Lectins for pest control
Transcript of Lectins for pest control
GENES OF INTEREST IN ENTOMOLOGICAL RESEARCH
Presentation by,
Mr. Guru P. N.Ph. D. 015/39
Cry toxin Bt: Cry1Ab, Cry1Ac, Cry2a, Cry9c, Cry2B, Vip I,
VipII etc.
Plant metabolites: Flavonoids, aklaloids, terpenoids.
Enzyme inhibitors: SbTi, CpTi.
Enzymes: Chitinase, Lipoxigenase.
Plant lectins: GNA.
Toxin from predators: Scorpion, spiders.
Insect hormones: Neuropeptides and peptidic hormones.
Genetic engineering of Plants for Insect Resistance
SEX DETERMINATIONSex determination is the process by which the gender of a bisexual organism becomes fixed, so that the individual progeny develops either as a son or a daughter.
Except diptera, sex determination in insects is cytological.
General aspects of sex determination in insects
Hermoproditism: same genotype producing male and female organs in same individualEg: Icerya
Gyanandromorphs/ intersexes: genetically abnormal individuals
1.XX/XY systems (1:1) Heterogametic sex: produce two gametically different types of
gametes
Homogametic sex: gametes of only one type
S1S1 X S1S2 (parents) 1:1 S1S1 and S1S2 (progeny)
Heterogametic : male in most and female of lepidoptera, trichoptera and diptera few.
When female is heterogametic sex, then named as Z & W i.e., ZW/ZZ
ZW (female) X ZZ (male) 1:1 ZZ and ZW (progeny)
2. XX/XO systems Y chromosome tends to degenerate leads having few or no
alleles functional
This is progressive evolutionary phenomena
XX (female) X XO (male) 1:1 XX and XO
It mainly based on genetic balance
It occurs in all type of insects both primitive and advanced
Ancestral form of sex deermination of orthopteroid insects
Neo-X systems: Orthopteroid, odonata, hemipteroid and coleoptera
3. Multiple sex chromosome system Difference between sexes involves larger number of
chromosomes
Most common has just two X’s
i.e., X1X2 Y or X1X2O and notation is
X1X1X2X2 / X1X2Y or X1X1X2X2 / X1X2O
Generally 1-6 X’s are present (extreme is 12)
Ex: hemiptera, dermaptera, diptera (multiple factor)
4. Multiple factor system
5. Haplodiploidy system Eg: Hymenoptera, Thysanoptera
Alternate alleles at a single locus- segregating in opposition
Females : S1S2, S1S3, S2S3 Males : S1S2S3 – if eggs are unfertilized
: S1S2, S2S2, S3S3 - inbred populations
This is intern also referred as “complementary sex determination mechanism”.
6. Molecular basis of sex determination Mainly restricted to Drosophila melanogaster
Sexlethal (Sxl) located on X-chromosome this is essential for determination of females
If, X:A is 1.0 (diploid with XX), Sxl produces an active product
that causes embryo to develop as female
X:A is 0.5 (diploid eggs with XY or XO) then Sxl is silent and embryo develops as male
OrthopteroidDictyoptera (Blattodea + Mantodea), Phasmida, Orthoptera, Embioptera XX/XO
Isoptera, Dermaptera XX/XYMost Heteroptera XX/XY or XOHomopteraAurenorhyncha XX/XOSternorhyncha Psyllodea : XX/XO
Coccoidea : XX/XOAphoidea : XX/XO
Coleoptera Diversified XX/XO, XX/XY, multiple, neo-X/neo-Y,……
Hymenoptera Haplodiploid
Lepidoptera Female heterogamy XY/XX or ZW/ZZ
Panarpoid XX/XO
Sex determination system in different insect orders
Model of JH III within a surface representation of the substrate-binding pocket of MsJHE.
Hormonal Control of Insect Metamorphosis
BrainTemperature, Light, Stress, etc.
