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Article

Diversity and Evolution of NLR Genes in Citrus Species

by
Zhiwei Xiong
1,
Wanshan Zhang
1,
Hui Yin
1,
Jiaxing Wan
1,
Zhuozhuo Wu
1 and
Yuxia Gao
1,2,*
1
National Navel Orange Engineering Research Center, Gannan Normal University, Ganzhou 341000, China
2
Jiangxi Provincial Key Laboratory of Pest and Disease Control of Featured Horticultural Plants (2024SSY04181), Ganzhou 341000, China
*
Author to whom correspondence should be addressed.
Biology 2024, 13(10), 822; https://doi.org/10.3390/biology13100822
Submission received: 7 September 2024 / Revised: 9 October 2024 / Accepted: 12 October 2024 / Published: 14 October 2024
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Abstract

:

Simple Summary

Citrus (Citrus spp.) is the world’s largest fruit crop, both in terms of cultivation area and production. In recent years, the citrus industry in countries such as China, Brazil, and the United States has suffered significant damage due to the increasing severity of citrus diseases, such as Citrus Huanglongbing (HLB). Plant nucleotide-binding leucine-rich repeat (NLR) genes play a critical role in the plant immune system by helping plants resist pathogen infections. A thorough understanding of the diversity and evolution of citrus NLR genes is of great significance for the development of disease-resistant cultivars. In this study, we systematically identified 1585 NLR genes across 10 citrus genomes and conducted an in-depth analysis of their origins and evolutionary processes.

Abstract

NLR genes are crucial components of the effector-triggered immunity (ETI) system, responsible for recognizing pathogens and initiating immune responses. Although NLR genes in many plant species have been extensively studied, the diversity of NLR genes in citrus remains largely unknown. Our analysis revealed significant variations in the copy numbers of NLR genes among these species. Gene duplication and recombination were identified as the major driving forces behind this diversity. Additionally, horizontal gene transfer (HGT) emerged as the principal mechanism responsible for the increase in NLR gene copy number in A. buxifolia. The citrus NLR genes were classified into four categories: TIR-NBS-LRR (TNL), CC-NBS-LRR (CNL), RPW8-NBS-LRR (RNL), and NL. Our findings indicate that TNL, RNL, and CNL genes originated from NL genes through the acquisition of TIR and RPW8 domains, along with CC motifs, followed by the random loss of corresponding domains. Phylogenetic analysis suggested that citrus NLR genes originated alongside the species and underwent adaptive evolution, potentially playing crucial roles in the global colonization of citrus. This study provides important insights into the diversity of citrus NLR genes and serves as a foundational dataset for future research aimed at breeding disease-resistant citrus varieties.

