Plant Growth and Culturing of Verticillium dahliae
Seeds of resistant cotton Zhongzhimian 2 (original strain no. GK44) were provided by the Cotton Research Institute, Chinese Academy of Agricultural Sciences (Henan, Anyang). The germinated seeds were cultured in nutrient soil and vermiculite (2:1, w/w) under a photoperiod of 16 h/8 h. After vernalization for three days at 4℃in Murashige-Skoog (MS) culture medium, both wild-type (Columbia-0) and transgenic Arabidopsis plants were grown in mixture of nutrient soil and vermiculite (1:1, w/w) under a controlled environment with 16-h light (25℃)/8-h dark (22℃). The aggressive defoliating isolate Vd991 of V. dahliae and F. oxysporum (strain AYF-1) were cultured on potato dextrose agar at 25℃for seven days and then inoculated in Czapek liquid medium. The experiments were performed using conidial suspensions of 107 conidia/mL.
cDNA Isolation and Sequence Analysis of GhPLP2
Total RNA was extracted from Gossypium hirsutum ‘Zhongzhimian 2’ plants using the RNA extraction kit [BIOMED GENE TECHNOLOGY (Beijing) CO., LTD.], and cDNA was synthesized using the manufacturer’s instructions with the FASTQuant cDNA RT Kit (TIANGEN Biotech Co., Ltd). The forward primer 5`-ACGCGTCGACATGGAAAAAAGT ACTGGAAAC-3`( Sal I, underlined restriction site) and reverse primer 5`-ATAGACTAGTTGGGCCAAGCTTGTGCA A-3`(Spe I, underlined restriction site) were designed to amplify the open reading frame (ORF) using the cDNA template. PCR product was transferred into the pMD18-T vector by manufacturer’s protocols (Takara, Dalian, China), and the positive clone was sequenced.
The theoretical isoelectric point (pI) and molecular mass were calculated with ProtParam (http://web.expasy.org/protpa ram/). Multiple amino acid sequence alignment was performed with Clustal Omega (http://www.ebi. ac.uk/Tools/msa/ clustalo/), and the multiple alignment file was shaded with BoxShade (http://www.ch.embnet.org/software/ BOX_form. html). The phylogenetic tree was constructed with the neighbor-joining method using MEGA 7 with bootstrap values from 1000 replicates indicated at the nodes, and motifs were annotated using MEME (http://meme-suite.org/tools /meme) (Bailey and Elkan 1994; Kumar, et al. 2016). The homology model of GhPLP2 was generated using SWISS-MODEL, and three-dimensional models were analyzed and visualized using EzMol, Version1.20(Bordoli, et al. 2009; Reynolds, et al. 2018). The crystal structure of SeMet Patatin (1oxw.1.B) was selected as a template to predict the theoretical model.
Analysis of GhPLP2 Expression Pattern
For abiotic stresses and hormone treatments, two-week-old cotton seedlings were gently uprooted and replanted in Hoagland medium containing 2.5% (w/v) PEG6000,100 µM JA, 2 mM ethrel respectively (Champion, et al. 2009; Pei, et al. 2019). For pathogen treatment, seedling roots were inoculated with V. dahliae and F. oxysporum conidial suspension for 5 min and then transplanted into sterile soil. Control samples were treated with sterile water. The inoculated samples were collected at appropriate time points for RNA extraction. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for GhPLP2 gene was conducted using SYBR Premix Ex Taq (Tli RNaseH Plus) (Takara Bio, Dalian, China) on an ABI 7500 thermocycler (Applied Biosystems, Foster City, CA, United States). The expression levels of GhPLP2 genes were normalized to GhUBQ7 (DQ116441), and relative gene expression was calculated using the 2−ΔΔCt method (Liu, et al. 2018; Livak and Schmittgen 2001). Data were analyzed in Origin 8 and presented as the mean ± standard error (SE) of three independent experiments. The primer sequences were listed in Supplementary Table S1.
Expression and Purification of Recombinant GhPLP2 Protein
GhPLP2 was cloned into the 6× His-Tagged protein expression vector pET-22b(+) (Novagen). The primers were listed in Supplementary Table S1. The recombinant vector was expressed in Escherichia coli BL21 (DE3). Single colonies were cultured at 37℃ in LB broth containing 100 µg/mL ampicillin when OD600 was reaching 0.6 induced with 0.4 mM IPTG for another 10 h at 22℃ with 200r/min. Protein was purified using 6×His-Tagged Protein Purification Kit (CW BIO) following manufacturer’s instructions and detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified protein concentration was determined using the Bradford Protein Assay Kit (Takara Bio, Dalian, China).
