SUPPLEMENTARY INFORMATION

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1 ARTICLE NUMBER: 643 DOI:.38/NMICROBIOL.6.43 A fungl pthogen secretes plnt lklinizing peptides to increse infection Sr Mschis, Dvid Segore, Dvid Turrà, Mercedes Leon-Ruiz, Ursul Fürst, Mennt El Ghlid, Guy Leonrd, Thoms A. Richrds, Georg Felix & Antonio Di Pietro NATURE MICROBIOLOGY

2 DOI:.38/NMICROBIOL Tomto root + Fusrium ph Tomto root Time (h) Supplementry Figure. F. oxysporum induces lkliniztion during infection of tomto roots. Roots of tomto plnts were immersed in er in the sence (empty circles) or presence (full circles) of F. oxysporum microconidi. Extrcellulr ph ws mesured t the indicted times (, P <., versus tomto root ccording to unpired Student s t-test) Error rs, s.d., n = 3 iologicl replictes. Experiments performed twice. NATURE MICROBIOLOGY

F-RALF inhiits root elongtion nd root hir groh in Aridopsis.. Primry sequence of F. oxysporum F-RALF.

Effect of F-RALF peptide on root groh nd morphology.

F-RALF peptide or with n equivlent volume of 5% (v/v) methnol (control).

3 DOI:.38/NMICROBIOL.6.43 MKFSIITLSLITLASAAPAAKPQSGEISYGALNRDHIPCSVKGASAANCRPGAEANPYNRGCNAIEKCRGGVGGN c MS+MethOH MS+Rlf µm Control µm F-RALF Control µm F-RALF ph 5.5 ph 6.8 ph 5.5 ph 6.8 Root length (mm) Control F-RALF Root length (mm) Control F-RALF ph 5.5 ph 6.8 Control µm F-RALF Supplementry Figure. F-RALF inhiits root elongtion nd root hir groh in Aridopsis.. Primry sequence of F. oxysporum F-RALF. Arrow indictes the clevge site of the secretion signl peptide predicted y SignlP., c. Effect of F-RALF peptide on root groh nd morphology. Seedlings of Aridopsis (Col-) were incuted for two dys in ½ MS medium supplemented with the indicted concentrtion of F-RALF peptide or with n equivlent volume of 5% (v/v) methnol (control). In (c) the medium ws supplemented with mm MES uffered to ph 5.5 or 6.8. Upper pnels: Representtive plnts from ech tretment were imged. Scle r mm. Middle pnels: Men length of roots ws mesured (, P <., versus control ccording to unpired Student s t-test). Error rs, s.d.; n = per tretment. Lower pnel: close up photogrphs of roots. Scle r.5 mm. Experiments performed twice. NATURE MICROBIOLOGY 3

trnscript level... # # #3 # #6 #7. 595 p +Ptef::f-rlf +Ptef::f-rlf(IA) Supplementry Figure 3. Genertion of f-rlf null mutnts, complemented nd overexpressing strins.

Genomic DNA of the wild type nd four independent trnsformnts ws treted with the restriction enzymes ScI (left pnel) or HindIII (right pnel), seprted on.

4 DOI:.38/NMICROBIOL.6.43 M ectopic #35 #4 #73 #5 M ectopic #35 #4 #73 # p 486 p 363 p 4839 p d Ptef-rlf-for tef- promoter 5p c +Ptef::f-rlf f-rlf f-rlf termintor Rlf-nest-rev() +Ptef::f-rlf(IA) f-rlf reltive trnscript level... # # #3 # #6 # p +Ptef::f-rlf +Ptef::f-rlf(IA) Supplementry Figure 3. Genertion of f-rlf null mutnts, complemented nd overexpressing strins.. Identifiction of deletion mutnts y Southern lot nlysis. Genomic DNA of the wild type nd four independent trnsformnts ws treted with the restriction enzymes ScI (left pnel) or HindIII (right pnel), seprted on.7% grose gel, trnsferred to nylon memrne nd hyridised with DNA proe corresponding to the 5' flnking region of the f-rlf gene. Trnsformnts showing nding ptterns consistent with ectopic (ectopic) or homologous integrtion () re indicted. Moleculr sizes of the hyridizing frgments re on the left.. Physicl mp of the Ptef::f-rlf overexpression construct. The promoter of the F. oxysporum tef- gene (Trnsltion Elongtion Fctor lph-) ws fused either to the wild type f-rlf llele with its termintor (Ptef::f-rlf), or to point-mutted f-rlf llele (Ptef::f-rlf(IA)). c, d. Anlysis of +Ptef::f-rlf (left pnel) nd +Ptef::f-rlf(IA) overexpressing strins. c. Genomic DNA of three independent trnsformnts for ech construct ws used s templte for PCR with the primer pir Ptef-rlf-for + Rlf-nest-rev() (indicted in ). Presence of n mplifiction product indictes the presence of the construct in the genome of the recipient strin. d. Trnscript levels of the f-rlf gene were mesured y rt RT-qPCR of cdna otined from mycelium of the indicted strins grown for h in liquid miniml medium. Trnscript levels were clculted y the ΔΔCt method nd normlized to the F. oxysporum ctin gene (, P <., versus wild type ccording to unpired t- test). Error rs, s.d., n = 3 iologicl replictes from one representtive experiment. Experiments performed twice. 4 NATURE MICROBIOLOGY

