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TOPIC

Crown gall disease in plants, Cancer as a developmental genetic disease.


 Part 1: Crown gall disease in plants.
 Part 2 : Cancer as a Developmental genetic disease:
Part 1: Crown Gall Disease in Plants:
Gene Transfer Methods: The gene transfer techniques in plant genetic transformation are
broadly grouped into two categories, Vector-mediated gene transfer & Direct or vector less
DNA transfer. Vector-mediated gene transfer is carried out either by Agrobacterium-mediated
transformation or by use of plant viruses as vectors.

What is crown gall?


Crown gall is a disease caused by bacterium Agrobacterium tumefaciens (synonym Rhizobium
radiobacter), which enters the plant through wounds in roots or stems and stimulates the plant
tissues to grow in a disorganized way, producing swollen galls. Galls are present all year.
Crown gall affects many plants, both woody and herbaceous. These are some of the plants on
which it is most commonly found:
 Fruit: Apples, cherries, currants, grapevines, blackberries, peaches, pears etc.
 Vegetables: Beetroot, courgette, runner beans and swedes
 Herbaceous plants: Alcea, Begonia, Dahlia, Lathyrus (sweet pea) and Phlox etc.
 Woody plants: Crataegus, Euonymus, Populus (poplar), Salix(willow), Rosa etc.
Swellings caused by crown gall should not be confused with the harmless nitrogen-fixing
nodules produced on the roots of many members of the pea family.
Symptoms: Swellings (galls) on the plant stems or roots. In severe cases almost all the root
system may be replaced by massive, swollen tissues Galls on herbaceous plants decay and soon
disintegrate, but those on woody plants may be hard and perennial Plant growth may be
affected, but often there is little apparent damage & root galls may go unnoticed for long time.
Agrobacterium-Mediated Gene Transfer:
Agrobacterium tumefaciens is a soil-borne, Gram-negative bacterium. It is rod shaped and
motile, and belongs to the bacterial family of Rhizobiaceae. A. tumefaciens is a phytopathogen,
and is treated as the nature’s most effective plant genetic engineer. Some workers consider this
bacterium as the natural expert of inter-kingdom gene transfer.
In fact, the major credit for the development of plant transformation techniques goes to the
natural unique capability of A. tumefaciens. Thus, this bacterium is the most beloved by plant
biotechnologists.
There are mainly two species of Agrobacterium:
 tumefaciens that induces crown gall disease.
 rhizogenes that induces hairy root disease.
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Crown Gall Disease and Ti Plasmid:
Almost 100 years ago (1907), Smith and Townsend postulated that a bacterium was the
causative agent of crown gall tumors, although its importance was recognized much later. As
A. tumefaciens infects wounded or damaged plant tissues, in induces the formation of a plant
tumor called crown gall. The entry of the bacterium into the plant tissues is facilitated by the
release of certain phenolic compounds (acetosyringone, hydroxy acetosyringone) by the
wounded sites.

Crown gall formation occurs when the bacterium releases its Ti plasmid (tumor- inducing
plasmid) into the plant cell cytoplasm. A fragment (segment) of Ti plasmid, referred to as T-
DNA, is actually transferred from the bacterium into the host where it gets integrated into the
plant cell chromosome (i.e. host genome). Thus, crown gall disease is a naturally evolved
genetic engineering process.
The T-DNA carries genes that code for proteins involved in the biosynthesis of growth
hormones (auxin and cytokinin) and novel plant metabolites namely opines amino acid
derivatives and sugar derivatives.
The growth hormones cause plant cells to proliferate and form the gall while opines and
agropines are utilized by A. tumefaciens as sources of carbon and energy. As such, opines and
agropines are not normally part of the plant metabolism (neither produced nor metabolized).
Thus, A. tumefaciens genetically transforms plant cells and creates a biosynthetic machinery
to produce nutrients for its own use.
As the bacteria multiply and continue infection, grown gall develops which is a visible mass of
the accumulated bacteria and plant material. Crown gall formation is the consequence of the
transfer, integration and expression of genes of T-DNA (or Ti plasmid) of A. tumefaciens in
the infected plant.
The genetic transformation leads to the formation of crown gall tumors, which interfere with
the normal growth of the plant. Several dicotyledonous plants (dicots) are affected by crown
gall disease e.g. grapes, roses, stone-fruit trees.
Organization of Ti plasmid: The Ti plasmids (approximate size 200 kb each) exist as
independent replicating circular DNA molecules within the Agrobacterium cells. The T-DNA
(transferred DNA) is variable in length in the range of 12 to 24 kb, which depends on the
bacterial strain from which Ti plasmids come. Nopaline strains of Ti plasmid have one T-DNA
with length of 20 kb while octopine strains have two T-DNA regions referred to as TL and
TR that are respectively 14 kb and 7 kb in length.