1. Introduction

When pathogens infect plants, the plants deploy two innate immune systems—pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI)—to defend against the invasion of pathogens [1,2]. PTI triggers fundamental immune mechanisms such as ethylene production, oxidative bursts, callose deposition, and the expression of defense-related proteins [3,4], thereby impeding the invasion and spread of pathogens. However, pathogens evolve various strategies to counteract or evade PTI defenses. When pathogens release effectors to suppress PTI, the plant’s resistance genes (R genes) can directly or indirectly recognize these effectors, activating ETI immune responses. ETI typically leads to a hypersensitive response (HR) at the infection site, where cells rapidly undergo programmed cell death, limiting the further spread of pathogens [5,6]. These two immune systems are interconnected, working together to maintain the health and survival of plants [2,7,8].
R genes are critical components of the ETI immune system, playing a key role [6]. R-proteins act as receptors for specific pathogens. Upon recognizing pathogen effectors, these proteins trigger complex signaling pathways that lead to various defense responses, such as the production of antimicrobial compounds, reinforcement of cell walls, and activation of programmed cell death. This rapid response not only limits pathogen spread but also induces systemic acquired resistance (SAR), providing long-lasting immunity against future infections [9]. The Rps2 gene in Arabidopsis thaliana confers resistance to the bacterial pathogen Pseudomonas syringae. The Rps2 protein detects the effector AvrRpt2, initiating a hypersensitive response characterized by localized cell death that restricts pathogen growth [10]. Similarly, the Pi-ta gene in rice (Oryza sativa) provides resistance to the rice blast fungus Magnaporthe oryzae. The Pi-ta protein recognizes the effector Avr-Pita, triggering defense responses and effectively curbing the disease [11].
Most R genes encode disease resistance proteins with nucleotide-binding site and leucine-rich repeat (NBS-LRR) domains; these genes are thus often termed NLR genes [12]. Beyond the canonical NBS and LRR domains, the N-termini of R proteins frequently feature additional domains or motifs, such as the toll/interleukin-1 receptor (TIR) domains, the powdery mildew resistance protein 8 (RPW8) domains, and coiled-coil (CC) motifs [1,13]. Proteins harboring the TIR and RPW8 domains are classified as TNL and RNL, respectively [13,14]. Similarly, proteins with CC motifs are designated as CNL [12]. A significant proportion of R proteins, possessing only the NBS and LRR domains, are collectively categorized as NL (NBS-LRR).
Comparative genomics research has shown that NLR genes are extensively distributed among land plants and charophytes, indicating a deep evolutionary origin [1,13,15]. Additionally, NLR genes have been identified in a limited number of green algae species, further supporting their ancient lineage [16]. Phylogenetic analyses suggest that the emergence of R genes preceded the evolutionary split between charophytes and land plants [1,13]. Following their origin, NLR genes have undergone adaptive evolution, significantly contributing to the transition of plants from aquatic to land habitats [13].
Citrus is one of the world’s most significant fruits, providing essential nutrients such as vitamin C and citric acid. The cultivation of citrus spans over 140 countries and regions globally, thriving in warm and moist climates below the northern latitudes [17]. In 2020, the global citrus cultivation area reached 521,000 hectares, yielding a production of 96.81 million metric tons [18]. However, citrus faces various threats from diseases, such as citrus HLB, a devastating ailment that severely impacts the yield and quality of citrus. Addressing this challenge necessitates a deep understanding of citrus’s mechanisms of disease resistance. The study of the diversity of disease-resistant genes is foundational for elucidating these defense mechanisms. Currently, there is no systematic research reported on citrus NLR genes.
This study integrates comparative genomics and phylogenetics to systematically explore the NLR genes in the citrus genome, unraveling the mechanisms underlying the diversity, origin, and evolutionary patterns of citrus NLR genes. We observed substantial diversity in NLR genes among different citrus species. Further in-depth investigation revealed that gene duplication and recombination are the primary mechanisms shaping the diversity of NLR genes in citrus. This study holds significant implications for a profound understanding of citrus NLR genes and for breeding disease-resistant citrus varieties.

2. Materials and Methods

2.1. Data Used in This Study

At the outset of this study (September 2020), genomic data for 10 citrus species were accessible, namely C. medica, C. ichangensis, C. hindsii, Atlantia buxifolia, C. trifoliata, C. grandis, C. mangshanensis, C. sinensis Osbeck cv. Newhall, C. sinensis Osbeck cv. Valencia, and C. clementina. The genome of C. sinensis Osbeck cv. Newhall was assembled and annotated by our research team [19] and available for download from the Zenodo database (https://zenodo.org/record/8176803, accessed on 1 January 2020). Genomic data for the remaining 9 citrus species can be obtained from the CPBD (http://citrus.hzau.edu.cn/download.php, accessed on 6 September 2024) [20].

