Genome-wide analysis of NPR1-like genes in citrus species and expression analysis in response to citrus canker (Xanthomonas axonopodis pv. citri)

Citrus fruits, revered for their nutritional value, face significant threats from diseases like citrus canker, particularly impacting global citrus cultivation, notably in Pakistan. This study delves into the critical role of NPR1-like genes, the true receptors for salicylic acid (SA), in the defense mechanisms of citrus against Xanthomonas axonopodis pv. citri (Xcc). By conducting a comprehensive genome-wide analysis and phylogenetic study, the evolutionary dynamics of Citrus limon genes across diverse citrus cultivars are elucidated. Structural predictions unveil conserved domains, such as the BTB domain and ankyrin repeat domains, crucial for the defense mechanism. Motif analysis reveals essential conserved patterns, while cis-regulatory elements indicate their involvement in transcription, growth, response to phytohormones, and stress. The predominantly cytoplasmic and nuclear localization of NPR1-like genes underscores their pivotal role in conferring resistance to various citrus species. Analysis of the Ks/Ka ratio indicates a purifying selection of NPR1-like genes, emphasizing their importance in different species. Synteny and chromosomal mapping provide insights into duplication events and orthologous links among citrus species. Notably, Xac infection stimulates the expression of NPR1-like genes, revealing their responsiveness to pathogenic challenges. Interestingly, qRT-PCR profiling post-Xac infection reveals cultivar-specific alterations in expression within susceptible and resistant citrus varieties. Beyond genetic factors, physiological parameters like peroxidase, total soluble protein, and secondary metabolites respond to SA-dependent PR genes, influencing plant characteristics. Examining the impact of defense genes (NPR1) and plant characteristics on disease resistance in citrus, this study marks the inaugural investigation into the correlation between NPR1-associated genes and various plant traits in both susceptible and resistant citrus varieties to citrus bacterial canker.


