The long non-coding RNA GhlncRNA149.1 improves cotton defense response to aphid damage as a positive regulator

Long non-coding RNAs (lncRNAs) play crucial roles in various plant biological regulatory processes and defense responses to all sorts of biotic and abiotic stresses. However, there are limited studies on the function and mechanism of lncRNAs in plant defense responses to phytophagous insects. In this research, a lncRNA GhlncRNA149.1 was identified from upland cotton (Gossypium hirsutum L.). Depending on the position relationship between lncRNA and mRNA, we identified CC-NBS-LRR family gene GhA01G0129 as the target gene of GhlncRNA149.1. Expression levels of GhlncRNA149.1 and its target gene GhA01G0129 were significantly down-regulated in cotton cotyledons upon cotton aphid infestation. The expression level of GhlncRNA149.1 was significantly down-regulated after the induction of methyl jasmonate and salicylic acid, whereas expression level of its target gene GhA01G0129 was significantly up-regulated. Overexpression of GhlncRNA149.1 in cotton cotyledons enhanced the tolerance of cotton plants to cotton aphid feeding. Expectedly, silencing GhlncRNA149.1 increased susceptibility of cotton plants to cotton aphids. The expression level of GhA01G0129, the target gene of GhlncRNA149.1, was significantly up-regulated in cotton plants overexpressing GhlncRNA149.1, and significantly down-regulated in cotton plants silencing GhlncRNA149.1. The activity and expression levels of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were up-regulated in cotton plants transiently overexpressing GhlncRNA149.1, while opposite results were obtained in cotton plants silencing GhlncRNA149.1. In addition, similar results were obtained in the expression levels of phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), enhanced disease susceptibility 1 (EDS1) and non-expression of pathogenesis-related gene 1 (NPR1) in cotton plants transiently overexpressing GhlncRNA149.1 and silencing GhlncRNA149.1. These results collectively suggest that GhlncRNA149.1 improve the plant defense response to aphid attack, and thus has potential for enforcing cotton aphid prevention and control. LncRNA GhlncRNA149.1 positively regulate the response of cotton to cotton aphid feeding.


