APPLICATION OF PLANT TRANSFORMATION FOR

PRODUCTIVITY AND PERFORMANCEBY ASHIKA RAVEENDRAN 2ND Msc BIOTECHNOLOGY

Fungi are extremely diverse group of organisms , which can grow in any surface.

Fungi are proficient at colonizing and using plants, humans and animals as substrate.

The fungal cell wall protects the organisms and relays signals for invasion and infection of a plant, animal or a human host.

ANTIFUNGAL PROTEINS

Plants are exposed to large number of pathogenic fungi

Although plants do not have an immune system, but they have a defense mechanism including antifungal activity

There are many of antifungal peptides and proteins known, with more being discovered almost daily

At least 14 families of PR proteins are recognized. The better known PR proteins are

PR1 proteins (antioomycete and antifungal), PR2 (b-1,3-glucanases), PR3 (chitinases), PR4 proteins (chitin binding), PR 5,

CLASSES OF ANTIFUNGAL PROTEINS

Proteinase inhibitors Defensins Cyclophilin like proteins, Glycine histidine rich proteins, RIPS LTPs Killer proteins

There are often numerous isoforms of each PR protein in various host plants.

Although healthy plants may contain trace amounts of several PR proteins, attack by pathogens, treatment with elicitors, wounding, or stress induce transcription of a battery of genes that code for PR proteins.

Plants exposed to pathogens such as fungi and viruses produce low molecular weight antimicrobial compounds called phytoalexins.

These plant proteins called pathogenesis related (PR) proteins , are divided into 5 groups, PR-1,-2,-3,-4,-5 based on serological and amino acid sequence analyses.

Recently, another 6 groups of proteins have been suggested for inclusion as PR proteins, bringing the total to 11 groups

PR PROTEINS

Pathogenesis-related proteins, often called PR proteins, are a structurally diverse group of plant proteins that are toxic to invading fungal pathogens.

They are widely distributed in plants in trace amounts, but are produced in much greater concentration in pathogen attack or stress.

Varying types of PR proteins have been isolated from each of several crop plants.

Different plant organs, e.g., leaves, seeds, and roots, may produce different sets of PR proteins.

The several groups of PR proteins have been classified according to their function, serological relationship, amino acid sequence, molecular weight, and certain other properties.

PR proteins are either extremely acidic or extremely basic and are highly soluble and reactive.

PR-1proteins have molecular massesof 15to 17 kDa

PR-1 proteins have been found in rice, wheat, maize, tobacco, Arabidopsis thaliana, barley, and many other plants.

PR-1 proteins have antifungal against a number of plant pathogenic fungi, including Uromyces fabae, and Phytophthora infestans

PR-1 PROTEINS

PR-2 have(1,3) beta glucanase activity in vitro & are grouped into 3 classes on the basis of aminoacid sequence analysis :

ClassI glucanasesare of 33 kDa Classes II and III are of about 36 kDa.

PR-2 proteins have been found in a wide variety of plants, including tobacco, A. thaliana, peas, grains, and fruits .

The proteins are active against pathogens such as

Rhizoctonia solani, C. albicans and Aspergillus fumigatus. PR-2 proteins hydrolyzes the structural (1,3)b- glucan

present in the fungal cell wall, resulting in a weak cell wall. This weakened cell wall results in cell lysis and cell death.

PR-2 PROTEINS

PR-3 proteins have molecular masses between 26 and 43 kDa

They have been divided into five groups.

Chitinases have been isolated from tobacco , cucumber, beans , peas, grains , and many others,

They have potent antifungal activity against Trichoderma reesei, Alternaria , Fusarium oxysporum etc

PR-3 proteins cleave cell wall chitin polymers, resulting in a weakened cell wall and rendering fungal cells osmotically sensitive.

PR-3 PROTEINS(chitinases).

PR-4 proteins are chitin-binding proteins, have molecular masses of 13-14.5kDa, and have been classified into two groups .

PR-4 proteins have been isolated from potato, tobacco, barley, tomato, and many other plants .

These proteins have potent antifungal activity against Trichoderma harzianum and Fusarium culmorum.

The antifungal activity these proteins is by binding to fungal cell wall b-chitin. This results in disrupted cell polarity and inhibition of growth.

PR-4 (chitin-binding) proteins.

PR-5 proteins are known as TL proteins (thaumatin like proteins).

TL proteins have been isolated from A. thaliana , corn, soybeans, rice, wheat, tobacco, tomato , pumpkin, beans and barley .

The majority of PR-5 proteins have molecular masses of 22 kDa and are stabilized by eight disulfide bonds.

These proteins they are fungicidal against a wide no: of plant pathogens

PR-5 PROTEINS

Defensins are low molecular mass cysteine rich proteins found in mammals, fungi ,insects & plants.

The treatment of hyphae with defensins from radish & dhalia cause k+ efflux & Ca+ uptake through binding to specific cell membrane receptors.

Defensins have the ability to inhibit pathogens including F.solani and C.albicans.

DEFENSINS

RIPs are RNA N- glucosidase that depurinates Rrna, result in arrest of protein synthesis due to ribosome damage.

RIPs have been isolated from Pisum sativum.

