Ipomoea batatas: papain propeptide inhibits cysteine protease in main plant parasites and enhances resistance of transgenic tomato to parasites

Abstract

Different parasites cause severe lose in quantity and quality of crops. Many parasites develop haustorial cells and stylets that penetrate the host using secreted enzymes and mechanical pressure. Cysteine proteases are pre-pro-enzyme produced by parasites that are essential for normal parasitism. Papain is also a kind of cysteine proteases such that its propeptide segment has inhibitory properties and limits the protease activity of papain. To investigate the inhibitory effects of papain propeptide on some parasite proteases, we cloned inhibitory propeptide of papain of Ipomoea batatas, and enzymatic fragments of Diabrotica virgifera cathepsin L-like protease-1, Meloidogyne incognita cathepsin L-like protease 1, Heterodera glycines cysteine protease-1, Cuscuta chinesis cysteine protease and Orobanche cernua cysteine protease. After purification of recombinant inhibitory propeptide and enzymatic fragments, the inhibition activity of propeptide on cysteine proteases was measured. Finally inhibitory propeptide was transformed into tomato and transgenic plants resistance to parasites (bioassay) were examined. We demonstrated papain-propeptide inhibits cysteine protease of mentioned parasites. In transgenic tomato plants, papain-inhibitory propeptide effectively interrupted haustoria development. Haustoria-digitate cells of dodder could not differentiate and develop into the phloem and xylem hyphae on transgenic tomatoes. Parasites grown on transgenic tomatoes showed reduction in vigor and productivity due to defective connection of haustoria. Lower ratio of female nematodes and a decrease of nematode egg mass per transgenic line indicated biocontrol of nematode. The changes in growth factors of parasite challenged transgenic lines relative to controls, indicates the efficacy of papain propeptide in control of parasitism.

Regulation of the Fasciola hepatica newly excysted juvenile cathepsin L3 (FhCL3) by its propeptide: a proposed ‘clamp-like’ mechanism of binding and inhibition

“…Similarly, Korde et al [ 41 ] found that a synthetic 15-mer residue peptide based on the propeptide sequence of falcipain-2, a Plasmodium falciparum cathepsin L, inhibited its cognate enzyme with 10,000 times less potency than the propeptide of falcipain-2 itself [ 41 , 42 ]. Therefore, despite the enthusiasm for exploiting the C-terminal portion of propeptides for small drug design against cathepsin-like peptidases, creating the 3-D configuration that binds the substrate cleft with high affinity will be challenging [ 13 , 18 , 22 ]. More creative design methods such a cyclisation of peptides to improve their conformation, stability and activity compared to linear peptides [ 43 , 44 ] may be a future option to develop specific propeptide-based inhibitors against the cathepsin peptidases of F. hepatica that could have applications as anti-parasitic reagents against this other medically-important parasites.…”

“…Since the N-terminal cathepsin propeptide of cysteine peptidases are highly specific and potent regulators of their cognate enzyme [13,18], they can act as useful structural templates to design active-site directed, selective, and efficient inhibitors of cathepsin peptidases [19][20][21][22]. Uncovering how propeptides bind to their cognate mature domain is, therefore, not only fundamental to understanding parasite virulence and infection, but also important in gaining information that could be exploited for anti-parasite drug design.…”