Introduction
Globally, citrus fruits stand out as the predominant category of commercially cultivated fruit within the Rutaceae family, offering a wealth of nutritional benefits and playing a crucial role in the world economy (Russo et al., 2021).Unfortunately, the citrus plant faces a significant threat from various diseases such as greening, citrus decline, and gummosis (Malik et al., 2021).Among these is Citrus Canker Disease (CCKD), caused by Xanthomonas axonopodis pv.citri (Xcc), which poses a major risk to citrus crop yield and quality worldwide, with particular severity in Pakistan (Malamud et al., 2011;Sami et al., 2023a;Sami et al., 2023b).The Sargodha region's citrus orchards contribute over 90% to Pakistan's national citrus production.Yield losses in Pakistan due to citrus canker can range from 10% to 50%, depending on infection severity and environmental conditions (Sabir et al., 2010;Almas et al., 2023).However, their potential is hampered by the onslaught of canker, evidenced by symptoms like discoloration, abrasions, and watersoaked lesions on fruit, stems, or leaves (Shahbaz et al., 2023).Several commercially important citrus varieties, including grapefruit, sweet oranges [C.sinensis C. sinensis (L.) Osbeck], lemons [C.limon (L.) Burm.F.], and key lime (Citrus aurantifolia Swingle), are susceptible to bacterial canker (Graham et al., 2004) impacting both the quantity and quality of fresh and processed fruit.This, in turn, leads to substantial economic losses for citrus growers (Spreen et al., 2003).The economic repercussions extend beyond the orchards, as limited fruit trade among states or internationally from canker-affected regions takes a severe toll on the overall economy (Duan et al., 2009;Khan et al., 2023).
Several alternative strategies for disease management have been devised with the aim of establishing enduring solutions for disease control in the long term.These methods encompass plant defense mechanisms specifically crafted to counteract various stressors, encompassing attacks from pathogens (Kushalappa et al., 2016;Manzoor et al., 2023).Resistance genes are considered one of the major factors in defense mechanisms against pathogenic attacks.Resistance (R) genes often trigger downstream signaling responses during plant disease resistance (Malamud et al., 2011;Khan et al., 2023).An immune response, SAR (Shahbaz et al., 2022), promotes the exploration of the resistant gene family to an infectious pathogen.SAR is considered a prime factor for activating numerous pathogenesis-related (PR) genes, such as NPR1 gene with its paralogues (NPR3 and NPR4) (Fu and Dong, 2013).Exogenous application of some chemicals, like benzol 1,2,3thiadiazole-7-carbothermic acid, 2,6-dichloroisonicotinic acid, salicylic acid, and S-methyl ester on plants are considered to be responsible for the stimulation of the SAR system (Durango et al., 2013).Additionally, it is noted that during specific hypersensitive responses, the signal transduction pathway(s) connecting HR to SAR involves the endogenous production of SA (Cao et al., 1997).
Based on structural motifs, transmembrane areas, interleukin-1 receptor domain, leucine rich repeat (LRR) domain, coiled coil (Spoel et al., 2003) domains, and nucleotide-binding sites (NBSs), resistant proteins are grouped into numerous superfamilies.The two most prevalent R genes (NBS-LRR) found in plants (Meyers et al., 2003) are further divided into sub-groups based on Nterminal CC or TIR domain (Li et al., 2018).NPR1 genes and their homologues were revealed to regulate the resistance of disease in the citrus family against various pathogens (Peng et al., 2021).NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1, along with their NPR3 and NPR4 as paralogues) serves as authentic SA receptors, actively participating in both local and systemic immunity by regulating SA-mediated processes (Birkenbihl et al., 2017;Sami et al., 2023c).
The investigation of the resistant gene family across various citrus species is vital in light of the aforementioned findings (Ahmad et al., 2023;Hussain et al., 2023).Thus, this resistant gene family was recognized and analyzed thoroughly in different species of citrus (Citrus sinensis, Citrus reticulata, Citrus fortunella, Citrus maxima, Citrus medica, Citrus ichangenesis, Atlanta buxifolia, and Poncirus trifoliata) that ranged from susceptible to highly resistant against canker.Phylogenic classification, characterization on molecular basis conserved motifs, residues of amino acids, distribution on chromosomes, and composition of protein domains were investigated.Furthermore, the stress-related expression pattern of citrus-resistant genes in different regions was also analyzed using citrus species sequencing of RNA (RNA-seq) datasets (Haider et al., 2023b).The findings underscore the need for substantial support to facilitate applied research and in-depth exploration of resistant genes in citrus species and related plant families.This exploration holds promise for influencing the creation of disease-resistant citrus varieties using various breeding methods such as double haploid technology, hybridization, tissue culture, and backcrossing.

Data retrieval of NPR1-like genes
The NPR reference sequences from A. thaliana were employed to identify homologous genes in the protein database of 11 citrus species via the https://www.citrusgenomedb.org/blastplatform, with an E-value threshold of < 1.2 × 10 −14 .Subsequently, genomic, protein, CDS, and promoter sequences for all NPR genes were retrieved.In-depth analysis included the thorough examination of protein sequences to pinpoint representative sequences within duplicated genes.This process involved the utilization of TBtools and the verification of the existence of distinctive NPR domains, encompassing ANK repeats and Nterminal BTB/POZ domains.Additionally, motif analysis was conducted on these protein sequences using the motif finder tool (https://www.genome.jp/tools-bin/search_motif_lib)(Arabbeigi et al., 2018).The identified motifs were then compared against the canonical pattern found in "AtNPR1"-like proteins.

Phylogeny analysis
Furthermore, the protein sequences of A. thaliana were retrieved from the GenBank (Sayers et al., 2022) database (https:// www.ncbi.nlm.nih.gov/genbank/) to find out the phylogenetic integration among these NPR1-like proteins and CsNPRs, CrNPRs, CfNPRs, CcNPRs, ClNPRs, CmdNPRs, CmNPRs, AbNPRs, PtNPRs, and CiNPRs-like proteins.The full-length sequences of Arabidopsis proteins and the protein sequences of NPR1-like genes in 10 different citrus species were aligned using the Clustal W method (Nakasu et al., 2019).As a result, an unrooted phylogenetic tree was generated using the maximum-likelihood (ML) algorithm, employing MEGA-X software, with the specified parameters including the bootstrap method (1,000 replicates), the Jones-Taylor-Thornton model, and the inclusion of all sites (Shafiq et al., 2024).

