GhPLP2 Positively Regulates Cotton Resistance to Verticillium Wilt by Modulating Fatty Acid Accumulation and Jasmonic Acid Signaling Pathway

Patatin-like proteins (PLPs) have non-specific lipid acyl hydrolysis (LAH) activity, which can hydrolyze membrane lipids into fatty acids and lysophospholipids. The vital role of PLPs in plant growth and abiotic stress has been well documented. However, the function of PLPs in plant defense responses against pathogens is still poorly understood. Here, we isolated and identified a novel cotton (Gossypium hirsutum) PLP gene GhPLP2. The expression of GhPLP2 was induced upon treatment with Verticillium dahliae, the signaling molecules jasmonic acid (JA) and ethylene (ETH) in cotton plants. Subcellular localization revealed that GhPLP2 was localized to the plasma membrane. GhPLP2-silenced cotton plants were more susceptible to infection by V. dahliae, while the overexpression of GhPLP2 in Arabidopsis enhanced its resistance to V. dahliae, which was apparent as mild symptoms, and a decrease in the disease index and fungal biomass. The hypersensitive response, deposition of callose, and H2O2 accumulation triggered by V. dahliae elicitor were reduced in GhPLP2-silenced cotton plants. The overexpression of GhPLP2 in Arabidopsis resulted in the accumulation of linoleic acid (LA, 18:2) and α-linolenic acid (ALA, 18:3) and facilitated the biosynthesis of JA and JA-mediated defensive responses. GhPLP2 silencing in cotton plants consistently reduced the accumulation of linoleic acid (LA, 18:2) and α-linolenic acid (ALA, 18:3) and suppressed the biosynthesis of JA and the defensive responses mediated by JA. These results indicate that GhPLP2 is involved in the resistance of cotton to V. dahliae by maintaining fatty acid metabolism pools for JA biosynthesis and activating the JA signaling pathway.


INTRODUCTION
Lipids in plants play a critical role in cell membrane components, the storage of sustainable energy, and signaling transduction in response to biotic and abiotic stress (Wang, 2004;Li et al., 2016;Lim et al., 2017). The accumulation of products of hydrolysis owing to the activity of lipid acyl hydrolase (LAH) in lipid metabolism affects pathogenesis and the mechanisms of resistance in plant-microbe interactions (Shah, 2005). As the key hydrolytic products of membrane lipids, fatty acids (FAs) participate in modulating the accumulation of endogenous nitric oxide and the biosynthesis of azelaic acid (AzA) which contribute to the development of systemic acquired resistance (SAR), production of reactive oxygen species (ROS), and subsequent defense responses in plants (Mandal et al., 2012;Yu et al., 2013;Wang et al., 2014). Oxidation and further conversions of polyunsaturated FAs by α-dioxygenase (α-DOX) and lipoxygenases (LOXs) generate various products that are collectively termed oxylipins (Savchenko et al., 2014;Cook et al., 2021). Jasmonates (JAs) constitute a crucial member of oxylipins, many of which are involved in plant defenses against pathogens, nematodes, and herbivores (Campos et al., 2014;Strnad, 2016, 2017). Moreover, oxylipins are related to the initiation of hypersensitive response (HR) in the interactions between plants and incompatible bacterial or fungal elicitors (Coll et al., 2011;García-Marcos et al., 2013;Sun et al., 2014).
Patatin proteins constitute a significant family of LAHs that possess non-specific LAH activity toward several membrane lipids; they were first identified and isolated as vacuolar storage proteins in potato tubers (Hendriks et al., 1991;Scherer et al., 2010). They generate free fatty acids and lysophospholipids by catalyzing the non-specific hydrolysis of membrane lipids, including phospholipids, glycolipids, sulfolipids, and mono-and diacylglycerols . Various functions of patatin proteins have been demonstrated in responses to auxin, the production of cellulose, salt tolerance, drought response, and plant growth Yang et al., 2012;Dong et al., 2014;. More importantly, patatin and patatinlike proteins (PLPs) play an essential role in defense response to various pathogens. In Arabidopsis, patatin-related phospholipase I (AtPLAI) with the activity to hydrolyze acyls plays a role in maintaining the homeostatic pool of basal free fatty acids and basal jasmonic acid production and conferring resistance to infection with Botrytis cinerea (Yang et al., 2007). The Arabidopsis patatin-like protein 2 (AtPLP2), a pathogen-induced patatinlike LAH, provides fatty acids precursors for the biosynthesis of oxylipins and differentially affects the resistance to pathogens with distinct lifestyles (La Camera et al., 2009). Overexpression of Arabidopsis patatin-related phospholipase IIIα (AtpPLAIIIα) enhanced resistance to turnip crinkle virus (TCV), which was accompanied by a higher ratio of salicylic acid to jasmonic acid, and increased the level of expression of defense gene PR1 (Jang et al., 2020). The silencing of pepper (Capsicum annuum) CaPLP1 compromised the defense responses to avirulent Xanthomonas campestris pv. vesicatoria (Xcv) (Kim et al., 2014). Several PLPs are involved in releasing FAs from lipid membranes and serving as biosynthetic precursors of jasmonic acid synthesis in the grapevine Vitis vinifera cv. Regent (resistant cultivars) after infection with Plasmopara viticola (Laureano et al., 2018). Furthermore, three lipid acyl hydrolases (NtPATs) were rapidly induced in tobacco during its HR response to the Tobacco mosaic virus (TMV) (Dhondt et al., 2000).
Cotton (Gossypium spp.) is widely cultivated around the world because of its significant economic value to the textile industry (Gao et al., 2013). However, the yield and quality of cotton can be seriously affected by Verticillium wilt, caused by the soil-borne pathogen Verticillium dahliae Kleb (Gong et al., 2017;Dhar et al., 2020). V. dahliae is a hemibiotrophic fungus that undergoes biotrophic and necrotrophic phases during the process of infecting the host (Pantelides et al., 2010;Lo Presti et al., 2015;Zhang L. et al., 2017). It colonizes the plants through young roots, the sites of lateral root formation or puncture wounds to the xylem and leads to the browning of vasculature, defoliation, and wilting (Chen et al., 2016;Shaban et al., 2018). This pathogen can infect more than 200 species of dicotyledonous plants and is particularly difficult to control by fungicides because the fungus resides in the woody vascular tissues (Fradin and Thomma, 2006;Mo et al., 2016). Moreover, the resting structures microsclerotia of V. dahliae can survive in the soil for up to 14 years even without a host (Short et al., 2015). The cultivation of resistant cotton varieties has proven to be an effective strategy to control Verticillium wilt (Gayoso et al., 2010). The identification and isolation of candidate gene involved in defense against Verticillium wilt are essential to study the mechanism of resistance to this disease. Therefore, studying the function and mechanism of patatin proteins in the resistance of cotton to infection by V. dahliae may provide a new strategy for controlling Verticillium wilt.
Jasmonic acid (JA) is a vital lipid-derived phytohormone that confers the host resistance to biotrophic and hemibiotrophic pathogens (Ghorbel et al., 2021). Previous genetic evidence indicates that the JA signaling pathway is integral to the resistance of cotton to V. dahliae He et al., 2016;Dhar et al., 2020). The homeodomain transcription factor HDTF1 identified from cotton plants infected with V. dahliae is a negative regulator of the JA signaling pathway. HDTF1 silencing resulted in the activation of the JA signaling pathway and enhanced the resistance to V. dahliae and B. cinerea (Gao et al., 2016). The GhJAZ2 protein can interact with GhbHLH171 and inhibit its transcriptional activity, resulting in an impaired JA-mediated defense response against V. dahliae (He et al., 2018). The calcium-dependent protein kinase GhCPK33 from upland cotton (Gossypium hirsutum) phosphorylates GhOPR3, which leads to the enhanced instability of GhOPR3, which consequently limits the biosynthesis of JA and the subsequent resistance to V. dahliae (Hu et al., 2018). Silencing of the BEL1-Like transcription factor GhBLH7-D06 enhanced the resistance of cotton to Verticillium wilt by activating the biosynthesis of lignin and the JA signaling pathway (Ma et al., 2020). The constitutive expression of the GSK3-like kinase BIN2 (Brassinosteroid insensitive 2) attenuated the resistance of plants to V. dahliae by regulating the endogenous content of JA and the expression of JA-responsive marker genes (Song et al., 2021).
In this study, we report the identification and characterization of PLP gene GhPLP2 in cotton (G. hirsutum) that is induced by V. dahliae. The patterns of transcriptional expression of GhPLP2 were studied in response to biotic and abiotic stress. Sequence analyses showed that GhPLP2 had conserved catalytic dyad residues, which are critical for the LAH activity of patatins. The recombinant GhPLP2 protein further confirmed its in vitro LAH activity. Reduced HR response during the V. dahliae elicitor infection was observed in GhPLP2-silenced plants, suggesting the role of GhPLP2 in HR-like cell death signaling. The positive role of GhPLP2 in regulating the resistance of cotton to V. dahliae was examined by virus-induced gene silencing (VIGS) technology in cotton plants and overexpression in Arabidopsis. Further experiments demonstrated that GhPLP2 positively regulates the resistance of cotton to V. dahliae by mediating the metabolism of fatty acids and activation of the JA signaling pathway. Taken together, this research reveals the molecular mechanism of GhPLP2 in the defense of cotton against V. dahliae and provides new insights to improve the resistance of cotton to Verticillium wilt.

