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Article

Comprehensive Bioinformatics and Expression Analysis of the TLP Gene Family Revealed Its Role in Regulating the Response of Oryza sativa to Nilaparvata lugens, Laodelphax striatellus, and Jinggangmycin

by
Sheraz Ahmad
,
Haowen Zhu
,
Yu Chen
,
Chuanyuan Xi
,
Amir Zaman Shah
and
Linquan Ge
*
School of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, China
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(6), 1297; https://doi.org/10.3390/agronomy12061297
Submission received: 3 May 2022 / Revised: 22 May 2022 / Accepted: 25 May 2022 / Published: 28 May 2022
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Abstract

:
Thaumatin-like proteins (TLPs), which belong to pathogenesis-related (PR) protein family 5 (PR5), are involved in the plant host defense and developmental processes. Detailed information regarding the TLP gene family in rice remains elusive. Here, we identified 43 OsTLP genes from the rice genome database. The in silico analysis comprised of the evolutionary tree, gene structure, conserved motifs, and chemical properties highlighted the key aspects of the OsTLP genes. By phylogenetic analysis, the OsTLP genes were classified into ten groups (OsTLPI-OsTLPX). Gene ontology (GO) analysis showed that OsTLP genes regulate plant growth and response to various environmental stimuli. Moreover, most of the OsTLP genes are expressed in almost all tissues. Microarray data showed differential expression trends of OsTLP genes under salinity stress and hormonal treatments, whereas under BPH, SSB, and RFL, only OsTLP1, OsTLP2 and OsTLP27 were expressed. The qRT-PCR analysis showed OsTLP27 as the prominent gene, displayed by its upregulated expression under JGM. Our results provide detailed knowledge of OsTLP gene resistance in rice plants, and we believe the current study will facilitate the development of cultivars resistant to biotic/abiotic stress, particularly stress caused by the chewing and sucking of insect pests.

1. Introduction

The static nature of plants entails the frequent endurance of numerous environmental stresses, such as drought, salinity, heat, and other biotic stressors [1]. Plants exhibit numerous key factors that provide response to various environmental stimuli. Among these are antioxidants enzymes, ROS (reactive oxygen species), anti-microbial compounds (e.g.,phytoalexins), proline, sugars, and pathogenesis-related (PR) proteins [2]. The serological and biochemical properties of PR proteins have been classified into 17 different families, including b-1,3-glucanases (PR-2), chitinases (PR-3, 4, 8, and 11), thaumatin-like proteins (TLPs), or osmotin (PR-5), proteinase-inhibitor (PR-6), endoproteinase (PR-7), peroxidase (PR-9), defense (PR-12), thionins (PR-13), and lipid transfer proteins (PR-14) [3].
Plant thaumatin-like proteins (TLPs) belong to the PR-5 proteins family, a sweet-tasting protein discovered as a combination of proteins isolated from the katemfe fruit, Thaumatococcus daniellii (Bennett), in West Africa [3,4]. The TLPs have 16 cysteine residues, which may evolve from eight disulfide linkages [5]. Further, these structures stabilize the protein that resists high pH, proteases, and temperature-induced denaturation of various biomolecules [5]. The small TLPs, with 10 conserved cysteine residues, were identified in various monocotyledonous and coniferous plant species [6,7].
TLPs play various key roles in the plant defense system against biotic/abiotic stresses and in various physiological and developmental processes [7]. For example, the over-expression of the Camellia sinensis (CsTLP) gene induced stress resistance in transgenic potatoes against late blight disease caused by the pathogenic fungi Phytophthora infestans and Macrophomina phaseolina [8]. TLP also plays a role in plant stress biology, such as that the TLP produces antifreeze protein in leaves of cereals during cold weather and collectively provides cold resistance [9]. However, the TLPs were also reported to have a vital role in biological processes such as seed germination [10], fruit ripening [11], and antimicrobial activity [12].
Rice (Oryza sativa L.) is one of the world’s staple foods and is widely cultivated, feeding almost half of the world’s population [13]. Rice is cultivated in more than 100 countries, with more than 700 million tons produced each year [14]. Because its easy availability and nutritional value lead to high consumption, it is estimated that rice production must be increased by 1% annually to fulfill the growing population’s needs [15]. However, the rice crop is exposed to various environmental stresses, and the combined effects lead to a huge yield loss [16]. The abiotic stresses include high/low temperature, drought, high salinity, low nutrient availability, flooding, etc., and biotic stresses include insect pests, herbicide toxicity, and bacterial, viral, and fungal infections that result in drastic yield loss [17].
More than 800 insect pests feed on rice, causing direct and indirect infections [18]. Among these, 65 species of planthoppers were recorded to be associated with the rice ecosystem in which the brown planthopper (BPH) Nilaparvata lugens Stål (Hemiptera: Delphacide) and small brown planthopper (SBPH) Laodelphax striatellus (Hemiptera: Delphacidae) are recognized as key pests [19,20]. The BPH and SBPH are severe pests in rice, causing a huge loss in annual rice production through direct infestation and transmission of several viruses, such as rice ragged stunt virus (RRSV), Southern rice black-streaked dwarf virus (SRBSDV), and rice grassy stunt virus (RGSV) [21,22,23]. The antibiotic jinggangmycin (JGM) was developed in China and is commonly applied 2–3 times per crop cycle in rice fields for controlling rice sheath blight disease (Rhizoctonia solani) [24]. However, JGM is also reported to induce BPH fecundity. In addition, several spraying treatments, such as root zone application, leaf spray, and topical application, among the JGM foliar sprays, are highly effective in inducing changes in rice physiology and biochemistry that lead to enhanced BPH fecundity [25]. In addition, JGM also enhances BPH flight capacity, body weight, thermotolerance, protein, and lipids contents [24].
Owing to the importance of the TLP gene family linked with developmental, physiological, various defense responses, and the diversity of TLP gene members in many plant species, it is critical to investigate the global status and evolution of the TLP gene family in rice and other cereals crops to maintain the food and brewing industry supply chain. However, little information exists regarding the role of the TLP gene family in rice, particularly the OsTLP genes response against the BPH and SBPH infestation and the JGM applications that stimulate the rice’s immune response. Herein, we performed comprehensive bioinformatics and expression analyses to investigate the involvement of OsTLP genes in rice growth and developmental processes. Additionally, the participation of OsTLP genes in boosting rice plant immunity against various stresses has been studied.

