VpRFP1, a novel C4C4-type RING finger protein gene from Chinese wild Vitis pseudoreticulata, functions as a transcriptional activator in defence response of grapevine
State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University
, Yangling 712100, Shaanxi,
Key Laboratory of Horticultural Plant Biology and Germplasm Innovation in Northwest China
, Ministry of Agriculture, Yangling, Shaanxi 712100,
College of Horticulture, Northwest A & F University
, Yangling 712100, Shaanxi,
RING finger proteins comprise a large family and play important roles in regulation of growth and development, hormone signalling, and responses to biotic and abiotic stresses in plants. In this study, the identification and functional characterization of a C4C4-type RING finger protein gene from the Chinese wild grapevine Vitis pseudoreticulata (designated VpRFP1) are reported. VpRFP1 was initially identified as an expressed sequence tag (EST) from a cDNA library constructed from leaves of V. pseudoreticulata inoculated with the grapevine powdery mildew Uncinula necator. Sequence analysis of the deduced VpRFP1 protein based on the full-length cDNA revealed an N-terminal nuclear localization signal (NLS) and a C-terminal C4C4-type RING finger motif with the consensus sequence Cys-X2-Cys-X13Cys-X1-Cys-X4-Cys-X2-Cys-X10-Cys-X2-Cys. Upon inoculation with U. necator, expression of VpRFP1 was rapidly induced to higher levels in mildew-resistant V. pseudoreticulata plants. In contrast, expression of VpRFP1 was downregulated in mildew-susceptible V. vinifera plants. Western blotting using an antibody raised against VpRFP1 showed that VpRFP1 was also induced to higher levels in V. pseudoreticulata plants at 12-48 hours post-inoculation (hpi). However, there was only slight increase in VpRFP in V. vinifera plants in the same time frame, even though a more significant increase was observed at 96-144 hpi in these plants. Results from transactivation assays in yeast showed that the RING finger motif of VpRFP1 exhibited some activity of transcriptional activation; however, no activity was seen with the full-length VpRFP1. Overexpression of VpRFP1 in Arabidopsis plants was found to enhance resistance to Arabidopsis powdery mildew Golovinomyces cichoracearum, which seemed to be correlated with increased transcript levels of AtPR1 and AtPR2 in the pathogen-infected tissues. In addition, the Arabidopsis transgenic lines showed enhanced resistance to a virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Taken together, the results suggested that VpRFP1 may be a transcriptional activator of defence-related genes in grapevines.
The capacity of plants to protect themselves against pathogens
depends on the detection mechanisms that recognize
pathogen-derived molecules to activate host defence responses.
Sophisticated plant defence responses, such as hypersensitive
cell death, oxidative burst, and synthesis of pathogen-related
(PR) proteins, are activated upon invasion by pathogens or
changes in environmental conditions (Wang et al., 2005; Koga
et al., 2006; Shabab et al., 2008). Subsequent to these events,
plants may develop a long-lasting and broad-spectrum
resistance known as systemic acquired resistance (SAR) (Wang
et al., 2005; Park et al., 2007). Plant transcription factors
(TFs) play crucial roles in the plant defence response to
pathogen infection (Jones et al., 2006; Mozoruk et al., 2006).
TFs contribute to the regulation of the plant defence response,
including the up-regulation of the PR genes via recognition of
specific DNA sequences in the promoter region (Rushton and
Somssich, 1998). So far, at least six major families of plant
TFs, including R2R3 MYB (Stracke et al., 2001), ERF
(Andriankaja et al., 2007), TGA bZIP (Jakoby et al., 2002),
WRKY (Zhang and Wang, 2005), NPR1 (Zhang et al., 1999),
and Whirly (Desveaux et al., 2004, 2005), have been reported
to participate in the regulation of plant defence responses.