Plant Growth and Culturing of Verticillium dahliae
Seeds of the upland cotton (G. hirsutum) resistant cultivar Zhongzhimian 2 (original strain no. GK44) were provided by the Cotton Research Institute, Chinese Academy of Agricultural Sciences (Anyang, Henan). 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 3 days at 4 • C in Murashige-Skoog (MS) culture medium, both the wild-type (Columbia-0) and transgenic Arabidopsis plants were grown in a mixture of nutrient soil and vermiculite (1:1, w/w) under a controlled environment with 16 h light (25 • C)/8-h dark (22 • C). The aggressive defoliating isolate Vd991 of V. dahliae was cultured on potato dextrose agar (PDA) at 25 • C for 7 days and then inoculated in Czapek liquid media. The experiments were performed using conidial suspensions of 1 × 10 7 conidia/mL. Briefly, the cotton plants (2 weeks after VIGS) and Arabidopsis plants (4-week-old) were gently uprooted and the roots were dipped in conidial suspensions (1 × 10 7 conidia/mL) for 5 min. Then they were replanted in a mixture of nutrient soil and vermiculite.

Isolation of GhPLP2 cDNA
Total RNA was extracted using an RNA extraction kit (Biomed Gene Technology Co., Ltd., Wuhan, China), and the cDNA was synthesized using the manufacturer's instructions with a FASTQuant cDNA RT Kit (Tiangen Biotech Co., Ltd., Beijing, China). The forward primer 5 -ACGCGTCGACATGGAAAAAAGTACTGGAAAC-3 (SalI, underlined restriction site) and the reverse primer 5 -ATAGACTAGTTGGGCCAAGCTTGTGCAA-3 (SpeI, underlined restriction site) were designed according to the GhPLP2 sequence (Gene ID: Gh_D02G0923) from cotton protein databases (G. hirsutum, NAU assembly) in the Cotton Functional Genomics Database 1 to amplify the open reading frame (ORF) using the cDNA template. The PCR product was transferred into the pMD18-T vector according to the manufacturer's instructions (TaKaRa, Dalian, China), and the positive clone was sequenced.

