New Phytologist Supporting Information Article title: Divergent evolution of multiple virus-resistance genes from a progenitor in Capsicum spp.

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1 New Phytologist Supporting Information Article title: Divergent evolution of multiple virus-resistance genes from a progenitor in Capsicum spp. Authors: Saet-Byul Kim, Won-Hee Kang, Hoang Ngoc Huy, Seon-In Yeom, Jeong-Tak An, Seungill Kim, Min-Young Kang, Hyun Jung Kim, Yeong Deuk Jo, Yeaseong Ha, Doil Choi and Byoung-Cheorl Kang Article acceptance date: 31 July 2016 The following Supporting Information is available for this article: Fig. S1 Comparative genetic map of pepper and tomato. The Pvr4 region including the TG420 marker is syntenic between pepper and tomato chromosome 10. Fig. S2 Analysis of the marker in F 2 population. The developed marker cosegregated with the phenotypic marker against PepMoV in F 2 population. Fig. S3 N. benthamiana leaves agroinfiltrated with Pvr4 candidates derived from CM334 and NIb of PepMoV. Fig. S4 N. benthamiana leaves agroinfiltrated with Pvr4 derived from CM334, susceptible pvr4 derived from ECW, and effectors from viruses. Fig. S5 Resistance of Pvr4 against PepMoV in N. benthamiana. The error bars represent standard deviations. Asterisks indicate statistically significant differences from virus accumulated in the leaves of N. benthamiana expressing empty vector and pvr4 (*, P<0.05; Duncan's multiple range test). Fig. S6 HR cell death induced by specific interaction between Pvr4 and NIbs of avirulent potyviruses (a) and accumulation of PVY-0 (b) and PepSMV (c) in N. benthamiana overexpressing Pvr4. The error bars represent standard deviations. Asterisks indicate statistically significant differences from virus accumulated in the leaves of N. benthamiana expressing empty vector (**, P<0.01; Duncan's multiple range test). Fig. S7 Protein sequence alignment of Pvr4 and pvr4 derived from resistant and susceptible alleles, respectively. Fig. S8 Identification of the avirulence factor for TSWV in the resistant pepper carrying Tsw. Fig. S9 Expression of candidate NBARC genes with Ns in N. benthamiana. Fig. S10 Identification of Tsw by co-infiltration into N. benthamiana of Tsw derived from PI152225, tsw, and effectors from viruses.

2 Fig. S11 Amino acid sequences of Tsw. Fig. S12 Alignment of amino acid sequences of the Tsw resistance allele derived from C. chinense PI and the Tsw susceptible allele (tsw). Fig. S13 HR induced by inoculation of TSWV onto N. benthamiana leaves transiently expressing Tsw. Fig. S14 Detection of TSWV accumulation on N. benthamiana leaves transiently expressing Tsw and tsw by ELISA. The error bars represent standard deviations. Asterisks indicate statistically significant differences from virus accumulated in the leaves of N. benthamiana expressing empty vector and tsw (**, P<0.01; Duncan's multiple range test). Fig. S15 Transgenic N. benthamiana plants expressing Pvr4. Fig. S16 HR cell death induced by PepMoV-NIb over-expression in transgenic N. benthamiana leaves carrying Pvr4. Fig. S17 Transgenic N. benthamiana plants expressing Tsw. Fig. S18 HR cell death induced by TSWV-NSs over-expression in transgenic N. benthamiana leaves carrying Tsw. Fig. S19 Dotplot of Pvr4/Tsw cluster in C. annuum CM334 and C. chinense PI Fig. S20 Comparison of Pvr4 and Tsw amino acid sequences. Fig. S21 Phylogenetic analysis of NLRs within the Pvr4 region from C. annuum CM334 and the Tsw region from C. chinense PI Fig. S22 Comparison of the nucleotide sequences of Pvr4, Tsw and NBARC genes. Fig. S23 Model of gene duplication and the date of the duplication events. Fig. S24 Phylogenetic relationships of the Pvr4/Tsw orthologous genes in pepper and tomato. Table S1 BLAST search to identify paralogues and orthologues pairs of sequences Table S2 The NLR genes in the Pvr4/Tsw region Table S3 Ortholgous genes in tomato genome Table S4 Microsynteny analysis of C. annuum 'CM334' containing Pvr4 and its collinear region in tomato. Blue box indicates NLR genes in the Pvr4 cluster region used in this study

