Plants have evolved diverse mechanisms to defend against fungal infections, as summarized in Fig. 1, with one important route utilizing the secretion of proteins to delay fungal infection or inhibit fungal growth. These plant anti-fungal proteins are promising candidates since they are biodegradable, generally nontoxic to humans and antagonistic microorganisms, and most importantly, have evolved for millions of years to combat phytopathogenic fungi with a narrow target range. In this mini review, we summarize and discuss plant defensive proteins that are promising candidates for the development of future anti-fungal agents for agricultural applications . Pathogenesis related proteins are a group of low molecular weight plant proteins involved in mitigating both biotic and abiotic stresses, and are often involved in triggering systemic acquired resistance in plants . There are 16 main groups of PRs , with each group classified based on different molecular and physiological properties. These proteins are often pathogen specific and involved in the transcriptional activation of plant defenses. Here, we will focus on some of the most promising candidates for the development of anti-fungal agents for agricultural applications: PR-3, PR-5, PR-6, and PR-12. One of the best known and most studied plant anti-fungal proteins is chitinase, which belongs to PR-3 . Chitinases are strongly induced when the host plants are under attack from pathogens and function as defense molecules against fungal infection. These proteins inhibit fungal growth by lysing hyphal tips in fungi and break down chitin into its oligomers.

Chitinases display strong anti-fungal activity against a wide range of phytopathogenic fungi. This includes Botrytis cinerea,hydroponic vertical farm a necrotrophic fungi that is considered one of the top fungal pathogens based on scientific and economic importance and infects over 200 species worldwide , as well as Rhizoctonia solani, which causes sheath blight in rice, one of the most widespread diseases of rice. While chitinases have been isolated from bacteria, fungi, humans, and plants , chitin has not been found in mammals and plants. As such, plant chitinases, are a valuable target for developing highly specific bio-control against phytopathogenic fungi in agriculture. While overexpressing plant chitinases in either native or heterologous plants have successfully enhanced plant resistance against phytopathogenic fungi ,plant chitinases have also been used to treat fungal infections as an exogenously applied pest control agent. One study extracted chitinase E from yam tubers and then sprayed it on strawberries infected with powdery mildew. The treatment using chitinase E was successful at preventing the disease for at least two weeks through damaging cell-wall components of the hyphae and conidia of the pathogenic fungi . Plant chitinase has been heterologously expressed in many microorganisms such the bacteria Escherichia coliand Bacillus sp. , the yeast Pichia pastoris, as well as in plants such as transgenic tobacco and cultured plant cells, which provides solid foundation to develop chitinases as anti-fungal agents. Meanwhile, fermentation optimization has also been explored to enhance the production of chitinase from microbial cell factories, and the strategies include but not limited to adjusting carbon sources, pH, aeration, and temperatures. Importantly, chitinases also display excellent protein stability that ensures reliable exogenous application.

Chitinase from Vitis vinifera exhibits a half-life of up to 4.7 days at 30 ◦C or 9 years at 15 ◦C, and the purified chitinase from Trichosanthes dioica, effective against Aspergillus niger and Trichoderma sp. In a fungal agar diffusion assay, remained stable between pH 5.0–11.0 and temperature 30–90 ◦C for at least 30 min . Likewise, the purified chitinase from Diospyros kaki, which inhibited the growth of T. viride, exhibits broad pH stability from pH 4.0–9.0, and retains more than 60% activity at pH as low as 3.0 and as high as 10.0. Due to its specificity in targeting chitin, success in Defensins belong to group PR-12, and exhibit broad-spectrum activities against different biotic agents including pathogenic fungi . They are named due to the structural and functional similarities to insect and mammalian defensins . Plant defensins are constitutively expressed in the extracellular space of most vegetative and reproductive plant tissues and can be specifically induced under pathogen stress condition. Typically, defensins are small soluble cationic proteins, 45–54 amino acid residues in size, exhibiting eight conserved cysteine residues with a conserved spacing pattern, and the tertiary structure is supported by at least four disulfide bonds .Defensins remain stable both under extreme temperatures and very acidic conditions . For instance, NRBAP, a defensin-like protein purified from Phaseolus vulgaris beans, retained its anti-fungal activity against Mycosphaerella arachidicola up to 100 ◦C, and in the pH range of 1–13. Defensins can interact with a significant diversity of biological targets . One common mechanism that defensins often adopt to inhibit fungal growth is through the disruption of cell plasma membranes. Plant defensins are usually positively charged proteins and interact with anionic moieties in the membrane, such as glycoproteins, sphingolipids, or phospholipids . The defensins cover the target membranes until it reaches a concentration threshold, and then disrupts the membrane integrity by affecting the bilayer curvature . One study provided evidence that NaD1 from Nicotiana alata, which displays anti-fungal activity against several agronomically important filamentous fungi , was able to bound to phospholipids phosphatidic acid .

