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

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)

Rust haustoria – a purifiable pathogenic niche! 3

Effectors and gene-for-gene resistance Virulence effector proteins are secreted by pathogens to enhance fitness. Bacteria have 20-30 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.

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.

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

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).

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

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.

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. 2011 Dec 6

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

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?

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