3 Fig. S1 Comparative genetic map of pepper and tomato. The Pvr4 region including the TG420 marker is syntenic between pepper and tomato chromosome 10.

4 Fig. S2 Analysis of the marker in F 2 population. The developed marker cosegregated with the phenotypic marker against PepMoV in F 2 population.

5 Fig. S3 N. benthamiana leaves agroinfiltrated with Pvr4 candidates derived from CM334 and NIb of PepMoV. Combination of R3a and Avr3a was used as positive control. Inoculated genes were depicted right panels. Five days post infiltration, N. benthamiana leaves were harvested and visualized under UV light. R3a, Phytophthora infestans resistance gene derived from potato; Avr3a, R3a effector of P. infestans; NIb, NIb derived from PepMoV; CaNBARC322, CaNBARC309, CaNBARC029a and CaNBARC575; the Pvr4 candidate genes derived from C. annuum CM334.

6 Fig. S4 N. benthamiana leaves agroinfiltrated with Pvr4 derived from CM334, susceptible pvr4 derived from ECW, and effectors from viruses. Combination of R3a and Avr3a was used as positive control. Seven days post infiltration, N. benthamiana leaves were harvested and visualized under UV light. R3a, P. infestans resistance gene derived from potato; Avr3a, R3a effector of P. infestans; NIP, necrosis-inducing protein derived from P. sojae; NS, TSWV- NSs gene; NIb, NIb derived from PepMoV; Pvr4, Pvr4 derived from CM334 ; pvr4, susceptible homolog derived from ECW.

7 Fig. S5 Resistance of Pvr4 against PepMoV in N. benthamiana. PepMoV-inoculated leaves of N. benthamiana transiently expressing genes were sampled at 1, 2, 3, 4, and 5 dpi. Empty vector was used as positive control. ELISA was performed using PepMoV antibody. It was performed five times as biological replicates. The error bars represent standard deviations. Asterisks indicate statistically significant differences from virus accumulated in the leaves of N. benthamiana expressing empty vector and pvr4 (*, P<0.05; Duncan's multiple range test).

8 Fig. S6 HR cell death induced by specific interaction between Pvr4 and NIbs of avirulent potyviruses (a) and accumulation of PVY-0 (b) and PepSMV (c) in N. benthamiana overexpressing Pvr4. Combination of R3a and Avr3a was used as positive control. Seven days post infiltration, N. benthamiana leaves were harvested and visualized under UV light. R3a, P. infestans resistance gene derived from potato; Avr3a, R3a effector of P. infestans; PVY-NIb, NIb derived from PVY-O; PepSMV-NIb, NIb derived from PepSMV; PepMoV- NIb, NIb derived from PepMoV; TEV-NIb, NIb derived from TEV; Pvr4, Pvr4 derived from CM334. Agrobacterium harboring Pvr4 was inoculated in N. benthamiana and PVY-O and PepSMV was rubbed, respectively. After rubbing virus, tobacco leaf discs of 5 plants were randomly sampled in 1, 2, 3, 4, 5 and 6 dpi. Empty vector was used as positive control. ELISA was performed using antibody to the corresponding virus. The experiment was performed three times. The error bars represent standard deviations. Asterisks indicate statistically significant differences from virus accumulated in the leaves of N. benthamiana expressing empty vector (**, P<0.01; Duncan's multiple range test).

