Almost 30 years ago, Broekaert and colleagues (1995) published the first articles describing plant defensins as part of the innate immune system of plants. From this discovery, we've learned about the role of these proteins in protecting plants against invading agents like fungi and bacteria. Today, these defensins are isolated or expressed and purified in laboratories for use as antimicrobial proteins (AMPs) against numerous pathogens. This is exemplified in Koo's (2019) work, which demonstrates the use of plant defensins not only in treating plants but also in combating pathogens that affect both animals and humans.
In our study, we expressed and purified PgD1, a defensin from P. glauca. This protein is rich in cysteines and has a molecular weight of 13.752 kDa, which is comparable to other pine defensins. For instance, Liu and colleagues (2022) found PaDef to have a molecular weight of 9.0665 kDa, while Picart and collaborators (2012) reported PgD5 at 18 kDa. Additionally, PsDef, another pine defensin, exhibits antifungal activity similar to PgD1. The work of Shalovylo and colleagues (2021) illustrates a close genetic relationship between PsDef and PgD1, which may lead to similar functional characteristics.
The similarity between these defensins is linked to their primary protein structures (Van Der Weerden et al., 2013). Research by Thomma (2002) and Shalovylo (2021) demonstrates that cysteine residues are essential for maintaining the stable conformation of plant defensins. The presence of disulfide bonds is also necessary for pore formation in membranes, which is fundamental to antimicrobial activity. Wang et al. (2019) confirmed that antimicrobial activity is closely associated with the stability of the β-hairpin structures, and removing disulfide bonds significantly diminishes their antimicrobial function. This was confirmed in our study through the DTT denaturation test (Fig. 12), which showed a complete loss of antifungal function after breaking disulfide bonds. Other amino acid residues in the primary protein structure are also crucial. Samblanx and collaborators (1997) observed that mutating specific amino acids in the RsAFP1 defensin led to the loss of function. Since PgD1 and RsAFP1 defensins share these residues (Fig. 4), part of their antifungal properties can be attributed to these amino acids (Shalovylo et al., 2021).
The recombinant defensin PgD1 exhibited antifungal activity against five phytopathogenic fungi: C. gloeosporioides, C. musae, C. graminicola, F. oxysporum, and B. cinerea. Pervieux and colleagues (2004) found that PgD1 also has activity against F. oxysporum. At the time of writing, no studies had examined PgD1 against C. and B. species. However, Liu et al. (2022) reported that PaDef, another pine defensin, also has antifungal activity against C. gloeosporioides and B. cinerea. Kovalera and collaborators (2011) and Picart et al. (2012) showed that the pine defensins PsDef and PgD5, respectively, are effective against B. cinerea and F. oxysporum. This confirms that pine defensins are effective antifungal agents. Further research (Parisi et al., 2024; Shahmiri et al., 2023; Wang et al., 2019) suggests that pine defensins interact with fungal membranes, forming pores that lead to cell death.
In this study, we've demonstrated the potent antifungal activity of plant defensins. However, large quantities are necessary for field applications. We opted for recombinant expression in E. coli cells, a highly efficient protocol for protein expression and purification. This method provides a fast, cost-effective, and reliable means to produce recombinant proteins at scale, making it an ideal strategy for manufacturing bioinputs (Deo et al., 2022; Kovalera, 2011).
Besides efficient expression, plant defensins are also resistant to degradation caused by temperature changes. This makes them suitable for various applications (Ermakova et al., 2016). The PgD1 defensin has shown stability under varying temperatures, which aligns with findings from other studies, such as those by Kovalera (2020) and Wu (2022).
Given its stability, relatively simple production, and excellent potential to inhibit phytopathogenic fungi, PgD1 defensin can be a viable alternative or complement to pesticides, reducing the socio-environmental impacts of food production. This potential is also seen in other plant defensins, such as MtDef4 (Tetorya & Li, 2023), PvDef (Mohamed et al., 2023), and GMA4CG_V6 (Djami-Tchatchou et al., 2023). Using defensins as antifungals represents a promising biotechnological innovation that warrants further research for improving environmental health.