Identification of the HbZAR1 Gene and Its Potential Role as a Minor Gene in Response to Powdery Mildew and Anthracnose of Hevea brasiliensis

Abstract

Powdery mildew and anthracnose are the main diseases of rubber trees. In recent years, there have been large outbreaks in the rubber-planting areas of Asia, seriously affecting the yield and quality of rubber latex. ZAR1 is a conserved and distinctive coiled-coil nucleotide-binding leucine-rich (CNL) repeat in the plant kingdom, playing a crucial role in disease-resistance processes. To elucidate the function of the HbZAR1 gene in rubber trees (Hevea brasiliensis), three candidate HbZAR1 genes were identified using bioinformatics methods and comprehensively analyzed. The results indicate that the HbZAR1 protein is conserved in different plant species. Examination of cis-regulatory element sequences of HbZAR1genes reveals that the HbZAR1 gene promoter exhibits a remarkable enrichment of stress, light, and hormone elements. An expression analysis shows that the expression levels of the three HbZAR1 genes are highest in the bark and lowest in latex. Three HbZAR1 genes can respond to both rubber tree Erysiphe quercicola and Colletotrichum siamense infection; especially, HbZAR1.1 and HbZAR1.2 show significant upregulation in expression levels during the early stages of infection. These findings suggest that the three HbZAR1 genes may be involved in rubber tree susceptibility to E. quercicola and C. siamense through different immune mechanisms. Subcellular localization results indicate that the HbZAR1 genes are expressed in the nucleus and plasma membrane. This study also shows that the three HbZAR1 genes and activated mutant HbZAR1.1^D481V do not induce stable ROS production and cell death, suggesting possible gene degradation, functional redundancy, or acting as minor genes in disease resistance. This research provides valuable insights for further studying the function of HbZAR1 genes in rubber trees and the mechanisms of immune molecules.

1. Introduction

NLR (nucleotide-binding domain and leucine-rich repeat containing receptor) is a key receptor protein that regulates ETI (effector-triggered immunity) [1]. Based on the different N-terminal structural domains of NLR proteins, they are classified into three types: CNL (N-terminal CC domain), TNL (N-terminal TIR domain), and RNL (N-terminal RPW8 domain) [2]. NLR receptor proteins have the capability to identify pathogen effector proteins either directly or indirectly, triggering targeted immune responses. This layer of immunity is characterized by its specificity, rapid response, and high intensity and can trigger systemically acquired immunity in plants [3,4,5]. Therefore, this form of immunity has garnered widespread attention in the fields of plant disease resistance and breeding.

ZAR1 is an atypically conserved CNL, with its origin dating back to the early flowering plants of the Jurassic period, around 220 to 150 million years ago [6]. A direct homologous sequence analysis reveals highly conserved characteristics of ZAR1, including pathogen-recognition regions and immune-activation regions. ZAR1 interacts with HopZ-ETI-DEFICIENT 1 (ZED1)-associated kinases (ZRKs) and AVRPPHB SUSCEPTIBLE 1 (PBS1)-like proteins, forming polymerized resistosomes that trigger immune responses [7,8,9,10,11,12]. Within plant cells, oligomeric ZAR1 resistosomes bind to the plasma membrane and form a calcium-permeable cation-selective channel, leading to an influx of calcium ions and further activation of downstream defense responses, including cell death [13]. Therefore, ZAR1 resistosomes serve as sensors of pathogen effectors and as effectors of downstream cell death and signal transduction [14]. Deciphering the mechanism of resistosome action is of guiding significance in designing broad-spectrum, long-lasting, novel, resistant proteins and in developing sustainable agriculture.