9 Fig. S7 Protein sequence alignment of Pvr4 and pvr4 derived from resistant and susceptible alleles, respectively. Black shading indicates identical sequences. Red, yellow, and blue lines indicate coiled-coil, NB-ARC, and LRR domains, respectively.

10 Fig. S8 Identification of the avirulence factor for TSWV in the resistant pepper carrying Tsw. Virus constructs derived from TSWV were infiltrated to C. chinense PI leaves. Pepper leaves were harvested and visualized at 4 d post infiltration. NIP, necrosis-inducing protein derived from P. sojae; EV, pcambia 2300 empty vector; NS, TSWV-NSs gene; N, TSWV-N gene.

11 Fig. S9 Expression of candidate NBARC genes with Ns in N. benthamiana. CcNBARC genes derived from PI and NSs were infiltrated in N. benthamiana leaves. R3a and Avr3a were used as positive control. Five days post infiltration, N. benthamiana leaves were harvested and visualized under UV light.

12 Fig. S10 Identification of Tsw by co-infiltration into N. benthamiana of Tsw derived from PI152225, tsw, and effectors from viruses. Combination of R3a and Avr3a was used as positive control. Seven days post infiltration, N. benthamiana leaves were harvested and visualized under UV light. R3a, P. infestans resistance gene derived from potato; Avr3a, R3a effector of P. infestans; NIP, necrosis-inducing protein derived from P. sojae; EV, pcambia 2300 empty vector; NS, TSWV-NSs gene; NIb, NIb derived from PepMoV; Tsw, Tsw derived from PI ; tsw, susceptible allele of Tsw.

13 Fig. S11 Amino acid sequences of Tsw. CC, coiled-coil; NBS, nucleotide-binding site; LRR, leucine rich repeat.

14 Fig. S12 Alignment of amino acid sequences of the Tsw resistance allele derived from C. chinense PI and the Tsw susceptible allele (tsw). Black shading indicates identical amino acid sequences.

15 Fig. S13 HR induced by inoculation of TSWV onto N. benthamiana leaves transiently expressing Tsw. TSWV-inoculated leaves of N. benthamiana transiently expressing genes were sampled and photos taken at 3 dpi. Upper panel is non-inoculated leaves and lower panel is TSWV-inoculated leaves of N. benthamiana after agroinfiltration. Empty vector, pcambia2300 vector; Tsw, Tsw derived from C. chinense PI52225 ; tsw, susceptible allele.

16 Fig. S14 Detection of TSWV accumulation on N. benthamiana leaves transiently expressing Tsw and tsw by ELISA. TSWV-inoculated leaves of N. benthamiana transiently expressing genes were sampled at 1, 2, 3, 4, and 5 dpi. As a control, the leaves were infiltrated with Agrobacterium harboring the empty vector pcambia Tsw, Tsw derived from C. chinense PI52225 ; tsw, susceptible allele of Tsw. The error bars represent standard deviations. Asterisks indicate statistically significant differences from virus accumulated in the leaves of N. benthamiana expressing empty vector and tsw (**, P<0.01; Duncan's multiple range test).

17 Fig. S15 Transgenic N. benthamiana plants expressing Pvr4. Empty denotes transgenic plants transformed with pcambia 2300 empty vector. Pvr4 #20, #27 and #50 denote T2 transgenic plants transformed with pc2300-pvr4.

18 Fig. S16 HR cell death induced by PepMoV-NIb over-expression in transgenic N. benthamiana leaves carrying Pvr4. Leaves of wild-type plants, empty vector transgenic plants and Pvr4 transgenic plants were sampled and photos taken at 5 dpi under light (left) and UV light (right).

19 Fig. S17 Transgenic N. benthamiana plants expressing Tsw. Empty denotes transgenic plants transformed with pcambia 2300 empty vector. Tsw #A, #E and #F denote T2 transgenic plants transformed with pc2300-tsw.