Zinc finger proteins are the most abundant proteins in
plants, which may be essential for fundamental plant growth
and development (Takatsuji, 1998). As a member of TFs, zinc
finger proteins can be grouped into the subfamilies of TFIIIA,
WRKY, Dof, GATA, RING finger, and PHD (Takatsuji,
1998). RING finger proteins are defined by the consensus
sequence containing cysteine (Cys) and histidine (His) residues
Cys-X2-Cys-X939-Cys-X13-His-X23-Cys/His-X2-Cys-X448Cys-X2-Cys, where X is any amino acid and the number of X
residues varies in different fingers (Borden and Freemont,
1996). Based on the variation in composition of the eight
zinccoordinating Cys (C) and His (H) residues and the distance
between the metal ligands, eight types of RING domain have
been identified in Arabidopsis thaliana (Stone et al., 2005). The
eight types include two canonical RING types: RING-HC
(C3HC4) and RING-H2 (C3H2C3), and six modified RING
domain types, namely RING-v (C4HC3), RING-C2 (C4C4),
RING-D (C3HDC3), RING-S/T (C3H2SC2 or CSCHCTC2),
RING-G (C3HGC3) (Albert et al., 2002; Hewitt et al., 2002;
Dasgupta et al., 2004; Stone et al., 2005), and C5HC2 (Song
et al., 2007). The RING finger domain has also been
documented with E3 ligase function, which plays a crucial
role in ubiquitin-dependent protein degradation (Smalle and
Genetic and biochemical studies have identified a number
of RING finger proteins that play key roles in various
biological processes. Initial functional evaluations have
indicated that RING finger proteins regulate
photomorphogenesis. For example, the regulatory protein COP1 was found to
represses photomorphogenesis in darkness (Deng et al., 1991,
1992; Ang and Deng, 1994). Previous studies also showed that
RING finger proteins are associated with plant growth and
development (Xu and Li, 2003; Disch et al., 2006; Zhang
et al., 2008). Moreover, other studies also demonstrated that
some RING finger proteins are directly involved in regulation
of hormone signalling pathways by targeting of specific
proteins for degradation (Zhang et al., 2005, 2007; Bu et al.,
2009). In particular, some reports indicated that an abundance
of RING finger proteins play key roles in regulating defence
responses against abiotic and biotic stresses. HOS1 is
a functional RING finger protein that has ubiquitin E3 ligase
activity, is a negative regulator of cold signal transduction,
physically interacts with ICE1, and is a TF activating the
expression of CBF genes (Lee et al., 2001; Dong et al., 2006).
RING finger proteins also seem to be involved in pathogen
response or plant defence. Arabidopsis ATL2 is induced
rapidly and momentarily by chitin and cellulose treatments
(Salinas-Mondragon et al., 1999), and the eca mutants with
constitutive expression of the ATL2 gene exhibited
upregulated expression of defence-related genes and salicylic acid
(SA)- and jasmonic acid (JA)-responsive genes (Serrano and
Guzman, 2004). A T-DNA insertion mutant of Arabidopsis
ATL9 results in increased susceptibility to powdery mildew
(Ramonell et al., 2005). Arabidopsis RIN2 and RIN3 and
tobacco ACRE132 regulate expression of disease resistance
genes specifically involved in the hypersensitive response
(Kawasaki et al., 2005).
Grapevine (Vitis vinifera L.) is economically the most
important fruit species worldwide. Vitis vinifera is currently
the major species cultivated for its high quality in producing
wine, juice, table grapes, and dried fruit. However, it is
susceptible to many fungal diseases including powdery
mildew [Uncinula necator (Schw.) Burr. or Erysiphe
cichoracearum], anthracnose [Elsinoe ampelina (de Bary) Shear],
and downy mildew [Plasmopara viticola (Berk. & Curt.) Berl.
& De Toni ] (Pavlousek, 2007). Fungal pathogens are a major
problem in grapevine globally; they cause huge losses in yield
and significant reduction in berry and wine quality. China is
one of the origins of Vitis species, and some Chinese wild
Vitis species possess desirable disease resistance to various
pathogens, such as extremely high resistance to anthracnose and
ripe rot [Glomerella cingulata (Ston.) Spauld et Schrenk], high
resistance to powdery mildew, and resistance to crown gall
(Agrobacterium vitis) (Chai et al., 1997; He, 1999). Baihe-35-1
is a unique accession of Chinese wild Vitis pseudoreticulata
W. T. Wang, which possesses a high resistance to multiple
fungi, particularly to U. necator (Wang et al., 1995).
