M. oryzae Yashiromochi (Pita) Pi No.4 (Pita2) Fukunishiki (Piz) M. oryzae Ina168
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1 Supplemental Data Yoshida et al., (2009) Association genetics reveals three novel avirulence genes from the rice blast fungal pathogen Magnaporthe oryzae. M. oryzae 70-15????????????? Sasanishiki (Pia) Kakehashi (Pii) Kanoto 51 (Pik) Tsuyuake (Pik-m) Fukunishiki (Piz) Yashiromochi (Pita) Pi No.4 (Pita2) Toride 1 (Piz-t) K60 (Pik-p) BL 1 (Pib) K59 (Pit) Moukoto (-) Shin-2 (-) R R R R R S R R S R R S S M. oryzae Ina168 Supplemental Figure 1. Ambiguous symptoms caused by isolate infected to various rice cultivars. Each isolate was sprayed onto the leaves of diagnostic rice cultivars differentially harboring eleven known resistance (R-) genes, Pia, Pii, Pik, Pik-m, Pik-p, Piz, Pita, Pita2, Piz-t, Pib, Pit cognate to the AVR genes (Kiyosawa, 1986) as well as susceptible cultivars Moukoto and Shin- 2, and compatible or incompatible interaction was observed among them. It is notable that in our experiments, the reaction between the isolate and these eleven rice R-genes could not be precisely defined since the disease symptoms caused by were not clear, and we could not assign each reaction as compatible or incompatible. In addition, inoculated Moukoto and Shin-2 also showed unclear symptoms. On the contrary, Ina168 induced typical susceptible spindleshaped lesions on Yahiromochi, K60, Moukoto and Shin-2 leaves, and typical resistance symptoms (few small brown spots) on the leaves of the other cultivars. Photos were taken 10 days after inoculation with conidia/ml (containing 0.01 % tween 20) of or conidia/ml (containing 0.01 % tween 20) of Ina168.
2 1000 Number of secreted protein genes AVR-Pita θ (x10-3 ) Supplemental Figure 2. Frequency distribution of nucleotide diversity among 1,032 loci of M. oryzae putative secreted protein genes. Nucleotide diversity (θ;watterson 1975) is given in x-axis and number of loci in the given θ range is shown in the y-axis. Out of 1,032 loci, 805 were monomorphic. of AVR-Pita (MG ) is
3 Supplemental Figure 3. Size distribution of predicted proteins of M. oryzae. Size 11,109 predicted proteins of Isolate (blue), 1,306 predicted secreted proteins of iso and 316 predicted secreted proteins in Ina168-unmapped regions (yellow).
