Discussion
As the risk of conventional fungicides continue to be a cause for a concern, there is increased demand for more eco-friendly pest management approaches. Regulation of fungicide use has increased in many countries and regions, and consumers are wary of chemical residues on their fruits and vegetables. However, more sustainable approaches to control fungal pathogens, especially at the post-harvest stage, are still in the early stages of development. PGIPs are naturally occurring plant proteins that can slow the rate of infection from pathogenic fungi by inhibiting one of the key steps in the infection process—the degradation of cell walls via PGs. While conventional fungicides continue to be key to ensuring food security and preventing crop loss across the world, our approach for developing and engineering PGIPs is a step towards providing an alternative strategy to fungal pest control, and could potentially serve as a platform for future engineering of a wider range of proteins for practical applications in agriculture and post-harvest fruit storage and transportation.
PGIPs are naturally occurring plant proteins that can slow the rate of infection from pathogenic fungi by inhibiting one of the methods fungi use for colonizing plants—the degradation of cell walls via PGs. The results of this research validate the utilization of Y2H to estimate the interactions between PvPGIPs and PGs. Both PvPGIP1 and PvPGIP2 have the strongest interactions with AnPG2 and BcPG1 compared to BcPG2 and FmPG3, as evidenced by both higher growth rate and stationary phase in growth curves. PvPGIP2 has greater levels of interactions with PGs compared to PvPGIP1, which supports the findings from previous studies (Bishop, 2005; D’Ovidio et al., 2004; Leckie et al., 1999; Manfredini et al., 2005). While conventional pesticides often leave residues in the soil and crop surfaces that can negatively impact non-target organisms, PGIPs are naturally occurring proteins that will likely degrade into harmless amino acids. The presence of these defensive proteins in plants increase disease resistance (De Lorenzo et al., 2001).
PGIPs are a family of LRR proteins with several highly conserved regions (Helft et al., 2011). In our study, we found that LRR5 resides in a critical region for recognizing PGs, and that regions of PvPGIP2 exclusive of LRR5 to LRR8 plays a very minor role in PG recognition. The successful truncation of PvPGIP2 to one-third of the original size with similar level of inhibition activity retained indicates the versatility of LRR proteins and the potential to engineer this group of proteins for altered recognition and activities. Although we were successful in demonstrating the interactions between PGIPs and PGs using a Y2H system, some previous studies report that PGIPs are difficult to express in soluble in microbial system. This could be due to a number of factors, such as protein instability or an aggregation of recombinant PGIPs (Haeger et al., 2020). Nevertheless, in this study we were able to reconstitute the PG-PGIP interaction using a Y2H system and identified a method to secrete functional PGIP from yeast into the medium. While a trace amount of PvPGIP1 was secreted without the Ost1 signal, utilizing the signal peptide allowed us to clearly confirm the presence of PGIP in the medium. The presence of the N-terminus domains, Gal4 AD or Ost1 signal peptide, may have enhanced the stability and folding of PvPGIP2 in the yeast environment.
Some challenges remain unsolved and further research is needed to develop truncated PGIP peptides into practical approach. First, there are beneficial fungi that play essential roles to promote plant growth, such as mycorrhizal fungi that mediate plant-soil feedback (Kadowaki et al., 2018). However, we do not know if the application of PvPGIP proteins will affect these nonpathogenic fungi. Second, while the full length PvPGIP2 is found in nature and is not known to negatively impact the environment, it is unknown if the truncated form has unwanted targets or toxic side products from its interactions. Although unlikely to be a toxic agent, in vivo studies are needed to test the toxicity of PGIPs before practical and large-scale applications. Although PGIPs are natural plant proteins, it may also be difficult to convince the public how they are different from GMOs. Education and improving laymen understanding of this potential fungal control tool will be important. PvPGIP2 may be the most studied PGIP, but other plant PGIPs are of interest as well. For example, CkPGIP1 from Cynanchum komarovii (Liu et al., 2016) and MdPGIP1 from Malus domestica(Oelofse et al., 2006) have shown to have activity against PGs that PvPGIP2 is not yet known to have, such as those from Rhizoctonia solani , Diaporthe ambigua, and Botryosphaeria obtusa (Liu et al., 2016; Oelofse et al., 2006). Studying how other PGIPs inhibit PGs and understanding what motifs are present that allow for that interaction may offer hints at further evolving our truncated PvPGIP2 to have a broader spectrum of activity.
As the long-term goal of this research is to develop a sustainable, alternative fungal treatment, another future direction and challenge to tackle includes improving the protein yield in the medium. S. cerevisiae is a well-studied model organism that is promising as a microbial host factory (Borodina & Nielsen, 2014; Siddiqui et al., 2012). Efficient protein production by yeast will require a combination of improved secretion signal peptides and fine-tuning yeast metabolism (Delic et al., 2013). By developing efficient synthesis, the cost of the proteins will decrease, enabling the peptides to be more viable and appealing as a product. This will allow for more widespread usage in agriculture. Furthermore, packaging PGIPs as a user-friendly product that can be applied with standard equipment is essential to the practical utilization of this strategy. One method is to freeze dry the PGIP-secreting yeast for application. Previous studies have found that freeze drying naturally occurring microbiota on apples and applying a resuspended solution on postharvest crops greatly reduces fungal disease and loss of crop during shipping (Navarta et al., 2020). AsSaccharomyces is natively found on wine grapes (Valero et al., n.d.), a fruit susceptible to rot from Botrytis (Elmer & Reglinski, 2006; Steel et al., 2013) utilizing cryonics on PGIP-secreting yeast is a possible developmental path though it would be a genetically modified Saccharomyces . Furthermore, thermostability of the products would also be an important trait for storage and utility purposes.
There are numerous innovative ways that plant proteins can be developed into tools for disease prevention in crops. With more research, they can be enhanced to further improve their efficacy and more functionality may be discovered. Here, PvPGIPs are proof that plant proteins can successfully be utilized as exogenously applied, sustainable, natural fungal control agent and reduce disease incidence in postharvest crops. This research is a stepping-stone towards developing an eco-friendly future where green products are an ever-growing industry.