2.2. Identification of NLR Genes

The nine seed sequences of the NB-ARC domain (PF00931) were obtained from the European Bioinformatics Institute (EBI) database and stored in Stockholm format. A hidden Markov model (HMM) of NB-ARC was constructed using HMMER package 3.1b2 [21]. Then, this model was used to search for proteins containing the NB-ARC domain among the protein sequences of 10 citrus genomes (Table S1). The Blastp algorithm was also employed to search for citrus proteins containing the NB-ARC domain, using the NLR proteins of Arabidopsis thaliana [13] as queries, with a cutoff E-value of 10−3. The results obtained from both methods were merged and redundant sequences were removed to obtain citrus proteins containing the NB-ARC domain. Multiple sequence alignment of citrus proteins containing the NB-ARC domain and previously published NB-ARC or NAT domain proteins from both eukaryotes and prokaryotes [22] was performed using MAFFT v7.526 software [23] with the G-INS-i algorithm, followed by manual editing using MEGA11 [24]. ModelFinder [25] was utilized to search for the best model, and IQ-TREE v2.3.6 software [26] was employed with the JTT+R10 model for phylogenetic analysis. Proteins with MalT and SWACOS domains were used as outgroups [22]. The protein domain architecture was annotated using CD-Search [27]. NLR genes were searched in the nucleotide sequences of 10 citrus genomes using NLR-Annotator software v2.1 [28] with default parameters for testing analysis of the identification results.

2.3. Phylogenetic Analysis

To investigate the origin of NLR genes in citrus, 1585 identified NLR proteins of citrus and 1583 published NLR proteins from the Anacardiaceae and Sapindaceae families were aligned using MAFFT v7.526 [23] with the G-INS-i algorithm, manually edited using MEGA11 [24]. ModelFinder [25] was used to research the best model, and then Q.mammal+R10 was employed for phylogenetic tree construction using IQ-TREE v2.3.6 [26].
The citrus NLR gene sequences were aligned using MAFFT v7.526 [23] for multiple sequence alignment, followed by manual editing using MEGA11 [24]. ModelFinder [25] was employed to identify the best model, and subsequently, JTT+F+R10 was used for phylogenetic tree construction using IQ-TREE v2.3.6 [26]. The phylogenetic analysis of citrus species based on whole genomes was conducted using the STAG algorithm implemented in OrthoFinder2 [29].

2.4. Positive Selection Analysis

Due to the significant divergence of NLR genes, alignment and positive selection analyses were not feasible for all sequences. Consequently, we divided them into 65 groups based on phylogenetic analysis. Nucleotide sequences were aligned using MAFFT v7.526 [23] and manually refined with MEGA11 [24]. A likelihood ratio test for positive selection was conducted by comparing the M7 (beta) and M8 (beta and v) models using the PAML v4.9 software package [30,31]. For gene groups demonstrating positive selection via the likelihood ratio test, positively selected sites were identified through the Bayes empirical Bayes (BEB) procedure.

2.5. Recombination Analysis

Recombination was detected using the RDP 5.05 package [32]. The aligned nucleotide sequences were imported into RDP 5.05 and analyzed with the RDP, GENECONV, Chimaera, MaxChi, BootScan, SiScan, and 3Seq methods. Potential recombination events were deemed significant only if supported by at least four methods with a p-value < 10−6 [33].

