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From Bt to RNAi: Current and Emerging Uses of

Insecticidal Toxins in Genetically Engineered Crops

ACMT Spring Conference

Phoenix, AZ Natural Toxins Pre-symposium

March 27, 2014

Daniel A. Goldstein, M.D., F.A.C.M.T. !Senior Science Fellow and Lead, Medical Sciences and Outreach""daniel.a.goldstein@monsanto.com 314-694-6469"

!!

people.cryst.bbk.ac.uk/ ~ubcg16z/pore/pore.html !

http://www.alnylam.com/rnai_primer/rna-interference-pg5.htm !

Disclaimer

•  I am a full time employee of the Monsanto Company, St. Louis, Missouri.

• Monsanto is the leading source of commercialized GM crops and leading producer of glyphosate-based herbicides (Roundup) commonly used in conjunction with genetically modified (herbicide) crops.

•  I will be talking today about technologies, not products. Specific Monsanto products are not mentioned in the slide set, and similar technology is offered by competing manufacturers.

• Any mention of specific products arising during the course of the lecture is intended to serve as a case-in-point example of the technology being discussed.

Outline

•  The quest for the Magic Bullet

•  100 Years of history: Bacillus thuringiensis

–  Agricultural use / specificity

–  Biological activity of Cry toxins

–  Pore Forming Toxins

–  Bt Cry Safety

•  The Cutting Edge: RNA mediated gene regulation –  Basics

–  RNA in GM plants •  Anti-viral

•  Gene regulatory

–  Insecticidal RNA- Specificity and the Magic Bullet

–  dsRNA Safety

• Questions/Discussion

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Insects and Nematodes:

• Coleoptera Beetles

•  Lepidoptera Butterflies and moths

• Diptera 2 winged “True” flies- including mosquitoes

• Hymenoptera Membrane winged- bees, wasps, ants

• Nematodes Small “worms” which are major root pathogens

The Quest for the Magic Bullet

• Antimicrobials and chemotherapeutics are pesticides for use in human beings.

•  The challenges in medicine and agriculture are all about achieving target specificity… kill the undesirable:

– Microbe

– Tumor Cell

– Plant

– Insect (nematode, fungus, etc..)

WITHOUT killing desirable species and without undue impact on the “environment”

• As a side note…. Both agriculture and medicine started with arsenic and tried to improve from there…

• … and it can be good business

Natural Toxins ! EPA “Biopesticides”

• Pesticides derived from such natural materials as animals, plants, bacteria, and certain minerals

– Microbial pesticides (historically, Bacillus thuringiensis / Bt)

–  “PIPs” or Plant Incorporated Protectants (pesticidal GM crops)

• What are the advantages of using biopesticides? – Usually inherently less toxic than conventional pesticides.

–  Generally affect only the target pest and closely related organisms.

– Often are effective in very small quantities and decompose quickly, resulting in lower exposures and largely avoiding the pollution problems.

– As component of Integrated Pest Management programs, can greatly decrease the use of conventional pesticides, while crop yields remain high.

• But- users do need to know a great deal about managing pests.

http://www.epa.gov/pesticides/biopesticides/whatarebiopesticides.htm!

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Bt - History 100 Years of Bacillus thuringiensis, Am Acad. Microbiology, 2002!

Bt- Biology

• Bt is part of a large clade of Gram positive spore-forming rods, obligate or faculatative anaerobes.

• Closely related to B. subtilis, B. cereus, anthrax, and more distantly to clostridium spp.

• Pathogenicity / target organisms defined largely by toxins “loaded” onto chassis…

• When stressed, produce durable spores which can survive for extended periods under dry or other adverse conditions.

ANTHRAX ENZYMES

Mom… or possibly dad…!

Bt- Biology

• Sporulation consists of nuclear condensation in conjunction with peri-nuclear crystalline toxin formation.

•  The organism may be utilized dry and intact OR

• Organism or crystal proteins can be isolated and utilized for pesticidal products

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Cry Toxins- a LARGE family

Soberon et al 2010!

Bt-Cry Proteins have limited specificity (Insecticidal proteins virtually non-toxic to non-insect genera)

Bt- Genetically Modified Crops

•  One (or more) Bt protein genes incorporated into the primary plant genome, inherited in a Mendelian fashion.

•  Optimized for plant expression and driven by plant promoter

•  In most instances this is hybrid seed (maize, cotton).