20 Fig. S18 HR cell death induced by TSWV-NSs over-expression in transgenic N. benthamiana leaves carrying Tsw. Leaves of wild-type plant, empty vector transgenic plants and Tsw transgenic plants were sampled and photos taken at 5 dpi under light.

21 Fig. S19 Dotplot of Pvr4/Tsw cluster in C. annuum CM334 and C. chinense PI C. annuum CM334 and C. chinense PI are shown on the horizontal axis and vertical axis, respectively. MUMmer version 3.23 was used to identify the syntenic region ( The forward matches are displayed in red, while the reverse matches are displayed in blue. Colored arrows indicate the positions of NLR genes. Enriched short blocks reveal repetitive sequence accumulation in this region.

22 Fig. S20 Comparison of Pvr4 and Tsw amino acid sequences. Conserved sequences are shown with black backgrounds. Conserved motifs described by Meyers et al. (1999) are underlined in green.

23 Fig. S21 Phylogenetic analysis of NLRs within the Pvr4 region from C. annuum CM334 and the Tsw region from C. chinense PI The NBARC domains were used for phylogenetic analysis. The evolutionary history was inferred using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model using MEGA6. Phylogenetic analysis revealed two distinct clades (Group A and Group B) showing paralogs and orthologs. Putative allelic pairs are shown in blue. Maximum bootstrap values are 500. Bootstrap scores of less than 50% are not shown.

24 Fig. S22 Comparison of the nucleotide sequences of Pvr4, Tsw and NBARC genes. The NBARC nucleotide sequences of Pvr4 (a) and Tsw (b) were compared with those of Pvr4/Tsw cluster genes (CaNBARC309, CcNBARC309, CcNBARC322a CcNBARC322, CaNBARC029 and CcNBARC029) using the VISTA program. The y axis indicates the percentage of nucleotide identity between the indicated reference sequence and each gene. The x axis represents the position in NBARC domain sequence. Group A and B indicate the distinct clade of NLRs as shown in Fig. S21.

25 Fig. S23 Model of gene duplication and the date of the duplication events. The different gene repertoires of Pvr4/Tsw homologs within Capsicum species and tomato have been involved in different type of gene duplication events. The Pvr4 region (a), Tsw region (b) and Pvr4/Tsw orthologs in the tomato genome (c). Branch length in each phylogenetic tree is proportional to the synonymous substitution rate (Ks). Ks calculation for paralogs were conducted by method described in Blanc and Wolfe, The date of the duplication event was estimated according to T = Ks/2λ. The clock-like rate (λ) was substitutions per site per year (tomato and pepper speciation time 19.2 MYA). MYA, million years ago. (d) The distribution of duplication events of Pvr4 and Tsw homologs on the horizontal lines. The small letters indicate the duplication events as shown in (a), (b), and (c). The blue and purple circles indicate local tandem gene duplication and segmental duplication, respectively. The vertical yellow box and green box indicate the speciation time between C. annuum and C. chinense (1.44 MYA) and between pepper and tomato (19.1MYA), respectively.

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27 Fig. S24 Phylogenetic relationships of the Pvr4/Tsw orthologous genes in pepper and tomato. The neighbor-joining tree is based on an amino acid alignment of the NB-ARC domains from CNL genes. Sequences from C. annuum CM334, C. chinense PI159236, and S. lycopersicum are prefixed with Ca, Cc, and Solyc, respectively. The genes from pepper and tomato are shown in red and pink, respectively. The triangle indicates genes in Group B used as outgroup. Branches corresponding to partitions reproduced in less than 50% of bootstrap replicates are collapsed. The evolutionary distances were computed using the JTT matrixbased method and are in the units of the number of amino acid substitutions per site. Evolutionary analyses were conducted in MEGA6.