4 a) pex22 ATCGCTTTGC CCTCATTGCA CACAACAACC TCCATTAAAA TACATACATA TTTCTATTCC 60 CTTTCCATAA CAGTATCCCT ACCCGCCAGC CCCATACACG AAAATTCTCA AAATGCATTT 120 TTCGACAATT TTCATCCCCT TTGCCTTAGC TGCTCTAAAA GTAAGCGCTG CGCCAGCTAG 180 ATTTTGCGTC TATTACGACG GCCACCTTCC CGCGACACGT GTCCTGCTTA TGTACGTTAG 240 AATCGGCACT ACAGCGACTA TTACGGCCCG TGGGCACGAA TTCGAAGTTG AAGCAAAAGA 300 CCAGAATTGC AAAGTTATTC TCACCAATGG CAAACAAGCA CCGGATTGGC TTGCTGCCGA 360 GCCTTACTAG GTTTAAAACG GAAGAGCAGC AGCTTGCTGG TTAAACTTCA AAATAAACGC 420 CAACTCAGTT ACTACTTACA ATACTATGAT TTCTGCCGAA AATTCCCTCT CCCCCTACCG 480 TACGTGTATA CCTGGGCTTT TACCAGGTTT ACTGTTTACG GGAGGAATCA AGTACCTAAA 540 TTTATTTTTT CCTTCAAAAT GATTACCAAA GTCGGAAACG CCCACAAGGC CTACACCACT 600 CGACGATGAA ACCAGGGTAG GCTTTTGTTA CCTGTGCAAT CATGGCAAAT TATCACTCTT 660 TAGCGAAAGT AAATACAAAC AAACGAGTTT CCAGG 695 Tag counts b) pex33 ATTTTTCATC TTAAAACTAA ACATTTAAAT TCAGCTTCAA ATATACCAAA ATCCCCTTTT 60 ATTCCTTCCA ATTTACCAAA ATGCAACTTT CCAAAATTAC TTTCGCTATT GCATTATATG 120 CAATCGGAAT CGCAGCACTT CCCACTCCGG CCAGCCTGAA TGGCAACACT GAGGTCGCAA 180 CCATCTCCGA CGTTAAACTT GAGGCCCGCA GCGACACCAC TTATCATAAA TGCTCCAAAT 240 GCGGTTATGG CAGCGATGAT TCCGACGCGT ATTTTAATCA TAAATGCAAC TAATCGCATA 300 CAAAGTAAGC TGGATTTTTC CAAACGAGCG GCGGACAGTT ATGGACTATT GCGGGAATTT 360 CAAGGATAGG AAAAGCCAAT TTGAAATATC CGGAATACTT TTTCCAAGCT TAACCATTAA 420 ATAACATGTC TTAAATCAAT CTACCTTTCG CGTTTAAA 458 Tag counts c) pex22 pex33 pex31 Supplemental Figure 4. pex22, pex33, pex31 transcripts are expressed during M. oryzae infection of rice leaf sheath as revealed by SuperSAGE and 3 -RACE RT-PCR. a) DNA sequence of the pex22 gene region. Pex22 ORF is indicated by red color. Location of the SuperSAGE tag Tag3294 is underlined. b) DNA sequence of the pex33 gene region. ORF is indicated by red color. Location of the SuperSAGE tag Tag1145 is underlined. c) 3 -RACE
5 pex22 Ina (+contig264) 1 2 Ina (+22p::pex22) 1 2 Ina Mg-Actin pex33 Mg-Actin Sasa2 (+contig389) Sasa2 (+22p::pex33) Sasa2 pex31 Sasa2 (+22p::pex31-D) Sasa2 (+pex31-dgenome) Sasa2 Mg-Actin Supplemental Figure 5. Confirmation of active transcription of pex transgenes by RT-PCR in M. oryzae transformants during infection.
6 a) O. sativa M. oryzae Sasanishiki Shin-2 (Pia + ) (Pia - ) TH TH (+contig264) b) O. sativa M. oryzae Kakehashi Shin-2 (Pii + ) (Pii - ) Ina Ina (+contig389) Ina (+22p::pex33) c) O. sativa M. oryzae Kanto51 (Pik + ) Tsuyuake (Pik-m + ) K60 (Pik-p + ) Shin-2 (Pik -, Pik-m -, Pik-p - ) Ina72 Ina72 (+pex31-d-genome) Ina72 (+22p::pex31-D) Supplemental Figure 6. Transformation of M. oryzae isolates with pex22, pex33 and pex31-d complements AVR-Pia, AVR-Pii and AVR-PIk/km/kp, respectively. Names of isolates and DNA fragments used for transformation are given in the left of each panel. (a) Transformation of TH with pex22 conferred the AVR-Pia phenotype. (b) Transformation of Ina with pex33 conferred the AVR-Pii phenotype. (c) Transformation of Ina72 with pex31 conferred the AVR-Pik/km/kp phenotypes.