•  Puts the pesticide where it is needed… and no place else.

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""""""""""

Bt’s"protect:""EAR""STEM""""ROOT"""

Bt / insect resistance

Bt proteins are"•  Insecticidal (gut specific)"•  Specific to sub-classes of insects"•  Virtually non-toxic to birds,

mammals, worms, etc."

GM Conventional!

Pore Forming Toxins- Nomenclature

• There are two general classes of pore forming toxins we will discuss today:

– Alpha-PFT: Intra-membrane portion composed of alpha-helices

– Beta-PFT: Intra-membrane portion consists of beta-pleated sheets

•  Includes CDCs- Cholesterol Dependant Cytolysins

• HOWEVER toxins may be described based upon:

– Hemolysis…. Staphlococcal alpha-hemolysin is a beta-PFT

– Order of discovery… the Bt proteins Cry (crystal) or Cyt (cytotoxic) are called delta-endotoxins, include variant of beta-hemolysin, and are both Alpha (Cry) and Beta (Cry) PFTs

• Most PFTs have 3 domains- but the relationship between domain number and function is not consistent outside of Cry protein family

Generalized mechanism of pore formation by PFTs. Soluble PFTs bind membrane receptors, which leads to oligomerization and insertion of an aqueous pore into the plasma membrane.

Los F C O et al. Microbiol. Mol. Biol. Rev. 2013;77:173-207

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Protein structures of various PFT classes.

Los F C O et al. Microbiol. Mol. Biol. Rev. 2013;77:173-207

Beta- PFTs"Trans-membrane!portion formed by!beta-pleated !sheet!

Alpha- PFTs"Trans-membrane!portion formed by!Alpha helices!

Pore forming toxins- a large class of microbial toxins

Pore forming toxins- a large class of microbial toxins

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Microbial PFTs

Activity of:

• Perfringolysin (PFO) from Clostridium perfringens

• Alpha-hemolytic toxin from Staphlococcus aureas

• Protective Antigen from binary anthrax toxin, Bacillus anthracis

•  Geny and Popoff 2006 http://www.biolcell.org/boc/098/0667/boc0980667.htm

Where have you seen this kind of thing before???

Nervous system: nAChR, GABA-A, GABA-C, Glycine, 5HT3, Glu R1-4 (AMPA), GluR6 (kainate), NMDA

Cardiac Ion Channels

http://www.nature.com/scitable/topicpage/ion-channels-and-excitable-cells-14406097#!

http://pubs.niaaa.nih.gov/publications/arh313/196-214.htm!

Beta-PFTs in Mammalian systems: Complement

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Da ‘shooms are out to get you with PFT’s

•  The Straw or Forest Child Mushroom (Volvariella volvacia) contains a heat labile beta-PFT, volvatoxin, closely related to Cyt2Aa1 (IP LD-50 1.2 mg/kg)

• Similar toxins are found in other mushroom species…. Many edible!

Ostreolysin (beta-PFT)!Pleurotus ostreatus!Oyster Mushroom!

Unnamed (alpha-PFT)!Laetiporus sulphureus!

Sulfur Shelf!

Flammutoxin!Flammulina velutipes!

Enoki!!

Pore Forming Toxins- General Mechanism (structure shown is actually S. aureas alpha-hemolysin, a beta-PFT)

Pro-toxin! Toxin!

Small-pore former!(alpha-hemolysin, N=7)!

Large-pore former!(CDC’s- N up to 70)!

Cry proteins require both primary and secondary binding sites (presumably contributes to selectivity)

• Cry-1Ab binds first to cadherin and secondarily to glycophosphorylinositol to achieve cell surface attachment prior to pore formation.

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Bt-Cry proteins: alpha-PFT family

•  Domain-1: Pore formation (note the alpha-helices)

•  Domain-2: Binding

–  Primary binding specificity usually in loops

–  Secondary binding may employ domains 2 and/or 3

•  Domain 3: Structure/ deployment and secondary binding

•  (Literature is not in full agreement regarding D2 and D3 binding function for all Cry proteins)

Pigott and Ellar Microbiol Mol Biol Rev 2007, 71:255!

Bt Crops- Safety

• Bt proteins used in GM crops have no discernible toxicity to humans (mammals/birds/reptiles)

– LD-50 > 5000 mg/kg in acute toxicity studies

– Long history of safe use in organic and conventional (non-GM) agriculture

•  Lack of effect is a function of:

– Need to bind to not one, but two sequential binding sites with species variability

– Lack of stability in animal vs. insect (alkaline) gut.

