GhMYB18 confers Aphis gossypii Glover resistance through regulating the synthesis of salicylic acid and flavonoids in cotton plants

R2R3 MYB transcription factor GhMYB18 is involved in the defense response to cotton aphid by participating in the synthesis of salicylic acid and flavonoids. R2R3 MYB transcription factors (TFs) play crucial roles in plant growth and development as well as response to abiotic and biotic stresses. However, the mechanism of R2R3 MYB TFs in cotton response to aphid infestation remains largely unknown. Here, an R2R3 MYB transcription factor GhMYB18 was identified as a gene up-regulated from upland cotton (Gossypium hirsutum L.) under cotton aphid (Aphis gossypii Glover) infestation. GhMYB18, which has transcription activity, was localized mainly to nucleus and cell membranes. Transient overexpression of GhMYB18 in cotton activates salicylic acid (SA) and phenylpropane signaling pathways and promoted the synthesis of salicylic acid and flavonoids, which leads to enhancing the tolerance to cotton aphid feeding. In contrast, silencing of GhMYB18 increased the susceptibility of G. hirsutum to aphid. Additionally, GhMYB18 significantly promoted the activities of defense-related enzymes including catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL). These results collectively suggest that GhMYB18 is involved in cotton defense response to cotton aphid attacks through regulating the synthesis of salicylic acid and flavonoids.


