Phenotyping the effect of mutant P. fluorescens on mycorrhizal development and function of L. bicolor with Populus

Swetha CharlesJessy Labbé

What’s coming?Plant Microbe Interface

Molecular communication between plants

and their microbial

environment

Genetic underpinnin

g

Myccorhizal Interaction

Populus species

Laccaria bicolor

Myccorhizal helper bacteria Pseudomonas

fluorescents

Which P. fluorescens genes are involved inthe helper effect?

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.

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

Why Populus?

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

What is mycorrhizal symbiosis?

A simple explanation:An interaction between

plants and fungi where both partners benefit [Barbieri]

Cusano, pg 204

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

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

Mycorrhizal Symbiosis

There are three main types of Mycorrhizal symbiosis [Smith] :

1. Arbuscular mycorrhiza (endomyccorhiza)

2. Ectomycorrhiza3. Ericoid, orchid and mycoheterotrophic

mycorrhiza (some are endomycorrhiza)

Populus trees form relationships with both endo- and ectomycorrhizas.

• The latter relationship is our focus.

Mycorrhizal helper bacteria

Mycorrhizal fungi are surrounded by microbial communities which affect mycorrhizal symbiosis. [Deveau]

Garbaye, 1994

The creation of the mycorrhizal relationship is substantially improved in the presence of certain soil bacteriaThe bacteria also help support the continuing symbiosis

Garbaye, 1994

Mycorrhizal helper bacteria

How do MHB help mycorrhizal formation?

1. Increase root receptivity for infection by fungi

2. Improve the recognition process between roots and fungi

3. Stimulate fungal growth before symbiosis occurs

4. Modify the rhizosphere making it more conducive for mycorrhizal infection

5. Induce fungal spore germination [Deveau]

Garbaye, 1994

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

● 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

Pseudomonas fluorescens characterisation

The T3SS system was checked by Cusano et al and proved important for MHB effect.

Pseudomonas fluorescens characterisation

Cusano, pg 204

Type three secretion system (T3SS)

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

http://www.cambio.co.uk/216/10/products/ez-tn5-andlt;t7/kan-2andgt;-promoter-insertion-kit/

Co-regulated flagellar system

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

Project 1:Laccaria Bicolor and P. fluorescens

Fungal plug in the centre and bacteria placed in four spots equidistant from the plug

Characteristics to measureFungal diameter of Laccaria bicolor with no bacteria

Day 18 Day 22 Day 25

Day 4 Day 8 Day 11 Day 14

Characteristics to measureFungal diameter of Laccaria bicolor with mtP464

Day 18 Day 22 Day 25

Day 4 Day 8 Day 11 Day 14

Fungal diameter of Laccaria bicolor with mtP464 vs. mt4delta1

mt4delta1 (Day 25) mtP464 (Day 25)

0 5 10 15 20 25 300.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00 Diameter of fungal growth

L. bicolorBBc6R8mt28D10mtP464mt4Δ1mt4Δ2

Days

Dia

mete

r

NB: This experiment is ongoing

0 5 10 15 20 25 300.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00Diameter of fungal growth

L. bicolorBBc6R8mt28D10mtP464mt4Δ1mt4Δ2

Days

Dia

mete

r

Receptor ResponseSignal

Higher than wild-type growth rate: bacteria as food source in nutrient limited environment

Receptor ResponseSignal

L. bicolor with no bacteria

Receptor ResponseSignal

L. bicolor with wild type bacteria

Receptor ResponseSignal

L. Bicolor with mutant bacteria

Branching density

BBc6R8 (Day 25) L. bicolor (Day 25)

Branching density

mtP464 (Day 25) mt4delta1 (Day 25)

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

Project 2:Testing a new phenotype by inoculating three species of poplar with L. Bicolor and 3 promising mutants

Populus species:

1. P. trichocarpa2. P. deltoides 3. P. tremuloides (P. tremula X P. alba)

P. fluorescens mutants:

1. mtP4642. mt4delta13. mt4delta2

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

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, 2007. 175, 732-755.

• 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, 2010. 3:203–210

• Smith, S., Read, D. Myccorhizal Symbiosis: third edition. 2008. • Garbaye J.  Helper bacteria: a new dimension to the

mycorrhizal symbiosis. New Phytologist, 1994. 128: 197–210

Acknowledgements

•Jessy Labbé•Lee Gunter•Zack Moore

•Julie Malicoat•Cheryl Brown

•Tim Sullivan

Questions?