Expression Profiling of Arabidopsis thaliana in Response

to Environmental Stresses
Arjun Sham
Postdoctoral Fellow
Department of Plant Science, Faculty of Plant Science, NMBU
 Plant Stress
A state where the plant grows in a non-ideal/non-favorable
conditions.

Leads to deficiencies in growth, crop yields, permanent damage or

death.

Stresses can be biotic (caused by living organisms) or abiotic

(caused by physical or non-living causes).
Types of Plant Stresses ?

BIOTIC HORMONE ABIOTIC

Botrytis cinerea

Fungal Salicylic Acid (SA) Cold

Alternaria brassicicola

Bacterial Pseudomonas syringae Abscisic Acid (ABA) Heat

Viral Tobacco mosaic virus Jasmonic acid (JA) Drought

Nematode Caenorhabditis elegans Salinity

Ethylene (ET)

Insects Peris rapae Osmotic

Auxin (IAA)

Weeds Cerastium vulgatum Cytokinin (CK) Oxidative

Gibberellin (GA) Wounding

 Pathogen of Interest
Botrytis cinerea BO5.10
Necrotrophic fungal pathogen.
Grey mold disease.
Affects ~500 crop varieties.

Images Obtained from: Strawberry: https://www.gardenersworld.com/how-to/solve-problems/grey-mould-on-soft-fruits; Tomato: https://www.nexles.com/articles/wp-

content/uploads/2019/10/Botrytis-cinerea-affected-plant-1-300x225.jpg; Grape: https://blog.pestprophet.com/managing-botrytis-grape-rot-on-grapes/
 Previous Studies- Sham et al., 2014
Stresses Used:
Biotic (Botrytis cinerea) and
Abiotic (Cold, Drought &
Oxidative).

Common Genes:
9 Up- & 28 Down-regulated

Sham. A., et al. (2014). Transcriptome analysis reveals genes commonly induced by Botrytis cinerea infection, cold, drought and oxidative stresses in Arabidopsis. PLoS
ONE, 9: e113718.
 Previous Studies- Sham et al., 2015
Stresses Used:
Biotic (Botrytis cinerea) and Abiotic
(Heat, Salinity & Osmotic) & Sham et
al., 2014.
Common Genes (This study):
13 Up- & 29 Down-regulated.

Common Genes (Both Studies):

3 Up- & 12 Down-regulated.

Sham, A., et al. (2015). Identification of Arabidopsis candidate genes in response to biotic and abiotic stresses using comparative microarrays. PLoS One, 10,
e0125666.
Outline of the Study

mRNA

Arabidopsis Infection/Stress Microarray Microarray

B. cinerea Chip Hybridization

?
Function
Candidate Genes
Data Analysis

Images obtained from: https://abrc.osu.edu/uploads/tinymce_asset/file/17/Arabidopsis-transparent-bkgr.gif; https://www.thermofisher.com/ae/en/home/life-science/microarray-analysis/affymetrix.html; Ingle

R.A., Roden L.C. (2014) Circadian Regulation of Plant Immunity to Pathogens. In: Staiger D. (eds) Plant Circadian Networks. Methods in Molecular Biology (Methods and Protocols), vol 1158. Humana Press, New
York, NY.
 CHAPTER 1:
Arabidopsis WRKY33 Mutants in Response to
Botrytis cinerea
 ArabidopsisWRKY33
Transcription factor.

Locus name: At2g38470.

Involved in defense responses.

Required for resistance to the necrotrophic fungal pathogens (e.g.,

Botrytis cinerea & Alternaria brassicicola) Zheng et al., 2006.

Zheng, Z., AbuQamar, S. F., Chen, Z., & Mengiste, T. (2006). Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens. The
Plant Journal, 48: 592–605.
Methodology
 PLANT GROWTH & DISEASE ASSAY
Seeds: Col-0 (Wt), wrky33 (T-DNA insertion mutant lines) and 35S:WRKY33
(Overexpression lines).

Age of Plants: 5 weeks old.

Botrytis cinerea infection: Drop inoculation (3×105 spores.mL-1).

Mock (0 hpi): Wt-0; wrky33-0; and 35S:WRKY33-0h.

Treated with B. cinerea (24 hpi): Wt-24; wrky33-24; and 35S:WRKY33-24.