Bioinformatic Analyses
The theoretical isoelectric point (pI) and molecular mass were calculated with ProtParam. 2 All the sequences were retrieved from the website of the National Center for Biotechnology Information database, 3 and the accession numbers of the patatin proteins are listed in Supplementary Table 1. Multiple amino acid sequence alignments were performed using Clustal Omega, 4 and the multiple alignment file was shaded with BoxShade. 5 A phylogenetic tree was constructed with the neighbor-joining method using MEGA 7 with bootstrap values from 1,000 replicates indicated at the nodes, and the motifs were annotated using MEME 6 (Bailey et al., 2009;Kumar et al., 2016). The homology model of GhPLP2 was generated using SWISS-MODEL, and three-dimensional models were analyzed and visualized using EzMol, Version 1.20 (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.
vector was expressed in Escherichia coli BL21 (DE3). Single colonies were cultured at 37 • C in LB broth that contained 100 µg/mL ampicillin. When the OD 600 reached 0.6, the culture was induced with 0.4 mM IPTG for another 12 h at 22 • C at 200 rpm. The protein was purified using a 6 × His-Tagged Protein Purification Kit (CWbio, Beijing, China) following the manufacturer's instructions and detected using SDS-PAGE. The concentration of purified protein was determined using a Bradford Protein Assay Kit (TaKaRa, Dalian, China).

GhPLP2 Enzyme Activity Assay Using the Substrates p-Nitrophenyl Palmitate and Phospholipids
The LAH activity of the GhPLP2 protein was assayed using the artificial substrate p-nitrophenyl palmitate (p-NPP) and phospholipids (Hong et al., 2008). Briefly, the reaction mixture was composed of 200 µL of Tris-HCl (pH 8.0), 10 mM CaCl 2 , 0.05% (v/v) Triton X-100, and 2 mM p-nitrophenyl palmitate to which 25 µg of purified protein was added and incubated at 37 • C for 30 min. A volume of 700 µL of absolute ethanol was added and centrifuged for 2 min at 7,000 rpm. The absorbance of p-nitrophenol (NP) liberated from p-NPP was measured at 405 nm.

Subcellular Localization and Generation of Transgenic Plants
The PCR product with a SalI/SpeI restriction site was inserted into a modified pCAMBIA 1300 vector that harbored hygromycin phosphor-transferase (hptII) and green fluorescent protein (GFP) under the control of the CaMV 35S promoter (Supplementary Figure 1; Wang et al., 2011). By particle bombardment (PDS-1000; Bio-Rad, United States), the CaMV 35S:GhPLP2-GFP and 35S:GFP empty vector were transformed into onion epidermal cells. The transformed cells were incubated on the MS medium (with 20 g/L sucrose) in the dark for 18-24 h at 22 • C.
For the generation of GhPLP2-transgenic plants, the construct was introduced into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method (Jyothishwaran et al., 2007), and Arabidopsis was transformed by the floral dip method (Clough and Bent, 1998). Transgenic Arabidopsis seeds were screened on MS plates (with 20 g/L sucrose) that contained 25 mg/mL hygromycin B and T3 homozygous lines that displayed 100% hygromycin resistance were used for additional experiments.

Construction of Virus-Induced Gene Silencing Vectors and Agrobacterium-Mediated Virus-Induced Gene Silencing
The virus-induced gene silencing (VIGS) transient expression methods were previously described by 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 the GhCLA1silenced plants showed clear signs of albinism in the leaves, the efficiency of GhPLP2 was evaluated by quantitative real-time reverse transcriptase-PCR (qRT-PCR). GhUBQ7 (DQ116441) was amplified as an internal control with the primers qUBQ7-F/R. All the primers are listed in Supplementary Table 2.

Verticillium dahliae Inoculation and Disease Investigation
Two weeks after VIGS, when the true leaves of GhCLA1-silenced cotton plants showed clear signs of albinism, the plants were inoculated with V. dahliae (1 × 10 7 conidia/mL) as previously described (Liu et al., 2018). The plant disease index (DI) was monitored using the following formula as described by Wang et al. (2020): DI = [ (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 inoculation with V. dahliae (level 0, 1, 2, 3, 4). For Arabidopsis plants, 4-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. The disease index and classification of symptoms were determined as previously reported . Data were collected from three independent technical replicates (n ≥ 30).
The V. dahliae recovery assay was conducted and modified as described by Fradin et al. (2009), and the antibiotic in the PDA is kanamycin in this study. The stem section above the cotyledons was taken from cotton plants 3 weeks after inoculation with V. dahliae. Six slices were sterilized with 75% ethanol and 10% sodium hypochlorite solution and transferred onto PDA supplemented with kanamycin (50 mg/L) after the slices were washed three times with sterile water. The V. dahliae biomass was quantified as previously described (Ellendorff et al., 2009). Inoculated cotton (first internode) and Arabidopsis plants (whole plants) were harvested at 21 days post-inoculation (dpi) and 14 dpi, respectively. DNA was extracted from 100 mg of the fine powder. V. dahliae biomass was determined by qRT-PCR using fungus-specific ITS1-F (Liguori et al., 2007) and V. dahliae-specific ST-Ve1-R primers (Fradin et al., 2011).
GhUBQ7-F/R (DQ116441) was used as a reference gene in the cotton plants, and AtEF1α-F/R (AT5G60390) was used as a reference gene in the Arabidopsis plants. All the primers are listed in Supplementary Table 2.

Analysis of Hypersensitive Response in Cotton Induced by the PevD1 Elicitor From Verticillium dahliae
The primers were designed to obtain the PevD1 fragment from V. dahliae (Supplementary Table 2), which was then cloned into the pET-28a (+) vector. The recombinant vector was expressed in E. coli BL21 (DE3), and the protein was induced with 0.1 mM IPTG for 10 h at 22 • C while shaken at 200 rpm. The protein was purified using a 6 × His-Tagged Protein Purification Kit (CWbio, Beijing, China) following the manufacturer's instructions and detected using SDS-PAGE. The concentration of purified protein was determined using a Bradford Protein Assay Kit (TaKaRa, Dalian, China). The treatment with PevD1 elicitor was performed as described by . The expression of HR marker genes GhHSR203 and GhHIN1 triggered by PevD1were detected in cotton leaves at 24 h after infection. Callose deposition was stained using 0.1% aniline blue as previously described . Images were observed using FLUOVIEW FV1000 on a confocal laser scanning microscope (Olympus, Tokyo, Japan). The level of H 2 O 2 was measured by freshly made FOX reagent (Fe[NH 4 ] 2 •[SO 4 ] 2 ) (250 mM), xylenol orange (100 µM), sorbitol (100 µM), and H 2 SO 4 (25 mM), and 1% ethanol as described by Pei et al. (2019).

