Abstract
BACKGROUND: Germin-like proteins (GLPs) are naturally occurring glycoproteins ubiquitously found in all plants. These are members of the cupin super family having defensive roles against pathogens causing biotic stresses. Before this study GLPs were not thoroughly investigated in Brassica juncea and its parent species particularly concerning biotic stress responses. This study aimed to identify and characterize the GLP family in B. juncea and its progenitor species, and to determine candidate B. juncea genes (GLPs) conferring resistance against biotic stresses using Alternaria leaf spot disease as model biotic stress. RESULTS: We have identified 102 GLPs in B. juncea, 51 in B. nigra and 48 in B. rapa using in-silico tools. Chromosomal localization revealed that the GLPs of these three species resides on all of their respective chromosomes. Motif, domain, and gene structures were conserved among all three species. Phylogenetic analysis of GLPs among B. juncea, its parental species (B. nigra and B. rapa), and the model plant Arabidopsis thaliana revealed 4 major clades divided into 21 subclades or members. Synteny analysis also revealed that all of the genes are similar and remain conserved in B. juncea as in its parental species. Furthermore, we have identified and characterized candidate genes in B. juncea that possess resistance properties against Alternaria alternata induced Alternaria leaf spot disease through RT-qPCR analysis. Gene duplication events were observed in B. juncea and B. nigra. Additionally, by analyzing RNA-Seq data we identified 25 GLPs at 2 DPI and 30 at 4 DPI in B. juncea responsive to biotic stresses, from which 10 genes having 2-fold change at both time points were selected for their RT-qPCR validation in laboratory under biotic stress conditions. For this, the expression of these differentially expressed GLPs was analyzed in B. juncea plants, infected with Alternaria leaf spot disease causing agent (A) alternata. RT-qPCR analysis in (B) juncea revealed that the expression of 5 GLPs (BjuGLP06, BjuGLP23, BjuGLP34, BjuGLP70, and BjuGLP97) was upregulated and the expression of 5 other GLPs (BjuGLP04, BjuGLP33, BjuGLP71, BjuGLP72, and BjuGLP91) was downregulated. All B. juncea GLPs expression trend observed in the in-silico analysis was confirmed in these in-vitro experiment. CONCLUSION: This study represents the first identification of GLP family in B. juncea and its parental species B. nigra and B. rapa, suggesting that these may contribute to plant defense, with a special focus on resistance to Alternaria leaf spot. This research provides a valuable foundation for further investigations into the functions of GLPs in Brassica and other economically important crops and their potential role for developing stress-tolerant crop varieties, better adaptable to changing environment.