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A figure of a Ti plasmid is depicted below. The Ti plasmid has three major regions.

T-DNA region: This region has the genes for the biosynthesis of auxin (aux), cytokinin (cyt)
and opine (ocs), and is flanked by left and right borders. These three genes-aux, cyto and ocs
are referred to as oncogenes, as they are the determinants of the tumor phenotype. T-DNA
borders — A set of 24 kb sequences present on either side (right and left) of T-DNA are also
transferred to the plant cells. It is now clearly established that the right border is more critical
for T-DNA transfer and tumorigenesis.
Virulence region: The genes responsible for the transfer of T-DNA into the host plant are
located outside T-DNA and the region is referred to as vir or virulence region. Vir region codes
for proteins involved in T-DNA transfer. At least nine vir-gene operons have been identified.
These include vir A, vir G, vir B1, vir C1, vir D1, D2 and D4, and vir E1, and E2.
Opine catabolism region: This region codes for proteins involved in the uptake and
metabolisms of opines. Besides the above three, there is ori region that is responsible for the
origin of DNA replication which permits the Ti plasmid to be stably maintained in A.
tumefaciens.
T-DNA transfer and integration:
Signal induction to Agrobacterium: The wounded plant cells release certain chemicals-
phenolic compounds and sugars which are recognized as signals by Agrobacterium. The signals
induced result in a sequence of biochemical events in Agrobacterium that ultimately helps in
the transfer of T-DNA of T-plasmid.
Attachment of Agrobacterium to plant cells: The Agrobacterium attaches to plant cells
through polysaccharides, particularly cellulose fibres produced by the bacterium. Several
chromosomal virulence (chv) genes responsible for the attachment of bacterial cells to plant
cells have been identified.
Production of virulence proteins: As the signal induction occurs in the Agrobacterium cells
attached to plant cells, a series of events take place that result in the production of virulence
proteins. To start with, signal induction by phenolics stimulates vir A which in turn activates
(by phosphorylation) vir C. This induces expression of virulence genes of Ti plasmid to
produce the corresponding virulence proteins (D1, D2, E2, B etc.). Certain sugars (e.g. glucose,
galactose, xylose) that induce virulence genes have been identified.
Production of T-DNA strand: The right and left borders of T-DNA are recognized by vir
D1/vir D2proteins. These proteins are involved in the production single-stranded T-DNA (ss
DNA), its protection and export to plant cells. The ss T-DNA gets attached to vir D2.