Introduction
Plants defend themselves against herbivores via various resistance strategies, including constitutive and inducible defenses (Karban and Baldwin 2007). Specific plant defense mechanisms rely on signaling networks, incorporating phytohormones, secondary metabolites, transcription factors, etc. (Erb and Reymond 2017). These transcription factors such as WRKY, NAC, bHLH, bZIP, ERF/AP2, and MYB families, play a crucial role in plant resistance to external biotic stresses by regulating the expression of defenserelated genes (Singh et al. 2002;Tsuda and Somssich 2015). MYB transcription factor is one of the most numerous and functional gene families in plants, with 51-52 amino acids at its N-terminal called MYB domain. The MYB domain is a highly conserved DNA-binding domain consisting of 1-4 imperfect amino acid sequence repeats (R), each forming three α-helices. The second and third helices of each repeat form a helix-turn-helix (HTH) structure required for binding to the promoters of the target genes (Dubos et al. 2010). Depending on the number of imperfect repeats, MYB proteins are split into four classes: R1-MYB, R2R3-MYB, 3R-MYB, and 4R-MYB, and most MYB transcription factors belong to R2R3-MYB (Stracke et al. 2001 (Arabidopsis thaliana), to explore the role of MYBs response to abiotic stresses and biotic stresses (Ambawat et al. 2013). Some MYB proteins have been proven to participate in plant defense against herbivores, including AtMYB102 (De Vos et al. 2006), GsMYB15 (Shen et al. 2018), AtMYB44 (Lu et al. 2013), NaMYB8 (Kaur et al. 2010), TaMYB19, TaMYB29, TaMYB44 (Zhai et al. 2017), yet the regulatory mechanisms and signaling pathways mediated by MYB proteins in defense responses remain largely unknown. Substantial researches have confirmed that MYB transcription factors contribute to the variation of reactive oxygen species (ROS) (Zhang et al. 2021a, b;Chen et al. 2016;Li et al. 2019). ROS, working as the one-electron reduction forms of atmospheric oxygen, including O 2− , H 2 O 2 , and OH − , are generally not harmful to organisms unless they are in adverse circumstances (Dautréaux and Toledano 2007). ROS scavenging enzymes take effect when excess ROS are accumulated in plants. ROS scavenging enzymes, including catalase (CAT), and peroxidase (POD), are essential to maintain the balance of ROS in organisms, which may relate to plant immunity (Zhang et al. 2021a, b). Additionally, polyphenol oxidase (PPO) is connected with the response of plants to diseases, pests, and environmental stresses (Jia et al. 2016;Thipyapong et al. 2004). It is capable of catalyzing the formation of lignin and other phenol oxides to form a defending shield to play a direct resisting role in the course of the defense response (Demeke and Morris 2002).
To date, different lines of evidence have indicated that SA interplays with ROS in stressed plants (Herrera-Vásquez et al. 2015). Salicylic acid (SA) plays a vital role in plant defense and activates resistance responses against external stresses. Mutants that are influenced in the accumulation of SA or insensitive to SA are more susceptive to abiotic and biotic stresses (Bari and Jones 2009). Protein arginine deiminase 4 (PAD4) and enhanced disease susceptibility 1 (EDS1) act upstream of SA to promote SA accumulation, and non-expressor of pathogenesis-related genes 1 (NPR1) is required for SA-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance (SAR) (Berrocal-Lobo and Molina 2004). Isochorismate synthase (ICS) pathway and the phenylalanine ammonia-lyase (PAL) pathway have been confirmed to be the main ways of SA synthesis (Bernal-Vicente et al. 2020). PAL is also the first key enzyme gene of the phenylpropane pathway, which can regulate the biosynthesis of flavonoids. Flavonoids are the most bioactive secondary metabolites among plants, which can eliminate reactive oxygen species by locating and neutralizing free radicals (Løvdal et al. 2010). The functions of flavonoids are diverse, which include defense against pathogens and pests, protection against UV light damage and oxidative stress, regulation of auxin transport, and allelopathy (Lloyd et al. 2017). The flavonoid biosynthesis pathway in plants has been extensively studied, the main catalytic enzymes are phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavonoid 3′-hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS) (Boudet 2007;Dong and Lin 2021). Those genes which relate to the structural flavonoid pathway are controlled by transcription factors (TFs) including MYBs (Zhai et al. 2016).
The aphid, Aphis gossypii Glover (Hemiptera: Aphididae) is the most common phloem-sucking pest in cotton. It can cause direct damage by feeding or indirect prejudice through transmission of plant viruses (Claude et al. 2018). Existing studies have only confirmed the MYB protein-mediated resistance mechanism to aphid in some plants. For example, TaMYB19, TaMYB29 and TaMYB44, which are regulated by the ethylene signaling pathway, cooperatively participate in wheat phloem defense and influence the feeding behavior of English grain aphid (Zhai et al. 2017). CmMYB15 and CmMYB19 provide chrysanthemum resistance to aphid by regulating the biosynthesis of lignin Wang et al. 2017). The Arabidopsis MYB transcription factors AtMYB102 and AtMYB44 regulate resistance to Myzus persicae Sulzer by activating ethylene defense in Arabidopsis (Lu et al. 2013;Zhu et al. 2018). In general, the roles of MYBs in cotton defense against aphid are not well known.
In this study, we identified an aphid-responsive gene GhMYB18 from upland cotton (Gossypium hirsutum). Transient expression and virus-induced gene silencing (VIGS) strategies were employed to clarify the role of GhMYB18 in plant response to Aphis gossypii infestation. Subsequent studies reveal that GhMYB18 can activate defense-related enzymes including CAT, POD, PPO and PAL, induce the synthesis of salicylic acid, and further accumulate flavonoids by regulating phenylpropanoid metabolic pathway in cotton for defense response against aphid.

Materials and culture condition
Cotton variety Zhongjixing 7 (Gossypium hirsutum L.), kindly provided by professor Dinguo Li from Yangtze University, was used in this study. Cotton and tobacco plants (Nicotiana benthamiana L.) were grown in pots (500 mL) filled with soil mix (vermiculite:humus, 1:1) at 25 °C under 16 h light/8 h dark conditions. Cotton aphid was collected from field-grown Zhongjixing 7 plants near Jingzhou, China. The colony was inoculated on cotton seedlings and transferred to fresh plants every 2 weeks (25 °C, 16 h/8 h light/dark).

Phylogenetic analysis of GhMYB18
The homologous sequences of GhMYB18 were retrieved and analyzed using online BLAST tools from the NCBI (National Center for Biotechnology Information, USA). The sequences of MYB proteins were aligned using DNA-MAN. A phylogenetic tree of GhMYB18 with its homologs was constructed using MEGA 7.0 with the maximum likelihood method (bootstraps = 1000).

RNA extraction and qPCR analysis
Total RNA was extracted using Spectrum™ plant total RNA kit (Sigma-Aldrich, USA) according to the manufacturer's protocol. Total RNA was reverse transcribed into first-strand cDNA with reverse transcriptase (Promega, USA), which was used as a template for quantitative realtime PCR (qPCR). Cotton gene GhUBI1 (GenBank accession number: EU604080) was used as an internal control. The relative expression level of each gene was calculated using the 2 −MMCT method (Livak and Schmittgen 2001). The relative expression value was shown as mean values of three independent tests, and three replicates were performed for each test. All the primer sequences are shown in Table S1.

Subcellular localization of GhMYB18
The CDS of GhMYB18 was cloned into the pBI121-GFP vector for overexpression under the control of the 35S promoter. The recombinant plasmid and empty vector (negative control) were separately transferred into Agrobacterium tumefaciens strain GV3101 competent cells, which were then injected into tobacco leaves. Fluorescence was detected with a confocal laser scanning microscope (Nikon, DS-Ri2, Japan) at 72 h after the injection (Ma et al. 2015).

Trans-activation activity assays
The full-length CDS of GhMYB18 together with the divided fragments nGhMYB18 (encoding 1-153 amino acids in the N-terminus of GhMYB18) and cGhMYB18 (encoding 154-327 amino acids in the C-terminus of GhMYB18) were ligated to the pGBKT7 vector (Clontech, Mountain View, CA, USA). Those resultant constructions which were designated as BD-GhMYB18, BD-nGhMYB18 and BD-cGhMYB18, were transferred into the Yeast strain AH109 (Clontech). The pGBKT7 empty vector was used as the negative control. The transformed strains were cultured on a medium at 28 °C for 3 days before observation. Four types of media were used: SD/-Trp, SD/-Trp with aureobasidin A (AbA), SD/-Trp with X-α-Gal, and SD/-Trp with AbA and X-α-Gal (Jiang et al. 2021).

Expression profile of GhMYB18 in response to aphid infestation and phytohormone treatment
Uniformly grown cotton seedlings (14 days old) were used in this experiment and each cotton seedling cotyledon was inoculated with 20 adult cotton aphids of the same size. The expression levels of GhMYB18 in roots, stems and cotyledons were analyzed at 24 h, 48 h, and 72 h after the cotton aphid infestation. A solution of methyl jasmonate (MeJA) and salicylic acid (SA) with a concentration of 0.05 mmol/L was prepared and sprayed evenly on cotton cotyledons. The expression levels of GhMYB18 were analyzed in cotton cotyledons at 24 h, 48 h, and 72 h after MEJA and SA treatment. Untreated cotton seedlings were used as a negative control, three biological replicates were used for the above treatments.

Transient expression
The vector pBI121-GhMYB18 and empty vector were ligated into Agrobacterium tumefaciens GV3101 (Sun et al. 2015). The transformed strains were grown in a lysogeny broth (LB) liquid medium (pH = 7.0) containing 50 ug/mL kanamycin and rifampin at 28 °C, 220 rpm. After centrifugation, the pelleted bacteria were resuspended (OD 600 = 0.8). The resuspension was injected into the cotton cotyledons by a needle-less syringe followed by the expression detection of GhMYB18 (Yue et al. 2012).

Virus-induced gene silencing
The pTRV1 and pTRV2 vectors were used for VIGS experiments. The specific fragment of GhMYB18 was inserted into pTRV2 (Liu et al. 2002). Agrobacterium tumefaciens cultures (OD 600 = 1.5) harboring pTRV1 and that containing pTRV2-GhMYB18 were mixed at a 1:1 ratio and inoculated into cotton plants by vacuum infiltration as described by Qu et al. (Qu et al. 2012).

The resistance of GhMYB18-overexpressing and GhMYB18-silenced cotton against cotton aphid
No-choice and choice assays were performed using wildtype plants, control plants and treatment group plants. For no-choice assay, ten adult cotton aphids of the same individual size were inoculated on the cotton cotyledons, and the cotyledons were fixed with insect cages to prevent the aphids from escaping. The number of cotton aphids on the cotyledons was counted at 24 h, 48 h, and 72 h after inoculation. For the choice assay, the detached cotton cotyledons leaves were placed symmetrically on the inner edge of a petri dish, and the petioles are soaked with MS nutrient solution to prevent the leaves from withering. The petri dish was inoculated with 30 adult cotton aphids of the same individual size and the number of aphids on the cotyledons was counted at 24 h after inoculation.