Prothoracic Gland
Ecdysteroid
Corpus Allatum
Prothoracicotropic hormone (PTTH)
Juvenile Hormone (JH)
LarvaPupa
Adult
Development of winged morph
Biosynthesis in the corpora allata glands and release into the hemolymph. The primary mode of JH metabolism in the hemolymph is through the action of JHE. JHE hydrolyzes
JH in the presence or absence of hemolymph
Juvenile hormone esterase
Family : hydrolases (carboxylic ester bonds)
Other names : JH esterase, juvenile hormone esterase and juvenile hormone carboxyesterase.
Important in the regulation of the JH titre
JHE's appear to be produced by the fat body
JH esterase induction
JH I being the most potent inducer
Factors present in the head of the insect are potent inducers of JH activity.
Starvation of lepidopteran larvae also induces appearance of JH esterase
The insect nervous system is the target of the majority of synthetic chemical insecticides that are in agricultural use.
JH analog insecticides, for example, do not target the endocrine system of mammals and can show selectivity within an insect order or even family, a level of selectivity that is not obtained with classical insecticides.
The jhe gene has potential as a highly insecticidal transgene given an appropriate vector for expression.
What we can do with JHE ?
What is the future ?
Insertion of transgenes into the baculovirus genome that express a peptide hormone or
enzyme such as JHE that can alter normal host physiology.
Recombinant protein is the JHE from Tyroglyphus molitor, a member of the order Coleoptera (beetles)
(Hinton and Hammock, 2003b).
A dipteran, Drosophila melanogaster (Campbell et al., 2001)
Four lepidopterans, Heliothis virescens
(Hammock et al., 1990; Bonning et al., 1992), Choristoneura fumiferana
(Feng et al., 1999), Manduca Sexta
(Hinton and Hammock, 2001), and Bombyx mori
(Hirai et al., 2002)
Tried in
Introduction
• More than 50 neuropeptides have been described – Behavior– Pain perception– Memory– Appetite– Thirst– Temperature– Homeostasis– Sleep
Neuropeptides: neurohormones or neurotransmitters?• Neurohormones: when neurons secrete their
peptides into the vascular system to be transported to a relatively distant target
• Neurotransmitter: Many axon terminals of neurosecretory cells secrete their products at the synapse to directly affect a post synaptic cell
• Neuropeptides can do both – depends on nerve terminal
Stages of action
• Synthesis (ER and Golgi apparatus)
• Packaging into large-dense core vesicles (with modifying enzymes)
• Transport (fast-axonal transport)
• Release• Action (prolonged)• Termination by diffusion
and degradation
Synthesis is sequential
Synthesis is controlled by alternative splicing
Neuropeptides
• The endogenous opiates• Neuropeptide Y• Galanin• Pituitary adenylate cyclase–activating peptide (PACAP) • Melanocyte-stimulating hormone (MSH)• Neurokinin A (NKA)• Substance P (SP)• Neurotensin• Calcitonin-gene–related protein (CGRP)• Vasoactive intestinal polypeptide (VIP)
Neuropeptide Y
• A 36 amino acid peptide• Acts via G-protein coupled receptors (decrease in
cAMP, decrease in Ca+2 , and increase in K+ conductance)
• Function: augments the vasoconstrictor effects of norepinephrine
Diet
MembranePL
Synthesis of acetylcholine
• Choline + acetylcoenzyme-A by choline acetyltransferase in cytoplasm
• Transported into and stored in vesicles.
• Removal: hydrolysis by acetylcholinesterase
PROTEASE INHIBITORSROLE IN INSECT CONTROL
Protease inhibitors (PIs)
Are one class of plant defense proteins against insect pest infestation.
Plant derived protease inhibitors inactivate proteases of animals and microbial origin
Inhibiting endogenous enzymes
They are involved in the protection of plants against pests and possibly pathogens.
Protease-inhibitor families in plant tissues.