To reveal the molecular mechanisms involved in the
defence response to pathogen infection in Chinese wild
V. pseudoreticulata, pathogen-induced genes have previously
been isolated from a cDNA library of V. pseudoreticulata
leaves inoculated with U. necator (Xu et al., 2009). Among
them, one was predicted to encode a putative RING finger
protein, and its full-length cDNA (GenBank accession no.
FJ356672) was obtained based on one expressed sequence
tag (EST) sequence (GenBank accession no. DQ354158).
This gene was designated as VpRFP1 (V. pseudoreticulata
RING-finger protein 1). In this study, the VpRFP1 gene
from Chinese wild V. pseudoreticulata accession Baihe-35-1
was cloned and its expression patterns were investigated. It
was shown that ectopic overexpression of VpRFP1 in
transgenic Arabidopsis resulted in enhanced resistance against both
the powdery mildew pathogen Golovinomyces cichoracearum
and the bacterial pathogen Pseudomonas syringae pv. tomato
Materials and methods
Grapevine (Chinese wild V. pseudoreticulata accession Baihe-35-1,
V. vinifera cv. Carignane) leaves were obtained from the Grape
Repository of Northwest A&F University, Yangling, Shaanxi, PR
China. Arabidopsis thaliana plants (ecotype Columbia, Col-0) were
grown in a soil mix of peat moss, perlite and vermiculite (3:1:1,
v/v/v) under a 12/12 h day/night cycle at 24 C with 60% humidity.
The pathogen U. necator, collected from leaves of field-grown
V. vinifera cv. Cabernet Sauvignon, was maintained in
greenhousegrown V. vinifera cv. Carignane plantlets. The pathogens were
collected and suspended in sterile water with a concentration of
53105 sporangia ml 1. The spore suspension were sprayed onto the
abaxial leaf surface of attached grapevine leaves. The inoculated
leaves were enclosed in plastic bags to maintain high humidity. After
inoculation for 0, 12, 24, 48, 72, 96, 120, and 144 h, leaves were
sampled, immediately frozen in liquid nitrogen, and stored at 80 C
The disease assay with G. cichoracearum isolate UCSC1 was
obtained from Dingzhong Tang and conducted as previously
described (Wilson et al., 2001). VpRFP1-overexpressing Arabidopsis
were inoculated by spraying leaves with the pathogen conidial
suspension (53105 conidia ml 1). The inoculated plants were placed
in a plant-growth box (25 C with 16 h of illumination per day
and 100% relative humidity). After inoculation for 0, 24, 48, 72, and
96 h, the leaves were sampled.
Pseudomonas syringae pv. tomato DC3000 was provided by
Dingzhong Tang, and grown at 28 C in Kings B medium
(supplemented with 100 mg l 1 rifampicin) overnight, then diluted
to ;107 cfu ml 1 with 10 mM MgCl2 solution. Approximately
10 ll of bacterial suspension was infiltrated into the abaxial side of
45 leaves per plant using a 1 ml needleless syringe. Quantification
of bacterial growth was performed as described (Wang et al.,
2007). Trypan blue staining was used to detect cell death as
described (Yang et al., 2009).
DNA sequencing and sequence analysis
The open reading frame (ORF) of VpRFP1 was cloned into the
pMD-19 vector (TaKaRa, Dalian, China) and sequenced
(SunBiotech Company, Beijing, China). DNA sequences were analysed using
BLASTN and BLASTX in the National Center for Biotechnology
Information (NCBI) databases (http://www.ncbi.nlm.nih.gov). The
protein conserved domain was analysed using Smart (http://
smart.emblheidelberg.de/smart/change_mode.pl) and ExPASy (http://
au.expasy org /tools/). The deduced amino acid sequence was aligned
and the phylogenetic tree was generated by ClustalW (http://
Preparation of fusion protein and polyclonal antibodies
The VpRFP1 gene from Chinese wild V. pseudoreticulata was PCR
cloned in-frame into the pGEX-4T-1 vector. The construct was
verified by DNA sequencing and transformed into Escherichia coli
BL21. Expression of the fusion protein glutathione S-transferase
(GST)VpRFP1 was induced by
isopropyl-b-D-thiogalactopyranoside (IPTG). The purification of the fusion protein was performed
by electrodialysis assay, and New Zealand rabbits were immunized
with the purificied product to obtain polyclonal antibodies. The
polyclonal antisera were used for western blot and subcellular
Gene expression analysis by real-time quantitative PCR and
GAPDH (glyceraldehyde phosphate dehydrogenase) sequences.