28 Table S1 BLAST search to identify paralogues and orthologues pairs of sequences Query Length (nt) C. annuum 'CM334' CaNBARC CaNBARC309 a CaNBARC CaNBARC029 a Max. identit y E- valu e Max. score Total Score C. chinense 'PI159236' Max. identit y E- Max. value score Total Score CcNBARC309b CcNBARC CaPvr CaNBARC CcNBARC322_F CaNBARC CaNBARC CcNBARC a CaNBARC CaNBARC CcNBARC CaNBARC CaNBARC CcNBARC a CaNBARC CaNBARC CcNBARC398_F a 4 CaNBARC CaNBARC CcNBARC

29 Table S2 The NB-LRR genes in the Pvr4/Tsw region Species Gene Name gene ID Scaffold start stop C. annuum 'CM334' cds size strand CaNBARC296 Ca10g21040 scaffold NBARC398 Ca10g21090 scaffold NBARC309a Ca10g21120 scaffold NBARC509 Ca10g21130 scaffold NBARC029a Ca10g21150 scaffold CaNBARC322 (Pvr4) Ca10g21170 scaffold NBARC309 Ca10g21190 scaffold NBARC029 Ca10g21180 scaffold CcNBARC575 (Tsw) Cc10g20770 scaffold CcNBARC029 Cc10g20750 scaffold CcNBARC509 Cc10g20720 scaffold CcNBARC309b Cc10g20710 scaffold CcNBARC309 Cc10g20700 scaffold C. CcNBARC398 Cc10g20680 scaffold chinense CcNBARC296a Cc10g20650 scaffold CcNBARC800 Cc10g20620 scaffold 'PI159236' CcNBARC029a Cc10g20610 scaffold CcNBARC322 Cc10g20600 scaffold CcNBARC309c Cc10g20599 scaffold CcNBARC322a Cc10g20598 scaffold CcNBARC398a Cc10g20597 scaffold CcNBARC296 Cc10g20596 scaffold

30 Table S3 Ortholgous genes in tomato genome. The gene set was determined using OrthoMCL clustering as shown in Kim et al. (2014). Subgroup no. indicates cluster no. classified by OrthoMCL Gene ID Subgroup no. Solyc12g cluster 11 Solyc12g cluster 11 Solyc12g cluster 11 Solyc12g cluster 11 Solyc02g cluster 11 Solyc04g cluster 11 Solyc04g cluster 11 Solyc10g cluster 11

31 Table S4 Microsynteny analysis of C. annuum 'CM334' containing Pvr4 and its collinear region in tomato. Blue box indicates NB-LRR genes in the Pvr4 cluster region used in this study. The gene set showing the best hit by the reciprocal BLAST was determined as described in Kim et al. (2014) NB-LRR gene in Pvr4 cluster NBARC296 NBARC398 NBARC309a NBARC509 NBARC029a Pvr4 NBARC029 NBARC309a *nd, not determined. C. annuum CM334 S. lycopersicum CA10g20940 Solyc10g CA10g20950 Solyc10g CA10g20960 Solyc10g CA10g20970 Solyc10g CA10g21020 Solyc10g CA10g21050 nd CA10g21070 nd CA10g21090 nd CA10g21110 Solyc03g CA10g21120 Solyc04g CA10g21130 nd CA10g21150 nd CA10g21170 nd CA10g21180 nd CA10g21190 nd CA10g21200 Solyc03g CA10g21210 Solyc10g CA10g21230 Solyc10g CA10g21240 Solyc10g CA10g21250 Solyc10g CA10g21260 Solyc10g CA10g21270 Solyc10g CA10g21250 Solyc10g CA10g21260 Solyc10g CA10g21270 Solyc10g References Blanc G, Wolfe KH Widespread paleopolyploidy in model plant species inferred from age distributions of duplicate genes. Plant Cell 16: Kim S, Park M, Yeom SI, Kim YM, Lee JM, Lee HA, Seo E, Choi J, Cheong K, Kim KT et al Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nature Genetics 46:

32 Meyers BC, Dickerman AW, Michelmore RW, Sivaramakrishnan S, Sobral BW, Young ND Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide binding superfamily. Plant Journal 20: