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

Stripe (yellow) Stem rust Leaf rust

P. striiformis P. graminis P. triticina

Stripe Rust is Dominant in the PNW, but Others Threaten

A transgenic approach would ideally address all three rusts

AND be durable

Effector proteins

Molecular interactions between rust and plant cells

PAMPs Plant cell

Rust hyphae

Antimicrobial responses &

Cell Death

Effector recognition

by R protein; e.g.

NB-LRR

PAMP triggered

defenses

PAMP recognition

Effector target

Can we affect the interaction

by manipulating the pathogen

proteins?

Haustorium

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

Expression of the Pst5a23 gene in nine

seedlings infected with the Pst5a23 construct

Seedling # VIGS/ virus control

1 0.63

2 0.14

3 0.38

4 1.38

5 0.42

6 0.37

7 0.28

8 0.12

9 0.43

• Haustorial EST; predicted 108 amino acid secreted protein (effector stereotype)

• Expression: infected leaf/urediniospores 3617:1

haustoria/infected leaf 29:1

Host- Induced Gene

Silencing (HIGS) ranged

from 0 to 8-fold in different

seedlings

Initial P. striiformis silencing result:

Jim Jurgenson, UNI

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±0.17 1.17±0.49

PSTha5A23 28 5/9 0.48±0.20 1.02±0.37

PSTha12H2 90 2/4, 2/6 0.64±0.11 1.20±0.24

PSTha2A5 92 1/6, 2/5, 0/6 0.44±0.18 1.73±2.54

PSTha5A1 >100 2/6, 5/6 0.55±0.16 1.15±0.28

PSTha12O3 12 6/6, 6/6 0.30±0.22 1.24±0.46

PSTha9F18 >100 2/6, 1/6 0.48±0.31 1.50±1.75

PST β-tubulin 1.0 0/6, 2/6 0.80±0.12 1.11±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±0.08 1.10±0.11

PGTEPSPS 2.8 0/6 - 1.00±0.13

Silencing results with additional P. striiformis genes

Yin et al. (2011) MPMI 24:554-

An explanation for the HIGS results

Photo by Zhensheng Kang

Silencing signal does

not enter hyphae

Silencing signal enters

haustorium

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

Puccinia Group Database

Puccinia genome projects

Genome Size %GC Genes

P. graminis tritici 88.64 Mb 43.35 20,567

P. triticina 162.95 Mb 46.34 11,638

P. striiformis coming soon

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:

• 1182 candidates for haustoria-enriched

proteins: 198 characterized proteins, the rest

predicted proteins

Purified haustoria

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

McNair

McNair + BSMV::MCS

McNair infected by pgt7A

McNair + pgt7A +BSMV::MCS

Sr31 + pgt7A

McNair + pgt7A + BSMV::PGTG_11658

McNair + pgt7A + BSMV::PGTG_03590

McNair + pgt7A + BSMV::PGTG_01136

Silencing essential genes of Puccinia graminis in wheat

(picture taken at 12 after rust infection)

Stable transformants are coming (slowly)

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

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-

Can VIGS be used to identify effectors with Avr function?

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

Transient silencing of an Avr gene by HIGS should

increase specific virulence

Loss of avirulence after effector silencing: (potential method of finding Avr genes)

No virus

After control BSMV

After BSMV::PSTha12O3

(different effector

construct)

After BSMV::PSTeTr

Leaves of cultivar Tres infected with rust race PST-78

No effects on Yr5 or Yr10 resistance or susceptible wheat lines

Resistance in Tres is not well characterized

Rpg1

Non

Inc.

Steptoe

rpg1

Morex

Rpg1 Rpg1 +

Virus

Rpg1 +

VPS9

HIGS

Rpg1 +

VPS9 &

RGD HIGS

Rpg1

+ RGD

HIGS

Loss of AvrRpg1 Avirulence from HIGS

Rxo1 Maize

Plasmids

GFP only

GFP and

AvrRxo1

Assays for transient expression of effectors

Biolistic delivery of AvrRxo1 Bacterial delivery

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

AvrRps4 N termini Effector HA

In planta

cleavage site

ATG 136 aa

Promoter

128 bp

Sohn et al. 2007, Plant Cell

Expression and Delivery of Rust Genes

Pseudomonas DC3000

HA AvrRPS4 Effector

Plant Cell

One Version of an Effector Detector Vector; pEDV6

HR cell death Callose, other defenses

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)

P. fluorescens strain EtHAn delivers bacterial effectors into wheat:

DAB staining showing H2O2 accumulation

P. fluorescens strain EtHAn delivering Stagonospora ToxA constructs;

effects detected by callose staining

pEDV3 vector control pEDV6::ToxA48-537

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

Cooperators

A. Kleinhofs

Stem rust effectors

Nirmala J.

Les Szabo

Chuntao Yin

Project Mgr.

Xianming Chen, P.

striiformis genomics,

genetics etc.

M. Pumphrey

Resistance eng.

S Ramachandran

Conserved effectors. Sam Downey

Wheat transf.