The combined role of WRKY and MYB TFs in the regulation of stilbene synthase genes in grapevine

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1 The combined role of WRKY and MYB TFs in the regulation of stilbene synthase genes in grapevine Alessandro Vannozzi

2 Big data is like teenage sex: everyone talks about it, nobody really knows how to do it, everyone thinks everyone else (ad is doing it, so everyone claims they are doing it (Dan Ariely)

3 Top 150 organisms in Gene Expression Omnibus database Grapevine is the 4 th most studied plant species for large-scale gene expression analyses!!! 149 series / experiments 36 platforms (Chip Arrays, NGS etc ) 3590 samples

4 How can we use all this information?

5 The regulation of the stilbene biosynthetic pathway A case study: IDENTIFYING NOVEL REGULATORS OF THE STILBENE PATHWAY IN GRAPEVINE Höll et Vannozzi; 2013 The Plant Cell

6 Roles and regulation of stilbene biosynthesis

7 Stilbene biosynthesis Vannozzi et al. (2012). BMC Plant Biology

8 chr5 chr10 The chr14 grapevine chr16 STS gene family 13,6 Chr 5 Chr 10 Chr 14 Chr 16 Proposed PN X V1 ORF Chr Closest prediction 12X V1 nomenclature Location predicted Vv10s0042g00840 VvSTS Vv10s0042g00850 Unsure Vv10s0042g00860 VvSTS Vv10s0042g00870 No VvSTS Vv10s0042g00880 Vv10s0042g00890 Unsure VvSTS Vv10s0042g00910 Unsure VvSTS Vv10s0042g00920 Yes VvSTS Vv10s0042g ,2 Yes VvSTS Vv16s0100g00750 Yes VvSTS Vv16s0100g00760 Unsure VvSTS Vv16s0100g00770 Yes VvSTS Vv16s0100g00780 Yes VvSTS ,3 No VvSTS Vv16s0100g00800 Unsure VvSTS Vv16s0100g00810 Unsure VvSTS No VvSTS Vv16s0100g00830 Yes VvSTS Vv16s0100g00840 Yes VvSTS Vv16s0100g00850 Yes VvSTS Vv16s0100g00860 Yes VvSTS Vv16s0100g00880 Yes VvSTS20 VvSTS1 VvCHS3 14, Vv16s0100g00900 Yes VvSTS21 16 VvSTS Vv16s0100g ,4 Yes VvSTS22 16 VvSTS Vv16s0100g00920 Yes VvSTS23 VvSTS4 14, Vv16s0100g00930 Yes VvSTS24 16 VvSTS Vv16s0100g00940 Yes VvSTS25 16 VvSTS Vv16s0100g00950 Unsure VvSTS Vv16s0100g00960 No VvSTS Vv16s0100g00990 Yes VvSTS Vv16s0100g01000 Yes VvSTS Vv16s0100g01010 Yes VvSTS Vv16s0100g ,5 Yes VvSTS Vv16s0100g01030 Yes VvSTS Vv16s0100g01040 Yes VvSTS Vv16s0100g01060 No VvSTS No VvSTS Vv16s0100g01070 Yes VvSTS Vv16s0100g01100 Yes VvSTS Vv16s0100g01110 Yes VvSTS Vv16s0100g01110 VvCHS2 Yes VvSTS ,7 Vv16s0100g ,6 VvCHS1 Yes VvSTS No VvSTS Vv16s0100g01130 Yes VvSTS Vv16s0100g01140 Yes VvSTS Vv16s0100g01150 Yes VvSTS No VvSTS Vv16s0100g ,7 Yes VvSTS Vv16s0100g01170 Yes VvSTS Vv16s0100g01190 Yes VvSTS Vv16s0100g01200 Yes VvSTS7 VvSTS8 VvSTS9 VvSTS10 VvSTS11 VvSTS12 VvSTS13 VvSTS14 VvSTS15 VvSTS16 VvSTS17 VvSTS18 VvSTS19 VvSTS20 VvSTS21 VvSTS22 VvSTS23 VvSTS24 VvSTS25 VvSTS26 VvSTS27 VvSTS28 VvSTS29 VvSTS30 VvSTS31 VvSTS32 VvSTS33 VvSTS34 VvSTS35 VvSTS36 VvSTS37 VvSTS38 VvSTS39 VvSTS40 VvSTS41 VvSTS42 VvSTS43 VvSTS44 VvSTS45 VvSTS46 VvSTS47 VvSTS48 1 Vannozzi et al BMC Plant Biol

9 The principle guilt-by-association genes involved in biologically related pathways or processes exhibit comparable expression dynamics across a wide range of experimental conditions.

10 Construction of a mutual-rank Gene Co-expression Network 29K NimbleGen 23 experiments, 359 conditions averaged from 914 NimbleGen arrays RNA-seq 21 experiments, 236 conditions averaged from 1353 replicates RMA-normalization of row intensity data mutual rank (MR) gene co-expression analysis VvSTS top 200 MR-ranked genes TF filtering based on Plant TFdb GCN network Row single/paired-end reads trimming/filtering Alignment vs 12X v1 PN40024 reference genome Estimation for FPKM transcript abundance mutual rank (MR) gene co-expression analysis VvSTS top 200 MR-ranked genes TF filtering based on Plant TFdb GCN network Merged GCN

11 Mutual Rank VS Pearson Correlation Coefficient Top 5 co-expressed list for gene A Rank PCC Locus Gene A Gene B Gene C Gene D Gene E Top 5 co-expressed list for gene B Rank PCC Locus Gene B Gene C Gene D Gene A Gene E Correlation rank is asymmetric: the rank of gene B from gene A is not the same as the rank of gene A from gene B!!!

