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

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?

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] : 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.

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

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

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

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

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

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

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

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

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

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

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

L. bicolor with no bacteria Receptor Response Signal

L. bicolor with wild type bacteria Receptor Response Signal

L. Bicolor with mutant bacteria Receptor Response Signal

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 P. fluorescens mutants: mtP464 mt4delta1 mt4delta2 Populus species: P. trichocarpa P. deltoides P. tremuloides (P. tremula X P. alba)

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 Myc

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

Questions?