1. John Rathjen and group ANU
Deep sequencing of the Puccinia striiformis genome and transcriptomes – strategies for pathogenesis
John Rathjen and group ANU

2. P. striiformis in Australia
BGYR
(1998)
Psd
Pst-1979
Psp
Pst-WA
(2002)
(~21 strains)
Puccinia striiformis f.sp. tritici Barley grass yellow rust
Psd– grows on Dactylis glomerata (Cocksfoot)
Psp – grows on Poa pratensis (Kentucky blue grass)
Stripe rust of Phalaris spp., Bromus spp., “wheat grass”, etc, etc
(~6 strains)

3. Rust haustoria – a purifiable pathogenic niche!3

4. Effectors and gene-for-gene resistance
Virulence effector proteins are secreted by pathogens to enhance fitness.
Bacteria have such genes, fungi such as rusts seem to have hundreds.
Mostly they are novel, uncharacterised proteins – some clearly act to inhibit plant disease resistance.
Some plants have learned to recognise pathogen effectors, which triggers resistance. In this case the effector is called an Avirulence (Avr) protein.
This type of resistance is short-lived.

5. Aims Create a draft genome of Pst containing all of its genes.
Create transcriptomes (= expressed genes) from key developmental stages of the fungus.
Use the expression data to annotate genes.
Use the sequenced genome as a basis for comparison with new virulent strains.

6. NGS datasets for stripe rust bioinformatics
Genome
Transcriptome
454 mate-pair
454 RNA-seq
Illumina mate-paired
Illumina pair-end (2)
Illumina RNA-seq
454 RNA-seq
Illumina RNA-seq
Transcriptome

7. Pst genome (1979 isolate)
7,800 contigs, N50 = 17 kb, 70 Mb assembled, total 96 Mb.
The rest is repetitive elements (transposons etc).
Can use this as a basis for gene prediction, particularly effector prediction.
We can ‘resequence’ the genomes of other Pst isolates to map variation, particularly with respect to virulence (BGYR, WA strains).

8. Haustoria transcriptome de novo assembled (454 reads)
437 secreted proteins predicted
100 screened for expression pattern
253 have no similarity to known genes
105 similar to hypothetical proteins of Puccinia graminis f.sp. tritici
40 partially annotated with BLAST2GO
15 glycoside hydrolases (chitinase activity and plant cell wall degradation)
4 polysaccharide deacetylases (chitin deacetylases)
21 other (proteolysis, oxidation-reduction regulation of transcription)
35 contain conserved domains
zinc finger domain
copper/zinc binding domain
cupredoxin domain
NUDIX hydrolase domain
thaumatin
Tobacco M
75%
Brendon Conlan & Will Jackson
Plant cell death inhibition
R-AvrR recognition
25%
RT-PCR

9. Effector gene variation correlates with virulence
437 candidate effector genes of Pst have been identified by three criteria.
Two candidate effector genes are missing in BGYR comparing with Pst.
We are currently testing these genes for recognition by barley grass.
Image courtesy of Diana.

10. Effector genes are highly polymorphic (copy no. variation)
Pst-79
Pst-79 5 5 4 4 3 3 2 2 6
Pst-130 (US) 1 1
Gene copy number
BGYR
Pst-BGYR 5 5 4 4
Gene copy number 3 3 2 2 1 1
Effector candidates, nominal rank
Effector candidates, nominal rank
Boeva V, et al. (2011) Control-FREEC: Bioinformatics Dec 6

11. What’s happening in haustoria?
Cell proliferation
SSP
NADPH vitamin B1 ribose
biosynthesis
amino acids
metal ions
hexoses

12. The pathogen has two points of weakness
Resistance genes
- Identify more resistance genes, eg non-host resistance genes. - this type of resistance may be intrinsically unstable.
Metabolism
- target essential metabolism genes in the fungus using host-induced gene silencing (HIGS).
- find genetic and chemical methods to restrict access to sugars and other nutrients.
- this may be how long-lasting disease resistance works?

13. Colin Wellings Robert Park
Acknowledgments
Diana Garnica
William Jackson
Colin Wellings Robert Park
Peter Dodds
Jeff Ellis
Narayana Upadhyaya
David Studholme 13