Introduction
LncRNAs are a class of poorly conserved RNAs with a length of more than 200 nucleotides and lack of proteincoding ability (Mercer et al. 2009). Most lncRNAs are transcribed by RNA polymerase II. However, some lncRNAs are transcribed by RNA polymerase III, and a small number of lncRNAs in plants are produced by plant-specific RNA polymerases IV and V . According to the localization of lncRNAs with reference to adjacent proteincoding genes in the genome, lncRNAs can be divided into five types: sense lncRNA, antisense lncRNA, bidirectional lncRNA, intronic lncRNA (incRNA), and large intergenic lncRNA (lincRNA) (Ponting et al. 2009). LncRNAs may interact with many large molecules, such as DNA, RNA, and proteins, and regulate protein modification, chromatin remodeling, protein functional activity, and RNA metabolism in vivo at the transcriptional, post-transcriptional and epigenetic levels (Lyu et al. 2019). The lncRNA action mechanisms have cis-and trans-associated functions. Cisacting lncRNAs are located in upstream or downstream of the encoded protein and interplay with cis-acting elements in the promoter or co-expressed genes, thereby modulating gene expression at the transcriptional or post-transcriptional level. Trans-acting lncRNAs may act as signals, guides or scaffolding to mediate the expression of target genes located in distant chromosomal domains or even on different chromosomes (Zhang et al. 2019a, b). Up to now, the regulatory roles of lncRNAs have been extensively studied in yeast and mammals, but little is known about their regulatory roles in plants .
With the rapid development of sequencing technology, the functions and mechanisms of plant lncRNAs have attracted more and more attention. Some studies have shown that lncRNAs are widely involved in plant growth and development through interaction or transcriptional regulation. For example, the lncRNA APOLO (AUXIN-REGULATED PROMOTER LOOP) is transcriptionally regulated by auxin and modulates later root development through mediating the formation of a chromatin loop (R-loop) encompassing the promoter of its neighboring gene PINOID (PID) and suppressing the PID transcript (Amor et al. 2009;Ariel et al. 2014). A lncRNA [termed COLD ASSISTED INTRONIC NONCODING RNA (COLDAIR)] recruits the protein complex, polycomb repressive complex 2 (PRC2) to activate tri-methylated histone H3 Lys 27 to maintain the lowlevel expression of the flowering repressor, FLOWERING LOCUS C (FLC) in Arabidopsis thaliana, while lncRNA COOLAIR involves alternative splicing (AS) and can indirectly inhibit FLC expression through transcription interference (Heo and Sung 2011;Swiezewski et al. 2009). More importantly, overexpressing lncRNA LAIR [LRK (leucinerich repeat receptor kinase) Antisense Intergenic RNA] transcribed from the antisense strand of the neighbouring gene LRK cluster increases grain yield and regulates neighbouring gene cluster expression in rice and it may be useful targets for crop breeding (Wang et al. 2018). Secondly, some studies have also proved that lncRNA is involved in plant response to all kinds of abiotic stresses. For instance, the lncRNA APOLO interacts with the transcription factor WRKY42 to trigger root hair cell expansion in response to cold (Moison et al. 2021). Potato central clock output transcription factor CYCLING DOF FACTOR 1 (StCDF1) and a lncRNA counterpart, named StFLORE also link tuber development and drought response. Both natural and CRISPR-Cas9 mutations in the StFLORE transcript produce plants with increased sensitivity to water-limiting conditions. Conversely, regulating expression of StFLORE, both by the overexpression of StFLORE or by the down-regulation of StCDF1, results in an increased tolerance to drought through reducing water loss (Ramírez Gonzales et al. 2021). LncRNA354 that was localized at the nucleus and cytoplasm in cotton functions as a competing endogenous RNA of miR160b to regulate the auxin response factor gene ARF in response to salt stress in upland cotton . LncRNA973 from cotton was localized in the nucleus and its expression was increased by salt treatment, lncRNA973-overexpression lines had increased salt tolerance compared with the wild type, the lncRNA973 knockdown plants had reduced salt tolerance (Zhang et al. 2019a, b). MSTRG.85814.11, a lncRNA from Malus domestica (apple), positively promotes the small auxin upregulated gene SAUR32 expression as a transcriptional enhancer of SAUR32 which then activate proton extrusion involved in the Fe-deficiency response (Sun et al. 2020). Additionally, plant lncRNAs are also important in responding to all sorts of biological stresses. For example, Sl-lncRNA15492 from tomato (Solanum lycopersicum Zaofen No. 2) plants, as a positive regulator, interacts with Sl-miR482a (miRNAs) and affects Solanum lycopersicum immunity against Phytophthora infestans . LncRNA39026 from tomato Zaofen No. 2 might function to decoy miR168a (miRNAs) and affect the expression of pathogenesis-related (PR) genes in tomato plants, increasing resistance to disease ). However, compared with studies on lncRNA function in plant response to pathogens, few studies have been conducted on plant lncRNA response to herbivores .
As an important cash crop, upland cotton (Gossypium hirsutum L.) produces a renewable natural fiber for the textile industry, provides edible protein for livestock feed and is a source of oil and biofuel. However, it is often damaged by cotton aphids in various stages of growth and development, which seriously affects the production of cotton (Barros et al. 2022;Jans et al. 2021;Tokel et al. 2021). It is well known that plant resistance is an environmentally friendly control method of cotton aphids. Therefore, it is imperative to study the functions and mechanisms of aphid resistance genes in cotton and aphid-resistant cultivated varieties. In our previous study, we identified differentially expressed lncRNAs in cotton leaves after aphid feeding (Zhang et al. 2019a, b). The results showed that the length of GhlncRNA149.1 gene was 740 bp, and the expression of GhlncRNA149.1 gene changed after cotton aphid infestation. Therefore, we hypothesized that GhlncRNA149.1 might affect the immunity of cotton to cotton aphid. On this basis, we analyzed the role and mechanism of GhlncRNA149.1 in the response of cotton to cotton aphid. Our results not only elucidate the function and molecular mechanism of GhlncRNA149.1 in cotton aphid resistance response, but also provide candidate genes for plant aphid resistance breeding.

Materials and growth conditions
Cotton variety Zhongjixing No.7 (Gossypium hirsutum L.) was provided by Professor Dinguo Li from Yangtze University. The seeds were soaked in sterile water for 24 h until the radicle penetrated the seed coat, then planted directly in plastic pots (25 cm in diameter) filled with vegetative soil (vermiculite, grass ash and soil mixed in at 1:1:2 ratio). The pots were placed in a room at 25 °C, 16 h/8 h light-dark cycle and 70% relative humidity. Seedlings with two cotyledons or four true leaves were used in this experiment. The cotton aphids used in this experiment were collected from the cotton field in the laboratory at Yangtze University. They were inoculated on Zhongjixing No.7 and reared in a culture room at 25°C, 16 h/8 h light-dark cycle and 70% relative humidity.

Cotton aphid and hormone treatment
Fifty adult cotton aphids were inoculated on the true leaves of 14 day-old cotton plants. The tissue samples from cotton leaves, stems, and roots of cotton cotyledons of 14 days were harvested at 24, 48, and 72 h after aphid infestation or no-cotton aphid infestation. Their total RNA was extracted using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, USA). In addition, salicylic acid (5 mmol/l), methyl jasmonate (0.5 mmol/l) and sterile water (CK) were sprayed onto the cotton plants until runoff. Each plant was covered with a clear plastic bag and removed after 6 h. Cotton cotyledons were collected at 24, 48 and 72 h after spraying, and total RNA was extracted using Spectrum™ Plant Total RNA Kit.

Carrier construction and transformation
The full length and specific fragment of cotton gene GhlncRNA149.1 were inserted into the expression vector pBI121 and virus-induced gene silencing vector pTRV2, respectively (Liu et al. 2002). The prepared vectors were transferred into Agrobacterium GV3101 by electric transfer method. Agrobacterium harboring pBI121-GhlncRNA149.1 and pBI121 were infiltrated into the cotyledons of 7-dayold cotton using a needless syringe (Norkunas et al. 2018;Sun et al. 2015). Similarly, the suspension containing pTRV2-GhlncRNA149.1, and the suspension mixed with pTRV2 and pTRV1 in the same proportion were injected into cotyledons of cotton seedlings by micro-injection on the 7th day after sowing (Degefu and Hanif 2003;Qu et al. 2012).

Resistance analysis of cotton with GhlncRNA149.1 overexpression and GhlncRNA149.1 silencing to cotton aphid
The wild-type plants, control plants and treated plants were used for no-choice assay and dual-choice assay. For nochoice assay, 10 adults aphids of the same size were inoculated on cotton cotyledons in cages to prevent aphids from escaping. The number of cotton aphids on cotton cotyledons was counted at 24, 48 and 72 h after inoculation. For dualchoice assay, isolated cotton cotyledons were placed symmetrically on the inner edge of the petri dish, and petioles were soaked in MS nutrient solution to prevent leaf wilting. Thirty adult cotton aphids of the same size were inoculated on cotton cotyledons, and the number of cotton aphids on cotyledons was counted 24 h after inoculation.

Quantification of cotton aphid excreted honeydew
Sterilized AGAR medium was poured into a glass petri dish with a diameter of 9 cm, and the adaxial side of the leaves was placed on the medium after it solidified, leaving no gap. Ten adult cotton aphids with of the same size were inoculated on cotton cotyledons, and the petri dish was inverted. Upon aphid feeding on cotton cotyledon, honeydew was collected with Whatman filter paper at 24, 48 and 72 h, and each treatment was repeated three times. The content of honeydew was determined by ninhydrin staining. The collected filter paper was immersed in 0.1% (W/V) acetone solution of ninhydrin, and dried in the oven at 65 °C for 30 min until the honeydew on the filter paper showed purple spots, and the amount of honeydew of cotton aphid was measured (Kim and Jander 2007). To quantify the honeydew stains, stained filter papers were cut into pieces and extracted with 1 ml of 90% (v/v) methanol for 1 h at 4 °C with continuous agitation. The supernatant absorbance was measured at 500 nm after centrifugation at 5000×g for 1 min, with 90% methanol used as a blank (Nisbet et al. 1994).

