A plastidic glucose-6-phosphate dehydrogenase is responsible for hypersensitive response cell death and reactive oxygen species production

Abstract

Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in the supply of reduced nicotine amide cofactors for resistance
to biotic and abiotic stresses. Here, we show participation of the plastidic P2 isoform of G6PDH in plant immunity. A cytosolic
isoform (NbG6PDH-Cyto) and two plastidic isoforms (NbG6PDH-P1 and NbG6PDH-P2) cloned from Nicotiana benthamiana were localized in cytosol and chloroplasts, respectively. Hypersensitive response (HR) cell death and NADPH oxidase (RBOH;
respiratory burst oxidase homolog)-dependent reactive oxygen species (ROS) production after recognition of INF1 elicitin,
secreted by oomycete Phytophthora infestans, decreased in NbG6PDH-P2-silenced plants, but not in NbG6PDH-Cyto- and NbG6PDH-P1-silenced plants. Silencing of the cytosolic NAD kinase NbNADK1, which phosphorylates NADH to form NADPH, compromised HR cell death and ROS production, and concomitant silencing with NbG6PDH-P2 reduced HR cell death and ROS to levels near those in NbG6PDH-P2-silenced plants. Similarly, silencing NbG6PDH-P2 and NbNADK1 resulted in high susceptibility to P. infestans. These results suggest that NADPH produced by the P2 isoform of G6PDH in chloroplasts is responsible for HR cell death and
ROS production mediated by RBOH and that NbNADK1 is involved in this pathway.

KeywordsG6PDH–HR cell death–ROS burst–NAD kinase–NADPH oxidase–
Phytophthora infestans

Full text

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HOST RESPONSES
A plastidic glucose-6-phosphate dehydrogenase is responsible
for hypersensitive response cell death and reactive oxygen species
production
Shuta Asai•Miki Yoshioka•Hironari Nomura•
Chiyori Tone•Kazumi Nakajima•Eiichi Nakane•
Noriyuki Doke•Hirofumi Yoshioka
Received: 11 December 2010/Accepted: 7 February 2011/Published online: 10 March 2011
? The Phytopathological Society of Japan and Springer 2011
Abstract
has been implicated in the supply of reduced nicotine amide
cofactors for resistance to biotic and abiotic stresses. Here,
we show participation of the plastidic P2 isoform of G6PDH
in plant immunity. A cytosolic isoform (NbG6PDH-Cyto)
and two plastidic isoforms (NbG6PDH-P1 and NbG6PDH-
P2) cloned from Nicotiana benthamiana were localized in
cytosol and chloroplasts, respectively. Hypersensitive
response (HR) cell death and NADPH oxidase (RBOH;
respiratory burst oxidase homolog)-dependent reactive
oxygen species (ROS) production after recognition of INF1
elicitin, secreted by oomycete Phytophthora infestans,
decreased in NbG6PDH-P2-silenced plants, but not in
NbG6PDH-Cyto-and
NbG6PDH-P1-silenced
Silencing of the cytosolic NAD kinase NbNADK1, which
phosphorylates NADH to form NADPH, compromised HR
cell death and ROS production, and concomitant silencing
with NbG6PDH-P2 reduced HR cell death and ROS to
levels near those in NbG6PDH-P2-silenced plants. Simi-
larly, silencing NbG6PDH-P2 and NbNADK1 resulted in
Glucose-6-phosphate dehydrogenase (G6PDH)
plants.
high susceptibility to P. infestans. These results suggest that
NADPH produced by the P2 isoform of G6PDH in chlo-
roplasts is responsible for HR cell death and ROS produc-
tion mediated by RBOH and that NbNADK1 is involved in
this pathway.
Keywords
kinase ? NADPH oxidase ? Phytophthora infestans
G6PDH ? HR cell death ? ROS burst ? NAD
Introduction
Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes
the first committed step of the oxidative pentose phosphate
pathway (OPPP), a major source of reducing power (as
NADPH). G6PDH has been reported to be involved in
resistance to biotic and abiotic stresses. Defective yeast
mutants were found to be vulnerable to oxidative stress
(Juhnke et al. 1996), whereas overexpressing mammalian
cells that display higher G6PDH activity are highly pro-
tected against oxidant-mediated cell death (Salvemini et al.
1999). In plants, there is substantial evidence that activity
and transcripts of G6PDH are modulated during responses
to salt, metals, herbicides and elicitor (Hauschild and von
Schaewen 2003; Nakane et al. 2003; Nemoto and Sasa-
kuma 2000; S´laski et al. 1996). G6PDH activity has been
detected in both cytosol and plastids (Schnarrenberger et al.
1973), and recent biochemical and genome-wide analyses
have provided evidence of a cytosolic isoform (Cyto) and
two plastidic isoforms (P1 and P2) (Wakao and Benning
2005; Wendt et al. 1999). In potato plants, the cytosolic
G6PDH isoform seems to be expressed ubiquitously, with
the highest mRNA and protein levels in tubers (von
Schaewen et al. 1995). By contrast, the plastidic isoforms
are expressed in leaves and roots rather than in tubers, and
The nucleotide sequence data reported are available in the DDBJ/
EMBL/GenBank databases as accessions AB603763-AB603768.
M. Yoshioka ? H. Nomura ? C. Tone ? K. Nakajima ?
E. Nakane ? N. Doke ? H. Yoshioka (&)
Graduate School of Bioagricultural Sciences,
Nagoya University, Nagoya 464-8601, Japan
e-mail: hyoshiok@agr.nagoya-u.ac.jp
Present Address:
S. Asai
The Sainsbury Laboratory, John Innes Centre,
Norwich Research Park, Norwich NR4 7UH, UK
123
J Gen Plant Pathol (2011) 77:152–162
DOI 10.1007/s10327-011-0304-3

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the levels of P2 transcripts are higher than those of P1
(Wendt et al. 2000). Two cysteine positions are highly
conserved in plastidic G6PDH isoforms, but not in cyto-
solic isoforms. Treatment with reduced dithiothreitol or
glutathione leads to inactivation of plastidic G6PDH,
whereas the activity of cytosolic isoform is not influenced
by reduction (Wenderoth et al. 1997). Recombinant P2
protein is more active than P1 and less susceptible to
feedback inhibition by its product NADPH (Wendt et al.
2000). P2-antisense tobacco (Nicotiana tabacum) has ele-
vated levels of reduced ascorbate and glutathione, provid-
ing greater protection from oxidative stress (Debnam et al.
2004). In Arabidopsis cytosolic G6PDH mutants, which
have a decrease in total cytosolic G6PDH activity, the
metabolism and oil content of developing seeds is affected
(Wakao et al. 2008). Altogether, each G6PDH isoform
seems to have a diversified regulatory mechanism and play
a diverse role in resistance to several stresses and devel-
opment. However, the role of G6PDH isoforms in plant
immunity is poorly understood.
NADPH, an important reducing-energy equivalent, is
also generated by NAD kinase, which catalyzes the phos-
phorylation of NAD(H) (Berrin et al. 2005; Kawai and
Murata 2008). Several studies have indicated that NAD
kinase is essential for the survival of certain organisms
(Kawai and Murata 2008). In most organisms, there is only
one NAD kinase, but in some organisms, such as yeast and
plant, several NAD kinase isoforms may exist. There are
threeNADkinasesin
Arabidopsis
AtNADK1 in cytosol, AtNADK2 in chloroplasts, and
AtNADK3 in peroxisomes (Chai et al. 2005, 2006; Turner
et al. 2004, 2005; Waller et al. 2010). The transcription of
AtNADK1 is upregulated after treatment with hydrogen
peroxide and gamma irradiation, and the deficient mutant is
vulnerable to irradiation and paraquat-induced oxidative
stress (Berrin et al. 2005). AtNADK2- and AtNADK3-defi-
cient mutants also exhibit hypersensitivity to oxidative
stress (Chai et al. 2005, 2006). These reports suggest that
plant NAD kinases play a role in protecting against oxi-
dative damage. However, little is known about the roles of
NAD kinases during plant–pathogen interactions.