Powdery mildew and anthracnose are the main diseases of rubber trees (Hevea brasiliensis) in China, significantly affecting the yield and quality of latex. The powdery mildew of rubber trees is caused by E. quercicola, mainly affecting the young leaves, shoots, and inflorescences, leading to premature shedding of new leaves, delayed tapping time, and reduced latex production [15,16]. Rubber tree anthracnose is mainly caused by C. gloeosporioides, C. acutatum, and C. siamense, affecting rubber seedlings, young trees, and tapped rubber trees, resulting in leaf shedding, shoot dieback, and fruit rot, causing delayed tapping time [17,18]. Currently, chemical control combined with prediction and forecasting is mainly used for rubber tree powdery mildew and anthracnose, but there are problems, such as polluting the environment, increasing pathogen resistance, time-consuming, laborious and few optional agents, difficulty in obtaining data, low prediction accuracy, and high subjectivity. The most cost-effective and green way to control powdery mildew and anthracnose is to develop disease-resistant asexual lines of rubber trees using existing asexual rubber tree lines and wild germplasm. Therefore, it is essential to study the molecular regulatory network of rubber trees in response to powdery mildew and anthracnose infections, as well as to explore and identify relevant disease-resistant genes. Elucidating the molecular mechanisms of how rubber trees develop immunity against powdery mildew and anthracnose pathogens holds greater significance.

Although the ZAR1 gene family has garnered considerable attention in the study of herbaceous plants, its examination in woody species remains sparse, with an absence of pertinent research in rubber trees. Previous studies have shown that the ZAR1 gene exhibits excellent disease-resistance functions in model crops. Currently, there is a lack of disease-resistant germplasm resources for rubber trees, especially powdery-mildew-resistant germplasms. Therefore, it is important to study the function and mechanism of the ZAR1 gene in rubber trees, which can provide effective genetic resources for screening and breeding highly disease-resistant rubber tree lines. Consequently, the identification of the HbZAR1 gene family in rubber trees and the analysis of its expression and function under biological stress are crucial. This study conducted a comprehensive analysis of the physicochemical properties, evolutionary kinships, exon–intron structure, conserved domains and motifs, and promoter cis-acting elements of three HbZAR1 genes in rubber trees. This study examined the gene expression profiles of HbZAR1 in different tissues of rubber trees, their expression after infection with E. quercicola and C. siamense, and the ROS burst and cell death after overexpression in tobacco. These results not only provide a foundational reference for further studying the function of the HbZAR1 gene family but also lay a foundation for comprehending the potential molecular mechanisms of HbZAR1 involved in the immune regulation of rubber trees.

2. Results

2.1. Identification and Physicochemical Properties of HbZAR1 Gene Family Members in Rubber Tree

By utilizing the Arabidopsis ZAR1 gene sequence to search the rubber tree genome database, three highly homologous HbZAR1 genes were identified through a sequence alignment analysis using default BLAST settings. Based on their level of homology, they were designated as HbZAR1.1, HbZAR1.2, and HbZAR1.3. The physicochemical properties, molecular weights, theoretical isoelectric points, and subcellular localization of these three genes and their encoded proteins are shown in

Table 1. Characterization and analysis of HbZAR1 gene family members in rubber tree.

Gene Name	Gene ID	Length of CDS (bp)	Number of Exons	Predicted Protein
				Siza (aa)	MW (Da)	PI	Subcellular Localization
HbZAR1.1	110668533	2532	1	843	96,350.36	6.37	M/N/Cyto
HbZAR1.2	110644474	2541	1	846	97,467.88	7.93	M/N/Cyto
HbZAR1.3	110671616	2514	1	837	95,889.91	8.06	M/N/Cyto

N represents nucleus; Cyto represents cytoplasm; M represents Cell membrane.

.

The coding-sequence lengths of these three HbZAR1 genes range from 2514 to 2541 bp, each containing only 1 exon. The protein sequences encoded by these members range from 837 to 846 amino acids, with molecular weights between 95,889.91 and 97,467.88 Da and isoelectric points between 6.37 and 8.06. In addition, the predicted subcellular localization results indicated that all three ZAR1 proteins could be localized in the nucleus, cytoplasm, and cell membrane. The variance in length and molecular mass among these HbZAR1 gene sequences is notably modest.

2.2. Phylogenetic Analysis of HbZAR1s

To understand the evolutionary relationship of the HbZAR1 gene in rubber trees, this study conducted multiple sequence alignments of three HbZAR1 protein sequences with 108 homologous ZAR1 proteins from 88 other species. A phylogenetic tree was constructed using a neighbor-joining method. According to the evolutionary tree, it is evident that the HbZAR1.1 protein clusters with ZAR1 homologous proteins from Euphorbiaceae plants such as castor bean (Ricinus communis) and cassava (Manihot esculenta). Interestingly, HbZAR1.2 and HbZAR1.3 show the highest similarity with ZAR1 proteins from Colocasia esculenta and Cinnamomum micranthum, respectively, (Figure 1). This indicates that the HbZAR1 is relatively conserved among various angiosperms.

