NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) is the central regulator of the pathogen defense reaction systemic acquired resistance (SAR). NPR1 acts by sensing the SAR signal molecule salicylic acid (SA) to induce expression of PATHOGENESIS-RELATED (PR) genes. Mechanistically, NPR1 is the core of a transcription complex interacting with TGA transcription factors and NIM1-INTERACTING (NIMIN) proteins. Arabidopsis NIMIN1 has been shown to suppress NPR1 activity in transgenic plants. The Arabidopsis NIMIN family comprises four structurally related, yet distinct members. Here, we show that NIMIN1, NIMIN2, and NIMIN3 are expressed differentially, and that the encoded proteins affect expression of the SAR marker PR-1 differentially. NIMIN3 is expressed constitutively at a low level, but NIMIN2 and NIMIN1 are both responsive to SA. While NIMIN2 is an immediate early SA-induced and NPR1-independent gene, NIMIN1 is activated after NIMIN2, but clearly before PR-1. Notably, NIMIN1, like PR-1, depends on NPR1. In a transient assay system, NIMIN3 suppresses SA-induced PR-1 expression, albeit to a lesser extent than NIMIN1, whereas NIMIN2 does not negatively affect PR-1 gene activation. Furthermore, although binding to the same domain in the C-terminus, NIMIN1 and NIMIN2 interact differentially with NPR1, thus providing a molecular basis for their opposing effects on NPR1. Together, our data suggest that the Arabidopsis NIMIN proteins are regulators of the SAR response. We propose that NIMINs act in a strictly consecutive and SA-regulated manner on the SA sensor protein NPR1, enabling NPR1 to monitor progressing threat by pathogens and to promote appropriate defense gene activation at distinct stages of SAR. In this scenario, the defense gene PR-1 is repressed at the onset of SAR by SA-induced, yet instable NIMIN1.

Potassium is the most important and abundant inorganic cation in plants and it can comprise up to 10% of a plant’s dry weight. Plants possess complex systems of transporters and channels for the transport of K+ from soil to numerous parts of plants. Cajanus cajan is cultivated in different regions of the world as an economical source of carbohydrates, fiber, proteins, and fodder for animals. In the current study, 39 K+ transport genes were identified in C. cajan, including 25 K+ transporters (17 carrier-like K+ transporters (KUP/HAK/KTs), 2 high-affinity potassium transporters (HKTs), and 6 K+ efflux transporters (KEAs) and 14 K+ channels (9 shakers and 5 tandem-pore K+ channels (TPKs). Chromosomal mapping indicated that these genes were randomly distributed among 10 chromosomes. A comparative phylogenetic analysis including protein sequences from Glycine max, Arabidopsis thaliana, Oryza sativa, Medicago truncatula Cicer arietinum, and C. cajan suggested vital conservation of K+ transport genes. Gene structure analysis showed that the intron/exon organization of K+ transporter and channel genes is highly conserved in a family-specific manner. In the promoter region, many cis-regulatory elements were identified related to abiotic stress, suggesting their role in abiotic stress response. Abiotic stresses (salt, heat, and drought) adversely affect chlorophyll, carotenoids contents, and total soluble proteins. Furthermore, the activities of catalase, superoxide, and peroxidase were altered in C. cajan leaves under applied stresses. Expression analysis (RNA-seq data and quantitative real-time PCR) revealed that several K+ transport genes were expressed in abiotic stress-responsive manners. The present study provides an in-depth understanding of K+ transport system genes in C. cajan and serves as a basis for further characterization of these genes.

Klebsiella oxytoca is a gram-negative bacterium. It is opportunistic in nature and causes hospital acquired infections. Subtractive proteomics and reverse vaccinology approaches were employed to screen out the best proteins for vaccine designing. Whole proteome of K. oxytoca strain ATCC 8724, consisting of 5483 proteins, was used for designing the vaccine. Total 1670 cytotoxic T lymphocyte (CTL) epitope were predicted through NetCTL while 1270 helper T lymphocyte (HTL) epitopes were predicted through IEDB server. The epitopes were screened for non-toxicity, allergenicity, antigenicity and water solubility. After epitope screening 300 CTL and 250 HTL epitopes were submitted to IFN-γ epitope server to predict their Interferon-γ induction response. The selected IFN-γ positive epitopes were tested for their binding affinity with MHCI-DRB1 by MHCPred. The 15 CTL and 13 HTL epitopes were joined by linkers AAY and GPGPG respectively in vaccine construct. Chain C of Pam3CSK4 (PDB ID; 2Z7X) was linked to the vaccine construct as an adjuvant. A 450aa long vaccine construct was submitted to I-TASSER server for 3D structure prediction. Thirteen Linear B cells were predicted by ABCPred server and 10 sets of discontinues epitopes for 3D vaccine structure were predicted by DiscoTope server. The modeled 3D vaccine construct was docked with human Toll-like receptor 2 (PDB ID: 6NIG) by PatchDock. The docked complexes were refined by FireDock. The selected docked complex showed five hydrogen bonds and one salt bridge. The vaccine sequence was reverse transcribed to get nucleotide sequence for In silico cloning. The reverse transcribed sequence strand was cloned in pET28a(+) expression vector. A clone containing 6586 bp was constructed including the 450 bp of query gene sequence.

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