 MICROARRAY DATA ANALYSIS
Botrytis cinerea infection: Spraying on leaf.

Affymetrix Arabidopsis ATH1 121501 Array.

Normalization: R Packages (Affy &MAS5).

Cut-off threshold: Only Present (P) values considered; Absence (A) & Medium (M) were
removed.

Fold Change: Treated/Control (Signal Intensity).

Up-regulated: ≥2 & Down-regulated: ≤0.5 (or-2); P-value: ≤ 0.05

Results
B. cinerea Infection in WRKY33 Transgenic Plants
DEGs are Dependent on Arabidopsis WRKY33

wrky33 35S:WRKY33
 Cyclopentenone Oxylipins in B. cinerea Infection

12-oxo-phytodienoic acid
(OPDA): Precursor of Jasmonic
Acid (JA).

Phytoprostane Type A1 (PPA1).

Formed via enzymatic and non-

enzymatic free radical-catalyzed
pathways.

Image obtained from: https://slideplayer.com/slide/10472636/

Regulation of Cyclopentenone During B. cinerea Infection
Validation of OBIGs and/or PBIGs Dependent on WRKY33 to
B. cinerea Infection

OBIGs & PBIGs

 Major Findings
Identification of potential defense-related genes that coordinate regulatory
pathways through WRKY33 in mediating resistance to B. cinerea.

The expression profile of OPDA- and PPA1-treated Arabidopsis plants in

response to B. cinerea revealed that cyclopentenones can also modulate
WRKY33 regulation upon inoculation with B. cinerea.

These results support the role of electrophilic oxylipins in mediating plant

responses to B. cinerea infection through the TGA transcription factor.
Sham et al. (2017) – PLoS ONE
 CHAPTER 2:
RAP2.4 Gene Associated Arabidopsis
Immunity to Botrytis cinerea
 Stresses Used in this Study
Abiotic Stress
Wounding
Biotic Stress Oxidative Stress
Hormonal Stress
Botrytis cinerea
Alternaria brassicicola Salicylic Acid
Pseudomonas syringae pv tomato DC3000 Methyl Jasmonates
P. syringae pv tomato DC3000 avrRpm1 Ethylene
Peris rapae Abscisic Acid

Image obtained from: https://abrc.osu.edu/uploads/tinymce_asset/file/17/Arabidopsis-transparent-bkgr.gif

 ArabidopsisRAP2.4 Gene
RELATED TO AP2.4
Locus Name: At1G22190.
GO Process Involved (TAIR):
 ET-activated signaling pathway.
 DNA depended-regulation of transcription.
 response to cold.
 response to fungus.
 response to osmotic stress.
 response to water deprivation.

Gene Ontology Data obtained from: TAIR (https://www.arabidopsis.org/servlets/TairObject?id=27983&type=locus)

Methodology
Datasets Used in This Study
 MICROARRAY DATA ANALYSIS
Affymetrix Arabidopsis ATH1 121501 Array.

Normalization: Affymetrix Expression Console or Transcriptome Analysis

Console (TAC).

Cut-off threshold: Only Present (P) values considered; Absence (A) &
Medium (M) were removed.

Fold Change: Treated/Control (Signal Intensity).

Up-regulated: ≥2 & Down-regulated: ≤0.5 (or -2); P-value: ≤ 0.05.

Results
Screening of DEGs in Response to Individual Stresses
Up-regulated Down-regulated

Bc Bc

Pst Pst

Pst avrRpm1 Pst avrRpm1

Ab Ab

Pr Pr

Ox Ox

W W

SA SA

MeJA MeJA

ACC ACC

ABA ABA
 Common BUGs/BDGs in Response to Biotic, Abiotic &
Hormonal Stresses
Co-upregulated genes Co-downregulated genes
 
Treatment
N° of genes Percentage N° of genes Percentage
 

1047 67.4 637 52.8

P. syringae pv. tomato DC3000  

1095 70.5 751 62.3

P. syringae pv. tomato avrRpm1  
A. brassicicola 635 40.9   491 40.7

P. rapae 761 49.0   548 45.4

Oxidative stress 193 12.4   65 5.4

Wounding 69 4.4   47 3.9

SA 528 34.0   532 44.1

MeJA 510 32.8   478 39.6

At1g22190 (RAP2.4)
ACC 443 28.5   429 35.6 At1g72060
At4g23600 (CORI3/JR2) At2g20670
ABA 562 36.2   511 42.4

