Genetic Engineering in Baculoviruses, Bacteria and

Entomopathogenic Fungi

Suman SanjtaA-2014-40-008ENT-611

Introduction• Insect pathogens have demonstrated to be

environmentally safe and economical alternative for the control of wide range of arthropod pests.

• But at present, less than 1% of the insecticides used worldwide for pest control are based on insect pathogens.

• Those used most widely are different subspecies of the bacterium, Bacillus thuringiensis (Bt), which constitute approximately 80% of the pathogens used as insecticides.

Limitations Although all of these products are effective when used properly, they have distinct

drawbacks which limit user acceptability. • The bacterial and viral agents must be ingested to be active, and their killing

action, especially the viruses, is slower than conventional chemical insecticides.

• These agents are also subject to rapid inactivation by exposure to sunlight and are readily washed off the foliage by rain.

• Viral products are expensive to produce since current methods require propagation in living insect larvae.

• Fungi are very intolerant of low humidity conditions or high temperature, and thus are generally used only in greenhouses or in cool climates.

Advent of genetic Engineering• The virulence and pathogenicity of pathogen is determined

by the microbial genome as a result of coordinated expression of a concert of genes.

• The acquisition of these domains or pathogenicity islands, may be sufficient to develop a transgenic virulent pathogen.

• The advent of recombinant DNA techniques—in essence, genetic engineering—has provided a myriad of opportunities to enhance the efficacy and thus cost-effectiveness of the insect pathogens as their control agents.

STEPS

1. Isolating a gene to be inserted

2. Inserting the gene in a Vector(Agent used to carry foreign gene)

3. Inserting Vector into the host.

4. Multiplication of host cells by cloning.

5. Extraction of desired product.

Baculoviruses

• These are arthropod specific viruses that infect species.

• The two genera– Nucleopolyhedrovirus

(NPV)( Multiple virions occluded in polyhedra

– Granulovirus (GV: single virions occluded in granules) .

Genetic engineering strategies:

1. Genetic Engineering to Optimize Speed of Kill

2. Genetic Engineering for Increased Virulence and modify host range.

1.Genetic Engineering to Optimize Speed of Kill

A. Gene Deletion

B. Gene Insertion

A.Gene Deletion

• EGT gene (Auxillary gene)

• Ecdysteroid UDP-glucosyl transferase (EGT), renders the ecdysteroids inactive, blocks molting of the host insect, thereby prolonging the actively feeding larval stage.

• Deletion of egt from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome resulted in more rapid death and an approximately 40% reduction in feeding damage caused by infected larvae of Trichoplusia ni and Spodoptera frugiperda compared to those infected with wild type AcMNPV. (O,Reilly and Miller, 1991)

(Han et al., 2015)

• Deletion of the gene encoding the polyhedral envelope protein that surrounds the OB of AcMNPV resulted in a 6-fold increase in infectivity against first instar Trichoplusia ni compared to that of wild type virus.

B.Gene Insertion• Insertion of a gene encoding a toxin, hormone or enzyme into

the baculovirus genome. • Several recombinant baculoviruses have been constructed for

overexpression of the host insect’s own hormones or enzymes such as diuretic hormone, eclosion hormone, prothoracicotrophic hormone and juvenile hormone esterase.

• A wide range of genes encoding insect-specific toxins isolated from various venomous creatures such as scorpions, spiders, parasitic wasps and sea anemones have been inserted into baculovirus genomes.

• Insertion of Diuretic hormone gene from Manduca sexta resulted in 20% increase in the insecticidal activity of a recombinant Bombax mori NPV. (Maeda, 1989)

• The insect selective toxin(LqhIT2) from yellow Israeli scorpion Leiurus quinquestriatus was inserted in HzSNPV for the control of Helicoverpa zea. (DuPont, 1996)

• The toxin from scorpion Androctonus australis was inserted in AcMNPV for the control of Helicoverpa zea. (Black et. Al., 1997)

• Another paralytic toxin that holds promise is the TxP I toxin, a component of the venom of ‐the predatory straw itch mite Pyemotes tritici.

• Korth and Levings (1993), inserted a toxin URF 13 from maize to AcMNPV. When the larvae of Trichoplusia ni were injected with this virus, all died by 60h postinjection.

• Two insect selective toxins ASII and Sh 1 from the sea anemones Anemonia sulcata and Stichadactyla helianthus resulted in 38% and 36% improvements in speed of kill in T. ni and S. frugiperda larvae.( Hughes et al., 1997)

• The expression of insect selective spider toxins µ-Aga-IV from Agelenopsis sperta and DTX9.2 and Ta1TX-1 from the spiders Diguetia canities and Tegenaria agrestis resulted in improved speeds of kill. (Prikhodko et al.,1996 , Hughes et al., 1997)

• Targeting basement membrane: Expression of Cathepsin L protease from flesh flies resulted in significant decrease in the survival time in the larvae of Autographa californica infected with AcMNPV. (Harrison and Bonning, 2012)

• One of the common factors associated with genetic optimization for increased speed of kill, is that the faster the virus kills the host insect, the fewer OB are produced . Hence, large scale production of these recombinant baculoviruses in vivo becomes a challenge

2. Genetic Engineering for Increased Virulence

• There are several examples of baculoviruses that have been genetically engineered to reduce the amount of virus required for a fatal infection of the targeted insect pest. Enhancin is a metalloprotease commonly expressed by baculoviruses that degrades insect intestinal mucin in the peritrophic membrane.

