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in the regulation of stilbene synthase genes in grapevine - - PowerPoint PPT Presentation

Jan 28, 2023 252 likes •526 views

The combined role of WRKY and MYB TFs in the regulation of stilbene synthase genes in grapevine Alessandro Vannozzi XII INTERNATIONAL CONFERENCE ON GRAPEVINE BREEDING AND GENETICS Big data is like teenage sex: everyone talks about it, nobody

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XII INTERNATIONAL CONFERENCE ON GRAPEVINE BREEDING AND GENETICS

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

Alessandro Vannozzi

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XII INTERNATIONAL CONFERENCE ON GRAPEVINE BREEDING AND GENETICS

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Big data is like teenage sex: everyone talks about it, nobody really knows how to do it, everyone thinks everyone else is doing it, so everyone claims they are doing it…

(Dan Ariely)

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XII INTERNATIONAL CONFERENCE ON GRAPEVINE BREEDING AND GENETICS

Top 150 organisms in Gene Expression Omnibus database

  • 149 series / experiments
  • 36 platforms (Chip Arrays, NGS etc…)
  • 3590 samples

Grapevine is the 4th most studied plant species for large-scale gene expression analyses!!!

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XII INTERNATIONAL CONFERENCE ON GRAPEVINE BREEDING AND GENETICS

How can we use all this information?

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The regulation of the stilbene biosynthetic pathway

Höll et Vannozzi; 2013 The Plant Cell

A case study: IDENTIFYING NOVEL REGULATORS OF THE STILBENE PATHWAY IN GRAPEVINE

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XII INTERNATIONAL CONFERENCE ON GRAPEVINE BREEDING AND GENETICS

Roles and regulation of stilbene biosynthesis

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Stilbene biosynthesis

Vannozzi et al. (2012). BMC Plant Biology

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The grapevine STS gene family

VvCHS3 13,6 VvSTS1 VvSTS2 14,2 VvSTS3 VvSTS4 VvSTS5 VvSTS6 14,3 VvCHS2 VvCHS1 24,7 VvSTS7 16,2 VvSTS8 VvSTS9 VvSTS10 VvSTS11 VvSTS12 VvSTS13 VvSTS14 VvSTS15 VvSTS16 VvSTS17 16,3 VvSTS18 VvSTS19 VvSTS20 VvSTS21 VvSTS22 VvSTS23 VvSTS24 VvSTS25 VvSTS26 16,4 VvSTS27 VvSTS28 VvSTS29 VvSTS30 VvSTS31 VvSTS32 VvSTS33 VvSTS34 VvSTS35 16,5 VvSTS36 VvSTS37 VvSTS38 VvSTS39 VvSTS40 VvSTS41 VvSTS42 VvSTS43 VvSTS44 16,6 VvSTS45 VvSTS46 VvSTS47 VvSTS48 16,7

Chr 5 Chr 10 Chr 14 Chr 16

Proposed nomenclature Chr PN40024 12X V1 Location Closest prediction 12X V1 ORF predicted VvSTS1 10 14216112..14217677 Vv10s0042g00840 Vv10s0042g00850 Vv10s0042g00860 Unsure VvSTS2 10 14246945..14248453 Vv10s0042g00870 No VvSTS3 10 14264038..14265601 Vv10s0042g00880 Vv10s0042g00890 Unsure VvSTS4 10 14284187..14285750 Vv10s0042g00910 Unsure VvSTS5 10 14298957..14300520 Vv10s0042g00920 Yes VvSTS6 10 14304787..14306350 Vv10s0042g00930 Yes VvSTS7 16 16239028..16240564 Vv16s0100g00750 Yes VvSTS8 16 16252494..16254029 Vv16s0100g00760 Unsure VvSTS9 16 16268816..16270352 Vv16s0100g00770 Yes VvSTS10 16 16276570..16278105 Vv16s0100g00780 Yes VvSTS11 16 16285039..16284807