7 a) O. sativa M. oryzae Sasa2 Hitomebore (Pii + ) Moukoto (Pii - ) WT +contig p::pex33 b) empty Relative Luciferase Activity pex22-ns pex33-ns 0 0 Akitakomachi (Pia +, Pii + ) Akitakomachi (Pia +, Pii + ) Supplemental Figure 7. Additional results of interactions between M. oryzae and rice. a) Results of interaction between M. oryzae and rice. The isolate Sasa2 is compatible with a rice cultivar Hitomebore harboring the R-gene Pii. Sasa2 transformed with contig389 (+contig389) as well as Sasa2 transformed with a fragment containing pex22 promoter fused with pex33 ORF (+22p::pex33) were incompatible with Hitomebore. Sasa2, [Sasa2(+contig389*)], [Sasa2 (+22p::pex33)] are all compatible with a rice cultivar Moukoto lacking Pii suggesting that the effect of transformation with contig389 and 22p::pex33 is Pii dependent. Photos were taken 10 days after inoculation with conidia/ml (containing 0.01 % tween 20) of each strain. b) Interaction
8 Ina168 GFSI1-7-2 Mb Supplemental Figure 8. Pulsed field gel electrophoresis images of chromosomes of Ina168 and GFSI1-7-2 isolates of M. oryzae. The typical 1.2 Mb supernumerary chromosome of GFSI1-7-2 is indicated by an arrowhead.
9 AVR-Pita Magnaporthe isolates?-? ????? ?? 1350 bp Magnaporthe iso?-? ????? bp 650 bp 600 bp 565 bp 530 bp 500 bp 495 bp 460 bp 400 bp 364 bp 350 bp 300 bp 255 bp 204 bp 200 bp 145 bp 100 bp IR DYE 700 IR DYE 8 Supplemental Figure 9. EcoTILLING result of AVR-Pita. The phenotypes of AVR-Pita (MG ) (top). The bands in red rectangles are significantly associated with the AVR-Pita phenotypes (Fisher s e
10 Supplemental Table isolates of M. oryzae used for EcoTILLING and phylogenetic analysis Code Code no. Isolate Host Race Country no. in Table Oryza sativa Guiana 2 GUY11 Oryza sativa Guiana Oryza sativa Guiana Br13 Oryza sativa 102 Brazil 5 15 Br18 Oryza sativa 176 Brazil 6 22 Br10 Oryza sativa 403 Brazil 7 Br15 Oryza sativa 507 Brazil 8 VHT6.1 Oryza sativa 002 Vietnam 9 VHG4.5 Oryza sativa 106 Vietnam 10 VTB6.1 Oryza sativa Vietnam 11 VHT3.3 Oryza sativa Vietnam 12 PO Oryza sativa Indonesia 13 PO Oryza sativa Indonesia 14 PO Oryza sativa Indonesia 15 PO Oryza sativa Indonesia 16 PO Oryza sativa Indonesia 17 TH3 Oryza sativa 135 Thailand 18 CHNOS Oryza sativa China 19 5 Shin85.86 Oryza sativa Japan 20 TH Oryza sativa Japan 21 Ken54-04 Oryza sativa Japan 22 Ken54-20 Oryza sativa Japan Oryza sativa 003 Japan 24 TH Oryza sativa Japan 25 Ina Oryza sativa Japan Ina Oryza sativa Japan Hoku1 Oryza sativa Japan Oryza sativa 007 Japan