Plant defenses are often initiated by a gene-for-gene
interaction between a dominant plant resistance gene and a
pathogen avirulence gene, which provides race-specific
resistance. In addition to the resistance-gene-mediated
pathways of plant resistance to specific pathogens, plants
have the capacity to recognize a number of microbial
surface-derived molecules known as microbe-associated
molecular patterns, which elicit a general immune response
in both host and nonhost plants. Following recognition of a
pathogen, a wealth of defense mechanisms (e.g., generation
thaliana, with
of reactive oxygen species [ROS] and nitric oxide [NO],
activation of mitogen-activated protein kinase [MAPK],
synthesis of pathogenesis-related proteins, and the hyper-
sensitive response [HR]) can efficiently limit pathogen
growth. The HR accompanied by ROS production and cell
death represents the most efficient mechanism of plant
defense (Doke 1983). NADPH oxidase (RBOH, respiratory
burst oxidase homolog), localized on the plasma membrane
(Kobayashi et al. 2006), is considered to be the main source
of extracellular ROS production during defense, the so-
called ROS burst. Down-regulation or elimination of
RBOH leads to suppression of the ROS burst and HR cell
death (Torres et al. 2002; Yoshioka et al. 2003).
In our previous study, we identified a G6PDH gene as an
elicitor-inducible gene in potato (Nakane et al. 2003). In
the present study, we investigated the role of G6PDH
isoforms in plant immunity in Nicotiana benthamiana.
Loss-of-functionanalyses
silencing (VIGS) showed that the plastidic P2 isoform of
G6PDH participates in regulating the HR cell death and
ROS burst induced by INF1 elicitin, produced by oomycete
Phytophthora infestans (Kamoun et al. 1997). We also
showed the involvement of the cytosolic NAD kinase,
NbNADK1, in this pathway.
usingvirus-induced gene
Materials and methods
Plant materials and growth conditions
Nicotiana benthamiana plants were grown at 25?C and
70% humidity with 16-h light/8-h dark in environmentally
controlled growth cabinets.
Agrobacterium tumefaciens-mediated transient
expression (agroinfiltration)
A cDNA fragment of b-glucuronidase (GUS), used as a
control for the effect of agroinfiltration, was cloned into
pGreen binary vector (Hellens et al. 2000) and was
expressed using A. tumefaciens strain GV3101 as described
by Asai et al. (2008). A. tumefaciens carrying inf1 was
prepared as described by Kamoun et al. (2003).
Virus-induced gene silencing
Virus-induced gene silencing (VIGS) was done as descri-
bed by Ratcliff et al. (2001). The following primers were
used to amplify cDNA fragments from N. benthamiana
using the N. benthamiana cDNA library as a template
(Yoshioka et al. 2003). Restriction sites were added to the
J Gen Plant Pathol (2011) 77:152–162153
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50-end of the forward and reverse primer for cloning into a
Tobacco rattle virus (TRV) vector pTV00 (RNA2):
NbG6PDH-Cyto-F-SalI (50-GCGTCGACCGTATCCGT
GGGTATCTTTCTA-30)and
(50-CCATCGATTAGTTCTCCTATCTGGGAACTT-30)
(restriction sites are underlined), which produced a 345-bp
fragment, NbG6PDH-P1-F-SalI (50-GCGTCGACGTGG
GCAGTTGCAATTGAATCC-30) and NbG6PDH-P1-R-
ClaI(50-CCATCGATTCTGGCATAGATTTTGACGCC
G-30), which produced a 259-bp fragment, NbG6PDH-P2-
F-SalI (50-GCGTCGACTGACCCTTTATTCTTCTCCTT
C-30) and NbG6PDH-P2-R-ClaI (50-CCATCGATTTCT
GCTCCTTTGGAGGTGCGA-30),
283-bp fragment, and NbNADK1-F-ClaI (50-CCATCGA
TTGGGGATGGAACCGTCCTTTGG-30) and NbNADK1-
R-BamHI (50-CCGGATCCACAAAAGAATTATCGCA
ATAGC-30), whichproduced
N. benthamiana was infected by viruses using Agrobacte-
rium-mediated transient expression of infectious constructs.
pBINTRA6 (RNA1) and pTV00 containing the inserts
(RNA2) were used separately to transform A. tumefaciens
strain GV3101, which includes the transformation helper
plasmid pSoup (Hellens et al. 2000) by electroporation. A
mixture of equal parts of Agrobacterium suspensions of
RNA1 and RNA2 was used to inoculate 2- to 3-week-old N.
benthamianaseedlings.Theinoculatedplantsweregrownin
16-h light/8-h dark at 23?C. The upper leaves of the inocu-
lated plants were used for assays 3 to 4 weeks after
inoculation.
NbG6PDH-Cyto-R-ClaI
which produceda
a315-bpfragment.
Microscopic observations
The following primers were used to amplify full-length
cDNA fragments of NbG6PDH-Cyto, NbG6PDH-P1 and
NbG6PDH-P2 from a N. benthamiana cDNA library as a
template (Yoshioka et al. 2003). Gene-specific primers
were as follows: NbG6PDH-Cyto (50-CACCATGGCAGC
ATCTTGGTGCAT-30, 50-TAATGTGGGAGGAATCCAT
A-30), NbG6PDH-P1 (50-CACCATGGGTGGGCAGTTGC
AATG-30,50-ATCATCACCAGACAGATCTC-30),
NbG6PDH-P2
(50-CACCATGGTGACCCTTTATTCTT
C-30, 50-CACAAGATCACCCCATCTTA-30). All PCR
products were cloned into pENTR D-TOPO, as described
by the manufacturer (Invitrogen, Carlsbad, CA, USA). The
transfer of the DNA fragment from the entry clone to
pGWB405 (Nakagawa et al. 2009) by LR reaction was
performed as described by the manufacturer (Invitrogen).
cDNA fragments of G6PDH isoforms including green
fluorescent protein (GFP) to the C-terminus were expressed
using A. tumefaciens strain GV3101 as already described.
Protoplasts were prepared 40 h after the inoculation as
described by D’Angelo et al. (2006). Fluorescence of GFP
and autofluorescence of chloroplasts were captured at
and
excitation 473 and 635 nm and emission 520/40 and
690/60 nm, respectively, using a confocal laser scanning
microscope(FluoviewFV1000-D,Olympus,Tokyo,Japan).
Reverse transcription (RT)-PCR
Total RNA samples were prepared from N. benthamiana
for RT-PCR. Gene-specific primers for RT-PCR in
N. benthamiana were: NbG6PDH-Cyto (50-CAGGGATTCC
TGCAATCCAATGAAGTTCAC-30,
ATTAAATAGTTCTCCTATCT-30), NbG6PDH-P1 (50-A
TGGGTGGGCAGTTGCAATTGAATCCTTG-30, 50-AG
GTCTTGCTGATCATGTTTCTCAGCTCTT-30), NbG6
PDH-P2 (50-ATGGTGACCCTTTATTCTTCTCCTTCAA
C-30, 50-TCATCTTACTCCGGGCATAGCCAAAGATA
G-30),
NbRBOHB
(50-TTTTCTCTGAGGTTTGCCAG
CCACCA-30, 50-GCCTTCATGTTGTTGACAATGTCTT
T-30), NbNADK1 (50-CAGCTGGAGGGTCAATGGTC-30,
50-CCCATGCATGACCTCTGCTA-30), NbNADK2 (50-G
CAAGATACCAGCGATCTTCATGAG-30, 50-GGTATC
TTTAATTCTAGTTTTGCAGAATCTG-30), NbNADK3
(50-CTTGAAAATCATGCTAGACCTTCTGAAGTC-30,
50-CCAGCCGCTGTTGACACTCTAAGTCC-30),
NbEF-1a (50-TGTGGAAGTTTGAGACCACC-30, 50-GC
AAGCAATGCGTGCTCAC-30).
