SUPPLEMENTAL INFORMATION

Transcription

1 SUPPLEMENTL INFORMTION P. syringae Genotype; relevant features a bbreviation Reference pv. tomato DC Wild Type; Rf R DC Cuppels, D.. 98 CUCP ΔhrcC::nptII, TSS -, Km R in hrp inducing media ΔhrcC Peñaloza- Vázquez et al., CUCP ΔavrE-hop-::ΩSp/Sm R ; Sp R Sm R ΔCEL lfano et al., CUCP8 pdc - pdc - ΔX uell et al., CUCP ΔhopH-hopC::FRT ΔII Wei et al., 7 CUCP ΔhopC-hopH::FRT, ΔhopD- ΔII, ΔIV, ΔIX Wei et al., CUCP CUCP9 CUCP8 CUCP CUCP9 CUCP8 hopr::frt, Δhop--hopG::FRT ΔhopC-hopH::FRT, ΔhopD- hopr::frt, Δhop--hopG::FRT, pdc - pdc - ΔhopU-hopF, ΔhopC-hopH::FRT, ΔhopD-hopR::FRT, Δhop-- hopg::frt, pdc - pdc - ΔhopC-hopH::FRT, ΔhopD- hopr::frt ΔhopU-hopF, ΔhopC-hopH::FRT, ΔhopD-hopR::FRT, ΔavrE-shcN, Δhop--hopG::FRT, pdc - pdc ΔhopC-hopH::FRT, ΔhopD- hopr::frt, ΔavrE-shcN, Δhop--hopG::FRT ΔhopC-hopH::FRT, ΔhopD- hopr::frt, ΔavrE-shcN, Δhop--hopG::FRT, pdc - pdc ΔII, ΔIV, ΔIX, ΔX ΔI, ΔII, ΔIV, ΔIX, ΔX ΔII, ΔIV ΔI, ΔII, ΔIV, ΔCEL, ΔIX, ΔX ΔII, ΔIV, ΔCEL, ΔIX, ΔII, ΔIV, ΔCEL, ΔIX, ΔX 7 Wei et al., 7 Kvitko et al., 9 Wei et al., 7 Kvitko et al., 9 Kvitko et al., 9 Kvitko et al., 9 CUCP9 ΔhopQ-, Δhop--hopG::FRT ΔIX Kvitko et al., 9 CUCP8 ΔhopU-hopF, ΔhopC-hopH::FRT ΔhopD-hopR::FRT, ΔavrE-shcN, Δhop--hopG::FRT, pdc - pdc -, ΔhopI, ΔhopM-, ΔhopF::FRT, ΔavrPto, ΔavrPto, ΔhopK, Δhop, ΔhopE, Δhop, hopy::frtsp R, rpslkr; Sp R Sm R D8E Cunnac et al., PSPRL ΔhopG ΔhopG This study a. Rf = Rifampicin, Km = Kanamycin, Sp = Spectinomycin, Sm = Streptomycin Supplemental Table S. Pst DC strains relevant to this study.

2 Name Sequence Kinesin F cacctggcttctcctctcggctggc Kinesin R CTCGTCCGCTGTCTCTTGGTTGG HopG_XhoIF caccctcgagtgctgcgtctc HopG-SpeIT7-R actagtctccctttgttgcccctgtcttggcct GCCGTTGTCTGCTT KO caccgaattcgttcccggcgtcttc KO TTgtcgacGCGTCTTGCCTCTC MR CGGCGCTTGC KO GTGCGCGCTGGTTTC KO CTTCTCCCCGTCTTGC UI- GCGGCTGTGCCTTTTC UI-S GGTTCTGTTTCGTTTCTGT Kinesin RT F TTGTCTCCGCCTGTGGC Kinesin RT R CGGGTGGCGGTTGCT FRK F CGGTCGTTTCCGTTTGTC FRK R TGCGGTTGGCCTGTTC PHI- F TTGGTTTGCGGGTGGTG PHI- R CTCCGTCGCCGTCC CYP8F F TGGGGGCCCTG CYP8F R TCGCCCTTCCTGTTC ROHD F CTGCGGGTGCCCTTT ROHD R TCCGCGGCTTCG FLS F CTCTCCTCCGGGGCTGGT FLS R GCTCGCTCTCCGGGTGG EFR F TGGTCTCGTCCGTGG EFR R CGTGGGTTCCTCCTGG NHL F TTCCTGTCCGTCCCC NHL R CCCTCGTGTGGCTGGC WRKY F GTGTTTGCTTCTTGCG WRKY R GTGGTTGTGCCTTGTGT WRKY F CGTGCTCTGTTTGCTCTGG WRKY R GTGTGGTCCTGCTGGG WRKY F CGCGTTGCCGTCTCC WRKY R CGTTTCTGGTTCTGTGGCTTT WRKY9 F CCCTTTCTCCCCCG WRKY9 R TGGGTTTCTGCCCGTTTT Supplementary Table S. DN primers used for cloning and qrt-pcr. Nucleotides denoted in bold italics indicate restriction enzyme sites added for cloning.

