Transcription factors play an important role in regulating plant gene expression and disease resistance. GhWRKY15 is a transcription factor that has been shown to enhance resistance to viruses and fungi in tobacco plants. Overexpression of GhWRKY15 in tobacco increased the expression of pathogenesis-related genes and activated antioxidant enzymes, reducing reactive oxygen species accumulation. GhWRKY15 was also found to affect plant growth and development, with transgenic tobacco plants exhibiting altered stem morphology and accelerated flowering. The presentation discussed the structure, mechanisms and important families of transcription factors in plants, using GhWRKY15 as a case study to illustrate how transcription factors regulate stress responses.

1. TRANSCRIPTION FACTORS AND THEIR ROLES
IN PLANT DISEASE RESISTANCE
Presentation by:
Bhor Sachin Ashok (PhD, II year)
Ehime University, Matsuyama, JAPAN

2. Outline of lecture
1) Introduction
2) Transcription factors
3) Mechanisms of gene expression regulation by TFs
4) How do transcription factors work?
5) Eukaryotic transcription factors (TFs)
a) General transcription factors (GTFs), and
b) Gene-specific transcription factors (activators)
6) Structure of transcription factors
7) Gene specific transcription factors / activators
8) Transcription factor families in plants
9) Case study

3. Introduction
The Basics of Life
Figure 1. Central dogma of molecular biology

4. Transcription and Translation are Regulated at Multiple
Steps
Figure 2. The flow of genetic information and their regulations at several
steps
B
A

5. Gene Expression-Regulation
DNA
pre-mRNA
mRNA
mRNA
Proteins
Metabolites
Nucleus
Cytoplasm
Figure 3. Regulation of gene expression at several stages
 The growth, development, and
function of an organism is a reflection
of gene expression.
 The timing, pattern, and quantity of
RNA and the production of proteins
from genes within the organism.
 Thus, gene regulation is essential to
life- from the simplest virus to the
most complex mammal.

6.  The transcription of DNA to make messenger RNA is highly
controlled by the cell.
 For higher organisms (plant or animal) to function, genes must be
turned on and off in coordinated groups in response to a variety
of situations.
 For a plant this may be “abiotic” (non-living) stress such as the
rising or setting sun, drought, or heat, “biotic” (living) stress such
as insects, viral or bacterial infection, or any of a limitless number
of other events.
 The job of coordinating the function of groups of genes falls to
proteins called transcription factors (TF’s).
 TFs are proteins that binds to specific sequence of DNA in
promoter region and regulate transcription.
Transcription Factors

7.  The three RNA polymerases (I, II and III) interact with their
promoters via transcription factors.
 Eukaryotic RNA polymerases, unlike their bacterial counterparts,
are incapable of binding by themselves to their respective
promoters.
 Some transcription factors (TFIIIA and TFIIIC for RNA polymerase
III) bind to specific recognition sequences within the coding
region.

8. Transcription factors use a variety of mechanisms for the regulation of
gene expression. These mechanisms include:
1)Stabilize or block the binding of RNA polymerase to DNA.
2)Catalyze the acetylation or deacetylation of histone proteins.
 histone acetyltransferase (HAT) activity –
acetylates histone proteins, which weakens the association of
DNA with histones, which make the DNA more accessible to
transcription, thereby up-regulating transcription
 histone deacetylase (HDAC) activity –
deacetylates histone proteins, which strengthens the association
of DNA with histones, which make the DNA less accessible to
transcription, thereby down-regulating transcription.
3) Recruit coactivator or corepressor proteins to the transcription factor
DNA complex.
Mechanisms of gene expression regulation by TFs

9. How Do Transcription Factors Work?
Formation of initiation
complex
 Eukaryotic promoter contains
 binding site for RNA
Polymerase,
 TATA box
 one or more enhancer sites.
 TF’s work by binding with DNA at
enhancer sites or other proteins in
initiation complex.
 TF’s help the cell to respond to
external and internal environment
by binding with ligand.
 Particular TF may bind to multiple
genes and each gene may be
controlled by multiple transcription
factors.
Figure 4. Schematic diagram showing the
coordinated gene function by transcription
factors