3. Results

3.1. Identification and Diversity of Citrus NLR Genes

This study employed comparative genomics and phylogenetics to identify NLR genes in the proteomes of 10 citrus species. Initially, protein sequences were screened for the NB-ARC domain using the HMM model, resulting in the identification of 1875 sequences containing this domain (Table S1). Subsequently, 1845 sequences with the NB-ARC domain were confirmed via Blastp homology searches (Table S1). After redundancy reduction, sequences containing the NB-ARC or NAT domains were used to construct a phylogenetic tree, along with reported homologous sequences from representative plants and other species (including animals, bacteria, fungi, and archaea). We identified citrus proteins with the NB-ARC domain that formed monophyletic groups with plant R genes, designating these as citrus NLR protein sequences (Figure 1A). Some sequences with the NB-ARC domain formed monophyletic groups with non-plant homologs and were therefore excluded. Ultimately, we identified 1585 NLR genes (Table S1).
Utilizing the HMM method and the Pfam database, we annotated the domain structures of the 1585 citrus NLR genes, identifying 183 as TNL and 35 as RNL (Figure 1B). Notably, we also annotated the Rx_N (PF18052) domain, predicted to be a CC motif but suggested by research to be a four-helical bundle fold [34]. The Rx_N domain is equivalent to the CC domain annotated in previous studies [34]. Thus, in this context, RxN-NBS-LRR corresponds to CNL, as identified in prior research [12,35]. We collectively termed the NLR proteins containing the Rx_N domain as CNL, totaling 272 proteins (Figure 1B). Additionally, a substantial number of NL proteins containing only NBS or NBS-LRR domains were annotated, totaling 1095 (Figure 1B).
Among the 10 citrus species, aside from the 108 TNL sequences in C. clementina, the number of TNL sequences in the other 9 species was relatively low. Only a few were found in C. medica, C. ichangensis, C. hindsii, and C. trifoliata, while they were absent in C. mangshanensis and C. grandis (Figure 1B). RNL was the least abundant among the three types, evenly distributed across all species, ranging from three to four sequences each (Figure 1B). There were six RNL sequences in C. sinensis Osbeck cv. Newhall, possibly due to its diploid nature, resulting in a doubling of the quantity.

3.2. The Origin of Citrus NLR Genes

This study utilized 1585 identified NLR proteins and 1583 published NLR proteins from the Anacardiaceae and Sapindaceae families (Table S2) to construct phylogenetic relationships (Figure 2). The results indicate that citrus NLR proteins fall within the diversity of Sapindaceae, suggesting that citrus NLR genes originated comparatively later than those in Sapindaceae. Within the Sapindales order, Rutaceae is the oldest, followed by Sapindaceae, with Aurantioideae (Citrus genus) being the most recent [36]. In most monophyletic groups, the phylogenetic relationships of NLR proteins align with the species relationships of Rutaceae, Sapindaceae, and the Citrus genus (Figure 2). The coevolutionary relationships between NLR proteins and species suggest that NLR genes originated concurrently with the species’ origins.

3.3. The Evolution of Citrus NLR Genes

We employed maximum likelihood methods to construct phylogenetic relationships among citrus NLR proteins. Integrating the domain architecture of NLRs, we identified five main clusters in the phylogenetic tree of citrus NLR proteins, comprising one cluster of TNLs, one cluster of CNLs, one cluster of RNLs, and two clusters of NLs (Figure 3). NLs exhibited the highest diversity, while TNLs, RNLs, and CNLs fell within the diversity of NLs, indicating their origin from NLs with the acquisition of TIR, RPW8 domains, and CC motifs, respectively. Additionally, NLs were found within both TNLs and CNLs clusters (Figure 3), indicating TNLs and CNLs lost their TIR and Rx_N domains, respectively, transforming into NLs. Overall, citrus NLR genes underwent frequent events of domain acquisition and loss during evolution.
To test whether citrus NLR genes underwent adaptive evolution after their origin, positive selection analysis was conducted on 65 groups selected based on phylogenetic tree. Amino acid residues under positive selection were detected in the majority of groups (52/65) (Figure 4). Among the 52 gene groups where positive selection was detected, the number of positively selected sites varied (Table S3). Our findings indicate that the vast majority of citrus NLR clusters underwent adaptive divergence.

