Supplementary Material: File 1
Figure 1
Representative
gel
pictures
showing
differential
expression
pattern
between
Control/mock treated (C) and P. personata treated (T) samples. Differential expression
pattern was observed using Genefishing DEG kit, involving isolation of total RNA from
control (C) and pathogen treated (T) leaves, cDNA synthesis followed by amplification
of cDNA with different random arbitrary primers (See Material and Methods for details).
Arrows indicate the differentially upregulated bands in pathogen treated (T) samples
compared with control(C).
1
Figure 2A
11.7%
Primary metabolism/ Development
Secondary metabolism
Defense
Stress response
Signal transduction
Transcriptional regulation
Transport/Secretion
Others/Uncharacterized
Unknown
6.67%
11.7%
5%
10%
16.7%
5%
10%
23.3%
A pie diagram showing the primary role known for/assigned to the proteins encoded by
the genes isolated in this study.
Figure 2B
28.3%
Reported role in defense or stress response
No known role in defense or stress response
Unknown or Uncharacterized
25%
46.7%
A pie diagram illustrating the number of isolated clones with reported role in plant stress
or defense responses.
2
Figure 3
Pair wise sequence alignment of DIR1 and DIR2 nucleotide sequences.
3
Table 1
Sequence information of gene specific primers utilized in this study.
Name
PAL
C4H
CAD
DIR1
DIR2
ACTIN
Forward
GGAACCGGTATGCTCACC
GCGAAATCAACGAAGACAAC
GTTCACTACTGTGGACTCC
CTACACAATGGTGACACAAC
CTACACAATGGTGACACAAC
TGGCATCACACTTTCTACAA
Reverse
GCATCATGTATTTCATTGGCA
CCAACGGCAATGACTTCAG
CGGGTTTATGCATAGCACC
AGTCAAATGCAGTTGTATTAAC
GCAGTTGTATTTACTTTATACG
CAACGGAATCTCTCAGCTCC
Established role of some of the cloned genes in plant defense.
Functional homologs of some of the cloned genes in other plant systems with
established role in defense include ADP-ribosylation factor (ARF) (AdDR-1), which was
shown to be up-regulated in responded to fungal and elicitor treatment. Constitutively
overexpressed rice ARF1 triggered spontaneous lesion mimic phenotype, induction of
pathogenosis-related (PR) genes, reduced susceptibility to a fungal pathogen, and caused
accumulation of SA (Lee et al. 2003). ADP-ribosylation factor induces cell death upon
overexpression in N. benthamiana and found to be a component of non-host resistance
and R-gene mediated resistance (Coemans et al. 2008). Sec61α subunit (AdDR-4), which
is part of Sec61 translocon complex, involved in the translocation of proteins to cross the
endoplasmic reticulum (ER) membrane. It was shown that protein secretory pathway is
required for systemic acquired resistance (SAR) and NPR1 regulates the expression of
several secretory pathway genes including Sec61α subunit gene in Arabidopsis (Wang et
al. 2005). T-DNA insertions in SEC61 α compromised the plant's ability to efficiently
secrete PR1 as well as impaired resistance against the pathogen Pseudomonas syringae
4
(Wang et al. 2005). CBL-interacting protein kinase (CIPK) (AdDR-5), which along with
calcineurin B-like protein (CBL) is emerging as a major calcium signaling network with
only reported role in abiotic stress responses, it would be interesting to study its role in
biotic stress responses. Papain-like cysteine protease (AdDR34) is a member of the
cysteine proteases, whihc have emerged as key enzymes in the regulation of animal PCD.
Rcr3 (papain-like cysteine endoprotease) is specifically required for the function of Cf-2,
a Lycopersicon pimpinellifolium gene bred into cultivated tomato (Lycopersicon
esculentum) for resistance to Cladosporium fulvum (Krüger et al. 2002). Zinc finger
proteins (AdDR-7) were shown to have role in defense against pathogens (Oh et al.
2005). Putative leucine rich repeat proteins (AdDR-10) are plant disease resistance genes
(R-genes), which mediate resistance to pathogens harboring corresponding avirulence
(Avr) gene (Dangl and Jones 2001).