Nonsynonymous (Ka) and synonymous (Ks) substitution rates calculation
The Ks and Ka values of genes from CsNPRs, CrNPRs, CfNPRs, CcNPRs, ClNPRs, CmdNPRs, CmNPRs, AbNPRs, PtNPRs, and CiNPRs were also determined.To achieve this, duplicate gene pairs resembling NPR1-like genes, originating from various duplication mechanisms, were employed to calculate substitution rates for Ka and Ks.TBtools were utilized to compute Ka and Ks values for these duplicated gene pairs and their respective Ka/Ks ratios using the simple Ka/Ks calculator feature.The Ka/Ks ratio was assessed to elucidate the molecular evolutionary rates of each gene pair.Generally, a Ka/Ks value <1 suggests purifying selection in evolution, Ka/Ks equals to 1 indicates neutral selection, while Ka/ Ks >1 signifies positive selection (Haider et al., 2023b).Furthermore, the calculation of the divergence time for these gene pairs was conducted using the formula t = Ks/2r, with r (1.5 × 10 −8 ) serving as the representative value for neutral substitution rates (Campos et al., 2023).

Cis-regulatory element and synteny data retrieval
PlantCARE database (http://bioinformatics.pAb.ugent.be/webtools/plantcare/htmL/) (Haider et al., 2023b) was used to identify Cis-regulatory elements.The promoter sequences of NPR1-like genes were extracted from genome sequences of 10 citrus species by TBtools software.Chromosome IDs and coordinates were sourced from the Citrus Genome Database, then Advanced Circos in TBtools was used to create the synteny graph.

Citrus NPR gene expression in diverse tissues and organs
The study of ESTs (expressed sequence tags) to find resistance genes in citrus species and characterize their expression levels across multiple citrus plant tissues and organs, leveraging the online NCBI Gene Expression Omnibus (GEO) database (Grechkin et al., 2017).The Blast P search was performed using a default parameter with an e-value of 1e−04.Various genes exhibited expression in distinct segments of the citrus plant, encompassing the flower, root, fruit, leaf, stem, and more, in response to diverse forms of biotic stress and various influencing factors.Heat maps were used to analyze the expression patterns of specific resistance genes across different tissues and organs in different citrus species.

Plant material
The four citrus cultivars C. sinensis (sweet orange), C. reticulata (mandarin), C. limon (lemon), and C. fortunella (kumquat) were inoculated with citrus bacterial culture (Xanthomonas axonopodis pv.citri) and chosen for gene expression.The experiment was conducted in a greenhouse (day/night temperature, 25°C; light/dark periods, 16/8) using the method of completely randomized design (CRD) and three replicates of each treatment.

RNA extraction and qRT-PCR analysis
mRNA was extracted using the triazole method (Fujino et al., 2016), and first-strand cDNA was synthesized using AB script II cDNA first-strand synthesis kit.SYBR Green (Wiz pure) was used to perform qRT-PCR.Standard curves were created to estimate the proportion of citrus NPR1-like genes, and housekeeping gene quantity in samples was determined using real-time PCR (Illumina) and software (https://biomolecularsystems.com/mic-qpcr/software/).To analyze the dynamics of expression, we conducted three biological and three technical replicates.Significance differences were assessed using Student's t-tests (p < 0.05).

Statistical analysis
The data were subjected to statistical analysis using a two-way factorial completely randomized design (CRD).Analysis of variance (ANOVA) was utilized to assess variances, and means were differentiated using the least significant difference (LSD) method, with a significance level set at p < 0.05.The entire statistical analysis was carried out using the Statistix 8.1 software suite.The mRNA lengths of these CsNPRs, CrNPRs, CfNPRs, CcNPRs, CmdNPRs, CmNPRs, AbNPRs, PtNPRs, and CiNPRs genes have nucleotides that range from 1,119 to 69,428, while the amino acid lengths of their respective proteins vary between 372 and 1,379 amino acids.Additionally, the overall pI values vary from 4.91 to 8.74, and the molecular weight of described genes CsNPRs, CrNPRs, ClNPRs, CfNPRs, CcNPRs, CmdNPRs, CmNPRs, AbNPRs, PtNPRs, and CiNPRs proteins vary from 118,053.42 to 46,773.06 (Table 1).