2. Materials and Methods

2.1. OsTLP Gene Family Member Identification and Sequence Analysis

The protein sequence of the TLP genes family was retrieved from the Arabidopsis thaliana [26] and rice [27] using the Hidden Markov model (HMM). Further, the extracted rice protein sequences were evaluated using the CD-search server (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, http://www.ebi.ac.uk/interpro/search/sequence/) (accessed on 10 February 2022) along with SMART databases (http://smart.embl-heidelberg.de/) (accessed on 10 February 2022). Protein that did not display thaumatin-like proteins (TLPs) was disregarded. Additionally, the chemical properties of OsTLP protein were examined by employing the (Swiss Institute of Bioinformatics, Zurich, Switzerland) originated Expasy online server (http://web.expasy.org/protparam/) (accessed on 10 February 2022). Finally, the OsTLP gene predicted subcellular location was displayed using the online server CELLO2GO [28].

2.2. Phylogenetic Tree, Motif, and Digital Expression Analysis

We used theClustalW 2.0 European Molecular Biology Laboratory, Cambridgeshire, CB10 1SD, UK (http://www.genome.jp/tools/clustalw/) (accessed on 16 February 2022) originated 01 July 2003 at Bioinformatics Center, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan, to analyze the sequences and construct the maximum-likelihood phylogenetic, and MEGA (Version 7.0) [29]. The coding sequences of each OsTLP gene were obtained from the rice genome database. The conserved motif of OsTLP genes was predicted with the online MEME server (Version 4.12.0) (http://meme-suite.org/tools/meme) (accessed on 16 February 2022). Further, for the heatmap, the TBtools developed by South China Agricultural University, Guangzhou, China, were used, and the transcriptomic data was retrieved from the rice genome database and GEO dataset platform in NCBI [27].

2.3. Cis-Elements and Gene Ontology of OsTLP Genes

For each OsTLP gene, 1.5 kb of upstream genomic DNA sequence with the starting codon (ATG) was obtained from the rice genome sequence database. We used the plantCARE database, Gent, Belgium (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 18 February 2022)to identify the cis-regulatory elements in the promoters of 10 OsTLP genes. Further, the gene ontology (GO) analysis of OsTLP protein sequences was obtained from the Blast2GO program, Valencia–ES-Spain (Version 2.7.2) (http://www.blast2go.com) (accessed on 18 February 2022); the resulting three groups, molecular functions, biological process, and cellular component GO classification, were recovered.

2.4. Interactive Protein Network Analysis of OsTLP Genes

The Swiss Institute of Bioinformatics, Zurich, Switzerland, developed online server string (https://string-db.org) was (accessed on 3 March 2022) to identify the interactive proteins with OsTLP proteins [30]. The Oryza sativa protein (OsTLP1) was selected as a reference protein, and the interactive proteins network was recovered.

2.5. Insect Rearing, Chemical and Stress Treatment

In the current study, we used the rice Ninjing4 and Wuyujing-3 varieties of Oryza sativa. These varieties are commonly planted in Jiangsu Province of China because they have no resistance to the Nilaparvata lugens (Stål) brown planthopper (BPH) and the Laodelphax striatellus (Fallén) small brown planthopper (SBPH) [31,32]. The seeds were sown indoors in a plastic tray in a standard size of ¼ 60 cm H _ 100 cm W _ 200 cm L), and after germination, the six-leaf seedlings were transplanted into plastic pots in dimensions R ¼ 16 cm. Next, the rice plants were used for further experiments at the tillering stage (40 ± 2 days).
We used the BPH and SBPH strains in the study, obtained from the China National Rice Research Institute Insect Repository (Hangzhou, China). The BPH strain was reared on the rice plants in cement tanks, covered with fine mesh, under natural conditions from April to October. Further, the BPH strain was overwintered in an insectary at the Ecological Laboratory of Yangzhou University on Ninjing4 variety rice seedlings under standard conditions of 26 ± 2 °C with the 16 h l: 8 h d in relative humidity of 80 ± 10% [32].
The SBPH strain was reared on 10–15 days old seedlings of the Wuyujing-3 rice variety in a laboratory. A plastic frame cage, with dimensions of 50 cm in length and width (L:W), was used in a growth chamber with standard temperature conditions of 26 ± 2 °C, 70–80% relative humidity (RH), and a 10:14 photoperiod. Further, a nylon mesh cloth with a zipper prevented the SBPH from escaping. The rice seedlings were replaced every 10–14 days to ensure sufficient nutrition for SBPH feeding [31].
Technical grade fungicide jinggangmycin (JGM) (61.7%) was purchased from the Qianjiang Biochemistry Co. Ltd. (Haining, Zhejiang Province, China). The 200 parts per million (PPM) JGM solution was prepared with Tween 20, purchased from Sinopsin Group Chemical Reagent Co. (Shanghai, China). As previously reported by Ge et al. (2010), the third instar of both BPH and SBPH was used in the experiments [32].

2.6. Expression Profiling of TLP Genes in Oryza sativa

Forty-day-old rice plants were exposed to BPH and SBPH stress, and samples were taken at 2, 4, and 8 days after infestation. Similarly, JGM was sprayed on the rice plants, and samples were taken at 2, 4, and 8 days after treatment. Following that, RNA was extracted from the samples using Trizol reagent (Invitrogen, Carlsbad, CA, USA). First, the DNA was removed using DNase I, the concentration and purity was measured with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Rockford, IL, USA), and the integrity was checked using 1.5% agarose gel electrophoresis. Following the manufacturer’s instructions, 1 μg of total RNA was reverse-transcribed on a thermocycler programmed at 37 °C for 15 min using the PrimeScriptTM RT Master Mix (TaKaRa, Dalian, China) containing oligo dT primer.
Two μL aliquots of cDNA were amplified by qPCR in 20 μL reaction volumes using the SYBR Premix Ex TaqTM II (TaKaRa, Dalian, China). The cDNAs were amplified at 95 °C for 2 min, followed by 35 cycles of 10 s at 95 °C for 30 s, and 72 °C for 30 s, with a final extension step of 72 °C for 10 min in a CFX96 real-time PCR system (Bio-Rad Co. Ltd., Hercules, CA, USA). The mRNA amounts of all genes were separately quantified with the stable expression of the constitutive reference gene, actin. The specific primers are detailed in Table S1. After amplification, the target gene cycle threshold (Ct) values were normalized to the reference gene using the 2−ΔΔCT method [33]. The data mean values of three biologically independent replicates were used for the final graphs.