  • No

VvSTS12 16 16287922..16286924 Vv16s0100g00800 Unsure VvSTS13 16 16290882..16289536 Vv16s0100g00810 Unsure VvSTS14 16 16324386..16323781

  • No

VvSTS15 16 16335697..16337233 Vv16s0100g00830 Yes VvSTS16 16 16344516..16343202 Vv16s0100g00840 Yes VvSTS17 16 16347949..16346580 Vv16s0100g00850 Yes VvSTS18 16 16351428..16350059 Vv16s0100g00860 Yes VvSTS19 16 16368410..16366907 Vv16s0100g00880 Yes VvSTS20 16 16387529..16386013 Vv16s0100g00900 Yes VvSTS21 16 16398234..16399770 Vv16s0100g00910 Yes VvSTS22 16 16406519..16405205 Vv16s0100g00920 Yes VvSTS23 16 16409837..16408469 Vv16s0100g00930 Yes VvSTS24 16 16413317..16411948 Vv16s0100g00940 Yes VvSTS25 16 16431392..16430833 Vv16s0100g00950 Unsure VvSTS26 16 16440652..16441618 Vv16s0100g00960 No VvSTS27 16 16468549..16467015 Vv16s0100g00990 Yes VvSTS28 16 16478613..16477097 Vv16s0100g01000 Yes VvSTS29 16 16493131..16491597 Vv16s0100g01010 Yes VvSTS30 16 16505168..16503636 Vv16s0100g01020 Yes VvSTS31 16 16509479..16507942 Vv16s0100g01030 Yes VvSTS32 16 16511216..16512602 Vv16s0100g01040 Yes VvSTS33 16 16521936..16520374 Vv16s0100g01060 No VvSTS34 16 16523861..16523409

  • No

VvSTS35 16 16527862..16526326 Vv16s0100g01070 Yes VvSTS36 16 16557435..16555945 Vv16s0100g01100 Yes VvSTS37 16 16588984..16587447 Vv16s0100g01110 Yes VvSTS38 16 16608730..16607176 Vv16s0100g01110 Yes VvSTS39 16 16617258..16615702 Vv16s0100g01120 Yes VvSTS40 16 16620545..16618991

  • No

VvSTS41 16 16624624..16623088 Vv16s0100g01130 Yes VvSTS42 16 16629091..16627536 Vv16s0100g01140 Yes VvSTS43 16 16645747..16644190 Vv16s0100g01150 Yes VvSTS44 16 16649027..16647473 No VvSTS45 16 16675524..16673986 Vv16s0100g01160 Yes VvSTS46 16 16684264..16682709 Vv16s0100g01170 Yes VvSTS47 16 16699842..16698303 Vv16s0100g01190 Yes VvSTS48 16 16711818..16710281 Vv16s0100g01200 Yes

Vannozzi et al. 2012 BMC Plant Biol

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The principle

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

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Construction of a mutual-rank Gene Co-expression Network RNA-seq

21 experiments, 236 conditions averaged from 1353 replicates

29K NimbleGen

23 experiments, 359 conditions averaged from 914 NimbleGen arrays mutual rank (MR) gene co-expression analysis VvSTS top 200 MR-ranked genes TF filtering based on Plant TFdb mutual rank (MR) gene co-expression analysis VvSTS top 200 MR-ranked genes TF filtering based on Plant TFdb GCN network GCN network RMA-normalization of row intensity data Row single/paired-end reads trimming/filtering Alignment vs 12X v1 PN40024 reference genome Estimation for FPKM transcript abundance

Merged GCN

mutual rank (MR) gene co-expression analysis mutual rank (MR) gene co-expression analysis

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Mutual Rank VS Pearson Correlation Coefficient

Rank PCC Locus 1 1.0 Gene A 2 0.93 Gene B 3 0.94 Gene C 4 0.90 Gene D 5 0.87 Gene E

Top 5 co-expressed list for gene A Top 5 co-expressed list for gene B

Rank PCC Locus 1 1.0 Gene B 2 0.99 Gene C 3 0.97 Gene D 4 0.93 Gene A 5 0.87 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!!!

