Introduction
Pathogenic fungi have long been a consistent source of devastation for the agricultural industry (De Lucca, 2007). Approximately 16% of the world’s crops are lost to microbial diseases, with an estimated 70-80% of these losses caused by fungi (Moore et al., 2011). Massive crop failures due to fungal diseases occur throughout history and threaten food security (Bebber & Gurr, 2015; Money, 2007). The range of tactics used to manage fungal pathogens include host resistance breeding (Melchers & Stuiver, 2000), cultural practices (Dekker, 1983), and plant protection (Möller & Stukenbrock, 2017; Wink, 1988). However, breeding for host resistance is a slow process and is not effective for certain pathogens, and cultural practices often do not provide complete control. Plant protection has greatly reduced the loss of crops while improving yields (Morton & Staub, 2008). For example, The United States agricultural industry applies over 108 million pounds of fungicides, costing roughly $880 million annually, but in turn, gains $12.8 billion due to the increased production value from the control of plant diseases (Gianessi & Reigner, 2006). However, the development of fungicide resistance in pathogen populations threatens to erode the efficacy of fungicides against several important pathogens (Ishii, 2006).
Although fungicides can efficiently reduce yield loss and improve food security, concerns about their non-target effects may affect their availability or utility in the future. Fungicides with relatively higher potential for mammalian toxicity may have limits placed on their use to mitigate exposure of applicators and field workers (Arcury & Quandt, 1998), and to ensure residue on harvested commodities remains below safe levels (Alavanja et al., 2004; Thabet et al., 2016). Fungicides may also negatively impact non-target organisms in agroecosystems, for example soil microbiota involved in nutrient cycling or plant symbionts (Yang et al., 2011), or insect symbionts (Wilson et al., 2014). To mitigate harm to pollinators the application of certain fungicides is not allowed during bloom, but this period is a critical opportunity to halt development of many diseases. Under certain conditions, fungicides may also be transported off-site following application via runoff or leaching and negatively impact aquatic organisms (Maltby et al., 2009). A more socially and environmentally sustainable method to control fungal plant pathogens is needed.
The plant cell wall is the first barrier a plant pathogen must overcome on its path to establishing a parasitic relationship (Hématy et al., 2009). Cell wall-degrading enzymes play an important role in the pathogenicity of many pathogenic fungi (Kubicek et al., 2014). To combat PGs, plants synthesize PG inhibiting proteins (PGIPs), which are highly conserved glycoproteins that contain leucine-rich repeat (LRR) regions and are located in plant cell walls (Adele Di Matteo et al., 2006; Hammond-Kosack & Jones, 1997). PGIPs can inhibit PGs through competitive inhibition, with the concave surface of the PGIP physically blocking the active site of PGs (A. Di Matteo et al., 2003; Kalunke et al., 2015). Additionally, it is believed that one of the ways in which PGIPs may play a role in plant protection is by reducing the rate at which the plant oligosaccharides are broken down (Frediani et al., 1993). A structural investigation of PGIPs indicates that the central LRR domain is flanked by the N- and C-terminal cysteine rich regions, and the residues involved in interacting with PGs are located in the concave surface between sheets B1 and B2 (Caprari et al., 1996), which are highly conserved across different PGIPs (Matsaunyane et al., 2015) (Figure 1a). These conserved regions are also known to be involved in the structural integrity of PGIPs (A. Di Matteo et al., 2003). It is believed that the central LRR pocket is responsible for recognizing the amino acids present at the active site of many PGs (Helft et al., 2011). This specificity allows PGIPs to differentiate fungal PGs from endogenous plant PGs (L. Federici et al., 2001). Prior studies indicate that overexpression of either endogenous or heterologous PGIPs in various plants enhanced their resistance towards fungal infection (Rathinam et al., 2020). However, whether this approach can be utilized as a tool for plant disease prevention remains unclear due to difficulties with public perception accepting transgenic plants.
Though there are numerous PGIPs studied for their potential in fungal protection, the best characterized PGIP is isoform 2 of PGIP from the common bean, Phaseolus vulgaris (PvPGIP2), which exhibits inhibitory activity toward a number of fungal PGs (Capodicasa et al., 2004; Leckie et al., 1999). Despite differing from isoform 1, PvPGIP1, by only 10 residues, PvPGIP2 confers resistance against a greater known number of fungal PGs (Kalunke et al., 2015; Maulik et al., 2009) (Figure 1). Here, we investigate the potential of using engineered PvPGIP2 as a fungal growth inhibitor. We utilized Saccharomyces cerevisiae as a microbial expression system, which has benefits such as the lack of background PGIP activity and a fast turnover time (Haeger et al., 2020). We reproduced previously reported interactions between PvPGIP2 and various PGs in a yeast two hybrid (Y2H) system, thus validating it as a handy approach to estimate PvPGIP2 activity. Y2H was then used to estimate the functions of various truncations and mutants of PvPGIP2. Different truncations resulted in varied levels of interaction with PGs, showing that some LRRs may be more important for recognizing certain PGs compared to others. We found that truncating the PvPGIP2 to only the region between LRR5 and LRR8 retained similar PG interaction levels to the full length PvPGIP2. Furthermore, mutants were created with amino acid residues that differed between PvPGIP1 and PvGIP2. Our approach also showed that Val172 of PvPGIP2 is the most critical residue that affects the recognition of fungal PGs, in agreement with previous research (Helft et al., 2011; Leckie et al., 1999; Sicilia et al., 2005). The interaction of PGIP2 variants with various PGs was demonstrated through assaying fungal growth on pectin agar, with or without the presence of engineered yeast strains. Our investigations highlight the potential of using engineered PGIPs as exogenous antifungal agent to inhibit the growth of phytopathogenic fungi as an environmentally and economically friendly approach.