The document discusses understanding the role of the WRKY gene family under stress conditions in pigeonpea. 94 WRKY genes were identified in pigeonpea that show a comparable number to other species. Analysis of the genes' phylogenetic distribution, exon-intron structure, and motifs provided insight into the evolution of the WRKY gene family. Expression analysis of the genes under drought and salt stress conditions using qRT-PCR suggested certain differentially expressed genes may play a role in stress responses. Further high-throughput studies at the protein level are needed to better understand the functional regulation of the WRKY gene family under stress.

1. Shreya Mandal
11733
PhD scholar
2020-21
UNDERSTANDING THE ROLE
OF THE WRKY GENE FAMILY
UNDER STRESS CONDITIONS
IN PIGEONPEA (CAJANUS
CAJAN L.)
BIO 604

2. Pigeonpea (Cajanus cajan L.), a
protein-rich legume, is a major
food component of the daily diet.
Drought-tolerant legume crop, is
grown in locations with less than
300 mm yearly rainfall due to its
extensive tap roots
Found in wide range of temperature
regimes from about 18 to 30◦C
(sometimes > 30◦C) (Kingwell et al.,
2014)

3.  WRKY family is known as the largest gene family across the higher plant
species (Karkute et al., 2018)
 WRKY family is known to have a significant role in stress (biotic as well as
abiotic stress) tolerance mechanism in plants (Jiang et al., 2017)
 The family is named WRKY due to the occurrence of the domain
containing well-conserved WRKYGQK domain at their N-terminus and a
zinc figure structure (Cx4–5Cx22–23HxH or Cx7Cx23HxC) at their C-
terminal.
 WRKY protein domain consists of 4-stranded β-sheet and a zinc finger
pocket that is responsible for the recognition with W-box sequence,
(C/T)TGAC(T/C) (Muthamilarasan et al., 2015)
 The length of the WRKY domain is approximately 60 amino acids long
(Rushton et al., 2011)
 The first WRKY gene was cloned and characterized from sweet potato
(Ishiguru et al., 1994)

4. MAPK pathway induces the activity of OsWRKY30 during drought
stress

5. WRKY proteins regulating plant responses against multiple abiotic stresses
like salinity, drought, heat, cold, nutrient starvation, light, radiation, and
oxidative stresses

6. Cross Talk between WRKY TF and ABA-Mediated Signaling

7. CASE STUDY

8. MATERIALS
Asha (ICPL 87119), a mild
period, high yield variety
wild variety (Rhynchosia
minima)
Genotypes

9. TREATMENTS
Drought treatment
control plants at about 80% of
relative water content (RWC),
while stressed plants were dried
up to 30% RWC
15 day-old plants
Salt treatment
hydroponic system
supplemented with 150 mM
NaCl solution (Electrical
Conductivity (EC) value 16.2)

10. METHODS Draft genome sequence data of the
pigeonpea genotype ‘Asha’ (ICPL 87119)
Identification of Genes Encoding WRKY
Transcription Factors (TFs)
BLASTP
Sequence Analysis and Phylogeny
Gene Structure, Motif Conservation, and
Regulatory Element Analysis
1.Gene Structure Display Server
2. MEME Suite v.5.0
3. Plant Cis-acting Regulatory DNA Elements (PLACE)
1. MSA- Clustal X v2.1
2. MEGA6.06

11. Contd. Gene Annotation and Co-expression
Network Analysis
Homology-Based CcWRKYs Protein
Structure Prediction
1. Blast2GO
2. CoExpNetViz
3. Cytoscape
Transcript Abundance Analysis
RNA Isolation, cDNA Synthesis, and qRT-PCR
I-Tasser
Heat map read density of mapped reads as ‘fragments per kilo base per
million mapped reads’ (FPKM)
a. RNA Isolation Kit (Sigma-Aldrich)
b. iScript cDNA Synthesis Kit (Bio-Rad)
c. Bio-Rad SYBR®Green master mixes on an Applied Biosystems™ 7500 Fast
Real-Time PCR System as per the ABI protocol

12. RESULT & DISCUSSION

13. Unrooted phylogenetic tree of 267 WRKY proteins including 94
WRKY proteins from pigeonpea and 173 from soybean
Phylogenetic Distribution of CcWRKY Proteins

14. Distribution of WRKY group members

15. Exon-Intron Organization
Among the 94 CcWRKY gene
No of genes constituting exons
43
4/5
13
11
3
6
5 7
groups IIc and IIe- 2-6 exons
groups III- 3
groupI- 2-7

16. Motif Composition of WRKY Proteins
WRKYGQK
heptapeptide
domain

17. Functional Annotation of WRKY Genes

18. Tertiary Structures of CcWRKY Proteins
Protein structure (A) CcWRKY1 (grp I), (B) CcWRKY2 (grp III) and (C) CcWRKY4 (grp
IIc ) were modeled with greater than 90% confidence score
C-score is basically based on the consequence of threading template
alignments and total coverage of the structure

19. Expression of CcWRKY Genes During Different Developmental Stages

20. Heat map showing the expression of CcWRKY genes at different
developmental stages

21. Expression of CcWRKYs Under Drought and Salt Conditions

22. Expression of CcWRKYs Under Drought and Salt Conditions

23.  94 WRKY genes are identified which are comparable to the number of
WRKY genes identified in the other species
 Chromosomal localization, phylogenetic distribution, exon-intron structure
analysis, and prospective motif composition provided a basis for the
understanding of WRKY gene family evolution
 Analysis of cis-regulatory elements in 2 Kb upstream regulatory regions
revealed the occurrence of some important motifs related to WRKY
functions under biotic and abiotic stress responses
 qRT-PCR analysis results suggested the possible role of differentially
expressed CcWRKY genes under different abiotic stresses
 Gene ontology analysis revealed gene distribution at a greater range of
biological processes, molecular functions as well as cellular components
CONCLUSION

24. High-throughput studies are needed with
proteomics to better understand
functional regulation of the WRKY gene
family at protein level in plants
PATH AHEAD