10. Eukaryotic transcription factors are classified into two classes:
1)General transcription factors (GTFs), and
2)Gene-specific transcription factors (activators)
 General transcription factors combine with RNA polymerase to
form a preinitiation complex
 This complex is able to initiate transcription when nucleotides are
available
 Tight binding involves formation of an open promoter complex
with DNA at the transcription start site that has melted
 The assembly of preinitiation complexes involving polymerase II
is quite complex
Eukaryotic Transcription Factors (TFs)

11.  Class II preinitiation complex contains:
• RNA polymerase II
• 6 general transcription factors:
 TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH
 The transcription factors (TF) and polymerase bind the
preinitiation complex in a specific order
Class II preinitiation complex

12. Preinitiation Complexes
Transcription factors bind to class II promoters in the
following order in vitro:
 TFIID with help from TFIIA binds to the TATA box forming
the DA complex
 TFIIB binds next generating the DAB complex
 TFIIF helps RNA polymerase bind to a region from -34 to
+17, now it is DABPolF complex
 Last the TFIIE then TFIIH bind to form the complete
preinitiation complex = DABPolFEH
 In vitro, the participation of TFIIA seems to be optional

13. Model of Formation of the DABPolF Complex
Fig. 5. Schematic figure showing the formation of DABPolF complex

14. Transcription factor motifs
bZIP Zinc fingers Helix loop helix
Fig. 6. Different types of transcription factor motifs in eukaryotic TF’s

15. Transcription factors are modular in structure and contain the
following domains:
 DNA-binding domain (DBD), which attaches to specific
sequences of DNA (enhancer or promoter).
 Trans-activating domain (TAD), which contains binding sites for
other proteins such as transcription co-regulators.
 An optional signal sensing domain (SSD) (e.g., a ligand binding
domain), which senses external signals and, in response,
transmits these signals to the rest of the transcription complex,
resulting in up- or down-regulation of gene expression.
Structure of TFs
Figure 8. Schematic diagram of the amino acid sequence

16. Some of the important functions and biological roles of
transcription factors are involved in:
 Basal transcription regulation
 Differential enhancement of transcription
 Development
 Response to intercellular signals
 Response to environment
 Cell cycle control
 Pathogenesis
Functions of TFs

17.  Gene specific transcription factors/ activators are proteins that
bind somewhere upstream of the initiation site to stimulate or
repress transcription.
Gene specific transcription factors/ activators
(Jin, et al., 2014, Nucleic Acid Research)

18. Transcription factor families in plants
PlantTFDB (Jin, et al., 2014, Nucleic Acid Research)
 PlantTFDB 3.0 has been published in 2014
 It contains 129288 TFs from 83 different species
 All plant TFs has been classified in to 58 families

19. Figure 9. Refined family assignment rules used for TF identification and assignment. Green ellipses
represent TF families and red rectangles represent DBDs. Blue rectangles denote auxiliary domains and
purple rectangles denote forbidden domains. Green solid lines link families and DBDs or auxiliary domains
and number ‘1’ or ‘2’ indicates number of DBDs.
(Jin et al. 2014, Nucleic Acid Research)
TF identification and assignment

20. 1) ERF / AP2
2) WRKY
3) bZIP
4) NAC
5) MYB
Examples of most extensively studied plant
transcription factor families in response to Biotic and
abiotic stresses

21. (Khong et al., 2008)
MeJA
OsWRKY03
NH1
bZIP
ZB8
POX22.3
PR1b
OsWRKY45
GST
Resistance to bacterial blight
x.oryza
Resistance to fungal blast
M.grisea
light
OsWRKY31
auxin
Mangnaportae griseaSA
BTH
PBZ1
OsSci2 Os1AA4 Arl1/Crl1
PAL peroxidase
Root development
wounding
RCl-1
Role of WRKY Transcription Factors in Resistance Against
Various Stresses
Figure 10. Role of OsWRKY in different biotic and abiotic stresses

23. Figure 1 A. Characterisation of WRKY transcription factors from various species.
Identical amino acids are highlighted in blue. The approximately 60-amino acid WRKY
domain and the C and H residues in the zinc-finger motif (C-X4-5-C-X22-23-H-X1-H) are
marked by the two-headed arrow and triangle, respectively. The short conserved HARF
structural motif and the highly conserved amino acid sequence WRKYGQK in the WRKY
domain are boxed.
Cloning and characterisation of GhWRKY15

24. Figure 4 B. Phylogenetic analysis of GhWRKY15 in relation to other plant WRKY
transcription factors

25. Figure 2 Subcellular localisation of the GhWRKY15::GFP fusion protein.
(A) Schematic representation of the 35 S-GhWRKY15::GFP fusionconstruct and the 35
S-GFP construct. GFP was fused in frame to the C terminus of GhWRKY15.
(B) Onion epidermal cells transiently expressing either the 35 S-GhWRKY15::GFP and
35 S-GFP construct were viewed using a confocal laser scanning microscope. The nuclei
of the onion cells were visualised using DAPI staining.
GhWRKY15 is localised to the nucleus

26. Analysis of partial putative cis-acting elements in the
GhWRKY15 promoter

27. GhWRKY15 expression in cotton following exposure to
diverse biotic stresses, SA, methyl jasmonate (MeJA)
and ET
Figure 3. Expression of GhWRKY15 in response to different fungal infections and
hormone treatments. Approximately one-week-old cotton seedlings were used for all
treatments. For the fungal inoculation, the roots of the cotton seedlings were dipped into
conidial suspensions of C. gossypii (A), F. oxysporum f. sp. vasinfectum (B) or R. solani (C)
(105 conidia/ml). The signalling molecules used were 2 mM SA (D), 100 μM MeJA (E) and ET
released from 5 mM ethephon (F). Whole seedling plants were collected for RNA extraction.
Ethidium bromide-stained rRNA was included as a loading control.

28. Figure 4 Enhanced resistance of GhWRKY15-overexpressing tobacco to viruses. (A)
Northern blot analysis of the expression levels of GhWRKY15 in T1 transgenic and WT tobacco
under normal conditions. Two leaves were tested for the GhWRKY15 transgenic tobacco. (B)
Leaf symptoms of tobacco plants infected with TMV (10 days post-inoculation) or CMV (14
days post-inoculation). OE: GhWRKY15- overexpressing tobacco; Mock: mock inoculation; CP:
coat proteins. (C) RT-PCR analysis of the expression levels of the CP gene in infected
transgenic lines (OE1, OE2 and OE3) and the WT line. (D) TMV and CMV titres in the
transgenic lines and the wild-type lines. The data are presented as the mean ± standard error
from three independent experiments.
Tobacco plants overexpressing GhWRKY15 exhibit
enhanced viral and fungal resistance

29. Overexpression of GhWRKY15 affects the expression of
PR genes and ethylene (ET) biosynthesis-related genes
Figure 5 Enhanced resistance of
GhWRKY15-overexpressing tobacco to
fungi.
(A) Leaf symptoms of tobacco plants infected
with fungi. The detached leaves in transgenic
and wild-type tobacco were inoculated with C.
gossypii or P. parasitica suspensions (106
conidia/ml) prepared in 1% glucose, and the
leaves were photographed 7 days after
inoculation.
(B) The diameters of the lesions on the
inoculated leaves. The diameters of the lesion
spots were recorded using the following
scoring system: 0, < 1 mm; 1, 1–2 mm; 2, > 2
mm.
(C) The numbers of lesions on the inoculated
leaves. The number of lesions per 10 cm2 was
counted on the inoculated leaves of three
independent transgenic and wild-type plants.
The values indicated by the different letters
are significantly different at P < 0.05, as
determined using Duncan’s multiple range

30. Figure 6. Expression of defence-related genes and ET biosynthesis genes.
(A) The expression of defence-related genes and ET biosynthesis genes was
examined following TMV infection. Next, qPCR was used to examine the expression
of defence-related genes, including PR1, PR2, PR4, PR5 and NPR1, and ET
biosynthesis genes, including ACO and ACS genes, in plants 10 days post infection
with TMV. (B) The expression of defence-related genes and ET biosynthesis genes
7 days post infection with a fungus. The actin gene was used to normalise the
amount of template in each reaction.