All stresses 1 0.1

  3 0.2
Selected Top Ranked DEGs
qRT-PCR Validation of Microarray Results
Mutation in RAP2.4 enhances resistance against B. cinerea
(C)
(B)

(A)

(D)
Down-regulation of RAP2.4 alters the expression of defense-
regulated genes in response to B. cinerea
 SA-associated genes
PR1, PR2 & PAD4
 JA/ET pathway defense genes
PDF1.2, PR3 & PR4
 JA and/or OPDA associated genes
VSP2, MYC2, OPR1 & OPR3
 Cyclopentenone associated genes
GST6 & GSTU19
 Major Findings
Plant responses to all tested stresses can be mediated by commonly
regulated genes.

Upon challenges to individual or multiple stress(es), accumulation of signaling

molecules (e.g., hormones) plays a major role in the activation of downstream
defense responses.

This is the first report demonstrating the role of RAP2.4 in plant defense against
B. cinerea.
Sham et al. (2019) – Sci. Rep.
 Impact of this study
 Findings from this study will have an impact on interactions involving many plant

pathogens and major abiotic stresses affecting economically significant crop species

(e.g., Solanaceae family).

 The identified target genes can be helpful in breeding programs to generate crop

varieties resistant to several pathogen attacks, climatic challenges and hormonal

imbalances.
Thank you!!
 Conclusions
 Identified potential defense-related genes that coordinate regulatory pathways through

WRKY33 in mediating resistance to B. cinerea.

 This comparison of expression profiling of the WRKY33 gene in response to B. cinerea helps

biotechnologist in developing transgenic crops resistant to this pathogen.

 There is a possibility to improve crop resistance by introducing the WRKY33 gene into other

crops (e.g. Solanaceae family) generating resistant/tolerant crops to B. cinerea.

 PLANT GROWTH & DISEASE ASSAY
Seeds: Col-0 (Wt), rap2.4-1 & rap2.4-2 (T-DNA insertion mutant lines).

Age of Plants: 5 weeks old.

Botrytis cinerea: 3μl drop inoculation (2.5×105 spores.mL-1).

Mock (0 hpi): Wt-0; rap2.4-0.

Treated with B. cinerea (18 hpi): Wt-18; rap2.4-18.

 BIOTIC & ABIOTIC TREATMENTS
A. brassicicola (24hpi): 3µl drops of 1 × 106 spores mL-1 (DeVos et al., 2005).

P. syringae pv. tomato DC3000 or avrRpm1 (24hpi): 1 × 108 CFU mL-1
(DeVos et al., 2005).

P. rapae (24hpi): 5 chewing insect larvae (DeVos et al., 2005).

Oxidative Stress (24hpt): exposed to 10 µM paraquat (Kilian et al., 2007).

Wounding (24hpt): punctured with pins (Kilian et al., 2007).

De Vos, et al. (2005). Signal signature and transcriptome changes in Arabidopsis upon pathogen and insect attack. Molecular Plant-Microbe Interactions, 18, 923–937.
Kilian, J. et al. (2007). The AtGenExpress global stress expression data set, protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses. The Plant Journal, 50, 347–363.
 HORMONAL TREATMENTS

Salicylic Acid (3hpt): 10 µM SA (Kilian et al., 2007).

Methyl Jasmonate (3hpt): 10 µM MeJA (Kilian et al., 2007).

Ethylene (3hpt): 10 µM ACC (Kilian et al., 2007).

Abscisic Acid (3hpt): 10 µM ABA (Goda et al., 2008).

Data obtained from: Kilian, J. et al. (2007). The AtGenExpress global stress expression data set, protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses. The Plant Journal, 50, 347–363.
Goda, H. et al. (2008). The AtGenExpress hormone and chemical treatment data set, experimental design, data evaluation, model data analysis and data access. The Plant Journal, 55, 526–542.
 Conclusions
 Identified potential defense-related genes that is common to B. cinerea, other biotic, abiotic

and hormonal stresses.

 CORI3/JR2 (up-regulated) and RAP2.4, At1g72060 and At2g20670 (down-regulated) were

found to be common across the 11 stresses.

 RAP2.4 has the potential to serve in plant defense through the regulation of endogenous signal

molecules and or pathogen‐derived effectors.