Determination of POD, CAT and SOD activity
After cotton aphid feeding, cotton leaves from wild-type, control and treated plants were ground to fine powder and frozen in liquid nitrogen. 100 mg of the sample was mixed in extraction buffer and the mixture was collected in a centrifuge tube. Centrifugation at 4 °C and 14,000×g for 10 min, and the supernatant was the crude enzyme solution. POD, CAT and SOD activities were measured according to the enzyme activity kit (Suzhou Comin Biotechnology Co. Ltd., Jiangsu, China).

Expression analysis of defense pathway related genes after transiently overexpressing and silencing of GhlncRNA149.1
The cDNA from plants transiently overexpressing and silencing GhlncRNA149.1 were used as qPCR template, and cotton gene GhUBI1 (accession number: EU604080) was used as internal reference. The expression levels of POD, CAT , SOD, PAL, PPO, EDS1 and NPR1 in the defense signal pathway were detected.

RNA isolation and quantitative real-time PCR (qPCR)
Total RNA was extracted from cotton roots, stems and cotyledons using the Fast pure plant total RNA isolation kit (Vazyme, Nanjing, China) according to the manufacturer's instructions. Total RNA from each sample was treated with RNase free DNaseI (Promega, USA) and reverse transcribed into cDNA with transcriptase (Promega, USA) and was used as a template for quantitative real-time polymerase chain reaction (qPCR). The cotton gene GhUBI1 (accession number: EU604080) served as an internal control. The GhUBI1 primer sequences are shown in Table S1. The relative value of each gene was showed as mean values of four independent tests, and three replicates were performed for each test.

Statistical analysis
The difference between each treatment and control was statistically analyzed with t-test for independent samples. Two significant levels were used (*, P < 0.05 and **, P < 0.01), three significance levels were used (different letters, P < 0.05), the statistical significance of the difference among treatments was analyzed using one-way ANOVA. The data was analyzed using statistical software SPSS 26.0 (SPSS Inc., Chicago, IL, United States of America). Statistical significance was considered when the p-value was less than 0.05.

Identification of GhlncRNA149.1 and its target GhA01G0129 in cotton
In our previous study, we sequenced the whole transcriptome of cotton leaves induced by cotton aphid feeding and identified lncRNAs whose expression levels changed significantly upon cotton aphid infestation (Zhang et al. 2019a, b). Among these lncRNAs, GhlncRNA149.1 was chosen for further study, because its target genes were specific. GhlncRNA149.1 is 740 bp in length and is located on the sense chain of chromosome 1 in the A genome of Gossypium hirsutum L.. A bioinformatic analysis (http:// cpc2. gao-lab. org/) revealed that GhlncRNA149.1 has got Fickett score 0.35472 with a pI 9.69085693359. Therefore, it was classified as a non-coding sequence with coding probability of 0.0117256 (Fig. 1). The ORF Finder online tool was used to predict the GhlncRNA149.1 ORFs that ranged from 26 to 31 amino acids. Furthermore, according to homology searches of large protein families and domain databases (Pfam and SMART, respectively), no functional domains matching any of the peptides were found. According to the positional relationship between lncRNA and mRNA, that is, neighboring genes within 100 kb of lncRNA were its target genes, it was found that GhlncRNA149.1 had 19 cis-target genes (Table S2). GhA01G0129 attracts our insights because it belongs to the CC-NBS-LRR family gene, which is involved in plant insect resistance. Therefore, it is speculated that GhlncRNA149.1 may improve cotton resistance to cotton aphids by regulating the expression level of GhA01G0129.