1. Soybean trypsin inhibitor (Kunitz) family
2. Bowman-Birk inhibitor family
3. Barley Trypsin inhibitor family
4. Potato Inhibitor I family
5. Potato Inhibitor II family
6. Squash Inhibitor family
7. Ragi 1-2/Maize bifunctional inhibitor family
8. Carboxypeptidase A. B inhibitor family
9. Cysteine proteinase inhibitor family (cystatins)
10. Aspartyl proteinase inhibitor family
Important ones are
Serine protease inhibitors
Cysteine proteinase inhibitor
Metallo- and aspartyl proteinase inhibitors
Aspartic and Metallo-Proteinase Inhibitors
Six families of Hemiptera,
pH optima =3-5
Serine Proteinase Inhibitors
Competitive inhibitors
Lepidoptera: (alkaline range of 9-11) : serine proteinases and
metalloexo peptidases are most active. (orders present: Lepidoptera,
Diptera, Orthoptera, Hymenoptera, and Coleoptera)
Antinutritional effects
Cysteine Proteinase Inhibitors (cystatins)
Most have been found in animals, but several isolated from plant
species, pineapple, potato, corn, rice, cowpea, etc.,
Found in midguts of several families of Hemiptera and Coleoptera
(digestion of food proteins).
Mildly acidic (pH optima of cysteine proteinases 5)
Plant PI’s
Mode of action
PI molecules inhibit protein digestive enzymes in insect guts, resulting in amino acid deficiencies and thereby developmental delay, mortality, and/or reduced fecundity
Hyperproduction of proteases to compensate for the loss of activity, causing the depletion of essential amino acids. The imposed nutritional stress would subsequently retard insect development.
Growth delay in a natural setting would also lengthen exposure of phytophagous insects to their predators and pathogens.
Increase in mortality.
Blocking insect midgut proteinases
The presence of inhibitor leads to the loss of nutrients particularly sulphur containing amino acids, and thereby weak and stunted growth and ultimate death
Insect resistant transgenic plants expressing PI’s
The first ever transgenic plants were Produced using cowpea trypsin inhibitor cDNA clone. The transgenic plants were resistant against herbivorous insects such as Collosobrchus maculatus, Heliothis spodoptera and Diabrotica and Tribolium sp.
Future prospects of inhibitory proteins
(i) Gene Combinations/Packaging/ Pyramiding
(ii) Protein Engineering
(iii) Single-chain Antibodies
(iv) Phage Display
Bowman-Birk Inhibitor (BBI) family
Closely related to serine protease inhibitors.
These proteins are classified as double-headed serine protease inhibitors due to the presence of two reactive site domains within the same polypeptide, one each for trypsin (Lys-Ser) and chymotrypsin (Leu-ser) molecules.
The cowpea trypsin inhibitor constitutes a some-what larger gene family of four major isoinhibitors.
Cowpea trypsin Inhibitor Gene (CpTi gene)
Isolated from cowpea plant (Vigna unguiculata).
This is the first plant-originated insect resistance gene to be successfully transferred into other plants species.
Against Lepidoptera, Coleoptera and Orthoptera
Inhibit the insect proteases involved in digestive processes.
Consequently, this will result in a depletion of essential amino acids required for growth, which in turn cause the larvae to die.
The CpTI gene is heritable to the next generation
LECTINS : Roles in Pests Control
“Lectins are a class of proteins of non-immune origin that possess at least one non-catalytic domain that specifically and reversibly bind to mono-or oligosaccharides”.
They are similar to antibodies in their ability to agglutinate red blood cells; however lectnis are not the product of immune system.
They may bind to a soluble carbohydrate or to a carbohydrate moiety that is a part of a glycoprotein or glycolipid.
“Based on the overall domain architecture of plant lectins, four major groups can be distinguished:
Merolectins, Hololectins, Chimerolectins and Superlectins
Entomotoxic lectins
Lectins are found to be effective on the insect orders like, Lepidoptera, Coleoptera, and Homoptera
Harmful effects of lectins
Larval weight decrease,
Mortality,
Feeding inhibition,
Delays in total developmental duration,
Delays in adult emergence and fecundity on the first and second
generation
Plant lectinsLectins are a group of proteins that are found in plants and they inhibit predation bybeing harmful to various types of insects and animals that eat plants.