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Transfer of T-DNA out of Agrobacterium: The ss T-DNA - vir D2 complex in association
with vir G is exported from the bacterial cell. Vir B products form the transport apparatus.
Transfer of T-DNA into plant cells and integration: The T-DNA-vir D2 complex crosses the
plant plasma membrane. In the plant cells, T-DNA gets covered with vir E2. This covering
protects the T-DNA from degradation by nucleases; vir D2 and vir E2interact with a variety of
plant proteins which influences T-DNA transport and integration.
The T-DNA-vir D2-vir E2 — plant protein complex enters the nucleus through nuclear pore
complex. Within the nucleus, the T-DNA gets integrated into the plant chromosome through a
process referred to illegitimate recombination. This is different from the homologous
recombination, as it does not depend on the sequence similarity.
Control
Non-chemical control: If crown gall is detected, lift and destroy affected plants. Grow crops
of potatoes or other vegetables (except beetroot, which are also susceptible) over the next one
or two years to help eliminate bacteria from soil, or grass the area over for one or more years.
Chemical control: There are no chemicals available for the control of crown gall.
Treatment
 Select resistant cultivars when possible and purchase plants from a reputable nursery.
 Do not buy plants that shows signs of swelling or galling.
 When caring for susceptible plants, avoid injury or pruning wounds that may come in
contact with the soil.
 Use Tree Wrap to protect against string trimmer damage and keep garden tools clean.
 Provide winter protection with natural burlap so bark does not crack.
In many cases, existing galls can be removed with a sharp pruning knife. Destroy the infected
plant tissue and treat the wound with pruning sealer. If the plant does not recover, remove and
destroy it.
Tip: To get rid of this problem on roses, remove the infested plant and prune out gall tissue.
Soak the entire root system and damaged areas for 15 minutes in a solution of 2 level Tbsp
of Actinovate per 2-1/2 gallons of water. Replant in healthy soil and apply 1/2 Tbsp per 2-1/2
gallons of water as a foliar spray at weekly intervals.