PCRs on serial dilutions of cDNA were performed at 57 C for 29
cycles to define semi-quantitative conditions that resulted in
amplification linear to RNA amounts. RT-PCR products were
separated on 1.2% agarose gels. The experiments were performed
three times with similar results. The special primers used for PCR
are listed in Supplementary Table S2 available at JXB online.
For real-time PCR, reactions were carried out on the Bio-Rad
IQ5 real-time PCR detection system (Bio-Rad, Hercules, CA,
USA). PCRs were carried out in triplicate in a reaction buffer
containing 13 SYBR Premix Ex Taq (TaKaRa), 0.2 lM of
forward and reverse primers, and 10 ng of reverse-transcribed
RNA in a final volume of 25 ll. Thermal cycling conditions were:
30 s at 95 C followed by 40 cycles of 15 s at 94 C, 30 s at 58 C,
and 30 s at 72 C. The specificity of the individual PCR
amplifications was checked using a heat dissociation curve from
55 C to 95 C following the final cycle of the PCR. The results
obtained for each gene of interest at each time point were
normalized against the grapevine GAPDH gene, which was used
as a reference gene. Mean values and SDs were obtained from
three duplicate experiments and are representative of three
independent experiments. Primers used for real-time quantitative
PCR are listed in Supplementary Table S2 at JXB online.
Plant protein extraction and protein gel blot analysis
Total protein was extracted from infected leaves as described (Wang
et al., 2003, 2006). The concentration of the protein extract was
determined with the Bradford protein assay (Bradford, 1976). SDS
PAGE was carried out according to standard procedures with 25 lg
of total proteins. Proteins were electrotransferred onto PVDF
membranes (Bio-Rad). The membrane was blocked for 3 h in
blocking buffer [phosphate-buffered saline (PBS), 0.1% (w/v)
Tween20, and 5% (w/v) skim milk] before incubating overnight in a 1:1000
dilution of polyclonal antiserum prepared in blocking buffer.
Detection of VpRFP1 was achieved with goat anti-rabbit IgG secondary
antibody and the BCIP/NBT chemiluminescent system according to
the manufacturers instructions (Sigma-Aldrich, Beijing, China). The
experiment was performed twice with similar results.
Analysis of VpRFP1 transcriptional activity
The deletion constructs of VpRFP1 for the transactivation assay
were generated by fusing the VpRFP1 gene downstream of the
GAL4 DNA-binding domain (BD). Fifteen VpRFP1 gene regions
were PCR amplified to introduce a BglII site at the 5# end and
a PstI site at the 3# end. The fragments were digested with BglII
and PstI, and cloned into the same sites of pGBKT7. The
genespecific primers are listed in Supplementary Table S1 at JXB
online. The pGBKT7 vector which expressed GAL4 BD alone
served as the negative control, and the GAL4-VpERF1 fusion
construct (designated as pGBKT7-VpERF1) was used as a positive
The 14 deletion constructs of VpRFP1 gene fused to the vector
pGBKT7 for the yeast GAL4 BD were generated. The fusion
constructs were transformed into the Saccharomyces cerevisiae
strain AH109 and cultured on YPDA plates at 28 C for 3 d. The
well-isolated colonies were transferred from YPDA plates to
SD/Ade /-His/-Trp/X-a-Gal plates to test the transcriptional activation
activity according to their growth and the activity of b-galactosidase
by observing the blue colour produced.