2.3. Conserved Domain and Motifs of HbZAR1 Proteins in Rubber Tree

The conserved structural domain analysis of the protein encoded by the HbZAR1 genes in rubber tree was conducted using the NCBI Batch CD website. The results were visualized using the TBtools, as shown in Figure 2A. All three proteins contain the CC, NB-ARC, and LRR structural domains, indicating that the structure and function of the HbZAR1 genes in rubber trees are relatively conserved during the evolutionary process of rubber trees.

Utilizing the MEME website, a conservative motif analysis of the HbZAR1 protein family members was conducted. A total of 12 conservative motifs were identified, with their specific conservative sequences shown in Figure 2B and Table S1. The distribution order is motif 11–motif 10–motif 2–motif 8–motif 7–motif 6–motif 12–motif1–motif 5–motif 9–motif 3–motif 4. Notably, motif 2 contains a high content of amino acid residues GMGGxGKTTLA, which can be predicted as a kinase-1 (also known as P-loop: GxxxGKT/S), with P-loop being crucial in recognizing CNL proteins. Based on the sequence, motif 10 is identified as the NBD–NBD interface, while motif 5 can be classified as the MHD conservative unit based on the accumulation of its M/LHDx amino acid residues. These are all conservative sequences of the NB-ARC domain, an indispensable part of the NB-ARC domain. Additionally, motif 9, motif 3, and motif 4 exhibit apparent leucine-rich repeat sequences, characterized by their long length and rich leucine residues, predicting a typical LRR structural region, depicted as xxLxLxx.

2.4. Structure and Putative Cis-Acting Element Analysis of HbZAR1s in Rubber Tree

To understand the tissue structure of the HbZAR1 genes, we have depicted a schematic diagram consisting of exons and introns (Figure 3A). It is evident from the diagram that all three genes contain only one exon, devoid of introns, indicating a significant level of conservation in the gene structure of HbZAR1s.

By analyzing the putative cis-acting elements in the promoter sequences of members of the HbZAR1 genes through the PlantCARE website and visualizing them using the TBtools, the results are shown in Figure 3B and Table S2. It can be observed from the figure that the promoter sequences of each HbZAR1 gene contain multiple cis-acting elements. Through statistical analysis, it was found that all three genes contain putative cis-acting elements related to light responses, such as AE-box, Box 4, and G-box. Furthermore, all three genes contain cis-acting elements related to stress responses, including MBS, which is associated with drought tolerance, and the LTR motif, indicative of a response to low-temperature stress, both present in HbZAR1.1. Additionally, TC-rich repeats, implicated in both defense and stress responses, are found in both HbZAR1.1 and HbZAR1.3. The analysis further disclosed that all three genes are replete with cis-acting elements associated with salicylic acid, denoted by the TCA-element. The promoter sequences of HbZAR1.1 and HbZAR1.3 are particularly enriched with elements related to auxin signaling, characterized by the AuxRR-core. HbZAR1.2 features putative cis-acting elements linked to abscisic acid, marked by the ABRE motif. HbZAR1.3 includes elements associated with gibberellin, identified by the TATC-box, while HbZAR1.1 encompasses elements related to jasmonic acid, signified by the CGTCA-motif. These findings suggest that the HbZAR1 genes may play different functions, potentially playing important roles in hormone regulation pathways, response to environmental stress, and growth and development.

2.5. Expression Patterns of HbZAR1s in Different Tissues

To investigate the expression profiles of the HbZAR1 gene in different tissues of rubber trees, this study involved the analysis of HbZAR1 genes in the roots, stems, leaves, flowers, and latex of rubber trees. The expression levels of HbZAR1 were found to be notably higher in the bark, followed by branches, leaves, and roots, while the lowest expression was observed in latex, as illustrated in Figure 4A. These findings suggest a potential pivotal function of the HbZAR1 genes within these specific plant tissues.

