Biology subjects

Muthusamy, S.

Publications and source records attributed to Muthusamy, S..

5 recordsLinked to original sources

bioRxiv2024

Heterologous Production of Cyprosin B in Nicotiana benthamiana: Unveiling the Role of the Plant-Specific Insert Domain in Protein Function and Subcellular Localization

The aqueous extract of Cynara cardunculus flowers is traditionally used in cheese production across Mediterranean countries. To meet the growing industrial demand for plant-based milk-clotting enzymes and to explore potential biotechnological applications, we initiated a study to heterologously produce cyprosin B (CYPB), a key milk-clotting enzyme from C. cardunculus, in Nicotiana benthamiana. We also investigated the role of its plant-specific insert (PSI) domain in the CYPBs activity and its localization. In this study, full-length CYPB and a PSI domain deleted CYPB (CYPB{Delta}PSI) were transiently expressed in N. benthamiana leaves using Agrobacterium-mediated infiltration. The leaves were harvested nine days post-infiltration, and proteins were purified, yielding approximately 81 mg/kg (CYPB) and 60 mg/kg (CYPB{Delta}PSI) fresh weight. CYPB{Delta}PSI showed significantly higher proteolytic activity (156.72 IU/mg) than CYPB (57.2 IU/mg), indicating that the PSI domain is not essential for enzymatic activity and that its removal results in enhanced enzymatic efficiency. In the milk-clotting activity assay, CYPB{Delta}PSI demonstrated a significantly faster clotting time than full-length CYPB, indicating enhanced milk-clotting efficiency for CYPB{Delta}PSI. Subcellular localization studies revealed that CYPB and PSI were localized in the vacuole and endocytic vesicles. In contrast, CYPB{Delta}PSI was primarily localized in the endoplasmic reticulum (ER) and the tonoplast, suggesting that the PSI domain is critical for vacuolar targeting and membrane permeabilization that affects overall protein yield. This study demonstrates the feasibility of using N. benthamiana as a platform for the scalable production of more efficient recombinant CYPB. It highlights the multifunctional role of the PSI domain in vacuolar sorting without impairing its functionality. These results underscore the potential of plant-based expression systems as a viable alternative for the industrial production of plant milk-clotting enzymes, with significant implications for sustainable cheese production.

molecular biology↗

bioRxiv2024

Genomic loci for sclerotinia stem rot resistance and chlorophyll stability in Brassica napus: integrating GWAS with microbiome insights

Sclerotinia Stem Rot (SSR) disease is one of the most serious diseases affecting the yield and quality of oilseed rape (Brassica napus). Understanding the genetic basis of the resistance trait in oilseed rape to SSR and microbiome composition for enhanced resistance is crucial for developing resistant varieties and sustainably mitigate the impact of the disease. In this study, in a panel of 168 oilseed rape accessions, most resistant (NGB 13503 and NGB 13834) and susceptible (NGB 13497 and NGB 13897) accessions are identified. A Genome-wide association study (GWAS) identified 47 SNPs linked to the SSR lesion length, lesion area, and lesion relative to the leaf area. Among the SNPs significantly linked to lesion length were Bn-A04-p10555408, Bn-A07-p12487549, Bn-A09-p4652268, Bn-A09-p4916858 and to our knowledge, these SNPs have not been previously linked to SSR resistance in oilseed rape. Moreover, the study identified 24 SNPs linked with chlorophyll content before SSR inoculation (SPADH), after the SSR inoculation (SPADI), and chlorophyll index (CI). Maintaining the chlorophyll level is correlated with the SSR resistance. Furthermore, bacterial taxa (e.g. Pseudomonas, Methylobacterium, and Aquabacterium) and fungal taxa (e.g. Mycosphaerellales, Thelebolales, and Akanthomyces) were enriched in the resistant compared to in the susceptible oilseed rape accessions. The SNPs linked to lesion length showed consistent haplotype variation between these selected accessions. Given the absence of complete resistance against SSR, the study provides insights into the significance of maintaining chlorophyll levels and considering microbiome composition for enhancing the level of existing partial resistance to SSR in oilseed rape.

plant biology↗

bioRxiv2024

Plant Development Drives Dynamic Shifts in the Root Compartment Microbiomes of Wild and Domesticated Finger Millet Cultivars