11 Oryza sativa 007 Japan Oryza sativa Japan 31 Naga Oryza sativa 007 Japan TH87-20-BII Oryza sativa Japan 33 6 Ina72 Oryza sativa Japan Oryza sativa 033 Japan TH Oryza sativa Japan 36 7 TH Oryza sativa Japan Oryza sativa Japan Oryza sativa Japan Sasa2 Oryza sativa Japan 40 8 TH69-8 Oryza sativa Japan 41 1 Ina168 Oryza sativa Japan 42 Ken53-33 Oryza sativa Japan TH78-15 Oryza sativa Japan P-2b Oryza sativa Japan A Oryza sativa 433 Japan 46 P2 Oryza sativa 003 Japan
12 Supplemental Table 2. A list of M. oryzae putative secreted proteins possessing the [RK]CxxCxxxxxxxxxxxxH] motif >gi_ _ref_xp_ _ predicted protein [Magnaporthe grisea 70-15] MQISVSQLVTLAGLIMGASARLHSAAVCVQNRSYGSTGNGTPQGITYGSYVDYEIDSAATQCACNFYRNR HTGNNQWDSCPDCVFDGIQCLSNGWHLGGDEITYYCEKLCNSQGAQAN >gi_ _ref_xp_ _ hypothetical protein MG [Magnaporthe grisea 70-15] MQIFNIVQVLGLLAVGASALPTPANVGAVQPVEGSQLQARSSNFYSAGWTQYPSANSGYPSNSASTYYYK CNHCGKHTKEESQQKYHQENSHKGKESNYSEVQA >gi_ _ref_xp_ _ hypothetical protein MG [Magnaporthe grisea 70-15] MQIFKIVQVLGLLAVGTSALPAPANPAAVQPAQGGQLAVHGQPASCPPECRDVQGAKPGHLQARSRFYAT SDTGRPLANSGYPTHGYRDAYRCLYCGAVRDDVSAVQDHITYRHSNRGGDTSNYDTTTVRDDR >gi_ _ref_xp_ _ predicted protein [Magnaporthe grisea 70-15] MHTYKFIQIALLFASVALAIPTPPSPPNPPPVPQLPNSETKSNRLVSHSCEFCGVVKPSGPAYLEHYHQN HREEVWGKLATPSPPNPPPVPTQKVETHAPKTHGCEWCNKVEPSGPAYIKHYKENHEDQVWGKWAGQDAK ASP >gi_ _ref_xp_ _ predicted protein [Magnaporthe grisea 70-15] MHLSTVSQILALFTAVASSAPTSHAAVRARHVSPPEADLLHVVKRGDDTDFSDWQRYSLWNKRVEALSRN PELVSRYGYKCNSCGKGRMDENGMRRHIMFDHYSKDIPLEKSAHVNKYIEFRNKGG >gi_ _ref_xp_ _ predicted protein [Magnaporthe grisea 70-15] MKYSKVSFTLALIALGATAAPTDLHKLVARGEVEVCYDPRDPRVPPLQNDPNVEKWIDDKTGHHCFALMR GVQPDHPCKACQGHQADLYLDHIHCLAAGHVHANKGDSCEATRQARLDRYKTALKGFRVPYGTDISKLET GGKGVTFNVNDPSHKLFSGDRSLTQVVKDGNVEFVRPHGEKYRGQGEDLYATIQRQWYALKAAKILKAAR AAKAARTPDASDAGKLEPPAKLATQQRRLDESRSAE >gi_ _ref_xp_ _ predicted protein [Magnaporthe grisea 70-15] MQLHNVCSILALLAAGVFALPAPVNPSEIQARSAADKAKPKPQTERVWMDEPEAAQYLSDPRWKLVKKKY SCGYCDSSSSKVDKINKHRDEVHGTRQAQVDHTIYPGETRLTFERVSGYP >gi_ _ref_xp_ _ predicted protein [Magnaporthe grisea 70-15] MLPLFLVTSLFLTQFLAAAHTENFEIVKGLADKARVKLDLTGQQSYAIYEKFPAKVYNPLDYHMRLIVGH VECKDDNDCDFAANAFHMHAHGGSCERVLGSKTNQHSRNRPWKANHYYDKKEDIDKPLPQKNRYQWAGPT RTLSYDEIYALGDKWCGSCFHSRYNKVFNNCHHFVYSLYNKIKQK >gi_ _ref_xp_ _ predicted protein [Magnaporthe grisea 70-15] MRITRALAPLGLLPLPAAAAAVVGIPLQAIDTTATASSAMEPRQAGDGSGGSLKQCRDVSLVQSRPGLSL NDTVSATCHVGTAEYVNTLNLNECLGVDPATLELGWGRAGQLSSFCWGCELSELETGGPDGHLAVGRVVF SCVCYDYGQERATSVRLDDGGIESKDGVLACRNGKRATAIRMNPDAPVRTEPGRCNLGLGDMGYMDCIPK LYDGKIVPVGERNRYGSVFNDWRGD
