Gene silencing approaches to understanding pathogenicity and virulence

Transcription

1 Gene silencing approaches to understanding pathogenicity and virulence The Palouse WSU rust workers (pathologists, breeders): X Chen, T Murray; K. Campbell; M. Pumphrey; Arron Carter; S. Jones; A. Klienhofs; J. Nirmala; C. Yin Active Emeritus: B. Allen, R. Line

2 Stripe Rust is Dominant in the PNW, but Others Threaten Stripe (yellow) Stem rust Leaf rust P. striiformis P. graminis P. triticina A transgenic approach would ideally address all three rusts AND be durable

3 Molecular interactions between rust and plant cells Rust hyphae Plant cell PAMPs PAMP recognition Haustorium PAMP triggered defenses Effector proteins Effector target Can we affect the interaction by manipulating the pathogen proteins? Antimicrobial responses & Cell Death Effector recognition by R protein; e.g. NB-LRR

4 Silencing Assay Methods Infect wheat seedlings (~2 leaves) with BSMV virus carrying rust gene fragment Inoculate with rust ~10 days later Isolate RNA from doubly infected plants ~7 days later Compare expression of gene in seedlings infected with recombinant VIGS construct to seedling infected with VIGS vector; use EF1 (β-tubulin, etc) gene as internal control Uninoculated Virus Rust Virus + Rust Chuntao Yin

5 Initial P. striiformis silencing result: Haustorial EST; predicted 108 amino acid secreted protein (effector stereotype) Expression: infected leaf/urediniospores 3617:1 haustoria/infected leaf 29:1 Expression of the Pst5a23 gene in nine seedlings infected with the Pst5a23 construct Seedling # VIGS/ virus control Jim Jurgenson, UNI Host- Induced Gene Silencing (HIGS) ranged from 0 to 8-fold in different seedlings

6 Silencing results with additional P. striiformis genes Gene Fold expression Haust/Infected leaves No. of silenced plants Average expression in silenced plants Average expression differences between control plants PSTha12J12 >100 5/6, 3/5 0.47± ±0.49 PSTha5A /9 0.48± ±0.37 PSTha12H2 90 2/4, 2/6 0.64± ±0.24 PSTha2A5 92 1/6, 2/5, 0/6 0.44± ±2.54 PSTha5A1 >100 2/6, 5/6 0.55± ±0.28 PSTha12O3 12 6/6, 6/6 0.30± ±0.46 PSTha9F18 >100 2/6, 1/6 0.48± ±1.75 PST β-tubulin 1.0 0/6, 2/6 0.80± ±0.37 PSTGAPDH 1.2 0/6-1.19±0.35 PSTActin 2.5 0/6-1.06±0.29 PSTEF1 control 1/6 0.80± ±0.11 PGTEPSPS 2.8 0/6-1.00±0.13 Yin et al. (2011) MPMI 24:554-

7 An explanation for the HIGS results Silencing signal does not enter hyphae Silencing signal enters haustorium Photo by Zhensheng Kang Haustoria-specific genes would therefore appear silenced in total-infected-leaf RNA, but constitutive genes would not. Haustoria-specific genes are likely better targets for engineering resistance

8 Puccinia genome projects Puccinia Group Database Genome Size %GC Genes P. graminis tritici Mb ,567 P. triticina Mb ,638 P. striiformis coming soon

9 Steps for predicting haustoria-specific (enriched) genes RNA-seq analysis of transcripts from purified P. graminis haustoria RNA-seq analysis of transcripts from wheat leaves infected with P. graminis Assemble sequences into genes, subtract out wheat genes Identify sequences 2+ enriched in haustorial samples than total samples Results: Purified haustoria 1182 candidates for haustoria-enriched proteins: 198 characterized proteins, the rest predicted proteins

10 Identifying essential haustoria-specific proteins Pick genes from haustoria-enriched list Make HIGS constructs Inoculate wheat with HIGS construct Inoculate with stem rust 10 days later Look for seedlings with reduced sporulation Repeat two more times to check consistency Results: PGT genes in which HIGS reduced sporulation 3/13 effector-like proteins 2/29 other haustorial proteins Next step: Demonstrate resistance in stable transgenic plant

