and JA act as important factors in contributing resistance of cotton to V. dahliae. For the first time, proteomics was combined with VIGS to discover and validate defense-related genes in cotton. Our research provides new insights into the molecular basis of cotton defense against V. dahliae. Seeds of G. barbadense cv7124 (resistant) and G. hirsutum cvYZ-1 (susceptible) were grown in a controlled environment chamber under a 14 h light/10 h dark cycle at 28 °C for 2 weeks. The defoliating isolate V991 of V. dahliae was grown on a potato-dextrose agar medium for 4 d; the fungus was then incubated in Czapek's medium (NaNO3, 0.3% w/v; MgSO4, 0.1% w/v; KH2PO4, 0.1% w/v; FeSO4, 0.0002% w/v; KCl, 0.1% w/v; sucrose, 3% w/v; pH 6.0) at 25 °C for 5 d. The concentration of spores was adjusted to ∼106 conidia per ml with deionized water for inoculation. The cotton seedlings were removed from the soil and dip-infected with the liquid containing V. dahliae spores. The seedlings were incubated at 25 °C under a 14 h/10 h light/dark photoperiod, and the roots were harvested at 1, 6, 12, 24, 48, and 72 h after inoculation. Seedlings treated with sterile distilled water in the same manner were used as a Mock treatment. Roots were stored at −80 °C until protein extraction was performed. For 2D-PAGE, proteins from cotton roots were prepared according to Yao and Pan (32Yao Y. Yang Y.W. Liu J.Y. An efficient protein preparation for proteomic analysis of developing cotton fibers by 2-DE.Electrophoresis. 2006; 27: 4559-4569Crossref PubMed Scopus (82) Google Scholar, 33Pang C.Y. Wang H. Pang Y. Xu C. Jiao Y. Qin Y.M. Western T.L. Yu S.X. Zhu Y.X. Comparative proteomics indicates that biosynthesis of pectic precursors is important for cotton fiber and Arabidopsis root hair elongation.Mol. Cell Proteomics. 2010; 9: 2019-2033Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) with minor modifications. Frozen root tissues were ground in liquid nitrogen to a fine powder and incubated in extraction buffer (−20 °C precooled acetone, 12% w/v trichloroacetic acid, and 0.07% w/v dithiothreitol (DTT)). After washing twice with cold acetone containing 0.07% (w/v) DTT, the vacuum-dried powder was suspended in extraction buffer [30% sucrose, 50 mm Tris-HCl (pH 8.0), 2% SDS, 2 mm PMSF, 0.07% DTT, and an equal volume of Tris-saturated phenol (pH 8.0)]. The phenol phase was then precipitated with 5 volumes of 0.1 m ammonium acetate in methanol at −20 °C. The collected protein pellets were washed twice with 80% methanol and 80% acetone. After air-drying, the pellets were dissolved in lysis buffer (8 m urea, 2 m thiourea, 2% CHAPS, 1% DTT, and 2% v/v immobilized pH gradient (IPG) buffer, pH 4–7). Protein concentration was determined using a 2-D quant kit (Bio-Rad, Hercules, CA). The first dimensional gel separation was performed according to the manufacturer's protocol with modifications (Bio-Rad). Total proteins (1.0 mg) were diluted in 450 μl rehydration buffer (8 m urea, 2 m thiourea, 2% CHAPS, 1% DTT, and 2% v/v IPG buffer, pH 4–7) and loaded on IPG strips (24 cm, pH 4–7 nonlinear) (Bio-Rad). The IPG strips were rehydrated for 12 h at room temperature and focused at gradient steps of 50 V for 1 h, 500 V for 1 h, 1000 V for 1 h, and 10,000 V for 4 h, with a final step of 10,000 V toward a total of 90 kVh. Before second dimension analysis, IPG strips were incubated in equilibration buffer (50 mm Tris-HCl, pH 8.8; 6 m urea; 30% glycerol; and 2% SDS) containing 1% DTT and then in equilibration buffer containing 2% (w/v) iodoacetamide. For the second dimension, IPG strips were fixed on 12.5% acrylamide gels. Then, 2D-PAGE was performed using the Ettan DALT six electrophoresis unit (GE Healthcare) at 5 W per gel for 1 h and then 15 W per gel for 6 h until the bromphenol blue dye front reached the bottom of the gels. Gels were stained with Coomassie brilliant blue solution (0.2% Coomassie brilliant blue G250, 20% methanol, 10% phosphoric acid, and 10% ammonium sulfate). The 2D gels were scanned using a GS-800 Calibrated Densitometer (Bio-Rad). Protein spots were detected using PDQuest software (Bio-Rad). After volumetric quantification and matching, differences in protein content between Mock and inoculated samples were analyzed using a Student t test and calculated as the fold ratio. Data was from three biological replicates; a threshold of p ≤ 0.05 and fold change of ≥2 or ≤0.5 was used to identify significantly differentially expressed protein spots. Differentially expressed proteins were excised from the gels and destained with 25 mm NH4HCO3 in 50% acetonitrile (ACN) until the Coomassie brilliant blue disappeared. After being washed twice with 100% ACN for 10 min, proteins were digested in-gel using trypsin (Promega, Madison, WI) overnight at 37 °C. The gel pieces were extracted once with extraction buffer (67% ACN and 5% TFA). After being dried completely, the samples were resuspended in 0.1% TFA and then mixed in a 1:1 ratio with a matrix consisting of a saturated solution of CHCA in 50% ACN containing 0.1% TFA. Samples were then spotted onto a freshly cleaned target plate. After air-drying, the crystallized spots were analyzed using an ABI 4800 MALDI-TOF/TOF Plus mass spectrometer (Applied Biosystems, Foster City, CA). Both the MS and MS/MS data were integrated and performed