2.6. Expression of HbZAR1 Genes Under Infection of E. quercicola and C. siamense

To delve deeper into the involvement of HbZAR1s in the response to biotic stress, we conducted an analysis on the expression of the HbZAR1 genes in rubber trees infected with E. quercicola and C. siamense. As shown in Figure 4B, the expression levels of all three HbZAR1 genes were upregulated after E. quercicola infection, with HbZAR1.1 showing significant upregulation at 4 h and 15 h post-infection. HbZAR1.2 exhibited an initial upregulation at 4 h post-E.-quercicola-infection followed by downregulation, while HbZAR1.3 displayed a downregulation trend, followed by upregulation, upon powdery mildew infection, suggesting that HbZAR1s may respond to rubber tree E. quercicola infection and potentially play a role in the defense against E. quercicola in rubber trees.

After infection by the rubber tree C. siamense, three HbZAR1 genes exhibited different expression patterns. HbZAR1.1 showed upregulation in the early stage of C. siamense infection, followed by downregulation in the middle stage, and then upregulation again towards stability in the later stage. HbZAR1.2 was upregulated at 4 h post-infection, followed by significant downregulation. HbZAR1.3 maintained a downward trend in expression levels after infection, reaching the lowest expression at 48 h post-infection (Figure 4C). This indicates that the HbZAR1 genes can respond to rubber tree anthracnose pathogen invasion and may participate in the rubber tree’s resistance to C. siamense through different mechanisms.

2.7. Subcellular Localization of the HbZAR1 Genes

The HbZAR1 genes’ intracellular distribution within rubber tree cells was examined to ascertain its cellular expression and function. As depicted in Figure 5, the subcellular localization of the three HbZAR1 genes was found to be consistent, located within the cell nucleus and plasma membrane. Therefore, the HbZAR1 gene may play a crucial role in pathogen recognition and signal transduction processes.

2.8. HbZAR1 Does Not Induce ROS Burst and Cell Death in Tobacco

Three HbZAR1 genes were transferred into N. benthamiana leaves via an agrobacterium-mediated transformation. Under the drive of the 35s promoter, HbZAR1s could be transiently expressed in the mesophyll cells of N. benthamiana leaves. As shown in Figure 6A, after 2 and 3 d of infection, partial injection of the positive control Inf1 resulted in ROS burst and cell death in tobacco, while injection of HbZAR1.1, HbZAR1.2, and HbZAR1.3 did not induce stable ROS burst and tobacco cell death.

Research indicates that in normally growing plants, the expression of NLRs is of low abundance, and the protein conformation is in an inactive state. To further verify whether the lack of activation of the HbZAR1 genes does not cause tobacco cell death, a mutated form of the HbZAR1.1 gene in an activated state (HbZAR1.1^D481V) was constructed and injected into tobacco (Figure 6B). It was observed that this also did not induce tobacco ROS burst and cell death, indicating that in the evolution of rubber trees, the disease-resistance function of the HbZAR1s may have degenerated, or there might be functional redundancy.

3. Discussion

3.1. Detailed Characterization and Evolution of HbZAR1s in Rubber Tree

Researchers have extensively studied the ZAR1 gene in model plants and other species, emphasizing their importance. Adachi identified 120 ZAR1-homologous genes from 88 angiosperm species, with 108 genes encoding typical CC-NLR proteins sharing 52.0% to 97.0% similarity with Arabidopsis ZAR1 [6,19]. It is noteworthy that HbZAR1.1 shows similarities of 57% and 53% with Arabidopsis and tobacco, respectively. Among these 120 ZAR1 homologous genes, a rubber tree HbZAR1.1-homologous protein was included, showing higher similarity to cassava’s ZAR1, while two others clustered with Cinnamomum micranthum ZAR1-homologous proteins, indicating a high homology of ZAR1 genes in woody plants among angiosperms.