50-TTTGGGGTTC
and
Cell death measurements
Cell death was quantified by measuring ion leakage as
described by del Pozo et al. (2004). For conductivity tests,
10 discs/leaf (8 mm diameter) were obtained from inocu-
lated areas of each leaf and were floated on 10 ml distilled
water for 4 h at room temperature with gentle shaking.
Conductivity was measured by using a multifunctional
meter D-54 (HORIBA, Kyoto, Japan).
ROS measurements
ROS measurements were done using L-012 (Wako Pure
Chemical Industries, Osaka, Japan) as described by
Kobayashi et al. (2007).
Pathogen inoculation and determination of the biomass
Phytophthora infestans race 1.2.3.4 zoospores were pre-
pared and inoculated as described by Asai et al. (2008).
Growth of P. infestans was measured using quantitative
real-time PCR and NbEF-1a-specific primers for RT-PCR
and the primers to detect the P. infestans-specific DNA
sequence (50-GAAAGGCATAGAAGGTAGA-30, 50-TAA
CCGACCAAGTAGTAAA-30) by the method of Asai et al.
(2008).
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Results
Cloning and subcellular localization of G6PDH
isoforms from N. benthamiana
Our previous study identified elicitor-inducible genes in
potato (Nakane et al. 2003). One of these encodes for
G6PDH, the key enzyme of OPPP that is responsible for
the generation of NADPH. The presence of cytosolic and
plastidic isoforms of G6PDH in plant tissues has been
reported (Schnarrenberger et al. 1973). To evaluate the role
of G6PDHs in plant immunity, we first isolated one cyto-
solic isoform (Cyto) and two plastidic isoforms (P1 and
P2) of G6PDH from N. benthamiana designated as
G6PDH-Cyto (AB603763), G6PDH-P1 (AB603764) and
G6PDH-P2 (AB603765), respectively. P1 and P2 share
51.0 and 51.5% identity with Cyto, respectively, and P1 is
82.6% identical to P2 (Fig. 1a). When comparing the
homologies among the sequences of N. benthamiana
G6PDH isoforms and the previously identified G6PDH
isoforms (Graeve et al. 1994; von Schaewen et al. 1995;
Wakao and Benning 2005; Wendt et al. 1999, 2000), Cyto,
P1 and P2 isoforms in N. benthamiana share high homol-
ogy with Cyto, P1 and P2 isolated from different plant
species, respectively (Fig. 1b). Transient expression of
N. benthamiana G6PDH isoforms fused with GFP in
N. benthamiana protoplasts clearly showed that the cyto-
solic isoform (Cyto) and plastidic isoforms (P1 and P2) are
localized in the cytosol and chloroplasts of N. benthamiana
cells, respectively (Fig. 2).
Silencing NbG6PDH-P2 compromises HR cell death
and ROS burst induced by INF1
G6PDH has been reported to be involved in several stress
responses in plants, because the activity and transcription
rate of G6PDH are modulated during stress responses such
as salt, metals, herbicides and elicitor (Hauschild and von
a
b
Solanum tuberosum
Nicotiana tabacum
Nicotiana benthamiana
Arabidopsis thaliana
Oryza sativa
Solanum tuberosum
Nicotiana tabacum
Arabidopsis thaliana2
Oryza sativa
Nicotiana tabacum
Arabidopsis thaliana2
Oryza sativa
Solanum tuberosum
Cyto
P1
P2
Nicotiana benthamiana
Nicotiana benthamiana
Arabidopsis thaliana1
Arabidopsis thaliana1
0.1
Cyto
P1
P2
1 ------------------------------------------------------------
1 M
1 M
Cyto
P1
P2
1 -------MAASWCIEKRGSLRLDS-------------FRENDNIPETGCLSIIVLGASGD
58-
61I
-- QVPLTELEN--AETTVSITVIGASGD
DTIDFDGNKAKSTVSITVVGASGD
Cyto
P1
P2
41 LAKKKTFPALFNLYRQGFLQSNEVHIFGYARTKISDDDLRS I
113 LAKKKIFPALFALFYEDCLPEN-FIVFGYSRTKMSDEELRNMISKTLTCRIDQRENCEAK
121 LAKKKIFPALFALYYEDCLPEH-FTIFGYARSKMTDVELRNMVSKTLTCRIDKRENCGEK
L ----KGKEYQEE
Cyto
P1
P2
97ARFA S PS
172 MDHFLERCFYQSGQYNSEDDFAELDYKLKAKEGCR-----VSNRLFYLSIPPNIFVDVVR
180 MEQFLERCFYHSGQYDSQDNFAELDKKLKEHEAGR-----FSNRLFYLSIPPNIFINAVR
Cyto
P1
P2
157
227 CAS---VKASSTSGWTRVIVEKPFGRDLESSSELTRCLKKYLTEEQIFRIDHYLGKELVE
235 CAS---LSASSAHGWTRVIVEKPFGRDSESSAALTRSLKQYLNEDQIFRIDHYLGKELVE
SIV FDEPQIYRIDHYLGKELVQ
Cyto
P1
P2
217 NLLVLRFANRFFLPLWNRDNIDNIQIVFREDFGTEGRGGYFDEYGIIRDIIQNHLLQVLC
284 NLSVLRFSNLVFEPLWSRNYIRNVQFIFSEDFGTEGRGGYFDNYGIIRDIMQNHLLQILA
292 NLSVLRFSNLIFEPLWSRQYIRNVQFIFSEDFGTEGRGGYFDHYGIIRDIMQNHLLQILA
Cyto
P1
P2
277 LVAMEKPVSLKPEHIRDEKVKVLQSMLPIKDEEVVLGQYEG----------YKDDPTVPD
344 LFAMETPVSMDAEDIRNEKVKVLRSMRPLQLEDVVLGQYKGHSKGGKLYPAYTDDPTVPN
352 LFAMETPVSLDAEDIRNEKVKVLRSMRPLQLDDVIIGQYKCHTKGDVTYPGYTDDKTVPK
Cyto
P1
P2
327 NSNTPTFATMVLRIHNERWEGVPFIMKAGKALNSRKAEIRVQFKDVPGDIFRCNK-----
404 GSITPTFSAAALFINNARWDGVPFLMKAGKALHTRRAEIRVQFRHVPGNLYKKNFGTDLD
412 DSLTPTFAAAALFIDNARWDGVPFLMKAGKALHTRSAEIRVQFRHVPGNLYNKNFGSDLD
Cyto
P1
P2
382 QGRNEFVIRLQPSEAMYMKLTVKKPGLEMSTVQSELDLSYGQRYQGVVIPEAYERLILDT
464 KATNELVLRLQPDEAIYLKINNKVPGLGMRLDRSDLNLLYKAKYR-GEIPDAYERLLLDA
472 QATNELVIRVQPNEAIYLKINNKVPGLGMRLDCSNLNLLYSARYS-KEIPDAYERLLLDA
Cyto
P1
P2
442 IRGDQQHFVRRDELKAAWEIFTPLLHRIDDGEVKPIPYKPGSRGPAEADELLQNVGYVQT
523 IEGERRLFIRSDELDAAWALFTPLLKELEEKKIAPELYPYGSRGPVGAHYLAA--KHNVR
531 IEGERRLFIRSDELDAAWSLFTPVLNELEDKKIVPEYYPYGSRGPIGAHYLAA--RYKVR
Cyto
P1
P2
502 HGYIWIPPTL
581 WGDLSGDD--
589 WGDLV-----
Fig. 1 Sequence alignment between G6PDH isoforms in Nicotiana
benthamiana and phylogenetic analysis of G6PDH isoforms. a Iden-
tical sequences are indicated in white on black, and similar amino
acids in hydrophobic or hydrophilic features are highlighted in black
on gray. Dashes indicate gaps introduced to maximize alignment.