13 Supplemental Table 3 A list of plasmids used for the genetic transformation of M. oryzae. Plasmid name Sources Description Construction methods M. oryzae isolates used for transformation pcb1004-pex22 pcb Kb fragment containing pex22 The 2.2 Kb fragment was amplified with the primers NotI-pex22-U1 (5 - ATAAGAATGCGGCCGCTTTCGTGACAGGC ACGTCGGG-3 ; NotI site is underlined) and XbaI-pex22-L1 (5 - GCTCTAGAACACGAGAATCAAAACCTGTA CAGACAGGTGGGTGGGC-3 ; XbaI site is underlined) using genomic DNA of Ina168 isolate as template, and inserted into pcb1004. TH and Ina isolates that do not have AVR- Pia locus. pcb1531-pex22p- EGFP pcb1531-pex22ppex22 pcb1531 EGFP fused to pex22 promoter The 1.4 Kb fragment containing pex22 gene promoter was amplified with the primers NotIpex22-U1 and XbaI-pex22p-L3 (5 - GCTCTAGAATTTTCGTGTATGGGGCTGGC GGGTAGGG-3 ; XbaI site is underlined) using pcb1004-pex22 as template, and inserted into pbagfp (Kimura et al., 2001). pcb1531 pex22 fused to pex22 promoter The pex22 ORF was amplified with the primers Xba1-pex22-U2 (5 - GCTCTAGACAAAATGCATTTTTCGACAATT TTC-3 ; XbaI site is underlined) and BamH1- pex22-l2 (5 - CGGGATCCTAGTAAGGCTCGGCAGCAAG CC-3 ; BamH1 site is underlined). EGFP in pcb1531-pex22p-egfp was replaced with pex22. pcb1531-pex33 pcb Kb fragment containing pex33 The 1.4 Kb fragment was amplified with the primers NotI-pex33-U1 (5 - ATAAGAATGCGGCCGCTTCGCTCTTTTGAT TAAATAC-3 ; NotI site is underlined) and XbaI-pex33-L1 (5 - GCTCTAGAAAACAGATTTGGAACTTTGGT GAAAACTAGAC-3 ; XbaI site is underlined) using genomic DNA of Ina168 isolate as template, and inserted into pcb1531. pcb1531-pex22ppex33 - Ina isolate that does not have AVR-Pia locus. Sasa2 and Ina isolates that do not have AVR-Pii locus. pcb1531 pex33 fused to pex22 promoter The pex33 ORF was amplified with the primers pbafp_kozak_pex33_xbai_f (5 - GCTCTAGACCAAAATGCAACTTTCCAAAAT TAC-3 ; XbaI site is underlined) and pbafp_pex33_bamhi_r (5 - CGGGATCCTTAGTTGCATTTATGATTAAAAT ACGC-3 ; BamHI site is underlined) using pcb1531-pex33 as template. EGFP in pcb1531-pex22p-egfp was replaced with pex33. Sasa2 and Ina isolates that do not have AVR-Pii locus. pcb1004-pex31-d pcb Kb fragment containing pex31- D pcb1531-pex22ppex31-d The 2.2 Kb fragment was amplified with the Sasa2, and Ina72 isolates that primers NotI-pex31-U1 (5 - do not have AVR-Pik, AVR-Pikm ATAAGAATGCGGCCGCAAAAGGAATAAGG and AVR-Pikp loci. CGGACCTCT-3 ; NotI site is underlined) and XbaI-pex31-L1 (5 - GCTCTAGATTAAAAGCCGGGCCTTTTTTTC CCCAA-3 ; XbaI site is underlined) using genomic DNA of Ina isolate as template, and inserted into pcb1004. pcb1531 pex31-d fused to pex22 promoter The pex31-d ORF was amplified with the Sasa2, and Ina72 isolates that primers