Quantification of aphid excreted honeydew
Sterilized agar medium was poured into a glass petri dish with a diameter of 9 cm, and the adaxial surface of the leaves were placed on the medium without leaving a gap. The cotyledons were inoculated with ten adult cotton aphids of the same individual size, the petri dish was inverted, and the aphid honeydew was collected with Whatman filter paper for 3 days. The determination method of aphid honeydew was as follows: the filter papers with collected honeydew were immersed in 0.1% (w/v) ninhydrin solution. After drying, the filter papers were torn into pieces, and stains were extracted in 1 mL of 90% (v/v) methanol for 1 h at 4 °C with continuous oscillation. After centrifugation at 5000×g for 1 min, the absorbance was measured at 500 nm (Kim and Jander 2007;Nisbet et al. 1994).

Determination of enzyme activity
Cotton leaves were frozen in liquid nitrogen and quickly ground into a fine powder. Samples of 100 mg were homogenized in extraction buffer and the mixture was collected in a centrifuge tube. After centrifugation at 4 °C and 14,000×g for 10 min, the supernatant was the crude enzyme solution. The activities of CAT, POD, PAL and PPO were determined according to the guidelines of the enzyme activity kit (Nanjing Jiancheng Bioengineering Institute, China).

Determination of salicylic acid content
The salicylic acid was extracted and purified according to the method in the previous study (Fu et al. 2012). 100 mg of fresh leaf tissue was fully ground in liquid nitrogen, suspended in 1 mL of methanol, and incubated at -20 °C overnight. The mixture was centrifuged at 14,000×g for 10 min at 4 °C and purified with a 0.25 um needle nylon filter. 200 μL of the filtrate was taken and dried with nitrogen gas, then 200 μL of sodium acetate buffer (0.1 M, pH = 5.5) was added. The mixture was shaken at 4 °C for 1 h and centrifuged at 14,000×g for 10 min at 4 °C. The suspension was treated with 10 μL of β-glucosidase, incubated in a water bath at 37 °C for 2 h, and then boiled in water for 5 min to stop the enzymatic reaction. After that, the mixture was centrifuged at 14,000×g for 10 min at 4 °C, and then the supernatant was subjected to LC-MS to analyze the total SA content.

Determination of total flavonoids and free gossypol content
100 mg of leaf powder and 1 ml 80% (v/v) methanol were placed in a centrifuge tube and then shook overnight at 4 °C. After centrifugation at 13,000×g at 4 °C for 15 min, the supernatant was collected, and the residual pellet was reextracted with 1 mL of 80% (v/v) methanol. The supernatant of the two extractions was combined for subsequent research. Total flavonoids were determined according to the method described previously (Sultana et al. 2009). 0.1 mL of supernatant was mixed with 0.5 mL distilled water into a test tube, to which was added 30 μL of 5% NaNO 2 (w/v) solution. After 6 min, 60 μL of 10% (w/v) AlCl 3 solution was added. 5 min later, 200 μL of 1 M NaOH was added to terminate the reaction. The volume was brought to 1 mL with distilled water and 200 μL of them was used to measure the absorbance at 420 nm.
The gossypol equivalents were extracted and quantitated following a previously reported phloroglucinol/HCl method . A standard curve was made using gossypol (Sinopharm Chemical Reagent Co LTD, China).

Statistical analysis
SPSS v26.0 was used for all statistical analysis, and all data were shown as mean ± SD of one representative experiment. The student's t test was used to compare the significant differences in GhMYB18 expression levels after aphid infestation and phytohormone treatment. The p value was shown as p < 0.05 or p < 0.01 to indicate significant differences. In the case of other experiments, Tukey's multiple range test (p < 0.05) and one-way analysis of variance was performed to determine the significance of the data. Different letters a, b and c indicated the significance levels (p < 0.05).