DOI:.38/NMICROBIOL.6.43 PDA ph 9 PDA ph 6.

response to osmotic nd cell wll stress or

. Colony phenotypes of the indicted strins

indicted ph with 5 mm MES, on yest peptone

soritol or of the cell wll perturing compounds

y growing fungl colonies for four dys on miniml

5 DOI:.38/NMICROBIOL.6.43 PDA ph 9 PDA ph 6.5 PDA ph 4 YPD YPD +Soritol YPD+CFW YPD+CR +f-rlf +f-rlf On top of cellophne ( d) Penetrted through cellophne (+ d) Supplementry Figure 4. Loss of F-RALF in F. oxysporum does not ffect vegettive groh, response to osmotic nd cell wll stress or cellophne invsion.. Colony phenotypes of the indicted strins grown on potto dextrose gr (PDA) djusted to the indicted ph with 5 mm MES, on yest peptone dextrose gr (YPD) in the sence or presence of M soritol or of the cell wll perturing compounds Clcofluor White (4 µg/ml) or Congo Red (5 µg/ml). Pltes were spot-inoculted, incuted for 4 dys t 8ºC nd scnned.. Penetrtion of cellophne memrnes ws determined y growing fungl colonies for four dys on miniml medium pltes covered y cellophne memrne (on top of cellophne). The cellophne with the fungl colony ws removed nd pltes were incuted for n dditionl dy to visulize the presence of the fungus (penetrted through cellophne). Experiments performed twice. Scle r 4 mm. NATURE MICROBIOLOGY 5

ph 7 #73 Dys fter infection 3 Nonuffered Uninoculted MES ph 7 Supplementry

Virulence of the mutnt is prtilly restored y exogenous lkliniztion.

suspension of 5 x 6 freshly otined microconidi/ml of the indicted strins,

6 % survivl DOI:.38/NMICROBIOL.6.43 Non-uffered control 8 Non-uffered 6 ph 7 control Non-uffered #73 ph 7 4 ph 7 #73 Dys fter infection 3 Nonuffered Uninoculted MES ph 7 Supplementry Figure 5. Virulence of the mutnt is prtilly restored y exogenous lkliniztion.. Kpln-Meier plot showing survivl of tomto plnts infected with F. oxysporum f. sp. lycopersici. Groups of plnts (cultivr Monik) were inoculted y dipping roots in suspension of 5 x 6 freshly otined microconidi/ml of the indicted strins, plnted in minipots nd ered either with unuffered er or with mm MES djusted to ph 7. Mortlity cused y the mutnt nd the wild type strin ws significntly higher t ph 7 (P <.) ccording to log-rnk test. Dt shown re from one representtive experiment. Experiments performed twice.. Representtive plnts from ech tretment were imged dys fter inocultion. 6 NATURE MICROBIOLOGY

7 DOI:.38/NMICROBIOL.6.43 Root Tomto, dy 5 Stem Rel. trnscript level 3 PR- GLUB CHI3 Reltive trnscript level PR- GLUB CHI3 H O Rel. trnscript level. CEVI- Rel. trnscript level. CEVI- #73 #5 +f-rlf Aridopsis, dy Rel. trnscript level WRKY53 Rel. trnscript level PDF. H O #73 +f-rlf Supplementry Figure 6. Loss of F-RALF in F. oxysporum results in incresed ctivtion of plnt defense responses.. Trnscript levels of tomto defense-relted genes PR- (Pthogenesis-relted protein ), GLUB (sic β-,3- glucnse), CHI3 (cidic chitinse) nd CEVI- (pthogen-induced nionic peroxidse) were mesured y rt RTqPCR of cdna otined from roots nd stems of tomto plnts t 5 dys fter inocultion with the indicted fungl strins or from the non-inoculted control (H O).. Trnscript levels of Aridopsis immunity mrker genes WRKY53 (slicylic cid-responsive) nd PDF. (jsmonic cid-responsive) were mesured y rt RTqPCR of cdna otined from plnts t dys fter inocultion with the indicted fungl strins or from the noninoculted control (H O). Trnscript levels for ech smple were clculted y the ΔΔCt method, normlized to the tomto GADPH or the Aridopsis ACTIN gene, respectively, nd expressed reltive to the non-inoculted control (H O) (, P <., versus ccording to unpired t-test). Error rs, s.d., n = 3 iologicl replictes from one representtive experiment. Experiments performed twice. NATURE MICROBIOLOGY 7