The expression level of GhA01G0129, the target gene of GhlncRNA149.1, was induced by SA and MeJA
To analyze the role of GhlncRNA149.1 and its target gene GhA01G0129 in the defense response of cotton to cotton aphid, qPCR was used to determine the expression levels of GhlncRNA149.1 and its target gene GhA01G0129 in cotton tissues (cotyledons, stems and roots) at 24, 48 and 72 h after cotton aphids infestation, salicylic acid (SA) and methyl jasmonate (MeJA) treatment, respectively (Fig. 2). The results showed that compared with the control group, the expression levels of GhlncRNA149.1 and its target gene GhA01G0129 were significantly up-regulated in the roots and stems of cotton plant infested by aphids for 24 and 48 h, but revealed the opposite trend in cotyledons (Fig. 2a, d). In addition, GhlncRNA149.1 expression level was down-regulated at 24, 48 and 72 h after SA and MeJA treatment (Fig. 2b, c), but the expression level of its target gene GhA01G0129 significantly up-regulated overally (Fig. 2e, f). These results indicated that both SA and MeJA could induce the expression level of GhA01G0129 and ofthe target gene of GhlncRNA149.1. Values are means ± SD (n = 3). Independent t tests were carried out to demonstrate whether there were significant (*P < 0.05) or very significant (**P < 0.01) differences between Aphid infestation and Mock 1 3

The expression level of GhlncRNA149.1 was positively correlated with the expression level of GhA01G0129
To further explore the relationship between GhlncRNA149.1 and its target gene GhA01G0129, we measured the expression levels of GhlncRNA149.1 and its target gene GhA01G0129 in transiently over-expressed cotton plants for 24, 48 and 72 h and in continuously silenced cotton plants for 8, 15 and 22 days (Fig. 3). After continuous transient overexpression of GhlncRNA149.1 in cotton for 24, 48 and 72 h, the overall expression level of GhlncRNA149.1 was significantly higher than that of WT and pBI121 plants (Fig. 3a), especially at 24 h, the expression level of GhlncRNA149.1 was 4.26 times higher than that of pBI121 plants. The expression level of GhA01G0129 in GhlncRNA149.1 transiently overexpressed cotton was about twice that of pBI121 (Fig. 3c). Moreover, the expression level of GhlncRNA149.1 in cotton was significantly lower than that in WT and TRV:00 cotton plants after 8, 15 and 22 days of continuous silencing of GhlncRNA149.1 (Fig. 3b). The expression level of its target gene GhA01G0129 was also down-regulated in cotton plants silencing GhlncRNA149.1 (Fig. 3d). These results suggest that the expression level of GhlncRNA149.1 may be positively correlated to the expression level of GhA01G0129.

The GhlncRNA149.1 enhanced the tolerance of cotton plants to cotton aphids
In order to further verify the regulatory role of GhlncRNA149.1 in the defense response of cotton to aphid feeding, we conducted aphid bio-assays on cotton plants transiently over-expressing and silencing GhlncRNA149.1. The results of no-choice assay and dual-choice assay showed that the number of aphids in cotton transiently overexpressing GhlncRNA149.1 was significantly lower than that in WT and pBI121 cotton at 24, 48 and 72 h after infestation (Fig. 4a, b). Moreso, the honeydew content from cotton GhlncRNA149.1. One-way ANOVA analyzed data. Error bars represent the SD of three biological replicates.Tukey's HSD test showed that different letters indicated significant differences among treatments (P < 0.05) aphids that fed on the leaves from pBI121-GhlncRNA149.1 cotton plants was very significantly less than that on WT and pBI121 leaves at 24, 48 and 72 h after aphid feeding (Fig. 4c,  d). These results indicated that GhlncRNA149.1 transiently overexpression could enhance cotton plant resistance to cotton aphids. In addition, this conclusion was further verified by the no-choice assay, dual-choice assay and aphid honeydew quantification of aphids feeding on GhlncRNA149.1silenced cotton plants. At 15 days after GhlncRNA149.1 gene silencing in cotton plants, the number of aphids on cotton plants at 24, 48 and 72 h after feeding was significantly higher than that in WT and TRV:00 cotton (Fig. 4e, f). At the same time, honeydew secretion at 24, 48 and 72 h after aphid feeding was also significantly higher than that of WT plants and TRV:00 cotton leaves (Fig. 4g, h).