Sequence analysis of GhMYB18
GhMYB18 was isolated from upland cotton 'Zhongjixing 7', and sequence analysis showed that it was a new cotton MYB family gene. The GhMYB18 gene is composed of 981 bp and encodes a polypeptide of 327 amino acids (File S1). GhMYB18 belongs to the R2R3-MYB subfamily, as it has two conserved MYB domains including an R2MYB domain between aa 13-63 and an R3MYB domain between aa 66-114. Alignment analysis indicated that this gene has 67% similarity with GhMYB30 (Fig. 1A). Phylogenetic tree of GhMYB18 and MYB proteins from other plant species shows that GhMYB18 are closely related to GhMYB30 (Fig. 1B).

GhMYB18 was induced by cotton aphid infestation, SA and MeJA
GhMYB18 was expressed in roots, stems and cotyledons in cotton plants. At 24 h, 48 h and 72 h after the sustained infestation of cotton aphid, the expression levels of GhMYB18 were significantly higher than that of the control in roots, stems and cotyledons of cotton plants. Furthermore,  GhMYB18 presented higher gene expression levels in roots, stems and cotyledons at 72 h under cotton aphid infestation. Their expression levels were, respectively, 4.03, 3.66, and 3.21 times more than that in the wild-type plants (WT) at 72 h ( Fig. 2A). In addition, GhMYB18 transcripts were significantly up-regulated in leaves at 24 h, 48 h and 72 h after salicylic acid treatment (Fig. 2B). In contrast, after methyl jasmonic acid treatment, the expression level of GhMYB18 was inhibited and was significantly down-regulated at 48 h compared with control (Fig. 2C). These results show that aphid damage and exogenous SA induction can promote the expression level of GhMYB18, while exogenous MeJA induction may decrease the expression level of GhMYB18.

GhMYB18 functions in nucleus and cell membranes, and has transcription activity
To determine the subcellular site where GhMYB18 functions, a subcellular localization assay was performed. Consistent with the control (35S-GFP) results, in the cells transferred into GhMYB18-GFP, the GFP signal was mainly detected in the cell membranes and nucleus (Fig. 3). For trans-activation activity assays, yeast harboring pGBKT7-GhMYB18 or pGBKT7 (negative control) grew normally on SD/-Trp medium (Fig. 4B). But on SD/-Trp with AbA medium, only the yeast strain of pGBKT7-GhMYB18 grew normally (Fig. 4C). At the same time, it was no more than yeast transferred into pGBKT7-GhMYB18 turned blue on the chromogenic medium (SD/-Trp with AbA and X-α-Gal medium) (Fig. 4D). To further explore the transcriptional activation domain (TAD), the CDs of GhMYB18 was incised into two fragments (N-terminal and C-terminal regions) based on its structural characteristics. As shown in Fig. S1, the BD-nGhMYB18 failed to grow on SD/-Trp with AbA and X-a-Gal medium, indicating that the transcriptional activation domains of GhMYB18 located in the C-terminal regions of GhMYB18. These results illustrate that GhMYB18, which has transcription activity, is mainly localized to nucleus and cell membranes.

Overexpression of GhMYB18 enhances cotton plants' tolerance to Aphis gossypii
The GhMYB18 expression level in transiently overexpressed GhMYB18 cotton seedlings was determined using qPCR. Compared with wild-type cotton plants (WT) and cotton plants containing pBI121(pBI121 plants), the expression level of GhMYB18 in cotton plants containing pBI121-GhMYB18 (pBI121-GhMYB18 plants) significantly increased at 24 h, 48 h 72 h. The pBI121-GhMYB18 plants presented a higher expression level of GhMYB18 at 48 h, and its expression level was 6.26 times higher than that of pBI121 plants (Fig. 5A). The choice assay showed that the cotton aphid was more widely distributed on pBI121 plants and WT than pBI121-GhMYB18 plants at 24 h, 48 h and 72 h after sustained feeding of cotton aphid (Fig. 5B). Obviously, WT and pBI121 plants were more attractive to cotton aphid. Simultaneously, we conducted a no-choice assay to examine the aphid resistance of pBI121-GhMYB18 plants. Consistently, compared with WT and pBI121 plants, the number of cotton aphids on pBI121-GhMYB18 plants was lower at 24 h, 48 h, and 72 h after persistent feeding of cotton aphid (Fig. 5C). Assays of honeydew secretion from Aphis gossypii was consistent with the above conclusions (Fig. S2). Results showed that the amount of honeydew secreted on pBI121-GhMYB18 plants was significantly lower than that of WT and pBI121 plants at 48 h and 72 h (Fig. 5D). Above all, GhMYB18 overexpression may enhance cotton plant tolerance to Aphis gossypii.

