Genome-Wide Characterization and Expression Analysis of Pathogenesis-Related 1 (PR-1) Gene Family in Tea Plant (Camellia sinensis (L.) O. Kuntze) in Response to Blister-Blight Disease Stress

Pathogenesis-related 1 (PR-1) proteins, which are defense proteins in plant–pathogen interactions, play an important role in the resistance and defense of plants against diseases. Blister blight disease is caused by Exobasidium vexans Massee and a major leaf disease of tea plants (Camellia sinensis (L.) O. Kuntze). However, the systematic characterization and analysis of the PR-1 gene family in tea plants is still lacking, and the defense mechanism of this family remains unknown. In this study, 17 CsPR-1 genes were identified from the tea plant genome and classified into five groups based on their signal peptide, isoelectric point, and C-terminus extension. Most of the CsPR-1 proteins contained an N-terminal signal peptide and a conserved PR-1 like domain. CsPR-1 genes comprised multiple cis-acting elements and were closely related to the signal-transduction pathways involving TCA, NPR1, EDS16, BGL2, PR4, and HCHIB. These characteristics imply an important role of the genes in the defense of the tea plant. In addition, the RNA-seq data and real-time PCR analysis demonstrated that the CsPR-1-2, -4, -6, -7, -8, -9, -10, -14, -15, and -17 genes were significantly upregulated under tea blister-blight stress. This study could help to increase understanding of CsPR-1 genes and their defense mechanism in response to tea blister blight.


Introduction
The plant immune system has multiple layers of defense responses to intercept the infection and damage caused by pathogenic microorganisms [1]. In the first barrier of the immune system, plants recognize the conserved pathogen-associated molecular patterns (PAMPs) of slowly evolving microorganisms or pathogens through the pattern-recognition receptors (PRRS) located on the surface of the cell membrane to activate pattern-triggered immunity (PTI) [2]. However, compatible pathogens can overcome this first layer of defense. Plants specifically recognize effectors via polymorphic nucleotide-binding-domain and leucine-rich repeat (NB-LRR) proteins encoded by most resistance genes (R genes) directly or indirectly to activate effector-triggered immunity (ETI) [3]. According to the products encoded by defense genes and their functions, these genes can be divided into secondary metabolite-synthesis genes, cell-wall-modification -related genes, protease-inhibitor genes, and plant-pathogenesis-related (PR) genes [4].
Numerous studies have shown that PR genes play important roles in the resistance and defense of plants against diseases [5][6][7]. PR proteins are induced and accumulated in host plants during attacks by oomycetes, fungi, bacteria, viruses, or insects. Seventeen families of PR proteins have been classified and characterized according to the homology of amino-acid sequences, serological relationships, and enzyme activities [8]. PR-1 proteins

Functional Interaction Networks of CsPR-1 Proteins
Functional interaction networks of the CsPR-1 proteins were constructed using STRING software on the basis of the homologous proteins in Arabidopsis (Table S3). The results show that the 17 CsPR-1 proteins participated in the interaction network, indicating a universal and complex interaction of CsPR-1 proteins (Figure 3). Most CsPR-1 proteins were involved in the SA-signaling pathway or JA/ET-signaling pathway by interacting with NPR1, EDS16, BGL2, PR4, and HCHIB. CsPR-1-12 was independent. Amino-acid alignment of the PR-1-like domain of the deduced CsPR-1 proteins. DNAMAN 7.0 was used to mark the amino-acid residues with an identity of more than 50% with black shade. The areas in the interior green boxes and the red solid line indicate the C1-C6 and CAPE peptide, respectively.

Predicted Secondary and 3D Structures of CsPR-1 Proteins
Secondary structures were analyzed using the SOPMA online server. The results show that α-helices, extended strands, beta turns, and random coils were found in the ranges of 23.82-39.88%, 8.24-22.45%, 1.73-7.05%, and 39.74-50.29%, respectively ( Table 2). The 3D structures of the CsPR-1 proteins were predicted using the Phyre2 online server.
The Ramachandran plot representing the residues in core, allowed, and generous regions exceeded 95%, revealing the quality and reliability of the protein structures ( Table 2). The predicted channel structures of CsPR-1 proteins ranged from 2 to 9, and the overall percentages of disordered regions were between 6.47% and 61.76% ( Table 2). The 3D structures of the CsPR-1 proteins were conserved, except for that of CsPR-1-2. The 3D structures and predicted pockets are shown in Figure 5.

