eCPMV NANOPARTICLES: THE POTENTIAL OF A BIO-INSPIRED STRATEGY FOR PLANT PROTECTION

Agricultural yield is dramatically affected by crop pests and pathogens, which are considered a major threat to food security. The intense and indiscriminate use of pesticides led to an increase in resistance occurrence in microorganisms and environmental issues, leading to the awareness towards the need for new alternative strategies respectful of both health and environment. Nanotechnology is one of the most promising technologies of the modern era. Indeed, its multidisciplinary nature makes it fascinating and exploitable in many fields, precisely including agriculture. Nanoagrochemicals can indeed provide a valuable alternative to common pesticides, due to a more targeted and enhanced efficacy, resulting in a lower dosage of active substances and a reduction of possible off-target effects. Although the biosafety of nanoagrochemicals is still not completely understood, their development for plant protection represents a promising strategy to achieve a sustainable and environmentally friendly crop production system. Plant viruses can play an important role in this context since they are naturally occurring proteinaceous nanoparticles able to carry, protect, and deliver a cargo. Moreover, the biosafety concerns about the use of plant viruses for crop protection have been exceeded by the development of nucleic acid-free virus nanoparticles, named empty virus-like particles (eVLPs). The Cowpea mosaic virus (CPMV) can be produced as eVLPs (eCPMV) through transient expression in Nicotiana benthamiana plants, at high titres and in an inexpensive manner. Additionally, eCPMV can be functionalized with peptides that can be displayed on the external surface by genetic manipulation or chemical conjugation, thus offering a wide range of possibilities for their application in plant protection. In this work, eCPMV nanoparticles have been exploited in an attempt to develop new products applicable for crop protection, using different approaches that rely on the physical stability of the eCPMV capsid to expose natural bioactive peptides or on the capacity of the empty eCPMV cavity to load natural molecules: (i) eCPMV as a scaffold for antimicrobial peptide (AMP) exposure, (ii), eCPMV as a plant immunity-triggering nanoparticle, and (iii) eCPMV as a functional nanocarrier.While it has not been possible to produce eCPMV harbouring AMPs on the surface, eCPMV functionalized with different elicitors were successfully expressed and purified and showed the capacity to induce plant molecular defence responses. Moreover, eCPMV nanoparticles were loaded with different molecules, efficiently further released in apoplastic-mimicking conditions, thus establishing the possibility for the cargo to be delivered within plant tissues. Overall, the data enforce to reconsider the paradigms regarding eCPMV functionalization, in particular in terms of peptide features required for genetical modification. Moreover, they support that eCPMV is a promising tool to develop new nanobiopesticides applicable in agriculture. Finally, they unveil unexpected effects of eCPMV nanoparticles, such as their capacity to disturb fungal growth and to fully protect plant from bacterial infection, which will certainly deserve attention for future research