3.4. Mechanism of NLR Gene Diversity Formation in Citrus

The phylogenetic tree of citrus was constructed using OrthoFinder2 software [29] based on the full genome of 10 citrus genomes (Figure 1A). Atlantia buxifolia is placed at the root of the tree, with the overall phylogenetic order largely consistent with previous reports [37]. Comparing the number of NLR genes across different citrus species on the phylogenetic tree reveals considerable diversity in NLR gene counts, even within the same subfamily. Among the 10 citrus species, substantial differences exist in the copy numbers of NLR genes. For instance, C. clementina has 405 copies, whereas C. ichangensis has only 56, making the former about 8 times more abundant than the latter. In the case of the sweet orange subspecies C. sinensis Osbeck cv. Newhall and C. sinensis Osbeck cv. Valencia, 178 and 107 copies were identified, respectively. Notably, while C. sinensis Osbeck cv. Newhall is a diploid genome, C. sinensis Osbeck cv. Valencia is a haploid genome. Although the genomic content of C. sinensis Osbeck cv. Newhall is double that of C. sinensis Osbeck cv. Valencia, the NLR gene copies are not proportionally higher. What evolutionary mechanisms have contributed to the genetic diversity of citrus NLR genes?
Based on the phylogenetic tree of citrus NLR proteins, we investigated horizontal gene transfer (HGT) and gene duplication events within citrus NLR genes. Gene duplication events were identified when NLR genes from the same species formed a monophyletic group on the phylogenetic tree. HGT events were identified when the genetic diversity of one species fell within that of another species.
Following statistical analysis, we generated a diagram depicting the levels of HGT between various citrus species (Figure 5A), revealing relatively frequent gene exchange among C. sinensis Osbeck cv. Newhall, C. sinensis Osbeck cv. Valencia, C. mangshanensis, and C. clementina. Notably, we observed gene exchange between C. sinensis Osbeck cv. Newhall and C. clementina with Atlantia buxifolia. Additionally, there was minimal gene exchange between C. sinensis Osbeck cv. Newhall and C. medica, while the remaining four citrus species (C. ichangensis, C. hindsii, C. trifoliata, and C. grandis) and other species showed no NLR gene exchange. Correlation analysis between the number of NLR genes and the frequency of HGT events revealed a non-significant positive relationship (Figure 5B), suggesting that horizontal gene transfer contributes insignificantly to citrus NLR gene diversity.
The number of gene duplications and recombinations in C. medica and A. buxifolia was the same, with 10 and 6 instances, respectively. HGT occurred nine times in A. buxifolia and only once in C. medica. The NLR gene copy number in A. buxifolia was 1.67 times that in C. medica. Therefore, HGT is the primary mechanism driving the increase in NLR gene copy number in A. buxifolia.
Recombination has long been considered a significant driver for NLR gene diversification [38]. We used seven algorithms within the RDP 5.05 software [32] to search for recombination signals within citrus NLR genes. A single gene recombination event was considered when four or more algorithms detected positive signals. In this study, 173 recombination events were identified, with C. clementina exhibiting the highest number at 81 occurrences (Table S4). Correlation analysis between the number of NLR genes and the number of recombination events across citrus species showed a significant positive relationship (Figure 5C), indicating that gene recombination is one of the mechanisms contributing to citrus NLR gene diversity.
A total of 293 gene duplication events were recorded in this study, with occurrences ranging from 6 to 116 across different citrus species. C. clementina once again exhibited the highest number of gene duplication events at 116 occurrences (Table S4). Correlation analysis between the number of NLR genes and the number of gene duplication events revealed a positive relationship, indicating that gene duplication also contributes to citrus NLR gene diversity.
The statistical analysis of recombination, HGT, and gene duplication reveals that Citrus clementina experienced gene duplication and recombination events as high as 116 and 81, respectively, resulting in a substantial increase in copy number. In contrast, Citrus ichangensis underwent only six instances of gene duplication and recombination, leading to a limited copy number. Thus, the stark difference in copy number between Citrus ichangensis and Citrus clementina can be attributed to the disparity in gene duplication and recombination events. In summary, gene recombination and gene duplication are the main mechanisms shaping NLR gene diversity in citrus.

