Transcript Study of Oryza Sativa genes in Arabidopsis To advance

STUDY OF ORYZA SATIVA GENES IN ARABIDOPSIS TO ADVANCE UNDERSTANDING OF PATHOGENIC DEFENSES IN BARLEY
Garrett Hall1, Jesse Wilcox2, Jackson Moeller3, Jaime Dittman3, and Steve Whitham3
1Southeast Polk High School, Pleasant Hill, Iowa ,2Plant Valley Southwoods High School, West Des Moines, Iowa, and 3 Department of Plant Pathology and Center for Plant Responses to
Environmental Stress, Iowa State University, Ames IA 50011
ABSTRACT
To find important differences between plants that are resistant to
pathogen attack and those that are susceptible, microarrays were
previously conducted to describe the response of the monocot plant
Hordeum vulgare, barley, to Blumeria graminis f. sp hordei
(Bgh), powdery mildew fungus. From these microarrays, over 200
genes were identified that may be associated with plant defenses due
to the mRNA accumulation in cases of resistance to the pathogen. To
determine the function of these genes, we are studying genes of highly
similar genetic sequence in Oryza sativa and the model plant
Arabidopsis. Although much is known about barley, the resources of
Arabidopsis allow for easier investigation of the genes of interest.
Through the over-expression of these genes we hope to gain a better
understanding of their function. With this approach using monocot and
dicot plants and their pathogens, we aim to find and characterize plant
genes that are of widespread importance to a plant's resistance to plant
pathogens. To get to this point a gene is inserted into an Agrobacterium
vector and is used to transform the Arabidopsis plant. These mutants
will then be challenged with strains of the bacterial pathogen
Pseudomonas syringae pv. tomato to assess whether or not these
genes have importance in limiting the growth and spread of this
pathogen. If these genes have importance in the ArabidopsisPseudomonas interaction, it is our expectation that the corresponding
barley genes will have similar functions in resistance to powdery
mildew as well.
MATERIALS AND METHODS
DISCUSSION
The processes used in our research focused on using different vectors
to move our target gene into Agrobacteria which would later be used
to transform Arabidopsis plants.
Genes that have potential pathogenic resistance functions were
sequenced following cloning into our plant over-expression vector.
We analyzed the nucleotide sequence and made a comparison to the
gene’s open reading frame to determine if the sequence generated
was correct. One problem that we encountered in this process was a
number of samples did not match the open reading frame, but the
forward strand matched the reverse strand. From this information, we
concluded that the forward and reverse strands were from the same
clone, but the clone that we had sequenced was not the clone we
expected. With a BLAST search of the Arabidopsis genome, some of
the samples were in the same gene family as the gene we were
interested in. With further investigation and testing, a likely cause of
the wrong clones was a contamination or labeling error from the
center they were purchased from.
The genes that were sequenced and matched the clones we expected
were electroporated into Agrobacteria. Arabidopsis florets were then
dipped with the Agrobacteria cultures. Unfortunately, with a six week
investigation, we were unable to ascertain if the clones were
incorporated and expressed in the Arabidopsis plants. Our prediction
is that a portion of the seeds from the dipped Arabidopsis plants
would express the herbicide resistance gene that is associated with
the gene of interest. RNA could then be extracted and analyzed to
determine if the gene of interest was incorporated into the plant and if
that gene is over-expressed compared to a control.
Oryza sativa and Arabidopsis homologues retrieved; cDNA clone
selected.
PCR gene amplification
BP Clonase Reaction & E. coli
transformation
LR Clonase Reaction & E. coli transformation
transformation of Agrobacteria
Growth of Agrobacteria
Transformation of Arabidopsis
Harvest Seed
Growth of seed to Check for Over-expression
A PCR product was obtained and sent to a BP Clonase reaction using
the pDONR207 vector. Once the BP had been quantified using a Nano
Drop it was then carried into the LR Clonase reaction using the
pCB2004 vector.
35S-Omega
attR1
rrnB T2 transcription terminator
NotI (131)
rrnB T1 transcription terminator
CaMV35S promoter
Cm R
pDONR™207 forward primer
ccdB
attP1
SalI (8865)
SalI (565)
bar
SalI (613)
Pc promoter
attR2
CaMV35S polyA
ccdB
pDONR207
SalI (1579)
T-Border (left)
5585 bp
Gm(R)
Nos poly-A
pCB2004 jrm
T-Border (right)
10872 bp
NotI (3009)
kanamycin (R)
pVS1 sta
Cm(R)
pBR322 ori
SalI (2508)
pBR322 bom
NotI (4541)
attP2
NotI (5831)
pDONR™207 reverse primer
BACKGROUND
Leaf spots, wilting, tumors, cankers, rotting, and rust are terms that
cause alarm to those involved in crop production. Large amounts of
resources are used to combat the pathogens that cause these
symptoms, but chemicals only help so much. Pathogens may adapt to
these chemicals over time. If new means of plant protection are
established, the agriculture industry may not lose as much yield each
year to pathogens. Recombinant DNA technology allows the
enhancement of plant responses to a pathogen. Genes of interest from
one plant may be transferred to another to increase the recipient
plants’ ability to defend itself. The Whitham lab at Iowa State University
is currently working on a model pathogen system in Arabidopsis to
discover plant genes responsible for pathogen defense. These results
can later be used to help protect barley and other crops. Emphasis is
placed on genes that are more likely to have large effects on pathogen
resistance such as signal transduction components. Around two
hundred genes of interest have been selected for over-expression to
determine gene function.
RESEARCH GOALS
The main goal of this research is to identify the role of specific genes in
pathogenic resistance. The results of this investigation could then be
applied to pathogen resistance in barley and other crops.
Our specific goal for this project was to clone genes of interest into a
plant over-expression vector for stable over-expression in Arabidopsis.
pVS1 rep
When the LR reaction had been validated by PCR it was then sent to
the Iowa State University DNA Facility for sequencing. Upon retrieving
the sequence data, the AlignX program was used to verify base pairs.
If the sequences matched, the LR reactions were transformed to
Agrobacteria cultures. These cultures were then used to dip
Arabidopsis flowers with the hope of transforming the future
generation.
RESULTS
During our six week summer experience, research that had been
occurring for 3 years was continued. The results from our
investigation included 35 total genes from Oryza sativa and
Arabidopsis. The genes were inserted into a BP vector after PCR
amplification. From this BP plasmid, pDONR207, 30 of those samples
were inserted in the LR plasmid, pCB2004. Once the samples were
verified, they were sequenced and analyzed. In total, 30 DNA clones
were taken from clones at the beginning of the process to DNA
sequencing during the six weeks. Arabidopsis plants were dipped
with 15 separate Agrobacteria cultures that were created from the LR
plasmid samples.
BP Clonase Reactions (pDONR207)
A total of 35 clonase reactions
were able to be sent to LR Reaction
LR Clonase Reactions (pCB2004)
A total of 30 clonase reactions
Were able to be sent to sequencing
Agrobacteria Solutions
15 solutions each with a separate gene of interest were used to
transform Arabidopsis.
REFERENCES
Caldo, R.A., Nettleton, D. and Wise, R.P. (2004) Interaction-dependent
gene expression in Mla-specified response to barley powdery mildew.
Plant Cell, 16, 2514–2528.
Clough S and Bent AF (1998) Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant
J 16: 735-43.
ACKNOWLEDGEMENT
We would like to thank the following people from the Whitham lab for
their graciousness and support during our experience: Jackson
Moeller, Jaime Dittman, Nathan Bestor, Chunling Yang, and Steve
Whitham.