Shades of red indicate amino acid sequence consistent with the
regions of insertion for virus-induced gene silencing. Multiple
alignments of the amino acid sequences were made using the Clustal
W2 method (www.ebi.ac.uk/Tools/clustalw2). b An unrooted tree
was constructed using the TreeView method (http://taxonomy.
zoology.gla.ac.uk/rod/treeview.html). The length of the lines con-
necting the sequences is proportional to the estimated genetic distance
between these sequences. The amino acid sequences can be found in
the GenBank/EMBL/DDBJ data libraries (Nicotiana benthamiana:
Cyto, AB603763; P1, AB603764; P2, AB603765; Arabidopsis tha-
liana: Cyto1, AT3G27300; Cyto2, AT5G40760; P1, AT5G35790; P21,
AT5G13110; P22, AT1G24280; Nicotiana tabacum: Cyto, AJ001769;
P1, AJ001772; P2, X99405; Solanum tuberosum: Cyto, X74421; P1,
X83923; P2, AJ010712; Oryza sativa: Cyto, AY078072; P1,
NM_001056891; P2, AY339367)
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Schaewen 2003; Nakane et al. 2003; Nemoto and Sasa-
kuma 2000; S´laski et al. 1996). For assessing the expres-
sion patterns of G6PDH isoforms during defense response,
INF1 and GUS as a control were expressed using agroin-
filtration in N. benthamiana leaves. The expression of INF1
led to increased expression of NbG6PDH-Cyto, but to a
decrease in NbG6PDH-P1. NbG6PDH-P2 was constitu-
tively expressed after INF1 treatment. We also checked the
induction of NbRBOHB, which is an inducible form of
NADPH oxidase of N. benthamiana (Yoshioka et al. 2003),
as a positive control (Fig. 3a).
ROS production has been implicated in HR cell death
and is mediated by NADPH oxidase (RBOH) (Torres
2010). G6PDH generates NADPH by oxidizing glucose-6-
phosphate in the first step of OPPP. These findings
prompted us to examine the possibility that G6PDH iso-
forms participate in HR cell death and the ROS burst. To
investigate this possibility, we conducted G6PDH isoform-
specific gene silencing in N. benthamiana by VIGS. We
cloned cDNA fragments of NbG6PDH-Cyto-, NbG6PDH-
P1- and NbG6PDH-P2-specific regions into TRV vector
(Fig. 1a) and introduced these into N. benthamiana by
agroinfiltration (Ratcliff et al. 2001). Three weeks after the
infiltration, the efficiency of silencing was confirmed using
RT-PCR (Fig. 3b). We also checked that silencing G6PDH
isoforms had no effect on NbRBOHB transcripts (Fig. 3b).
Silenced leaves were inoculated with A. tumefaciens con-
taining inf1 or GUS as a control. Cell death was quantified
by measuring ion leakage from inoculated leaf areas.
Silencing NbG6PDH-Cyto and NbG6PDH-P1 did not
affect INF1-inducible cell death. By contrast, the cell death
was compromised in NbG6PDH-P2- and NbG6PDH-P1/
P2-silenced leaves (Fig. 3c, e). ROS production was
measured 24 h after the inoculation using L-012 solution, a
highly sensitive agent to detect ROS (Asai et al. 2008).
This method detects RBOH-generated ROS in N. benth-
amiana (Asai et al. 2008; Kobayashi et al. 2007). Like HR
cell death, silencing NbG6PDH-P2 and NbG6PDH-P1/P2,
but not NbG6PDH-Cyto and NbG6PDH-P1, significantly
decreased ROS production induced by INF1 (Fig. 3d, f).
These results suggest that the P2 isoform is responsible for
HR cell death and RBOH-dependent ROS burst.
NbNADK1 participates in NbG6PDH-P2-related HR
cell death and ROS burst
As already shown, the plastidic G6PDH-P2, which is
localized in chloroplasts, seems to be related to HR cell
Cyto
P1
P2
GFPChloroplastMerge
Fig. 2 Localization of
NbG6PDH-Cyto-GFP (Cyto),
NbG6PDH-P1-GFP (P1), and
NbG6PDH-P2-GFP (P2) in
Nicotiana benthamiana
protoplasts. Confocal images of
protoplasts prepared from
leaves inoculated with
Agrobacterium tumefaciens
containing NbG6PDH-Cyto-
GFP, NbG6PDH-P1-GFP, or
NbG6PDH-P2-GFP for 40 h.
Scale bar represents 10 lm
156J Gen Plant Pathol (2011) 77:152–162
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Page 6

death and to ROS production that is dependent on RBOH
localized on plasma membrane (Kobayashi et al. 2006).
The findings reminded us that NADPH produced by the P2
isoform in chloroplasts might be transported to RBOH on
plasmamembrane.Indirecttransportof reducing
equivalents from chloroplasts to cytosol can be achieved by
malate-oxaloacetate (OAA) shuttles, involving malate
dehydrogenase (MDH) for the interconversion (Scheibe
2004). In the shuttle system, NADH equivalent to NADPH
in chloroplasts can be transported to the cytosol. NAD
TRVTRV:CytoTRV:P1 TRV:P2 TRV:P1/P2
TRV
TRV:P1/P2
TRV:P1 TRV:P2
TRV:Cyto
Cyto
P1
P2
NbRBOHB
NbEF-1α
a
c
d
GUS
GUS
INF1
INF1
GUS
GUS
INF1
INF1
GUS
GUS
INF1
GUS
INF1
GUS
INF1
f
1000
TRV
1500
0
TRV:Cyto TRV:P1
GUS
INF1
*
TRV:P2
500
GUS
INF1
*
TRV:P1/P2
Chemiluminescence
(counts/min/pixel)
INF1
GUS
INF1
GUS
INF1
e
Conductivity (μS)
200
TRV
300
0
TRV:Cyto TRV:P1
*
TRV:P2
100
*
TRV:P1/P2
C24 36
GUS
24 36
INF1
Cyto
P1
P2
NbRBOHB
NbNADK1
NbEF-1α
(h)
b
Fig. 3 Expression profiles of G6PDH isoforms and effects of
silencing on HR cell death and ROS burst. Total RNA was extracted
from intact leaves (C) and from leaves at the indicated time after
inoculation with Agrobacterium tumefaciens containing the indicated
gene constructs (a) or from silenced leaves 24 h after inoculation with
A. tumefaciens containing inf1 (b). RNA was used for RT-PCR with
specific primers for NbG6PDH-Cyto, NbG6PDH-P1, NbG6PDH-P2,
NbRBOHB, and NbNADK1. Equal loads of cDNA were monitored by
amplification of constitutively expressed NbEF-1a. c Silenced leaves
inoculated with A. tumefaciens containing inf1 at the right half of
leaves were photographed 3 d after inoculation. d At 24 h after
inoculation with the indicated gene constructs, ROS were detected
using L-012. White circles indicate areas infiltrated with L-012. e Cell
death was quantified by ion leakage of the inoculated leaves in (c).
