Detoxification of fusarium fusarium toxins in toxins in - - PowerPoint PPT Presentation

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Detoxification of Detoxification of fusarium fusarium toxins in toxins in transgenic crop plants transgenic crop plants

RAKESH KHADKA M.Sc. Agrobiotechnology

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Lay out Lay out

General introduction General introduction

Fusarium Fusarium spp. spp. Mycotoxins Mycotoxins

Chemical Names of major Chemical Names of major Fusarium Fusarium Mycotoxins Mycotoxins Importance Importance

Detoxification of mycotoxins Detoxification of mycotoxins Methods for identifying organisms capable of degrading Methods for identifying organisms capable of degrading mycotoxins. mycotoxins. Case Study Case Study-

• on Detoxification of Fusarium Toxin
• n Detoxification of Fusarium Toxin

Fusarium Fusarium trichothecenes trichothecenes biosynthesis pathway biosynthesis pathway Transformation of Transformation of Tri101 Tri101 Gene Gene in Rice. in Rice. Detection of in vitro trichothecene 3 Detection of in vitro trichothecene 3-

• O

O-

• acetyltransferase activity in

acetyltransferase activity in transgenic lines. transgenic lines.

Discussion Discussion Reference Reference

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Fusarium Fusarium spp. spp.

Taxonomic Classification Taxonomic Classification

Kingdom: Fungi Kingdom: Fungi Phylum: Ascomycota Phylum: Ascomycota Order: Hypocreales Order: Hypocreales Family: Hypocreaceae Family: Hypocreaceae Genus: Genus: Fusarium Fusarium

Contains over 20 species. Contains over 20 species. Filamentous fungus widely distributed on plants and in the soil. Filamentous fungus widely distributed on plants and in the soil. Fusarium species are toxigenic. Fusarium species are toxigenic. Common contaminant and a well Common contaminant and a well-

• known plant pathogen causing

known plant pathogen causing destructive and agriculturally important diseases of small grain destructive and agriculturally important diseases of small grain, , cereals and maize. cereals and maize. Mycotoxins produced are often associated with animal and human Mycotoxins produced are often associated with animal and human diseases diseases.

.

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Mycotoxins in General: Mycotoxins in General:

Mycotoxins are the toxic chemicals produced by fungi for a varie Mycotoxins are the toxic chemicals produced by fungi for a variety of ty of reasons. reasons.

1. 1. To attack or gain access to hosts by helping to dissolve cell me To attack or gain access to hosts by helping to dissolve cell membranes. mbranes. 2. 2. For as protective measures against encroaching organisms. For as protective measures against encroaching organisms.

Mycotoxins, such as Mycotoxins, such as Mycotoxin T2 (Fusariotoxin) Mycotoxin T2 (Fusariotoxin) or the Amanita

• r the Amanita-
• toxins can be lethal to animals.

toxins can be lethal to animals. Most Most-

• studied mycotoxins in Fusarium are toxic to both plants and

studied mycotoxins in Fusarium are toxic to both plants and

• animals. including hemorrhagic, estrogenic, emetic, and feed ref
• animals. including hemorrhagic, estrogenic, emetic, and feed refusal

usal syndromes, fescue foot, degnala disease etc etc. syndromes, fescue foot, degnala disease etc etc. Disease caused by fusarium toxin do not only severely reduce Disease caused by fusarium toxin do not only severely reduce yield,but also results in contamination of grain with unacceptab yield,but also results in contamination of grain with unacceptable high le high amounts of mycotoxins, a problem of world wide significance. amounts of mycotoxins, a problem of world wide significance.

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Chemical Names of major Chemical Names of major Fusarium Fusarium Mycotoxins; Marasas Mycotoxins; Marasas et al. et al.

Some of the names are redundant, and some are Some of the names are redundant, and some are the result of research in different countries. the result of research in different countries.