Identification, description, and evolutionary analysis of NPR1 homologues in citrus varieties
The evolutionary tree from the protein sequences of NPR1-like was constructed among the various species of citrus (C.sinensis, C. reticulata, C. fortunella, C. maxima, C. medica, C. ichangenesis, C. limon), and A. buxifolia and P. trifoliata, and references the A. thaliana species as a model (Cao et al., 1997;Spoel et al., 2003;Peng et al., 2021).A 1,000 bootstrap tree was constructed using the UPGMA model in MegaX software.The phylogeny analysis demonstrated the presence of NPR-like proteins categorized into three distinct classes or clades, matching patterns found in other species.Clade I include 41 NPR-related proteins, and clade II comprised 16 NPR-related proteins.Proteins from the same clade appear to be structurally and functionally related (Sami et al., 2024).

Gene duplication analysis
To estimate the approximate gene divergence time, the TBtools program was utilized, and it furnished values for Ks, Ka, and Ka/Ks ratios as specified.The value of synonymous substitutions per synonymous site was denoted by Ks, while the value of nonsynonymous substitutions per non-synonymous site was quantified by Ka.The balance between synonymous and nonsynonymous mutations can be seen in the Ka/Ks ratio.The evolutionary investigation revealed six gene pairs associated with gene duplication within the NPR family.The first pair included ClNPR4/CmNPR5, the second pair CsNPR4/CrNPR5, the third pair CrNPR3/CsNPR8, the fourth pair CmNPR6/CrNPR6, the fifth pair AtNPR6/AtNPR5, and the sixth pair AbNPR1/AtNPR2 (Table 2).
A measure that evaluates the selection pressure affecting amino acid substitutions was the Ka/Ks ratio.When the Ka/Ks ratio declines below 1, it denotes an evolutionary process of purifying selection

Syntenic relationship of NPR gene of different plants
To determine the collinearity relationships among orthologous genes of C. sinensis, C. fortunella, C. reticulata, C. medica, C. maxima, C. limon, A. buxifolia, P. trifoliata, C. ichangenesis, and C. climentina, a synteny analysis was conducted.Notably, scaffolds 1, 2, and 4 of P. trifoliata did not exhibit orthologous gene counterparts.In A. buxifolia, AbNPR1 genes located on scaffold 10 shared orthologs with chromosome 4 of A. thaliana, which also houses AtNPR4 and AtNPR3.Similarly, in A. thaliana, the gene AtNPR5 found on chromosome 2 exhibited orthologs on chromosome 3, including AtNPR6.Moreover, genes located on scaffold 86 of C. reticulata-specifically, CrNPR3, CrNPR4, CrNPR5, and CrNPR6 showed orthology with CmNPR6 on chromosome 9 of C. maxima and with genes CsNPR3 through CsNPR9 on chromosome 7 of C. sinensis (Figure 7).

Expression analysis of NPR genes in citrus
CmNPR1 and CmNPR5 were derived from previously generated in silico data that focused on NPR1 expression during the flavonoid biosynthesis process in both red and white cultivars of C. maxima.The results did not indicate any substantial upregulation or downregulation of CmNPR1 or CmNPR5, implying that NPR1 might not have a significant influence on flavonoid biosynthesis (Zhao et al., 2020) within C. maxima.

Sweet orange fruit expression data across developmental stages
The role of CsNPR1 in the developmental phases of C. sinensis was scrutinized, revealing its active involvement across various stages.Notably, the expression of CsNPR1 remained consistent across all four treatments, suggesting a stable pattern without significant fluctuations in up-or downregulation.This constancy in expression implies that CsNPR1 maintains a steady involvement throughout the developmental processes, indicating a potential regulatory role that remains relatively unaltered across the conditions tested.The observed consistency in expression underscores the significance of CsNPR1 in the intricate orchestration of developmental events within C. sinensis.An abundance of putative NPR1-like genes in various species of citrus, connected to a phylogeny tree, enables a better understanding of gene function and analogy.Cytoplasm exhibited the highest abundance of CsNPR1, CrNPR1, CcNPR2, PtNPR7, CiNPR1, and PtNPR1, while various organelles had distinct but lower quantities of these proteins, indicating diverse subcellular distributions.