• Insertion of the enhancin gene derived from Trichoplusia ni GV enhanced AcMNPV virulence by 2 to 14-fold in various insect species.

• Conversely, deletion of two enhancin genes from Lymantria dispar MNPV reduced viral potency 12-fold compared to wild type virus.

• AcMNPV expressing an algal virus pyrimidine dimer-specifi c glycosylase, cv-PDG, is less susceptible to UV inactivation, signifi cantly increased the virulence to kill S. frugiperda larvae bu16-fold.

Bacteria

• Bacillus thuringiensis (Bt) has been the most successful commercial microbial insecticide and also has been the subject of the overwhelming majority of genetic engineering studies to improve efficacy.

• Bts are characterized by the production of a parasporal body during sporulation that contains one or more protein endotoxins in a crystalline form

The immediate challenge for genetic engineering of bacteria is to:

1. increase the potency of the toxin(s), 2. broaden the activity spectrum,3. improve the persistence under field

conditions, and 4. reduce the production costs.

• The cryIAc gene from Bacillus thuringiensis was integrated into Pseudomonas fluorescens P303-1 by electroporation and the engineered bacteria were highly insecticidal to cotton bollworm, H. armigera.

(Duan et al., 2002),

• A toxin gene from B. thuringiensis subsp. israelensis inserted into Bradyrhizobium species that fix nitrogen in nodules of pigeonpea.

• Experiments in a greenhouse indicated that this provided protection against root nodule damage by larvae of Rivellia angulata

Nambiar, Ma, and Iyer (1990)

• Bacillus thuringiensis subsp israelensis expressing the binary toxin gene from B. sphaericus showed high toxicity against different species of mosquitoes.

(Yuan et al.,1999)

• The mosquitocidal proteins from three different species; Bin from Bacillus sphaericus 2362, Cry11B—a protein B. thuringiensis subsp. jegathesan and Cyt1A from Bt subspp israelensis.

• The resulting recombinant B. thuringiensis produced three distinct crystals and was 3 to 5 times as toxic to Culex species as either Bti IPS-82 or Bs 2362

• IPS-82 strain of Bti, which produces the complement of toxins characteristic of this species, was transformed with pPHSP-1, the pcyt1A/STAB plasmid that produces a high level of Bs Bin toxin. This recombinant was more than ten-fold more toxic than either of the parental strains to larvae of Cx. quinquefasciatus and Cx. tarsalis.

• The B. thuringiensis crystal genes have been introduced into E. coli, B. subtilis, B. megaterium, and P. fluorescens and form biopesticide formulations consisting of encapsulated Cry inclusions . These encapsulated forms of the Cry proteins have shown improved persistence in the environment.

(Gawron-Burke and Baum, 1991)

• Cry1Aa gene from B thuringiensis subspp kurstaki HD1 was inserted into maize root colonizer Pseudomonas flourescence. Recombinant strains were stable under environmental conditions and gave 100% mortality against Manduca sexta.

(Obukowicz et al., 1986)

• The cry1Aa1 gene encoding insecticidal crystal protein (ICP) was transferred into three isolates (Eh4, Eh5, and Eh6) of, Erwinia herbicola (Lohnis).

• The transformed E. herbicola strains expressed the toxin protein and conferred insecticidal activity and resulted in 94 to 100% mortality of diamondback moth, P. xylostella.

Lin et al. (2002)

Entomopathogenic fungi

• Insect pathogenic fungi are key regulatory factors in insect pest populations.

• Most attention has focused on the ascomycetes Metarhizium anisopliae and Beauveria bassiana.

• The major drawbacks associated with fungal pesticides include relative instability, requirement for moist conditions for spore germination, invasion, and growth, and slow rates of mortality.

• Paecilomyces fumosoroseus and P. lilacinus have been transformed using a Benomyl-resistant b-tubulin gene from Neurospora crassa .

• Benomyl-resistant transformants of P. lilacinus were obtained that could tolerate greater than 30 µg/ml benomyl and P. fumosoroseus transformants were obtained that could tolerate 20 µg/ml benomyl.

(Inglis et al., 1999)

• Bernier et al. (1989) introduced benomyl resistance (beta-tubulin) gene from Neurospora crassa (encoding resistance to benomyl) into M. anisopliae. The transformants were mitotically stable when subcultured on nonselective agar and retained the ability to infect and kill larvae of M. sexta.

• A hybrid chitinase containing the chitin binding domain from the silkworm Bombyx mori chitinase fused to the B. bassiana chitinase showed the greatest ability to bind to chitin.

• Constitutive expression of this hybrid chitinase gene by B. bassiana reduced time to death of insects by 23% compared to the wild-type fungus.

Fan et al. (2007)

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