Deoxynivalenol Deoxynivalenol Fumonisin B1 Fumonisin B1 Moniliformin Moniliformin Neosolaniol Neosolaniol Nivalenol Nivalenol Scirpentriol Scirpentriol Vomitoxin Vomitoxin Zearalenol Zearalenol Zearalenone Zearalenone Trichothecenes Trichothecenes

Fusarium mycotoxins may leach into the soil, causing damage to plants and animals through leaching even after the fungus is no longer active.

Head blight of wheat caused by Fusarium graminearum

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Importance Importance

Fungal diseases are common problems in crop agriculture. Fungal diseases are common problems in crop agriculture. Mycotoxins, such as those produced by fusaria fungi for instance Mycotoxins, such as those produced by fusaria fungi for instance, are in fact a major , are in fact a major food safety issue. food safety issue. Many strides have been made against plant diseases as exemplifie Many strides have been made against plant diseases as exemplified by the use of d by the use of hybrid plants, pesticides and improved agricultural practices. hybrid plants, pesticides and improved agricultural practices. However, the problems of fungal plant disease continue to cause However, the problems of fungal plant disease continue to cause difficulties in plant difficulties in plant cultivation. cultivation. Thus, there is a continuing need for new methods and materials f Thus, there is a continuing need for new methods and materials for solving the

• r solving the

problems problems.

.

These problems can be met through a variety of approaches: These problems can be met through a variety of approaches:

1. 1. the infectious organisms can be controlled through the use of ag the infectious organisms can be controlled through the use of agents that are selectively ents that are selectively biocidal for the pathogens. biocidal for the pathogens. 2. 2. interference with the mechanism by which the pathogen invades th interference with the mechanism by which the pathogen invades the host crop plant. e host crop plant. 3. 3. interference with the mechanism by which the pathogen causes inj interference with the mechanism by which the pathogen causes injury to the host crop plant. ury to the host crop plant. 4. 4. is interference with toxin production, storage, or activity. is interference with toxin production, storage, or activity.

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Detoxification of mycotoxins Detoxification of mycotoxins

Although reducing fungal infections is the most desirable method Although reducing fungal infections is the most desirable method of

• f

eliminating mycotoxins. eliminating mycotoxins. Cultural practices and genetic approaches have achieved limited Cultural practices and genetic approaches have achieved limited success in disease control in terms of effectiveness and cost. success in disease control in terms of effectiveness and cost. Efforts to produce GM crops with enhanced disease resistance hav Efforts to produce GM crops with enhanced disease resistance have e not been successful on a practical level. not been successful on a practical level. An alternative solution, transgene An alternative solution, transgene-

• mediated detoxification of mycotoxins

mediated detoxification of mycotoxins has been proposed. has been proposed. A prerequisite for this is the availability of genes encoding en A prerequisite for this is the availability of genes encoding enzymes with zymes with detoxification activities in plants by the deployment of anti detoxification activities in plants by the deployment of anti-

• mycotoxin

mycotoxin antibodies (plantibodies) or mycotoxin antibodies (plantibodies) or mycotoxin-

• degrading enzymes.

degrading enzymes.

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Methods for identifying organisms capable of Methods for identifying organisms capable of degrading mycotoxins. degrading mycotoxins.

Mycotoxin is incorporated into culture medium for selection of o Mycotoxin is incorporated into culture medium for selection of organisms. rganisms. These organisms are used to isolate the enzyme and the gene resp These organisms are used to isolate the enzyme and the gene responsible for

• nsible for

conferring mycotoxin conferring mycotoxin-

• resistance.

resistance. The gene is cloned and inserted into a suitable expression vecto The gene is cloned and inserted into a suitable expression vector for the r for the further characterization of protein. further characterization of protein. The DNA encoding for mycotoxin The DNA encoding for mycotoxin-

• resistance can be used to transform plant

resistance can be used to transform plant cells normally susceptible to Fusarium or other toxin cells normally susceptible to Fusarium or other toxin-

• producing fungus

producing fungus infection. infection. Plants can be regenerated from the transformed plant cells. Plants can be regenerated from the transformed plant cells. A transgenic plant can be produced with the capability of degrad A transgenic plant can be produced with the capability of degrading mycotoxin, ing mycotoxin, as well as with the capability of producing the degrading enzyme as well as with the capability of producing the degrading enzymes. s.

