1. Swetha Charles Jessy Labbé
Phenotyping the effect of mutant P. fluorescens on mycorrhizal development and function of L. bicolor with Populus
Swetha Charles
Jessy Labbé

2. What’s coming? Plant Microbe Interface Myccorhizal Interaction
Molecular communication between plants and their microbial environment
Genetic underpinning
Myccorhizal Interaction
Populus species
Laccaria bicolor
Myccorhizal helper bacteria
Pseudomonas fluorescents
Which P. fluorescens genes are involved inthe helper effect?

3. Plant Microbe Interface (PMI)
The PMI group is studying the relationship that exists between plants and their microbial environment.
One key area of study is to understand the genetic foundation that underpins molecular communication between Populus trees and their microbial system.

4. Why Populus?
It is the first tree genome that was sequenced. Many tools are available for genetic manipulation.
It’s one of the prime candidates for bioenergy production and for studying carbon sequestration.
It is found in many places in the USA

5. Why Populus?
A good understanding of microbial interactions with Populus will be useful across many areas.

6. What is mycorrhizal symbiosis?
A simple explanation:
An interaction between plants and fungi where both partners benefit [Barbieri]
Cusano, pg 204

7. Mycorrhizal Symbiosis
Mycorrhizas, rather than roots, are responsible for nutrient uptake in land based plants [Smith]
Mycorrhizal symbiosis is the normal state for most plants

8. Mycorrhizal Symbiosis
Except in a few cases, the fungi obtains most of its organic C from the plant [Smith]
With this advantage, the mycorrhiza can compete with other organisms in the soil for nitrogen, phosphate and other nutrients
ECM fungi prefer sources of inorganic N
This increases plant access to nutrients and allows growth in suboptimal conditions

9. Mycorrhizal Symbiosis
There are three main types of Mycorrhizal symbiosis [Smith] :
Arbuscular mycorrhiza (endomyccorhiza)
Ectomycorrhiza
Ericoid, orchid and mycoheterotrophic mycorrhiza (some are endomycorrhiza)
Populus trees form relationships with both endo- and ectomycorrhizas.
The latter relationship is our focus.

10. Mycorrhizal helper bacteria
Mycorrhizal fungi are surrounded by microbial communities which affect mycorrhizal symbiosis. [Deveau]
Garbaye, 1994

11. Mycorrhizal helper bacteria
The creation of the mycorrhizal relationship is substantially improved in the presence of certain soil bacteria
The bacteria also help support the continuing symbiosis
Garbaye, 1994

12. How do MHB help mycorrhizal formation?
Increase root receptivity for infection by fungi
Improve the recognition process between roots and fungi
Stimulate fungal growth before symbiosis occurs
Modify the rhizosphere making it more conducive for mycorrhizal infection
Induce fungal spore germination [Deveau]
Garbaye, 1994

13. Pseudomonas fluorescens characterisation
The P. fluorescens strain BBc6R8 has been used to study mycorrhizal formation in Douglas fir trees
It was initially recovered from a sporocarp of Laccaria bicolor
A test of the microbial communities in Populus soil showed the presence of Laccaria bicolor

14. Pseudomonas fluorescens characterisation
After transcriptome and genome analysis, two sets of gene clusters in P. fluorescens (BBc6R8) were found to be necessary for optimum mycorrhizal development with Laccaria bicolor
T3SS system
Co-regulated flagellar system

15. Type three secretion system (T3SS)
Pseudomonas fluorescens characterisation
Type three secretion system (T3SS)
The T3SS system was checked by Cusano et al and proved important for MHB effect.
Cusano, pg 204

16. Co-regulated flagellar system
For the flagellar system, the cluster underwent targeted mutagenesis to create different mutant strains.
Mutants are created by tn5 insertions
elements known as transposons are inserted into the gene cluster, disabling certain genes

17. Populus, Laccaria bicolor and Pseudomonas fluorescens (BBc6R8)
My project will be in parts and will focus on a model system:
Project 1: co-cultivation of L. bicolor and P. fluorescens
Project 2: co-cultivation of L. bicolor and P. fluorescens mutants with Populus species

18. Project 1: Laccaria Bicolor and P. fluorescens
Fungal plug in the centre and bacteria placed in four spots equidistant from the plug

19. Characteristics to measure
Fungal diameter of Laccaria bicolor with no bacteria
Day 4
Day 8
Day 11
Day 14
Day 18
Day 22
Day 25

20. Characteristics to measure
Fungal diameter of Laccaria bicolor with mtP464
Day 4
Day 8
Day 11
Day 14
Day 18
Day 22
Day 25

21. Fungal diameter of Laccaria bicolor with mtP464 vs. mt4delta1
mt4delta1 (Day 25)
mtP464 (Day 25)

23. Higher than wild-type growth rate: bacteria as food source in nutrient limited environment
Receptor
Response
Signal

24. L. bicolor with no bacteria
Receptor
Response
Signal

25. L. bicolor with wild type bacteria
Receptor
Response
Signal

26. L. Bicolor with mutant bacteria
Receptor
Response
Signal

27. Branching density
BBc6R8 (Day 25)
L. bicolor (Day 25)

28. Branching density
mtP464 (Day 25)
mt4delta1 (Day 25)

29. Expected Results
We hope shed more light on the P. fluorescens genes that are important for myccorhizal development and function:
The mutants which have no helper effect may point to genes of interest
Mutants which have a higher than wt growth rate may point to the fungus gaining nutrients by eating the bacteria
Previous proof of concept project highlighted mtP464 as a possible focus

30. Project 2:
Testing a new phenotype by inoculating three species of poplar with L. Bicolor and 3 promising mutants
P. fluorescens mutants:
mtP464
mt4delta1
mt4delta2
Populus species:
P. trichocarpa
P. deltoides
P. tremuloides (P. tremula X P. alba)

31. Other contributions
BESC – Inoculating promising P. fluorescens mutants with P. deltoides mutants
The Populus mutants are aimed at reducing recalcitrance to increase the ease ethanol production
Bioinformatics:
Look for known transposable elements in the genome sequence of 17 fungal isolates:
Check for polymorphisms in the transposable elements across the isolates
Highlight areas where the transposable elements and symbiosis genes overlap or occur in close proximity
Search for polymorphisms in symbiosis genes across the 17 isolates
Methods paper:
Summary of techniques used for phenotyping experiments

32. References
Deveau, A., Palin B., et al. The myccorhiza helper Pseudomonas fluorescens BBc6R8 has a specific priming effect on the growth, morphology and gene expression of the ectromycorrhizal fungus Laccaria bicolor S23N. New Phytologist, ,
Barbieri, E., Ceccaroli, P., et al. Ectomycorrhiza associated bacteria: the third partner in the symbiosis.
Cusano, A.M., Burlinson, P., et al.  Pseudomonas fluorescens BBc6R8 type III secretion mutants no longer promote ectomycorrhizal symbiosis.  Environmental Microbiology Reports, :203–210
Smith, S., Read, D. Myccorhizal Symbiosis: third edition
Garbaye J.  Helper bacteria: a new dimension to the mycorrhizal symbiosis. New Phytologist, 1994. 128: 197–210
Myc

33. Acknowledgements Jessy Labbé Lee Gunter Zack Moore Julie Malicoat
Cheryl Brown
Tim Sullivan

34. Questions?