FIGURE 5
Different citrus species chromosomes location and duplication occurrences to NPR1-like genes revealed 5 orthologous pairs, e.g., CrNPR6 in C. reticulata aligns with CsNPR4 in C. sinensis.Additionally, AbNPR1 in A. buxifolia corresponds to AtNPR2 in A. thaliana, while AtNPR5 aligns with AtNPR6 within A. thaliana.

RNA-seq and targeted metabolomics reveal C. sinensis leaf responses to prolonged low pH exposure
In the face of diverse low pH conditions ranging from 2 to 6, tested at intervals of 1 h, 2 h, 3 h, 4 h, and 10 h, CsNPR1 displayed a remarkable resilience.Importantly, throughout these varying acidic stress conditions, there was no discernible alteration in the expression of CsNPR1.This steadfastness in expression levels strongly suggests that CsNPR1 remains unaffected by acidic stress within the tested pH range and exposure durations.The consistent expression of CsNPR1 despite the challenging acidic conditions underscores its robust nature and resilience to environmental stressors, highlighting its potential role as a key player in mitigating the impact of low pH stress on the biological processes it governs NPR1 (Zhang et al., 2022).

Analyzing cuticle regulation in Newhall navel oranges during fruit development and ripening
CsNPR1 expression was identified in the regulation of cuticle formation during two distinct developmental stages, a finding consistent across two separate replicates.Importantly, there were no significant fluctuations observed in the expression levels of CsNPR1 during these stages.This stability in expression implies that CsNPR1 consistently plays a role in the control of cuticle formation at these specific developmental points.The lack of Illustration of cis elements of NPR1 in different citrus spiecies.notable changes in CsNPR1 expression levels underscores its reliability as a regulator in the intricate processes involved in cuticle development, emphasizing its potential importance in maintaining the integrity and protective function of the cuticle across these specific developmental milestones.
3.9.4The expression levels of CNPRs genes in different citrus species in response to citrus canker using qRT-PCR The quantitative analysis using qRT-PCR revealed the presence of NPR1 genes expression in C. sinensis following inoculation with Xanthomonas axonopodis pv.citri.Out of the nine CsNPR1 genes, statistical analysis showed that three genes (CsNPR1, CsNPR4, and CsNPR5) did not exhibit significant differences (Figure 8).Conversely, the remaining six genes (CsNPR2, CsNPR3, CsNPR6, CsNPR7, CsNPR8, and CsNPR9) displayed statistically significant responses.Notably, all samples that were treated with infected citrus bacterial canker exhibited higher relative expression of the NPR1 gene compared to the control group.Among them, CsNPR7-and CsNPR9-treated samples demonstrated the highest gene expression levels.However, it is important to note that control samples displayed higher expression levels than the other control samples.
The results obtained from qRT-PCR analysis indicated the expression of NPR1 genes across all treatments in C. reticulata.Notably, all NPR1 genes in C. reticulata displayed statistically significant expression, except for CrNPR1, which exhibited varying results.Among these genes, CrNPR3, CrNPR4, and CrNPR6 demonstrated the highest levels of expression in the treated samples compared to the control group, while CrNPR2 and CrNPR5 exhibited higher expression levels in the control samples than in other genes (Figure 9).
In the case of C. fortunella, the real-time qPCR findings showed significant expression of NPR1 genes, particularly CfNPR1 and CfNPR2.Interestingly, CfNPR2 displayed increased expression in the control samples compared to the treated (infected with citrus bacterial canker) samples (Figure 10).
Similarly, NPR1 genes were found to be expressed in all treated samples of C. limon compared to the control.Among these, ClNPR1, ClNPR3, and ClNPR6 yielded statistically non-significant results, while ClNPR2, ClNPR4, and ClNPR5 exhibited statistically significant responses.Notably, ClNPR2 and ClNPR4 upregulated the expression of the NPR1 gene in the treated samples, whereas ClNPR5 showed higher expression in the control samples compared to the treated ones (Figure 11).

