New approaches for diagnosing bacterial pathovars

Transcription

1 New approaches for diagnosing bacterial pathovars A Brendan Rodoni, B Jim Stack, C Deb Hailstones, D Matthew Bellgard and A Jane, Moran A DPI, Knoxfield, Victoria, Australia B Department of Plant Pathology, Kansas State University, USA C I&I, EMAI, NSW, Australia D School of Information Technology, Murdoch University, WA. Rachel Powney (CRCNPB/Vic DPI) Cooperative Research Centre for National Plant Biosecurity

2 Biosecurity Story: Diagnostics of Erwinia amylovora (1997) Erwinia billingiae Marker H20control Ea 322A Endemi c sp. 1 Endemi c sp. 2 Endemi c sp. 3 Ea- specific PCR 1 Ea- specific PCR 2 AB primers (Bereswell 1992) AJ Nested Primers (Mc Mannus & Janes 1996) 1 kb- 500 bp- 1 kb- 500 bp- Rhanella sp.

3 The Microbiome Microbial communities are more diverse than previously thought Next Generation sequencing/metagenomics Very cheap, generate gigabases of sequence in one run Can identify both culturable and non-culturable species of bacteria Metagenomic analysis of the Human Distal Gut Microbiome 22% of the microbial species identified in the human gut were unknown bacterial species. 60% of the bacteria represented uncultivated species

4 Horizontal Gene Transfer The Canopy of Life Evidence is emerging that the rate of lateral gene flow or horizontal gene transfer is at far higher rates than previously thought (Achtman and Wagner 2008). Doubt has been cast on the existence of microbial species among environmental isolates. The Mobilome

5 The genetic components of a bacterial species is now referred to as the pan-genome Pan-genome (pan, from the Greek word παν, meaning whole) Consists of: - Core genome genes present in all strains of the species - Flexible genome - composed of genes absent from one or more strains and genes that are unique to each strain 500 genes Core Genome 4000 genes 600 genes The core pan-genome Flexible genome concept needs can to provide is be a means targeted of insight describing into by differences diagnostics intra-species between to genetic strains identify diversity (i.e. pathovars) all strains and of can a species also be targeted for detection of specific strains Flexible Genome 1100 genes

6 The composition of the Pan-genome can vary between bacterial species Core % Dispensable % P. aeruginosa Streptococcus pneumoniae Escherichia coli E. carotovora subsp. atroseptica

7 Development of improved diagnostics targeting the core genome

8 Diagnostic development pipeline for bacteria Step 1 Compare genomes of target species to identify core genome Step 2 Compare target species core genome to closely related species to identify potential regions for diagnostic development Step 3 Compare potential regions of the target species to sequence data at NCBI to identify specific targets for diagnostic development Step 4 - Analyse specific targets for further diagnostic development Step 5 - Design multiplex primers from chosen regions at different locations in the genome Step 6 - Validate in house with multiple strains of target species, closely related spp. and environmental samples Step 7 - Validate tests globally

9 Erwinia amylovora Fire Blight Spiraeoideae strains Rubus strains

10 E. amylovora genomics Identify the pan-genome of E. amylovora in order to: gain insight into hostspecificity and pathogenplant interactions develop improved diagnostics Powney et al (PhD thesis)

11 Genome sequencing Sequence Size Strain Syn. Host Origin Virulence type coverage (Mb) Ea 495 CFBP2585 Sorbus sp. Ireland Ea 266 E4001A Malus domestica Canada High Ea 356 EA 1/79 Malus domestica Germany Low - Moderate MR1 Rubus sp. USA Low - Moderate Ea644 Rubus sp. USA Ea 246 ATCC BAA Rubus sp. USA High Ea 273 ATCC Malus domestica USA Moderate Sanger CFBP1430 Malus domestica France Moderate CFBP1232T Pyrus communis UK Illumina UPN527 Malus domestica Spain Illumina SFR-BO Sorbus sp. Italy Illumina ACW56400 Pyrus communis Switzerland Illumina Powney et al (PhD thesis)

12 Diagnostic Development IL5 MR1 Ea 266 Ea 644 Ea 356 CFBP1430 CFBP1232 Ea 495 ACW56400 Step 1 Compare genomes of E. amylovora to identify core genome Ea 273 Ea01SFRBO UPN527 Core genome = sequence present in all strains of E. amylovora Ea core genome Powney et al (PhD thesis)

13 Diagnostic Development IL5 MR1 Step 2 Compare E. amylovora core genome to closely related species Ea 356 Ea 273 CFBP1430 Ea01SFRB Ea 266 CFBP1232 Ea 495 UPN527 Ea 644 ACW56400 Ea core genome vs E. tasmaniensis E. pyrifoliae Ea-potential targets Reduced dataset of core sequence that can potentially be targeted for diagnostics Powney et al (PhD thesis)

14 Diagnostic Development Step 3 Compare E. amylovora potential targets to sequence data at NCBI to identify E. amylovora specific targets Ea 356 Ea regions of sequence collectively spanning bps IL5 CFBP1430 Ea01SFRB Ea 266 CFBP1232 Ea core genome MR1 Ea 495 UPN527 vs Ea 644 ACW56400 E. tasmaniensis E. pyrifoliae Ea-potential targets NCBI database Ea-specific targets Powney et al (PhD thesis)

15 Diagnostic Development Step 4 - Analyse Ea specific targets Functional significance Likelihood of being present in other (unsequenced organisms) Location on genome Transposase

16 Diagnostic Development Step 5 - Design multiplex primers from chosen targets at different locations in the genome Step 6 - Validate in house with multiple strains of E. amylovora, closely related spp. and environmental samples Step 7 - Validate globally Step 8 - Publish diagnostic protocol Lang et al Genomics-Based Diagnostic Marker Development for Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola. Plant Disease 94 (3):

17 Thank you