2.7. Statistical Analysis

The SPSS software (Version 25.0, SPSS Inc., Chicago, IL, USA) was used for statistical analysis (ANOVA) and statistical significance, and a 95% confidence interval (p ≤ 0.05) was used. The data were analyzed and expressed as means ± standard deviation (SD) of the three biologically independent replicates in all measured parameters, and GraphPad Prism (Version 8.0.2) (GraphPad Software, Inc., LA Jolla, CA, USA) was used for graphical representation.

3. Results

3.1. Identification and Sequence Analysis of TLP Genes in Oryza sativa

We retrieved 43 OsTLP genes encoding the TLPs protein sequence from the rice genome database [27]. Genes that are not possessing THAUMATIN domain were excluded from the list. The domain structure of OsTLP genes has been presented in (Table S2). All the genes were nomenclatured as OsTLP1 to OsTLP43 (Table 1). Among 43 OsTLP genes, 37 OsTLP genes were located extracellularly, four resided in the nucleus, and only two were found in the plasma membrane. Additionally, various other features of Oryza sativa OsTLP protein were identified, such as chromosomal coordinates, molecular weight, chemical properties, and isoelectric point (PI) and are listed in Table 1.

3.2. Sequence Alignment and Evolutionary Relationship of TLP Genes in Oryza sativa

A maximum-likelihood phylogenetic tree was created following alignment to learn more about the evolutionary relationship between Oryza sativa and Arabidopsis thaliana thaumatin-like proteins (Figure 1). The protein sequence of 43 OsTLP and 28 AtTLP [26,27] genes constructed the phylogenetic tree. First, all the sequences were aligned using ClustalX software, with default parameters, then the phylogenetic tree was constructed using MEGA6 software, and the final tree was programmed using the Biobyte Solutions Company; Heidelberg, Germany, developed Interactive Tree Of Life (iTOL) (version 5) https://itol.embl.de/ (accessed on 10 March 2022) [34]. Additionally, the OsTLP protein was clustered into ten subgroups based on their phylogenetic relationships (Group I, Group II, Group III, Group IV, Group V, Group VI, Group VII, Group VIII, Group IX, and Group X). We found that among the ten sub-groups, the rice TLP protein was present in all subgroups, with different numbers of proteins, such as Group X, which contained 8 proteins, followed by Group I, Group III, and Group IX, which had 6 proteins. Further, Group II and Group VII had the third-highest number, at 5 proteins, whereas Group V, Group VI, and Group VII were found to have the least 2, while Group IV had only a single protein.

3.3. Gene Structure and Motif Compositions Analysis of TLPs in Oryza sativa

We identified the exons-introns distribution through CDS and genomic sequence of OsTLP genes using gene structure display (GSDS 2.0) [35]. The analysis revealed that the TLP genes family contains multiple exons and has varied intron lengths (Figure 2). Furthermore, the OsTLP11, OsTLP15, and OsTLP18 have the highest number of 5 exons and 4 introns, followed by OsTLP26, OsTLP27, OsTLP6, and OsTLP7, having 5 exons and 2 introns. In addition, OsTLP12, OsTLP19, OsTLP20, and OsTLP29 have 4 exons with a single intron, whereas OsTLP22, OsTLP24, and OsTLP25 possess 3 exons and 2 introns. Furthermore, OsTLP3, OsTLP8, OsTLP9, OsTLP10, OsTLP16, OsTLP31, OsTLP32, OsTLP35, OsTLP36, OsTLP37, OsTLP40, OsTLP41, and OsTLP42 have 3 exons without introns, whereas OsTLP28 has 3 exons with a single intron. Furthermore, the 4 OsTLP genes, such as OsTLP4, OsTLP5, OsTLP14, and OsTLP33, were found to have 2 exons and a single intron, whereas OsTLP5, OsTLP38, OsTLP39, and OsTLP43 had 2 exons. Three genes, including OsTLP13, had 4 and 3 exons-introns, followed by OsTLP17, with 4-2, and OsTLP, with 3-1 exons and introns, whereas OsTLP34 possessed only a single exon.
To further study the phylogenetic relationship of OsTLP in Oryza sativa, conserved motifs were identified using the University of Nevada, Reno, Nevada, USA; University of Washington, Seattle, WA, USA; University of Queensland, Brisbane, Australia and University of California, San Diego, CA, USA) developed MEME server (Version 5.4.1) https://meme-suite.org/meme/ (accessed on 15 march 2022) [36] (Figure 3) and identified motif of OsTLP genes in Oryza sativa (Table S3). The motifs were variously allocated on the OsTLP genes. Among these, OsTLP25, OsTLP28, OsTLP7, OsTLP6, OsTLP27, OsTLP17, OsTLP23, OsTLP19, OsTLP26, OsTLP20, OsTLP22, OsTLP24, OsTLP33 exhibit 9 motifs each, followed by OsTLP10, OsTLP8, OsTLP1, OsTLP29, OsTLP43, and OsTLP38 with 8 of motifs. Further, OsTLP36, OsTLP32, OsTLP42, OsTLP40, and OsTLP39 were observed with 6 motifs. Several members of the OsTLP gene family were found with fewer motifs, such as OsTLP18, OsTLP13, and OsTLP4 with 4 motifs, OsTLP4 with 2 motifs, and OsTLP5 with a single motif.

