In planta detection of Puccinia horiana by in situ hybridization

Presented by: Mitchell A. Ellison

Puccinia horiana

• Causes Chrysanthemum White Rust (CWR) Disease

• Obligate Parasite • Microcyclic• Two Spore types• Basidiospores• Teliospores

• Quarantine Pest

Life Cycle

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.

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

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

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

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.

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.

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.

Protocol Overview1. Fix (48hrs)2. Clear (72hrs)3. Embed, Section, and Mount

(American HistoLabs)4. Pre-Hybridization (3hrs)5. Hybridization (Overnight)6. Post-Hybridization Wash (1hr)7. Detection (1.5hrs)8. Staining (30min)9. Permanent Mounting (30min)

100µm50µm

OptimizationThe signal intensity and specificity seen in these photos was accomplished by optimizing key steps of a general in-situ hybridization protocol.

Proteinase K Digestion

10µg/mL

No digestion

10µg/mL 20µg/mL

40µg/mL 80µg/mL

Experiment 1 Experiment 2

Formamide Concentration & Final Wash Temperature

42°CFinalWash

10% 30% 50% 70%

52°CFinalWash

All samples were hybridized overnight at 42°C

Formamide Concentration

Wash Temperature & Specificity TestAnti-Sense Probe

Infected Tissue52°C Wash

Healthy Tissue42°C Wash

Sense Probe

Infected Tissue42°C Wash

Results Summary

Protocol Step Optimal Condition

Proteinase K Digestion 10 µg/mL

Formamide Concentration 10 %

Final Wash Temperature 52 °C

Trypan Blue In Situ Hybridization

Conclusion

• In situ hybridization offers more intense signal and a higher degree of specificity than our previous methods.

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.

Questions

Collaborators:Michael B. McMahonOney P. SmithDouglas G. LusterSusan E. NesterMorris R. BondeCristi L. Palmer

Thank you:Jonas King

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

Plant Tissue

Fungal Cell18S rRNA

DNA Probe

BiotinStreptaviden

HRPChromogen

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