using GPS Explorer V3.6 software (Applied Biosystems). Proteins were successfully identified with a 95% or higher confidence interval using the MASCOT V2.3 search engine (Matrix Science, London, UK) and searching in the Gossypium EST database (release data 20120128; 2476590 sequences; 555009942 residues). The other search parameters were the enzyme trypsin; one missed cleavage site; partial modifications of cysteine carbamido methylation and methionine oxidization; no fixed modifications; peptide tolerance of 100 ppm; and fragment mass tolerance of 0.3 Da. Total RNA was isolated from cotton as previously described (34Zhu L. Tu L. Zeng F. Liu D. Zhang X. An improved simple protocol for isolation of high quality RNA from Gossypium spp. suitable for cDNA library construction.Acta Agronomic Sinica. 2005; 31: 1657-1659Google Scholar). First strand cDNA was synthesized from 5 μg of total RNA using the Superscript first-strand synthesis system (Invitrogen, Foster City, CA). For RT-PCR, an aliquot of the reverse transcription product was used as the template. The ubiquitin7 gene (DQ116441) of cotton was used as an internal control. Primers were designed as shown in supplemental Table S1. qPCR was performed according to the guidelines of the Minimum Information for Publication of Quantitative Real Time PCR Experiments (35Bustin S.A. Benes V. Garson J.A. Hellemans J. Huggett J. Kubista M. Mueller R. Nolan T. Pfaffl M.W. Shipley G.L. Vandesompele J. Wittwer C.T. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.Clin. Chem. 2009; 55: 611-622Crossref PubMed Scopus (10347) Google Scholar). Diluted cDNA was used for qPCR with SYBR green using ABI 7500 Real Time PCR system (Applied Biosystems). The expression analysis data are presented as the means ± S.D. from three biologically independent experiments. All primers for qPCR were designed using Primer Express 5.0 software (Applied Biosystems) and are shown in supplemental Table S2. The TRV vectors and Agrobacterium tumefaciens for VIGS were prepared according to Fradin et al. (17Fradin E.F. Zhang Z. Juarez Ayala J.C. Castroverde C.D. Nazar R.N. Robb J. Liu C.M. Thomma B.P. Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1.Plant Physiol. 2009; 150: 320-332Crossref PubMed Scopus (341) Google Scholar). Inserts to generate TRV:GbCAD1, TRV:Gb14-3-3c, TRV:Gb14-3-3d, TRV:GbSSI2 and positive control TRV:GbCLA1 (cloroplastos alterados 1) were amplified from the cDNA of G. barbadense cv7124. Primer pairs to generate TRV vectors are shown in supplemental Table S3. PCR fragments were digested with BamHI and KpnI and then ligated into the TRV:00 plasmid (36Liu Y. Schiff M. Marathe R. Dinesh-Kumar S.P. Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus.Plant J. 2002; 30: 415-429Crossref PubMed Scopus (763) Google Scholar). The constructs were transformed into A. tumefaciens GV3101 by electroporation. TRV vectors were agro-infiltrated as described (31Gao X. Wheeler T. Li Z. Kenerley C.M. He P. Shan L. Silencing GhNDR1 and GhMKK2 compromises cotton resistance to Verticillium wilt.Plant J. 2011; 66: 293-305Crossref PubMed Scopus (185) Google Scholar) into the cotyledons of 10-day-old seedlings of G. barbadense cv7124 or G. hirsutum cvYZ-1. The seedlings were then grown at 25 °C with a 16 h/8 h light/dark photoperiod cycle in a controlled environment chamber. As shown in supplemental Fig. S1, the leaf bleaching phenotype was observed 2 weeks after infiltration in TRV:GbCLA1 plants. Therefore, inoculation with V. dahliae isolate V991 was also performed 2 weeks after infiltration. The rate of diseased plants and disease index were scored with at least 16 plants per treatment and repeated at least for three times. Plant disease index (DI) is calculated as the following formula: INSERT EQUATION HERE, n denotes disease level, cotton seedlings were divided into five levels based on their disease severity after V. dahliae inoculation (level 0, 1, 2, 3, 4) according to Xu et al. (37Xu F. Yang L. Zhang J. Guo X. Zhang X. Li G. Prevalence of the defoliating pathotype of Verticillium dahliae on cotton in central china and virulence on selected cotton cultivars.J. Phytopathol. 2012; 160: 369-376Crossref Scopus (24) Google Scholar), DI reflects the disease infection status of a population, not an individual plant; higher DI means more serious infection. For treatments with MeJA and BL, the concentrations were 200 μm and 5 μg/pot, respectively. Cotton seedlings were cultured in a pot (four or five plants per pot) in a greenhouse and three-leaf stage plants were treated with MeJA or BL by soil drench application. The extraction and measurement of the endogenous SA and JA of cotton seedlings were performed as described (38Bowling S.A. Guo A. Cao H. Gordon A.S. Klessig D.F. Dong X. A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance.Plant Cell. 1994; 6: 1845-1857Crossref PubMed Scopus (534) Google Scholar). Three replicates of each frozen sample (∼100 mg for each replicate) were ground to a fine powder in liquid nitrogen and mixed with 750 μl cold extraction buffer (methanol/water/acetic acid, 80:19:1, v/v/v). After shaking for 16 h at 4 °C in the dark, the supernatants were collected and then filtered using a syringe-facilitated 13-mm diameter nylon filter with a pore size of 0.22 μm (Nylon 66; Jin Teng Ex
Referência(s)