To estimate the effect of the total net charge of defensins on the anti-fungal activity, a mutagenesis analysis was performed on Rs-AFP2 from radish, and the interaction between the defensins and membrane lipids was improved when the net charge of the protein increased. Plant defensin anti-fungal activity may not be restricted to targeting the membrane of pathogenic fungi. Indeed, the exogenous application of NaD1 is also associated with the entrance of the protein into fungal intracellular space, resulting in granulation of the cytoplasm and cell death . This suggests that plant defensins could also interact with fungal intracellular targets and possibly with DNA, as already demonstrated by ostrich β-defensins, where E. coli growth was inhibited in assays due to interactions between peptides and cytoplasmic targets that curbed DNA, RNA, and protein synthesis. The diversity of anti-fungal mechanisms and effectiveness of defensins against a wide range of pathogens implies the potential of this protein family as a promising resource for fighting plant pathogens. Thaumatin-like proteins belong to PR-5 family . TLPs are named so due to their structural similarity to thaumatin, a sweet-tasting, non-toxic protein that was first discovered from the fruit of the tropical plant Thaumatococcus daniellii . TLPs exhibit a broad range of biological activities, including anti-fungal activity. Different TLPs inhibit fungal growth through different mechanisms, including but not limited to disrupting fungal membrane ,nft vertical farming inhibiting fungal enzymes such as xylanase, inducing apoptosis by binding to specific fungal membrane receptors, and hydrolyzing β-1,3-glucans.Osmotin and osmotin-like proteins are among the most studied TLPs of anti-fungal activity. Osmotin and orthologs have been shown to exhibit broad-spectrum anti-fungal inhibitory effects .Additionally, an osmotin-like protein from Solanum nigrum L. var indica was shown to inhibit fungal spore germination and permeabilize fungal hyphae in vitro. This protein is also stable and retains its anti-fungal activity at temperatures as high as 75 ◦C for 30 min and pH 3–8. Further functional exploration of TLPs under various stress conditions for in planta assays will be necessary before its development into a reliable anti-fungal tool . Plant protease inhibitors , also called PR-6, are important proteins involved in many plant biological processes, including seed germination, protease-related house-keeping functions, and defense against biotic and abiotic stresses. PIs are normally found in ample quantities in seeds and tubers, and plants in the Solanaceae family generally have exceptionally high levels of PIs , including some that can be promising candidates of anti-fungal agents.

For instance, potatoes encode several PIs ranging from 4.1 to 39 kDa that exhibit broad-spectrum anti-fungal activities. Potide-G, a Kunitz-type PI isolated from potato tubers of size 5.5 kDA, inhibits pathogenic fungi Candida ablicans and Rhizoctania solani in vitro even when heated to 70 ◦C for 20 min, and also exhibits antiviral and antibacterial activities. Similarly, the potato protease inhibitors I and II can inhibit the growth of various fungi, including B. cinerea, Fusarium solani, and Fusarium oxysporum. Both PPI-I and II are heat stable, which can maintain their ability to inhibit F. solani and F. oxysporum growth in vitro under temperature as high as 100 ◦C. PPI-I and II are also nontoxic, as they have previously been utilized in human clinical trials for appetite control .The extraction of bio-active PPIs from potatoes is laborious and of low yields . They have also been heterologously expressed in Saccharomyces cerevisiae, yet the anti-fungal activity of the purified protein was not examined . A more economic production method is needed to enable the development of PPIs as anti-fungal agents for agriculture applications. Another PI of interest is the Bowman-Birk protease inhibitor , which is typically under 20 kDA , contains seven conserved disulfide bonds, and inhibits trypsin and chymotrypsin, which are common enzymes pathogenic fungi utilize when infecting plants . The BBI gene is induced during plant immune responses and over expression of this gene in plants confers improved disease resistance against both insect and fungal pathogens . BBIs from the legume or cereal family have a double or single inhibitory loop respectively, and synthetic peptides that contain only the disulfide-linked, 9-residue long loop have shown to retain their trypsin and chymotrypsin inhibitory activity. This short, truncated form of the protein may be of interest for the development of anti-fungal agents of smaller molecular weight for easier production and higher stability, compared with larger protein agents. Aside from the small size, BBI is thermostable with the ability to withstand 100 ◦C for 10 min, tolerates a wide pH range from 1.6 to 8.0, is not allergenic, and is approved by the FDA for human consumption . Additionally, unlike some other candidates to be engineered as anti-fungal agent, BBI has passed phase II human clinical trials and is highly unlikely to be toxic, especially given its prevalence in soy products . BBIs have already been successfully utilized as an exogenously applied anti-fungal agent in vitro. One study identified that a BBI-type trypsin-chymotrypsin inhibitor purified from broad beans can inhibit the growth of B. cinerea, F. oxysporum, and M. arachidicola at a dose as low as 60 μg per plate . Plant BBIs have often been isolated from a variety of seeds such as those from Vigna mungo, Cajanus cajan, and Clitoria fairchildiana and have been tested for their insecticidal properties. Rice BBI has also been expressed in E. coli and retained the inhibitory activity. However, the titer is relatively low at 20 mg/L, likely due to the presence of the disulfide bonds that make it prone to forming inclusion bodies. In addition, care should be taken when developing BBI as an anti-fungal agent, as it is a multifunctional PI with a relatively broad activity towards various proteases, and may affect beneficial microbiota and fungi in the soil and plants. In addition to PRs, antimicrobial peptides are another protein group of interest. AMPs, also known as host defense peptides , can be derived from a variety of organisms, including plants, bacteria, and fungi. In plants, AMPs play a role in the plant innate immune system . AMPs that work specifically against fungi are known as anti-fungal peptides, and feature a wide range of functions that are of interest to both pharmaceutical and agricultural industries . Here, we will only discuss AFPs that have shown potential for agricultural applications. One AFP of interest is tomato systemin, a small peptide of only 18 amino acids long and is involved in inducing the synthesis of PIs in response to plant wounding and damage from herbivores. Research suggests that systemin moves through the plant phloem and helps amplify the signaling process and allows for distal leaves to respond to the wounding . Tomato plants that overexpress prosystemin, the precursor of systemin, are found to induce high levels of PI proteins even without wounding. Additionally, transgenic plants expressing prosystemin reduce lesions by at least 50% from Phytophthora infestans, a pathogen that causes late blight. Systemin peptides have been successfully isolated from tomato, sprayed onto grapevine and eggplant plants infected with B. cinerea at a concentration of 100 pM, and efficiently delayed necrosis of the infected plants. Snakins are cell wall-associated defensins that are also classified as AMP and believed to play a role in plant growth, signaling, and defense.