Chapter 2

Plant transformation

Plant transformation:It is the production of a plant containing a new gene (foreign gene) or a DNA sequence which is added to the plant genome and this sequence must be stable and can be inherited with the rest of the plant genomeThere are many ways to perform plant transformation but all of them must follow the same main steps:

1- Preparing the plant tissue needed to be transformed.2- Inserting the foreign DNA to the plant tissue3- Screening cells and selecting the transformed ones4- The production of a whole plant using tissue culture5- Examining the produced plant and studying the ability of the transformed character to be transferred successfully from one generation to another

1- Preparing the plant tissue needed to be transformed:-Not all tissues or plant cells are equal in their ability to accept a new DNA.-The selected plant tissue must has the ability to regenerate and differentiate to a full plant.-For the transformation to occur the plant tissue must be wounded, and the response of the different plant to wounds differ (Dicotyledonous plants when wounded a callus tissues are formed at the site of injury and this callus can be transformed, while monocotyledonous plants when wounded a site of dead cells are formed around the site of injury which cannot be transformed)That’s why Embryogenic tissues are used when monocot. Plants are needed to be transformed

Examples for the tissues used in plant transformation:- Protoplast: which is a plant cell without its cell wall,

transformation of a nude cell is much easier than cells with cell wall.

- Leaf disks: leaf disks are taken from the mother plant and the main idea of taking the disks is to injure the edges of the leaf disks.

- Cotyledon sections: Used in Soybean- Embryogenic tissues: It is used in transforming monocot.

Plants.

2- Inserting the foreign DNA to the plant tissue:1- Direct DNA insertion: Plant protoplasts are incubated with the foreign DNA in the presence of Polyethyleneglycol or using Electroporation, both will cause the increase in permeability of the plasma membrane allowing the DNA to pass through it to the inside of the cell.

2- Microinjection: The nucleus or the cell is injected under microscope with the foreign DNA. Performing this technique is very difficult and takes a long time to inject a number of cells but one of its benefits is the ability of the micro-injector to penetrate the cell wall.

3- The Gene gun or microprojectile: The easiest way to perform transformation to a plant tissue, its idea is built on the fast movement of DNA coated golden particles toward the target tissues, the gold particles will penetrate the cells causing transformation.

4- Agrobacterium mediated transformation : Agrobacterium tumefaciens is a bacteria abundant in the soil related to family Rhizobium this becteria causes the formation of tumors on the stem at the soil surface those tumors are called Crawn galls.

The main cause of the tumor is the transfer of a DNA sequence from the bacteria to the plant tissues, this sequence has the ability to combine with the plant DNA and express its characters.

-The Agrobacterium contains a large plasmid called the Ti plasmid (Tumor inducing), this plasmid contains genes which interfere with the plant hormones production. The bacteria infects the plant through wounds at the soil surface.-The bacterial genes cause an abnormal growth and the formation of tumors at the site of injury. -The tumor cells can grow on a medium free of cytokinins and auxins.- This means that the infected cells had been naturally transformed.

The Ti plasmid consists of many regions:1- T-DNA or (Transferred DNA) which is the region that is transferred to the plant genome, this region is confined between 2 DNA sequences, one from the left and is called the left border L and one to the right and is called the right border R. (any sequence between the L and the R can be transferred to the plant genome)

-In the wild type of the bacteria the T-DNA contains Opine gene which is responsible for the catabolism of Octopine and Nopaline and use them as a source of N and carbohydrates also this region contains the gene responsible for the production of auxin and cytokinins (Oncogenes)

2- Origin of replication3- Virulence region: which is responsible for the cause of infection

-Scientists were able to remove the genes responsible for the production of auxins and cytokinins aldo the genes responsible for the Opine catabolism and a Binary plasmid was formed.- A marker gene (mostly antibiotic resistance gene) and the foreign gene can be added to the T-DNA by the use of restriction and ligating enzymes.

Mechanism of Agrobacterium infection:First if the plant is injured at the soil surface phenolic exudates are excreted from the wound in order to protect the wound from external infection. The phenolic compounds are the main trigger that activates the virulence genes ( VirA, VirB, VirC, VirD, VirE, VirF, VirG) at the virulence region, The virulence genes cause the formation of a protein membrane between the bacteria and the host plant.

-The Bacteria forms a single strand of the T-DNA and it moves toward the plant cell through the protein membrane. - The T-DNA (single strand) enters the plant nucleus and then becomes a double helix and binds with the plant DNA.

- Auxin and cytokinin are produced in large quantities leading to the production of tumors and abnormal growth.