Fatty Acids Analysis and the Quantification of Hormones
Four-week-old Arabidopsis and 2-week GhPLP2-silenced cotton plants after VIGS were used to analyze the composition of fatty acids and quantify the hormones. The hormones were quantified as previously described (Li D. et al., 2020). Fresh samples were ground into fine powder in liquid nitrogen. A total of 100 mg powder was extracted with 1 mL of 80% aqueous methanol that contained 1% formic acid using an ultrasonic treatment at 4 • C for 10 min and centrifuged at 12,000 rpm at 4 • C for 10 min. The supernatant was transferred into a 2 mL centrifuge tube that contained 50 mg of primary secondary amine (CNW Technologies GmbH, Dusseldorf, Germany), dried with nitrogen, and added to 100 µL of 80% aqueous methanol that contained 1% formic acid. The samples were analyzed by an Agilent 6410B Triple Quadrupole HPLC-MS/MS (Agilent Technologies), 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 the mobile phases A and B, respectively. Isocratic elution was conducted 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 as described by Li D. et al. (2020).
The fatty acid composition was analyzed by gas chromatography (GC) on Agilent 7890A (Agilent Technologies, Santa Clara, CA, United States) with a flame ionization detector (FID). Fatty acid methyl esterification was performed as described by Liu et al. (2019) with slight modification. The plant samples were dried at 90 • C to a constant weight, and 50 mg of dry powder was weighed. Free fatty acids were methylated in 1 mL 0.5 mol/L KOH-methanol at 60 • C for 2 h. Next, 1 mL of hexane that contained 0.01% butylated hydroxytoluene (BHT) and 0.1 mg methyl non-adecanoate was added. After shaking and standing for stratification, the supernatant that contained the FAMEs was separated. The GC parameters were as follows: 180 • C for 10 min followed by a ramp to 190 • C at 1 • C/min, a hold at 190 • C for 3 min, and then heating up to 220 • C with a gradient at 4 • C/min. The final temperature was maintained for 3 min.

Plant Drought Treatment
Arabidopsis drought treatment was carried out as previously described . Briefly, water was withheld from 3week-old Arabidopsis plants for 16 days, followed by watering for 2 days from the bottom. All the Arabidopsis plants were grown in a controlled environment with 16 h light (25 • C)/8-h dark (22 • C) and a relative humidity at 80%.

Gene Expression Analysis by Quantitative Real-Time PCR
Total RNA was obtained from the plant samples with an RNA extraction kit with DNase I (Biomed Gene Technology, Co., Ltd., Beijing, China). The cDNA was synthesized using a PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa Bio, Dalian, China). qRT-PCR was performed using SYBR Premix Ex Taq (Tli RNaseH Plus) (TaKaRa, Dalian, China) on an ABI 7500 thermocycler (Applied Biosystems, Foster City, CA, United States). AtEF1a (AT5G60390) and GhUBQ7 (DQ116441) were utilized as internal standard genes in Arabidopsis and cotton, respectively. The relative gene expression was calculated using the 2 − Ct method (Livak and Schmittgen, 2001). The melting curves for each gene studied (with a no template control) are provided in Supplementary Figures 2, 3

Data Analysis
The data were obtained from three independent technical or biological replicates per treatment and presented as the mean ± standard error. The statistical analyses were performed by Student's t-test. Asterisks indicates a significant difference between the groups ( * P < 0.05, * * P < 0.01).

Identification and Sequence Analysis of GhPLP2
From the transcriptome analysis of the cotton cultivar Zhongzhimian 2 that is resistant to infection by V. dahliae Vd991, the PLP gene GhPLP2 was highly induced when the plants were infected with V. dahliae. We isolated the full-length clone of GhPLP2 that consists of a 68 bp 5 untranslated region (5  UTR that encoded a 405 amino acid protein with a theoretical pI of 8.21 and a molecular weight of 44.44 kDa. The GhPLP2 protein contains the conserved serine (S) hydrolase motif GXSXG at residues 63-67 and a conserved aspartic acid (D) residue at 214 within the patatin domain (residues 21-221) (Supplementary  Figure 4). The catalytic dyad of S and D residues is critical for the LAH activity of patatin (Rydel et al., 2003;Rietz et al., 2010). Using SeMet patatin, an isozyme of patatin 17 from Solanum cardiophyllum as the template, the homology modeling results indicate that the Ser-Asp catalytic dyad is essential for the LAH activity of GhPLP2 (Supplementary Figure 5). GhPLP2 lacks the transmembrane domain or signal peptide. To study the possible role of GhPLP2 protein in cotton plants, a phylogenetic analysis was generated with homologous patatin proteins with precise functions that had been previously reported from other species. The phylogenetic analysis showed that plant patatins were divided into three groups, and GhPLP2 is located in group II that contains NtPat1 (Cacas et al., 2005), GhPat1 (Cacas et al., 2009), CaPLP1 (Kim et al., 2014), and AtPLP2 (La Camera et al., 2009; Figure 1A). The patatin homologs were divided into three groups based on the conserved motif alignments, which are consistent with the phylogenetic analysis result ( Figure 1B).
Group II members have the canonical S-D dyad esterase motif constituted by GxSxG and DGG/A in patatin catalytic centers, a conserved anion binding element DGGGxxG and the proline motif APP. GhPLP2 is a patatin group II member with the conserved catalytic dyad residues.

Lipid Acyl Hydrolase Activity Assay of GhPLP2
The LAH activity of patatin proteins can hydrolyze membrane lipids into free fatty acids and lysophosphatidic acid, which lead to a series of signaling pathways involved in growth development and defense responses (Ryu, 2004). To understand the role of GhPLP2 in more detail, the LAH activity of the recombinant GhPLP2 protein was studied. The recombinant protein GhPLP2 was produced and purified from E. coli BL21 (DE3). The GhPLP2 protein was induced with 0.4 mM IPTG at 22 • C and as early as 1 h after induction (Figure 2A). The GhPLP2 fusion protein was purified using Ni columns, and the elution fractions were confirmed by SDS-PAGE ( Figure 2B). When p-NPP was used as the substrate, the His-GhPLP2 protein exhibited high LAH activity (97 nmol min −1 mg −1 ), and the vector control was totally deficient in enzyme activity ( Figure 2C). In addition, when the phospholipids PC, PE, and PG were used as substrates, the release of free fatty acids was quantified. The purified GhPLP2 protein released fatty acids from these phospholipids and exhibited variant enzyme activities in different phospholipid substrates ( Figure 2D). The results indicate that GhPLP2 may function as a LAH to release free fatty acids in plants.

