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

Abstract

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.

Keywords: biotic stress; citrus; expression profile of NPR1; phylogenetic analysis; salicylic acid.

Figure 1

Polygenetic tree of NPR1 like proteins in CsNPRs, CrNPRs, CfNPRs, CcNPRs, CmdNPRs, CmNPRs, sbNPRs, PtNPRs and CiNPRs and its homologous in, A. thaliana (At).

Figure 2

Phylogenetic relationship and gene structure of NPR1 genes from CsNPRs, CrNPRs, CfNPRs, CcNPRs, CmdNPRs, CmNPRs, sbNPRs, PtNPRs and CiNPRs. The phylogenetic tree was constructed using full length sequences of CsNPRs, CrNPRs, CfNPRs, CcNPRs, CmdNPRs, CmNPRs, sbNPRs, PtNPRs and CiNPRs-like genes. Yellow boxes indicate exons; and black lines indicate introns.

Figure 3

The domain arrangements of putative NPR1-like genes in C. sinensis, C. reticulata, C. fortunella, C. limon, C. medica, C. maxima, C. clementina, C. ichangensis, P. trifoliate, A. buxifolia, and A. thaliana connected with the phylogenetic tree.

Figure 4

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.

Figure 6

Illustration of cis elements of NPR1 in different citrus spiecies.

Figure 7

A 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

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

Figure 9

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

Figure 10

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

Figure 11

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