GhPLP2 Enzyme Activity Assay Using p-Nitrophenyl palmitate and Phospholipids As Substrates
Lipid acyl hydrolase (LAH) activity assay of GhPLP2 protein was determined using artificial substrate p-Nitrophenyl palmitate (p-NPP) and phospholipids (Camera, et al. 2010; Hong, et al. 2008). Briefly, the reaction mixture comprised 200µL of Tris-HCl (pH 8.0), 10 mM CaCl2, 0.05% (v/v) Triton X-100 and 2 mM p-Nitrophenyl palmitate, 25 µg purified protein were added and incubated at 37 ℃for 30 min. Then 700 µL absolute ethanol was added and centrifuged for 2 min at 7000r/min. The absorbance of p-nitrophenol (NP) liberated from p-Nitrophenyl palmitate was measured at 405 nm.
Phospholipases substrates were 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1,2-distearoyl-sn-glycerol-3-phosphocholine, or L-α-phosphatidylglycerol (Aladdin ®, China). The reaction mixture comprised 50 mM Tris-HCl (pH 8.0), 10 mM CaCl2, 0.05% (v/v) Triton X-100, 500 µg substrate and 10 µg purified protein in a final volume of 600 µl. Reactions were performed at 37 ℃for 1 h and termination by adding 500 µL chloroform/methanol (2:1, v/v). The product fatty acids released from the substrates were separated and analyzed according to the method described before (Camera, et al. 2010). Free fatty acids are methylated in 1 mL 0.5 mol/L KOH-methanol at 60℃ for 2 h. Then 1 mL hexane containing 0.01% butylated hydroxytoluene (BHT) and 0.1 mg methyl nonadecanoate was added. After shaking and standing for stratification, the supernatant containing fatty acid methyl esters (FAMEs) was separated. Quantitation of individual FAMEs was analyzed by gas chromatography (GC) equipped with an Agilent column (USA) (30 m by 0.25 mm, 0.25 um film) and a flame ionization detector (FID).
Generation of Transgenic Plants
The PCR product with Sal I/Spe I restriction site was inserted into a modified pCAMBIA 1300 vector harboring a hygromycin phosphor-transferase (hptII) gene and the green fluorescent protein (GFP) (Wang, et al. 2011). The construct was introduced into Agrobacterium tumefaciens strain GV3101 by freeze-thaw method, and transformation of Arabidopsis was performed by the floral dip method (Clough and Bent 1998). Transgenic Arabidopsis seeds were screened on MS plates containing 25 mg/mL hygromycin B and T3 homozygous lines displaying 100% hygromycin resistance were used for further experiments.
Construction of VIGS Vectors and Agrobacterium-mediated VIGS
The VIGS transient expression methods were followed by Gao (Gao, et al. 2011). The silenced fragments of GhCLA1(Cloroplastos alterados 1) and GhPLP2 were amplified from cotton cDNA and inserted into the TRV:00 vector to generate the TRV: GhCLA1 and TRV: GhPLP2 vectors. Then plasmids of TRV: GhCLA1 and TRV: GhPLP2 were transformed into A. tumefaciens strain GV3101 by heat shock (Dong, et al. 2007). TRV:GhCLA1 plants were used as positive controls. Two weeks after infiltration, when GhCLA1-silenced plants showed clear signs of albinism in leaves, the efficiency of GhPLP2 was evaluated by RT-PCR. GhUBQ7 was amplified as internal control with primers qUBQ-F/R. The primers used in vector construction were listed in Supplementary Table S1
V. dahliae Inoculation and Disease Investigation
Two weeks after VIGS, when true leaves of GhCLA1-silenced cotton plants showed clear signs of albinism, inoculation with V. dahliae (107 conidia/mL) was performed previously reported (Liu, et al. 2018). Plant disease index (DI) was monitored as the following formula according to Wang (Wang, et al. 2020): DI = [Σ(n×the number of seedlings at level n)/(4×the number of total seedlings)]×100, 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). For Arabidopsis plants, four-week-old GhPLP2-transgenic and wild-type (WT) Arabidopsis plants were inoculated with V. dahliae spores as previously described (Gao, et al. 2013). The controls were dipped in sterilized water. Disease index and symptom classification were performed as previously report (Pei, et al. 2019). Data were collected from three independent replicates (n ≥ 30).