31. Overexpression of GhWRKY15 decreases the
accumulation of ROS and activates the expression of
oxidation-related genes
Figure 7. Expression of GhWRKY15 in tobacco decreased the accumulation of ROS,
and MV enhanced GhWRKY15 expression. (A), (B) and (C) show that the expression of
GhWRKY15 in tobacco decreased the accumulation of ROS after TMV, CMV or C. gossypii
treatment, respectively. The level of H2O2 in the tobacco leaves was determined using 1
mg/ml DAB as substrate. The top figure indicates the visualisation of the H2O2
accumulation, and the bottom figure shows the microscopic observations of the brown
precipitate. (D) MV enhances GhWRKY15 expression. Approximately one-week-old cotton
seedlings were used for the 0.5 mM MV treatment.

32. Figure 8 Expression of antioxidant enzymes in transgenic lines. (A) The
expression of antioxidant enzymes under normal conditions. (B) The expression of
antioxidant enzymes during MV treatment. Also, qPCR analysis was performed to
detect the levels of the antioxidant enzymes (NtSOD, NtGPX, NtAPX1, NtAPX2,
NtCAT1 and NtCA). Approximately three-week-old transgenic and wild-type
tobacco plants were used for the expression analysis. For the MV treatment, the
tobacco seedlings were sprayed with 0.5 mM MV and analyzed 6 h after treatment.

33. Figure 9. Effect of virus infection on the SOD, POD, CAT and APX
activities. (A) and (B) present the SOD, POD, CAT and APX activities 7 days
post inoculation with TMV and CMV.

34. Expression of GhWRKY15 affects plant growth and
development
Figure 10. Comparison of the growth and development of the
transgenic and wild-type tobacco. (A) Seed germination and
growth phenotype of transgenic and wild-type tobacco. (B) The
growth phenotype of transgenic and wild-type tobacco at
approximately 10 weeks. Differences in stem elongation are
clearly observable. (C) The height of transgenic and wild-type
tobacco from the shooting stage to the flowering stage. (D)
Premature flowering of the transgenic plants relative to the wild-
type plants. The growth phenotype was photographed at
approximately 22 weeks. (E) The phenotype of the bottom leaves
of the transgenic and wild-type tobacco at approximately 18
weeks. The figures are a magnification of the red boxes in (E).

35. Figure 11. Comparison of stems between transgenic and wild-type tobacco.
(A) Transverse section of the stems of transgenic and wild-type tobacco at the
shooting stage. (B) Vertical section of the stems of transgenic and wild-type
tobacco at the shooting stage. (C) Magnification of the red boxes on the left in (A).
(D) Magnification of the red boxes on the right in (A). The left and right red boxes
primarily indicate cells of the cortex, vascular bundle and pith. Bar: 100 μm. (E)
Visual differences in the stems of transgenic and wild-type tobacco at the shooting
stage.

36. Tissue-specific expression of GhWRKY15 and the
effects of abiotic stresses on GhWRKY15 expression
Figure 12 Tissue-specific expression of GhWRKY15 and expression analysis of
GhWRKY15 in response to abiotic stresses. Total RNA was extracted from the roots
(R), stems (S) and leaves (L) for the tissue-specific expression analysis (A). Total RNA
was extracted from the leaves at the indicated time points after treatment with cold (4
°C) (B), 200 mM NaCl (C), wounding (D), 15% (w/v) PEG6000 (E), 500 μM GA3 (F) or
100 μM ABA (G). Ethidium bromide-stained rRNA was included as a loading control.

37. Thank you