Biology subjects

Kanagarajan, S.

Publications and source records attributed to Kanagarajan, S..

3 recordsLinked to original sources

bioRxiv2026

A family of metabolite damage-control phosphatases modulates ROS-induced autophagy and plant stress resilience

Autophagy is an intracellular recycling pathway with profound impacts on development, growth, and stress tolerance in eukaryotes. Therefore, unravelling signalling mechanisms that modulate the process has broad applicability. Here, we characterised a small organic molecule as an enhancer of autophagy across diverse plant lineages. Through an in vivo photoaffinity proteomics approach in Arabidopsis thaliana seedlings, we identified Domain of Unknown Function 89 (DUF89) proteins as molecular targets. Hereafter, we refer to these proteins as Reactive Metabolite Damage-Control Phosphatases (RMDPs), which are metabolite-repair phosphatases that clear reactive products. The new autophagy modulator is called RMDP inhbitor-1 (RMDPi-1) due to its demonstrated inhibitory effect on AtRMDP1 activity in vitro. Binding was further confirmed through co-incubation that thermostabilised the protein. Crystal structures of AtRMDP1 were obtained via X-ray diffraction, confirming binding of RMDPi-1 in the catalytic site and revealing a flexible region near the binding pocket that was absent in previous models. Molecular docking predicted two possible binding modes consistent with the observed electron density. RMDPi-1 treatment and the phosphatase gene knockout increased autophagic flux in Arabidopsis and Chlamydomonas reinhardtii, establishing a conserved effect. The observed autophagy response is linked to a spike in reactive oxygen species (ROS). AtRMDP knockout also results in higher ROS tolerance and greater biomass in Arabidopsis seedlings. Targeted and untargeted metabolomic experiments revealed that both pharmacological and genetic suppression of RMDP leads to accumulation of glycating agents and phosphate sugars, associating loss of RMDP activity to the ROS stress that induces alternate ROS-scavenging mechanisms, and autophagy. Taken together, our integrated chemical genetics approach reveals enhanced autophagy via inhibition of RMDP family members, which impacts on plant stress tolerance via conserved metabolite damage-control functions.

plant biology↗

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↗

bioRxiv2022

Biodiversity of the Genus Trichoderma in the Rhizosphere of Coffee (Coffea arabica) Plants in Ethiopia and their Potential Use in Biocontrol of Coffee Wilt Disease

The present study investigated the distribution status and biodiversity of Trichoderma species surveyed from coffee rhizosphere soil samples from Ethiopia and their potential for biocontrol of coffee wilt disease (CWD) caused by Fusarium xylarioides. Trichoderma isolates were identified based on molecular approaches and morphological characteristics followed by biodiversity analysis using different biodiversity indices. The antagonistic potential of Trichoderma isolates was evaluated against F. xylarioides using the dual confrontation technique and agar diffusion bioassays. A relatively high diversity of species was observed including 16 taxa and 11 undescribed isolates. Trichoderma asperellum, T. asperelloides and T. longibrachiatum were classified as abundant species, with dominance (Y) values of 0.062, 0.056 and 0.034, respectively. Trichoderma asperellum was the most abundant species (comprising 39.6% of all isolates) in all investigated coffee ecosystems. Shannons biodiversity index (H), the evenness (E), Simpsons biodiversity index (D), and the abundance index (J) were calculated for each coffee ecosystem, revealing that species diversity and evenness were highest in the Jimma zone (H =1.97, E = 0.76, D = 0.91, J = 2.73). The average diversity values for Trichoderma species originating from the coffee ecosystem were H = 1.77, D = 0.7, E = 0.75 and J = 2.4. In vitro confrontation experiments revealed that T. asperellum AU131 and T. longibrachiatum AU158 reduced the mycelial growth of F. xylarioides by over 80%. The potential use of these Trichoderma species for disease management of F. xylarioides and to reduce its impact on coffee cultivation is discussed in relation to Ethiopias ongoing coffee wilt disease crisis.

microbiology↗