3.4. Interactive Analysis of OsTLP in Rice

The interactive protein study unfolds the numerous interacting partners of OSTLP1 (Figure 4) and their interactive protein partners (Table S4). The ERF2 had high interactions with our reference gene OSTLP1, which contributes to petunia waterlogging tolerance by suppressing programmed cell death and an alcoholic fermentation system [37]. Additionally, our reference OSTLP1 protein was highly associated with the LTP family and involved in numerous biological processes, including plant growth, development, and response to biotic and abiotic stresses [38]. Additionally, the polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the pectin-depolymerizing activity of polygalacturonases secreted by microbial pathogens and insects [39]. These ubiquitous inhibitors have a leucine-rich repeat structure that is strongly conserved in monocot and dicot plants. Finally, the interaction between OsTLP1 with NBS-LRR proteins was observed. The NBS-LRR are plant stress-specific proteins that play a vital role in regulating immunity [40].

3.5. Gene Ontology (GO) Analysis

Gene ontology (GO) enrichment pathway analysis showed various key functions of OsTLP genes in Oryza sativa. Three different functional parameters were analyzed (biological, molecular, and cellular processes). According to the predicted biological processes, OsTLP genes play a crucial role in growth-related activities via hormonal and metabolic modulation (Figure 5). Additionally, the response to external stimuli and the cellular analysis proved that most of the OsTLP genes reside extracellularly, and several nuclear genes were present in OsTLP gene family and could be key to various activities. At the same time, OsTLP genes are involved in DNA binding activities, as shown in the molecular functions pie chart.

3.6. Identification of OsTLP Genes Cis-Elements

The upstream region of OsTLP genes possesses various stress, hormones, light, and growth related cis elements. Here we identified forty-three cis-regulatory elements. Among these 43 cis-elements, 4 were related to light response, 17 were related to stress response, 12 were related to hormonal response, and 10 to growth and development (Table S5).

3.7. Microarray Expression Analysis of TLP Genes in Rice Tissues in Developmental Stages Biotic/Abiotic Stresses and under Hormonal Applications

3.7.1. Expression Analysis of OsTLP Genes in Developmental Stages

We examined the different developmental stages and tissue-specific expressions to study the biological roles of OsTLP genes in rice plant growth and development based on a set of transcriptomic data retrieved from the National Institute of Agrobiological Sciences Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan RiceXPro expression database (Version 3.0) https://ricexpro.dna.affrc.go.jp/ (accessed on 20 March 2022) [41]. The microarray expression data analysis of the OsTLP gene family members in twelve different tissues is presented in a heatmap, with blue to red colors reflecting the expression percentage (Figure 6). Among OsTLP 43 genes, nine members (OsTLP10, OsTLP36, OsTLP47, OsTLP38, OsTLP39, OsTLP40, OsTLP41, OsTLP42, and OsTLP43) were dominantly expressed in roots, leaf blades, and leaf sheaths, whereas no expressions were found for OsTLP21, OsTLP23, and OsTLP25 in any of the developmental stages. However, we found eight OsTLP family members (OsTLP7, OsTLP8, OsTLP9, OsTLP10, OsTLP12, OsTLP16, OsTLP40, and OsTLP43) with a high transcript in the roots. Furthermore, a deficient transcript level was detected for OsTLP3, OsTLP5, OsTLP9, OsTLP12, OsTLP16, OsTLP26, OsTLP27, and OsTLP28, among all the studied tissues. Moreover, we observed the expression profile of OsTLP3, OsTLP10, OsTLP42, and OsTLP43 among all tissues, apart from the pistil, anther, and endosperms. The expressions analysis found that most of the OsTLP gene family members were expressed in the roots, except for OsTLP19, OsTLP21, OsTLP23, and OsTLP25.

3.7.2. Expression Analysis of OsTLP Genes under Salinity

To further examine the response of OsTLP genes to saline conditions, the expression profile obtained from the publicly available NCBI database (GSE102152) [42] was analyzed in the form of a heatmap. As presented in the heatmap, various OsTLP genes displayed high/low transcription under SWR_CK (Seawater) and SWR_NaCl. The dark orange color on the scale bar represents high expression in the heatmap, whereas the light orange is moderate, and the blue color represents extremely low or no expression (Figure 7A). In the OsTLP genes family, several members such as OsTLP3, OsTLP9, OsTLP11, OsTLP12, OsTLP13, OsTLP17, OsTLP19, OsTLP26, OsTLP29, and OsTLP40 were recorded with the highest expressions, and the OsTLP4, OsTLP22, OsTLP23, OsTLP31, OsTLP32, OsTLP33, OsTLP34, OsTLP39, OsTLP41, and OsTLP42 were found with moderate expressions. Furthermore, the SWR_NaCl was found to have upregulated expressions in eight genes, including OsTLP1, OsTLP5, OsTLP6, OsTLP16, OsTLP20, OsTLP24, OsTLP25, OsTLP27; while OsTLP36, and OsTLP12, OsTLP13, OsTLP17, and OsTLP29 were found to have moderate expressions in comparison with control, whereas half of the OsTLP genes displayed no transcription in response to saline conditions.

3.7.3. Expression Analysis of OsTLP Genes under Nilaparvata lugens and Chilo suppressalis

The transcriptomic expression data were obtained from NCBI (GSE167872) [43], and the analysis provided the possible role of OsTLP genes involvement in rice plant defense against brown planthopper (BPH), rice striped stem borer (SSB), Chilo suppressalis, and the combined SSB_BPH stresses (Figure 7B). Most of the OsTLP genes did not show expressions in response to the BPH infestation and SSB feeding stress. However, in response to BPH and SSB combined stress, only two genes (OsTLP2 and OsTLP27) showed dominant expressions, whereas the rest of the genes displayed extremely low or no transcriptions. The expression analysis unfolds the role of the TLP gene family in rice plants’ defense against pest infestations.

3.7.4. Expression Analysis of OsTLP Genes under Cnaphalocrocis medinalis

To explore the expression level of OsTLP genes under rice leaffolder (RLF) Cnaphalocrocis medinalis Guenée (Lepidoptera: Crambidae) feeding stress at 0, 1, 6, 12, and 24 h time points, the transcriptomic expression data were obtained from NCBI (GSE159259) [44], and the analysis unfolded that most of the OsTLP gene expression was unaffected, except OsTLP1 and OsTLP27, which show dominant expressions (Figure 7C). These expressions showed that the TLP gene family plays a vital role in the rice defense system against feeding insects.

