Genome-Wide Identification, Genomic Organization, and Characterization of Potassium Transport-Related Genes in Cajanus cajan and Their Role in Abiotic Stress.

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.

Subtractive Proteomics and Immuno-informatics Approaches for Multi-peptide Vaccine Prediction Against Klebsiella oxytoca and Validation Through In Silico Expression.

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.

First report of post-harvest Fusarium rot of mandarin Citrus reticulata cv. 'Kinnow' caused by Fusarium equiseti in Pakistan.

Citrus reticulata cv. 'Kinnow' mandarin is the most popular and widely grown fruit crop in Pakistan. During 2017, a survey was conducted to the local citrus fruit markets of Faisalabad, Pakistan. Citrus fruits (n=50) exhibiting stem end rot and fruit rot were collected with 15% disease incidence. The stem end region showed light to dark brown lesions and white fungal growth was also observed in the severely infected fruit. Infected fruit were excised into 2mm2 segments, surface disinfected with 1% NaClO, rinsed with sterilized water and dried. Later, these tissues were placed on potato dextrose agar (PDA) medium and subsequently incubated at 25 °C. Purified isolates produced white colonies with beige pigmentation. The frequency of fungal isolation was 47%. Microscopic observations revealed that macroconidia (n=50) had 5 to 6 septations, with a prominent dorsiventral curvature, tapered and elongated apical cell, and a foot shape basal cell. The macroconidia were measuring 22 to 45 × 2.9 to 4.3 µm with an average of 31 × 3.6 µm. However, microconidia were not observed. Chlamydospores were globose, intercalary, solitary, or in pairs, appearing in chains (Leslie and Summerell 2006). For molecular identification, DNA was extracted from all isolates. The internal transcribed spacer region (ITS) ITS1/4 (White et al. 1990), translation elongation factor-1 alpha (TEF) EF1/2 (O'Donnell et al. 1998), and RNA polymerase II subunit 1 (RPB1) (O'Donnell et al. 2013) were amplified using PCR and the product was subsequently sequenced. Based on BLAST analysis, the isolate was identified as Fusarium equiseti (FUS-21). The sequences of the representative isolate FUS-21 were deposited in the GenBank with accession numbers (ITS, MH581300), (TEF, MK203749), and (RPB1, MW596599) showing more than 99% similarity with ITS accession GQ505683, TEF accession GQ505594, and 100% to RPB1 accession JX171481. To determine the pathogenicity, 40 healthy surface disinfested citrus fruit were taken. The fruit were inoculated by creating artificial wounds on the surface with a sterilized needle and 10 µL of 105 spores/mL was deposited in the wounds. In case of control fruit were inoculated with 10 µL sterilized distilled water only, and incubated at 25 °C. All fruit inoculated with the putative pathogen, developed symptoms like the original fruit from which they were isolated. The pathogenicity test was repeated twice. Visible white mycelium appeared at the stem end region and the fruits became dried as the infection progressed. However, the control fruit remained asymptomatic. The pathogen was re-isolated from infected fruit and identified based on morphometric and molecular analysis. Previously we have reported F. oxysporum causing citrus fruit rot in Pakistan (Moosa et al. 2020). This is the first report of F. equiseti causing post-harvest rot of citrus fruits in Pakistan. Kinnow is an important fruit crop of Pakistan with huge export value the management of Fusarium rot is quite important to save the loss of fresh produce.

Hematological and toxicological effects of aqueous leaf extract of Stevia rebaudiana Bertoni in normal rat modals.

Stevia rebaudiana Bertoni a non-caloric, safe and natural sweetener has been shown pharmaceutically important in the management of blood disorders. This study was designed to investigate hematology and safety of stevia aqueous extract through animal modeling. For this purpose, fifty albino rats were categorized into 5 groups and all the groups were received aqueous stevia extract at different dosage levels (200, 300, 400 and 500 ppm/kg b. wt) for 8 weeks except control group. Hematological and toxicological analyses were conducted using standard recommended procedures. The results indicated that biochemical parameters (RBC, HB, HCT, MCV, MCH, MCHC, WBC, eosinophils, lymphocytes and neutrophils) of albino rats significantly (P<0.05) increased and PLT, MPV and monocytes levels non-significantly decreased by using aqueous extract of stevia at different levels after eight weeks of study. Furthermore, Stevia aqueous extracts had non-toxic effect on liver functioning tests. However, stevia aqueous extracts were insignificant in their impression regarding organ to body weight ratios. The stevia aqueous extract has positive effect on hematological parameters of albino rats and is toxicologically safe. Therefore it could be used as a natural remedy for the management of hematological disorders without any health hazards.

The Arabidopsis NIMIN proteins affect NPR1 differentially.

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.