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Strategies for the production engineered plants resistant against fungi (resistant transgenic plants) can be achieved through the production of transgenic plants with antifungal molecules like proteins and toxins, ...


Master commun: Microbiologie appliquée-Gestion et valorisation des phytoressources, Module 'Phytopathologie et méthodes de lutte'

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Strategies for development of fungus-resistant transgenic plants


Strategies for development of fungus-resistant transgenic plants


In vitro studies have shown that chitinases have an inhibitory effect upon the growth of chitin-containing fungi. Considering its potential antifungal properties, it is possible to constitutively over-express a chitinase gene in transgenic plants resulting in enhanced resistance to one or more fungal pathogens.

Strategies for the production of fungus resistant transgenic plants can be basically classified into two categories:

1/ Production of transgenic plants with antifungal molecules like proteins and toxins (like phytoalexins).

2/ Generation of a hypersensitive response through R genes or by manipulating genes of the systemic acquired resistance (SAR) pathway.

Salicylic acid synthesis

Diseases caused by bacterial pathogens are also covered wherever appropriate as there is considerable commonality in modes of pathogenesis and plant responses in fungal and bacterial diseases.


1/ Production of transgenic plants with antifungal molecules like proteins and toxins (like phytoalexins).

Some of these proteins are: Pathogenesis-related proteins, Ribosome-inactivating proteins, Small cystein-rich proteins, Lipid transfer proteins, Storage albumins, Polygalacturonase inhibitor proteins (PGIPS), Antiviral proteins, and Non-plant antifungal proteins.

-- Pathogenesis-related proteins (PR proteins) as example:

PR proteins were shown to be induced not only by pathogens but also by wounding, fungal cell wall elicitors, ethylene, UV light, heavy metals, etc. PR proteins are induced during hypersensitive response (HR) and also during systemic acquired resistance (SAR) and therefore are thought to have a role in natural defense or resistance of plants against pathogens. PR proteins have been grouped into five families based on primary structure, serological relatedness and enzymatic and biological activities. Members of all the five PR families (PR-1 to PR-5) have been shown to have antifungal activity

---- Family of PR-1 proteins consists of low molecular weight (15–17 kDa) proteins. Their biological function is not known, nevertheless, constitutive expression of PR1A gene in tobacco enhances resistance of the plant to Peronospora tabacina12.

---- PR2 and PR3 type proteins are the fungal cell wall hydrolysing enzymes, glucanase and chitinase, respectively. These proteins can inhibit the fungal growth in vitro by causing lysis of hyphal tips.

---- Proteins of PR4 families are also low molecular weight and similar to potato win proteins. They show in vitro antifungal activity particularly in combination with other antifungal proteins.

---- PR5 proteins (thaumatin-like or AP24 or osmotin), in all probability, cause lysis of the pathogen by permeabilizing the fungal cell wall.



Livre 'Sciences de la vie. Protéines et Enzymes' (+ DVD), Baaziz, 2013: QCM corrigés, Exercices corrigés, Contrôles corrigés en Biologie cellulaire et Biochimie pour S1, S3, S4 et S5.

Protéines et Enzymes, Baaziz 2013



Transgenic plants expressing more than one PR protein genes in a constitutive manner were developed . Such transgenics showed better resistance levels than transgenics having a single gene.

RESISTANCE. TRANSGENIC PLANTS

-- Phytoalexins as an example of metabolites.

Phytoalexins are antimicrobial low molecular weight secondary metabolites produced in plants following pathogen attack and are believed to have a role in plant defense. Biosynthesis of phytoalexins is often complex involving many pathways and hence, several substrates and enzymes. Nevertheless, there has been success in developing transgenics which synthesize new phytoalexins by simply introducing the gene for the last enzyme of the pathway.

Example: introduction of the gene encoding stilbene synthase in a host plant. The expression of stilbene synthase (or resveratrol synthase) gene results in the production of resveratrol, a stilbene-type phytoalexin. Such transgenics showed enhanced resistance to many fungi.

On utilise des promoteurs de transcription.

 

transgenesis. Transformation sens

La surexpression d'un gène codant une chitinase peut conduire à une résistance accrue des plantes à des champignons pathogènes

A bean vacuolar chitinase gene under thecontrol of the strong constitutive promoter from t he cauliflower mosaic virus (CaMV)35S was transferred to tobacco and Brassica napus.