Silencing of GhMYB18 increased cotton plant susceptibility to Aphis gossypii
The VIGS method was also employed to further clarify the role of GhMYB18 in cotton response to the infestation of Aphis gossypii. Compared with the wild-type plants (WT) and control group plants (TRV:00), the expression level of GhMYB18 was reduced in GhMYB18-silenced plants (TRV:GhMYB18) at 18 d and 24 d after Agrobacterium tumefaciens infiltration (Fig. 6A). The choice tests showed that cotton aphid was more inclined to feed on GhMYB18silenced cottons than WT and control group plants at 18 d (Fig. 6B). In the no-choice assay, the number of cotton aphid in the GhMYB18-silenced plants was significantly higher than that of WT and control group plants after 18 d (Fig. 6C). The quantity of honeydew secreted on GhMYB18silenced cotton was significantly higher than others at 18 d and 24 d after aphid infestation (Fig. 6D). These results suggest that silencing of GhMYB18 increased cotton plant susceptibility to Aphis gossypii.

Influence of altering GhMYB18 expression on enzyme activities
As a specific secondary metabolite in cotton, gossypol, conduce to the defense response against cotton aphid (Du et al. 2004). We first measured the content of free gossypol, there was no significant difference between treated and control plants (Fig. S3). Enzyme activities of PAL, CAT, PPO and POD go hand in hand with plant resistance to insect pests. Therefore, these enzyme activities and their gene expression levels were measured. The gene expression level and activities of PAL, CAT, PPO and POD in pBI121-GhMYB18 plants were always higher than those in pBI121 plants (Fig. 7A, B). In the same way, the gene expression levels and activities of these enzymes in GhMYB18-silenced plants were always lower than those in WT and control group plants (TRV:00) (Fig. 7C, D). These results prove that the expression level of GhMYB18 is closely related to PAL, CAT, PPO and POD.

GhMYB18 acts as a positive regulator in SA signaling pathway
Salicylic acid is irreplaceable in plant response to aphid infestation. Therefore, we analyzed the expression levels of the genes (EDS1, PAD4, ICS, PAL, NPR1) related to the salicylic acid signaling pathway and the content of salicylic acid in GhMYB18 transiently overexpressed cotton plants and GhMYB18-silenced cotton plants. Compared with WT and pBI121 plants, EDS1 and PAL transcripts were significantly higher in the GhMYB18-overexpressing plants, but the PAD4 transcripts had no significant difference. Moreover, the expressed levels of ICS and NPR1 decreased in the GhMYB18overexpressing plants than in WT plants (Fig. 8A). A similar pattern was observed in GhMYB18-silenced plants, the expression levels of EDS1, ICS, PAL were down-regulated than that WT and control group plants (TRV:00), but the expression levels of PAD4 and NPR1 had no significant difference (Fig. 8B). The SA content was significantly increased in plants overexpressing GhMYB18, but decreased in GhMYB18-silenced plants (Fig. 8C, D). These results show that GhMYB18 may participate in the defense response to cotton aphid by regulating the SA signaling pathway.

GhMYB18 stimulates the production of total flavonoids by the PAL pathway
Previous studies have shown that PAL is involved in the biosynthesis of polyphenolic compounds, including flavonoids and lignin in plants ( Rohde et al. 2004). To further explore the causal relationship between flavonoids or lignin content and expression level of GhMYB18, we detected related indicators in GhMYB18 transiently overexpressed cottons and GhMYB18-silenced cottons. Firstly, we examined the expression levels of genes related to flavonoids and lignin synthesis pathway. The expression level of CHS, CHI, FLS and ANS was up-regulated significantly in plants overexpressing GhMYB18, but compared with WT plants, the transcripts of F3'H and FLS significantly decreased in plants overexpressing GhMYB18. More interestingly all these genes were significantly down-regulated as expectedly in GhMYB18-silenced plants (Fig. 9A, B). Furthermore, it seems that GhMYB18 is not needed in lignin synthesis pathway (Fig. S4). At the same time, the total flavonoid content was significantly increased in plants overexpressing GhMYB18, but decreased in GhMYB18silenced plants (Fig. 9C, D). These results indicate that GhMYB18 regulates the defense response of cotton to Aphis gossypii by influencing the synthesis of flavonoids.