BackgroundPlant-microbe interactions in two root compartments - the rhizosphere and endosphere - play vital roles in maintaining plant health and ecosystem dynamics. The microbial communities in these niches are shaped in complex ways by factors including the plants developmental stage and cultivar, and the compartment where the interactions occur. Different plant cultivars provide distinct nutritional and ecological niches and may selectively enrich specific microbial populations through the secretion of root exudates. This gives rise to complex and dynamic plant-microbe interactions; some cultivars promote the recruitment of beneficial symbionts while others may deter pathogens. To clarify these processes, this work investigated the structure of the endosphere and rhizosphere microbial communities of wild type finger millet and five domesticated cultivars across two plant developmental stages. ResultsOur results showed that the plant developmental stage, compartment, and cultivar have varying degrees of impact on root-associated microbiomes. The dominant bacterial phyla in all samples were Proteobacteria, Actinobacteria, and Bacteroidetes, while the dominant fungal phyla were Ascomycota and Basidiomycota. All of these phyla exhibited pronounced variations in abundance. In general, an increased abundance of Actinobacteria in the endosphere was accompanied by a reduced abundance of Proteobacteria. The most pronounced changes in microbial community structure were observed in the rhizosphere during the flowering stage. Changes in the microbiome patterns of the rhizosphere were driven predominantly by the genus Pseudomonas. Moreover, the host plants developmental stage strongly influenced the microbial communities, suggesting that plants can recruit specific taxa based on their need for particular soil consortia. ConclusionsOur results show that both host developmental stage and domestication strongly affect the assembly and structure of the plant microbiome. Moreover, plant root compartments can selectively recruit specific taxa from associated core microbial communities to fulfill their needs in a manner that depends on both the plants developmental stage and the specific root compartment that is involved. These findings show that deterministic selection pressures exerted by plants during their growth and development can significantly affect their microbial communities and have important implications for efforts to create tools for manipulating the microbiome to sustainably improve primary productivity.

microbiology↗

bioRxiv2022

Multi-omic Analysis of Primary Human Kidney Tissues Identifies Medulla-Specific Gene Expression Patterns

The renal medulla is a specialized region of the kidney with important homeostatic functions. It has also been implicated in genetic and developmental disorders and ischemic and drug-induced injuries. Despite its role in kidney function and disease, the medullas baseline gene expression and epigenomic signatures have not been well described in the adult human kidney. Here we generate and analyze gene expression (RNA-seq), chromatin accessibility (ATAC-seq) and chromatin conformation (Hi-C) data from adult human kidney cortex and medulla. Using data from our carefully annotated specimens, we assign samples in the larger public GTEx database to cortex and medulla, thereby identifying several misassignments and extracting meaningful medullary gene expression signatures. Using integrated analysis of gene expression, chromatin accessibility and conformation profiles, we reveal insights into medulla development and function. Our datasets will also provide a valuable resource for researchers in the GWAS community for functional annotation of genetic variants.

genetics↗

bioRxiv2021

Multi-omic Characterization of Human Tubular Epithelial Cell Response to Serum

Proteinuria, the spillage of serum proteins into the urine, is a feature of glomerulonephritides, podocyte disorders and diabetic nephropathy. However, the response of tubular epithelial cells to serum protein exposure has not been systematically characterized. Using transcriptomic profiling we studied serum-induced changes in primary human tubular epithelial cells cultured in 3D microphysiological devices. Serum proteins induced cellular proliferation, cytokine secretion and activated a coordinated stress response. We orthogonally confirmed our findings by comparing the transcriptomic and epigenomic landscapes of intact human kidney cortex and isolated tubular epithelial cells cultured in fetal bovine serum. Importantly, key transcriptomic programs in response to either type of serum exposure remained consistent, including comparisons to an established mouse model of kidney injury. This serum-induced transcriptional response was dominated by switching off of nuclear receptor-driven programs and activation of AP-1 and NF-{kappa}B signatures in the tubular epigenomic landscape. These features of active regulation were seen at canonical kidney injury genes (HAVCR1) and genes associated with COVID-19 (ACE2, IL6). Our data provide a reference map for dissecting the regulatory and transcriptional response of kidney tubular epithelial cells injury induced by serum.

cell biology↗