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Case Study Case Study-

• on Detoxification of Fusarium
• n Detoxification of Fusarium

Toxin Toxin

The necrotrophic fungal pathogens The necrotrophic fungal pathogens Fusarium graminearum Fusarium graminearum and and Fusarium culmorum Fusarium culmorum cause Fusarium head blight (FHB). cause Fusarium head blight (FHB). A loss of yield. A loss of yield. Serious threats to humans and animals by contaminating Serious threats to humans and animals by contaminating grains with the tri-chothecene mycotoxin deoxynivalenol grains with the tri-chothecene mycotoxin deoxynivalenol (DON). (DON). DON inhibits protein synthesis in eukaryotes. DON inhibits protein synthesis in eukaryotes. Stimulates the development of plant diseases as a Stimulates the development of plant diseases as a phytotoxin presumably by interfering with the expression of phytotoxin presumably by interfering with the expression of defense defense-

• related genes

related genes. . Fusarium Fusarium species have species have Tri101 Tri101 gene gene for self protection. for self protection. Tri101 Tri101 gene gene encodes trichothecene 3 encodes trichothecene 3-

• O

O-

• acetyltransferase.

acetyltransferase.

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Fusarium Fusarium trichothecenes trichothecenes biosynthesis pathway biosynthesis pathway

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Transformation of Transformation of Tri101 Tri101 Gene Gene in Rice. in Rice.

Total DNA and RNA were extracted from leaves using a Nu-cleon Ph Total DNA and RNA were extracted from leaves using a Nu-cleon PhytoPure plant ytoPure plant and fungal DNA extraction kit and RNeasy Plant Mini kit. and fungal DNA extraction kit and RNeasy Plant Mini kit. DNA and RNA probes were prepared using a PCR DIG probe synthesis DNA and RNA probes were prepared using a PCR DIG probe synthesis kit and a kit and a DIG RNA labeling kit. DIG RNA labeling kit.

Construction of pEU Construction of pEU-

• Tri101

Tri101

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Transformation of Transformation of Tri101 Tri101 Gene Gene in Rice. in Rice.

Mature seeds of japonica rice, Mature seeds of japonica rice, Oryza sativa Oryza sativa L. cv. Nip-ponbare, were used

• L. cv. Nip-ponbare, were used

for callus induction. for callus induction. Scutellum tissues were excised from a 1 Scutellum tissues were excised from a 1-

• week culture on LS medium

week culture on LS medium solidi solidifi fied by 0.25% gellan gum containing 2 mg/l of 2,4 ed by 0.25% gellan gum containing 2 mg/l of 2,4-

• dichlorophnoxyacetic acid (2,4

dichlorophnoxyacetic acid (2,4-

• D).

D). Bombarded with plasmid Bombarded with plasmid-

• coated gold particles (0.6

coated gold particles (0.6 µ µm). m). pAct1 pAct1-

• gfbsd1

gfbsd1 containing an enhanced green containing an enhanced green fl fluorescence protein gene uorescence protein gene ( (egfp egfp) fused to the blasticidin S (BS) resistance gene ( ) fused to the blasticidin S (BS) resistance gene (bsd bsd) ) was used as a was used as a transformation vector. transformation vector. Plantlets were regenerated from transgenic calli and grown in a Plantlets were regenerated from transgenic calli and grown in a greenhouse greenhouse under natural light. under natural light.