Discussion
As one of the largest transcription factors families in plants, MYBs play a vital role in regulating abundant plant-specific physiological processes (Jiang and Rao 2020). Compared with extensive studies on the MYB transcription factors in plant growth and abiotic stress (Loguercio et al. 1999;Lu et al. 2017), little is known about MYB proteins regarding their functions in resisting agriculturally pests such as aphid. In this work, a novel resistance-related R2R3-MYB gene, GhMYB18, was identified and cloned. Subsequent researches indicate that GhMYB18 confers Aphis gossypii resistance by regulating the defense-related enzyme activities, salicylic acid and phenylalanine ammonia-lyase pathways in cotton.
Unlike most MYB transcription factors, GhMYB18 has transcription activity, not only localized to the nucleus, but also the cell membranes. Phylogenetic analysis showed that GhMYB18 is closely related to GhMYB30, AtMYB94, GhMYB18-silenced cottons (pTRV2-GhMYB18). C The salicylic acid content of wild-type cottons, control group cottons and GhMYB18overexpressing/ silenced plants. D The salicylic acid content of wildtype cottons, control group cottons and GhMYB18-silenced plants.
Those bars indicate the standard errors, the alphabets represent the level of significant difference (p < 0.05) and GhMYB60, which are related to the plant immune system (Zhou et al. 2022). Moreso, the expression levels of GhMYB18 were significantly up-regulated after aphid infestation. To investigate the role of GhMYB18 in the defense response to the aphid, transient overexpression and VIGS methods were applied, and there were no phenotypic differences among wild-type, control and treatment plants (Fig. S5). Overexpression of GhMYB18 increased resistance in plants against aphid, reduced the reproduction and feeding rate of aphid. The opposite results were shown after silencing GhMYB18 in cotton plants. These results indicated that GhMYB18 contributes to the resistance of cotton aphid.
In response to aphid infestation, plants have evolved multiple strategies to resist aphid, including trichome formation, callose deposition, and the production of secondary metabolites such as phenols (Goggin 2007). Although there are many related studies, our understanding of plant defense mechanisms against aphid is far from enough (Nalam et al. 2019). Prior researches confirm the potential role of CAT, POD, PAL and PPO, in plant defense against aphid infestation, such as the activities of the POD and CAT enzyme in tobacco were enhanced to respond to aphid attacks (Ren et al. 2014); the resistant cultivars exhibit greater constitutive activities of POD, PPO and PAL than in susceptible cultivars following aphid infestation (Han et al. 2009); four defenserelated enzymes including CAT, POD, PPO increased under high densities of aphid infestation stress . In this study, we found that overexpression of GhMYB18 increased the activities and expression levels of PAL, CAT, PPO and POD in cotton seedlings. When the expression of GhMYB18 is suppressed, these results were opposite. Consistent with our findings, the cotton gene GhRac6 improves the plant defense response to aphid feeding by improving activities of PAL and CAT ); overexpression of GhChi6 increases the activity of PPO, thus increasing plant immunity to aphid (Zhong et al. 2021).
Salicylic acid (SA) plays a crucial role in minimizing infections and the effects of biotic and abiotic stresses (Zhang and Li 2019). The salicylic acid signaling pathway is activated when plants are exposed to piercing-sucking insects, such as Diuraphis noxia Mordvilko (Mohase and Westhuizen 2002), Nilaparvata lugens Stal (He et al. 2020), Aphis gossypii Glover (Zhong et al. 2021) and Myzus persicae Sulzer (Moran and Thompson 2001). Exogenous treatment with plant hormones on cotton seedlings resulted in significant up-regulation of GhMYB18 after SA induction, but MeJA induction down-regulated the expression level of Fig. 9 The expression level of genes related to PAL signaling pathway and total flavonoids content in GhMYB18 transiently expressed cotton and GhMYB18-silenced cotton. A The expression level of six genes related to the flavonoids synthesis pathway in wild-type cottons (WT), control group cottons (pBI121) and GhMYB18-overexpressing cottons (pBI121-GhMYB18). B Six gene transcripts in flavonoids