Discussion
This study marks the inaugural genome-wide exploration of NPR1-like gene families and their corresponding expression patterns in reaction to Xanthomonas axonopodis pv.citri infection within four distinct citrus species (Cao et al., 1997;Spoel et al., 2003;Li et al., 2018;Peng et al., 2021).
NPR1 (Non-expressor of pathogenesis related genes 1) serves as the authentic receptor for salicylic acid, playing a crucial role in plant immunity (Cao et al., 1997;Spoel et al., 2003;Li et al., 2018;Peng et al., 2021).NPR1 plays a pivotal role in the signaling pathway of salicylic acid and systemic acquired resistance (SAR) in plants.Additionally, other members of the NPR1 gene family are associated with various reactions to both biotic and abiotic stresses (Cao et al., 1997;Peng et al., 2021).The basic significance of studies on NPR1 is due to its extensive role in plant defense (Cao et al., 1997;Peng et al., 2021).It is generally postulated that salicylic acid-based defense shields plants against host-or semi-host-dependent pathogens, while jasmonic acid-based defense protects against necrotrophic pathogens (Wally et al., 2009).This study represents the first comprehensive examination of the NPR1-like gene family in various citrus species, identifying a total of 63 NPR1-like genes across 10 citrus species (Irfan et al., 2023).
The phylogenetic tree illustrates three distinct clades of NPR genes, with members within the same clade grouping together based on their presumed similar functions.Gene structure predictions using intron-exon displays and motif analysis revealed that most NPR1 sequences lacked introns, although some had between 1 and 7.In motif analysis, it was observed that motifs 8, 2, 7, 9, 5, 1, 4, 3,   and 10 were commonly found in the majority of sequences.Nevertheless, variations, including the presence or absence of specific motifs, were evident, even among sequences within the same clade.These variations suggest that the NPR1 gene has undergone structural mutations, including insertions and deletions, over time.The orthologous relationships inferred from gene mapping and syntenic analysis support the notion of a shared evolutionary history among NPR sequences.For NPR1 to upregulate plant resistance genes, it must localize in the nucleus (Kinkema et al., 2000).Changes in redox potentials influence its nuclear-cytoplasmic localization (Bassene et al., 2011).Typically, NPR1 exists as a cytoplasmic oligomer under normal conditions.However, upon detecting pathogen infection and salicylic acid accumulation, which alters cellular redox potential, NPR1 translocates to the nucleus as an active monomer, where it interacts with bZIP transcription factors from the TGA family (Despreś et al., 2000).The results of subcellular localization confirm that NPR1 is primarily located in both the cytoplasm and the nucleus, thereby substantiating its involvement in conferring resistance among various citrus species (Spoel et al., 2003;Wally et al., 2009;Despreś et al., 2000).
Examining selective pressure unveils the benefits of particular amino acid sequence modifications within proteins, offering insights into functional residues and alterations in protein function.The analysis of synteny and chromosomal mapping of NPR1-like genes reveals evidence of tandem and segmental duplications, along with the identification of orthologous relationships among different citrus species.This information provides valuable insights into gene duplications, rearrangements, and the evolutionary history of these species.C. sinensis and C. reticulata share a common ancestor, supported by the presence of various orthologs on chromosome 7 of C. sinensis and scaffold 86 of C. reticulata.A duplication event occurred in scaffold 86 of C. reticulata and chromosome 9 of C. maxima, A. buxifolia, and A. thaliana diverged from a common ancestor, sharing an ortholog.