11 Silencing essential genes of Puccinia graminis in wheat McNair McNair + BSMV::MCS McNair infected by pgt7a McNair + pgt7a +BSMV::MCS Sr31 + pgt7a McNair + pgt7a + BSMV::PGTG_11658 McNair + pgt7a + BSMV::PGTG_03590 (picture taken at 12 after rust infection) McNair + pgt7a + BSMV::PGTG_01136

12 Stable transformants are coming (slowly)

13 Considerations for transgenic plants for disease resistance No transgenic wheat production yet Approvals for deregulation of every single transgenic event is very costly High levels of disease control probably essential Resistance should be durable Multiple diseases would be preferable Additional RNAi transgene considerations No non-target effects on host (no host homology) No non-target effects on environment or people Avoid genes with homology in animals? Transgene made from multiple genes or single gene? Are there rust genes conserved enough to silence three species? 432 of 1182 haustoria-enriched transcripts have clear P. triticina homologs (236 of these in Melampsora laricis-populina) 57/432 have predicted secretion signals 53/432 have predicted trans-membrane domains

14 Methods for effector phenotype characterization Phenotypes of interest: Defenses triggered (in host or non-host) Specific avirulence phenotype Essentiality for pathogenicity, fitness, development etc. Informatic approaches to identifying candidates Prediction methods are flawed, e.g. avrrpg1 genes don t look like our stereotype Avr proteins (no secretion peptide, not small, not cysteine-rich)* Sequence (transcriptome or genomic) associations with races to predict candidate Avr genes is difficult * Nirmala et al. (2011) Concerted action of two avirulent spore effectors PNAS 108:14676-

15 Can VIGS be used to identify effectors with Avr function? Transient silencing of an Avr gene by HIGS should increase specific virulence Resistant wheat lines Inoculate wheat with VIGS construct carrying putative Avr gene Inoculate with avirulent rust and look for increased virulence on a specific resistant lines

16 Loss of avirulence after effector silencing: (potential method of finding Avr genes) Leaves of cultivar Tres infected with rust race PST-78 No virus After control BSMV After BSMV::PSTha12O3 (different effector construct) After BSMV::PSTeTr No effects on Yr5 or Yr10 resistance or susceptible wheat lines Resistance in Tres is not well characterized

17 Loss of AvrRpg1 Avirulence from HIGS Steptoe rpg1 Morex Rpg1 Rpg1 + Virus Rpg1 + VPS9 & RGD HIGS Rpg1 + VPS9 HIGS Rpg1 + RGD HIGS Rpg1 Non Inc.

18 Assays for transient expression of effectors Biolistic delivery of AvrRxo1 Rxo1 Maize Bacterial delivery Plasmids GFP only GFP and AvrRxo1 Rxo1 rxo1 Other possibilities: Agrobacterium delivery? Protoplast transformation? Bacterial delivery via TTSS BSMV viral delivery Advantage of transforming a patch of cells: Can monitor cell death easily Can monitor gene expression or biochemical changes

19 Sohn et al. 2007, Plant Cell Expression and Delivery of Rust Genes One Version of an Effector Detector Vector; pedv6 Promoter 128 bp In planta cleavage site AvrRps4 N termini HA Effector ATG 136 aa Pseudomonas DC3000 AvrRPS4 HA Effector Plant Cell HR cell death Callose, other defenses

20 P. fluorescens strain EtHAn delivers bacterial effectors into wheat: DAB staining showing H 2 O 2 accumulation EtHAn strain EtHAn w/ genomic copy of avrrpt2 EtHAn w/ genomic copy of avrrpm1 EtHAn w/ pedv6 empty vector pedv6:: avrrpt2 pedv6:: avrrpm1 Mock (water)

21 P. fluorescens strain EtHAn delivering Stagonospora ToxA constructs; effects detected by callose staining pedv3 vector control pedv6::toxa Conclusions from Bacterial delivery experiments: Some Pseudomonas strains, like DC300, cause HR in wheat Pseudomonas species can deliver proteins to wheat cells Not clear if they will deliver enough protein of effectors to monitor HR We need more rust Avr genes to test the systems

22 Cooperators Nirmala J. A. Kleinhofs Stem rust effectors Chuntao Yin Project Mgr. Xianming Chen, P. striiformis genomics, genetics etc. Les Szabo S Ramachandran Conserved effectors. Sam Downey Wheat transf. M. Pumphrey Resistance eng.