3. Projected Cost of CWR Establishment
• Chrysanthemum wholesale market value in 2013 was
$149 million.
• Total annual impact of endemic CWR estimated at
$33.5 million.
The Bonde Lab studies the overwintering of P. horiana
in Chrysanthemum x morifolium.
4. Attempts to Assay Samples for
Infection
• PCR, Nested PCR, and qPCR
– Too sensitive for inoculation methods
– False positives
• ELISA, Western Blot
– Not sensitive enough
• Chemical Stains
– Not specific
– Difficult to interpret results
5. Trypan Blue Trypan Blue
100µm 100µm
• Common fungal stains were not providing easily
interpretable results
Chemical Staining Example
• In-situ hybridization can provide a more specific
staining
6. 1. Modify a generalized in situ hybridization protocol
for the detection P. horiana in sections of
Chrysanthemum leaf.
2. Diagnose P. horiana infection and track disease
progress in plant tissue other than leaf.
Objectives
7. In-situ hybridization
• Hybridization is the process of combining two
complementary single stranded nucleotide molecules
to make one hybrid duplex.
• In this case we use
a DNA probe which
targets fungal RNA.
• This hybridization is
carried out in situ
or in place on tissue
samples.
8. 18S rRNA Targeting
• Ribosomal RNA accounts for about 85% of total
cellular RNA.
• We target a variable
region of the 18S rRNA
subunit.
• With probes generated
by molecular and
bioinformatics methods.
9. Leaf Tissue
• Easy to generate and
inoculate in large
amounts.
• Easy to sample.
• Primary site of P.
horiana infection.
• P. horiana infection
can be easily
identified by pustules.
12. Optimization
The signal intensity and specificity seen in these
photos was accomplished by optimizing key steps of
a general in-situ hybridization protocol.
14. Formamide Concentration &
Final Wash Temperature
42°C
Final
Wash
10% 30% 50% 70%
52°C
Final
Wash
All samples were hybridized overnight at 42°C
Formamide Concentration
15. Wash Temperature & Specificity Test
Anti-Sense Probe
Infected
Tissue
52°C
Wash
Healthy
Tissue
42°C
Wash
Sense Probe
Infected
Tissue
42°C
Wash
16. Results Summary
Protocol Step Optimal Condition
Proteinase K Digestion 10 µg/mL
Formamide Concentration 10 %
Final Wash Temperature 52 °C
17. Trypan Blue In Situ Hybridization
Conclusion
• In situ hybridization offers more intense signal
and a higher degree of specificity than our
previous methods.
18. Future Directions
• Begin work with tissue from other parts of the
plant.
• Assay samples from overwintering
experiments.
• Test protocol on other rust species such as
Soybean rust and Gladiolas rust.
• Develop probes for diagnostic assays.
20. Sense vs Anti-Sense
Anti-Sense Probe
• Cellular rRNA:5’-UGUCUAUACACGACG-3’
• AS-Probe: 3’-ACAGATATGTGCTGC-5’
Sense Probe
• Cellular rRNA:5’-UGUCUAUACACGACG-3’
• S-Probe: 3’-GCAGCTCATATCTGT-5’
The reverse compliment of our target results
in hydrogen bonding between base pairs
An exact duplicate of our target does not result in
hydrogen bonding between base pairs
21. Plant Tissue
Fungal Cell
18S rRNA
DNA Probe
Biotin
Streptaviden
Chromogen
DNA probes marked with biotin are hybridized to the 18S
rRNA of the pathogen.
We target ribosomal RNA because of its abundance
in the cell.
Streptaviden conjugated horseradish peroxidase (HRP) is
applied and binds to the biotin.
A chromagen is applied which reacts with HRP to produce
a color change in the sample.The color change is collocated with the pathogen.
Proteinase K
Fixative causes protein cross-linking resulting in a web
of proteins which help to maintain tissue morphology,
but can inhibit probe access
Proteinase K is an enzyme that degrades proteins.
This allows us to create gaps in the web generated by the
fixative allowing our probes to gain access to the tissue
Editor's Notes
Hello and thank you for the opportunity to share some of my research with you.
My name is Mitch Ellison. I am a graduate student at Johns Hopkins University and a laboratory technician at USDA-ARS in Fort Detrick Maryland.
Today I am going to be discussing the “In planta detection of Puccinia horiana by in situ hybridization.
Puccinia horiana is the causal agent of chrysanthemum white rust
It is an obligate parasite meaning that it can only live on its host and cannot be cultured in vitro
It is a microcyclic rust fungus meaning that it only possesses two spore types: basidiospores and teliospores
CWR is classified as a quarintine pest by AIPHIS
Need a permit and our BSL-3 greenhouse
Fig. 1 black backing = infected mum
Fig. 2 below = leaf showing pustules
Fig.3 right = life cycle
Explain life cycle briefly
Don’t be so static
We study CWR because of its impact on the chrysanthemum industry and the potential for loss of revinue in the US
The PERAL group at USDA estimated cost of P. horiana becoming endemic in the US at 4 million dollars
PPD = USDA Policy and Program Development
PERAL = USDA Plant Epidemiology and Risk Analysis Laboratory (PERAL) CWR risk assessment
The two photos show greenhouse production of chrysanthemums
Close quarters make disease spread rapidly if infected cuttings are brought in
I work in the Bonde Lab and we study the overwintering of P.horiana on Chrysanthemum x morifolium
Hypothesize that pathogen may overwinter in plant tissue
http://www.yamashitaflowerfarm.com/photos/greenhouse-1663-718x477.jpg
https://agroecologyaz.files.wordpress.com/2013/11/vrijdag-deliflor-1262.jpg
Explain assays and their points of failure
Different assays to ID pathogen (not molecular assays)
http://iws.collin.edu/biopage/faculty/mcculloch/1406/outlines/chapter%207/ribosome1.jpg
http://www.anatomybox.com/wp-content/uploads/2011/07/Ribosomes.jpg
Mention fungal target and specificity
Here are the results of two experiments that helped us to first find out that a proteinase K step was necessary in the protocol and second to determine the proper amount to use as we went forward.
Experiment 1 (on the left) simply tested the protocol with a proteinase K digestion at a concentration of 10ug/mL against the protocol with no proteinase K digestion were samples were placed in proteinase K buffer with no enzyme. This demonstrated the need for a proteinase K digestion in our protocol.
Experiment 2 (on the right) asked the question “will higher concentrations of proteinase K allow us to obtain more signal. The answer as you can see was no and we found as one would expect that at higher proteinase K concentrations we saw more damaged tissue than at the lower concentrations.