Analyzing gene expression patterns of NPR1-like genes using existing data reveals variations in their expression levels under different stresses (biotic and abiotic).The expression of CsNPR1 exhibits a remarkable consistency and stability across diverse conditions, reflecting its pivotal role in C.sinensis development and stress response.In developmental phases, CsNPR1 maintains a steady presence, suggesting its regulatory involvement throughout these processes.Its resilience to varying low pH conditions, tested over different time intervals, underscores its robust nature in withstanding acidic stress.Notably, CsNPR1 expression remains unaffected, implying a key role in mitigating the impact of low pH stress on biological processes.Furthermore, in the regulation of cuticle formation, CsNPR1 demonstrates reliability with no significant fluctuations in expression levels across distinct developmental stages.Collectively, these findings emphasize the versatility and importance of CsNPR1, positioning it as a central player in orchestrating developmental events, responding to environmental stressors, and regulating key processes like cuticle formation in C. sinensis.A joint transcriptome analysis was conducted to indicate changes in gene expression of CmNPR1 and CmNPR5 during flavonoid biosynthesis, although the differences were not significant (Chen et al., 2022).When C. sinensis is subjected to acidic stress for a long time, the expression of CsNPR1 modulates in the leaves, although this variation is minimal.Similar variations in CsNPR1 expression were detected during cuticle development in Newhall navel orange fruits (Liu et al., 2022).In C. sinensis, all NPR1-like genes exhibited upregulated expression in response to Xac infection compared to the control.CsNPR3, CsNPR7, CsNPR8, and CsNPR9 lacked significant expression in the control group.Except for CrNPR2 and CrNPR5, which exhibited no significant expression in Xacinfected samples, all NPR1-like genes in C. reticulata showed higher expression.Conversely, NPR1-like genes in C. fortunella exhibited downregulated expression in response to Xac treatment compared to control samples, while in C. limon, NPR1-like genes were downregulated except for ClNPR4 and ClNPR6.
Citrus bacterial canker (Xanthomonas axonopodis pv.citri) stress was studied in terms of NPR1-like genes and their associated expression.Citrus species were picked based on their reported resistance to citrus bacterial canker.Two resilient species, C. sinensis and C. limon, and two vulnerable species, C. sinensis and C. limon, were picked for a complete comparative study.C. sinensis and C. limon are vulnerable to citrus bacterial canker, although C. reticulata and C. fortunella are resistant (Licciardello et al., 2022).The first identified and characterized citrus bacterial canker through cultural, biochemical, and molecular analysis, confirming the presence of Xanthomonas axonopodis pv.citri, a Gram-negative bacterium (Chaudhari et al., 2022).PCR results using Xanthomonas axonopodis pv.citri-specific primers XACF (5′-CGTCGCAATACGATTGGAAC-3′) and reverse primer XACR (5′−CGGAGGCATTGTCGAAGGAA-3′) yielded a 581-bp product.
Previous investigations into NPR1-like gene families in model organism A. thaliana had already established the importance of these families of genes in enhancing tolerance to both biotic and abiotic stress factors (Cao et al., 1997;Despreś et al., 2000;Kinkema et al., 2000;Spoel et al., 2003;Wally et al., 2009;Peng et al., 2021).This current study unveils alterations in the expression analysis of NPR1-like genes in response to an attack by Xac bacteria, with varying responses observed across different citrus species.Notably, in C. limon and C. sinensis, there was an upregulation in the expression of NPR1-like genes.Conversely, in C. fortunella, a lower expression of NPR1-like genes was observed under Xac attack, rendering it more resistant to the pathogen.C. reticulata exhibited varying NPR1-like gene expression, indicating a moderate level of resistance to Xac attack.These findings were further corroborated by physiological and biochemical analyses in C. limon, which showed decreased levels of chlorophyll a and b, alongside an increase in flavonoid and carotenoid compounds, known for their roles in defense mechanisms against biotic stress.However, the production of secondary metabolites like phenols and soluble proteins was suppressed.Additionally, the activity of vital enzymes, such as catalase and peroxidase, was found to decrease, subsequently impeding the plant's growth and development.This groundbreaking research addresses a critical gap by conducting the inaugural analysis of the correlation between NPR1-associated genes and diverse physiological plant parameters in both citrus cultivars susceptible and resistant to citrus bacterial canker.The findings offer potential implications for the development of diseaseresistant citrus varieties through employing multiple breeding techniques, including double haploid technology, hybridization, tissue culture, and backcrossing.