3.7.5. Expression Analysis of OsTLP Genes under Jasmonic Acid and Brassinosteroids

Jasmonic acid (JA) and brassinosteroids (BRs) are important regulators of plant growth and developmental processes, and the role of OsTLP proteins in the hormonal signaling pathway is of great concern. To discover the potential role of OsTLP genes in response to JA and BRs, the expression data for the OsTLP genes were obtained from the National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan RiceXPro expression database (Version 3.0) https://ricexpro.dna.affrc.go.jp/ (accessed on 23 March 2022) [41]. In the expression analysis of JA, among 43 members of OsTLPs, 32 genes displayed expression levels at all time points (0 h, 1 h, 6 h, and 12 h) (Figure 8). Furthermore, OsTLP6, OsTLP10, OsTLP32, and OsTLP34 showed downregulated expressions at the beginning of the treatment, but gradually increased during the treatment time. Additionally, a higher expression was detected at 6 h, followed by 12 h (OsTLP7, OsTLP9, OsTLP23, and OsTLP28), 3 h, and 1 h (OsTLP12, OsTLP13, OsTLP17, OsTLP18, OsTLP20, OsTLP24, OsTLP25, and OsTLP26) with moderate expression (OsTLP3, OsTLP12, OsTLP14, OsTLP16, OsTLP29, and OsTLP32) with extremely low expressions in 0 h.
BRs are a group of steroid phytohormones with a wide range of biological activities. Here, the expression profile of the OsTLP genes under BRs unfolded the biological role of OsTLP genes (Figure 9). Among the 43 OsTLP gene family members, only 22 OsTLP showed expressions at all time points, suggesting the importance of the TLP gene family to hormonal changes. Among the OsTLP gene family, the OsTLP8, OsTLP10, OsTLP33, and OsTLP35 showed dominant indications at 6 h treatment, followed by OsTLP7, OsTLP9, OsTLP23, and OsTLP28 at 12hr, OsTLP12, OsTLP13 and OsTLP21 at 1 h, and OsTLP12, OsTLP27, OsTLP29, and OsTLP32 at 0 h, with moderate regulations. However, no transcription was observed for OsTLP10, OsTLP28, OsTLP29, OsTLP30, and OsTLP32 expressed in 1 h.

3.8. Differential Expression of OsTLP Genes in Response to Nilaparvata lugens, Laodelphax Striatellus Infestations, and JGM Spraying

To further study the response of OsTLP genes to BPH and SBPH infestations, and botanical fungicides applications, we performed the qRT-PCR to analyze the expression patterns at three time points of 2 d, 4 d, and 8 d treatments under BPH and SBPH infestation, and JGM spraying (Figure 10). The results unfolded that the response of ten candidate genes were expressed under all stress conditions. Under BPH infestation, these 10 OsTLP genes were found with moderate and high transcription under all time points. Particularly, the OsTLP36 at 4 d with high expressions of 20-fold, followed by OsTLP2, OsTLP22, and OsTLP29. Further, the OsTLP12 and OsTLP29 at 2 d infestations were observed with a dominant expressions level. However, OsTLP2, OsTLP10, OsTLP22, OsTLP27, and OsTLP33 showed moderate expressions. In addition, a similar expression pattern was observed under the SBPH infestation, in which the OsTLP2 at the 2 d time point showed high transcriptions of 11-fold, followed by OsTLP10, with a 0.5-fold expression; however, OsTLP19 at 4 d and OsTLP33 at 8 d showed upregulated expressions, and the rest of the genes were observed to have low transcriptions. Furthermore, the response of OsTLP genes was relatively higher than the BPH and SBPH at all time points, specifically of OsTLP27, OsTLP36, OsTLP22, OsTLP10, and OsTLP39, with dominant expressions, whereas the rest of the genes were observed with moderate transcription.
JGM is a synthetic antibiotic applied to treat rice sheath blight disease, and it is also reported to enhance BPH fecundity; therefore, we performed the qRT-PCR analysis to validate the OsTLP gene family response under JGM treatment (Figure 10). The obtained results revealed that the OsTLP genes, such as OsTLP36, OsTLP27, OsTLP29, and OsTLP22, presented upregulated expressions, whereas the OsTLP12 and OsTLP33 showed moderate expressions, and a single gene, OsTLP2, had a negligible expression pattern. These results suggest that the OsTLP genes participated in rice immunity regulation against the JGM spraying applications.

4. Discussion

Plants are often exposed to various harsh environmental conditions, such as biotic and abiotic stresses, during their entire life cycle, impairing their growth and development. In response to these environmental stimuli, plants activate various built-in immune machinery. Key stress marker genes are crucial in limiting the negative impact of stresses. Earlier, the thaumatin-like protein (TLPs) genes were reported to enhance plant immunity against pathogens and growth [7,16,17].

4.1. OsTLP Genes Are Widely Distributed in the Rice Genome

We identified 43 OsTLP genes in the rice genome, which is comparatively high compared to those identified in Arabidopsis thaliana 28 AtTLP [26]. The OsTLP were further grouped into ten subgroups based on their phylogenetic relationship (Figure 1). The concurrent results were previously reported by [45]. Further, the exon and intron structural differences provide indications of the divergence and duplications of a specific gene family [46]. In this current study, we found that the intron sizes of most OsTLP genes varied across subgroups. This OsTLP gene intron size variation could be linked to tendon duplication [47]. Furthermore, the size and number of exons were shown to be conserved among OsTLP gene subgroups (Figure 2). OsTLP genes have a conserved exon size, indicating that they have undergone few structural changes and may have conserved functional characteristics [47,48].