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Part 2 : Cancer as a Developmental genetic disease:
Cancer is a term for diseases in which abnormal cells divide without control and can invade
nearby tissues. To become cancerous, a cell must acquire mutations in several growth-
controlling genes.
Cancer is a genetic disease, consisting of uncontrolled cell division into large tumors. There
are many different types of cancers, for example: lung cancer and cancer of the blood
(leukemia). Cancers begin with the development of small benign tumors, which can develop
into malignant ones, spreading to other parts of the body. Cancerous cells have lost control of
their cell cycle, are immune to apoptosis (programmed cell death) and are not fixed in place
meaning they can spread.
Genes are in the DNA of each cell in your body. They control how the cell functions, Including:
How quickly it grows, How often it divides, How long it lives etc. Researchers estimate that
each cell contains 30,000 different genes. In each cell, genes are located on chromosomes.
About chromosomes: Chromosomes are the thread-like structures in cells that contain genes.
There are 46 chromosomes, arranged in 2 sets of 23. You inherit one set from your mother and
one from your father. One chromosome in each set determines whether you are female or male.
The other 22 chromosome pairs determine other physical characteristics. These chromosome
pairs are called autosomes.
How genes work: Genes control how your cells work by making proteins. The proteins have
specific functions and act as messengers for the cell. Each gene must have the correct
instructions for making its protein. This allows the protein to perform the correct function for
the cell. All cancers begin when one or more genes in a cell mutate. A mutation is a change. It
creates an abnormal protein. Or it may prevent a protein’s formation. An abnormal protein
provides different information than a normal protein. This can cause cells to multiply
uncontrollably and become cancerous.
About genetic mutations: There are 2 basic types of genetic mutations:
Acquired mutations: These are the most common cause of cancer. They occur from damage
to genes in a particular cell during a person’s life. For example, this could be a breast cell or a
colon cell, which then goes on to divide many times and form a tumor. A tumor is an abnormal
mass. Cancer that occurs because of acquired mutations is called sporadic cancer. Acquired
mutations are not found in every cell in the body and they are not passed from parent to child.
Factors that cause these mutations include: Tobacco, Ultraviolet (UV) radiation, Viruses, Age.
Germline mutations: These are less common. A germline mutation occurs in a sperm cell or
egg cell. It passes directly from a parent to a child at the time of conception. As the embryo
grows into a baby, the mutation from the initial sperm or egg cell is copied into every cell
within the body. Because the mutation affects reproductive cells, it can pass from generation
to generation. Cancer caused by germline mutations is called inherited cancer. It accounts for
about 5% to 20% of all cancers.
Mutations and cancer: Mutations happen often. A mutation may be beneficial, harmful, or
neutral. This depends where in gene, change occurs. Typically, body corrects most
mutations. A single mutation will likely not cause cancer. Usually, cancer occurs from
multiple mutations over a lifetime. That is why cancer occurs more often in older people. They
have had more opportunities for mutations to build up.
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Genes carry the instructions to make proteins, which do much of the work in our cells. Certain
gene changes can cause cells to evade normal growth controls and become cancer. For example,
some cancer-causing gene changes increase production of a protein that makes cells grow.
Others result in the production of a misshapen, and therefore nonfunctional, form of a protein
that normally repairs cellular damage. Genetic changes that promote cancer can be inherited
from our parents if the changes are present in germ cells, which are reproductive cells of body
(eggs & sperm). Such changes, called germline changes, are found in each cell of offspring.
Cancer-causing genetic changes can also be acquired during one’s lifetime, as the result of
errors that occur as cells divide or from exposure to carcinogenic substances that damage DNA,
such as certain chemicals in tobacco smoke, and radiation, such as ultraviolet rays from the
sun. Genetic changes that occur after conception are called somatic (or acquired) changes.
There are many different kinds of DNA changes. Some changes affect just one unit of DNA,
called a nucleotide. One nucleotide may be replaced by another, or it may be missing entirely.
Other changes involve larger stretches of DNA and may include rearrangements, deletions, or
duplications of long stretches of DNA.
Sometimes the changes are not in the actual sequence of DNA. For example, the addition or
removal of chemical marks, called epigenetic modifications, on DNA can influence whether
the gene is “expressed”—that is, whether and how much messenger RNA is produced.
(Messenger RNA in turn is translated to produce the proteins encoded by the DNA.)
In general, cancer cells have more genetic changes than normal cells. But each person’s cancer
has a unique combination of genetic alterations. Some of these changes may be the result of
cancer, rather than the cause. As the cancer continues to grow, additional changes will occur.
Even within the same tumor, cancer cells may have different genetic changes.
A series of mutations are required to develop cancer: Throughout our lives, we are constantly
getting mutations in our DNA. Mutations are what drive adaptation and evolution, so in the
long run many of these are beneficial. Whilst some mutations are beneficial, many are
detrimental to our health and cause 'genetic disease'. Cancer is one such example of a genetic
disease. For cancer to develop, one mutation is not enough. A series of mutations must
accumulate over our lives in order for cells to become malignant. Sometimes these mutations
can occur early, or late, and sometimes we may never get mutations required to develop cancer.
Mutation rate: Mutations in the DNA of living cells occur at a given, stochastic rate. This is
because DNA replication is not an entirely fool proof process. The rate of mutation in
humans is roughly 0.32 substitutions per complete replication of the genome. The rate of
mutation depends on several different risk factors.
Risk factors: The development of cancer is down to chance. We require a sequence of
mutations to develop it, but whether or not this happens is pot luck! There are however some
risk factors and behaviours that can increase the mutation rate of our cells, and as a result make
us more likely to get the mutations responsible for cancer development. These include exposure
to high levels of background radiation and smoking.
Researchers found the effects in leaves of the bandicoot berry (Leea indica), South African leaf
(Vernonia amygdalina), and simpleleaf chastetree (Vitex trifolia). Three other medicinal plants
also demonstrated anti-cancer properties. Medicinal plants have been used for the treatment of
diverse ailments since ancient times, but their anti-cancer properties have not been well studied.

