Transcript The (Indirect) Costs of Conducting Research: A study of

Study of plant pathogen responsive genes in Arabidopsis to further knowledge of gene function
Sara Kate Howe1, Jesse Wilcox2, Jackson Moeller3, Jaime Dittman3, and Steve Whitham3
1Johnston High School, Johnston, Iowa ,2 Valley Southwoods Freshman 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
PROJECT 1:
ABSTRACT
PROJECT 2:
Knockout and Silenced Gene Analysis
From microarrays designed to test the response of barley to powdery
mildew fungus, over 200 genes were identified with patterns of
expression suggestive of involvement in pathogen defense. To
determine the function of some of these genes, we are studying
homologous genes of the model plant Arabidopsis (Project 1). These
genes were identified by using BLAST. Although much is known about
barley, the resources of Arabidopsis allow for easier investigation of
the genes of interest. Through the knock-out, silencing, and
overexpression of these genes we hope to gain a better understanding
of their function. These lines are screened for altered gene expression,
then challenged with strains of the bacterial pathogen Pseudomonas
syringae pv. syringae 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 Arabidopsis-Pseudomonas interaction, it
is our expectation that the corresponding barley genes will have
similar functions in resistance to powdery mildew. Correspondingly,
for barley genes shown to function in plant defense we expect that
their expression in Arabidopsis will also cause a change in plant
defense phenotype (Project 2). 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.
Testing conserved function in plant defenses
RESEARCH GOAL
The end goal of this project is to identify the role of specific genes in
pathogen resistance. Our task was to first determine if the expression
of the genes of interest was altered from wildtype in our
Arabidopsis lines.
PROCEDURES
Barley microarray data was analyzed to determine which genes were
expressed differently in the Blumeria graminis infected array versus
the uninfected array (Caldo et al., 2004). Arabidopsis orthologs to the
genes of interest were found using BLAST. Information from the
Genevestigator database (figure 1.1) indicates the usual expression of
these select Arabidopsis thaliana orthologs. RNAi & TDNA insertion
lines of Arabidopsis thaliana were obtained for these genes in the
hopes of finding lowered or no expression. At the end of our
program, we were attempting to detect the altered expression lines.
The following procedures were performed to make the comparison
between the differentiated lines and a wildtype control:
RNA was extracted, converted to cDNA, and analyzed by PCR to
determine if the gene of interest was expressed differently than
wildtype controls (figure 1.2).
BACKGROUND
Numerous pathogens are responsible for decreased yields in crop
production. Large amounts of resources are used to combat the
pathogens that cause these symptoms, but the use of chemicals has
numerous drawbacks. Pathogens may adapt to these chemicals over
time and the chemicals can be detrimental to the environment . 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.
However, before these practical applications of research can be
realized, more needs to be known about gene function.
The Whitham Lab at Iowa State University is currently working on a
model pathogen system in Arabidopsis to investigate plant gene
function associated with pathogen defense. These results may have
implications of gene function for 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.
FIGURE 1.1
Genes of Interest
Yellow: 1G30360
Blue: 1G53390
Green: 4G35150
Forest: 1G77210
Figure 1.1- Expression of genes of interest from a compilation of
microarray data at the Genevestigator DB. This allows us to determine
the expected level of wildtype expression for these genes prior to
testing for knockout or lowered expression in our lines.
https://www.genevestigator.ethz.ch/
FIGURE 1.2
Hordeum
vulgare
genes of
interest
Figure 1.3- Semi-quantitative
RT-PCR gel results showing
equivalent expression of the
control gene Actin for our
lines of interest.
Arabidopsis
thaliana
ortholog
gene
COMPLETED
TO BE
COMPLETED
Confirm
silencing
through
RNA
isolation
and PCR
Expression
profile
(Fig 1.1)
Altered
expression
lines
Confirm
knockout
through
RNA
isolation
and PCR
Infect
knockout lines
with
Pseudomonas
syringae pv.
syringae as
pathogen
challenge
A barley defensin gene identified from microarrays has been shown
to act to condition susceptibility to barley powdery mildew
fungus. This protein also localizes to the cell wall at the point of
fungal attack. By expressing this gene in Nicotiana benthamiana and
Arabidopsis thaliana, and applying fluorescent tags, we hope to find
conserved function & localization in plant defenses across these
taxonomic boundaries.
PROCEDURES
Primers were designed for cloning the barley defensin gene into
separate plant expression vectors. The PCR products were then
purified and recombined with the pDONR207 vector, en route to
recombination with the plant expression vectors pEARLEYGATE103 &
104. Agrobacteria containing these constructs were then used along
with Gus controls in Agro-infiltration of Nicotiana benthamiana for
transient expression, or in floral-dip transformation of Arabidopsis
(Clough & Bent, 1998). Observations of whole leaves pre and postinfection with appropriate pathogenic Pseudomonas strains were
made and bacterial multiplication recorded.
FIGURE 2.1
FIGURE 2.2
www.arabadopsis.org
GRAPHIC DESCRIPTION OF PROJECT 1
Infect silenced
lines with
Pseudomonas
syringae pv.
syringae as
pathogen
challenge
RESEARCH GOAL
Quantify
Pseudomonas
colonies to
determine
gene role in
pathogen
resistance
Figure 2.1- Vector utilized to insert barley
defensin gene into N. Benthamiana.
Figure 2.2- Transient expression of
barley defensin gene fused with GFP
and co-inoculated with p19 construct.
Photos were taken 6 days after
agroinfiltration.
REFERENCES
Caldo, R.A., Nettleton, D. & Wise, R.P. (2004) Interaction-dependent
gene expression in Mla-specified response to barley powdery mildew.
Plant Cell, 16, 2514–2528.
Clough S & Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of
Arabidopsis thaliana. Plant J 16: 735-43.
Quantify
Pseudomonas
colonies to
determine
gene role in
pathogen
resistance
Pikaard, C., & Earley, K. (2004). Vectors. Retrieved July 22, 2008, from The Arabidopsis Information
Resource Web site: www.arabidopsis.org
Zimmermann P, Hennig L, and W Gruissem (2005) Gene expression analysis and network discovery using
Genevestigator Trends in Plant Science 9 10 , 407-409 [PubMed ]
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, Ashley Stabenow, Chunling Yang, and Steve
Whitham.