The diversity of gene structure often plays an important role in the evolutionary history of gene families. The three HbZAR1 genes’ structures in rubber trees are highly similar, with no introns, only one exon, and similar exon lengths. Additionally, all three HbZAR1 proteins contain the CC-, NB-ARC-, and LRR-conserved domains, which have been confirmed to contain multiple conserved motifs that play a key role in protein function. Arabidopsis ZAR1 primarily recognizes effector molecules through the LRR domain, reshapes molecular interactions through the ADP/ATP switch, oligomerizes through the NBD–NBD interface, and activates hypersensitive cell death through the α1 helix/MADA motif [14,20,21]. The three HbZAR1s in rubber trees contain these related motifs as well. Adachi and others reconstructed the evolutionary history of ZAR1, indicating it is an atypical conservative NLR whose origins can be traced back to approximately 220 to 150 million years ago in the early Jurassic period of angiosperms [6,22]. ZAR1 stands out among the NLRs in angiosperms, having undergone relatively limited gene duplication and expansion throughout its deep evolutionary history [6]. These findings suggest that the HbZAR1 gene in rubber trees may also perform functions similar to those of the Arabidopsis ZAR1 gene.

Predicting the location and function of putative gene-promoter cis-acting elements is helpful for a deeper understanding of gene transcriptional regulation processes, revealing details and mechanisms of gene expression regulation [23]. In this study, three HbZAR1 genes promoter sequences contain different types and numbers of putative cis-acting elements, with the HbZAR1.1 promoter clearly having the most diverse types and numbers of putative cis-acting elements, followed by HbZAR1.3, and HbZAR1.2 having the least. This indicates that the three genes may play different roles in rubber trees, suggesting that the HbZAR1 genes may have different functions, potentially exerting significant influence on hormonal regulatory pathways, stress responses, and the developmental processes. Subsequently, further research on this subject can be conducted through the addition of exogenous hormones, artificial manipulation of biological or abiotic stresses, elucidating the manner in which the HbZAR1 gene is involved in hormone regulatory pathways and stress response processes.

3.2. The HbZAR1s Responds to Biological Stress

ZAR1 is a unique CNL that encodes immune receptors capable of recognizing multiple effector proteins of different pathogenic bacteria through various adaptor proteins, such as the effector protein AvrAC from Xanthomonas campestris pv. campestris and HopZ1a from Pseudomonas syringae, exhibiting broad-spectrum disease resistance [9,24,25,26,27]. Bi and others discovered that ZAR1 forms a preformed resting complex RKS1–ZAR1 with the resistance-related kinase RKS1 to perceive the effector protein AvrAC from X. campestris [13]. In 2022, Zhou’s research indicates that ZAR1 from non-host plants N. benthamiana and Arabidopsis can recognize the effector protein HopZ5 from the pathogen P. syringae, participating in kiwi-fruit resistance against P. syringae infection [28]. These results all indicate that the ZAR1 gene plays an important role in the process of antibacterial disease resistance. Additionally, studies have shown that plant ZAR1 is involved in antifungal disease resistance, such as Thatcher’s research indicating that the ZAR1 homolog gene Ht1 is involved in the resistance process against Northern corn leaf blight [29]. This research evinces that the HbZAR1 genes exhibit a responsive phenotype to infections incited by E. quercicola and C. siamense in rubber trees. This study indicates that HbZAR1.1 and HbZAR1.2 can be upregulated 2–3 times in the early stage of powdery mildew infection, while HbZAR1.3 shows significant downregulation. This suggests that the three HbZAR1 genes may be involved in the rubber tree’s response to powdery mildew in different ways. Notably, after 4 h of powdery mildew infection, the expression of HbZAR1.1 was significantly upregulated, which suggests a rapid upregulation of HbZAR1.1 expression to identify the powdery mildew pathogen. Previous studies have shown that pathogens adapted to host plants secrete a large number of effectors into the host’s extracellular space or enter host cells. These effectors can interfere with the immune response or hijack the host’s metabolism to promote pathogenicity [30]. The sudden decrease at 8 h may be due to the powdery mildew adapting to rubber trees and secreting a large number of effectors into the extracellular space of rubber trees or entering host cells. These effectors interfere with the immune response, leading to the sudden downregulation of the HbZAR1.1 gene. Subsequently, the expression level of HbZAR1.1 increased in the late stage of powdery mildew infection, which may be due to rubber trees increasing the expression of immune genes like HbZAR1.1 to eliminate this interference and maintain a normal immune response. This indicates that the HbZAR1.1 gene may be involved in rubber trees’ response to powdery mildew infection at different stages through different mechanisms. Upon infection by anthracnose fungus, the expression of HbZAR1.1 and HbZAR1.2 is upregulated in the early stage and downregulated in the later stage. Interestingly, the expression levels of HbZAR1.2 and HbZAR1.3 show extremely significant downregulation at 48 h post anthracnose infection, with fold reductions reaching 35 and 11, respectively. This also suggests that the HbZAR1 genes may be involved in the response to anthracnose at different infection stages through different mechanisms. Bi et al. demonstrated that Arabidopsis ZAR1 is localized on the plasma membrane, and its activation leads to Ca²⁺ influx dependent on Glu11, generation of reactive oxygen species, disruption of plasma membrane integrity, and ultimately results in cell death [13]. In this study, subcellular localization detection results indicate that the HbZAR1 gene can also be located on the plasma membrane, suggesting that the rubber tree HbZAR1 may have similar functions to Arabidopsis ZAR1. Notably, the rubber tree HbZAR1 gene can also be located on the subcellular nucleus, indicating the potential existence of different functions compared to Arabidopsis ZAR1 gene, possibly due to interspecies differences. These findings imply that the HbZAR1 gene may play a crucial role in pathogen recognition and immune signal transduction processes. Thereby underscoring their potential involvement in the tree’s defensive mechanisms against these pathogens.

