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.