1,6. Tu18 1,2 0,8 0,4 0,0. Tu29. >10x down * 1,6. Tu43 1,2 0,8 0,4 0,0 8 0,0 1,6. Tu80 0,8 0,4 0,0

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Dwayne Cummings
5 years ago
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1 Reltive expression Tu14,9,6,3 2, Tu16 1,5 1,,5 Tu18, Tu19 >1x down * Reltive expression 3,2 Tu23 2, Tu28 >1x down * 4 Tu Tu31,9,6,3 Tu32 3,2 Tu33 Tu43 Tu57 Reltive expression Reltive expression Reltive expression,4 3,2 Tu58 2, Tu9 2,4 8 Tu >1x down Tu128 >1x down * * 14 7 *,9,6,4,3 Tu8 75 Tu ,4 Intct mite Anterior body dissected mite >1x down * Figure S1. Screening for potentil T. urtice slivry-glnd specific genes. Expression vlues of 18 T. urtice genes in intct mites (grey brs) nd in nterior body dissected mites (blck brs). For ech gene the brs were scled to the lowest verge i.e. the lowest br in ech plot is 1. Cndidtes showing n t lest 1-fold sttisticlly-significnt lower expression in dissected mites were considered s potentil slivry-glnd specific genes (Tu19, Tu28, Tu84, Tu9, nd Tu128). Sttisticl differences were nlyzed using Student s t-test (* = p <.5).

2 ARM-type fold GS L N K R S F L D D L QN N T QN A F H A F E QF GQN F N E K V QE A L K N L F N A F GN K T N N S S L N K R S F I D D I QN N T QN A F H A F E QF GQT F N E K V QE A L K N L L S A F GN K N S S Repet S E V S V I V S K R QT N P L QL I N D L GD P A K L A QD L L K V L A QMA T GQGK R K R E I F D A E A S V V V E K R A T N P L QL I N D L D D P A QF A QT F L K V L L D L A T GQGR R K R D I A E D L K K F S E E A K H N A E E A L K K L F S L F E QL K N K P S E S S D A - V N V I K R QT N P L QL D L K K F S E E A K H N A E E A L K K L F S F L E QF K S QS S GS T E A S V V V E K R A T N P L QL Repet 2 I N D I GD P A K F A QD L L QV L A QMA T GQGK R K R D A A E E L K K F A E A A K QN A QE GL I N D L D D P A QF A QT L L K V L A D I A T GQGR R K R D I A E D L K K F S D E A K H N A E E A L K K L F E F L E QF K N QF A H D E K D A E K V V S K R D I QE T L E N F N K L A QT K A QE A F K N K K L F S F L E QF K P QS S E S S D A V T V A K R A T N P L V L F N D L S QQD L GK F A QD I QK A L E D L F K S S P F F QS V S S QS N N GE N S I E K P I S K R G I F D T V E K F S R N A F D F L K V L A D I A T A QG~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ D F MN A L D K L F N R F GGD D D E E Y D E I I V E P E T E H Y ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Figure S2. Amino cid sequence lignment of Te28 nd Tu28. Alignment of mture mino cid sequences (minus predicted signl peptide) of Te28 nd Tu28. Clustl Omeg ws used to lign both sequences. Blck nd grey shding indicte identicl nd similr residues, respectively. Lines bove the lignment indicte the ARMADILLO domin (IPR1624) in Te28 (upper) nd Tu28 (lower). The conserved 8-mino cid repets in Te28 nd Tu28 re indicted with line below the lignment.

3 Figure S3. Detection of Te28 nd Te84 gene expression in slivry glnds of T. evnsi using whole-mount in situ hybridiztion. DIG-lbeled ntisense riboprobes (left pnel) were used for hybridiztion, while corresponding sense probes (right pnel) served s negtive controls. Signls were detected with lkline phosphtse linked to n nti-dig ntibody. In situ hybridiztion experiments for Te28 nd Te84 were repeted using both nitro-blue tetrzolium/5-bromo-4-chloro-3'-indolyphosphte (NBT/BCIP) (top row) or Fst Red (bottom row) s substrte. The pictures show mites representtive for the smples. Specific coloriztion (rrows) in slivry glnds ws only seen in ntisense probe smples nd never in sense probe smples. Some bckground stining in e.g. stylet nd legs ws common in ll smples. Scle brs indicte.5 mm. The blck drk spots re spider mite midgut cells contining excretory mteril (e.g. chlorophyll) (first described by Wiesmnn 1968, Bluvelt 1945).

4 5 DPI Te28 EV Figure S4. Cndidte effector Te28 cuses tissue necrosis fter groinfiltrtion. Agroinfiltrted leves expressing Te28 or with the EV. The rrow indictes necrotic zone in the Te28-groinfiltrted re. Necrosis fter expression of Te28 occsionlly occurred.

Agroinfiltrted leves fter expression of cndidtes from fmilies 19, 9, or 128.