4.2. Biological and Molecular Characteristics of OsTLP Genes

The thaumatin-like protein gene family is involved in various biological processes such as responding to immune responses and biotic/abiotic stresses, hormonal signaling pathways transduction, protein binding, enzyme regulation, and hydrolase activities.
The gene ontology revealed the importance of OsTLP genes in an array of physiological and molecular functions (Figure 4). For instance, 71% of the genes modulated the biosynthesis and development of the secondary cell wall, whereas 29% of the genes occupied the extracellular location, regulating plant immunity, providing the first line of defense in biotic/abiotic factor suppression [49,50]. The study reveals that the thaumatin-like protein gene family is important for maintaining appropriate rice plant development under both normal and stressed situations.
The cis-elements, including enhancers and promoters, have been proven to be the regulatory mechanism of gene expression and function [51,52]. By identifying the cis-element, it is critical to provide the source of morphological evolution influenced by gene function diversity [53,54]. In this study, several cis-elements of TLP genes were predicted in Oryza sativa. The majority of the key physiological activities are directly or indirectly controlled by cis-regulatory elements [55], such as fine tunning hormones or programming the trade-offs between stress and the growth of plants (Table S5). The major hormonal regulating elements, such as ABRE, ERE, TATC-box, TCA-element, ARR1, CARE, and NTBBF, were found in abundance in the promoter region of the OsTLP genes, thus implying their participation in growth-related activities. On the other hand, the presence of stress-related cis-elements such as MYC and WRKY, CTR/DRE, MYB, SEBF, SEBF, and BIHD1 increased the importance of OsTLP genes many times. These cis-elements indicate the stress and hormonal responsive nature of OsTLP genes and could be used as a potential biomarker to develop stress-resilient rice cultivars.
According to the protein–protein interaction network analysis, the OsTLP genes are homodimers and heterodimers at the same time, as they interact with other proteins to influence rice plant development. For instance, our reference gene, OsTLP1, interacts with ERF2; interestingly, ERF2 played a key role in multiple SA, JA, and ROS signaling pathways to confer stress resistance. In addition, the ERF2 may directly or indirectly regulate Pto, PR1b1, and PR-P2 expression and enhance resistance to pathogens [56]. Additionally, our reference OSTLP1 protein is highly associated with the LTP family and involved in numerous biological processes, including plant growth, development, and responses to biotic and abiotic stresses [38]. Finally, the interaction between OsTLP1 and NBS-LRR proteins was observed. The NBS-LRR are plant stress-specific proteins that play a vital role in regulating immunity [40].

4.3. OsTLP Genes Are Important to Rice Plant Growth and Development

The thaumatin-like proteins have been characterized as a highly complex protein family associated with plant host defense and developmental processes. TLPs exhibit various properties associated with their structural diversity, being mostly associated with responses against biotic stress, besides some predicted activity under drought and osmotic stresses [57]. In our study, most of the OsTLP genes showed expression in all tissues, highlighting their crucial role in plant growth and development (Figure 6). For instance, based on expression analysis, various members of the OsTLP gene family were found to be crucial in seed germination [10]. Additionally, the current study revealed that most of the OsTLP genes were dominantly expressed in root tissues, conferring their role in root developmental activity [41]. Similarly, the leaf blade, leaf sheet, and stem show regulated expressions, suggesting their critical role in development. Here, we observed most of the OsTLP genes in rice stem tissues, indicating the significant importance of this gene family in the organogenesis of leaves in rice plants. Moreover, the agronomic traits, including plant height, leaf angle, grain size, and tiller number, determine the fate of overall yield [58]. Modulations of these traits can significantly influence the yield [59]. In the present study, the majority of the OsTLP genes displayed expressions in all rice developmental stages, including the stem, panicle, anther, pistil, lemma, ovary, embryo, and endosperm; therefore, they is essential for regulating the growth and developmental activities of rice plants.

4.4. OsTLP Genes Regulate Plant Response to Chewing Insects and Other Abiotic Stresses

The TLP genes are the key players capable of rapidly accumulating to high levels in response to biotic/abiotic stress and exhibiting antifungal activity in various plant species [7]. Additionally, these biomolecules showed regulated responses to various exogenous chemicals, hormones, and biotic/abiotic stresses. However, the response of OsTLP genes against chewing and sucking insects remains elusive.
The BPH and SBPH are serious rice pests, inflicting damage on a massive scale across Asia [19,21]. Their nymphs and adults cause direct damage by feeding on phloem sap from the tillering to milking stages of rice and, in the process, transmit viral pathogens, such as rice ragged stunt virus (RRSV) and rice grassy stunt virus (RGSV) [60,61]. To study the response of OsTLP genes to BPH and SBPH infestations, we performed qRT-PCR analyses of ten candidate genes (a single gene from each subgroup) to validate their expressions. The results revealed the obvious response of OsTLP genes. Furthermore, among these ten candidate genes, OsTLP36, OsTLP12, OsTLP2, and OsTLP39 were observed with dominant expressions, and the rest of the genes with moderate expressions (Figure 10). These results uncovered the crucial role of OsTLPs in rice plant immunity in response to insect pest infestations.
The antibiotic jinggangmycin (JGM) was developed in China and is commonly applied 2–3 times in rice fields during the crop cycle for controlling rice sheath blight disease (Rhizoctonia solani) [24]. Furthermore, JGM has achieved obvious results in controlling sheath blight disease; however, it is also reported to induce BPH fecundity [24]. For example, JGM was applied to the rice plants at the rate of 200 parts per million (ppm), and the results revealed that JGM increased the resistance to rice sheath blight disease by disrupting the fungal cell wall and reducing sporulation [24]. However, JGM was also reported to enhance BPH fecundity [24]. In the current study, the OsTLP genes showed a differential expression pattern where the candidate genes from each subgroup, including OsTLP2, OsTLP22, OsTLP36, OsTLP23, OsTLP29, and OsTLP39, were dominantly expressed, whereas OsTLP10, OsTLP12, OsTLP19 and OsTLP33 were moderately expressed (Figure 10). We speculate that these OsTLP genes are essential for inducing plant immunity to fungal pathogens in rice plants. Further studies are required to elucidate the underlying mechanisms of OsTLP genes under insects pests infestations.
Jasmonic acid (JA) signaling plays a central role in plant defenses against necrotrophic pathogens and herbivorous insects, afflicting roots and shoots. This pathway is also activated following the interaction with beneficial microbes that may lead to induced systemic resistance [62]. Here, the OsTLP8, OsTLP10, OsTLP34, and OsTLP35 displayed upregulated expressions; on the other hand, most genes show a sharp decline in expression (Figure 8). Our results suggest that the OsTLP gene may be involved in JA-mediated plant growth activities; however, this area requires further study.
Brassinosteroids (BRs) are a group of polyhydroxylated steroidal phytohormones that are mandatory for plant development, growth, and productivity. Besides their significant involvement in growth-related activities, BRs are considered a key stress hormone [63,64,65]. Herein, the upregulated expressions of OsTLP8, OsTLP10, and OsTLP21 suggest the essential role of the OsTLP gene family and its possible participation in the immunity regulation of the rice plant (Figure 8).