3 Fold Col- Untreat Mock flg undling, skewness Mock flg Mock flg Supplemental Figure S. FRK mrn expression is induced in -day-old WT Col- seedlings expressing GFP-fD when spray inoculated with flg, but changes in filament architecture are not., The mrn expression level of FRK in Col-/GFP-fD seedlings was determined following spray inoculation with µm flg. ll expression values were determined by quantitative real-time PCR, using amplification of UI as a control. Error bars, indicating mean ± SEM, are representative of two technical replicates of two biological replicates (n = ),, p <.., ctin skewness and density analysis in Col-/GFP-fD cotyledons at h after flg treatment. Error bars represent mean ± SEM from the skewness and density values of two independent biological replicates (n = ). Statistical significance was determined using a Student s t-test, as compared to mock treatment. DN primer sequences used for real-time PCR analysis are shown in Supplemental Table S.

4 DC WT DC EV Log cfu/mg f.w. dpi dpi undling, skewness DC WT DC EV DC WT DC EV Supplemental Figure S. rabidopsis seedlings inoculated with Pst DC WT and Pst DC EV shows a similar actin skewness/density response., In planta bacterial growth was measured and -day post-infection (dpi) in seedlings infected with Pst DC WT and Pst DC EVV (pvsp) at a concentration of x 7 cfu ml -. Data shown are from two biological repeats (n = )., ctin skewness and density were quantified in Col-/GFP-fD seedlings at hpi with Pst DC WT and Pst DC EV. Error bars represent mean ± SEM from the skewness and density values of three independent biological repeats (n = 8). Statistical significance was determined using a Student s t-test. EV = pvsp.

5 undling, skewness DC (.) CUCP DC (.) CUCP undling, skewness DC (.) CUCP DC (.) CUCP C undling, skewness DC (.) CUCP8 DC (.) CUCP8 D undling, skewness DC (.) CUCP DC (.) CUCP E undling, skewness DC (.) CUCP8 DC (.) CUCP8 F undling, skewness DC (.) CUCP9 DC (.) * CUCP9 Supplemental Figure S. ctin skewness and density in rabidopsis inoculated with Pst DC mutants lacking clustered TE genes. ctin skewness and density in Col-/GFP-fD seedlings was observed at hpi inoculated with the Pst DC at concentrations of x 7 cfu ml -, x cfu ml -, and cfu ml -. Pst DC polymutant strains were all inoculated at a concentration of x 7 cfu ml -., CUCP;, CUCP; C, CUCP8; D, CUCP; E, CUCP8; and F, CUCP9. ctin skewness and density values were determined at hours after dip-inoculation. Error bars represent mean ± SEM from the skewness and density values of three independent biological repeats (n = 7-9). Statistical significance was determined using a Student s t-test, as compared to Pst DC treatment. *, p <.;, p <..

