Laor, D. | bioRxiv Science

bioRxiv2022

Engineered Riboswitch Nano-carriers as a Possible Disease-Modifying Treatment for Metabolic Disorders

Both DNA- and RNA-based nanotechnologies are remarkably useful for in vitro molecular-scale device engineering and are applied in a vast array of applications. However, while the function of nucleic acid nanostructures is robust under in vitro settings, their implementation in real-world conditions requires overcoming their inherent degradation sensitivity and subsequent loss of function. Viruses are minimalistic yet sophisticated supramolecular assemblies, able to protect their nucleic acid content in inhospitable biological environments. Inspired by this natural ability, we engineered RNA-virus-like particles (VLPs) nanocarriers (NCs). We showed that the VLPs can serve as an excellent protective shell against nuclease-mediated degradation. We then harnessed biological recognition elements and demonstrated how engineered riboswitch NCs can act as a possible disease-modifying treatment for genetic metabolic disorders. The functional riboswitch is capable of selectively and specifically binding metabolites and preventing their self-assembly process and its downstream effects. When applying the riboswitch nano-carriers to an in vivo yeast model of adenine accumulation and self-assembly, significant inhibition of the sensitivity to adenine feeding was observed. In addition, using an amyloid-specific dye, we proved the riboswitch nano-carriers ability to reduce the level of intracellular amyloid-like cytotoxic structures. The potential of this RNA therapeutic technology does not stop at metabolic disorders, as it can be easily fine-tuned to be applied to other conditions and diseases.

bioengineering↗

bioRxiv2020

Cytokinin inhibits fungal development and virulence by targeting cellular trafficking

Cytokinin (CK) is an important plant developmental regulator, having activities in many aspects of plant life and its response to the environment. CKs are involved in diverse processes in the plant, including stem-cell maintenance, vascular differentiation, growth and branching of roots and shoots, leaf senescence, nutrient balance and stress tolerance. In some cases, phytopathogens secrete CKs. It has been suggested that to achieve pathogenesis in the host, CK-secreting biotrophs manipulate CK signaling to regulate the host cell cycle and nutrient allocation. CK is known to induce host plant resistance to several classes of phytopathogens from a handful of works, with induced host immunity via salicylic acid signaling suggested to be the prevalent mechanism for this host resistance. Here, we show that CK directly inhibits the growth, development, and virulence of fungal phytopathogens. Focusing on Botrytis cinerea (Bc), we demonstrate that various aspects of fungal development can be reversibly inhibited by CK. We also found that CK affects both budding and fission yeast in a similar manner. Investigating the mechanism by which CK influences fungal development, we conducted RNA-NGS on mock and CK treated B. cinerea samples, finding that CK inhibits the cell cycle, cytoskeleton, and endocytosis. Cell biology experiments demonstrated that CK affects cytoskeleton structure and cellular trafficking in Bc, lowering endocytic rates and endomembrane compartment sizes, likely leading to reduced growth rates and arrested developmental programs. Mutant analyses in yeast confirmed that the endocytic pathway is altered by CK. Our work uncovers a remarkably conserved role for a plant growth hormone in fungal biology, suggesting that pathogen-host interactions resulted in fascinating molecular adaptations on fundamental processes in eukaryotic biology. ImportanceCytokinins (CKs), important plant growth/ developmental hormones, have previously been associated with host disease resistance. Here, we demonstrate that CK directly inhibits the growth, development, and virulence of B. cinerea (Bc) and many additional phytopathogenic fungi. Molecular and cellular analyses revealed that CK is not toxic to Bc, but rather, Bc likely recognizes CK and responds to it, resulting in cell cycle and individual cell growth retardation, via downregulation of cytoskeletal components and endocytic trafficking. Mutant analyses in yeast confirmed that the endocytic pathway is a CK target. Our work demonstrates a conserved role for CK in yeast and fungal biology, suggesting that suggesting that pathogen-host interactions may cause molecular adaptations on fundamental processes in eukaryotic biology.

microbiology↗