Function of VtPGIP in pathogenic fungus resistance of Vitis thunbergii

In plants, polygalacturonase inhibitor proteins (PGIPs) are very important to inactivate polygalacturonases secreted by pathogens. Vitis thunbergii Sieb. et Zucc. polygalacturonase inhibitor proteins (VtPGIP) was first isolated from the wild grape Vitis thunbergii Sieb. et Zucc., which exhibits high resistance to disease. VtPGIP is sublocated in the plant cell wall, and this location is consistent with the function of PGIPs in the first line of host defense. The promoter of VtPGIP contains salicylic acid (SA), abscisic acid (ABA), and fungus infection response elements. Results from real-time quantitative reverse transcriptase (RT)-PCR analysis showed that VtPGIP expression was induced by SA, ABA, and fungi. The results indicated that VtPGIP may have important functions in defense-related responses of V. thunbergii against pathogenic fungi.


INTRODUCTION
Epiphytes cause significant losses as destructive pathogens of many fruits and vegetables worldwide; such organisms particularly affect the post-harvest industry.The main methods used to control fungal diseases include field and equipment sanitation, crop rotation, soil fumigation (Wheeler et al., 1994), and fungicide application; however, fungicides are deleterious to the environment.Other strategies are more promising and have been shown to decrease the incidence of fungal diseases by enhancing natural plant defense capabilities (Kessmann et al., 1994).A very important aspect is to determine potential molecules that exhibit defense functions in plants, that is, the natural defense system, and the molecules that respond to induction.
Fungal disease is caused by the secretion of hydrolytic enzymes to pectin substrates (Fish, 2005).Pathogens can release several types of enzymes, such as exopolygalacturonases and endopolygalacturonases (endo-PGs), to breach this barrier and function with pectin methyl and acetyl esterases (Prade et al., 1999) to degrade pectin.Fungal endo-PGs, the first enzymes secreted by fungal plant pathogens, have important functions during the early stages of plant pathogenesis (English et al., 1971) to separate and macerate host tissues, thereby facilitating pathogen penetration and colonization of plant tissues.Subsequently, the products of this degradation process are used as a nutrient source for fungal growth (Karr et al., 1970).
Polygalacturonase-inhibiting proteins (PGIPs) are basic proteins present in the cell walls of most plants; PGIPs are specific, saturable, reversible, and high-affinity "receptor" endo-PGs of fungi (Cervone et al., 1987(Cervone et al., , 1990(Cervone et al., , 1989)).PGIPs can directly impede pathogen invasion by inactivating polygalacturonases (PGs) (Hegedus et al., 2008).The inhibition of PG activities by PGIPs has been proposed to prolong the accumulation of oligogalacturonides and improve defense response (Federici et al., 2008).For instance, PGIPs of Pyrus communis (Tamura et al., 2004) and Gossypium hirsutum (James et al., 2001) inhibit fungal PG activity.However, field trials with the pear PGIP-expressing tomato lines provide contradictting results of the functions of PGIP in resistance.Hence, PGIPs from different plants vary in terms of inhibitory activity; PGIPs from a single plant also inhibit PGs from different fungi or different PGs from the same fungus with various strengths (Desiderio et al., 1997).For instance, a PG from Aspergillus niger is inhibited by PvPGIP1 and PvPGIP2 (Leckie et al., 1999) but not by PvPGIP3 and PvPGIP4 (D" Ovidio et al., 2004).Fusarium moniliforme PG is inhibited only by PvPGIP2 (Leckie et al., 1999 (Ferrari et al., 2003).
Vitis thunbergii Sieb.et Zucc.(V.thunbergii) is native to China and grown throughout the central and southern parts of China as well as in Korea and Japan.V. thunbergii has a strong resistance to Coniothyrium diplodiella, Glomerella cigulata, and other diseasecausing pathogens.In addition, the plant hormones salicylic acid (SA) and abscisic acid (ABA) have been implicated in various plant responses (Rao et al., 2000;Borsani et al., 2001;Turner et al., 2002;Xiong et al., 2002).The cloned VtPGIP promoter sequence contains SA and ABA-related elements components and allows the study of the expression and regulation of VtPGIP by the corresponding SA or ABA treatment.In this study, VtPGIP was characterized to determine whether or not the PGIP gene in V. thunbergii is responsible for disease resistance.We also described VtPGIP expression in response to applied SA, ABA, and fungal infection.VtPGIP may have important functions in the disease resistance of V. thunbergii.

Plant materials and growth conditions
V. thunbergii plantlets were grown at 25°C under a 16 h/8 h (day/night) photoperiod by tissue culture.The plantlets were cultivated in the medium of 3/4 MS supplemented with 0.35 mg•L -1 indole-3-butyric acid.