Data are means ± SD from three experiments. f Chemiluminescence
intensities mediated by L-012 in (d) were quantified with a photon
image processor. Data are means ± SD from four experiments. Data
were analyzed with Student’s t-test. *P\0.01 versus TRV control
plants
J Gen Plant Pathol (2011) 77:152–162157
123

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kinase catalyzes the phosphorylation of NAD(H) to form
NADP(H) (Berrin et al. 2005; Kawai and Murata 2008).
Three NAD kinases, AtNADK1, AtNADK2, and At-
NADK3, were found in Arabidopsis thaliana and were
localizedincytosol,chloroplasts
respectively (Chai et al. 2005, 2006; Turner et al. 2004,
2005; Waller et al. 2010). These findings prompt us to
examine the possibility that NADK1 also participates in
HR cell death and ROS burst mediated via G6PDH-P2. To
confirm this possibility, we isolated the NADK homologs
from
N.benthamiana
designated
(AB603766), NbNADK2 (AB603767) and NbNADK3
(AB603768) and silenced NbNADK1, NbG6PDH-P2 or
both using the TRV-VIGS system. The transcripts in
silenced plants decreased compared with TRV control
plants(Fig. 4a). Wealso
NbNADK1 did not affect transcript levels of NbNADK2,
NbNADK3 and NbRBOHB using NbNADK2-, NbNADK3-
and NbRBOHB-specific primers, respectively (Fig. 4a).
Silencing NbNADK1 significantly compromised the HR
cell death and ROS burst induced by INF1, and double
silencing NbNADK1 and NbG6PDH-P2 reduced these
close to the levels in NbG6PDH-P2-silenced plants
(Fig. 4b, c). These results suggest the involvement of
NbNADK1 in NbG6PDH-P2-related HR cell death and
ROS burst.
andperoxisomes,
as
NbNADK1
confirmed thatsilencing
Silencing NbG6PDH-P2 and NbNADK1 causes high
susceptibility to P. infestans
ROS burst plays an important role in resistance to several
pathogens, such as P. infestans (Asai and Yoshioka 2008),
which is a hemibiotroph but a near-obligate pathogen (Fry
2008) as well as a potent pathogen of N. benthamiana
(Kamoun et al. 1998). The death of host cells is disad-
vantageous for obligate pathogens, that is, biotrophs feed-
ing on living host tissue. NbG6PDH-P2-, NbNADK1- or
both-silenced plants showed the reduction of HR cell death
and ROS burst induced by INF1 derived from P. infestans
(Figs. 3, 4). To evaluate the effects of silencing G6PDH
isoforms and NbNADK1 on disease resistance to P. infe-
stans, silenced leaves were inoculated with a P. infestans
zoosporesuspension. Silencing
NbNADK1 led to high susceptibility to P. infestans com-
pared with TRV control leaves, although NbG6PDH-
Cyto- and NbG6PDH-P1-silenced leaves had no effect on
susceptibility (Fig. 5a). Similarly, analyses of P. infestans
biomass using real-time PCR showed that the growth rate
of P. infestans in NbG6PDH-P2- and NbNADK1-silenced
leaves increased by approximately three-fold compared
with that in TRV control leaves (Fig. 5b). These results
indicate the participation of the P2 isoform and NbNADK1
in the basal defense against P. infestans.
NbG6PDH-P2
and
Discussion
In this study, we provided evidence that plastidic
NbG6PDH-P2 is responsible for HR cell death and RBOH-
dependent ROS burst induced by INF1 elicitin and for
resistance to P. infestans. We also showed that the gene for
cytosolic NAD kinase, NbNADK1, participates in this
pathway.
It has been reported that NADPH is oxidized during the
ROS burst caused by cryptogein, an elicitin produced by
the oomycete Phytophthora cryptogea, and that the ROS
burst is suppressed by a G6PDH inhibitor in tobacco cells
a
TRV
TRV:P2
TRV:N/P2
TRV:N
NbNADK1
P2
NbEF-1α
Chemiluminescence
(counts/min/pixel)
500
0
TRV
TRV:N TRV:P2 TRV:N/P2
*
*
*
b
c
Conductivity (μS)
200
300
100
0
*
TRVTRV:NTRV:P2 TRV:N/P2
GUS
INF1
*
*
NbNADK2
NbNADK3
NbRBOHB
GUS
INF1
1500
1000
Fig. 4 Effects of silencing NbNADK1 on HR cell death and ROS
burst in Nicotiana benthamiana. At 3 weeks after inoculation with
TRVgene-silencingconstructs
TRV:NbG6PDH-P2 (P2), TRV:NbNADK1/P2 (N/P2), and TRV as
a control, silenced leaves were inoculated with Agrobacterium
tumefaciens containing inf1 or GUS. a Total RNA extracted from
silenced leaves 24 h after inoculation with A. tumefaciens containing
inf1 was used for RT-PCR with specific primers for NbNADK1,
NbNADK2, NbNADK3, NbG6PDH-P2, and NbRBOHB. Equal loads
of cDNA were monitored by amplification of constitutively expressed
NbEF-1a. b At 3 d after inoculation, ion leakage was measured. Data
are means ± SD from three experiments. c At 24 h after the
inoculation, ROS were measured as described in Fig. 3. Data are
means ± SD from four experiments. Data were analyzed with
Student’s t-test. *P\0.01 versus TRV control plants
TRV:NbNADK1(N),
158J Gen Plant Pathol (2011) 77:152–162
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Page 8

(Pugin et al. 1997). In this study, we revealed that P2
isoform of G6PDH, but not Cyto and P1 isoforms, plays a
key role in the ROS burst during plant–pathogen interac-
tions. Similarly, P2-knockdowned tobacco has an increase
in antioxidants and a decrease in oxidative damage caused
by paraquat (Debnam et al. 2004). Despite conservation of
the cysteine positions responsible for redox regulation in
both P1 and P2 isoforms, P2 isoform displays relaxed
redox regulation and a higher activity than P1 isoform
(Wendt et al. 2000). P2 also has enhanced tolerance toward
NADPH and seems to be needed in the stroma during the
dark phase to sustain ferredoxin-dependent reactions,
especially in heterotrophic plastids (Bowsher et al. 1992;
Wendt et al. 2000). These findings indicate that P2 isoform
is a stronger G6PDH and has an important role in several
redox reactions in plants. The P2 isoform may thus be
active in resistance to several stresses. In fact, it was
reported recently that ectopic expression of P2 isoform in
cytosol leads to enhanced resistance to pathogens and
drought stress (Scharte et al. 2009).
As reported previously (Wendt et al. 2000), GFP-fused
NbG6PDH-P2 was localized in chloroplasts (Fig. 2). It was
surprisingthatsilencing
NbG6PDH-Cyto, which is localized in the cytosol, reduced
production of ROS detected with the chemiluminescence
probe L-012, because the method is suitable for detecting
NbG6PDH-P2, butnot
apoplastic ROS generated by RBOH localized on the
plasma membrane in plant cells (Asai et al. 2008;
Kobayashi et al. 2007). We also showed the involvement of
NbNADK1, which is localized in the cytosol and catalyzes
the phosphorylation of NADH to form NADPH, in
NbG6PDH-P2-related ROS
results support the idea, as shown in Fig. 6, that reducing
power of NADPH produced by the P2 isoform in chloro-
plasts is transported to the cytosol as NADH via the
malate-OAA shuttle (Scheibe 2004), then NADK1 phos-
phorylates NADH in the cytosol to form NADPH, which is
transported to the plasma membrane to serve as a substrate
of RBOH.