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1) Mathematical: smaller samples produce larger amplitude of correlation between any two genes

Why mutual ranking instead of PCC?

Obayashi & Kinoshita, DNA RESEARCH 2009, 16:249-260

PCC rank distribution aligns in the diagonal indicating linear correspondence between the PCC ranks between different samples 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.

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XII INTERNATIONAL CONFERENCE ON GRAPEVINE BREEDING AND GENETICS

Construction of a mutual-rank Gene Co-expression Network RNA-seq

21 experiments, 236 conditions averaged from 1353 replicates

29K NimbleGen

23 experiments, 359 conditions averaged from 914 NimbleGen arrays mutual rank (MR) gene co-expression analysis VvSTS top 200 MR-ranked genes TF filtering based on Plant TFdb mutual rank (MR) gene co-expression analysis VvSTS top 200 MR-ranked genes TF filtering based on Plant TFdb GCN network GCN network RMA-normalization of row intensity data Row single/paired-end reads trimming/filtering Alignment vs 12X v1 PN40024 reference genome Estimation for FPKM transcript abundance

Merged GCN

VvSTS top 200 MR-ranked genes VvSTS top 200 MR-ranked genes

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XII INTERNATIONAL CONFERENCE ON GRAPEVINE BREEDING AND GENETICS

Why the top 200 ranking genes?

Array NGS 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

  • bserved and random GCN
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XII INTERNATIONAL CONFERENCE ON GRAPEVINE BREEDING AND GENETICS

Construction of a mutual-rank Gene Co-expression Network RNA-seq

21 experiments, 236 conditions averaged from 1353 replicates

29K NimbleGen

23 experiments, 359 conditions averaged from 914 NimbleGen arrays mutual rank (MR) gene co-expression analysis VvSTS top 200 MR-ranked genes TF filtering based on Plant TFdb mutual rank (MR) gene co-expression analysis VvSTS top 200 MR-ranked genes TF filtering based on Plant TFdb GCN network GCN network RMA-normalization of row intensity data Row single/paired-end reads trimming/filtering Alignment vs 12X v1 PN40024 reference genome Estimation for FPKM transcript abundance

Merged GCN

TF filtering based on Plant TFdb TF filtering based on Plant TFdb GCN network GCN network

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Condition-independent STS-TF Gene Co-expression Network

Top 200 MR genes Degree > 5

42VvSTSs and 30 TFs (564 edges)

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VviMYB14 and VviMYB15 regulate two VviSTS genes

Höll et Vannozzi; 2013 The Plant Cell

Dual reporter luciferase assay VviMYB15 overexpression in hairy roots

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XII INTERNATIONAL CONFERENCE ON GRAPEVINE BREEDING AND GENETICS

Condition-independent STS-TF Gene Co.expression Network

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Heat-map of VvSTSs and candidate TFs under stress conditions

biotic abiotic

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VvSTS and TFs expression under abiotic stresses

wound UV-C

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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. 2016 The Plant Cell

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TF-TF CGN

1 2 3 4 5 6 7 8 9 WRKY03 WRKY24 WRKY43 WRKY53 MYB15 MYBA1 control WRKY03 WRKY24 WRKY43 WRKY53 MYB14 MYBA1 control

luciferase fold change

VviMYB14 promoter VviMYB15 promoter

VvWRKY3 and VvWRKY53 regulate the VviMYB14 expression

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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.

  • Confirm the role of WRKY3, VvWRKY24 and VvWRKY53 by functional assays in planta
  • To test the interaction between VvWRKY3 (VvWRKY53) and VviMYB14 (and VviMYB15)

proteins by Yeast-2-hybrid and BiFC assays.

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Thanks to

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

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Thank you

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Roles and regulation of stilbene biosynthesis