Subcellular immunogold labelling
Total RNA was extracted from grapevine leaves as previously
described (Asif et al., 2000). First-strand cDNA was synthesized
from 2 lg of DNase-treated total RNA using PrimeScript RTase
(TaKaRa). Semi-quantitative RT-PCR was performed by using
a PrimeScript RT-PCR Kit (TaKaRa). Control reactions to
normalize RT-PCR were done with primers derived from grapevine
Ultrathin sections were prepared from Lowicryl K4M-embedded
specimens according to the method previously described (Peng
et al., 2003). The ultrathin sections were first blocked for 30 min at
room temperature by floating the grids on droplets of PBS
containing 8 mM Na2HPO4, 1.5 mM KH2PO4, 3 mM KCl, and
500 mM NaCl (pH 7.4) supplemented with 50 mM glycine and
continuously blocked with PBS supplemented with 0.1% (w/v)
gelatin, 0.5% (w/v) bovine serum albumin (BSA), and 0.1% (v/v)
Tween-20 (pH 7.4, PBGT). Without rinsing, the sections were
incubated with New Zealand rabbit antiserum directed against
VpRFP1 (all diluted 1:100 in PBGT buffer) overnight at 4 C.
Following extensive washes with PBGT buffer, the sections were
incubated with secondary antibody (goat anti-rabbit IgG antibody
conjugated with 10 nm gold) at a 1:100 dilution in PBGT buffer
for 2 h at room temperature. The sections were rinsed consecutively
with PBGT and double-distilled water, followed by staining with 2%
uranyl acetate in 50% ethanol for 25 min at 25 C and with alkaline
lead citrate for 15 min. After washing extensively with
doubledistilled water, the ultrathin sections were examined with an electron
The specificity and reliability of the immunogold labelling were
tested by two negative controls. In the first one, the antiserum was
omitted to test possible unspecific labelling of the goat anti-rabbit
IgG antibodygold conjugate. In the second, rabbit pre-immune
serum was used instead of the rabbit antiserum before
immunogold labelling to test the specificity of the antiserum. At least three
repetitions for each control were conducted for each sample.
Plant expression vector construction and Arabidopsis
To generate the VpRFP1-overexpressing gene construct, the
VpRFP1 coding region including the termination codon was
amplified using the following primers: VpRFP1 forward primer:
5#-GGGGTCGACATGATTACCGATTCGATCAC-3# (SalI site
underlined), and VpRFP1 reverse primer:
5#-GGGGGTACCCTAAGACCTTGCAATCATGC-3# (KpnI site underlined). The
PCR products were inserted in the pMD-19-T vector (TaKaRa)
and identified by DNA sequencing. The digested VpRFP1
fragments were subcloned in the same sites of the plant expression
vector pWRII (Lei et al., 2009) in which the VpRFP1 was driven
by the ehanced Cauliflower mosaic virus (CaMV) 35S promoter
and possesses hygromycin resistance in plants. The recombinant
construct harbouring VpRFP1 was introduced into Agrobacterium
tumefaciens strain EHA105 via electroporation. Arabidopsis
transformation of the VpRFP1 gene was carried out according to the
floral dipping method (Clough and Bent, 1998). For selection of
Arabidopsis transgenic lines, seeds were surface-sterilized with 70%
ethanol for 2 min, then 2% sodium hypochlorite (NaOCl) solution
for 6 min and rinsed five times in sterile water. Seeds were plated
on basal MS salt medium containing 1% sucrose and 20 mg l 1
hygromycin. Plates were kept at 4 C for 24 d in darkness to
synchronize germination, then transferred to growth chambers,
and maintained under the environmental conditions described
above for plant growth in pots. Fourteen-day-old seedlings were
transferred to the soil mix. Wild-type plants and transgenic
Arabidopsis T3 lines were used for further analysis.