Histomorphological Observation of Tea Leaves
Healthy tea leaves had a complete cell structure, including cuticle, upper epidermis, palisade tissue, sponge tissue, down epidermis, and cuticle, and the glycogen and neutral mucus in the palisade tissue cells were red (Figure 6a,e). However, the cell structure of leaves invaded by tea blister blight disease was infected to varying degrees. When the pathogen of tea blister blight disease invaded in the early stage, an obvious hyphal invasion was observed in the gap of the tea plant in down-epidermal cells, but the down-epidermal cells were still intact (Figure 6b,f). In the middle stage of pathogen invasion, the hyphae proliferated rapidly in the gap between tea cells. Some down-epidermal cells of the tea leaves were broken by hyphae, and a small amount of basidiospores broke through the stratum corneum in clusters (Figure 6c,g). In the late stage of pathogen invasion, the hyphae occupied and destroyed most of the sponge-tissue cells, and a large number of basidiospores grew outward in clusters, while the down-epidermal cells of the tea leaves were almost invisible (Figure 6d,h).

Expression Patterns of CsPR-1 Genes
To further investigate the response of CsPR-1 genes to blister blight disease and to verify the accuracy of the RNA-seq data, the expression of 17 CsPR-1 genes in uninoculated (healthy) and inoculated (early, middle, and late stages) E. vexans was measured by qRT-PCR. The results show that the change trend of the expression pattern of most CsPR-1 genes after E. vexans invasion in tea plants was basically similar to that of transcriptome sequencing (Figure 7). A strong response of the CsPR-1 genes was observed, in which 10 . Predicted 3D structure of the CsPR-1 proteins using the Phyre2 server and pockets using the Pasaccca server. The top five predicted pockets are indicated as yellow, blue, green, red, and grey, respectively.

Histomorphological Observation of Tea Leaves
Healthy tea leaves had a complete cell structure, including cuticle, upper epidermis, palisade tissue, sponge tissue, down epidermis, and cuticle, and the glycogen and neutral mucus in the palisade tissue cells were red (Figure 6a,e). However, the cell structure of leaves invaded by tea blister blight disease was infected to varying degrees. When the pathogen of tea blister blight disease invaded in the early stage, an obvious hyphal invasion was observed in the gap of the tea plant in down-epidermal cells, but the down-epidermal cells were still intact (Figure 6b,f). In the middle stage of pathogen invasion, the Figure 5. Predicted 3D structure of the CsPR-1 proteins using the Phyre2 server and pockets using the Pasaccca server. The top five predicted pockets are indicated as yellow, blue, green, red, and grey, respectively. hyphae proliferated rapidly in the gap between tea cells. Some down-epidermal cells of the tea leaves were broken by hyphae, and a small amount of basidiospores broke through the stratum corneum in clusters (Figure 6c,g). In the late stage of pathogen invasion, the hyphae occupied and destroyed most of the sponge-tissue cells, and a large number of basidiospores grew outward in clusters, while the down-epidermal cells of the tea leaves were almost invisible (Figure 6d,h).

