Apprendre du pathogène pour établir des résistances durables et locales : Le cas de la bactériose vasculaire du riz en Afrique Boris SZUREK Montpellier France

1.Mini-intro 2.TAL Effectors : function and models 3.Biotech implications 4.The Rice – Xanthomonas pathosystem Outline

Jones et Dangl, The plant immune system. 444: Nature. Plant defense level HR PAMPS Basal resistance Specific resistance PAMP Loss of or acquisition of or modification of effectors Avr R receptor Avr R effectors Evolution Time Effector-Triggered Susceptibility (ETS) Effector-Triggered Immunity (ETI) Plant – Bacteria co-evolution Zig-Zag model

localize to host nucleus induce expression of host genes some are essential virulence factors TAL effectors TAL (Transcription Activator-Like) Type III effectors

Xanthomonas spp.TALs per strain X. campestris pv. campestris0-4 X. campestris pv. vesicatoria0-1 X. campestris pv. armoraciae0-3 X. citri1-4 X. campestris pv. malvacearum6-10 X. oryzae pv. oryzae7-16 X. oryzae pv. oryzicola12-26 Ralstonia solanacearum 1 TAL (Transcription Activator-Like) Type III effectors

Ineffective TAL- ETS Resistance Susceptibility } TAL effectors roles in plant disease and resistance

Boch et al. (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326: ; published online 29 October 2009 ( /science ). Moscou and Bogdanove (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326:1501; published online 29 October 2009 ( /science ). The TAL code revealed

: “hypervariable” 4 repeat types in AvrBs3 HD, NI, NG, NS modified from: Schornack et al. (2006) J. Plant Physiol. 163:256. AvrBs3 repeat types

Correlation between repeat types and UPA box UPA box consensus AvrBs3 COOHNH x 34 aa LTPEQVVAIASHDGGKQALETVQRLLPVLCQAH G NIN G NS 112/1334 T A T A T A A A C C T N N C C C T C T DNA HD NG NS NG NI NI NI HD HD NG NS NS HD HD HD NG HD NG Repeats 1.) 1 AvrBs3-repeat corresponds to 1 bp 2.) Most repeat types favor a specific bp

"Breaking the code" test the model! Effector – target sites AvrBs3 – Bs3, UPAs AvrBs3  rep109 – Bs3 AvrHah1 – Bs3 AvrBs3  rep16 – Bs3E AvrXa27 – Xa27 PthXo1 – Os8N3/Xa13 PthXo6 – OsTFX1 PthXo7 – OsTFIIA  1

Hax2, Hax3, and Hax4 from X. campestris Kay et al. (2005) MPMI 18: TAL effectors from Arabidopsis-pathogenic Xc, unknown specificity Hax3 and Hax4: 34-aa repeats Hax2: 35-aa repeats

Target specificity of Hax2, Hax3, and Hax4

Target specificity of repeat types

Artifical TAL effectors with novel specificities

Summary TAL effectors recognize target DNA with novel DNA-binding domain 1 repeat corresponds to 1 bp 2 hypervariable amino acids / repeat determine specificity Rearranging repeats yield new DNA-binding specificities (biotech)

The Crystal Structure of TAL Effector PthXo1 Bound to Its DNA Target Mak et al., Science, 2012

. TAL effectors are highly customizable DNA binding proteins. They can be fused to a nuclease and used for genome engineering (TALEN). They can be used as custom transcriptional activators. Other uses, including gene repression, point mutagenesis, and modification of epigenetic marks, can be envisioned. Tools exist for quick, cheap, and easy synthesis of TAL effector constructs. Many questions remain: - contribution of individual RVD-nucleotide associations to affinity - relation of length to specificity - biophysical nature of interactions (minimal DNA binding domain) Summary

The Rice-Xoo interaction

BLB infection areas Emergent infection areas Geographical distribution of BLB and BLS 1884 BLS infection areas Emergent infection areas 1918

Virulence - Susceptibility mechanisms Xo Diversity Improve Plant Resistance Molecular Typing tools Genomics Germplasm screening Genome-editing TAL effectors targets Pathotyping Field surveys  Develop locally-adapted disease control strategies

Les souches africaines de Xoo sont distinctes de celles d’Asie (Gonzalez et al., 2007, MPMI) Trois nouvelles races ont été identifiées en Afrique : A1, A2 and A3 Leur TALome est réduit par rapport à celui des souches asiatiques Xoo Asia Xoc Asia Xoc Africa Xoo Africa Caractéristiques des souches Africaines de Xoo AsiaAfrica Poulin L., 2014