4. Discussion

In this study, we employed comparative genomics and phylogenetic methods to identify NLR genes across 10 citrus species. To validate the effectiveness of our mining approach, we used NLR-Annotator software [28] to search nucleotide genomes, resulting in the retrieval of 1847 citrus NLR gene sequences. The NLR counts extracted by NLR-Annotator across various citrus species closely matched the results obtained from Blastp and HMM of this study. Comparison with previously reported citrus NLR gene numbers in the ANNA database [12] revealed close correspondence between the quantities in A. buxifolia, C. grandis, C. medica, and C. ichangensis and the results obtained in this study through Blastp and HMM. Thus, the identification methods utilizing Blastp and HMM employed in this study are validated.
The ANNA database reports 529 NLR genes of C. sinensis Osbeck cv. Valencia [12], whereas our study’s Blastp and HMM analyses yielded 179 and 178, respectively, and the NLR-Annotator software result was 181. The substantial variance can be attributed to the utilization of the C. sinensis Osbeck cv. Valencia v2.0 genome [39] in prior studies, while we employed the v3.0 genome version [40]. Factors such as genome assembly quality and annotation precision between the two genome versions may influence the NLR gene number. We conducted NLR gene mining on the nucleotide sequences of both sweet orange genome versions using the tblastn algorithm. We found comparable outcomes, with no significant differences between the two genome versions. This underscores annotation precision as the primary factor influencing the quantity of NLR genes.
We integrated the results from Blastp and HMM, eliminating redundancies. Subsequently, we employed phylogenetic analysis to further screen the sequences, remove false positives, and discard NLR sequences generated by alternative splicing, thereby obtaining the NLRs for each citrus species in this study. We employed a more stringent identification method to ensure the accuracy of the results.
The distribution of TNLs varies significantly among citrus species. TNLs are sparsely present or absent in C. medica, C. ichangensis, C. hindsii, C. trifoliata, C. grandis, and C. mangshanensis, while in C. clementina, their number can be as high as 108. To exclude the impact of gene annotation on the results, we used the tblastn algorithm to mine the genomes of C. medica, C. ichangensis, C. hindsii, C. trifoliata, C. grandis, and C. mangshanensis for TNLs. The identification results were consistent with our study, thus eliminating the influence of genome annotation on the identification results. The evolutionary fate of NLR genes is mainly determined by natural selection and genetic drift [21]. In this study, positive selection amino acid residues were detected in all four clusters of TNLs selected, suggesting that the loss of TNLs in species such as C. mangshanensis may be due to genetic drift.
In-depth analysis revealed that the expansion of TNL gene copies in C. clementina is due to frequent gene recombination and duplication events. On one hand, previous studies have speculated multiple origins of TNLs [41], while loss has been reported in Aquilegia coerulea and monocots [42]. On the other hand, research has indicated that the evolutionary rate of TNLs is higher than that of non-TNLs [41], suggesting that, compared with CNLs and RNLs, TNLs may be more prone to acquiring or losing the TIR domain, thus undergoing a more complex evolutionary process.
Differences in NLR gene copy numbers among citrus species are not uncommon, as seen in the Rosaceae family, where the wild strawberry (Fragaria vesca) has 144 copies, while the apple (Malus pumila) has 748 copies [41]. This study further analyzes gene recombination and duplication as the primary mechanisms shaping NLR gene diversity in citrus. While HGT did not exhibit significant impact across all citrus species, it was the major driver of the NLR gene copy number increase in A. buxifolia. The number of gene duplications and recombinations in C. medica and A. buxifolia was the same, with 10 and 6 instances, respectively. HGT occurred nine times in A. buxifolia and only once in C. medica. The NLR gene copy number in A. buxifolia was 1.67 times that in C. medica. Therefore, HGT is the primary mechanism driving the increase in NLR gene copy number in A. buxifolia.
Previous studies have indicated that the reduction in NLR genes in angiosperms is linked to ecological specialization and the loss of signal transduction components [12]. This contraction of NLR genes has been associated with adaptations to aquatic, parasitic, and carnivorous lifestyles [12]. The convergent reduction of NLRs in aquatic plants bears resemblance to the absence of NLR expansion throughout the prolonged evolutionary history of green algae prior to their terrestrial colonization [12]. Among citrus species, Citrus sinensis and Citrus clementina are the most widely cultivated, thriving in complex environments, which may account for their relatively higher copy number of RNL genes.
Approximately 30 million years ago, the orange subfamily (Rutaceae: Aurantioideae) originated from the ancient Indian plate, which was then connected to Africa and Australia. As the plates collided and drifted, the Rutaceae subfamily gradually diversified, with the citrus genus originating over 8~9 million years ago in southern China [36]. During the colonization of terrestrial environments, plants encountered new pathogen species and infection levels [43]. Positively selected NLR genes play a crucial role in plant–pathogen interactions. This evolutionary process involved frequent gains and losses of TIR, RPW8, and LRR domains, enhancing R-protein diversity and enabling adaptation to various environmental challenges [13]. The discovery of numerous adaptively evolved NLR genes in citrus suggests their significant role in global citrus colonization.