5 Tu19 Tu128 Te19 Te128 Tu9 EV Te28 Figure S5. Agroinfiltrtion of cndidte effectors from fmilies 19, 9, or 128 does not induce chlorosis in N. benthmin. Agroinfiltrted leves fter expression of cndidtes from fmilies 19, 9, or 128. Infiltrted res were delineted with blck mrker. Leves with the EV or expressing the cndidte Te28 re shown s negtive nd positive control for tissue chlorosis, respectively. Pictures were tken t 5 DPI.

6 5 DPI Reltive expression PR1 1 b 13 b 56 c c c Mock GFP Te28 Tu28 Te84 Tu84 Figure S6. Reltive gene expression of the SA-mrker gene PR1 fter groinfiltrtion of 4 cndidte effectors or 35s:GFP s control. Reltive expression of the SA-relted mrker PR1 t 5 DPI fter groinfiltrtion of Te28, Tu28, Te84, Tu84, nd 35s:GFP or infiltrtion of the mock control. Error brs denote SE. Sttisticl differences were nlyzed using Generl Liner Model, nd re denoted s different letters (p<.5).

7 2 DPI 5 DPI Reltive expression Reltive expression b c bc bc c. Mock EV Te28 Tu28 Te84 Tu84 3 TPI 2 1 TPI Mock EV Te28 Tu28 Te84 Tu84 Figure S7. Reltive gene expression of the JA-mrker gene TPI fter groinfiltrtion of 4 cndidte effectors. Reltive expression of the JA-relted mrker TPI fter groinfiltrtion of Te28, Tu28, Te84, Tu84, nd EV or infiltrtion of the mock control, t 2 nd 5 DPI. Error brs denote SE. Sttisticl differences were nlyzed using Generl Liner Model, nd re denoted s different letters (p<.5). The dt re representtive for 2 experiments.

8 EV Te28 Tu28 Te84 Tu84 Figure S8. Lef-discs expressing cndidte Te28 show intense chlorotic symptoms. The picture shows lef-discs from groinfiltrted N. benthmin leves expressing the cndidte effectors or the control EV t the end of T. urtice performnce ssy (6 DPI, 4 dys of spider mite infesttion).

9 T e 8 4 T u 8 4 K S N S E L L D QL H D L F V E A S K R H N QA A L D L V N D A I S T V N MA L V QMT S A K D QA T S S N S E L L D K L D D ML V E A S QK N N K A A A D A V S E A I D T V GL A L S QMS - GK D E A T T e 8 4 T u 8 4 K D L F QS H I D F V QD D L S K L E K MV K QE I K T R GF MS R MH GA F N Y A S S K A A L L T E K N ML QS H I D A V R S D L N K L E K MV N QQ I K S R A F S - - V GGA F N F V A S R A S L L T E T e 8 4 T u 8 4 K V H S I F S H A T K S GL GGL V A L F E P F F E K V I N N P N I K QM I GGL E T I T S A L E QL K V QS MF T A V QK S D L GA L I A L I K P F L D N A MN D P QL K S MV GN L E T V T S A L E QL T e 8 4 T u 8 4 N K MT GGMF QP L N A V T D I L S N I E A S GK L F E E MS A A N S S D A I A D A M I N T L A S A D K MT GGMF E P L T H V K D I L A N I D S T GK L F E S MGS A K S S D E I A D A L I N T L A N A T e 8 4 T u 8 4 K S S H A H R I E K A L D Y V C N GL E D A E QS L K S S H A N K I E K A L D Y V C N GL A D V E K S A Figure S9. Amino cid sequence lignment of Te84 nd Tu84. Alignment of predicted mino cid sequences (minus predicted signl peptide) of Te84 nd Tu84. Clustl Omeg ws used to lign both sequences. Blck nd grey shding indicte identicl nd similr residues, respectively. No known motifs were identified in Te84 or Tu84.

10 (A) (B) Figure S1. Agroinfiltrted N. benthmin leves 6er lef- surfce wshing. (A) Picture of n intct groinfiltrted N. benthmin lef showing high density of glndulr trichomes (rrows) on the dxil side. (B) Glndulr trichomes were removed with >ssue pper soked in wter.

11 Number of mites MM NhG time (dys) Figure S11. Exponenl growth of two hypothecl populons growing on the SA- impired nhg tomto plnts or Moneymker (MM) tomto plnts. Despite the moderte differences in oviposi2on rtes observed on both genotypes (Fig. 6), this hs big impct on popul2on level. The intrinsic rte of increse (r m ) ws es2mted using the regression model proposed in Jnsen nd Sbelis (1992) for T. ur&ce: r m = x; where x is the oviposi2on rte (i.e. number of eggs per femle, per dy). The intrinsic rtes of increse for mites rered on Moneymker (MM) nd nhg plnts were: r m MM =.212; nd r m nhg =.22. The vlues were used to prmetrize themodel: n = 1 (t*rm) ; where n is the number of individuls; t is 2me in dys; nd r m is the intrinsic rte of increse.

supplementary information

supplementary information DOI: 1.138/nc8 Top-GFP!-ctenin GFP Intensity 3 1 1 3 5 6 7 8 Nucler Bet-Ctenin c MUC FABP KRT 8 5 3 6 15 1 5 LGR5 ASCL AXIN 15 5 15 5 Figure S1 TOP-GFP expression nd reltion with nucler β-ctenin, wnt trgets