Post on 22-Jan-2016
description
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?