The malate-OAA shuttle involving MDH is known to be
a well-controlled indirect export system for reducing
equivalents (Scheibe 2004). Isoforms of MDH are present
in each cellular compartment. Chloroplasts contain the
redox-controlled NADP-MDH. The activation of NADP-
MDH leads to consumption of excess NADPH in chloro-
plasts to convert OAA to malate, that is exported to the
cytosol. The malate is dehydrated to OAA by cytosolic
NAD-MDH. Accordingly, reducing equivalents can be
transported as NADH from the chloroplasts to the cytosol.
This pathway likely functions only in the light, because
NADP-MDH is only active in the light (Johnson and Hatch
1970). Liu et al. (2007a) reported that light is required for a
production(Fig. 4). The
TRVTRV:Cyto TRV:P1 TRV:P2
b
a
TRV:P1/P2 TRV:N TRV:N/P2
Growth of P. infestans
(rel. units) 300
TRV
400
100
0
*
TRV:CytoTRV:P1
*
TRV:P2
200
*
TRV:P1/P2TRV:N TRV:N/P2
*
Fig. 5 Effects of silencing
G6PDH isoforms and
NbNADK1 on susceptibility of
Nicotiana benthamiana to
Phytophthora infestans. At
3 weeks after inoculation with
TRV gene-silencing constructs
TRV:NbG6PDH-Cyto (Cyto),
TRV: NbG6PDH-P1 (P1), TRV:
NbG6PDH-P2 (P2), TRV:
NbG6PDH-P1/NbG6PDH-P2
(P1/P2), TRV:NbNADK1 (N),
TRV:NbNADK1/NbG6PDH-P2
(N/P2), and TRV as a control,
silenced leaves were inoculated
with drops of a P. infestans
zoospore suspension (2 9 105
zoospores/mL). a Inoculated
leaves 5 d after inoculation.
b Real-time PCR with
P. infestans-specific primers
using DNA isolated from the
inoculated leaves in a to
determine biomass of
P. infestans. Data are
means ± SD from three
experiments. Data were
analyzed with Student’s t-test.
*P\0.01 versus TRV control
plants
J Gen Plant Pathol (2011) 77:152–162 159
123

Page 9

constitutively active form of Nicotiana tabacum MEK2
(NtMEK2DD)-inducible HR-like cell death and ROS pro-
duction intobacco.Silencing
NbNADK1 compromised HR-like cell death and ROS
production induced by an active form of potato ortholog of
NtMEK2, StMEK2DD(data not shown), as well as those by
INF1 (Figs. 3, 4). These findings also support the model in
Fig. 6.
Defense-related RBOHs, which play a key role in ROS
production during the interactions between plant and
pathogen, seem to be regulated at the transcriptional and
post-translational levels (Yoshioka et al. 2009). The cata-
lytic core of RBOH contains two hemes in the N-terminal
transmembrane region and NADPH-binding and FAD-
binding domains in the C-terminal cytosolic region. Elec-
trons are transferred from the cytosolic NADPH, through
FAD, and across the membrane via the hemes to molecular
oxygen, leading to superoxide production (Sumimoto
2008). RBOH also carries a cytosolic N-terminal extension
with Ca2?-binding EF-hand motifs. Sagi and Fluhr (2001)
showed that RBOH is stimulated directly by Ca2?. Recent
work has demonstrated that elicitor-responsive phosphor-
ylation of the N-terminal region of RBOH leads to its
activation (Kobayashi et al. 2007; Nu ¨hse et al. 2007),
which is mediated by Ca2?-dependent protein kinases
(Kobayashi et al. 2007), and that Ca2?-binding and phos-
phorylation synergistically activate ROS production by
AtRBOHD (Ogasawara et al. 2008). Activation of RBOH
is also regulated by Rac1, a small GTPase that interacts
with the N-terminal extension of RBOH (Wong et al.
2007). The crystal structure of N-terminal region of
NbG6PDH-P2
and
OsRBOHB revealed that Ca2?binding to the EF hand
causes conformational change of N-terminus of OsRBOHB
and that key residues for the interaction of OsRBOHB and
OsRac1 are located in the coiled-coil region created by
dimerization (Oda et al. 2010). Our most recent study
showed that the key enzyme for flavin synthesis is neces-
sary for ROS production via RBOH, which is responsible
for supplying FAD as a cofactor (Asai et al. 2010). In
N. benthamiana, two MAPK cascades, MEK2-SIPK and
MEK1-NTF6, regulate the transcription of NbRBOHB
which is required for ROS production caused by INF1
(Asai et al. 2008). Silencing NbG6PDH-P2 and NbNADK1
did not affect the transcript levels of NbRBOHB induced by
INF1, although the silenced plants showed significant
reduction in the ROS burst (Figs. 3, 4). The results indicate
that NbG6PDH-P2 and NbNADK1 participates in regula-
tion of RBOH at the post-translational level, that is, by
supplying NADPH as a substrate.
The effect of ROS burst on disease resistance seems
to be diverse in plant–pathogen interactions (Asai and
Yoshioka 2008). In N. benthamiana, silencing NbRBOHs
reduces HR and enhances susceptibility to infection with
P. infestans (Asai et al. 2008; Yoshioka et al. 2003).
Similarly, NbG6PDH-P2- and NbNADK1-silenced plants
in which INF1-inducible ROS burst and HR cell death
were compromised (Figs. 3, 4) were highly susceptible to
P. infestans (Fig. 5). By contrast, depletion of the ROS
burst by an NADPH oxidase inhibitor or silencing
NbRBOHB leads to reduction in disease lesions by Botrytis
cinerea, a necrotroph that kills host cells and then uses the
dead tissues for its own growth (Asai and Yoshioka 2009).
Increased levels of ROS in plants cause more aggressive
infection by necrotrophs such as B. cinerea and Sclerotinia
sclerotiorum and result in accelerated colonization of host
tissue (Govrin and Levine 2000; von Tiedemann 1997).
A reduction in production of nitric oxide (NO), another
reactive oxygen derivative produced after pathogen rec-
ognition, also induces high susceptibility to B. cinerea
(Asai and Yoshioka 2009). These studies suggest that NO
plays a key role, but that ROS have an opposite effect in
disease resistance against necrotrophs. Liu et al. (2007b)
reported that G6PDH participates in NO production via
nitrate reductase, which contributes to tolerance to salt
stress. A fine balance between ROS and NO might be
required for HR cell death (Delledonne et al. 2001).
Investigating the effects of silencing NbG6PDH isoforms
and NbNADK1 on NO production and disease resistance to
diverse pathogens might lead to curious findings.
In conclusion, this study showed that the plastidic P2
isoform of G6PDH is responsible for RBOH-dependent
ROS production and HR cell death. On the basis of the
present results and previous reports, we propose the model
shown in Fig. 6. The reducing power of NADPH produced
G6PDH (P2)
NADPHNADP
OAA
Malate
NADK1
NADNADH
OAA
Malate
NADP-MDH
NADPH
Chloroplast
Cytosol
RBOH
O2
O2-
H2O2
Fig. 6 Hypothetical model of the role of plastidic G6PDH in ROS
production and cell death. Reducing power of NADPH produced by
the plastidic G6PDH (P2) in chloroplasts is conveyed from chloro-
plasts to cytosol as NADH via the malate-OAA shuttle including
MDH. NADH is converted to NADPH by a cytosolic NAD kinase
NADK1 in cytosol. NADPH is served to RBOH, leading to ROS
production
160J Gen Plant Pathol (2011) 77:152–162
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Page 10

by the P2 isoform of G6PDH in chloroplasts is conveyed to
the cytosol as NADH via the malate-OAA shuttle involving
MDH, then NADH is converted to NADPH by a cytosolic
NAD kinase NADK1 in cytosol. Supplying NADPH as a
substrate is necessary for the activation of RBOH and HR
cell death induced by INF1. Although there is some evi-
dence for a partnership between H2O2and NO during the
induction of cell death (Delledonne et al. 2001; Zago et al.