GhlncRNA149.1 regulates the expression of ROS scavenging genes
Since CAT, SOD and POD are closely related to insect resistance in plants (Liu et al. 2021;Qasim et al. 2021), we measured the changes of enzyme activities and expression levels of CAT, SOD and POD in cotton strains transiently overexpressing and silencing GhlncRNA149.1. In cotton plants transiently overexpressing GhlncRNA149.1, the expression levels of SOD, POD and CAT and enzymatic activities showed an up-regulated trend (Fig. 5a, c). On the other hand, the expression levels and enzyme activities of SOD, POD and CAT in cotton plants silencing GhlncRNA149.1 showed a downward trend (Fig. 5b, d). These results showed that GhlncRNA149.1 may regulate the expression of SOD, POD and CAT of ROS scavenging genes in cotton response to cotton aphid damage.

GhlncRNA149.1 regulates the expression of genes related to defense pathway in cotton
In order to clarify the defense mechanism of GhlncRNA149.1 in cotton defense response to cotton aphids, the expression levels of genes involved in plant defense response to aphids such as PAL, PPO, NPR1 and EDS1 were detected by qPCR.
The experimental results showed that the transcript levels of PAL, PPO, EDS1 and NPR1 in GhlncRNA149.1 cotton were significantly higher than those in WT cotton and pBI121 cotton (Fig. 6a). The transcription levels of PAL, PPO, EDS1 and NPR1 in silenced GhlncRNA149.1 cotton were down-regulated compared with WT cotton and TRV:00 cotton. (Fig. 6b). These results indicate that GhlncRNA149.1 may regulate the expression of PAL, PPO, NPR1 and EDS1 related genes in cotton defense pathway.

