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The Nucleotide-binding site domain (NBD) of plant resistance (R) genes plays a vital role during plant defense signaling. The functional significance of CC-NBS-LRR (Coiled coil-NBS-Leucine Rich Repeat) class of R gene designated ZzR1, characterized from Zingiber zerumbet in earlier studies, was determined by molecular modeling and docking studies. Docked complex showed the ligand GTP interacts with amino acid residues in the cleft made by GLPL and P-loop motifs of the NBD. Heterologous expression of ZzR1 NBS protein was optimized using expression vectors, pEcoli-Nterm-6xHN and pET Directional TOPO and transformed in five Escherichia coli strains namely DH5α, TOP10, BL21DE3, BL21DE3 star and BL21plysS cells. The NBS protein of 36 kDa molecular size was expressed in E. coli BL21DE3 strain using pET TOPO vector. Optimum induction was detected at 30 °C using isopropyl-1-thio-β-D-galactopyranoside (IPTG) (1 mM). The present study provides valuable information on ligand interactions and heterologous expression of ZzR1 NBD protein. [ABSTRACT FROM AUTHOR]

Proteins containing nucleotide binding sites (NBS) encoded by plant resistance genes play an important role in the response of plants to a wide array of pathogens. In this paper, an in silico search was conducted in order to identify and characterize members of NBS-encoding gene family in the tribe of Triticeae. A final dataset of 199 sequences was obtained by four search methods. Motif analysis confirmed the general structural organization of the NBS domain in cereals, characterized by the presence of the six commonly conserved motifs: P-loop, RNBS-A, Kinase-2, Kinase-3a, RNBS-C and GLPL. We revealed the existence of 11 distinct distribution patterns of these motifs along the NBS domain. Four additional conserved motifs were shown to be significantly present in all 199 sequences. Phylogenetic analyses, based on genetic distance and parsimony, revealed a significant overlap between Triticeae sequences and Coiled coil-Nucleotide binding site-Leucine rich repeat (CNL)-type functional genes from monocotyledons. Furthermore, several Triticeae sequences belonged to clades containing functional homologs from non Triticeae species, which has allowed for these sequences to be functionally assigned. The findings reported, in this study, will provide a strong groundwork for the isolation of candidate R-genes in Triticeae crops and the understanding of their evolution.

Plant disease-resistance response is a complex process which is regulated by multiple genes. Plant resistance genes(R genes)play an important role in this process. R genes can be divided into different types based on their N-terminal and transmembrane domains. The majority of disease resistance genes in plants encode nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins. The TIR-NB-LRR(TNL)type is a large family of plant NB-LRR genes. And it is also the current hot topics in the studies of plant disease resistance genes. We summarized the related research progresses of different domains of TNL proteins comprehensively in this review. TIR domain in resistance signaling via homodimerization or heterodimerization is a major role of plant TNL proteins. However, emerging roles for pathogen recognition for the plant TIR domain were identified in some researches. The NBS domain in NBS-LRR proteins was proposed to function as a molecular switch. It can adjust the conformation changes of plant R proteins through binding ADP or ATP, which regulates resistance signal conduction downstream. The LRRs of plant TIR-NB-LRR proteins were predicted to interact directly or indirectly with their avirulent effectors to recognize the pathogen specifically. The information provides a good theory basis for study of plant disease-resistance mechanisms, and also provides new insights and choices for crop disease-resistance breeding directionally by gene editing technology in the future. [ABSTRACT FROM AUTHOR]

Plants defend themselves against pathogens via the expressions of disease resistance (R) genes. Many plant R gene products contain the characteristic nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains. There are highly conserved motifs within the NBS domain which could be targeted for polymerase chain reaction (PCR) cloning of R genes. Here, we report a 4-week undergraduate laboratory exercise that mimics the research environment to PCR-clone partial ribs sequences using degenerate primers corresponding to the phosphate-binding loop (P-loop) and GLPL motifs within the NBS domain of potential R gene products from the North American grape, Vitis aestivalis Michx. Students were able to complete the laboratory procedures successfully and obtained four different clones, among which three are new. Through the laboratory exercise, students learned a variety of important molecular techniques including genomic DNA isolation, DNA quantification, PCR, agarose gel electrophoresis, DNA extraction from agarose gel, ligation, bacterial transformation, and plasmid DNA isolation and purification. They also used currently available web-based bioinformatic programs for sequence analysis. The laboratory exercise provides students the hands-on experience on PCR cloning and shows them how it is done in a research environment. The clones obtained may be further tested for their potential use as markers to differentiate resistant cultivars from the susceptible ones, a useful tool in breeding programs. [ABSTRACT FROM AUTHOR]