Papaya trees are rich in papain, a cysteine protease that has been shown to
mediate plant defense against pathogens and insects. However, the pathogen
Phytophthora palmivora infects all parts of papaya plants, suggesting that
it has evolved cysteine protease inhibitors to counter papain and enable
infection.

Out of several cystatin-like cysteine protease inhibitors from P. palmivora,
PpalEPIC8 appeared to be unique to P. palmivora and was highly induced
during infection of papaya. Analysis revealed that PpalEPIC8 strongly
inhibited papain enzyme activity, suggesting that it is a cysteine protease
inhibitor. Phytophthora palmivora mutants with knocked-out PpalEPIC8 were
the generated using CISPR-Cas9 -mediated gene editing.

The mutant strains exhibited increased papain sensitivity in vitro and had
reduced pathogenicity during infection of papaya fruits compared to the
wild-type strain. This strongly suggests that PpalEPIC8 indeed plays a role
in P. palmivora virulence by inhibiting papain.

This study provides evidence of pathogens secreting cystatins to invade
plants. It also established a gene-editing system for P. palmivora using
CRISPR-Cas9.



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