Interactions between PG and PGIP can be monitored using yeast two hybrid system.
Because PGIPs can physically inhibit PGs via competitive inhibition (Sicilia et al., 2005; Spinelli et al., 2009; Stotz et al., 2000), we tested whether the Y2H system could be used to interrogate the binding between the two proteins. The crystal structure of PvPGIP2 shows a large, negatively charged pocket on the inner LRR concave surface believed to bind to the positively charged residues on the active site of PGs (A. Di Matteo et al., 2003) (Figure 1). This physical contact between the two proteins led us to believe that Y2H can be a reasonable approach to observe PG-PGIP interactions, which were mainly monitored by surface plasmon resonance (SPR) (Cervone, 1997; L. Federici et al., 2001; Leckie et al., 1999). However, unlike Y2H, SPR requires expression and purification of each component. Although Y2H has rarely been used to estimate PG-PGIP activity, it was adopted in one previous investigation to conclude that a PGIP family protein, the carrot antifreeze protein, does not exhibit PGIP activity (Zhang et al., 2006).
We chose four PGs that play important roles in infection by their corresponding pathogens: PG2 from A. niger (AnPG2), PG1 and PG2 from B. cinerea (BcPG1 and BcPG2), and PG from F. moniliforme FmPG3). Fusarium moniliforme (synonym = F. verticillioides ), Aspergillus niger, and Botrytis cinereaall exhibit widespread impact on food crops. F. moniliforme(Pamphile & Azevedo, 2002) can produce mycotoxins with toxic secondary metabolites to both humans and animals (Schoeman et al., 2018). It is one of the most significant fungal pathogens to infect corn (Munkvold, 2003). Aspergillus niger is one of the most significant causes of postharvest decay due to its ability to affect many common crops, such as onions (Gherbawy et al., 2015), grapes (Logrieco et al., 2009), peanuts (Xu et al., 2015), and maize (Palencia et al., 2010). Due to its wide range of pH tolerance and quick growth, A. niger is considered to be one of the most significant fungi associated with postharvest decay (Sharma, 2012). Botrytis cinerea is a necrotrophic fungus with a wide host range of over 200 species, resulting in an economic loss upwards of $10 billion globally every year (Weiberg et al., 2013). For example, it is considered the most economically significant pathogen of strawberry worldwide (Petrasch et al., 2019).
Previous investigations indicate that PvPGIP2 can inhibit the function of AnPG2 (Leckie et al., 1999; Spadoni et al., 2006; Spinelli et al., 2009) and BcPG1 (Leckie et al., 1999; Manfredini et al., 2005; Sicilia et al., 2005), weakly inhibits FmPG3 (L. Federici et al., 2001; Leckie et al., 1999), and possibly inhibits BcPG2 (Leckie et al., 1999). In contrast, PvPGIP1 inhibits the activity of AnPG2 (Leckie et al., 1999; Spadoni et al., 2006), weakly inhibits BcPG1 (Leckie et al., 1999) and does not inhibit FmPG3 (Leckie et al., 1999; Maulik et al., 2009). The activity of PvPGIP1 against BcPG2 is unknown. In our experiments, PGs were fused to the GAL4 binding domain (GAL4-BD), and PvPGIP1 and PvPGIP2 were fused to GAL4 activation domain (GAL4-AD). The bait and prey constructs, each containing leu2 and trp1 gene respectively, were co-transformed into a Y2H reporter strain S. cerevisiae PJ69-4A (Shaffer et al., 2012) (Figure S1). PJ69-4A encodeshis3 gene regulated by the GAL4 promoter, and interaction between the Gal4-AD and Gal4-BD fusion proteins results in growth of yeast cells in synthetic dropout (SD) medium lacking histidine (H), tryptophan (T), and leucine (L). PG-PGIP interactions were estimated using OD600 of Y2H yeast strains at stationary phase. Growth of yeast harboring Gal4 AD-PvPGIP2 and Gal4 BD-AnPG2 was grown in -LT and used as a positive control, while yeast harboring Gal4 AD-HAB1, a protein not known to interact with PGs, and Gal4 BD-BcPG2 was grown in -HTL media and used as the negative control. With Gal4 AD-PvPGIP2, yeast containing Gal4 BD-AnPG2 or BcPG1 exhibited robust growth in -HTL medium, indicating strong PG-PGIP interactions (Figure 2). Yeast with Gal4 AD-PvPGIP2 harboring either Gal4 BD-BcPG2 or Gal4 BD-FmPG3 grows much slower than the positive control (Figure 2), which implies weak PG-PGIP interactions. The distinct PG-PGIP interactions may be due to the different specificity of the protein recognition site at the concave surface of the PGIPs (A. Di Matteo et al., 2003; Sicilia et al., 2005). Yeast harboring Gal4 AD-PvPGIP1 and Gal4 BD-AnPG2 grew at a similar level to the positive control (Figure 2). Yeast harboring FmPG3 exhibits the slowest growth that is slightly higher than the negative control indicating little to no Y2H interaction. The PG-PGIP interactions implied from Y2H assay overall are reflective of findings in previous studies (Luca Federici et al., 2006; Leckie et al., 1999; Manfredini et al., 2005; Maulik et al., 2009; Sella et al., 2004; Spadoni et al., 2006; Spinelli et al., 2009)(L. Federici et al., 2001; Leckie et al., 1999; Manfredini et al., 2005; Sella et al., 2004; Spadoni et al., 2006; Spinelli et al., 2009) and suggests that it is feasible to use Y2H assay to functionally map PG-PGIP interactions.