-Screening and selecting transformed cells - Producing a whole plant from the successfully transformed cells

Analyze transformants :1- Southern hybridization: by this way the existence of the gene and its number of copies can be detected.2- mRNA hybridization: the gene expression can be detected because some times the gene is transferred to the plant genome but it doesn’t express its self as protein.3- Studying the enzymes and proteins (proteomics): some times the mRNA is produced but not translated to protein due to mutations.4- Biochemical and morphological studies: must be done in more than one generation in order to make sure of the stability of the transferred gene.5- Reporter gene: It is a gene added to the T-DNA with the marker gene responsible for showing a morphological character (radiate or glowing under UV)

Chapter 4

Plant pathogen interaction

Introduction:- The relation ship between plant and pathogen is one of the most important plant studies due to its effect on plant growth and crop productivity. - Most of the pathogens are very specific to a certain plant or species, but after the full infection the pathogen will cause a great damage to the cultivated crop.-Pathogens are either Necrotrophic pathogen (Affects the dead tissues and had a wide range of hosts) or Biotrophic pathogen (Affects living cells and had a very narrow range of hosts)- The outcome of the plant pathogen relationship is either plant infection or plant resistance, plant resistance appears as natural borders (increasing cuticle, increasing cell wall thickness, production of antioxidants, phenolic compounds…etc.)

The relationship between plant and pathogen

plant pathogen Relationship Result

Susceptible Virulent Compatible Infection

Resistant Avirulent Incompatible Resistance

The resistance to a pathogen refers to the speed of the immunity system of the plant to respond toward the pathogen and activate the pathways leading to the formation of thick cell wall, cell death, phenolic compounds production…

Gene to gene hypothesis (the old theory):“ In each plant and pathogen there are genes for the resistance and virulence and they are either dominant or recessive, depending on the relationship between the genes whether it is compatible or incompatible the infection or the plant resistance occur” The plant is resistant if it has the dominant resistant gene R which interact with the gene of the pathogen which is also dominant gene Avr (Avirulence gene) - Each of the R gene and Avr gene produces an intermediate protein compound and they are responsible for the activation of the resistance pathways.

The new theory for plant resistance:“ the relationship between the R gene and the Avr gene is not a direct relation, but it needs an intermediate compound which is a protein produced by the host plant and this protein activates the NB-LRR region”Example:The R gene in Arabidopsis (RPM1) which resists Pseudomonas syringae the Pseudomonas produces a protein called RIN4 (RPM1- interacting protein) which is the intermediate protein responsible for causing the infection. The resistant plants deactivate the RIN4 and signal the plant to produce salicylic acid (SA) to increase cell wall thickness.

Plant defense mechanisms:1- Phytoalexins: they are compounds with small molecular weight. Phytoalexins are produced by large quantities during stress conditions which cam reach 10% of the plant dry weight.The genes responsible for the production of phytoalexins becomes active only during infection or stress since the phytoalexins are poisonous substances for pathogen and plant cells as well. One of the genes responsible for the phytoalexin production is the PAL gene (phenylalanine ammonia lyase)

PR protein:Some proteins are produced by the plants and they are specific to attack certain pathogen and they are called related protein pathogenesis.Example: the chitinase and glucanase are enzymes responsible for the analysis of the chinitin and glucane forming the fungal cell wall.Those proteins are not poisonous to plant cells and they are stored in the cell vacuole therefore scientists are trying to transfer the gene responsible for the production of the RP protein to different plat types.

3- Cell wall:Most pathogens produce cellulase and pectinase enzymes to analyze the plant cell wall, after infection and the beginning of the cell wall analysis some genes become active and start to produce Callose synthase enzyme which increase the cell wall thickness.

4- Salicylic acid and (SAR) system acquired resistance: The salicylic acid is a natural compound produced by the plant, during infection the conc. of salicylic acid increases and translocated to different plant tissues through the xylem and the phloem causing a signal and activates the PR protein production.

Also it was found in some plants that after infection other parts of the plant become resistant to the pathogen also the neighboring plants become resistant to that pathogen .. By studying this they found that the plant produces a compound called Jasmonic acid which causes signal to the plant and the other neighboring plants to become resistant to that pathogen (acquired resistance)

Example:Resistance gene in tomato plant is called Pto this gene can produce a protein called Serine-threonine protein kinase which can cause phosphorylation to other amino acids. The phosphorylation will cause the stop of the AA action.The binding of the Pto and the AvrPto caused a signal and the beginning of the resistance pathways

So what is the reason of the existence of the Avrgene in the pathogen?-It is an important gene for the life and the existence of the pathogen.-Selection of the weak population.

R gene :Consists of 3 main parts:1- Leucine rich repeats LLR: 24 AA it is important in the binding of the protein product of the R and Avr genes also it plays a role in the activation of the resistance pathways.2- Nucleotide binding site NB: Resposible for the production of ATP’s and any mutation occurs at that part will cause the deactivation of the gene.3- Toll interleukin receptor similarity TI: Activates the resistance pathways leading to the production of antioxidants, phenols, salsilic acid…