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Shen et al., 2002 Mol MicroGoes da Silva et al., 2004 MPMIPruitt, Bahar et al., unpublished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