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Above is an image from the national cancer institute that illustrates the process of cancer
development. Several mutations in both oncogenes and tumour suppressor genes are required
for the development of cancerous, malignant tumours.
Mutations leading to cancer often occur in two different types of genes:
Proto-oncogenes: Proto-oncogenes are those which have the potential to mutate into cancer
causing genes (oncogenes). In tumour cells they are mutated, or expressed in larger quantity
than healthy cells. Overactive oncogenes have many functions including: speeding passage
through the cell cycle uncontrollably.
Tumour suppressor genes: Tumour suppressor genes (TSGs) protect cells from damage and
from becoming cancerous. For example the TSG, p53 is involved in a variety of processes
including DNA repair and programmed cell death. Mutations in TSGs stop these protective
functions and allow cells to become cancerous.

Oncogenes push the development of cancer, whereas tumour suppressor genes try to prevent
cancer from developing. For a tumour to develop into a full blown cancer, mutations must
occur in both oncogenes and TSGs. Different cancer are a result of different mutations, in
different oncogenes and TSGs.
The cancer cycle
Uncontrolled cell growth is the hallmark of cancer. The rate and timing of cell division in the
body is normally highly regulated. However, if the genes that directly regulate the cell cycle
are mutated, this regulation is lost and uncontrolled cell division occurs: Numerous mutations
in genes controlling the cell cycle enable cells to multiply out of control. This leads to a mass
of cells that continues to grow and grow, eventually developing into a tumour.
Initially the tumour is confined to the tissue in which it is located, for example, the breast tissue.
However, as the tumour gets bigger it requires more nutrients, so starts to develop its own blood
vessel network. This process is called angiogenesis.
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The cancer cells then develop the ability to migrate around the body via the bloodstream or
lymphatic vessels. This process is called metastasis.
Cancer is a genetic disease. Cancer is a genetic disease, and this should not be confused with
the statement that cancer is a hereditary disease. The two statements are profoundly different.
A hereditary disease is one that is passed from the parents to a child through the inheritance of
a defective gene. Although in some rare instances, such as retinoblastoma (a rare childhood
tumor of the eye), cancer is hereditary, this is the exception rather than the rule. Most cancers
are not obviously hereditary, although for certain cancers, like breast cancer, there may be a
hereditary component to the disease (a "susceptibility"). However, all cancers are genetic,
meaning that they result from the unnatural function of one or more genes.
Cancer forms when genes within a normal cell are damaged and mutated. Mutations in DNA
can occur for many reasons. Cigarette smoke contains chemicals that will damage DNA. Solar
radiation from the sun contains ultraviolet (or UV) rays that will damage DNA. In most
instances, the DNA damage will not lead to cancer or other diseases, but in some cases the
damaged DNA does lead to cancer. There are about 25,000 genes in each human cell, but, in
most cases, a mutation within a gene will not lead to the development of cancer. It is only when
mutations occur in certain key genes that cancer develops. These key genes can be grouped
into three classes:
 Growth promoting genes (called proto-oncogenes) that normally tell the cell when to
grow and divide.
 Growth inhibiting genes (called tumor suppressor genes) whose normal function is to
maintain the cell in a non-dividing state.
 Genes whose function is to repair damage to DNA (called DNA repair genes).
Multiple genes are defective in cancers. Cancer does not occur from a single gene mutation in
a single gene. Instead, the development of cancer involves multiple mutations within several
key genes, including mutations in proto-oncogenes, tumor suppressor genes, and DNA repair
genes (Figure 2). The process of accumulating mutations in several genes like this normally
takes many years, and this is why cancer is more frequently seen in older individuals.

Environmental factors:
Cancer is a complex disease?, which means it is influenced by environmental and lifestyle
factors as well as genetic factors. Environmental exposures can include factors such as UV
light, chemicals (for example in cigarette smoke) and radiation. Lifestyle factors include
cigarette smoking, excessive alcohol consumption and diet
Certain types of exposures are linked to specific cancers. For example, exposure to carcinogens
from tobacco is linked to several cancers, including lung, bladder, mouth and throat cancers.
The link between environmental factors & cancer are complex & vary depending on individual.
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