Xba1_kozak_pex31_U1 (5 - do not have AVR-Pik, AVR-Pikm GCTCTAGAAAAGTCAATATGCGTGTTACCA and AVR-Pikp loci. CTTT-3 ; XbaI site is underlined) and pbafp_pex31_bamhi_r (5 - CGGATCCTCGTCAAACCTCCCTACG-3 ; BamHI site is underlined) using genomic DNA of Ina isolate as template. EGFP in pcb1531-pex22p-egfp was replaced with pex31-d. Kimura, A, Takano, Y, Furusawa, I, Okuno, T. (2001) Peroxisomal metabolic function is required for appressorium-mediated plant infection by Colletotrichum lagenarium. Plant Cell 13:
14 Supplemental Table 4. A list of transposons analyzed for the linkage with secreted protein genes Name Accession number DNA transposons Pot 2 Z Pot 3 U Occan AB LTR retrotransposons MAGGY L Pyret AB MGLR-3 AF Inago1 AB Inago2 AB Non-LTR retrotransposons MGL (MGR583) AF Mg-SINE MGU35313 Other retrotransposons Tf2 AY MINE-C EF MINE-B EF MINE-A EF MINE AJ
15 Supplemental Table 5. Primers used for plasmid construction and RT-PCR. Primer name Sequences (5' - 3') Description NotI-pex22-U1 ATAAGAATGCGGCCGCTTTCGTGACAGGCACGTCGGG Constructions of the plasmids pcb1004-pex22 and pcb1531-pex22p-egfp XbaI-pex22-L1 XbaI-pex22p-L3 Xba1-pex22-U2 BamH1-pex22-L2 GCTCTAGAACACGAGAATCAAAACCTGTACAGACAGGTGGGTGG GC GCTCTAGAATTTTCGTGTATGGGGCTGGCGGGTAGGG GCTCTAGACAAAATGCATTTTTCGACAATTTTC CGGGATCCTAGTAAGGCTCGGCAGCAAGCC Constructions of the plasmid pcb1004-pex22 Constructions of the plasmid pcb1531-pex22p-egfp Constructions of the plasmid pcb1531-pex22p-pex22 NotI-pex33-U1 XbaI-pex33-L1 ATAAGAATGCGGCCGCTTCGCTCTTTTGATTAAATAC GCTCTAGAAAACAGATTTGGAACTTTGGTGAAAACTAGAC Constructions of the plasmid pcb1531-pex33 pbafp_kozak_pex33_xb ai_f GCTCTAGACCAAAATGCAACTTTCCAAAATTAC pbafp_pex33_bamhi_r CGGGATCCTTAGTTGCATTTATGATTAAAATACGC Constructions of the plasmid pcb1531-pex22p-pex33 NotI-pex31-U1 XbaI-pex31-L1 ATAAGAATGCGGCCGCAAAAGGAATAAGGCGGACCTCT GCTCTAGATTAAAAGCCGGGCCTTTTTTTCCCCAA Constructions of the plasmid pcb1004-pex31-d Xba1_kozak_pex31_U1 GCTCTAGAAAAGTCAATATGCGTGTTACCACTTT pbafp_pex31_bamhi_r CGGATCCTCGTCAAACCTCCCTACG Constructions of the plasmid pcb1531-pex22p-pex31-d RTpex22-U1 RTpex22-L1 CCGCTCGAGCAAAATGGCGCCAGCTAGATTTTGCG CTAGTAAGGCTCGGCAGCAA RT-PCR for pex22 RTpex33-U1 RTpex33-L1 CTTCCCACTCCGGCCAGCCTGAATGGCA ATACTAGTTTAGTTGCATTTATGATTAA RT-PCR for pex33 RTpex31-U1 RTpex31-L1 CGACCTAAGTCGAGAGCGAGACCCTAAC TTAAAAGCCGGGCCTTTTTTTCCCCAA RT-PCR for pex31 RiceActin-U1 RiceActin-L1 CTGAAGAGCATCCTGTATTG GAACCTTTCTGCTCCGATGG RT-PCR for rice actin RAc7 MgActin-U1 MgActin-L1 TCGACGTCCGAAAGGATCTGT ACTCCTGCTTCGAGATCCACATC RT-PCR for Magnaprothe oryzae Actin
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