Instead of using a constitutive promoter such as 35S to ensure the expression of chitinase genes in the transgenic plants, it is possible to use tissue specific promoter to direct the location of the chitinase gene expression. Since roots are the organ of the plant that are most subject to pathogen attack, it would be very beneficial to over-express chitinase specifically in roots via a root-specific promoter.

2/ Production of transgenic plants engineered for hypersensitive response

Genes encoding antifungal proteins provide resistance to only a limited level and to only a limited number of fungi. For example, over-expressing the chitinase gene did not provide resistance against fungi lacking chitin. Moreover, a fungus can modify its cell wall by biosynthesis of more chitosan or glucan in place of chitin and, therefore, may become pathogenic again or it can evolve mechanisms to detoxify certain phytoalexins.

During the last decade more than 30 resistance genes which confer resistance against a wide range of pathogens, including viruses, bacteria, fungi, nematode and even aphids have been cloned from both monocots and dicots. Interestingly, different resistance genes are highly homologous to each other and their products are remarkably similar. All the R proteins contain leucine-rich repeat (LRR) domain, with only one exception of R protein of tomato, potato..

Other approaches to induce cell death.

One of the earliest events in incompatible plant pathogen interaction is oxidative burst during which active oxygen species such as H2O2 are produced. H2O2 triggers production of phytoalexins, PR proteins and other HR related processes.
glucose-oxidase

H2O2 also has a direct inhibitory effect on microbial growth. Glucose oxidase (GO), an enzyme occurring in some bacteria and fungi, brings about the oxidation of b-D-glucose, yielding gluconic acid and H2O2. GO has not been found in animals and plants. Expressing a GO gene from a fungus Aspergillus niger in potato showed increased level of H2O2. Such transgenics had reduced susceptibility to Erwinia carotovora subspecies carotovora (bacterium), Phytophtora infestans (fungus, mildew) and Verticillim dahliae (fungus).


Engineered plants with overexpression of peroxidase
A high level of constitutive peroxidase expression (as well as other defence-related enzymes) in a hybrid between Nicotiana glutmosu/N debneyi was found to be associated with resistance to a number of tobacco pathogens including Phytophthora parasitica var nicotiana. High levels of peroxidase activity has been used as a marker for resistance to downy mildew in muskmelon. Peroxidase enzymes can generate toxic radicals which are inhibitory to the growth of fungal pathogens in vitro.
Engineered plants with overexpression of chitinase. Towards biocontrol strategies

The plant defense system against microbial pathogens may be modified to produce high constitutive levels of antimicrobial compounds. Plant genes encoding cell wall degrading enzymes, especially chitinases, have been used to alter plant resistance to fungal pathogens, but no single genes have produced an adequate level of resistance, and almost no papers report resistance to multiple pathogens. Reasons for this may be that plant chitinases: (i) usually affect only the hyphal tip and are unable to effectively degrade harder chitin structures, (ii) have weak antifungal activity alone, (iii) are inhibitory only to a limited number of fungal species, and (iv) have no effect on several important pathogens.


Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens. Lorito et al. Proc. Natl. Acad. Sci. USA 95 (1998).

In terms of antifungal activity, chitinase genes from biocontrol fungi such as Trichoderma are clearly an improvement over corresponding plant genes. These fungal genes encode for chitinolytic enzymes that can reach the antifungal activity level of some chemical fungicides. Furthermore, extensive testing in vitro has shown that there are virtually no chitinous pathogens resistant to Trichoderma chitinases. Trichoderma spp. are fungi that are present in nearly all soils and other diverse habitats. Endochitinase gene of Trichoderma harzianum was transferred to tobacco and potato. High expression levels of the fungal gene were obtained in different plant tissues. Resistance to the foliar pathogens Alternaria alternata, A. solani, Botrytis
cinerea
, and the soilborne pathogen Rhizoctonia solani was tested.

Therefore, it is expected that the transgenic use of these enzymes should produce a high level of resistance in crop plants against a variety of fungal pathogens and, in contrast to plant genes, could be accomplished with a single fungal gene.

Bacteria against fungi. An example of biocontrol strategy

Chitinase effect on conidium
Fungi against fungi. An example of biocontrol strategy Chitinase-engineered tobacco

Contrôles: Master exam June 10, 2016 (chitinase) -------------- Contrôle S6 sur transformation génétique du tabac et de la pomme de terre par le gène de la chitinase de Trichoderma harzianum
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