Protease Inhibitors (PIs): Candidate Molecules for Crop Protection Formulations against Necrotrophs.

Necrotrophic phytopathogens pose a serious challenge to the productivity of several crops causing seedling damage, pre- and post-emergence damping-off and root rot thus reducing plant growth and yield. They are known to gain nutrition by secreting a diverse array of hydrolytic enzymes and thereby causing extensive host plant tissue maceration. Amongst the diverse hydrolases, proteases play a pivotal role in the necrotrophic mode of nutrition and thereby in determining pathogenic virulence. Host plants often counteract the necrotrophic proteolysis events by proteins (peptides), particularly through protease inhibitors (PIs). PIs play an important role in host innate immunity function by functioning as anti-metabolic proteins inhibiting the activity of phytopathogenic secretory proteases. Their abundance in plant storage organs explains their anti-nutritional interaction which stalls pathogenic invasion. PIs, therefore, constitute potential candidates that can be deployed as effective antimicrobials in agriculture, particularly against necrotrophic soil-borne pathogens. The present review traces the progress made in the identification of PIs from plants, and their inhibitory potential against necrotrophic phytopathogens and explores prospects of utilizing these molecules as effective anti-necrotrophic formulations for disease management.

Anti-microbial and cytotoxic activity of ZzAMP, a serine protease inhibitor (SPI) with nutraceutical potential from rhizomes of medicinal plant, Zingiber zerumbet.

Serine protease Inhibitors (SPIs) abundantly reported in plant storage organs constitute important candidate molecules for antimicrobial and anticancer therapeutics. Our earlier studies had identified antimicrobial protein/or peptides (AMP) from Zingiber zerumbet rhizomes designated ZzAMP inhibiting serine protease (SP) of necrotrophic phytopathogen, Pythium myriotylum. Considering the high ethno-medicinal applications of Z. zerumbet rhizomes, present study evaluated the anti-bacterial, anti-oxidant and cytotoxic properties of ZzAMP. Though ZzAMP displayed low radical scavenging activity (IC50 1000 µg/ml), it exhibited considerable anti-bacterial activity towards the nosocomial pathogen Klebsiella pneumonia (93%), which produced maximal extracellular protease (30.6 ± 1.47 U/ml) amongst the pathogens screened. Evaluation of cytotoxic activity of ZzAMP revealed decrease in viability of cancer cell line, HeLa (IC50 115.09 µg/ml) compared to normal cells, L929 (IC50 299.95 µg/ml). Present experiments showing antimicrobial and cytotoxic activities of ZzAMP with minimal damage to normal cells are indicative of its potential as a promising nutraceutical protein.

Antimicrobial peptide (AMP) from Zingiber zerumbet rhizomes with inhibitory effect on Pythium myriotylum secretory proteases and zoospore viability.

Protease mediated proteolysis has been widely implicated in virulence of necrotrophic fungal pathogens. This is counteracted in plants by evolving new and effective antimicrobial peptides (AMP) that constitute important components of innate immune system. Peptide extraction from rhizome of Zingiber zerumbet was optimized using ammonium sulphate (50-80% w/v) and acetone (60 and 100% v/v) with maximal protein recovery of 1.2 ± 0.4 mg/g obtained using 100% acetone. Evaluation of inhibitory potential of Z. zerumbet rhizome protein extract to prominent hydrolases of necrotrophic Pythium myriotylum revealed maximal inhibition of proteases (75.8%) compared to other hydrolytic enzymes. Protein was purified by Sephacryl S200HR resin resulting in twofold purification and protease inhibition of 84.4%. Non-reducing polyacrylamide gel electrophoresis (PAGE) of the fractions yielded two bands of 75 kDa and 25 kDa molecular size. Peptide mass fingerprint of the protein bands using matrix assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectroscopy (MS) and subsequent MASCOT searches revealed peptide match to methylesterase from Arabidopsis thaliana (15%) and to hypothetical protein from Oryza sativa (98%) respectively. Further centrifugal filter purification using Amicon Ultra (10,000 MW cut-off) filter, yielded a prominent band of 25 kDa size. Concentration dependent inhibition of zoospore viability by Z. zerumbet AMP designated as ZzAMP was observed with maximal inhibition of 89.5% at 4 µg protein and an IC50 value of 0.59 µg. Studies are of particular relevance in the context of identifying the molecules involved in imparting below ground defense in Z. zerumbet as well in development of AMPs as potential candidate molecules for control of necrotrophic pathogens of agricultural relevance.