8 DOI:.38/NMICROBIOL.6.43 fer-4 Col- ph 6.8 ph 5.5 ph 6.8 Root length (mm) ph ph 5.5 Col- ph 6.8 fer4 Time (h) : : 4: 6: 8: 9: : :3 : Col- DIC GFP AF GFP AF fer-4 DIC GFP AF GFP AF Supplementry Figure 7. FERONIA medites F-RALF-triggered inhiition of root elongtion nd of plnt immunity.. FERONIA medites F-RALF-triggered root groh rrest vi lkliniztion. Seedlings of A. thlin wild type (Col-) nd fer-4 mutnt were incuted for two dys in ½ MS medium uffered t the indicted ph with mm MES. Left pnel: representtive plnts from ech tretment were imged. Right pnel: Men length of roots ws mesured (, P <., versus control ccording to unpired t-test). Brs, s.d.; n = per tretment. Experiments performed twice. Scle r, mm.. F. oxysporum infection in the fer-4 mutnt is locked y the plnt immune response. Roots of Aridopsis Col- nd fer-4 plnts were inoculted with microconidi of Foc constitutively expressing green fluorescent protein (GFP) nd imged over the indicted time period strting t h post-inocultion (Time ). DIC, differentil interference contrst; AF, red root utofluorescence. Arrows point to ppressorium-like fungl structures formed t sites of cell wll penetrtion. Arrowhed points to region of the fungl hyph undergoing cell deth cused y the plnt defense response (visile y red AF). Experiments performed twice. Scle r, µm. 8 NATURE MICROBIOLOGY

ntiody: nti-mouse-α-tuulin 55 α-tu 4 6 proe: 5' ﬂnking region of f-rlf 7 5 4 3 7 5 4 3

Full imges of western nd Southern lots included in figures.

Arrows point to hyridising nds corresponding to the indicted proteins.

Note tht the nti-phospho-p44/4 MAPK ntiody lso recognizes the phosphorylted form of the

solution with the nti-fus3 ntiody lso contined nti-mpk ntiody for simultneous detection of

9 DOI:.38/NMICROBIOL.6.43 ntiody: nti-phospho-p44/4 MAPK 55 P-Fmk 4 6 ntiodies: nti-fus3 + nti-mpk 55 Fmk 4 6 ntiody: nti-mouse-α-tuulin 55 α-tu 4 6 proe: 5' ﬂnking region of f-rlf NATURE MICROBIOLOGY Supplementry Figure 8. Full imges of western nd Southern lots included in figures.. Full imges of western lots included in Figure c. Antiodies used re indicted on the right. Arrows point to hyridising nds corresponding to the indicted proteins. Reltive positions of moleculr size mrkers (kd) re on the left. Note tht the nti-phospho-p44/4 MAPK ntiody lso recognizes the phosphorylted form of the cell wll integrity MAPK Mpk which hs higher moleculr weight thn Fmk; tht the hyridiztion solution with the nti-fus3 ntiody lso contined nti-mpk ntiody for simultneous detection of oth MAPKs; nd tht the nti-mouse-αtuulin ntiody used s loding control detects numer of unspecific nds in the F. oxysporum protein extrct, esides the 5 kd α-tuulin nd.. Full imges of the Southern lots included in Supplementry Dt Figure 3. Moleculr size mrkers (k) re on the left. 9

