The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a gram-negative bacterium

Benjamin SchwessingerAustralian National University

@schwessingerCOMBIO Melbourne

29/09/2015

Plants are constantly surrounded by microbes

VirusesBacteria

FungiOomycetes

Genetic control of Xanthomonas oryzae pv. oryzae

Gurdev Kush

UC Davis andInternational Rice Research Institutein Philippines (IRRI)

At least 37 major disease resistance loci against Xoo in rice-> Xa21 first cloned resistance gene in rice (Song et al. 1995)

1995: XA21 a rice receptor kinase conferring qualitative disease resistance to Xoo

WT XA21

Xoo infected rice

2014: XA21 defense signaling at the plasma-membrane

Park et al., PLoSBiology 2008Chen et al., PNAS 2010Chen, Zhou, Schwessinger et al., Molecular Plant 2014adapted from Macho and Zipfel, Molecular Cell 2014

Hypothesis: The XA21 recognized molecule encodes a small tyrosine sulfated type-I secreted protein

42-

Shen et al., 2002 Mol MicroGoes da Silva et al., 2004 MPMIPruitt, Bahar et al., unpublished

RaxX is REQUIRED to trigger XA21-mediated immunity

Pruitt, Schwessinger et al. 2015 Science Advances

Rice-Xoo disease assays

S S S

RaxX is sulfated and sulfation is required to trigger XA21 mediated immunity

Pruitt, Schwessinger et al. 2015 Science Advances

MS analysis of RaxX sulfation status in vivoRice-Xoo disease assays

S S

Pruitt, Schwessinger et al. 2015 Science Advances

+ RaxX-sY/Y heterolgously expressed or chemically synthesized

Defense gene expression Reactive oxygen production

RaxX is SUFFICIENT to trigger XA21-dependent defense responses in rice

Natural variant of RaxX evades XA21 mediated immunity

Pruitt, Schwessinger et al. 2015 Science Advances

Rice-Xoo disease assays

P44/P48are important for RaxX recognition by

XA21

SS S

Pruitt, Schwessinger et al. 2015 Science Advances

Credit Kelsey Wood

2015: RaxX triggers XA21 mediated immunity in rice

What is RaxX? What does it do?Can we engineer XA21 do recognize other RaxX variants?

Xoo population monitoring in the field

Acknowledgment

Ronald Lab at UC DavisRory Pruitt

Nick ThomasFurong LiuAnna Joe

DeeDee LuuOfir Bahar (now ARO, Bet-Dagan, Israel)

Arsalan Daudi (now bio-protocol.org, USA)Maw Sheng Chern

Randy Ruan….

Pamela Ronald

RaxX contributors and collaboratorsJürg Felix, Hubert Kalbacher and Markus Albert

(University of Tübingen, Germany)

Chris Petzold and Josh Heazelwood(JBEI/LBNL Berkeley, USA)

Jenny Brodbelt(UT Austin, USA)

Ramesh Sonti et al. (CCMB, Hyderabad, India)

Chang Liu (UC Irvine, USA)

Bing Yang and Kai-Jun Zhao(Iowa State University, USA,

and CAAS Beijing, China)

Jillian Lang and Jan Leach(Colorado State University, USA)

Silke Robatzek and Cyril Zipfel(Sainsbury Laboratory, UK)

Rathjen Lab at ANU

RaxX is SUFFICIENT to trigger XA21-dependent defense responses in rice

How to obtain sulfated and unsulfated RaxX peptide:-> express it in E.coli and purify to high-homogeneity -> order a short epitope produced by chemical synthesis

Collaboration with Chang Liu at UC Irvine

mRNA UAG

= Sulfotyrosine

= An engineered tRNA that decodes only the UAG codon and is charged only by the sulfotyrosine-specific synthetase

Translationinside E. coli

Site-specifically sulfated protein

Liu & Schultz, Nature Biotech., 2006Liu et al., Nature Protocols, 2009

= An engineered aminoacyl-tRNA synthetase specific for sulfotyrosine and the cognate engineered tRNA

RaxX is SUFFICIENT to trigger XA21-dependent defense responses in rice

Direct site-specific incorporation of sulfotyrosine

Xiang Li

UAGUAAUGA RF2

sulfotyrosine

UAGUAAUGA

ΔRF1

R2

RF2

Translation

Translation

Enhanced decoding of UAG codons in a recoded E. coli

AUC

RF2RF2

Lajoie et al., Science 2013

AUG UAA

UAAUAGAUG

RaxX is SUFFICIENT to trigger XA21-dependent defense responses in rice