Tonoplast intrinsic protein (TIP) (AdDR-12) was found to have role in H2O2
transport as well as detoxification of light induced ROS and suggested to have involved
in intracellular signal propagation (Maurel et al. 2009). Enzyme 1-deoxy-D-xylulose 5phosphate synthase (AdDR-17) is part of terpenoid biosynthesis pathway. Glutathione Stransferase (GST) (AdDR-19) was proposed to be involved in detoxification and
controlling the progress of cell death during hypersensitive reaction (Wagner et al. 2002).
Putative translation initiation protein (AdDR-20) was reported as a key element of the
signal transduction pathway resulting in plant programmed cell death (Hopkins et al.
2008). Lysin motif-type receptor-like kinase (AdDR-31) plays a role in recognizing both
friends and foes (Knogge and Scheel 2006). AdDR32 encodes Pyridoxine biosynthesis
protein, Pyridoxine (Vitamin B6) is a strong antioxidant with potential role in the plant-
5
pathogen defense response. Proline is the most diversely used osmolyte that accumulates
in osmotically stressed organisms. Proline dehydrogenase (ProDH) (AdDR-33) was
transiently induced both in tobacco and in Arabidopsis upon R. fascians infection
(Simón-mateo et al. 2006). Heat shock protein 70 (HSP70) (AdDR-37) along with HSP90
were found to be essential components of hypersensitive response and non-host
resistance in N. benthamiana plants (Kanzaki et al. 2003). Thaumatin like proteins
(AdDR-11) are class-V pathogenesis related proteins with antimicrobial activity (van
Loon et al. 2006). Lipid transfer proteins (LTPs) (AdDR-51) belong to PR-14 family and
known for antifungal and anti microbial activity (van Loon et al. 2006).
References
Coemans B, Takahashi Y, Berberich T, Ito A, Kanzaki H, Matsumura H et al. (2008)
High-throughput in planta expression screening identifies an ADP-ribosylation
factor (ARF1) involved in non-host resistance and R gene-mediated resistance.
Mol Plant Pathol 9:25-36
Dangl JL, Jones JD (2001) Plant pathogens and integrated defence responses to infection.
Nature 411:826-833
Hopkins MT, Lampi Y, Wang TW, Liu Z, Thompson JE (2008) Eukaryotic translation
initiation factor 5A is involved in pathogen-induced cell death and development
of disease symptoms in Arabidopsis. Plant Physiol 148:479-489
Kanzaki H, Saitoh H, Ito A, Fujisawa S, Kamoun S, Katou S et al. (2003) Cytosolic
HSP90 and HSP70 are essential components of INF1-mediated hypersensitive
response and non-host resistance to pseudomonas cichorii in Nicotiana
benthamiana. Mol Plant Pathol 4:383-391
Knogge W, Scheel D (2006) LysM receptors recognize friend and foe. Proc Natl Acad
Sci USA 103:10829-10830
6
Krüger J, Thomas CM, Golstein C, Dixon MS, Smoker M, Tang S et al. (2002) A tomato
cysteine protease required for Cf-2-dependent disease resistance and suppression
of autonecrosis. Science 296:744-747
Lee WY, Hong JK, Kim CY, Chun HJ, Park HC, Kim JC et al. (2003) Over-expressed
rice ADP-ribosylation factor 1 (RARF1) induces pathogenesis-related genes and
pathogen resistance in tobacco plants. Physiol Plant 119:573-581
Maurel C, Santoni V, Luu D-T, Wudick MM, Verdoucq L (2009) The cellular dynamics
of plant aquaporin expression and functions. Curr Opin Plant Biol 12:690-698
Oh S-K, Jeong MP, Young HJ, Lee S, Chung E, Kim S-Y et al. (2005) A plant EPF-type
zinc-finger protein, CaPIF1, involved in defence against pathogens. Mol Plant
Pathol 6:269-285
Simón-Mateo C, Depuydt S, De Oliveira Manes CL, Cnudde F, Holsters M, Goethals K
et al. (2006) The phytopathogen Rhodococcus fascians breaks apical dominance
and activates axillary meristems by inducing plant genes involved in hormone
metabolism. Mol Plant Pathol 7:103-112
Wagner U, Edwards R, Dixon DP, Mauch F (2002) Probing the diversity of the
Arabidopsis glutathione S-transferase gene family. Plant Mol Biol 49:515-532
Wang D, Weaver ND, Kesarwani M, Dong X (2005) Induction of protein secretory
pathway is required for systemic acquired resistance. Science 308:1036-1040
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