Lectin (plant source) Insect Mannose specific:
ASA (Allium sativum) Myzus persicaeDysdercus cingulatus
ASA I, II Dysdercus cingulatusD. Koenigii
ASAL (Allium sativum leaf) D. CingulatusLiphaphis erysimi
Mannose/ glucose specificConA (Canavalia ensiformis) Aphids
LCA (Lens culinaris) Acyrthosipum pisum
PSA (Pisum sativum) A. PisumClover leaf weevil
N-acetyl-D-glucosamine specificACA (Amaranthus caudatus) A. Pisum
WGA (Triticum aestivum) Diabroticus undecimpuctataL. Erysimi
The first lectin to be purified on a large scale and was available on a commercial basis was Concanavalin A; which is now the most well- known lectin to control of some pest insects
Canavalia ensiformis, or Jack-bean, is used for animal fodder and human nutrition.
It is also the source of concanavalin A lectin.
Galanthus nivalis or snowdrop is the best-known and most widespread plant. (Fam:
Amaryllidaceae).
Currently, the two major groups of plant derived genes used to confer insect resistance on crops are lectins and inhibitors of digestive enzymes (proteases and amylase inhibitors)
Transgenic tobacco expressing Pinellia ternata agglutinin (pta) gene induced enhance level of resistance to M. persicae.
Transgenic plants with insecticidal lectin gene
Transgenic plants with lectin genes to confer resistance against insects
Transformed plant Lectin Target pest
Maize WGA Ostrinia nubilalisDiabrotica undecimpunctata
Rice GNANilaparvata lugensNephotettix virescensCnaphalocrocis medinalis
Tobacco PSA Heliothis virescens (tobacco bud worm)
Tobacco GNA Helicoverpa zeae (cotton bollworm)
Fungal lectins with insecticidal activity
•Mushrooms contain various potential interesting proteins, including lectins in their organs such as mycelium, spores and fruiting bodies
Some lectins from fungi including Xerocomus chrysenteron (XCL), Arthrobotrys oligospora (AOL) and Agaricus bisprous (ABL)
While, XCL is having higher insecticidal activity on Dipteran (Drosophila melanogaster) Homopteran (Myzus persicae and Acyrthosipon pisum)
reversible antiproliferative effects
Action mechanism of lectin at the tissue level of insects (MoA)Binding of the lectin to the midgut tract causing disruption of the epithelial cells including
Elongation of the striated border microvilli,
Swelling of the epithelial cells into the lumen of the gut lead to complete closure of
the lumen,
Permeability of cell membrane to allow the harmful substances penetrations from
lumen towards haemolymph and
Impaired nutrient assimilation by cells,
Allowing absorption of potentially harmful substances from lumen into circulatory
system, fat bodies, ovarioles and throughout the haemolymph
Fungal lectins with insecticidal activity
Lectin (fungal source) Insect Host
XCL (Xerocomus chrysenteron)
M. persicae, Acyrthosipon siphum
Peach, potato
Drosophila melanogaster Pea
SSA (Sclerotinia sclerotium)
A. Pisum Pea
RSA (Rhizoctonia solanii agglutinin)
Spodoptera littoralolis Cotton
PeCL (Penicillium chrysogenum)
M. persicae, A. pisum
Peach, potato, pea
(A) Xercomus chrysenteron
(B) Penicillums chrysogenum
Indirect effects of lectins
Interaction with virus transmission
Mannose-binding lectins are able to bind to carbohydrate on micro-organisms.
Circulatory viruses contain numerous N-linked glycosylation sites on their surface cells. Many of these sites contain high-mannose glycans which could interact with mannose-binding lectin such as ConA
Synergistic effects on other proteins
The insecticidal activity of protease inhibitor and α-amilase inhibitors were significantly increased when these inhibitors enzymes incorporated with lectin
Rice transgenic plant carrying three insecticidal genes including lectin gene (encoding gene GNA), cry1A and cry 2A, have enhanced levels of resistance to a wide range of different rice pests
THANK YOU
GENES OF INTEREST IN ENTOMOLOGICAL RESEARCH
Presented by, Mr. Guru P. N.Ph. D. 015/39
Dept. of Agril Entomology