Fungi, are especially destructive crop pathogens and the search for new fungal-

resistance genes forms a valuable part of plant biotechnology research. Recent

progress in approaches that exploit proteins and molecules that either
kill fungi or kill infected plant cells.

Fungi are responsible for a range of serious plant diseases such as blight, grey
mould, bunts, powdery mildew, and downy mildew.

Crops of all kinds often suffer heavy losses.

Fungal plant diseases are usually managed with applications of chemical

fungicides or heavy metals. In some cases, conventional breeding has provided
fungus resistant cultivars.

Besides combating yield losses, preventing fungal infection keeps crops free of
toxic compounds produced by some pathogenic fungi. These compounds, often
referred to as mycotoxins, can affect affect the immune system and disrupt
hormone balances. Some mycotoxins are carcinogenic.
As a defense strategy against the invading pathogens (fungi and bacteria) the
plants accumulate low molecular weight proteins which are collectively known
as pathogenesis-related (PR) proteins.

Several transgenic crop plants with increased resistance to fungal pathogens

are being raised with genes coding for the different compounds.

One of the examples is the Glucanase enzyme that degrades the cell wall
of many fungi. The most widely used glucanase is beta-1,4-glucanase.

Lysozyme degrades chitin and peptidoglycan of cell wall, and in this way fungal
infection can be reduced. Transgenic potato plants with lysozyme gene providing
resistance to Eswinia carotovora have been developed.
 Fungus resistant GM plants

Genetic engineering enables new ways of managing fungal infections.

Several approaches have been taken:

Introducing genes from other plants or bacteria encoding enzymes like

chitinase or glucanase: These enzymes break down chitin or glucan,
respectively, which are essential components of fungal cell walls.

Introducing plant genes to enhance innate plant defense mechanisms

(e.g. activing phytoalexins, proteinase inhibitors, or toxic proteins).

Invoking the hypersensitive reaction: Plants varieties that are naturally

resistant to specific types of fungal diseases are often programmed to
have individual cells quickly die at the site of fungal infection.

This response, known as the hypersensitive reaction, effectively stops

an infection in its tracks. Genetic engineering can help plant cells
'know' when a fungus is attacking.
Several new approaches have been applied to use fungal proteins like Ag-AFP,
from Aspergillus giganteus, ribosome inactivating protein (RIP), thaumatin-like
protein (PR-5) and human lyzosome in plants to combat fungal diseases.

Chitinase and AFP increase resistance in wheat.

However better results were obtained with integration of novel cDNA for
acidic chitinase and beta 1,3 glucanase as these enzymes were more effective.

Introduction of infection related chitinase and rice thaumatin like protein in rice
gave stable resistance against sheath blight.

Studies on carrot transformation with human lyzosome which can cleave beta
1, 4 glycosidic bond of bacterial cell wall and chitin in fungal cell wall is found
to be very effective against both pathogen.

Alfa-alfa antifungal peptide defensin from seeds of Medicago sativa was shown
to have significant activity against Verticillium dahliae.
The transgenic potato expressing the peptide showed to reduce area of infection.
Potato was transformed with Agrobacterium tumefaciens strain EHA101 harboring
chitinase, (ChiC) isolated from Streptomyces griseus. The transgenic plants demonstrated
enhanced resistance against the fungal pathogen Alternaria solani (causal agent of early
blight).

By introduction of ribosome inactivation protein (RIP) gene from the seeds of barley,
produced tobacco plants with enhanced resistance to the fungal pathogen R. Solani.
 HERBICIDE RESISTANCE

Excessive weed growth forces crops to compete for sunlight and

nutrients, often leading to significant losses.

Because herbicides cannot differentiate between plants that are

crops and plants that are weeds, conventional agricultural
systems can only use 'selective' herbicides. Such herbicides do
not harm the crop, but are not effective at removing all types of
weeds. If farmers use herbicide resistant crops, 'non-selective'
herbicides can be used to remove all weeds in a single, quick
application. This means less spraying, less traffic on the field,
and lower operating costs.