Subcellular Localization of the GhPLP2 Protein
To determine the subcellular localization of GhPLP2 protein, the GFP signals of GhPLP2-GFP fusion protein and 35S:GFP empty vector were monitored by Nikon A1R SI Confocal (Nikon, Japan). The GFP signal of 35S:GFP empty vector was diffused throughout the cell (Figures 3A-C), whereas the GhPLP2-GFP fusion protein was found to accumulate exclusively in the cell wall or plasma membrane (Figures 3D-F). To further clarify the location of GhPLP2, the onion epidermal cells were treated with 0.8 M mannitol for 10 min. After plasmolysis, the GFP signals of GhPLP2-GFP fusion protein was found in the plasma membrane (Figures 3G-I). These results revealed that GhPLP2 was localized to the plasma membrane.

Induction of GhPLP2 by Various Stresses
Expression of the GhPLP2 gene in cotton was examined by qRT-PCR. GhPLP2 was the most abundant in cotton roots (Figure 4A), which are the first physical barrier in cotton plants against infection by V. dahliae. The change in expression of GhPLP2 in response to various stresses was investigated. The expression of GhPLP2 was significantly increased by 9.4-and 7.1-fold at 0.5 h and 7 days after inoculation with V. dahliae, respectively ( Figure 4B). We investigated the expression of GhPLP2 after treatment with the defense-related signaling molecules methyl jasmonate and ethylene in more detail. The expression of GhPLP2 was significantly higher at 3 h by 6.2-fold, and another 6.0-fold peak appeared at 24 h after treatment with JA ( Figure 4C). In contrast, ethylene increased the expression of GhPLP2 at 0.5 h, with a maximum level of 4.7-fold observed at 3 h ( Figure 4C). The treatment with 2.5% PEG 6000 (w/v) markedly increased the gene expression at 1 and 3 h with 5.6-and 3.7-fold, respectively ( Figure 4D).

Enhanced Disease Susceptibility of GhPLP2-Silenced Cotton Plants to Verticillium dahliae
The GhPLP2 gene was silenced by VIGS in cotton plants to clarify its function by Gao et al. (2013). The cotton gene GhCLA1, which is involved in chloroplast development, was used as a positive control (Gao et al., 2011). To assess its gene silencing efficiency, the expression of GhPLP2 was monitored by semi-quantitative PCR and qRT-PCR in TRV:00 and TRV:GhPLP2 cotton true leaves after 2 weeks of VIGS ( Figure 5G and Supplementary Figure 6). The results indicated that the expression of GhPLP2 was effectively reduced in GhPLP2-silenced cotton plants.
To analyze the involvement of GhPLP2 in the interaction between cotton and V. dahliae, cotton plants were inoculated with this pathogen. After inoculation, typical disease symptoms appeared at 10 days in GhPLP2-silenced cotton. At 14 days, leaf chlorosis and necrosis were more severe in the GhPLP2-silenced plants (Figures 5A,B). Dark and necrotic vascular bundles of the dissected stems were more apparent in GhPLP2-silenced plants at 21 days (Figures 5C,D). The fungal renewal cultivation showed that more fungi were recovered from GhPLP2-silenced plants compared with the control (Figures 5E,F). The plant disease index of the silenced plants was higher than that of the control plants ( Figure 5H). In addition, the fungal biomass of the stems from the GhPLP2-silenced plants were higher than that in the control plants determined by qRT-PCR analysis ( Figure 5I). These results suggest that the silencing of GhPLP2 attenuates the resistance of cotton to V. dahliae.

Silencing of GhPLP2 Compromises the Hypersensitive Response Symptoms Triggered by Verticillium dahliae Elicitor PevD1
The HR is an efficient and immediate resistance reaction during the attack of pathogens, which rapidly causes cell death around the attempted infection site that restricts the spread of pathogens (Vailleau et al., 2002). During the HR, plant immune responses, including the accumulation of H 2 O 2 , deposition of callose, and the expression of HR-related marker genes were rapidly induced (Hamada et al., 2005;Simon et al., 2010;Rossi et al., 2011). PLPs have been shown to participate in HR in response to infection by the Gram-negative bacterial pathogen Xanthomonas campestris pv. vesicatoria in pepper plants and the response to Tobacco mosaic virus (TMV) in tobacco plants (Dhondt et al., 2000;Choi and Hwang, 2011;Kim et al., 2014). Therefore, GhPLP2 may regulate the resistance to V. dahliae by mediating an HR induced by V. dahliae elicitors. To test our hypothesis, we expressed and purified PevD1 from V. dahliae (Supplementary Figure 7), which triggered HR and resistance responses in cotton, Arabidopsis, and tobacco (Wang et al., 2012;Bu et al., 2014;Liu et al., 2016). The HR-like cell death was initiated at 24 h after injection with PevD1, while it was significantly reduced in GhPLP2-silenced leaves ( Figure 6A). Moreover, PevD1 triggered the deposition of callose, and the expression of HR marker genes GhHIN1 and GhHSR203 were compromised in the GhPLP2-silenced leaves at 24 h after the infiltration (Figures 6B-D). In addition, the silencing of GhPLP2 significantly compromised the production of H 2 O 2 induced by PevD1 (Figure 6E).