5. Conclusions

The current study identified and analyzed 43 OsTLP genes in rice from the genome database; additionally, we performed a comprehensive analysis of OsTLPs that included gene identification, phylogenetic analysis, chromosomal location, interactive protein network analysis, cis-regulatory elements, gene differential expression analysis, etc. Based on their domain and structural characteristics, 43 TLPs from the thaumatin-like protein family were identified and classified into ten subgroups. Additionally, the heatmap analysis revealed the response of OsTLPs to biological and hormonal stress. The qRT-PCR analysis demonstrated the expression patterns under BPH and SBPH infestation and JGM applications in rice, suggesting that these genes may influence resistance to pest infestations. Stress regulation is also a complicated mechanism to understand. Moreover, stress regulation is a complex mechanism, but the in silico analysis provides valuable information for future functional studies in stress biology. As a result, further functional studies are required to fully understand the regulation and pathway mechanism of OsTLPs in Oryza sativa L.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12061297/s1, Table S1: Primer sequences for RT-qPCR; Table S2: Conserved domains of OsTLP gene family; Table S3: The identified motif of OsTLP genes in Oryza sativa; Table S4: Predictive interactive partners of OsTLP1 gene; Table S5: The predicted cis-acting elements in the 1.5 kb promoter region of OsTLP genes.