2006), we cannot rule out the possibility that, other than
being supplied to RBOH as a substrate, NADPH produced
by the P2 isoform of G6PDH takes part in the pathway
leading to cell death. Although further investigations are
required for establishing the model, we have shown that the
plastidic P2 isoform of G6PDH is involved in plant defense
responses.
Acknowledgments
pSLJ4K1 vector, Phil Mullineaux and Roger Hellens for the pGreen
binary vector, Sophien Kamoun for INF1 elicitin, David Baulcombe
for the pTV00 vector, and the Leaf Tobacco Research Center, Japan
Tobacco Inc., for N. benthamiana seeds. This work was supported
by the Program for Promotion of Basic Research Activities for
Innovative Biosciences (PROBRAIN) and by a Grant-in-Aid for
Scientific Research (A) from the Japan Society of the Promotion of
Science.
WethankJonathanD.G.Jones forthe
References
Asai S, Yoshioka H (2008) The role of radical burst via MAPK
signaling in plant immunity. Plant Signal Behav 3:920–922
Asai S, Yoshioka H (2009) Nitric oxide as a partner of reactive
oxygen species participates in disease resistance to necrotrophic
pathogen Botrytis cinerea in Nicotiana benthamiana. Mol Plant
Microbe Interact 22:619–629
Asai S, Ohta K, Yoshioka H (2008) MAPK signaling regulates nitric
oxide and NADPH oxidase-dependent oxidative bursts in
Nicotiana benthamiana. Plant Cell 20:1390–1406
Asai S, Mase K, Yoshioka H (2010) A key enzyme for flavin
synthesis is required for nitric oxide and reactive oxygen species
production in disease resistance. Plant J 62:911–924
Berrin JG, Pierrugues O, Brutesco C, Alonso B, Montillet JL, Roby
D, Kazmaier M (2005) Stress induces the expression of
AtNADK-1, a gene encoding a NAD(H) kinase in Arabidopsis
thaliana. Mol Gen Genet 273:10–19
Bowsher CG, Boulton EL, Rose J, Nayagam S, Emes MJ (1992)
Reductant for glutamate synthase is generated by the oxidative
pentose phosphate pathway in nonphotosynthetic root plastids.
Plant J 2:893–898
Chai MF, Chen QJ, An R, Chen YM, Chen J, Wang XC (2005)
NADK2, an Arabidopsis chloroplastic NAD kinase, plays a vital
role in both chlorophyll synthesis and chloroplast protection.
Plant Mol Biol 59:553–564
Chai MF, Wei PC, Chen QJ, An R, Chen J, Yang S, Wang XC (2006)
NADK3, a novel cytoplasmic source of NADPH, is required
under conditions of oxidative stress and modulates abscisic acid
responses in Arabidopsis. Plant J 47:665–674
D’Angelo C, Weinl S, Batistic O, Pandey GK, Cheong YH, Schu ¨ltke
S, Albrecht V, Ehlert B, Schulz B, Harter K, Luan S, Bock R,
Kudla J (2006) Alternative complex formation of the Ca2?-
regulated protein kinase CIPK1 controls abscisic acid-dependent
and independent stress responses in Arabidopsis. Plant J
48:857–872
Debnam PM, Fernie AR, Leisse A, Golding A, Bowsher CG,
Grimshaw C, Knight JS, Emes MJ (2004) Altered activity of the
P2 isoform of plastidic glucose 6-phosphate dehydrogenase
in tobacco (Nicotiana tabacum cv. Samsun) causes changes in
carbohydrate metabolism and response to oxidative stress in
leaves. Plant J 38:49–59
del Pozo O, Pedley KF, Martin GB (2004) MAPKKKa is a positive
regulator of cell death associated with both plant immunity and
disease. EMBO J 23:3072–3082
Delledonne M, Zeier J, Marocco A, Lamb C (2001) Signal
interactions between nitric oxide and reactive oxygen interme-
diates in the plant hypersensitive disease resistance response.
Proc Natl Acad Sci USA 98:13454–13459
Doke N (1983) Involvement of superoxide anion generation in the
hypersensitive response of potato tuber tissues to infection with
an incompatible race of Phytophthora infestans and to the hyphal
wall components. Physiol Plant Pathol 23:345–357
Fry W (2008) Phytophthora infestans: the plant (and R gene)
destroyer. Mol Plant Pathol 9:385–402
Govrin EM, Levine A (2000) The hypersensitive response facilitates
plant infection by the necrotrophic pathogen Botrytis cinerea.
Curr Biol 10:751–757
Graeve K, von Schaewen A, Scheibe R (1994) Purification, charac-
terization, and cDNA sequence of glucose-6-phosphate dehy-
drogenase from potato (Solanum tuberosum L.). Plant J
5:353–361
Hauschild R, von Schaewen A (2003) Differential regulation of
glucose-6-phosphate dehydrogenase isoenzyme activities in
potato. Plant Physiol 133:47–62
Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM
(2000) pGreen: a versatile and flexible binary Ti vector for
Agrobacterium-mediated plant transformation. Plant Mol Biol
42:819–832
Johnson HS, Hatch MD (1970) Properties and regulation of leaf
nicotinamide-adenine dinucleotide phosphate-malate dehydro-
genase and ‘malic’ enzyme in plants with C4-dicarboxylic acid
pathway of photosynthesis. Biochem J 119:273–280
Juhnke H, Krems B, Ko ¨tter P, Entian KD (1996) Mutants that show
increased sensitivity to hydrogen peroxide reveal an important
role for the pentose phosphate pathway in protection of yeast
against oxidative stress. Mol Gen Genet 252:456–464
Kamoun S, van West P, de Jong AJ, de Groot KE, Vleeshouwers
VGAA, Govers F (1997) A gene encoding a protein elicitor of
Phytophthora infestans is down-regulated during infection of
potato. Mol Plant Microbe Interact 10:13–20
Kamoun S, van West P, Vleeshouwers VGAA, de Groot KE, Govers
F (1998) Resistance of Nicotiana benthamiana to Phytophthora
infestans is mediated by the recognition of the elicitor protein
INF1. Plant Cell 10:1413–1425
Kamoun S, Hamada W, Huitema E (2003) Agrosuppression: a
bioassay for the hypersensitive response suited to high-through-
put screening. Mol Plant Microbe Interact 16:7–13
Kawai S, Murata K (2008) Structure and function of NAD kinase and
NADP phosphatase: key enzymes that regulate the intracellular
balance of NAD(H) and NADP(H). Biosci Biotechnol Biochem
72:919–930
Kobayashi M, Kawakita K, Maeshima M, Doke N, Yoshioka H
(2006) Subcellular localization of Strboh proteins and NADPH-
dependent O2-generating activity in potato tuber tissues. J Exp
Bot 57:1373–1379
Kobayashi M, Ohura I, Kawakita K, Yokota N, Fujiwara M,
Shimamoto K, Doke N, Yoshioka H (2007) Calcium-dependent
protein kinases regulate the production of reactive oxygen
species by potato NADPH oxidase. Plant Cell 19:1065–1080
J Gen Plant Pathol (2011) 77:152–162161
123

Page 11

Liu YD, Ren DT, Pike S, Pallardy S, Gassmann W, Zhang SQ
(2007a) Chloroplast-generated reactive oxygen species are
involved in hypersensitive response-like cell death mediated by
a mitogen-activated protein kinase cascade. Plant J 51:941–954
Liu YG, Wu RR, Wan Q, Xie GQ, Bi YR (2007b) Glucose-6-
phosphate dehydrogenase plays a pivotal role in nitric oxide-
involved defense against oxidative stress under salt stress in red
kidney bean roots. Plant Cell Physiol 48:511–522
Nakagawa T, Ishiguro S, Kimura T (2009) Gateway vectors for plant
transformation. Plant Biotechnol 26:275–284
Nakane E, Kawakita K, Doke N, Yoshioka H (2003) Elicitation of
primary and secondary metabolism during defense in the potato.
