Questions we can address with bioinformatic analysis and genome sequence comparison:

1. Questions we can address with bioinformatic analysis and genome sequence comparison: • Why is a given pathogen more virulent? • What is the geographic range of different pathogen strains and how are they changing with time? • Why does a given pathogen attack one host but not another? Closely related strains caused different symptoms on the same host Distantly related strains are pathogens of woody plants Distantly related strains are pathogens of the same herbaceous species

2. Pseudomonas syringae is a plant pathogenic bacterium divided into “pathovars” depending largely on the host plant from which they were isolated MLST analysis reveals a population structure composed of 4 major clades Host specificity is only partially related to phylogenetic relationship

3. Three strains representing three of the major clades sequenced to completion bean (Pph 1448A) bean (Psy B728a) tomato (Pto DC3000)

4. More recently draft genome sequences have become available for three P. syringae pathogens of woody plants bean (Pph 1448A) olive (Psv NCPPB 3335) horse chestnut (Pae 2250) bean (Psy B728a) kiwi (Pan M303091) tomato (Pto DC3000)

5. Pathogens of woody plants: P. savastanoi olive P. aesculi horse chestnut P. actinidae kiwi What enables these strains to be pathogens of woody plants? Are the properties shared (or not) between different clades? Candidate determinants: 1. Type III effectors 2. iron acquisition capabilities 3. metabolism of compounds associated with woody tissue

6. 1. Type III effectors • Translocated into the plant cell by the Type III secretion system • Regulated by the hrpL alternative sigma factor • Impact host range chiefly through suppression of plant defenses • Tools for finding type III effector genes: • Look for genes named as Type III effectors by the automated annotation pipeline • Look for genes associated with predicted binding sites for HrpL • Look for regions showing BLAST similarity to known Type III effectors • Compare genomes and examine regions known to be enriched for Type III effectors in other strains

7. 2. Siderophores: • extracellular iron-chelating compounds used by microbes to scavenge iron from their environment • Synthesized by non-ribosomal peptides synthases – enormous modular enzymes • Tools for finding siderophores: • Look for genes named with the following keywords by the automated annotation pipeline • non-ribosomal • siderophore • pyoverdine, achromobactin, yersiniabactin • Compare genomes (sometimes helps in identification of unannotated fragments • Look for REALLY big genes

8. 3. Metabolism: • Are these strains able to thrive in woody hosts because they can derive nutrition from wood while others can’t? • Challenge: • Metabolic modeling from sequence data and comparison of metabolic pathways is not easily automated.

9. Rodriguez-Palenzuela et al found that P. savastanoi encodes genes allowing degradation of aromatic compounds (assoc with woody plants) to readily metabolizable compounds Genes shaded gray are present in P. savastanoi but not the three pathovars with closed genome sequences (pathogens of herbaceous plants) Read more about aromatic metabolism here: http://www.microbialcellfactories.com/content/5/1/1

10. Metabolic questions • Are similar genes present in P. aesculi and P. actinidae? • Do the genes appear in a genomic island when compared to the related herbaceous pathogen P syringae phaseolicola 1448A?

11. Materials for genome analysis • Annotated pseudomolecule: • contigs concatenated into a single string of nucleotides • contigs within scaffolds delineated by 50 “Ns” • Scaffolds delineated with TIGR linker NNNNNAATTAATTAATTNNNNN • gene calls and functional assignments generated by RAST • 2. HrpL binding sites predicted • 3. Regions similar to effector genes IDed using BLAST • Sequence/annotation visualization: • Artemis • Artemis Comparison Tool • MAUVE (see Dave) • RAST

12. http://www.pseudomonas-syringae.org/ Scroll to the bottom of the home page: Teaching materials

13. Handout with instructions for using Artemis and ACT Open files and save as text

14. Alternative to handout: Go to “View genomes” at PPI website

15. Sequences in the P. syringae Hop database can also be used for BLAST analysis of Genbank nr

16. Artemis output Overview window + - + DNA view window - Feature annotation window

17. Artemis Comparison Tool output (for three genomes) Note usefulness for visualizing variable regions

18. To work on: • 1. Select one of the three “tree” pathogens • 2. Make an inventory of the effector genes using the tools available • Do the genes appear to be complete? • Do they have hrp boxes? • Are there hrp boxes with good scores (>15) near the starts of genes that do not appear to be effectors? • 3. How many siderophores and non-ribosomal peptide synthases do you find? • 4. Are genes linked to catechol/anthranilate metabolism and similar to those in P. savastanoi present? • Are they in a conserved location in the three tree pathogens? • Are they in a genomic island relative to sequenced herbaceous pathogens?