Discussion
PR-1 is a kind of PR protein induced by the host against a pathogen and encoded by multigene families in plants [8]. Seventeen members of the PR-1 gene family from the tea genome were identified in the current study, consistent with similar investigations in other plant species. Comprehensive analyses of the gene structures, conserved motifs, cis elements, protein-interaction networks, and expression patterns were performed, and the results could provide a scientific basis for future functional research.
The PR-1 family is highly conserved and exists in all plants studied. This family has been confirmed to possess an antifungal function. According to pI values, PR-1 proteins can be broadly classified as either acidic or basic proteins [12]. These two protein types have different inhibitory activities against fungal pathogens. In tomato plants, basic PR-1c and PR-1g exhibit greater antifungal activity against Phytophthora infestans than acidic PR-1a and PR-1b [32]. Overexpression of TMV-inducible basic pepper PR-1 gene in tobacco leaves could significantly enhance the resistance to heavy-metal stresses and pathogens [33]. In the current study, most basic CsPR-1 proteins were found to be more rapidly and strongly expressed than acidic CsPR-1 proteins in tea plants during interaction with E. vexans. This finding is similar to those reported by previous studies. The basic CsPR-1-2, -4, -7, -8, -9, -14, and -17 genes were obviously upregulated, and most of these genes responded rapidly during the early or middle stages of infection. These characteristics indicate that these genes may play an important role in the early pathogenesis of tea blister blight disease. Acidic CsPR-1-6, -10, and -15 were also upregulated. The expression trends of CsPR-1 genes were diverse, indicating a complex defense mechanism to tea blister blight in tea plants.
Notably, all deduced amino-acid sequences of CsPR-1 genes, except for those of CsPR-1-11 and-12, contained an SP at the N terminal. An SP is an RNA region encoding hydrophobic amino-acid sequences, which, in most cases, is a transient extension to the amino terminus of the protein and is responsible for guiding proteins into subcellular organelles with different membrane structures [34]. Previous studies on Triticum aestivum and Vitis showed that all PR-1 proteins contain an SP at the N terminal, which could secrete PR-1 proteins into the extracellular environment [14,24]. Two PnPR-1 proteins without SPs were also found in Piper nigrum, which is consistent with the present results [13]. Most CsPR-1 proteins containing an SP at the N terminal might be guided into special subcellular organelles for their biological functions. The CTE is supposed to be functionally similar to the carboxyl-terminal propeptide encoded by PR-2 and PR-3 genes, which is known to serve as a vacuolar targeting signal [16,35]. The CTE domain has been found in both dicot and monocot species, such as tobacco PR-1b and tomato PR-1a1 [36,37]. In this study, only CsPR-1-2 was found to contain the CTE domain, which may play an important role in guiding CsPR-1 proteins into vacuolar targeting. The 3D structures of CsPR-1 proteins are also conserved, except for those of CsPR-1-2. The structural variation was significantly connected with the CTE domain of the CsPR-1-2 gene and resulted in different function of PR-1 proteins in tea plants. Disordered regions contain flexibility-intrinsic conformation to bind multiple partners in transient interactions and are closely related to transcriptional regulation, signaling cascade, cell-cycle control, and chaperone activity [38]. The presence of disordered regions in CsPR-1-2 was up to 61.76%, which might be related to the metabolic roles of these proteins in cellular regulation. Further characterization may provide a theoretical basis for the exploration of the structural and functional diversity of CsPR-1 proteins in tea plants.
Previous studies have shown that the multigene family was formed by duplication and mutation of an ancestral gene [39]. The survival of duplicate genes made the regulatory and coding regions tend to differentiate, leading to differences in the structure and function of genes in the same family [39]. In the current study, the most closely related CsPR-1 genes in the same group were found to share a similar conserved motif and gene structures, indicating that analysis of the motif and gene structures was contributed to understanding of the evolutionary relationships of CsPR-1 family genes. Furthermore, introns are less conserved compared with exons. Intron density tended to decline during evolution in some genes that need to rapidly activate in response to stress, such as heat-shock proteins [40]. Using molecular characterization and sequence analysis, investigators in a previous study found that PR-1 genes lack introns [41]. In the current investigation, a majority of CsPR-1 genes were found to have no introns or only between one and three introns, which was conducive to the transcriptional regulation of these genes under stress. CsPR-1-7, -9, and -14 without introns in the same group responded more rapidly to tea blister blight disease stress than CsPR-1-1, which contains three introns.
Although substantial evidence has demonstrated that PR-1 acts as a defense protein in plant-pathogen interactions, the biological functions of PR-1 proteins remain obscure. A previous study showed that SA and JA can induce the expression of up-and downregulated PR genes, respectively [42]. PR-1 gene in wheat was strongly induced after ABA and SA treatments [43]. Similarly, under cold treatment and exogenous application of SA, the expression of PR-1 genes in Arabidopsis was significantly upregulated [44]. In the interaction networks of CsPR-1, many key genes engaged in activating and mediating diverse defense responses were involved. AT5G66590 (CsPR-1-5 and -11), AT5G57625 (CsPR-1-7), and AT4G33720 (CsPR-1-3, -6, -10, and -12) interacted with PR4, which was highly related to the defense of grapevine against downy mildew resistance and related to the JA/ET-signaling pathway [45]. HCHIB was basic endochitinase B, related to the JA/ET-mediated signaling pathway during systemic acquired resistance, which is an enzyme known for antifungal activity and close interaction with AT1G50060 (CsPR-1-1, -4, -9, and -14) and AT4G25780 (CsPR-1-13) [40]. CsPR-1-15 and -17 were involved in the defense of SA response. NPR1 was found to positively modulate SA signaling in plants and to interact with PRB1 (CsPR-1-8) [46]. CsPR-1 proteins were found to be involved in diverse disease resistance and to play a connecting role in the complex response network of different signal pathways.