5dpi 1cm Mock BAI3 BAI3ΔtalC [Os11N3ArTal1] BAI3ΔtalC [Os11N3ArTal2] Search for susceptibility genes of rice to Xoo 5 kb 4 kb 2 kb MAI1BAI3 3 kb Yu et al., MPMI, 2011 Major virulence TAL ? Systematic K.O. of BAI3 TALome

Suicide vector Kan R B3B3-1-1 ATG uidA Kan R Target gene Disappearance of a 3.7 kb-band in mutant B3-1-1

B3 B3-1-1 B3 Kitaake Moroberekan IR24 1: H20 2: BAI3 (WT) 3: dpi Azucena Loss of B3-1-1 virulence on susceptible rice lines

talC IS5 family Transposase ISXo17 family Transposase EcoRI IS1595 family Transposase oxidoreductase TalC is a new TAL with 21,5 repeats

5dpi 1cm Mock BAI3ΔtalC BAI3 BAI3ΔtalC [talC] BAI3ΔtalC [Os11N3ArTal1] BAI3ΔtalC [Os11N3ArTal2] talC is a major virulence TAL in Xoo strain BAI3 What are TalC targets ? 5 kb 4 kb 2 kb MAI1BAI3 3 kb talC Search for susceptibility genes of rice to Xoo

(JP Renou & S Balzergue, URGV, Evry) Affymetrix - 24 hpi - OD = 0,2 Validation by sqRT-PCR Search for TalC direct targets Microarray analysis of rice leaves challenged with : Xoo BAI3 (WT) vs. Xoo BAI3  talC Microarray analysis of rice leaves challenged with : Xoo BAI3 (WT) vs. Xoo BAI3  talC 128 genes are up-regulated 20 genes are down-regulated 128 genes are up-regulated 20 genes are down-regulated

Rank Search for TalC direct targets

SWEET/nodulin-3 proteins. ~20 members in rice (xa13 and xa25). Membrane Transporters. Glucose & sucrose export Talbot., 2010, Nature

pthXo3 SWEET14 avrXa7 talC TalC BAI3 PthXo3 PXO61 tal5 Tal5 MAI1 AvrXa7 PXO86 Philippines Burkina Faso …of different geographic origins and lineages Mali Susceptibility SWEET14 is targeted by multiple strains..

SWEET14 TalC BAI3 PthXo3 PXO61 Tal5 MAI1 AvrXa7 PXO86 Philippines Burkina Faso Mali ??? Loss of Susceptibility

Unique collection of 169 wild and cultivated rice accessions Sequencing of a 400-bp PCR product pthXo3 SWEET14 avrXa7 talCtal Search for polymorphism in the SWEET14 promoter

PATTERN 1 PATTERN 2 PATTERN 3 AvrXa7Tal5 Search for polymorphism in the SWEET14 promoter

Diapos sur le Système de Sécrétion de type III

Hrp : « hypersensitive reaction and pathogenicity » Essentiels pour: – les symptômes de la maladie chez la plante sensible – la croissance in planta Jours après inoculation 1 hrp + hrp - Log(bactéries/cm 2 ) Courbe de croissance dans une feuille de poivron sensible - la réaction d'hypersensibilité (HR) chez la plante résistante Poivron résistant HR hrp + hrp - Le système de sécrétion de type III

0BDEFHJKNQUVWXTCAI hrp genes hpaP Y CTNJ U V QRS hrc genes Cluster de gènes hrp de R. solanacearum : ~26 kb CTNJ U V QRS yscYersinia Les gènes hrp et ysc sont conservés Le système de sécrétion de type III

Anticorps : anti-HrpA marqué par anti-HrpA marqué par billes d’or 10 nm Anticorps : anti-HrpA marqué par anti-HrpA marqué par billes d’or 10 nm Anticorps : anti-HrpW marqué par anti-HrpW marqué par billes d’or 10 nm billes d’or 10 nm Anticorps : anti-HrpW marqué par anti-HrpW marqué par billes d’or 10 nm billes d’or 10 nm Le pilus hrp de Pseudomonas syringae pv. tomato (HrpA et HrpW) et la sécrétion de AvrPto Le pilus hrp de Pseudomonas syringae pv. tomato (HrpA et HrpW) et la sécrétion de AvrPto Anticorps : anti-HrpA (10 nm) anti-AvrPto (15 nm) Anticorps : anti-HrpA (10 nm) anti-AvrPto (15 nm) Les pili Hrp chez les bactéries phytopathogènes Roine et al. (1997) PNAS 94 : 3459; Jin et He (2001) Science 294: 2556_2558 Le système de sécrétion de type III

Structure du « Needle complex » (NC) de Shigella Negative staining of isolated NCs Blocker et al Mol. Microbiol. 39: Le système de sécrétion de type III

Paroi bactérienne Paroi végétale Devenir des effecteurs de type III dans la plante