Isolation and sequence analysis of a novel RING finger
protein (VpRFP1) gene from Chinese wild
Based on the EST sequence (DQ354158) obtained previously
(Xu et al., 2009), the full-length cDNA sequence (FJ356672)
has now been obtained. The VpRFP1 cDNA of 1415 nt
contains a 170 nt untranslated region (UTR) at the 5 end
and a 192 nt 3-UTR. The genomic sequence of the VpRFP1
gene was also cloned directly from the V. pseudoreticulata
genomic DNA (GeneBank accession no. GU446678),
VpRFP1 is located on chromosome 17 of the published Pinot
Noir whole genome (Jaillon et al., 2007). Alignment of the
genomic DNA sequence with the VpRFP1 cDNA indicated
that the VpRFP1 gene contained two introns and three
exons. The predicted ORF encodes a RING finger protein of
350 amino acids, which has a theoretical pI value of 5.83 and
a deduced molecular mass of 38 005 Da. The deduced amino
acid sequence of VpRFP1 contained a nuclear localization
signal (NLS) at its N-terminus and the RING finger motif at
its C-terminus. Compared with other species, the RING
finger motifs belong to a novel RING finger variant of the
C4C4 type, with the consensus sequence
Cys-X2-Cys-X13Cys-X1-Cys-X4-Cys-X2-Cys-X10-Cys-X2-Cys (X indicates any
amino acid, and subscript number indicate the number of
amino acids) (Fig. 1A). AtPEX10 is a typical C3HC4-type
RING finger (Schumann et al., 2007) which possesses the
C-X2-C-X939-C-X13-H-X23-C-X2-CX448-C-X2-C (Stone et al., 2005) (Fig. 1A). Comparative
analysis of these two motifs revealed that both are
remarkably similar, but with the histidine of the C3HC4 type
replaced by a cysteine residue.
Comparisons of the amino acid sequences among VpRFP1
and other putative proteins are presented in Fig. 1B. The
VpRFP1 protein shared 50% identical amino acids with
a Solanum lycopersicum (formerly Lycopersicon esculentum)
hypothetical protein (accession no. ABI34275), 46% homology
with an A. thaliana RING finger family protein (accession no.
AAY57618), and 38% homology with an Oryza sativa
hypothetical protein (accession no. EAZ23574). Phylogenetic
tree analysis showed that they were classified into three
subfamilies. Vitis pseudoreticulata (accession no. FJ356672),
V. vinifera (accession no. XP_002281744), and S. lycopersium
(accession no. ABI34275) were part of the same subfamily
Expression patterns of VpRFP1 in grapevine resistance
To investigate whether the expression pattern of RFP1 was
similar among different Vitis species, the expression levels of
RFP1 in a resistant grapevine (accession Baihe-35-1 of V.
pseudoreticulata) and a susceptible variety (V. vinifera cv.
Carignane) inoculated with U. necator were determined using
real-time PCR and western blot. The results showed that the
expression of RFP1 was induced by U. necator at the
transcriptional level. For resistant grapevine accession
Baihe35-1 of V. pseudoreticulata, the up-regulated expression of
VpRFP1 was detected at 12 hours post-inoculation (hpi) (Fig.
2A), peaked at 24 hpi, and returned to low baseline levels at
48 hpi. In contrast, the susceptible V. vinifera cv. Carignane
displayed a high transcription level at 0 h, but this decreased
immediately after inoculation, and reached the lowest levels
at 24 hpi and 48 hpi, respectively.
To further determine whether RFP1 was induced by the
pathogen at the translational level, western blot assay was
carried out using a specific antiserum. The result showed that
the expression of RFP1 at the translational level was induced
by U. necator both in the resistant accession of
V. pseudoreticulata and in the susceptible V. vinifera cv.
Carignane (Fig. 2B). The expression of RFP1 in V. vinifera
VpRFP1 as a transcriptional activator in defence response of grapevine | 5675
RFP1 is induced by U. necator in both V. pseudoreticulata
and V. vinifera cv. Carignane; the expression pattern of
RFP1, however, was different between the two grapevine
genotypes. This prompted the further elucidation of the
function of VpRFP1 in response to pathogen attack.
Immunogold labelling localization of VpRFP1
VpRFP1 fused with a GST tag was induced in E. coli BL21
(Supplementary Fig. S1A at JXB online), and then purified
by an electrodialysis procedure (Supplementary Fig. S1B).