– Ready digestibility of Bt Cry proteins in humans

•  Limited off-target species effects (no impact on birds, reptiles, mammals…)

•  Limited environmental persistence

• Additionally- reduces need for small molecule chemical insecticides which have greater toxicity and are less specific to target organism(s)

Double Stranded RNA, Gene Regulation and Pest Control

The road to the Magic Bullet?

November 2007, Volume 25 No 11 pp1187-1328 !

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Cell Biology….. 101

DNA"

mRNA"

Polymerase"DNA"Transcription

mRNA"

Protein"Ribosome"

Amino"Acids"

Translation

RNA Mediated Gene Regulation

RNAi!

(PRE-) microRNA! (miRNA)!

Dicer "DCL-4"

Dicer "DCL-1"

Dicer "DCL-3"

siRNA "duplex"

Cross-talk! Cross-talk!

Viral!Transgenic!Endogenous!Topical!

Mainly!Endogenous!Vs.!

“Classical” RNAi!RNA inhibition!

mRNA"

Message"destroyed"

miRNA- PTGS!Post-Transcriptional !

Gene Supression!

X"mRNA"

Translation"repressed"

miRNA- TGS!Transcriptional Gene!

Suppression!

Me" Me" Me"

X"

DNA"

DNA"Methylation"

Transcriptional suppression"

Early Applications- “Anti-sense Suppression” (RNAi not known at the time)

Potato"

Papaya"

Control" Transgenic"

Squash"

Anti-sense RNA (approved 1994)"Expression of an inverted DNA segment turned expression off (polygalacturonase)"

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Engineering in the Soybean Seed

RNAi and endogenous gene regulation

Two Suppression Genes"

Monounsaturate"="

Polyunsaturate"= = Omega 6"

Fatty Acid"Synthase"

Precursors"

Omega 6 Desaturase"

Omega 3 Desaturase"

Saturated"Fatty Acids"

Polyunsaturate"= = = Omega 3"

Low Lin Breeding Cross"

Soybean "Oil"

"20%"18:1"

55%"18:2"

8% 18:3"

15%"16:0 18:0"

Low Saturate"High Oleic Oil"

"74%"

17%"

2.5%"

6%"

Thioesterase"

RNAi: Topical Applications-

• Glyphosate resistant Palmer Pigweed (Amaranth) has replicated the target gene (for EPSPS protein)…. In some cases over 200 copies.

•  dsRNA directed at EPSPS restores glyphosate sensitivity!!

GR Palmer with 16-copy EPSPS at 9-Days post Rx (holes are punch samples)

Untreated Control" dsRNA only" 2X Rdup

only"dsRNA"

+ 2X Rdup"

Topically Applied dsRNA Restores Roundup Activity on Resistant Palmer

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Insect Control using dsRNA

Coleopterin…

• Corn Rootworm

Lepidopterin...

• Cotton Bollworm

Specificity- The Magic Bullet!!

Limited genetic divergence profoundly impacts efficacy!!

Western CRW!

Southern CRW!

Bachman et al, ibid.!

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Sequence divergence over 240 bp dsRNA Bachman et al, ibid.!

Safety dsRNA….. The magic bullet?

• GRAS (Generally Regarded as Safe)

• DNA and RNA a normal, digestible component of the diet.

– Food crops (grains) known to contain large amounts of dsRNA homologous to human genes with no discernible effect. (Ivashuta et al Food Chem Tox 2009)

• Readily digestible

• Multiple barriers to GI uptake (membranes, cells)

• Pharmacological activity has been impossible to achieve with oral DNA/RNA

–  In fact, very little activity can be demonstrated even with IV injection of homologous dsRNA

•  Limited environmental stability

• High degree of genus/species specificity

• Reduction in small-molecule chemical pesticides with less specificity

Review: Parrott et al Food Chem Tox 2010, 48:1773)!

Questions / Discussion…..

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0066673 http://www-mslmb.niddk.nih.gov/davies/research.html !

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What’s Buggin’ U?

Putative targets of Cry proteins

• Primary binding sites of insecticidal Bt (cry) proteins:

• Unique to insects

• Virtually no toxicity is seen in mammalian species