10 DOI:.38/NMICROBIOL.6.43 Supplementry Tle 3. Oligonucleotides used in this study. Primer Sequence 5 à3 Use M3-for CGCCAGGGTTTTCCCAGTCACGAC HygB/Phleo cssettes M3-rev AGCGGATAACAATTTCACACAGGA HygB/Phleo cssettes PHL AGTTGACCAGTCCGTTCCG Phleomycin resistnce LEO GCCACGAAGTGCACGCAGTT Phleomycin resistnce HygY CGTTGCAAGACCTGCCTGAA Split mrker HygG GGATGCCTCCGCTCGAAGTA Split mrker rlf-for ATCCCTCATCACTCTCGCTTC f-rlf knockout, rel time qpcr rlf-rev AATGTCGTGGTGGTGTTGGTG f-rlf knockout rlfup-for CACTCCTTGAACTCCTCTTGC f-rlf knockout rlfup-rev GTCGTGACTGGGAAAACCCTGGCGGAAGAGGCT f-rlf knockout GCTGAGTGAAAG rlfdo-for TCCTGTGTGAAATTGTTATCCGCTGAGAGGACCC f-rlf knockout ATCAAGTTTGG rlfdo-rev GACGCCAACAGTGAAAGAAGC f-rlf knockout rlf-for-nested TGGCGTGGTTTCATGTTGCTG f-rlf knockout rlf-rev-nested CCGCCACTTTAGTCTTTCCGA f-rlf knockout rlf-forrace ATGAAGTTCTCTATCATTACATTAT 3 RACE PCR Poly-T-Rce GCTCGCGAGCGCGTTTAAACGCGCACGCGTTTTT 3 RACE PCR TTTTTTTTTTTTT Rev-Rce-A GCTCGCGAGCGCGTTTAAAC 3 RACE PCR Rev-Rce-B GCGTTTAAACGCGCACGCGT 3 RACE PCR Ptef-for TCCACAGTGGCTGGACATGAT Ptef-rev TGTCGAGGAGAGTACTCACAG Ptef-RALF-for TGTGAGTACTCTCCTCGACAATGAAGTTCTCTATC ATTACATTATC Genertion of +Ptef::f-rlf nd Rlf-rev AATGTCGTGGTGGTGTTGGTG +Ptef::f-rlf(IA) strins Pteft-for () AACACACAGGCGCAAGACCAA Rlf-nest-rev CCGCCACTTTAGTCTTTCCGA Mut-rlf-for CGGTGAGGCCTCATATGGTGC Mut-rlf-rev CCATATGAGCGCTCACCGCTC rlf-rt-rev GTCCTCTCTTAGTTGCCACCA Rel time qpcr primer (f-rlf) ct-q7 ATGTCACCACCTTCAACTCCA Rel time qpcr primer (ctin) ct-q8 CTCTCGTCGTACTCCTGCTT Rel time qpcr primer (ctin) pr-7 GCATCCCGAGCACAAAACTA Rel time qpcr primer (PR) pr-8 TGGTAGCGTAGTTATATCTG Rel time qpcr primer (PR) glub-7 ATTCTGTTTATGCTGCGATGG Rel time qpcr primer (GLUB) glub-8 CTTTCTCGGACTACCTTCTTT Rel time qpcr primer (GLUB) chi3-5 TCTTGTCTCTTTTTCTTGTTCC Rel time qpcr primer (CHI3) chi3-6 GCAGTATCATCACCAGCAGT Rel time qpcr primer (CHI3) gdph- TGATTTGAACTCGTCGCAG Rel time qpcr primer (GADPH) gdph- CCAAAAACAGTAACAGTAACAGCCTTC Rel time qpcr primer (GADPH) six-- ATAGCATGGTACTCCTTGGCG Rel time qpcr primer (six) six-- CCTGATGGTGACGGTTACGAA Rel time qpcr primer (six) cevi-forrtpcr TCCATTTGAAAGCCT Rel time qpcr primer (CEVI) cevi-revrtpcr AAGTCTTTGTTGAAA Rel time qpcr primer (CEVI) Actin-for TCCCTCAGCACATTCCAGCAGAT Rel time qpcr primer (ACTIN) Actin-rev AACGATTCCTGGACCTGCCTCATC Rel time qpcr primer (ACTIN) PDF.-for TGTTCTCTTTGCTGCTTTCGACGC Rel time qpcr primer (PDF.) PDF.-rev TGTGTGCTGGGAAGACATAGTTGC Rel time qpcr primer (PDT.) WRKY53-for GCGACAAGACACCAGAGTCA Rel time qpcr primer (WRKY53) WRKY53-rev ACCGTTGGATTGAACCAGTC Rel time qpcr primer (WRKY53) FRK-for GGAAGCGGTCAGATTTCAAC Rel time qpcr primer (FRK) FRK-rev AGCTTGCAATAGCAGGTTGG Rel time qpcr primer (FRK) NATURE MICROBIOLOGY

Interplay between NS3 protease and human La protein---- by Ray and Das Supplementary fig 1. NS3 pro

Interplay between NS3 protease and human La protein---- by Ray and Das Supplementary fig 1. NS3 pro Interply etween tese nd humn L protein---- y Ry nd Ds Supplementry fig 1 1 2 3 4 UV crosslinking ssy: α[ 32 P]UTP leled HCV IRES RNA ws UV-crosslinked to incresing concentrtions (0.1, 0.2 nd 0.4µM) in