Novel Protease Inhibitors Provide Disease Resistance in Potatoes

Anti-microbial products for food safety
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Available for Licensing
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Inventors

Adam Heuberger
Amy Charkowski
Janak Joshi

Reference No: 2020-064
Licensing Manager

Jessy McGowan
Jessy.McGowan@colostate.edu

970-491-7100

At a Glance

Researchers at Colorado State University have identified novel protease inhibitors from a wild potato species that provide disease resistance and may be used for anti-microbial applications in food production. Methods and compositions are proprietary.

 

Background

Potato is a major staple crop, and necrotrophic bacterial pathogens such as Pectobacterium sp. and Dickeya sp. are major threats to potato production and therefore, global food security. These diseases cause over $40 million in annual loss to the US potato industry alone. Most lines of cultivated potato (Solanum tuberosum) are susceptible to soft rot caused by these pathogens, however, certain lines of wild potato have been found to be resistant.

It is understood that soft rot bacteria such as Pectobacterium sp. have several known pathogenicity factors, including the production of cell wall degrading enzymes (cellulases, pectinases, proteases, etc.) that break open plant cells and ultimately enable the bacteria to establish and grow. However, specific resistance genes have yet to be discovered. In addition, there are currently no reliable chemistries available to prevent these diseases.

Overview

Using a multi-omic approach, protein extracts isolated from resistant stem and tuber tissues have been found to inhibit the ability for bacteria to produce cellulases, pectases, and proteases. Subsequent proteomic characterization of these extracts have indicated an abundance of protease inhibitors. Genomic analysis of resistant and non-resistant potato lines further support that the resistant variety encodes several protease inhibitor genes absent in non-resistant varieties, as well as coding sequence differences in the protease inhibitors that are common between the species. Taken together, these data support that the protein extract discovered here is not only novel, but also that the trait can be transferred into domestic potato varieties to provide resistance to soft rot pathogens.

Furthermore, these protease inhibitors can be isolated and manufactured for applications in food safety and agricultural production as anti-microbial compounds. Research is underway to determine the impact on human bacterial pathogens. 

This novel protein extract inhibited bacterial protease activity, motility, and affected cell morphology, see Figure 1. 

Figure 2 Bar graphs and observation of Pb1692 cells under compound microscope.
Figure 1. Protease and motility inhibition and cell morphology effects of M6 potato protein on Pectobacterium brasiliense Pb1692. Pb1692 cultures were incubated with 400 μg ml−1 DM1 or M6 protein and were compared with cultures with protein extraction buffer alone as a negative control and with buffer with a protease inhibitor cocktail (cPI). A, Trypsin inhibition activity, B, exo-protease inhibition, and C, motility inhibition. Y axis area measurements indicate spread of activity halo on agar plates. Data are presented as mean ± standard error of the mean and are a combination of two independent experiments, with n = 8 for A and B and n = 5 for C. Asterisks indicate differences between treatment and buffer control (analysis of variance Dunnett’s post hoc, P < 0.05). D, Example observations of Pb1692 under compound microscope (1,000×). Bacterial cells were exposed to controls or potato protein extracts, were fixed on glass slides, and were stained with crystal violet. Representative filamentous cells (>5 μm) are marked with yellow arrows. Microscopy experiments were repeated three times with similar results.

Recombinant protease inhibitors reduced Pectobacterium motility, protease activity, and disease symptoms.

Figure 2 Bar graphs and observation of Pb1692 cells under compound microscope.
Figure 2. Effect of cloned and purified M6 protease inhibitors (PIs) on Pectobacterium brasiliense Pb1692 virulence factors and disease. A, PIs were cloned from Solanum chacoense M6, were purified, and were tested for effects on trypsin activity, and B, Pb1692 bacterial exo-protease activity and C, motility. Data is presented as mean ± standard error of the mean, which is a combination of two independent biological replicates, with n = 8 for (A and B) and n = 5 for (C). Asterisks indicate significant differences between treatment and empty vector control (analysis of variance Dunnett’s post hoc P < 0.05). D, Observation of Pb1692 cells under compound microscope (1,000×). Bacterial cells were exposed to empty vector protein extract or cloned and purified M6 PIs, were fixed onto a glass slide, and were stained with crystal violet. Representative filamentous cells (>5 μm) are marked with yellow arrows. Experiments were repeated three times and each replicate demonstrated similar results. E, Representative images of disease symptoms on potato tubers co-inoculated with Pb1692 and each of the purified M6 PIs. F, Quantitation of tuber disease severity caused by Pb1692, measured as amount of decayed tissue collected from the tuber.
 
Benefits
  • Baseline genetic resistance to soft rot in potatoes
  • Anti-microbial products for human, plant and animal pathogens
Benefits
  • Baseline genetic resistance to soft rot in potatoes
  • Anti-microbial products for human, plant and animal pathogens
Applications
  • Integrating genetic resistance to soft rot pathogens into potato and related crops
  • Anti-microbial products for food safety