Prediction and expression analysis of deleterious nonsynonymous SNPs of Arabidopsis ACD11 gene by combining computational algorithms and molecular docking approach.

Accelerated cell death 11 (ACD11) is an autoimmune gene that suppresses pathogen infection in plants by preventing plant cells from becoming infected by any pathogen. This gene is widely known for growth inhibition, premature leaf chlorosis, and defense-related programmed cell death (PCD) in seedlings before flowering in Arabidopsis plant. Specific amino acid changes in the ACD11 protein's highly conserved domains are linked to autoimmune symptoms including constitutive defensive responses and necrosis without pathogen awareness. The molecular aspect of the aberrant activity of the ACD11 protein is difficult to ascertain. The purpose of our study was to find the most deleterious mutation position in the ACD11 protein and correlate them with their abnormal expression pattern. Using several computational methods, we discovered PCD vulnerable single nucleotide polymorphisms (SNPs) in ACD11. We analysed the RNA-Seq data, identified the detrimental nonsynonymous SNPs (nsSNP), built genetically mutated protein structures and used molecular docking to assess the impact of mutation. Our results demonstrated that the A15T and A39D mutations in the GLTP domain were likely to be extremely detrimental mutations that inhibit the expression of the ACD11 protein domain by destabilizing its composition, as well as disrupt its catalytic effectiveness. When compared to the A15T mutant, the A39D mutant was more likely to destabilize the protein structure. In conclusion, these mutants can aid in the better understanding of the vast pool of PCD susceptibilities connected to ACD11 gene GLTP domain activation.

Gene editing of putative cAMP and Ca2+ -regulated proteins using an efficient cloning-free CRISPR/Cas9 system in Trypanosoma cruzi.

Trypanosoma cruzi, the agent of Chagas disease, must adapt to a diversity of environmental conditions that it faces during its life cycle. The adaptation to these changes is mediated by signaling pathways that coordinate the cellular responses to the new environmental settings. Cyclic AMP (cAMP) and Calcium (Ca2+ ) signaling pathways regulate critical cellular processes in this parasite, such as differentiation, osmoregulation, host cell invasion and cell bioenergetics. Although the use of CRISPR/Cas9 technology prompted reverse genetics approaches for functional analysis in T. cruzi, it is still necessary to expand the toolbox for genome editing in this parasite, as for example to perform multigene analysis. Here we used an efficient T7RNAP/Cas9 strategy to tag and delete three genes predicted to be involved in cAMP and Ca2+ signaling pathways: a putative Ca2+ /calmodulin-dependent protein kinase (CAMK), Flagellar Member 6 (FLAM6) and Cyclic nucleotide-binding domain/C2 domain-containing protein (CC2CP). We endogenously tagged these three genes and determined the subcellular localization of the tagged proteins. Furthermore, the strategy used to knockout these genes allows us to presume that TcCC2CP is an essential gene in T. cruzi epimastigotes. Our results will open new venues for future research on the role of these proteins in T. cruzi.

Gene editing of putative cAMP and Ca 2+ -regulated proteins using an efficient cloning-free CRISPR/Cas9 system in Trypanosoma cruzi.

Trypanosoma cruzi , the agent of Chagas disease, must adapt to a diversity of environmental conditions that it faces during its life cycle. The adaptation to these changes is mediated by signaling pathways that coordinate the cellular responses to the new environmental settings. Cyclic AMP (cAMP) and Calcium (Ca 2+ ) signaling pathways regulate critical cellular processes in this parasite, such as differentiation, osmoregulation, host cell invasion and cell bioenergetics. Although the use of CRISPR/Cas9 technology prompted reverse genetics approaches for functional analysis in T. cruzi , it is still necessary to expand the toolbox for genome editing in this parasite, as for example to perform multigene analysis. Here we used an efficient T7RNAP/Cas9 strategy to tag and delete three genes predicted to be involved in cAMP and Ca 2+ signaling pathways: a putative Ca 2+ /calmodulin-dependent protein kinase ( CAMK ), Flagellar Member 6 ( FLAM6 ) and Cyclic nucleotide-binding domain/C2 domain-containing protein ( CC2CP ). We endogenously tagged these three genes and determined the subcellular localization of the tagged proteins. Furthermore, the strategy used to knockout these genes allow us to presume that TcCC2CP is an essential gene in T. cruzi epimastigotes. Our results will open new venues for future research on the role of these proteins in T. cruzi .

In silico identification of conserved miRNAs in the genome of fibre biogenesis crop Corchorus capsularis.

Corchorus capsularis, commonly known as jute occupies the leading position in the production of natural fibre alongside lower environmental threat. Small noncoding ~21 to 24 nucleotides long microRNAs play significant roles in regulating the gene expression as well as different functions in cellular growth and development. Here, the study adopted a comprehensive in silico approach to identify and characterize the conserved miRNAs in the genome of C. capsularis including functional annotation of specific gene targets. Expressed Sequence Tags (ESTs) based homology search of 3350 known miRNAs of dicotyledons were allowed against 763 non-redundant ESTs of jute genome, resulted in the prediction of 5 potential miRNA candidates belonging five different miRNA families (miR1536, miR9567-3p, miR4391, miR11300, and miR8689). The putative miRNAs were composed of 18 nucleotides having a range of -0.49 to -1.56 MFEI values and 55%-61% of (A + U) content in their pre-miRNAs. A total of 1052 gene targets of putative miRNAs were identified and their functions were extensively analyzed. Most of the gene targets were involved in plant growth, cell cycle regulation, organelle synthesis, developmental process and environmental responses. Five gene targets, namely, NAC Domain Containing Protein, WRKY DNA binding protein, 3-dehydroquinate synthase, S-adenosyl-L-Met-dependent methyl transferase and Vascular-related NAC-Domain were found to be involved in the lignin biosynthesis, phenylpropanoid pathways and secondary wall formation. The present study might accelerate the more miRNA discovery, strengthening the complete understanding of miRNAs association in the cellular basis of lignin biosynthesis towards the production of high standard jute products.