6 DC avre Skewness DC hopm Occupancy (%) DC avre DC avre DC hopm Occupancy (%) Skewness DC hopm Supplemental Figure S. The type three secreted effector vre suppresses actin bundling and a concomitant reduction in filament density in rabidopsis epidermal cells. ctin filament architecture in -day-old Col-/GFP-fD seedlings was observed at hours post-inoculation (hpi)., Pst DC avre and, Pst DC hopm were inoculated at a concentration of 7 cfu ml-. Pst DC was inoculated at a concentration of cfu ml- to compare and adjust for increased bacterial density at hpi, as compared to Pst DC avre. ctin skewness and density values were determined in -day-old Col-/GFP-fD seedlings at hours after dipinoculation with Pst DC ( and ), Pst DC avre (), and Pst DC hopm (). Error bars represent mean ± SEM from the skewness and density values of two independent biological repeats (n=-). Statistical significance was determined using a Student s t-test, as compared to Pst DC treatment., p <.. ar = µm.

7 Log cfu/mg f.w. DC IX phopq- IX phopo IX phopv IX phopg IX phop- Supplemental Figure S. Pst DC complemented cluster IX polymutants in planta growth. acterial growth was measured at hpi in seedlings infected with Pst DC and Pst DC polymutants at a concentration of x 7 cfu ml -. Data shown for Pst DC is from two biological replicates (n = ); data for Pst DC polymutants are from four biological replicates (n = ). Statistical significance was determined using a one-way NOV, Tukey test; P <..

8 Mock +CD +JSP Col- Supplemental Figure S. Cytochalasin-D and jasplakinolide do not induce chlorosis in WT Col- plants. Five-week-old plants were infiltrated with either MgCl (Mock), cytochalasin- D (CD), or jasplakinolide (Jasp).

9 . Col- SLK_788 Fold Col-.7.. Mock +CD +JSP SLK_ 788 Supplemental Figure S7. Genetic (qrt-pcr) and actin pharmacological evaluation of the rabidopsis kinesin mutant, SLK_788., mrn expression of tg9 (kinesin) was measured from total RN isolated from WT Col- and the kinesin mutant. ll expression values were determined by qrt-pcr, using amplification of UI as an endogenous control. Error bars, indicating mean ± SEM, are representative of two technical replicates of two biological replicates (n = ). Statistical significance was determined using a two-way NOV;, p <.., Treatment of kinesin mutant plants with the actin modifying agents cytochalasin-d or jasplakinolide, alone, does not induce chlorosis. Five-week-old plants were infiltrated with either MgCl (Mock), cytochalasin-d (CD), or jasplakinolide (Jasp).

10 Fold Col- 7 PHI- Col- SLK_788 UT hr hr hr 8 7 CYP8F UT hr hr hr hr ROHD UT hr hr hr 8 7 FLS UT hr hr hr hr Fold Col- 8 EFR WRKY WRKY WRKY UT hr hr hr hr UT hr hr hr UT hr hr hr hr UT hr hr hr Fold Col- 7 7 WRKY9 UT hr hr hr hr 9 NHL UT hr hr hr hr FRK UT hr hr hr hr % maximal phosphorylation 7 min min min min Col- SLK_788 Supplemental Figure S8. Quantitative real-time PCR analysis of defense signaling marker genes from WT Col- and the rabidopsis kinesin mutant (SLK_788)., mrn expression of PHI- (tg), CYP8F (tg7), ROHD (tg79), FLS (tg), EFR (tg8), WRKY (tg87), WRKY (tg), WRKY (tg), NHL (tg98), and FRK (tg99) were measured from total RN from WT Col- and the rabidopsis kinesin mutant line (SLK_788). ll expression values were determined by qrt-pcr, using amplification of UI as an endogenous control. Error bars, indicating mean ± SEM, are representative of two technical replicates of two biological replicates (n = ). Statistical significance was determined using a two-way NOV;, p <.., Percent maximal phosphorylation of the MPK/ TEY motif in WT Col- and the kinesin mutant following µm flg spray treatment. No statistically significant differences were observed between WT Col- and the kinesin mutant using two-way NOV analysis as compared to untreated WT Col-, with onferroni post-test.

11 Log cfu/cm Day Day Col- SLK_788 Supplemental Figure S9. The Pst DC ΔhopG mutant does not show an altered in planta growth phenotype in the kinesin mutant (SLK_788) as compared to WT Col-. In planta bacterial growth was measured - and dpi in -week-old leaves infiltrated with the Pst DC ΔhopG mutant at a concentration of x cfu ml -. Data shown are from three technical replicates (n = 9) of three independent biological repeats. Statistical significance was determined using Student s t-test; p <..