5. Conclusions

This study provides a comprehensive understanding of the diversity and evolution of NLR genes in citrus species, which were previously underexplored compared with other plants. This study identified 1875 NLR genes across 10 citrus species and analyzed their variations, origins, and adaptive evolution. The findings are particularly significant for their potential application in breeding disease-resistant citrus varieties, making this study a foundational resource for future citrus genetic research and crop improvement efforts.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biology13100822/s1, Table S1: The identification of citrus NLR genes; Table S2: This study used NLR genes of Anacardiaceae and Sapindaceae; Table S3: Positive selection analysis of NLR gene groups in citrus; Table S4: Statistics of three events occurring in citrus NLR genes. Figure S1: Phylogenetic analysis of citrus NLR proteins. The Clades boxed by the green box is selected for the positive selection analysis.

Author Contributions

Z.X. participated in designing this study and obtained and analyzed the data. W.Z., H.Y. and J.W. participated in interpreting the results. Z.W. contributed technical assistance. Corresponding author Y.G. designed this study, interpreted the results, wrote the manuscript, and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the National Natural Science Foundation of China (32060615), and Double Thousand Plan of Jiangxi Province (jxsq2020102134).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The amino acid sequence of NLR protein in citrus species is accessible in the Science Data Bank (https://doi.org/10.57760/sciencedb.11629).

