Genome wide identification of the NPR1 gene family in plant defense mechanisms against biotic stress in chili (Capsicum annuum L.)

Abstract

Chili pepper cultivation in the Indian subcontinent is severely affected by viral diseases, prompting the need for environmentally friendly disease control methods. To achieve this, it is essential to understand the molecular mechanisms of viral resistance in chili pepper. The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) genes are known to provide broad-spectrum resistance to various phytopathogens by activating systemic acquired resistance (SAR). An in-depth understanding of NPR1 gene expression during begomovirus infection and its correlation with different biochemical and physiological parameters is crucial for enhancing resistance against begomoviruses in chili pepper. Nevertheless, limited information on chili CaNPR genes and their role in biotic stress constrains their potential in breeding for biotic stress resistance. By employing bioinformatics for genome mining, we identify 5 CaNPR genes in chili. The promoter regions of 1,500 bp of CaNPR genes contained cis-elements associated with biotic stress responses, signifying their involvement in biotic stress responses. Furthermore, these gene promoters harbored components linked to light, development, and hormone responsiveness, suggesting their roles in plant hormone responses and development. MicroRNAs played a vital role in regulating these five CaNPR genes, highlighting their significance in the regulation of chili genes. Inoculation with the begomovirus "cotton leaf curl Khokhran virus (CLCuKV)" had a detrimental effect on chili plant growth, resulting in stunted development, fibrous roots, and evident virus symptoms. The qRT-PCR analysis of two local chili varieties inoculated with CLCuKV, one resistant (V1) and the other susceptible (V2) to begomoviruses, indicated that CaNPR1 likely provides extended resistance and plays a role in chili plant defense mechanisms, while the remaining genes are activated during the early stages of infection. These findings shed light on the function of chili's CaNPR in biotic stress responses and identify potential genes for biotic stress-resistant breeding. However, further research, including gene cloning and functional analysis, is needed to confirm the role of these genes in various physiological and biological processes. This in-silico analysis enhances our genome-wide understanding of how chili CaNPR genes respond during begomovirus infection.

Keywords: NPR1; begomovirus; biotic stress; chili pepper; geminivirus; virus titer.

Figure 1

A phylogenetic analysis was conducted on NPR1 homolog proteins derived from various plant species, and the resulting tree was constructed using the maximum likelihood (ML) algorithm in MEGA v7.0. The study involved NPR1-related proteins from 10 different plant species, namely A. thaliana (At), S. tuberosum (St), S. lycopersicum (Sl), B. Rapa (Br), C. papaya (Cp), T. aestivum (Ta), G. max (Gm), G, hirsutum (Gh), O. sativa (Os), V. vinifera (Vv), and Z. mays (Zm). In this analysis, the C. annuum genes, known as CaNPR, were specifically designated as CaNPR1 through CaNPR5. The study’s results identified three distinct clades, each represented by a unique color, and the NPRs from C. annuum and Arabidopsis were differentiated by green and red markers, respectively.

Figure 2

The subcellular localization prediction analysis of CaNPR1 proteins indicated their predominant localization in chloroplasts, cytoplasm and nucleus. The analysis highlighted that the maximum number of proteins showed a red color, representing their localization in these cellular compartments.

Figure 3

Examination of cis-regulatory elements in chili’s CaNPR gene promoters unveiled their involvement in diverse plant developmental processes. This analysis offered data on the occurrence of these elements within each CaNPR gene, and their spatial distribution across the promoters was also explored, providing insights into their arrangement.

Figure 4

The analysis examined conserved domains and motifs in CaNPR1 genes and their comparison with A. thaliana NPRs, along with the establishment of their phylogenetic relationships. This was visually represented through a color-coded bar graph, generated using MEME version 5.5.2, revealing 20 distinct motifs. By linking this graph to a phylogenetic tree, it offered valuable insights into the evolutionary patterns and functional connections among the CaNPR proteins.

Figure 5

The exon-intron structures were depicted using shapes colored in yellow and black to represent the exons contained within the genes.

Figure 6

The chromosomal distribution of the CaNPR gene family in chili is illustrated in the figure. The figure’s green bars represent chili’s chromosomes, while the green lines indicate the functional relationships or co-regulation between CaNPR genes. This analysis provides insights into the spatial arrangement and potential interactions among CaNPR genes within the chili genome.

Figure 7

Ks (synonymous substitution rate) and Ka (nonsynonymous substitution rate) were computed through TB-tools. The rectangular rate (λ) for chili was assessed at 7.85 × 10 ⁻⁹ . By employing the formula T = Ks/2λ, we determined the date of the duplication event. This analysis yields valuable information about the temporal and evolutionary aspects of gene duplications in chili.

Figure 8

Single synteny illustrates how CaNPR genes are distributed across chromosomes, with lines connecting genes on separate chromosomes to indicate potential gene duplications.

Figure 9

Dual Synteny plot of putative NPR1 genes between C. annuum and 10 other plant species (red lines indicates the orthologous of CaNPR1 in other plants).

Figure 10

Visualization of the protein–protein interaction network among CaNPR proteins, illustrating their connectivity and interaction patterns.

Figure 11

Virus titer quantification in both varieties; V1 (Resistant) and V2 (Susceptible) varieties.

Figure 12

Relative expression of each identified CaNPR1 genes in both varieties upon geminivirus infection.