3.3. Function and Mechanism of HbZAR1 Genes

The NLR immune receptor, as an essential anti-disease protein, has long been discovered, but its activation mechanism and working principles remain unclear [31]. It was not until 2019 that Chai and others made a breakthrough in the molecular mechanism research of anti-disease protein activation. They found that the anti-disease protein ZAR1 forms a multi-protein complex with related proteins after activation, and using cryo-electron microscopy technology, they resolved the complex structures in different states, thereby revealing the core molecular mechanism of NLR activation [32]. ZAR1 can interact with multiple members of the receptor-like protein kinase (RLCK) subfamily to form an immune receptor complex, perceiving pathogenic microbial effector proteins and triggering an immune response [32,33,34,35]. ZAR1 can form a complex with the RLCK family member, RKS1, known as RKS1–ZAR1, to perceive the effector protein AvrAC. Structural analysis by cryo-electron microscopy revealed that intramolecular interactions within ZAR1 maintain its inactive state, which is further stabilized by ADP binding. AvrAC induces uridylation of the kinase PBL2, forming PBL2^UMP. PBL2^UMP acts as a ligand recruited to the RKS1–ZAR1 complex, leading to the formation of the recognition–activation complex PBL2^UMP–RKS1–ZAR1 [35]. Interactions during PBL2^UMP binding involve spatial clashes between RKS1 and ZAR1’s NB-ARC domains, triggering conformational changes that release ADP, activating ZAR1 to bind dATP or ATP, resulting in the assembly of the ZAR1 resistosome [32]. The oligomeric ZAR1 resistosome binds to the plasma membrane and forms a cation-selective channel, allowing for calcium permeation, causing inward flow of calcium, and further activating downstream defense responses, including cell death [36]. Therefore, the ZAR1 resistosome acts as both a sensor for pathogenic effectors and an executor of downstream cell death and signal transduction [37,38,39]. Unlike Arabidopsis and tobacco ZAR1, this study found that HbZAR1s and HbZAR1.1-activated mutants do not induce stable ROS production and cell death. Previous research by the team showed that overexpression of rubber tree CNL gene HbCNL2 in tobacco can lead to stable cell death and trigger higher levels of ROS in rubber tree leaves [40]. It is speculated that during the evolution of rubber trees, the disease-resistant function of the HbZAR1 gene might have degenerated or there could be functional redundancy, possibly acting as a minor-effect gene during disease resistance in rubber trees. In the future, more HbZAR1-like minor genes or main-effective disease resistance genes can be identified to provide effective genetic resources for screening and breeding of highly powdery-mildew-resistant rubber tree lines. We can also identify HbZAR1-interacting proteins, explore its regulatory network, and analyze the molecular regulatory mechanism of ZAR1 gene in rubber trees. To lay the foundation for breeding rubber trees against powdery mildew and anthracnose.