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Detection of in vitro trichothecene 3 Detection of in vitro trichothecene 3-

• O

O-

• acetyltransferase activity in transgenic lines

acetyltransferase activity in transgenic lines

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Detection of in vitro trichothecene 3 Detection of in vitro trichothecene 3-

• O

O-

• acetyltransferase activity in transgenic

acetyltransferase activity in transgenic lines lines

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Discussion Discussion

Fungal diseases are common problems in crop agriculture. Many st Fungal diseases are common problems in crop agriculture. Many strides have rides have been made against plant diseases as exemplified by the use of hy been made against plant diseases as exemplified by the use of hybrid plants, brid plants, pesticides and improved agricultural practices. pesticides and improved agricultural practices. The problems of fungal plant disease continue to cause difficult The problems of fungal plant disease continue to cause difficulties in plant ies in plant cultivation. cultivation. There is a continuing need for new methods and materials for so There is a continuing need for new methods and materials for solving the lving the problems problems . . Efforts to produce GM crops with enhanced disease resistance hav Efforts to produce GM crops with enhanced disease resistance have not been e not been successful on a practical level. successful on a practical level. An alternative solution, transgene An alternative solution, transgene-

• mediated detoxification of mycotoxins has

mediated detoxification of mycotoxins has been effective to control somewhat.. been effective to control somewhat..

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References References

Bruins MBM, Bruins MBM, Karsai Karsai I, I, Schepers Schepers J, J, Snijders Snijders CHA (1993) CHA (1993) Phyto-toxicity Phyto-toxicity of

• f

deoxynivalenol to wheat tissue with regard to in vitro selection deoxynivalenol to wheat tissue with regard to in vitro selection for Fusarium for Fusarium head blight resistance. Plant head blight resistance. Plant Sci Sci 94:195 94:195 Dahleen Dahleen LS, McCormick SP (2001) Trichothecene toxin effects on barley LS, McCormick SP (2001) Trichothecene toxin effects on barley callus and seedling growth. Cereal callus and seedling growth. Cereal Res Res Commun Commun 29:115 29:115– – 120 120 Eudes Eudes F, F, Comeau Comeau A, A, Rioux Rioux S, Collin J (2000) S, Collin J (2000) Phytotoxicity Phytotoxicity of eight

• f eight mycotoxins

mycotoxins associated with Fusarium in wheat head blight. Can J Plant associated with Fusarium in wheat head blight. Can J Plant Pathol Pathol 22:286 22:286– –292 292 Kimura M, Kaneko I, Komiyama M, Kimura M, Kaneko I, Komiyama M, Takatsuki Takatsuki A, A, Koshino Koshino H, H, Yoneyama Yoneyama K, K, Yamaguchi I (1998) Trichothecene 3 Yamaguchi I (1998) Trichothecene 3-

• O

O-

• acetyltransferase protects both the

acetyltransferase protects both the producing organism and trans-formed yeast from related producing organism and trans-formed yeast from related mycotoxins

• mycotoxins. Cloning

. Cloning and characteriza-tion of and characteriza-tion of Tri101

• Tri101. J

. J Biol Biol Chem Chem 273:1654 273:1654– –1661 1661 McCormick SP, Alexander NJ, Trapp SE, McCormick SP, Alexander NJ, Trapp SE, Hohn Hohn TM (1999) Dis-ruption of TM (1999) Dis-ruption of TRI101 TRI101, the gene encoding trichothecene 3 , the gene encoding trichothecene 3-

• O

O-

• acetyltransferase, from

acetyltransferase, from Fusarium Fusarium sporotrichioides sporotrichioides. . Appl Appl Environ Environ Microbiol Microbiol 65:5252 65:5252– –5256 5256 Zamir Zamir LO, LO, Nikolakakis Nikolakakis A, A, Devor Devor KA, KA, Sauriol Sauriol F (1996) Biosynthesis of the F (1996) Biosynthesis of the trichothecene 3 trichothecene 3-

• acetyldeoxynivalenol.
• acetyldeoxynivalenol. Is isotrichodermin a biosynthetic

Is isotrichodermin a biosynthetic precursor? J Biol Chem 271:27353 precursor? J Biol Chem 271:27353– –27359 27359

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