12 Why mutual ranking instead of PCC? 1) Mathematical: smaller samples produce larger amplitude of correlation between any two genes 2) Biological: gene expression changes amongst developmental samples are far larger than those induced by abiotic stresses. Changes in gene expression amplitude decrease experimental noises that decrease any gene correlation. PCC rank distribution aligns in the diagonal indicating linear correspondence between the PCC ranks between different samples Obayashi & Kinoshita, DNA RESEARCH 2009, 16:

13 Construction of a mutual-rank Gene Co-expression Network 29K NimbleGen 23 experiments, 359 conditions averaged from 914 NimbleGen arrays RNA-seq 21 experiments, 236 conditions averaged from 1353 replicates RMA-normalization of row intensity data mutual rank (MR) gene co-expression analysis VvSTS top 200 MR-ranked genes TF filtering based on Plant TFdb GCN network Row single/paired-end reads trimming/filtering Alignment vs 12X v1 PN40024 reference genome Estimation for FPKM transcript abundance mutual rank (MR) gene co-expression analysis VvSTS top 200 MR-ranked genes TF filtering based on Plant TFdb GCN network Merged GCN

14 Why the top 200 ranking genes? 1) Based on the relationship of different k thresholds (100 1,000) to the distribution of PCC values for each member of the grapevine STS family in observed and random GCN Array NGS

15 Construction of a mutual-rank Gene Co-expression Network 29K NimbleGen 23 experiments, 359 conditions averaged from 914 NimbleGen arrays RNA-seq 21 experiments, 236 conditions averaged from 1353 replicates RMA-normalization of row intensity data mutual rank (MR) gene co-expression analysis VvSTS top 200 MR-ranked genes TF filtering based on Plant TFdb GCN network Row single/paired-end reads trimming/filtering Alignment vs 12X v1 PN40024 reference genome Estimation for FPKM transcript abundance mutual rank (MR) gene co-expression analysis VvSTS top 200 MR-ranked genes TF filtering based on Plant TFdb GCN network Merged GCN

16 Condition-independent STS-TF Gene Co-expression Network Top 200 MR genes Degree > 5 42VvSTSs and 30 TFs (564 edges)

17 VviMYB14 and VviMYB15 regulate two VviSTS genes Dual reporter luciferase assay VviMYB15 overexpression in hairy roots Höll et Vannozzi; 2013 The Plant Cell

18 Condition-independent STS-TF Gene Co.expression Network

19 Heat-map of VvSTSs and candidate TFs under stress conditions biotic abiotic

20 UV-C wound VvSTS and TFs expression under abiotic stresses

21 Single and combined effector Dual reporter luciferase assay VvWRKY24 effector increases the luciferase activity as much as VviMYB14 and VvMYB15 VvWRKY3 shows a combinatorial effect in association with VviMYB14 leading to a promoter induction higher than MYB14 alone. TF-TF interaction? W W Modified from Verweij et al The Plant Cell

22 luciferase fold change VvWRKY3 and VvWRKY53 regulate the VviMYB14 expression 9 TF-TF CGN WRKY03 WRKY24 WRKY43 WRKY53 MYB15 MYBA1 control WRKY03 WRKY24 WRKY43 WRKY53 MYB14 MYBA1 control VviMYB14 promoter VviMYB15 promoter

23 Conclusions We identify a number of TFs representing good candidates for further studies on the regulation of the stilbene pathway in grapevine including R2R3-MYBs, WRKYs, ERF and NAC genes and a bhlh gene. We confirmed the validity of the GCN guilt by association approach for inferring the biological function of genes (confirmed the role of VviMYB14 and VviMYB15 TFs). We identified a novel regulator, namely VvWRKY24 which appears to induce the promoter activity of at least one VvSTS gene in transfected grapevine cells. We described a combinatorial effect of VvWRKY3 and VviMYB14 in the regulation of the promoter of a VvSTS gene. Preliminary results suggest that VvWRKY3 (and VvWRKY53) could act upper level (regulatory circuitry?) in the STS regulation controlling the transcription of VviMYB14 TF. To test the interaction between VvWRKY3 (VvWRKY53) and VviMYB14 (and VviMYB15) proteins by Yeast-2-hybrid and BiFC assays. Confirm the role of WRKY3, VvWRKY24 and VvWRKY53 by functional assays in planta

24 Thanks to DAFNAE University of Padova Prof Margherita Lucchin Prof Gianni Barcaccia Dr Ibrahim Hmmam Petra Dona delle Rose COS University of Heidelberg Prof Jochen Bogs Dr Janine Höll Dr Stefan Czemmel CSIRO Plant Industry Adelaide SA Dr Ian Dry Dr Paul Boss Dr Angelica Jermakow CSIC-IRTA-UAB-UB Barcelona Dr Tomàs J. Matùs Australian National University Acton Dr Darren C.J. Wong

25 Thank you XII International Conference on GRAPEVINE BREEDING and GENETICS

26 Roles and regulation of stilbene biosynthesis