'Non-selective' herbicides: Not always useful

'Broad-spectrum', or non-selective herbicides are effective at killing a
wide range of weeds. The problem is, they can also kill valuable crops.
Therefore, broad-spectrum herbicides are only useful before
seedlings emerge or in special cases like fruit orchards, vineyards, and
tree nurseries.
 everal biotechnological strategies for weed control are being used e.g. the over-
roduction of herbicide target enzyme in the plant which makes the plant insensitive
o the herbicide. This is done by the introduction of a modified gene that encodes for
resistant form of the enzyme targeted by the herbicide in weeds and crop plants.
oundup Ready crop plants tolerant to herbicide-Roundup, is already being used
ommercially.

he biological manipulations using genetic engineering to develop herbicide

esistant plants are:
a) over-expression of the target protein by integrating multiple copies of the gene
or by using a strong promoter.,
b) enhancing the plant detoxification system which helps in reducing the effect of
herbicide.,
c) detoxifying the herbicide by using a foreign gene
d) modification of the target protein by mutation.

Examples of herbicide are: glyphosate, chlorsulfuron, imazapur, DL-phosphinothricin,

romoxynil, atrazine.
Glyphosate resistance - Glyphosate is a glycine derivative and is a herbicide which
is found to be effective against the 76 of the world’s worst 78 weeds. It kills the plant
by being the competitive inhibitor of the enzyme 5-enoyl-pyruvylshikimate 3-
phosphate synthase (EPSPS) in the shikimic acid pathway. Due to it’s structural
similarity with the substrate phosphoenol pyruvate, glyphosate binds more tightly
with EPSPS and thus blocks the shikimic acid pathway.

Certain strategies were used to provide glyphosate resistance to plants.

(a) It was found that EPSPS gene was overexpressed in Petunia due to gene
amplification. EPSPS gene was isolated from Petunia
and introduced in to the other plants. These plants could tolerate glyphosate at a
dose of 2- 4 times higher than that required to kill wild type plants.

(b) By using mutant EPSPS genes- A single base substitution from C to T

resulted in the change of an amino acid from proline to serine in EPSPS.
The modified enzyme cannot bind to glyphosate and thus provides resistance.
c) The detoxification of glyphosate by introducing the gene (isolated from soil
organism- Ochrobactrum anthropi) encoding for glyphosate oxidase into crop
plants. The enzyme glyphosate oxidase converts glyphosate to glyoxylate and
aminomethylphosponic acid. The transgenic plants exhibited very good glyphosate
resistance in the field.

Another example is of Phosphinothricin resistance

Phosphinothricin is a broad spectrum herbicide and is effective against broad-leafed

weeds. It acts as a competitive inhibitor
of the enzyme glutamine synthase which results in the inhibition of the enzyme
glutamine synthase and accumulation of ammonia and finally the death of the plant.
The disturbance in the glutamine synthesis also inhibits the photosynthetic activity.

The enzyme phosphinothricin acetyl transferase ( which was first observed in

Streptomyces sp in natural detoxifying mechanism against phosphinothricin)
acetylates phosphinothricin, and thus inactivates the herbicide. The gene
encoding for phosphinothricin acetyl transferase (bar gene) was introduced in
transgenic maize and oil seed rape to provide resistance against phosphinothricin.
 Herbicide resistant crops are changing weed management

Several crops have been genetically modified to be

resistant to non-selective herbicides. These transgenic crops contain genes
that enable them to degrade the active ingredient in an herbicide, rendering it
harmless. Farmers can thereby easily control weeds during the entire growing
season and have more flexibility in choosing times for spraying.

Herbicide resistant crops also facilitate low or no tillage cultural practices, which
many consider to be more sustainable. Another advantage is that farmers can
manage weeds without turning to some of the more environmentally suspect
types of herbicides.

Critics claim that in some cases, the use of herbicide resistant crops can lead to
an increase in herbicide use, promote the development of herbicide resistant
weeds, and damage biodiversity on the farm. Extensive ecological impact
assessments have been addressing these issues.
Among the field trials conducted on herbicide resistant crops, studies in the
United Kingdom have shown that different herbicides and different herbicide
application practices can affect the amount of wild plants on the farm. In
comparison with conventional cropping systems, weed and animal populations
were negatively affected by herbicide tolerant sugar beet and rapeseed, but
biodiversity was increased with the use of herbicide tolerant maize.