CYCLOPHILIN LIKE PROTEINS

Cyclophilins are conserved group of proteins – that are intracellular receptors for cyclosporin & is found in bacteria, plant, animal,fungi

RIPs

Proteins secreted by yeast that are sensitive to fungal cells.

Killer proteins can disrupt cell wall synthesis ,DNA synthesis and k+ channel activity – any of these effect leads to inhibition of fungal cell growth

KILLER PROTEINS

Protein inhibitors of serine & cysteine protease have emerged as antifungal proteins that have potent activity against plant & animal pathogens.

LTPs LTPs are small proteins(8.7kDa) of 90 amino acids

stabilized by four disulfide bonds.

PROTEIN INHIBITORS

Thionins are a family of small proteins present in the endosperm of wheat and related species

A thionin consists of 45–48 amino acid residues. 6–8 of these are cysteine forming 3–4 disulfide bonds

Thionins are mainly found in seeds where they may act as a defence against consumption by animals.

THIONINS

SYNTHESIS AND DEPOSITION:Endosperm thionins: Accumulation in developing barely endosperm. synthesis takes place in membrane bound

polysomes.

Leaf thionins: The mRNA leaf thionins when translated yield

precursors similar to endosperm thionins.

Toxicity.

Alteration in membrane permeability.

Inhibitory properties on RNA,DNA and protein synthesis.

In plant pathogen interaction.

BIOLOGICAL ACTIVITIES

Thionin genes are targets for disease resistance in plants.

Tomato plant carrying gene encoding wheat and barely thionins with different promoters

THIONINS IN PLANT BREEDING

Parasitic nematodes infect thousands of plant species but some plants harbor specific resistance genes that defend against these pests.

Several nematode resistance genes have been cloned in plants, and most resemble other plant resistance genes

Nematode resistance is generally characterized by host plant cell death near or at the feeding site of the endoparasitic worm.

The timing and localization of the resistance response varies with the particular resistance gene and nematode interaction.

Nematode resistance

Plant parasitic nematodes feed on living plant tissues. All have some form of oral stylet or spear, which is used somewhat like a hypodermic needle to puncture the host cell wall.

Many plant nematodes inject enzymes into the host cell before feeding.

These enzymes partially digest the cell contents before they are sucked into the gut. Most of the injury that nematodes cause to plants is related in some way to the feeding process.

Nematodes may feed on plant tissues from outside the plant (ectoparasitic) or inside the tissues (endoparasitic).

If the adult female moves freely through the soil or plant tissues, the species is said to be "migratory.“

Species in which the adult females become swollen and permanently immobile in one place in or on a root are termed "sedentary".

NEMATODE RESISTANCE

These soil-dwelling pests parasitize plant roots, taking up the nutritional resources of their hosts while evading or suppressing host defenses.

Although nematicides have been successfully used to control nematodes, host resistance is a preferable alternative because of the expense and environmental toxicity of nematicides.

To protect themselves from invading pathogens, hosts have evolved defense mechanisms including chemical and physical barriers and a highly specialized resistance (R) gene-mediated defense

In recent years, it has been revealed that plants use similar R-gene- based resistance mechanisms to protect themselves from nematodes.

POTATO CYST

NEMATODE LIFE CYCLE

ROOT KNOT NEMATODE DISEASE CYCLE

ROOT KNOT IN TOMATO

Several nematode resistance (Nem-R) genes have been isolated from plants, all conferring resistance against sedentary endoparasites.

The first nematode resistance gene to be cloned was Hs1pro-1 from sugar beet , which confers resistance against the sugar beet cyst nematode. The encoded protein does not have obvious similarities to known plant genes.

Other cloned Nem-R genes closely resemble known plant

R-genes in their domain structure. Four of these genes, Mi-1, Hero A, Gpa2 and Gro1-4, all cloned from tomato or potato relatives.

NEMATODE RESISTANCE GENE

The tomato genes Mi-1 and Hero A confer broad-spectrum resistance against several root knot nematode species and against several pathotypes of two potato cyst nematode species.

By contrast , the potato genes Gpa2 and Gro1-4confer resistance to a narrow range of pathotypes of a single potato cyst nematode species

Resistance to nematodes in plants is generally characterized by failure of the nematodes to produce functional feeding sites in the host after invasion and to develop subsequently as reproducing females.

In nematode-resistant tomatoes, neither the feeding site nor the nematodes develop.

For example, Mi-1 mediated resistance is characterized by a rapid localized cell death that occurs near the anterior end of the nematode in the region of the root where feeding site initiation occurs

RESISTANCE PHENOTYPES

However, for other interactions such as Hero A-mediated resistance, the response seems to be initiated after feeding site induction and leads to atrophy or abnormal development of the feeding site .

The nematodes that do develop are mostly males, resulting in severe reduction in reproduction of the invading nematodes.

Resistance coupled with a high ratio of males to females is commonly observed against cyst-forming nematodes.

Another example, the pepper genes Me1 and Me3 each control the main root-knot nematode species that affect this plant.

Function of Nem-R-genes in heterologous plants: A practical goal of isolating Nem-R genes is to

transfer these genes to economically important crop plants where resistance is not available.

Transgene-based resistance against severely damaging nematodes will have a clear benefit of reduced use of highly toxic nematicides.

So far intraspecific transfer of Nem-R genes by transgenic techniques has been successful, but there has been limited success in transfering these genes to new species.

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