Functional Characterization of a Rice Thioredoxin Protein OsTrxm and Its Cysteine Mutant Variant with Antifungal Activity

Abstract

Although there are many antimicrobial proteins in plants, they are not well-explored. Understanding the mechanism of action of plant antifungal proteins (AFPs) may help combat fungal infections that impact crop yields. In this study, we aimed to address this gap by screening Oryza sativa leaves to isolate novel AFPs. We identified a thioredoxin protein with antioxidant properties. Being ubiquitous, thioredoxins (Trxs) function in the redox balance of all living organisms. Sequencing by Edman degradation method revealed the AFP to be O. sativa Thioredoxin m-type isoform (OsTrxm). We purified the recombinant OsTrxm and its cysteine mutant proteins (OsTrxm C/S) in Escherichia coli. The recombinant OsTrxm proteins inhibited the growth of various pathogenic fungal cells. Interestingly, OsTrxm C/S mutant showed higher antifungal activity than OsTrxm. A growth inhibitory assay against various fungal pathogens and yeasts confirmed the pertinent role of cysteine residues. The OsTrxm protein variants penetrated the fungal cell wall and membrane, accumulated in the cells and generated reactive oxygen species. Although the role of OsTrxm in chloroplast development is known, its biochemical and molecular functions have not been elucidated. These findings suggest that in addition to redox regulation, OsTrxm also functions as an antimicrobial agent.

Antifungal Mechanism of Vip3Aa, a Vegetative Insecticidal Protein, against Pathogenic Fungal Strains

“…The mechanisms of action of numerous AMPs are broadly classified into the following two categories: membranolytic actions such as membrane potential changes and the formation of pores in the cell membrane; and intracellular inhibiting actions such as interfering gene expression, inhibiting enzyme activity, generating reactive oxygen species (ROS), and inducing osmotic pressure [ 28 , 29 ]. There is growing evidence of AMPs inducing cell death by stimulating the production of ROS [ 30 , 31 , 32 , 33 , 34 ]. Although fungal cells repeat the cycle of generating and eliminating intracellular ROS during metabolic pathways, the high levels of ROS damage intracellular lipids, proteins, DNA, organelles, and cell walls [ 35 , 36 ].…”

“…The inhibitory concentration 50 (IC 50 ) against each fungus was defined as the lowest concentration of a sample that inhibited 50% visible growth. All assays were performed in triplicate [ 30 , 31 , 32 ].…”

Aldehyde dehydrogenase 3I1gene is recruited in conferring multiple abiotic stress tolerance in plants

“…However, the underlying biochemical mechanism needs further studies on its interaction with the ROS scavenging system, such as thioredoxins or enzymatic antioxidant proteins (Lu & Holmgren 2014; Park et al . 2019; Zafar et al . 2020a).…”

“…A similar response for MDA was observed in cold, drought and salt stresses, which led to higher MDA levels in WT as compared to transgenic plants, suggesting that ALDH3I1 plays a crucial role in alleviating oxidative stress in plants. However, the underlying biochemical mechanism needs further studies on its interaction with the ROS scavenging system, such as thioredoxins or enzymatic antioxidant proteins (Lu & Holmgren 2014;Park et al 2019;Zafar et al 2020a).…”

Genome-Wide Association Study for Cold Tolerance in Rice Seedlings under Cold-Water Treatment

“…In maize, cupredoxin domain-containing protein was reported to be involved in modulation of redox homeostasis in kernel development (Zhang et al 2021). Os10g0482900 encodes a thioredoxin fold domain containing protein, which is an antioxidants in reactive oxygen species (ROS) scavenging in plant responses against stresses (Park et al 2019). The increase of Os10g0482900 under the nitrogen starving stress condition was reported (Shin et al 2018).…”