Discussion
With the rapid development of sequencing technology, more and more plant lncRNAs have been identified and studied. However, there are few studies on the functions and Error bars represent the SD of three biological replicates.One-way ANOVA analyzed data. Error bars represent the SD of three biological replicates. Tukey's HSD test showed that different letters indicated significant differences among treatments (P < 0.05) mechanisms of lncRNAs in plant response to phytophagous insect damage. Therefore, in this study, on the basis of the whole transcriptome sequencing of cotton leaves induced by cotton aphid feeding, a cotton lncRNA GhlncRNA149.1 was cloned and performed a preliminary study of its function and mechanism in cotton defense response to cotton aphid feeding. The results are of great significance for enriching the functions and mechanisms of lncRNAs.
It is known that the most important characteristic of lncR-NAs is that they lack encoding protein potentials. Therefore, it is very important to determine whether lncRNA has coded potential. CPC (Coding Potential Calculator), CNCI (Coding-Non-Coding Index), CPAT (Coding Potential Assessment Tool), Pfam Microsoft are the most widely used methods for coding potential analysis (Kong et al. 2007;Sun et al. 2013;Wang et al. 2013;Finn et al. 2014). In the previous study, GhlncRNA149.1 could be analyzed and predicted using these four methods. The result showed that GhlncRNA149.1 is a lncRNA without proteinencoding function. In addition, according to the positional relationship between lncRNA and mRNA, we also found that GhA01G0129 was a cis-target gene of GhlncRNA149.1 (Zhang et al. 2019a, b). Analysis of GhA01G0129 sequence showed that GhA01G0129 was a gene of a plant NBS-LRR family. The previous studies revealed that plant NBS-LRR proteins were associated with plant resistance to pathogens and pests. And what's more, many NBS-LRR proteins could recognize effectors secreted by pathogens directly or indirectly that in turn activate downstream signaling pathways leading to activation of plant defense response against various classes of pathogens, including bacterial, fungal, viral and nematode (Dubey and Singh 2018;Du et al. 2009). Therefore, it is possible that GhlncRNA149.1 may improve the cotton defense response to cotton aphids.
The various gene expression levels in plants often changes greatly in the face of all sorts of environmental stresses (Santamaria et al. 2018). Aphid, as a worldwide insect pest, often poses a serious threat to the safe production of crop (Jiao et al. 2021). Under aphid infestation, plant genes must respond to the stress caused by aphids (Züst and Agrawal 2016). Our study showed that the expression levels of GhlncRNA149.1 and its target gene GhA01G0129 in cotton root, stem and leaves were influenced to different extents under cotton aphid infestation, SA or MeJA treatment. These preliminary results show that GhlncRNA149.1 and its target gene GhA01G0129 do respond to the induction of cotton aphids, SA and MeJA. It is worth noting that their expression levels in cotyledons were inhibited by aphid feeding and these results are consistent with previous studies Zhong et al. 2021).
Previously thought to be dark matters of the genome, lncRNAs have been gradually recognized as crucial gene regulators in plant growth and response to all kinds of biotic and abiotic stresses Zhang and Chen 2013). Functional studies on lncRANs involves the gene knockdown and overexpression of lncRNA. For example, overexpressing lncRNA LAIR increases grain yield and regulates neighbouring gene cluster expression in rice (Wang et al. 2018). In our studies, the function of GhlncRNA149.1 were verified from positive and negative aspects of using overexpression and virus-induced gene silencing (VIGS) methods. The results from dualchoice, no-choice and honeydew secretion assays showed that overexpression of GhlncRNA149.1 enhanced cotton plant resistance to cotton aphids. Conversely, silencing of GhlncRNA149.1 weakened the resistance of cotton plants to cotton aphids. In short, the results indicated that GhlncRNA149.1 positively regulated cotton defense response to cotton aphids.
LncRNAs can not only directly regulate the expression of DNA, protein and miRNA, but also indirectly regulate the expression of other genes in plants. For instance, ELENA1 has also been found to bind to MED19a to regulate PRI expression in response to pathogen stress and to increase plant immune response (Seo et al. 2019). In our study, we demonstrated that the expression level of GhlncRNA149.1 was positively correlated to the expression level of its target gene GhA01G0129. The activities and expression levels of SOD, POD and CAT of ROS scavenging genes were up-regulated in pBI121-GhlncRNA149.1 cotton plants. On the other hand, activities and expression levels of SOD, POD and CAT in plants silencing GhlncRNA149.1 showed a downward trend. In addition, the transcript levels of PAL, PPO, EDS1 and NPR1 were significantly up-regulated in leaves from pBI121-GhlncRNA149.1 cotton plants, and the opposite results were obtained in cotton plants silencing GhlncRNA149.1. These results showed that GhlncRNA149.1 may regulate the expression of many defense-related genes in cotton response to cotton aphid damage.
In short, our results indicated that transient overexpression of GhlncRNA149.1 could increase the cotton plant resistance to cotton aphids, while silencing of GhlncRNA149.1 can reduce the tolerance of cotton plants to cotton aphids. GhlncRNA149.1 and its target gene GhA01G0129 positively regulated the cotton response to cotton aphid feeding together. In addition, some ROS-scavenging genes and PAL, PPO, EDS1 and NPR1 were regulated in GhlncRNA149.1 transiently overexpressed cotton plants and GhlncRNA149.1 silenced cotton plants. In this study, the role of GhlncRNA149.1 in the defense response of cotton to aphid damage was clarified, and the regulatory mechanism of GhlncRNA149.1 in the defense response of plants to aphids was initially explored, which provided candidate genes for breeding aphid resistant cotton and promoted the development of aphid control. However, due to the complex 1 3 interactions between plants and aphids, there is need to further study the regulatory network of GhlncRNA149.1 and its effect on cotton aphids.

Conclusion
This study showed that GhlncRNA149.1 is a positive regulator of cotton defense response to cotton aphid. GhA01G0129 from NBS-LRR family is the cis-target gene of GhlncRNA149.1. Exogenous SA and MeJA treatment significantly increased the expression level of GhA01G0129. Expression levels of GhlncRNA149.1 and its target gene GhA01G0129 were positively correlated. The results of choice assay, no-choice assay and cotton aphid honeydew assay showed that GhlncRNA149.1 response to cotton aphid infestation. Subsequent analysis showed that GhlncRNA149.1 not only enhanced PAL, PPO, EDS1, NPR1 and other defense-related enzymes, but also participated in the antioxidant enzyme system, which improved the resistance of upland cotton to cotton aphids. Our results provide an important reference in investigating the role of GhlncRNA149.1 in cotton aphid resistance.