V. dahliae recovery assay was conducted at 21 d after infection (Fradin, et al. 2009). Stem section above the cotyledons was taken from inoculated cotton plants, six slices were transferred onto potato dextrose agar supplemented with kanamycin (50 mg/L) after surface sterilized. V. dahliae biomass was quantified following a previously described protocol (Ellendorff, et al. 2009). Inoculated cotton and Arabidopsis plants were harvested at 21 days and 14 days, respectively. DNA was extracted from 100 mg of the fine powder. V. dahliae biomass was determined by qPCR using fungus-specific ITS1-F and V.dahliae-specific ST-Ve1-R primers (Fradin, et al. 2011). The GhUBQ-F/R and AtEF1α-F/R primers were used as reference genes. All primers were listed in Supplementary Table 1.
Analysis of HR In Cotton Induced by PevD1 Elicitor From V. dahliae
Primers are designed to obtain PevD1 fragment from V.dahliae (Supplementary Table S1) and cloned into the pET-28a(+) vector (Novagen). The recombinant vector was expressed in E. coli BL21 (DE3), and protein was induced with 0.1 mM IPTG for 10 h at 22℃ with 200r/min. Protein purification and concentration assay performed as described above. The treatment of PevD1elicitor was performed as Li (Li, et al. 2019). Expression of HR marker genes GhHSR203 and GhHIN1 triggered by PevD1were detected in cotton leaves at 24h after infection. Callose deposition was stained by 0.1% aniline blue as previously (Wang, et al. 2018). Images were observed using FLUOVIEW FV1000 confocal laser scanning microscope (OLYPUS, Tokyo, Japan). The level of H2O2 was measured by freshly FOX reagent [Fe(NH4)2•(SO4)2(250 mM), xylenol orange (100 µM), sorbitol (100 µM), H2SO4 (25 mM), 1% ethanol] according to Pei (Pei, et al. 2019).
Fatty Acid Profile Analysis and Hormone Quantitation
Four-week-old Arabidopsis and two-week GhPLP21-silenced cotton plants after VIGS were used to analyze the fatty acid profile and hormone quantification. Hormone quantitation was detected as previously reported (Li, et al. 2020). Fresh samples were ground into fine powder in liquid nitrogen. 100 mg powder was extracted with 1 mL of 80% methanol-water (containing 1% formic acid) using ultrasound at 4°C for 10 min and centrifuged at 4°C at 12,000 rpm for 10 min. The supernatant was transferred to a 2mL centrifuge tube containing 50mg primary secondary amine (CNW Technologies GmbH, Germany), dried with nitrogen, and added 100 µL 80% methanol-water (containing 1% formic acid). The samples were analyzed by Agilent 6410B Triple Quadrupole HPLC − MS/MS (Agilent Technologies, USA), which is equipped with an HPLC reverse phase C18 column (Athena C18-WP 2.1 × 50 mm, 3 µm). The flow rate was 0.3 mL/min. Methanol and ultrapure water were used as mobile phases A and B, respectively. The isocratic elution was carried out for 5 min at a 65(A):35(B) ratio. MS was performed using the multiple reaction monitoring modes and negative electrospray ionization. All the parameters were performed according to Li (Li, et al. 2020).
For fatty acids profile analysis, plant samples were dried to a constant weight under 90 ℃, and 50 mg of dry powder was weighed. Methyl esterification of fatty acids and analysis were performed as described above. GC parameters were as follows: 180°C for 10 min followed by a ramp to 190°C at 1°C min− 1, holding 190°C for 3 min and then heating up to 220°C with a gradient at 4°C min− 1, final temperature was maintained for 3 min.
Gene Expression Profiling of Jasmonic Acid Signaling Pathway In Arabidopsis and Cotton Plants
Total RNA was obtained from non-inoculated plants with an RNA extraction kit [BIOMED GENE TECHNOLOGY (Beijing) CO., LTD.]. The synthesis of cDNA and qRT-PCR assays was conducted as mentioned above. AtEF1a and GhUBQ7 were employed as internal standards genes in Arabidopsis and cotton, respectively. Relative gene expression was calculated using the 2−ΔΔCt method. Data were presented as the mean ± standard error (SE) of three independent experiments. The primer sequences are listed in Supplementary Table S1.
Data are obtained from three independent replicates per treatment and presented as mean ± standard error. Significant differences between groups were analyzed by ANOVA using statistical software IBM SPSS statistics 20 followed by Student's t-test. Asterisks indicates a significant difference compared with control (*P < 0.05, **P < 0.01).