4. Materials and Methods

4.1. Plant Material

This experiment used the seedlings of ‘Renyan 73397’ as the experimental material. Budded seedlings were obtained from the experimental base of the Rubber Research Institute, Chinese Academy of Tropical Agriculture Sciences (19°51′51 N; 109°55′63 E) and grown in a seedling room until the leaves reached the bronzestage. Collect 20 g of roots, 20 g of branches, 20 g of leaves, 10 g of flowers, 5 g of bark, and 500 mL of latex sample from three healthy 18-year-old rubber trees for expression analysis in different tissues. Fresh spore suspension of E. quercicola HO-1 strain was used to infect the leaves of rubber tree seedlings at the bronzestage, followed by placing the treated rubber tree seedlings in a stable environment with a temperature of 22–24 °C, 60%–80% RH, and a light–dark cycle of 14 h/10 h. Fresh spore suspension of rubber tree C. siamense CS23 strain was used to infect the leaves of rubber tree seedlings at the bronze stage, followed by placing the treated rubber tree seedlings in a stable environment with a temperature of 26 °C, 90% RH, with untreated plants as controls, each experiment was conducted three times, and all collected samples were immediately frozen in liquid nitrogen and stored at −80 °C for further analysis. Nicotiana tabacum seeds were used in this study, stored in our laboratory, and 4–5-week-old Nicotiana tabacum was used for cell death and subcellular localization experiments.

4.2. Identification and Physicochemical Properties of HbZAR1 Gene Family Members in Rubber Tree

Download gene and protein sequences of corn, rubber tree, and other plants from NCBI database (https://www.ncbi.nlm.nih.gov/orffinder/, 22 June 2024), the Arabidopsis gene and its encoded protein sequences are derived from the Arabidopsis database (http://www.arabidopsis.org/, 22 June 2024), and the rice sequences originate from the rice database (http://rice.plantbiology.msu.edu/analyses_search_locus.shtml, 22 June 2024). The A. thaliana ZAR1 sequences served as templates for searching for HbZAR1 sequences within the rubber tree genome (ASM165405v1), analyzing the aligned sequences using BLAST default settings, and manually removing redundant sequences. Analyze gene open-reading frames (ORFs) using NCBI ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/, 22 June 2024), predict candidate gene amino acid sequences using NCBI Conserved Domain Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, 22 June 2024). Predict physical and chemical information, such as molecular weight (MW) and isoelectric point (PI), using ExPASy (http://web.expasy.org/protparam/, 22 June 2024). Subcellular localization of three HbZAR1 proteins using DeepLoc—2.1 (DeepLoc 2.1—DTU Health Tech—Bioinformatic Services, 22 June 2024).

4.3. Phylogenetic Analysis of HbZAR1s

MEGA 7.0 is used for conducting phylogenetic analysis. The purpose of 1000 bootstrap replicates are to check the branch reliability of the phylogenetic tree by repeatedly sampling the dataset. Three ZAR1-homologous proteins from the rubber tree and 108 ZAR1 homologous proteins from 88 other plant species were used for phylogenetic analysis [6].

4.4. Conserved Domain and Motifs of HbZAR1 Proteins in Rubber Tree

Analyzing the conserved domain of the HbZAR1 proteins using NCBI Conserved Domains. Predicting protein motifs using Multiple Em for Motif Elicitation (MEME, http://meme-suite.org/tools/meme, 24 June 2024), with an optimal width setting of 16–100 residues and visualizing them using the TBtoos tool.

4.5. Structure and Putative Cis-Acting Element Analysis of HbZAR1 Genes in Rubber Tree

By aligning the coding sequence with the genomic sequence, the Gene Structure Display Server (GSDS, gsds.gao-lab.org, 26 June 2024) is used to illustrate the exon–intron structure. Analyzing putative cis-acting elements in the promoter region involves obtaining the upstream 2 kb sequence from the start codon in the genomic sequence, using the plantCARE software (http://bioinformatics.psb.ugent.be/, 24 June 2024) to search for cis-acting elements, confirming their presence in the promoter region, and visualizing them with TBtools. For detailed website information, please refer to Table 1.