Author Contributions

S.A. and L.G. designed the research; S.A. and Y.C. conducted the experimental work; H.Z. and C.X. contributed to the preparation of biological materials; A.Z.S. performed the bioinformatics analysis, and S.A. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32072415), Agricultural Science and Technology Independent Innovation Project of Jiangsu Province (CX (20) 3123), and the Yangzhou University Interdisciplinary Research Foundation for Agronomy Discipline of Targeted Support (yzuxk202005).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We thank Rahat Sharif, Department of Horticulture, School of Horticulture and Plant Protection, Yangzhou University, for his critical manuscript revision.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic analysis of TLP. The phylogenetic tree was generated using the amino-acid sequences of selected TLPs via NJ methods. All O. sativa TLPs and their A. thaliana counterparts were classified into 10 groups.
Figure 1. Phylogenetic analysis of TLP. The phylogenetic tree was generated using the amino-acid sequences of selected TLPs via NJ methods. All O. sativa TLPs and their A. thaliana counterparts were classified into 10 groups.
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Figure 2. Schematic representation of the gene structure of TLP genes in Oryza sativa.
Figure 2. Schematic representation of the gene structure of TLP genes in Oryza sativa.
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Figure 3. Schematic representation of the conserved motif of TLP genes in Oryza sativa.
Figure 3. Schematic representation of the conserved motif of TLP genes in Oryza sativa.
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Figure 4. Interactive network of OsTLP1 protein.
Figure 4. Interactive network of OsTLP1 protein.
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Figure 5. Gene ontology (GO) enrichment analysis of TLP genes in Oryza sativa. The data are represented as biological processes, molecular processes, and cellular processes.
Figure 5. Gene ontology (GO) enrichment analysis of TLP genes in Oryza sativa. The data are represented as biological processes, molecular processes, and cellular processes.
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Figure 6. Heatmap of OsTLP genes representation in developmental stages, including leaf blade, leaf sheath, root, stem, inflorescence, pistil, lemma, plea, ovary, embryo, and endosperm. The log2 transformation method normalized and converted the RPKM values displayed.
Figure 6. Heatmap of OsTLP genes representation in developmental stages, including leaf blade, leaf sheath, root, stem, inflorescence, pistil, lemma, plea, ovary, embryo, and endosperm. The log2 transformation method normalized and converted the RPKM values displayed.
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Figure 7. Heatmap under salinity and biotic stresses. Heatmap (A) represents OsTLP gene expression in CK, SWR_CK, and SWR_NaCl. Heatmap (B) represents the OsTLP gene expressions in shoot tissues under BPH, SSB, and SSB_BPH. Heatmap (C) represents response to Cnaphalocrocis medinalis stress at different time points in Oryza sativa. The log2 transformation method normalized and converted the RPKM values displayed.
Figure 7. Heatmap under salinity and biotic stresses. Heatmap (A) represents OsTLP gene expression in CK, SWR_CK, and SWR_NaCl. Heatmap (B) represents the OsTLP gene expressions in shoot tissues under BPH, SSB, and SSB_BPH. Heatmap (C) represents response to Cnaphalocrocis medinalis stress at different time points in Oryza sativa. The log2 transformation method normalized and converted the RPKM values displayed.
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Figure 8. Heatmap of OsTLP genes representation under jasmonic acid. The log2 transformation method normalized and converted the RPKM values displayed.
Figure 8. Heatmap of OsTLP genes representation under jasmonic acid. The log2 transformation method normalized and converted the RPKM values displayed.
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Figure 9. Heatmap of OsTLP genes representation under brassinosteroids. The log2 transformation method normalized and converted the RPKM values displayed.
Figure 9. Heatmap of OsTLP genes representation under brassinosteroids. The log2 transformation method normalized and converted the RPKM values displayed.
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Figure 10. Differential expression analysis of OsTLP genes (OsTLP2, OsTLP10, OsTLP12, OsTLP19, OsTLP22, OsTLP27, OsTLP29, OsTLP33, OsTLP36, and OsTLP39) under Nilaparvata lugens and Laodelphax striatellus infestations and Jinggangmycin (JGM) spraying treatment in Oryza sativa. Different letters indicate significant difference.
Figure 10. Differential expression analysis of OsTLP genes (OsTLP2, OsTLP10, OsTLP12, OsTLP19, OsTLP22, OsTLP27, OsTLP29, OsTLP33, OsTLP36, and OsTLP39) under Nilaparvata lugens and Laodelphax striatellus infestations and Jinggangmycin (JGM) spraying treatment in Oryza sativa. Different letters indicate significant difference.
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Table
Table 1. The gene and protein features of TLP gene family members in Oryza sativa.
Table 1. The gene and protein features of TLP gene family members in Oryza sativa.
NameLocus IdSub-GroupChrStartEndCDSProteins (aa)Mw (kDa)PISL
OsTLP1LOC_Os01g02310.141728,977730,930188462868,677.46.6086EC
OsTLP2LOC_Os01g12150.1116,618,8236,616,73243214415,466.35.2417N
OsTLP3LOC_Os01g62260.12136,030,96936,029,78574424826,188.87.7208EC
OsTLP4LOC_Os02g02200.112676,466677,74273524525,910.410.5619PM
OsTLP5LOC_Os03g13070.1837,067,4317,066,55179826626,828.94.3398EC
OsTLP6LOC_Os03g14030.1937,614,0837,610,990101133733,963.57.3287EC
OsTLP7LOC_Os03g14050.1937,626,4617,629,68299933333,013.34.6412EC
OsTLP8LOC_Os03g45960.14325,973,47025,972,22569923324,090.16.1897EC
OsTLP9LOC_Os03g46060.14326,033,80226,032,94066922322,765.97.4059EC
OsTLP10LOC_Os03g46070.14326,035,3572,603,442669023023,769.74.3373EC
OsTLP11LOC_Os04g24130.11413,797,44713,794,142130243447,0937.8092PM
OsTLP12LOC_Os04g59370.18435,287,78135,289,15683727929,8637.6934EC
OsTLP13LOC_Os05g46110.11526,742,72326,739,397141347149,260.86.7655EC
OsTLP14LOC_Os06g16990.1169,858,16998,57,52336912313,156.99.3203EC
OsTLP15LOC_Os06g19250.11610,955,71010,957,53767222424,069.19.0395N
OsTLP16LOC_Os06g47600.16628,825,83028,827,03675625225,634.28.3328EC
OsTLP17LOC_Os06g50240.110630,420,07830,422,99895731931,8575.1652EC
OsTLP18LOC_Os07g04730.1172,103,6332,099,058208869670,7828.7916N
OsTLP19LOC_Os07g23470.110713,258,67013,256,62375025025,690.84.6436EC
OsTLP20LOC_Os07g23730.110713,388,82613,392,09183127728,194.77.0885EC
OsTLP21LOC_Os08g29320.11817,988,59617,988,94634211412,090.97.8139EC
OsTLP22LOC_Os08g40600.17825,701,62325,700,147115238438,359.94.6931EC
OsTLP23LOC_Os08g43510.19827,519,46227,522,32997232433,940.94.5857EC
OsTLP24LOC_Os09g32280.17919,268,70419,270,43299633234,151.75.0408EC
OsTLP25LOC_Os09g36560.110921,088,84621,090,58299333132,724.84.6955EC
OsTLP26LOC_Os09g36580.19921,098,26721,101,21393931332,347.24.5469EC
OsTLP27LOC_Os10g05600.19102,796,6782,794,499117039038,581.54.3722EC
OsTLP28LOC_Os10g05660.110102,829,1512,830,79897832631,969.64.3195EC
OsTLP29LOC_Os10g27280.151014,400,69514,399,00882827628,365.58.2043EC
OsTLP30LOC_Os10g38100.191020,408,00120,407,41159119719,003.97.2119N
OsTLP31LOC_Os11g47670.121128,781,16128,782,20263921321,3125.5156EC
OsTLP32LOC_Os11g47680.121128,783,15828,783,99262120720,704.75.6546EC
OsTLP33LOC_Os11g47944.161128,919,76028,918,91676225425,770.37.4395EC
OsTLP34LOC_Os12g38120.121223,410,05223,411,14269323122,994.25.0605EC
OsTLP35LOC_Os12g38150.121223,427,67923,428,88170223423,430.18.0885EC
OsTLP36LOC_Os12g38170.121223,435,45823,436,59470223423,787.46.7116EC
OsTLP37LOC_Os12g43380.131226,913,85126,912,97453417817,992.14.9096EC
OsTLP38LOC_Os12g43390.131226,922,69926,922,17552517517,607.74.3415EC
OsTLP39LOC_Os12g43430.131226,922,76126,913,12653717918,105.34.9096EC
OsTLP40LOC_Os12g43410.131226,933,29226,932,27345315115,454.34.5204EC
OsTLP41LOC_Os12g43440.131226,956,89426,956,14254918318,629.98.073EC
OsTLP42LOC_Os12g43450.141226,960,48726,961,40571723925,008.48.1132EC
OsTLP43LOC_Os12g43490.141226,981,08726,979,99177725926,519.85.0243EC
CDS: coding sequence; MW: molecular weight; CL: chromosome location; SL: sub-cellular location; EC: extracellular; N: nuclear; PM: plasma membrane; PI: isoelectric point.
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Ahmad, S.; Zhu, H.; Chen, Y.; Xi, C.; Shah, A.Z.; Ge, L. Comprehensive Bioinformatics and Expression Analysis of the TLP Gene Family Revealed Its Role in Regulating the Response of Oryza sativa to Nilaparvata lugens, Laodelphax striatellus, and Jinggangmycin. Agronomy 2022, 12, 1297. https://doi.org/10.3390/agronomy12061297

AMA Style

Ahmad S, Zhu H, Chen Y, Xi C, Shah AZ, Ge L. Comprehensive Bioinformatics and Expression Analysis of the TLP Gene Family Revealed Its Role in Regulating the Response of Oryza sativa to Nilaparvata lugens, Laodelphax striatellus, and Jinggangmycin. Agronomy. 2022; 12(6):1297. https://doi.org/10.3390/agronomy12061297

Chicago/Turabian Style

Ahmad, Sheraz, Haowen Zhu, Yu Chen, Chuanyuan Xi, Amir Zaman Shah, and Linquan Ge. 2022. "Comprehensive Bioinformatics and Expression Analysis of the TLP Gene Family Revealed Its Role in Regulating the Response of Oryza sativa to Nilaparvata lugens, Laodelphax striatellus, and Jinggangmycin" Agronomy 12, no. 6: 1297. https://doi.org/10.3390/agronomy12061297

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