Conclusion
In conclusion, the comprehensive genome-wide analysis of NPR1-like gene families across diverse citrus species provides compelling evidence of their pivotal role in citrus plants resistance mechanisms against biotic stress factors.This exploration not only enhances our understanding of the evolutionary relationships within these gene families but also offers promising avenues for addressing challenges in citrus production and elevating fruit quality.Our hypothesis proposes the targeted modification of the NPR1 gene through CRISPR Cas9 technology, with the potential to yield a resilient C. limon variant resistant to Xac infection.Moreover, a thorough investigation into the roles played by SA and NPR1 during biotic stress emerges as a promising direction for future research.By integrating modern natural breeding techniques-such as double haploids, hybridization, tissue culture, and backcrossing-we aim to fortify susceptibility-prone varieties, bolstering their resilience against both biotic and abiotic stresses.In this way, our holistic approach not only advances citrus resilience but also contributes to a broader understanding of plant resistance mechanisms, marking a significant step forward in the pursuit of sustainable and robust citrus cultivation.
Approximately 63 non-redundant NPR1-like genes were retrieved from the genome of C. sinensis, C. reticulata, C. fortunella, C. limon, C. maxima, C. ichangenesis, C. maxima, P. trifoliata, C. climentina, A. buxifolia, and C. medica.Utilizing bioinformatics tools like BLAST search, 63 NPR1-like genes were discovered in the genome and named as CsNPRs, CrNPRs, CfNPRs, ClNPRs, CcNPRs, CmdNPRs, CmNPRs, AbNPRs, PtNPRs, and CiNPRs.Using NCBI CDD Domain analysis, all the proteins that lack N-terminal BTB/POZ domain or ANK repeats in the middle region were excluded.As a result, different species have different number of NPR1-like genes, like nine in C. sinensis; seven in C. reticulata; two in C. fortunella; six in C. limon, A. thaliana, and C. medica; 10 in C. climentina and C. ichangensis; seven in C. maxima and P. trifoliata; and three in A. boxifolia (Figure1).
Comparative chromosomal mapping of several citrus species, including C. sinensis, C. climentina, C. medica, C. maxima, C. ichangenesis, C. limon, C. fortunella, A. buxifolia, and P. trifoliata, about A. thaliana, revealed the presence of five pairs of orthologous genes among these citrus species.Specifically, Scaffold 86 of C. reticulata contained CrNPR6, which had an ortholog, CsNPR4, on chromosome 7 of C. sinensis.Additionally, CrNPR3 on Scaffold 86 of C. reticulata exhibited orthology with CsNPR7 on chromosome 7 of C. sinensis, while CrNPR5 on Scaffold 86 of C. reticulata shared orthology with CmNPR6 on chromosome 9 of C. maxima.Furthermore, AbNPR1 on Scaffold 15601 of A. buxifolia was found to be orthologous to AtNPR2 on chromosome 4 of A. thaliana, and AtNPR5 on Chromosome 2 of A. thaliana displayed orthologous relationships with AtNPR6 on chromosome 3 of A. thaliana (Figure 5).

FIGURE 7 A
FIGURE 7A Circos plot depicting the NPR1-like gene family in citrus species.The red connections indicate instances of gene duplications occurring both within and between various species.Synteny analysis revealed collinearity relationships among orthologs from various citrus species and identified specific gene matches, such as AbNPR1 in A. buxifolia, AtNPR5 in A. thaliana, and CrNPR3, CrNPR4, CrNPR5, CrNPR6 in C. reticulata with their respective orthologs in related species.

FIGURE 8
FIGURE 8Relative expression of NPR1 genes in C. sinensis infected and healthy plants.

FIGURE 9
FIGURE 9Relative expression of NPR1 genes in C. reticulata infected and healthy plants.

FIGURE 10
FIGURE 10Relative expression of NPR1 genes in C. fortunella infected and healthy plants.

FIGURE 11
FIGURE 11Relative expression of NPR1 genes in C. limon-infected and healthy plants.

TABLE 1
Physiochemical properties of NPR1 gene family in different citrus species.

TABLE 2
The expression ka/ks represents the ratio of mutations involving synonymous substitutions (ks) to mutations involving non-synonymous substitutions (ka).