synthesis pathway in wild-type cottons (WT), control group cottons (pTRV2) and GhMYB18-silenced cottons (pTRV2-GhMYB18). C Total flavonoids content of wild-type cottons, control group cottons and GhMYB18-overexpressing plants. D Total flavonoids content of wild-type cottons, control group cottons and GhMYB18-silenced plants. Those bars indicate the standard errors, the alphabets represent the level of significant difference (p < 0.05) GhMYB18. The reciprocal antagonism of SA and jasmonic acid (JA) signaling pathways has been demonstrated in numerous studies (Koornneef and Pieterse 2008). For example, when Bemisia tabaci Gennadius nymphs fed on Arabidopsis, JA-responsive defenses were suppressed downstream by inducing SA (Zarate et al. 2007); exogenous SA reduced the amount of JA that Egyptian cotton worm induced in both NPR1 mutant and wild-type Arabidopsis (Stotz et al. 2002). In this research, overexpressing of GhMYB18 in cotton plants increased the SA content and the expression levels of EDS1, ICS and PAL in the SA signaling pathway. By inhibiting the expression of GhMYB18, we then concluded that: GhMYB18 may be involved in the defense response to aphid through regulating the SA signal pathway and further research is still needed to know whether the expression of GhMYB18 affects the JA signaling pathway.
PAL is the first enzyme induced by the phenylpropanoid pathway in the biosynthesis of various phenolic compounds with structural and defense-related functions such as lignin, pigment, flavonoid, and phytoalexin (Solekha et al. 2020). These compounds have a significant effect on plant growth, development, reproduction, defense, and environmental responses (Qian et al. 2019). Different studies have reported the potential role of flavonoids and lignin in plant defense response to aphid. For example, when cotton plants are subjected to salt stress, the content of the flavonoids in the cotton will increase to promote the cotton's defense response against aphid ; more flavonoids and phenol will accumulate followed by aphid infestation in melon (Zahedi et al. 2019); overexpression of GhLac1 increased the lignin content in cotton associated with increased tolerance to aphid (Hu et al. 2018). Moreso, we also determined the content of lignin and flavonoids in GhMYB18-overexpressed and GhMYB18-silenced plants. In our findings, the expression of GhMYB18 did not affect the synthesis of lignin, but the content of total flavonoids and the expression levels of flavonoid biosynthetic pathway-related genes increased significantly in the plants overexpressing GhMYB18, but decreased in the plants silencing GhMYB18. Above all, the results suggested that GhMYB18 may regulate the defense response against aphid through the synthesis of flavonoids.
In summary, our research reveals the GhMYB18-mediated plant defense response against aphid. GhMYB18 expression was induced by aphid infestation and activates defenserelated enzyme activities (CAT, POD, PPO and PAL). Moreover, GhMYB18 acts as a positive regulator in SA signaling pathway and flavonoid synthesis pathway to improve cotton resistance to aphid. Collectively, our research provides new insight into the mechanism of transcriptional regulation in plant defense response to aphid, offering valuable evidence of upland cotton breeding. However, on account of the complex interaction between plants and aphid, more studies on GhMYB18 response to cotton aphid will be performed in the future.

Conclusion
This study suggests that GhMYB18 functions as a positive regulator of aphid tolerance. GhMYB18 acted as an R2R3 MYB transcription factor and was localized in the cell membrane, cytoplasm and nucleus. Exogenous salicylic acid treatment and aphid treatment significantly increased GhMYB18 expression levels, except for JA induction. The results of the choice assay, no-choice assay and assays of honeydew secretion from Aphis gossypii correlated to the expression level of GhMYB18 upon aphid infestation. Subsequent analysis showed that GhMYB18 not only activated defense-related enzymes such as CAT, POD, PPO and PAL, but also participated in salicylic acid and phenylpropane signaling pathways to improve upland cotton resistance to Aphis gossypii. Our results provide a significant advancement in figuring out the functions of R2R3-MYB transcription factors in aphid resistance.