commonly recognized as a significant component of natural plant defense, as they inactivate digestive proteases upon insect herbivory.

1. Protease inhibitor (PIs) and
Molecular Interaction in Insect
M. M. Mawtham
Ph.D. Scholar
Tamil Nadu Agricultural University

2. Protease and Protease inhibitors (PIs)
 Protease - an enzyme that catalyzes (increases the rate of)
proteolysis, the breakdown of proteins into smaller polypeptides
or single amino acids.
 Protease inhibitors (PIs) - commonly recognized as a
significant component of natural plant defense, as they inactivate
digestive proteases upon insect herbivory.
 Protease inhibitors (PIs) in plant protection- Mickel and Standish
(1947)
 Peptidase, Exopeptidases, Endopeptidases, Protease
(NC-IUBMB)

3.  Serine proteases - insect orders - Diptera, Orthoptera,
Hymenoptera and Coleoptera. Larval midgut extracts of 12
lepidopteran species indicated that most of them use a trypsin-
and elastase-based digestive system and others depend largely
on chymotrypsins. (Shu and Guo, 2005)
SPIs are classified
Bowman– Birk serine protease inhibitors
Cereal trypsin/-amylase inhibitors
Mustard trypsin inhibitors
Potato type I protease inhibitors
Potato type II protease inhibitors
Serpins
Kunitz type inhibitors
Squash serine inhibitors

4. Protease enzyme
 The enzymes - membrane associated forms and sequestered in
vesicles (cytoskeleton) - ectoperitrophic space between the
epithelium - move transversely into the lumen of the gut.
 Two mechanisms- i) Direct effect of food components (proteins)
on the midgut epithelial cells ii) Hormonal effect triggered by food
consumption
 The proteinases are group of hydrolytic enzymes in insects and
are included in digestive processes, proenzyme activation,
freedom of physiologically dynamic peptides, supplement
initiation, and aggravation forms among others.

5. (Keyan and Rensen, 2015)

6. Direct Defense in Plants
In tomato and potato plants, the 18-amino-acid-long peptide systemin
and the hormones abscisic acid and jasmonic acid (and its derivative
methyl jasmonate) act as wound signals in the activation of proteinase
inhibitor genes in response to mechanical wounding.
(Tamayo et al., 2000)
 DAMPs (damage-
associated molecular
pattern)- triggered
immunity (DTI)
 Efector-Triggered
Immunity (ETI)

7. Functions of Protease Inhibitors
 Plant
 plant SPIs are involved in the mobilization of storage proteins,
regulation and stabilization of endogenous enzymatic activities,
morphogenesis, flower development, modulation of apoptosis,
and cell death and in plant defense mechanisms against insects.
 Insect
 Interfering with important biochemical or physiological processes
of insects such as the proteolytic activation of enzymes and
molting of insects.
 It has been proposed that the N- and C-terminal peptide domains
of the PIs bind to cellular receptors lining the insect gut to
antagonize peptidehormone- regulated protease production.
(Gutierrez et al., 1999)

8. Mechanisms of protease-inhibitor interactions
(A)Irreversible reactions. The protease–inhibitor interaction induces the
cleavage of an internal peptide bond in the inhibitor triggering a
conformational change.
(B)Reversible interactions. The inhibitor interacts with the protease
active site in a similar way to the enzyme-substrate interaction.
(Clemente et al., 2019)
 Reversible interactions
 Competitive
 Non-competitive
 Uncompetitive
 Irreversible interaction

9. Molecular Farming
 Molecular farming refers to the production of recombinant
proteins in plants, including pharmaceutical products, industrial
proteins and other secondary metabolites.
Plant protease inhibitors with
potential application in agriculture
and molecular farming
(Clemente et al., 2019)

10. Commercial Applications of Plant PIs

11. Adaptations of insect pests to plant PIs (Akbar et al., 2018)
H. armigera regulates its digestive proteinase levels against different types of PIs
of Albizia lebbeck seeds by constitutive hyper-production of existing enzymes,
trypsin, chymotrypsin and aminopeptidase activities to overcome the
antinutritional effects of the inhibitor (Hivrale et al., 2013).

12. Molecular Investigation of Protease-Based
Counterdefense
Summary of sequence annotation of genes
expressed differentially in response to dietary
soybean cystatin (scN) based on (A) biological
process and (B) molecular function (Chi et al.,
2009). Suppression Subtractive Hybridization (SSH)
Microarray

13. Regulation of Protease Expression
 Cowpea bruchid (Callosobruchus maculatus) controls midgut CatB
expression by manipulating the intracellular balance of the transcription
activator HNF4 and transcription repressor Svp.
 Abbreviations: CatB, cathepsin B; COUP, chicken ovalbumin upstream
promoter; GTF, general transcription factor; HNF4, hepatocyte nuclear
factor 4; RNA pol II, RNA polymerase II; scN, soybea cysteine protease
inhibitor N; Svp, Seven-up; TATA, TATA box (Broadway, 1997).

14. Conclusions
 Host Plant Resistance
 Genes have been used for the construction of transgenic crop
plants to be incorporated in integrated pest management
programs.
 The coevolution between plants and insects - developed several
strategies to avoid plant defense systems.
 Using RNAi targeting PI induced insect digestive enzymes and
other counterdefense genes may prevent insect adaptation.
 Use of transgenic plants may also have some effects on
environmental, public health, antibiotic resistance ,
gastrointestinal problems and nutritional effects of proteinase
inhibition on mammals.