Plant Materials
Tea plants (Camellia sinensis cv. Fuding dabai) were grown in the gardens of Northwest A&F University Tea Experimental Station (Xixiang, Shaanxi, China, 32 • 57 43" N, 107 • 40 12" E). Tea leaves (from the third leaf from the top of the plant) with typical tea blister-blight symptoms were sampled within 4 h. Each tea leaf only had one blister. The severity was classified into three grades on the basis of the course of the disease, as previously reported: early stage (S1, the formation of yellow, transparent patches with a diameter of 2-4 mm), middle stage (S2, the formation of features blisters), and late stage (S3, the formation of necrotic spots) [47]. Healthy tea leaves (H) free of any infestation at the same leaf positions were used as control. All samples were immediately frozen in liquid nitrogen and stored at −80 • C for periodic acid-Schiff (PAS) staining, RNA-seq, and real-time PCR (qRT-PCR) with three biological replicates.

Database Mining and Identification of CsPR-1 Genes
Published Arabidopsis PR-1 sequences were used as queries for BLASTP searches against the Tea Plant Genome Database (http://tpia.teaplant.org/index.html, accessed on 16 September 2021) [48]. All output genes were then verified using Pfam (http://pfam.

Analysis of the Conserved Motifs, Gene Structures, and Protein Functional Networks
The conserved motifs were analyzed with the MEME platform (http://meme-suite.org/ tools/meme, v4.9.0, accessed on 20 September 2021). The number of motifs was set to 20, and other parameters were set to default values [49]. The exon-intron structures were retrieved from the gene-annotation file (http://www.plantkingd omgdb.com/tea_tree/data/gff3/, accessed on 20 September 2021), and the diagrams were drawn by using TBtools v1.098661 software [50]. The functional-interaction networks of the PR-1 proteins in tea plants were analyzed based on the STRING protein-interaction database (http://string-db.org/, accessed on 21 September 2021).

PAS Staining
The samples were fixed with formaldehyde/acetic acid/ethanol fixative (containing 50% ethanol, 5% acetic acid, and 10% formaldehyde in H 2 O), dehydrated, transparented, waxed, and embedded. The paraffin sections were then routinely dewaxed in water and put into periodic acid solution for 10-15 min. Then, the slides were stained with PAS for 10 min. After being washed with water and dehydrated, the slices were covered with paraffin for further observation using the Olympus BX51 microscope (Olympus, Tokyo, Japan).

RNA Extraction and Quantitative RT-PCR Analysis
Total RNA was extracted using the Plant RNA Kit (Omega, Norcross, GA, USA). cDNA was synthesized by using the 5 × All-In-One RT MasterMix Kit (ABM, Richmond, BC, Canada) according to the manufacturer's protocol, and the cDNA was diluted to 200 ng/µL for subsequent analysis. Then, qRT-PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) on an iQ5 real-time PCR platform (Bio-Rad, Hercules, CA, USA) with the following PCR parameters: 95 • C for 30 s, followed by 40 cycles of 95 • C for 5 s and 60 • C for 30 s. Three independent biological replicates were performed, and the qPCR of each replicate was performed in triplicate. Relative transcript abundances of the PR-1 genes were calculated via the 2 -∆∆CT method [51]. All primers were designed using Primer5 software, and primer sequences are listed in Table S4.

Conclusions
In the present study, 17 CsPR-1 genes were identified in tea plants, and bioinformatics and expression-profile analyses were performed to determine their potential functions ( Figure 8). The CsPR-1 genes were found to be actively involved in the response to tea blister blight stress, and these processes are closely related to the signal transduction pathways involving TCA, NPR1, EDS16, BGL2, PR4, and HCHIB. The results provide new insights into the response to tea blister blight stress and also contribute to an important basis for subsequent functional studies investigating CsPR-1 in tea plants.