The purified protein was used to immunize New Zealand
rabbits to obtain polyclonal antiserum. Immunogold
labelling with VpRFP1 antiserum showed that gold particles
were mainly localized in the nucleus (Fig. 3). No substantial
signal was detected in controls (Fig 3A, B), confirming the
specificity of the immunolocalization. Non-infected leaves
treated with the specific antiserum against VpRFP1 showed
few gold particles in the nucleus and fewer in the cytoplasm
(Fig. 3C). At 24 hpi, pathogen-infected samples showed
a high density of gold particles in the nucleus (Fig. 3D),
while similar signals were found in the cytoplasm of
Transcriptional activation activity of VpRFP1 in yeast
To examine whether VpRFP1 protein could function as
a transcriptional activator in yeast, a series of deletion
constructs of the VpRFP1 gene were fused to the GAL4 BD
domain. The constructs were then transformed into yeast
(strain AH109), and screened on SD medium. The VpERF1
cloned from Chinese wild grapevine has been previously
confirmed to be active in yeast (unpublished data).
Therefore, VpERF1::GAL4 was used as the positive control, and
the empty vector pGBKT7 as the negative control.
AH109 competent cells harbouring the recombinant
plasmid or the empty vector pGBKT7 were able to grow on
SD/Trp medium, indicating that all recombinant plasmids or the
empty vector pGBKT7 were transferred into AH109 (Fig.
4BII). All yeast cells carrying constructs pGBKT7-A, pGB
KT7-B, pGBKT7-C, pGBKT7-E, pGBKT7-F, pGBKT7-G,
pGBKT7-H, pGBKT7-I, pGBKT7-K, pGBKT7-L, pGB
KT7-M, pGBKT7-N, and pGBKT7-O could not grow on
the SD/-Trp/-Ade/-His medium, or yield the colour reaction
on the SD/-Trp/-Ade/-His/+X-a-gal medium. Cells expressing
the constructs pGBKT7-D and pGBKT7-J, and the positive
control caused yeast growth on the SD/-Trp/-Ade/-His
medium, and induced the blue colour on the
SD/-Trp/-Ade/His/+X-a-gal medium (Fig. 4BIII, IV), indicating that
portions D and J of VpRFP1 have potential transcriptional
activity in yeast. The effector constructs pGBKT7-D and
pGBKT7-J (Fig. 4A) shared the C-terminal region with the
RING finger motif, and displayed obvious transcriptional
activities. This result suggested that the C-terminal region
with the RING finger motif plays a significant role in the
transcriptional activity. Interestingly, the full-length VpRFP1
has no transcriptional activity in yeast.
VpRFP1 as a transcriptional activator in defence response of grapevine | 5677
Overexpression of VpRFP1 in Arabidopsis enhanced
resistance to pathogens
To further study the biological role of VpRFP1 in defence
responses, functional analyses of VpRFP1 were performed in
transgenic Arabidopsis plants transformed with the
overexpression construct pWRII/VpRFP1 (Fig. 5A). Upon inoculation,
VpRFP1 transgenic plants were more resistant against the
pathogen than the wild type (Fig. 5B, C). At 8 days
postinoculation (dpi) with G. cichoracearum, wild-type leaves
showed some disease symptoms, while transgenic plants
remained disease free with no visible fungal colonies. The
microscopic images showed that the transgenic Arabidopsis
plant inhibited the spread of G. cichoracearum (Fig. 5D). At
12 dpi, the disease symptoms in wild-type leaves became more
severe than in the transgenic plants, and only a few symptoms
occurred in transgenic plant leaves (Fig. 5C). As is shown (Fig.
5E), VpRFP1 was constitutively expressed in the T3
generation, but was also affected by G. cichoracearum inoculation.
Interestingly, PR1 and PR2 displayed the same expression
pattern as VpRFP1, whose expression increased gradually, and
began to decrease after reaching a peak at 72 hpi. In contrast,
the transcripts of PR3 and PDF1.2 were suppressed at 24 hpi,
and reached maximum expression at 96 hpi.
Experiments were also carried out to examine whether or
not the transgenic plants were resistant to P. syringae pv.
tomato DC3000 infection. The result indicated that none of
the VpRFP1 transgenic plants exhibited spreading maceration
in the infected leaves, and displayed clearly enhanced
resistance (Fig. 6A). The growth of P. syringae pv. tomato
DC3000 in inoculated plants was measured and it was found
that the bacterial titres in transgenic lines were significantly
lower than in the wild-type plants at 3 dpi (Fig. 6B).