4.6. Expression of HbZAR1s Using qRT–PCR

RNA was extracted from all samples following the protocols outlined in a TIANGEN Polysaccharide Polyphenol Plant Total RNA Extraction Kit, provided by Takara in Tokyo, Japan. The isolated RNA samples, each 1 µg, underwent reverse transcription using the RevertAid First Strand cDNA Synthesis Kit from Thermo Fisher in Beijing, China. The synthesized cDNA was then employed for qRT–PCR experiments.

The primers for qRT-PCR were meticulously designed using Primer 5 software (Version 5.00), with the HbActin (GenBank accession number HO004792) serving as the internal control (Table S3). The qRT–PCR reactions were performed using the SYBR Premix Ex Taq II Kit from Takara, Japan, in a 20 µL reaction volume. The qRT–PCR detection-operating conditions include an initial denaturation at 95 °C for 3 min, followed by forty cycles of amplification with specific conditions of 95 °C for 10 s and 60 °C for 30 s. Each sample was subjected to three biological replicates and three technical replicates per treatment.

The expression patterns were analyzed using a comparative 2^−ΔΔCT method. Statistical significance was determined through one-way ANOVA, with post hoc multiple comparisons conducted using Tukey’s test at a significance threshold of p < 0.05.

4.7. The HbZAR1 Genes Subcellular Localization

The pCAMBIA1300 vector carries the 35s×2pro strong promoter and GFP tag, enabling transient expression of the target gene and emitting green fluorescence signal under specific excitation light within a specific range, chosen for use in the subcellular localization study of the HbZAR1s. The target fragment of HbZAR1 genes was cloned from rubber tree leaf cDNA as template, and the linear vector was obtained by double cleavage of the vector with KpnI and XbaI restriction endonucleases. The HbZAR1-coding sequence was constructed into pCAMBIA1300 vector by homologous recombination. Sequencing was performed using Sangyo Bioengineering (Shanghai) Co. Then, the empty and positive cloned recombinant vector was transformed into Agrobacterium tumefaciens GV3101 using heat-excited methods, respectively. The positive PCR test was continued until OD600 = 0.8, and the organisms were collected. The bacteria were washed, and then the OD600 = 0.6 was adjusted with the infestation solution (10 mmol L⁻¹ MES, 10 mmol L⁻¹ MgCl₂, 150 μmol L⁻¹ acetosyringone). In addition, an H2B-mCherry vector was selected as a nucleus marker. Tobacco leaves aged 3–4 weeks with consistent growth are selected, and the recombinant vector liquid is aseptically aspirated from the leaf backside into the plant using a sterile syringe. After injection, the plants are kept in the dark for 12 h, then maintained normally for 48 h before the leaves are harvested and observed for green- and red-fluorescence signals under excitation light at a wavelength of 488 nm and 543 nm using laser-scanning confocal microscopy (Zeiss LSM 880 0, Jena, Germany) [41].

4.8. The HbZAR1 Genes Transiently Overexpressed in Tobacco

Primers were designed using Primer5.0 software to clone the HbZAR1s using rubber tree leaf cDNA as a template. A cell necrosis assay was performed using a pBin vector. The target fragment was amplified using HbZAR1–pBin primers, and the desired fragment was obtained through gel extraction. Subsequently, the vector was linearized using a Smal restriction endonuclease, and the linear vector was recovered from the gel. The vector construction was achieved through homologous recombination, resulting in the HbZAR1–pBin recombinant vector. The MHD motif of HbZAR1.1 gene was found by protein sequence comparison [42], and the target fragment was cloned using HbZAR1–pBin and HbZAR1–DV–pBin primers for the target fragment, and the HbZAR1–DV–pBin recombinant vector was constructed according to the above method. The recombinant vector was transformed into agrobacterium tumefaciens GV3101-competent cells using a freeze–thaw method and injected into tobacco leaves. Simultaneously, the pBin vector was injected as a negative control, and inf1 was used as a positive control. After injection, the leaves were cultured in the dark for 12 h followed by normal cultivation. The reactive oxygen species (ROS) burst was observed using DAB staining [43], and the cell necrosis phenomena were observed under UV light.