Overexpression of GhPLP2 in Arabidopsis Confers Enhanced Resistance to Verticillium dahliae
To further evaluate the role of GhPLP2 in response to V. dahliae, we examined the resistance of wild-type (WT) and transgenic Arabidopsis seedlings to V. dahliae infection. Three homozygous transgenic (T3 generation) lines with the highest levels of (D) Expression of GhPLP2 after treatment with 2.5% PEG 6000 (w/v). Data were obtained from three independent biological replicates and presented as the mean ± standard error. Asterisks indicate a significant difference compared with the control. (*P < 0.05, **P < 0.01, Student's t-test). expression of GhPLP2 (Lines 1, 4, and 8) were selected for additional experiments (Supplementary Figure 8).
Four-week-old transgenic and WT plants were infected with V. dahliae spores by the root dipping method . After inoculation, the WT plants showed more serious wilting, yellowish coloring and necrosis compared with the transgenic plants at 14 dpi ( Figure 7A); this was consistent with the disease index investigation (Figure 7B). The fungal biomass of the WT plants was remarkably high compared with that on the transgenic plants as determined by qRT-PCR analysis ( Figure 7C). These results indicate that the overexpression of GhPLP2 in Arabidopsis plants confers enhanced resistance to V. dahliae.

GhPLP2 Involved in the Accumulation of Fatty Acids and Jasmonic Acid Signaling Pathway
Free fatty acids (FFAs) are among the hydrolyzates of membrane lipids owing to PLPs with LAH activity (Rivas and Heitz, 2014). The endogenous LAH activity of crude proteins from different genotypes plants was assayed using p-NPP as the substrate (Lin et al., 2011). The LAH activity of GhPLP2overexpressed Arabidopsis lines was higher than that of the WT plants, whereas the LAH activity decreased in TRV:GhPLP2 cotton plants compared with the TRV:00 plants ( Supplementary   Figure 9). To further assess the metabolic differences between different genotypes of plants, the composition of fatty acids was examined in Arabidopsis and cotton plants 3 days after the inoculation of V. dahliae and water (mock) (Figure 8). Figure 8A, the analyses of the composition of fatty acids indicated that the levels of linoleic acid (LA, 18:2) and α-linolenic acid (ALA, 18:3) were higher in GhPLP2overexpressed Arabidopsis plants than in the WT plants under treatment with water. After inoculation with V. dahliae, the content of different types of fatty acids increased in both genotypes of Arabidopsis plants, and the increase in GhPLP2overexpressed Arabidopsis plants was more apparent compared with the WT (Figure 8A). Moreover, GhPLP2-silenced cotton plants exhibited a significant reduction in their contents of LA and ALA regardless of the treatment with V. dahliae (Figure 8B). In addition, the content of palmitic acid (16:0) and stearic acid (18:0) also increased significantly after the inoculation of cotton plants with V. dahliae.

As shown in
It has been reported that the oxidation and further conversion of C18 polyunsaturated FAs with lipoxygenases (LOXs) may generate the production of JA (Walley et al., 2013;Sun et al., 2014;Fu et al., 2015). We then investigated the JA contents in GhPLP2-overexpressed Arabidopsis and GhPLP2-silenced cotton plants. The contents of JA in GhPLP2-overexpressed Arabidopsis lines 4 and 8 were 84.97 and 101.99% higher than that of the WT, respectively, when they had not been inoculated with V. dahliae. After inoculation with V. dahliae, the JA contents of GhPLP2-overexpressed Arabidopsis lines 4 and 8 were 51.63 and 74.42% higher than that of the WT, respectively ( Figure 8C). The levels of JA in the TRV:GhPLP2 cotton plants were 41.21 and 33.83% lower than that of the TRV:00 cotton plants under mock and V. dahliae inoculations, respectively ( Figure 8D).
We also conducted qRT-PCR to detect the expression of a series of the JA-synthesis and -signaling genes from Arabidopsis and cotton plants, namely AOS, LOX2, OPR3, and PDF1.2. All of these genes were highly induced by infection with V. dahliae in both Arabidopsis and cotton at 3 dpi (Figures 8E,F). Consistent with the contents of JA, we found that the expression of these genes was significantly upregulated in GhPLP2-overexpressed Arabidopsis plants compared with the WT and downregulated in TRV:GhPLP2 cotton plants relative to the TRV:00 plants in both treatments (Figures 8E,F). Taken together, the results showed that GhPLP2 contributes to the accumulation of fatty acids and is involved in the JA signaling pathway in both Arabidopsis and cotton plants.

GhPLP2 Overexpression Has No Effect on Arabidopsis Tolerance to Drought
Based on our observation that PEG 6000 induced the expression of GhPLP2, we investigated the drought tolerance of GhPLP2-overexpressed Arabidopsis plants. However, the GhPLP2-overexpressed Arabidopsis did not show the distinct phenotype of drought sensitivity compared with the WT plants (Supplementary Figure 10).