Acknowledgments

We thank Nian Wang (University of Florida), Han-Guan Zhu (Nanjing Normal University), and Gong Zhen (Nanjing Normal University) for guidance during the study research process.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The identification and distribution of NLR genes in citrus. (A) Phylogenetic analysis of NB-ARC domain-containing proteins. Phylogenetic analysis was conducted using an approximate maximum likelihood method with the JTT+R10 model. Proteins with MalT and SWACOS domains were used as outgroups. UFBoot values are shown near the selected nodes. (B) The distribution of NLR proteins in 10 citrus species. The distribution and number of NLR proteins, including TNL, CNL, RNL, and NL, in various citrus species are displayed in the right columns. The phylogenetic relationships among citrus species were constructed based on all citrus genes.
Figure 1. The identification and distribution of NLR genes in citrus. (A) Phylogenetic analysis of NB-ARC domain-containing proteins. Phylogenetic analysis was conducted using an approximate maximum likelihood method with the JTT+R10 model. Proteins with MalT and SWACOS domains were used as outgroups. UFBoot values are shown near the selected nodes. (B) The distribution of NLR proteins in 10 citrus species. The distribution and number of NLR proteins, including TNL, CNL, RNL, and NL, in various citrus species are displayed in the right columns. The phylogenetic relationships among citrus species were constructed based on all citrus genes.
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Figure 2. Phylogenetic analysis of NLR proteins in Sapindales. On the left, phylogenetic analysis was performed using an approximate maximum likelihood method with Q.mammal+R10. Proteins with MalT and SWACOS domains were used as outgroups. The SH-aLRT/bootstrap values are depicted near the selected nodes. The expanded section represents a part of the whole tree. The phylogenetic relationships between Citrus, Anacardiaceae, and Sapindaceae are based on Joyce [36]. Dashed lines indicate the one-to-one correspondence of NLR genes and species.
Figure 2. Phylogenetic analysis of NLR proteins in Sapindales. On the left, phylogenetic analysis was performed using an approximate maximum likelihood method with Q.mammal+R10. Proteins with MalT and SWACOS domains were used as outgroups. The SH-aLRT/bootstrap values are depicted near the selected nodes. The expanded section represents a part of the whole tree. The phylogenetic relationships between Citrus, Anacardiaceae, and Sapindaceae are based on Joyce [36]. Dashed lines indicate the one-to-one correspondence of NLR genes and species.
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Figure 3. Phylogenetic analysis of citrus NLR proteins. The SH-aLRT/bootstrap values are depicted near the selected nodes. The green, red, purple, and black branches are TNL, CNL, RNL, and NL proteins, respectively. The NL clade is collapsed.
Figure 3. Phylogenetic analysis of citrus NLR proteins. The SH-aLRT/bootstrap values are depicted near the selected nodes. The green, red, purple, and black branches are TNL, CNL, RNL, and NL proteins, respectively. The NL clade is collapsed.
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Figure 4. Positive selection analysis of citrus NLR genes. The phylogenetic tree is one shown in Figure 3. There are 65 groups for positive selection analysis as indicated. The red and green groups are detected NLR proteins containing amino acid residues under positive selection and negative selection, respectively.
Figure 4. Positive selection analysis of citrus NLR genes. The phylogenetic tree is one shown in Figure 3. There are 65 groups for positive selection analysis as indicated. The red and green groups are detected NLR proteins containing amino acid residues under positive selection and negative selection, respectively.
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Figure 5. Mechanism analysis of NLR gene diversity. (A) Horizontal gene transfer events in NLR genes between citrus species. Arrows indicate the direction of transfer, and numbers indicate the number of HGT events. (B) The relationship between the number of NLR genes and HGT events. (C) The relationship between the number of NLR genes and gene recombination events. (D) The relationship between the number of NLR genes and gene duplication events.
Figure 5. Mechanism analysis of NLR gene diversity. (A) Horizontal gene transfer events in NLR genes between citrus species. Arrows indicate the direction of transfer, and numbers indicate the number of HGT events. (B) The relationship between the number of NLR genes and HGT events. (C) The relationship between the number of NLR genes and gene recombination events. (D) The relationship between the number of NLR genes and gene duplication events.
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Xiong, Z.; Zhang, W.; Yin, H.; Wan, J.; Wu, Z.; Gao, Y. Diversity and Evolution of NLR Genes in Citrus Species. Biology 2024, 13, 822. https://doi.org/10.3390/biology13100822

AMA Style

Xiong Z, Zhang W, Yin H, Wan J, Wu Z, Gao Y. Diversity and Evolution of NLR Genes in Citrus Species. Biology. 2024; 13(10):822. https://doi.org/10.3390/biology13100822

Chicago/Turabian Style

Xiong, Zhiwei, Wanshan Zhang, Hui Yin, Jiaxing Wan, Zhuozhuo Wu, and Yuxia Gao. 2024. "Diversity and Evolution of NLR Genes in Citrus Species" Biology 13, no. 10: 822. https://doi.org/10.3390/biology13100822

APA Style

Xiong, Z., Zhang, W., Yin, H., Wan, J., Wu, Z., & Gao, Y. (2024). Diversity and Evolution of NLR Genes in Citrus Species. Biology, 13(10), 822. https://doi.org/10.3390/biology13100822

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