Two cDNAs encoding cysteine proteinases were isolated from a cDNA
library constructed from feeding females of
Heterodera glycines. The library was screened with a cysteine
proteinase gene fragment originally amplified from cDNA
of H. glycines. Database searches predict that 1 cDNA
(hgcp-I) encodes a cathepsin L-like proteinase, while the second
(hgcp-II) encodes a cathepsin S-like enzyme. Both predicted proteins
contain a short secretion signal sequence, a long pro-peptide and a mature
protein of 219 amino acids. Southern blot analysis suggests that the
cathepsin S-like enzyme, HGCP-II, is encoded by a single-copy gene in contrast
to the cathepsin L-like proteinase, HGCP-I which may have 2 homologues.
The regions encoding the mature proteinases were cloned into an expression
vector and recombinant protein produced
in E. coli. HGCP-I was shown, after refolding, to cleave the
synthetic peptide Z-Phe-Arg-AMC, and this activity could
be inhibited by the engineered rice cystatin Oc-IΔD86. HGCP-II showed
no activity against the synthetic substrates
tested. The knowledge gained from these studies will improve our
understanding of plant nematode proteinases and aid
the development of a rational proteinase inhibitor-based approach to plant
nematode resistance.