Macroscopic and microscopic analyses were also performed
to detect the cell death. The results indicated that at the
macroscopic level, cell death in infected mature leaves
significantly increased in the transgenic lines compared with
the wild-type plants (Fig. 6C), and at the microsopic level, the
extent of visible lesions also increased significantly in the
transgenic lines (Fig. 6D).
VpRFP1 as a transcriptional activator in defence response of grapevine | 5679
Based on previous findings from studies of other plant
C4C4type RING finger (RING finger-C2) genes (Albert et al., 2002;
Stone et al., 2005), it was hypothesized that VpRFP1 isolated
from the resistant accession Baihe-35-1 of Chinese wild
V. pseudoreticulata may be a novel C4C4-type RING finger
protein involed in grapevinepowdery mildew interaction. The
gene was found to show strong evidence for transcriptional
change in the susceptible V. vinifera and a significantly
different response in the disease-resistant V. pseudoreticulata
(Fig. 2). This rapid and strong transcriptome response to
U. necator infection is consistent with recent findings of the
C3HC4-type RING finger protein in pepperpathogen
interaction (Hong et al., 2007). It is possible that this
induced expression pattern of defence-related transcripts
plays a critical role in the regulation of early defence singal
pathways in the leaves of disease-resistant V.
pseudoreticulata. Further investigations will be required to provide
evidence for the relationship between the expression pattern
of this gene and U. necator resistance.
The translocation of TFs from the cytoplasm into the
nucleus is considered to be an important step in
posttranslational control (Laskey and Dingwall, 1993), and
requires an NLS for selective transportation into the
nucleus (Raikhel, 1992). The present data suggest that the
VpRFP1 amino acid sequence from residues 18 to 46
contained a putative NLS, thus suggesting that VpRFP1
can function as a TF and may be targeted to the nucleus.
Evidence from immunolocalization analyses indicated that
VpRFP1 was not only located in the nucleus (Fig. 3), but
was also induced by the pathogen. It is interesting to note
that the full-length VpRFP1 and most of the deletion
mutants showed no transcriptional activation activity,
whereas the RING finger motif connected to the C-terminal
region of VpRFP1 (pGBKT7-D and pGBKT7-J) presented
transactivation activity in yeast (Fig. 4). A possible
explanation is that the transcriptional activation activity of
VpRFP1 may require an environmental stimulus to become
activated, such as pathogen induction. This induced
activation seems to be coordinated with the phosphorylation state
of the TF gene involved in response to extracellular signals
via the modulation of the activities of protein kinases and
protein phosphatases (Whitmarsh and Davis, 2000). It has
also been documented that the activation function of TFs in
planta was considerably different from that in yeast systems
(Sprenger-Haussels and Weisshaar, 2000; Heinekamp et al.,
2002). Therefore, another possible explanation is that the
novel VpRFP1 could function as a transcriptional repressor
in yeast, but as a transcriptional activator in grapevine.
Functional analyses revealed that the expression of the
novel VpRFP1 was regulated by U. necator at both the
transcriptional and translational levels, and also enhanced
the resistance to G. cichoracearum and P. syringae pv.
tomato DC3000 in transgenic Arabidopsis plants. Further
functional characterization of VpRFP1 in interacting with
other proteins, such as ubiquitin ligases activity in vitro and
the degradation substrate, is required to provide more
comprehensive information about the disease-resistant
specifities of the novel C4C4-type RING finger protein
VpRFP1, and will also be helpful in understanding the
molecular mechanism of disease resistance in Chinese wild
V. pseudoreticulata species.
Supplementary data are available at JXB online.
Figure S1. VpRFP1 expression in E. coli and purification
of GSTVpRFP1 fusion protein.
Table S1. Primers used for amplifying the target gene.
Table S2. Primers used for RT-PCR and real-time PCR
analysis of target gene expression.
We would like to thank Dr Wenping Qiu (Division of Plant
Sciences, Missouri State University), Dr Hily Jean-michel
(Cornell University College of Agriculture and Life
Sciences), and Dr. Shunyuan Xiao for critical review and
comments on the manuscript. This study was supported by
National Natural Science Foundation of China (grant nos
30771493 and 30971972).
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