DISCUSSION
Patatin-like proteins (PLPs) are a significant family of lipases with LAH activity and play an essential role in lipid metabolism during the immunity that arises from plant defenses (Rivas and Heitz, 2014). In this study, we identified the novel PLP gene GhPLP2 from cotton and demonstrated its role in fatty acid metabolism and the JA signaling pathway that confers resistance to V. dahliae in cotton.
PLPs have a conserved S-D catalytic dyad that is composed of serine of esterase motifs (GxSxG) and a central aspartic acid, which is essential for the LAH activity (Rydel et al., 2003). These residues were also identified in human Ca 2+ -independent phospholipase A2 (iPLA2s) and many microbial proteins (Burke and Dennis, 2009;Mansfeld, 2009;Heller et al., 2018). Similar to other patatin proteins in plants, GhPLP2 has the canonical esterase motif and the conserved S-D catalytic dyad for its LAH activity (Figure 1B). GhPLP2 is localized in the plasma membrane (Figure 3), which was consistent with the localization of several PLPs (Holk et al., 2002;Kim et al., 2014). The localization on the plasma membrane suggests that GhPLP2 may be involved in plant physiology and defense responses through the activation of lipid signaling by the hydrolysis of membrane lipids. The in vitro enzyme activity assay confirmed that GhPLP2 has LAH activity (Figures 2C,D) as it has been reported for other plant PLPs (Holk et al., 2002;Li and Wang, 2014). The GhPLP2overexpressed Arabidopsis plants and GhPLP2-silenced cotton plants were utilized to investigate the in vivo function of GhPLP2 on plant fatty acid metabolism. The overexpression of GhPLP2 in Arabidopsis enhanced the levels of linoleic acid (LA, 18:2) and α-linolenic acid (ALA, 18:3) relative to the WT (Figure 8A) not only after inoculation with V. dahliae but also after the water treatment. In contrast, the knockdown of GhPLP2 in cotton plants reduced the contents of both types of FAs ( Figure 8B). The functional LAH activity of GhPLP2 indicates its ability to release fatty acids from membrane lipids.
Many studies have suggested that PLPs with soluble LAH activity are necessary for the response of plants to various environmental stresses, such as drought, wounding, or pathogen infections (Matos and Pham-Thi, 2009;Rivas and Heitz, 2014). PLP proteins in tobacco, Arabidopsis, pepper, and grape show distinct resistance mechanisms in different hostpathogen interactions (Cacas et al., 2005;Yang et al., 2007;  La Camera et al., 2009;Kim et al., 2014;Laureano et al., 2018). The strong upregulation of the GhPLP2 gene upon infection by V. dahliae ( Figure 4B) suggests its crucial role in the interaction of cotton plants with V. dahliae. The silencing of GhPLP2 in cotton plants enhanced their susceptibility to infection by V. dahliae (Figure 5), and the overexpression of GhPLP2 in Arabidopsis plants visibly enhanced their resistance to disease through the appearance of merely mild symptoms of disease (Figure 7), demonstrating that GhPLP2 played a positive role in the plant defense to infection with V. dahliae.
Previous studies have shown that the JA biosynthetic and signaling pathway are important for plant resistance against V. dahliae He et al., 2016;Dhar et al., 2020). GhPLP2 was highly induced after JA treatment (Figure 4C), suggesting that GhPLP2 is likely to involve JA-dependent defense pathway. Several studies have shown that PLPs mediate plant immune responses through the regulation of FA metabolism and the JA signaling pathway. In tobacco, PLP was rapidly induced before the accumulation of JA in response to infection by the Tobacco mosaic virus (TMV) (Dhondt et al., 2002). AtPLAI targets the chloroplasts and plays a critical role in maintaining the homeostatic pool of free FA and basal JA, thus, increasing resistance to B. cinerea (Yang et al., 2007). The branch of the JA family (OPDA, JA, and dinor-OPDA) are fed by the primary cytoplasmic localization protein AtPLP2 (La Camera et al., 2009). The overexpression of the PLP gene OSAG78 in Arabidopsis increased the amounts of linoleic acid and linolenic acid and induced the expression levels of the JA-related defense genes PDF 1.2 and PR4 (Lin et al., 2011). In this study, the content of JA and the levels of expression of the JA biosyntheticrelated genes (LOX2, AOS, OPR3) were increased in GhPLP2overexpressed plants and decreased when GhPLP2 was knocked down (Figures 8C-F). As expected, the JA-responsive marker gene PDF1.2 exhibited a similar trend, which is upregulated upon infection with V. dahliae and positively related to the content of JA and the level of expression of GhPLP2 in plants. Therefore, GhPLP2 may be involved in the biosynthesis of JA in Arabidopsis and cotton, which contributed to the plant defenses against V. dahliae.
JA is known to take part in various physiological processes including salinity, drought, and temperature (low/high) stress (Ghorbel et al., 2021). The application of exogenous JA not only increased the total carbohydrate, soluble sugar, free amino acid, total proline, and protein contents, but also the activities The relative level of expression of the genes associated with the JA signaling pathway in WT and GhPLP2-overexpressed Arabidopsis plants 3 days after inoculation with water (mock) and V. dahliae. AtEF1α (AT5G60390) was used as the standard internal gene. (F) The relative expression of genes associated with the JA signaling pathway in TRV:00 and GhPLP2-silenced cotton plants 3 days after inoculation with water (mock) and V. dahliae. GhUBQ7 (DQ116441) was used as the standard internal gene. Data represent the means ± standard error of three independent biological repeats. Asterisks indicate a significant difference compared with WT or TRV:00, respectively. (*P < 0.05, **P < 0.01, Student's t-test).
of catalase (CAT), POD, and SOD in maize plants (Zea mays) (Ali and Baek, 2020). In pearl millet, the exogenous application JA weakens drought stress effects on pearl millet plantlets by increasing the fresh weight of seedlings, root length and shoot length (Awan et al., 2021). In this study, the phenotype of drought sensitivity has no significant difference between GhPLP2-overexpressed and WT Arabidopsis plants (Supplementary Figure 10). It seems that GhPLP2 altered JA level is not related to the plant drought response. Whether it plays a role in other abiotic stresses sharing a similar pathway with PEG 6000 treatment needs to be further determined.
Moreover, the alteration in JA-mediated pathways can also affect the vegetative and reproductive growth of plants (Qi et al., 2016;Ghorbel et al., 2021). The Arabidopsis mutant defective in anther dehiscence1 (dad1) displays the reduced accumulation of JA and defects in anther dehiscence, pollen maturation, and flower opening. The exogenous application of JA or linolenic acid could rescue the defects (Ishiguro et al., 2001). In rice (Oryza sativa), a plastid-targeted lipase EXTRA GLUME 1 (EG1) participates in the biosynthesis of JA during the development of rice spikelets. The eg1 mutant exhibits altered spikelet morphology with changes in floral organ identity and number, as well as defective floral meristem determinacy, resulting in a reduction in reproductive success and the yield of grains (Cai et al., 2014). In this study, GhPLP2-silenced cotton plants and GhPLP2-overexpressed Arabidopsis plants have altered JA content under mock treatment but no changes in the growing phenotype (Supplementary Figures 11A,B). This suggests that an alteration in the JA pathway through the modification of GhPLP2 does not affect the normal growth of plants. Additionally, the overexpression of GhPLP2 in Arabidopsis does not affect the weight of the grains (Supplementary Figure 11C). Whether the change in the JA-mediated pathway related to GhPLP2 affects fertility and reproductive output in cotton plants is yet to be determined.
Fatty acids are not only major membrane components of the cell, but they also directly or indirectly participate in a series of immune responses in plants (Walley et al., 2013;Lim et al., 2017;He and Ding, 2020). In fact, C16:0 and 18:0 FAs serve as precursors for the synthesis of cutin and wax polymers, which provide a physical barrier to defend against insects and pathogens (Raffaele et al., 2009;Yu et al., 2013). Moreover, C18:0 fatty acid can be converted to unsaturated fatty acids including oleic acid (18:1), linoleic acid (18:2), and α-linolenic acid (18:3) after a series of desaturation reactions (Kachroo and Kachroo, 2009). Previous studies confirmed that linoleic acid (18:2) and α-linolenic acid (18:3) are precursors for JA biosynthesis (Hung and Kao, 1998;Li et al., 2003;Banilas et al., 2007). The Arabidopsis defective in anther dehiscence1 (DAD1) protein is a chloroplastic phospholipase A1 that catalyzes the initial step of JA biosynthesis by the release of JA precursor α-linolenic acid (Ishiguro et al., 2001). In cotton plants, cytochrome P450 CYP82D (SSN) competes for C18 fatty acids substrates with LOXs. The inhibition of SSN leads to free fatty acids that enter the metabolism of LOXs to synthesize JA and enhance the levels of JA and disease resistance to V. dahliae (Sun et al., 2014). The long non-coding RNA (lncRNA) GhlncLOX3 positively regulates the resistance to V. dahliae through the modulation of the expression of GhLOX3 implicated in α-linolenic acid (18:3) metabolism associated with JA biosynthesis (Wang et al., 2021). In this study, the contents of palmitic acid (16:0) and stearic acid (18:0) significantly increased after inoculation with V. dahliae in cotton and increased slightly in Arabidopsis plants. However, the difference in the contents between GhPLP2silenced and control cotton plants was not significant. It is not clear whether GhPLP2 enhances the resistance of cotton against V. dahliae via the regulation of accumulation of palmitic acid (16:0) and stearic acid (18:0). However, the contents of linoleic acid (18:2) and α-linolenic acid (18:3) in both Arabidopsis and cotton plants were not only closely related to the expression of GhPLP2 under mock treatment but were also increased following infection with V. dahliae (Figures 8A,B). These findings suggest that GhPLP2 mediates the production of linoleic acid (18:2) and α-linolenic acid (18:3), which are involved in plant defense against V. dahliae. Free C18:2 and C18:3 was reduced to stable hydroxy FA in the cytosol or plastids and then converted to oxylipins which including JAs (Feussner and Wasternack, 2002;La Camera et al., 2009). Therefore, we hypothesize that the production of linoleic acid and α-linolenic acid by GhPLP2 may promote the biosynthesis and signaling of JA.
HR is one of the defense mechanisms that confers the plant with the ability to inhibit pathogen infection and further induce a rapid defense response (Kombrink and Schmelzer, 2001;Zhang Y. et al., 2017). Interestingly, several oxylipins that are derived from fatty acids in the 9-and 13-LOX pathways were found to be sufficient to initiate HR in different pathosystems (García-Marcos et al., 2013;Sun et al., 2014). Increasing amounts of evidence showed that the PLPs play an essential role in the execution of cell death in HR in plants. In Arabidopsis, AtPLP2 contributes to the resistance response to Cucumber mosaic virus (CMV) upon HRdependent process (La Camera et al., 2009). CaPLP1 regulates the production of phospholipid-derived molecules, which leads to the HR-like cell death during the incompatible interaction of pepper plants with avirulent Xcv (Kim et al., 2014). The elicitor protein PevD1 from V. dahliae can induce typical HR-like necrosis in tobacco and trigger innate immunity in cotton plants. GhPLP2 silencing has reduced the degree of HR and expression of the HR marker gene GhHIN1 and GhHSR203 during the induction of V. dahliae elicitor PevD1 in cotton leaves (Figure 6). This suggests that GhPLP2 is required for resistance (R) genemediated disease resistance in cotton plants. However, further research is merited to reveal the relationship between GhPLP2 and PevD1-triggered HR during the interaction between cotton and V. dahliae.
Taken together, we propose a potential model of the involvement of GhPLP2 in the resistance of plants against V. dahliae (Figure 9). GhPLP2 contributes to the build-up of free fatty acids pools, including linoleic acid and α-linolenic acid. The accumulation of linoleic acid and α-linolenic acid may directly or indirectly lead to the activation of the JA-synthesis and -signaling pathway in plants; GhPLP2 is also involved in PevD1-triggered HR. Both in concert enhance the resistance of cotton plants to disease caused by infection with V. dahliae.