J Gen Plant Pathol 69:378–384
Nemoto Y, Sasakuma T (2000) Specific expression of glucose-6-
phosphate dehydrogenase (G6PDH) gene by salt stress in wheat
(Triticum aestivum L.). Plant Sci 158:53–60
Nu ¨hse TS, Bottrill AR, Jones AME, Peck SC (2007) Quantitative
phosphoproteomic analysis of plasma membrane proteins reveals
regulatory mechanisms of plant innate immune responses. Plant
J 51:931–940
Oda T, Hashimoto H, Kuwabara N, Akashi S, Hayashi K, Kojima C,
Wong HL, Kawasaki T, Shimamoto K, Sato M, Shimizu T
(2010) Structure of the N-terminal regulatory domain of a plant
NADPH oxidase and its functional implications. J Biol Chem
285:1435–1445
Ogasawara Y, Kaya H, Hiraoka G, Yumoto F, Kimura S, Kadota Y,
Hishinuma H, Senzaki E, Yamagoe S, Nagata K, Nara M, Suzuki
K, Tanokura M, Kuchitsu K (2008) Synergistic activation of the
Arabidopsis NADPH oxidase AtrbohD by Ca2?and phosphor-
ylation. J Biol Chem 283:8885–8892
Pugin A, Frachisse JM, Tavernier E, Bligny R, Gout E, Douce R,
Guern J (1997) Early events induced by the elicitor cryptogein in
tobacco cells: involvement of a plasma membrane NADPH
oxidase and activation of glycolysis and the pentose phosphate
pathway. Plant Cell 9:2077–2091
Ratcliff F, Martin-Hernandez AM, Baulcombe DC (2001) Tobacco
rattle virus as a vector for analysis of gene function by silencing.
Plant J 25:237–245
Sagi M, Fluhr R (2001) Superoxide production by plant homologues
of the gp91phoxNADPH oxidase. Modulation of activity by
calcium and by tobacco mosaic virus infection. Plant Physiol
126:1281–1290
Salvemini F, Franze ´ A, Iervolino A, Filosa S, Salzano S, Ursini MV
(1999) Enhanced glutathione levels and oxidoresistance medi-
ated by increased glucose-6-phosphate dehydrogenase expres-
sion. J Biol Chem 274:2750–2757
Scharte J, Scho ¨n H, Tjaden Z, Weis E, von Schaewen A (2009)
Isoenzyme replacement of glucose-6-phosphate dehydrogenase
in the cytosol improves stress tolerance in plants. Proc Natl Acad
Sci USA 106:8061–8066
Scheibe R (2004) Malate valves to balance cellular energy supply.
Physiol Plant 120:21–26
Schnarrenberger C, Oeser A, Tolbert NE (1973) Two isoenzymes
each of glucose-6-phosphate dehydrogenase and 6-phosphoglu-
conate dehydrogenase in spinach leaves. Arch Biochem Biophys
154:438–448
S´laski JJ, Zhang GC, Basu U, Stephens JL, Taylor GJ (1996)
Aluminum resistance in wheat (Triticum aestivum) is associated
with rapid, Al-induced changes in activities of glucose-6-
phosphate dehydrogenase and 6-phosphogluconate dehydroge-
nase in root apices. Physiol Plant 98:477–484
Sumimoto H (2008) Structure, regulation and evolution of Nox-
family NADPH oxidases that produce reactive oxygen species.
FEBS J 275:3249–3277
Torres MA (2010) ROS in biotic interactions. Physiol Plant
138:414–429
Torres MA, Dangl JL, Jones JDG (2002) Arabidopsis gp91phox
homologues AtrbohD and AtrbohF are required for accumulation
of reactive oxygen intermediates in the plant defense response.
Proc Natl Acad Sci USA 99:517–522
Turner WL, Waller JC, Vanderbeld B, Snedden WA (2004) Cloning
and characterization of two NAD kinases from Arabidopsis.
Identification of a calmodulin binding isoform. Plant Physiol
135:1243–1255
Turner WL, Waller JC, Snedden WA (2005) Identification, molecular
cloning and functional characterization of a novel NADH kinase
from Arabidopsis thaliana (thale cress). Biochem J 385:217–223
von Schaewen A, Langenkamper G, Graeve K, Wenderoth I, Scheibe
R (1995) Molecular characterization of the plastidic glucose-6-
phosphate dehydrogenase from potato in comparison to its
cytosolic counterpart. Plant Physiol 109:1327–1335
von Tiedemann A (1997) Evidence for a primary role of active
oxygen species in induction of host cell death during infection of
bean leaves with Botrytis cinerea. Physiol Mol Plant Pathol
50:151–166
Wakao S, Benning C (2005) Genome-wide analysis of glucose-6-
phosphate dehydrogenases in Arabidopsis. Plant J 41:243–256
Wakao S, Andre C, Benning C (2008) Functional analyses of
cytosolic glucose-6-phosphate dehydrogenases and their contri-
bution to seed oil accumulation in Arabidopsis. Plant Physiol
146:277–288
Waller JC, Dhanoa PK, Schumann U, Mullen RT, Snedden WA
(2010) Subcellular and tissue localization of NAD kinases from
Arabidopsis: Compartmentalization of de novo NADP biosyn-
thesis. Planta 231:305–317
Wenderoth I, Scheibe R, von Schaewen A (1997) Identification of the
cysteine residues involved in redox modification of plant
plastidic glucose-6-phosphate dehydrogenase. J Biol Chem
272:26985–26990
Wendt UK, Hauschild R, Lange C, Pietersma M, Wenderoth I, von
Schaewen A (1999) Evidence for functional convergence of
redox regulation in G6PDH isoforms of cyanobacteria and
higher plants. Plant Mol Biol 40:487–494
Wendt UK, Wenderoth I, Tegeler A, von Schaewen A (2000)
Molecular characterization of a novel glucose-6-phosphate
dehydrogenase from potato (Solanum tuberosum L.). Plant J
23:723–733
Wong HL, Pinontoan R, Hayashi K, Tabata R, Yaeno T, Hasegawa K,
Kojima C, Yoshioka H, Iba K, Kawasaki T, Shimamoto K
(2007) Regulation of rice NADPH oxidase by binding of Rac
GTPase to its N-terminal extension. Plant Cell 19:4022–4034
Yoshioka H, Numata N, Nakajima K, Katou S, Kawakita K, Rowland
O, Jones JDG, Doke N (2003) Nicotiana benthamiana gp91phox
homologs NbrbohA and NbrbohB participate in H2O2accumu-
lation and resistance to Phytophthora infestans. Plant Cell
15:706–718
Yoshioka H, Asai S, Yoshioka M, Kobayashi M (2009) Molecular
mechanisms of generation for nitric oxide and reactive oxygen
species, and role of the radical burst in plant immunity. Mol
Cells 28:321–329
Zago E, Morsa S, Dat JF, Alard P, Ferrarini A, Inze ´ D, Delledonne M,
van Breusegem F (2006) Nitric oxide- and hydrogen peroxide-
responsive gene regulation during cell death induction in
tobacco. Plant Physiol 141:404–411
162J Gen Plant Pathol (2011) 77:152–162
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