CONCLUSION
Our study provides evidence that the patatin-like protein GhPLP2 with LAH activity plays a positive role in the enhancement of plant disease resistance. The silencing of GhPLP2 in cotton plants attenuated resistance against V. dahliae, and the overexpression of GhPLP2 in Arabidopsis enhanced resistance against infection with V. dahliae. The resistance mechanism of GhPLP2 may be owing to its release of the synthetic precursors to jasmonic acid, including linoleic acid and α-linolenic acid, via its LAH activity, followed by the accumulation of JA and activation of the JA signaling pathway. In addition, GhPLP2 is involved in triggering a hypersensitive response for disease resistance by a V. dahliae elicitor. These results stressed some insights for the mechanism of GhPLP2 in the response of plant defenses and highlighted the potential application of GhPLP2 in genetic engineering to increase the disease resistance of cotton germplasm.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

AUTHOR CONTRIBUTIONS
YH, FL, and YZ conceived and designed the study. YZ conducted most of the experiments and wrote the manuscript. XH, LG, and ZG provided technical assistance to YZ. YP and PW provided analysis tools. XG contributed reagents and materials. All authors reviewed and approved the final manuscript.

ACKNOWLEDGMENTS
We are grateful to Yule Liu from Tsinghua University for the VIGS constructs. We also thank Dong Li (College of Science, China Agricultural University) and Chunran Zhou (College of Science, China Agricultural University) for hormone quantitation.