Plant treatments
The leaves of seven-week-old intact tissue culture plantlets were selected to examine the effect of exposure to different defense response activators (signaling molecules).SA (Sigma, St. Louis) and ABA (Sigma, St. Louis) were dissolved in water, and the aerial parts of the plants were sprayed with 5 mM SA (Ferrari et al., 2003) or 50 mM ABA (Yuasa et al., 2007).The plants of the same age and treated with distilled water were used as control plants.VtPGIP expression on the leaves of tissue culture plantlets was analyzed at 0, 2, 6, 16, 24, 48, and 72 h after ABA and SA treatments.After harvest, the leaves were immediately frozen in liquid nitrogen and stored at -70°C for subsequent RNA extraction.
The leaves of V. thunbergii tissue culture plantlets were inoculated with pathogenic fungi (C. diplodiella, Erysiphe cichoracearumr, G. cigulata, and B. cinerea).The plantlets of the same age and treated with water were used as control plants.After 3 d, the leaves treated with pathogenic fungi and water were harvested, immediately frozen in liquid nitrogen, and stored at -70°C for subsequent RNA extraction.

Isolating the VtPGIP gene
Genomic plant DNA was isolated using the CTAB extraction method described in a previous study (Chang et al., 1993).Total RNA was extracted by scaling down the experimental conditions previously described (Chang et al., 1993).Plant leaves (200 mg) were ground in liquid nitrogen to a fine powder by using mortar and pestle.Approximately, 900 μl of extraction buffer was added and the mixture was mixed thoroughly.The mixture was extracted with 900 μl of chloroform/isoamyl alcohol (24:1 v/v).The upper phase was transferred to a fresh tube, and the hydroxybenzene/chloroform/isoamylalcohol (25: 24:1 v/v/v) was added.The mixture was vortexed vigorously and centrifuged at 12,000 rpm for 10 min at 4°C.The clear supernatant was carefully transferred to a clean tube and extracted again with the chloroform/isoamylalcohol (24:1 v/v).The RNA was precipitated with 10 M LiCl for 8 h at -20°C, washed with 70% cold ethanol, air dried for 20 min, and resuspended RNA precipitate in 30 μl DEPCtreated ddH2O.CDNA was synthesized using the ReverTra Ace-a-TM kit (ToYoBo, Osaka, Japan) according to the manufacturer"s instructions.
The obtained amino acid sequence was aligned with related genes by using BioXM, and the alignment was analyzed using PROSITE (http://ca.expasy.org/prosite/).The three-dimensional structure of the VtPGIP protein and molecular modeling were analyzed using Swiss-Pdb Viewer 3.7.

Cloning of the VtPGIP promoter
The primers [F1: 5′-TGGGGAGGCTCTACCCAGGA-3′ (forward) and Q1: 5′-GGACGAGTGGCGAGTAAGAGTAGGAGG-3′ (reverse)] were designed based on the sequencing of Vitis (Pinot noir, PN40024).The promoter of the PGIP gene sequence was cloned by PCR.PCR was performed using genomic DNA extracted from V. thunbergii leaves with the primers p-pgip-F1/-Q1.A 36-cycle PCR was conducted (one cycle was performed under the following conditions: 1 min at 94°C; 2 min at 58°C; and 2 min at 72°C) with a final extension of 10 min at 72°C.The products were cloned into a pMD19-T vector (TaKaRa), and the two clones were sequenced.
The promoter of the VtPGIP gene was analyzed using promoter prediction software programs (Promoter Scan, Neural Network and CpG Island) and transcription factor prediction software programs (TF Search, Match 1.0-public and Plantcare).

Subcellular localization of the VtPGIP protein
To determine the subcellular distribution of the VtPGIP protein, we generated a plasmid that enabled the expression of VtPGIP fused to EGFP.This plasmid was used as control and was transformed into onion epidermal cells.The entire coding region of the VtPGIP gene was cloned into the plasmid of Pyk2784-EGFP (enhanced green fluorescent protein), generating the p35S:VtPGIP-EGFP plasmid, which was designed to express the in-frame fusion protein of VtPGIP-EGFP.Onion epidermal cells were subsequently transformed with p35S: VtPGIP-EGFP or the control plasmid p35S:EGFP according to the particle bombardment method by using a particle delivery system (Bio-Rad Biolistic PDS-1000/He; Bio-Rad, Hercules, CA).The transformed onion epidermal cells were incubated at 25°C in the dark for 12 h, and VtPGIP-EGFP expression was then examined under a fluorescent microscope (Axio Imager A1; Carl Zeiss, Germany).
The PCR efficiency of each target mRNA was obtained; the absence of primer-dimer formation, which could interfere with the specific amplification, was checked in no template control sample.Each time point was determined as an average of the data obtained from triplicate trials.Relative gene expression was calculated using the following equation: Relative expression = [Etarget ∆CPtarget (control -sample) ]/[ Ereference ∆CPref (control - sample) ].
The relative levels of VtPGIP used to control PGIP mRNA were analyzed using the 2-∆∆Ct method (Livak and Schmittgen, 2001).The threshold cycle (Ct) value represents the PCR cycle in which the copy number passes the fixed threshold and can be first detected.

Analysis of the VtPGIP gene
A PGIP gene (EU037367) with a length of 1002 bp was isolated from V. thunbergii VtPGIP cDNA sequence was compared with the DNA sequence and showed no introns (unpublished).The amino acid sequences of some PGIPs were also compared and the results revealed sequence similarities as indicated by a phylogenetic tree of PGIPs from plants (Figure 1).Using sequence comparison data, we found that the species of the same genus were classified into the same group except Eucalyptus grandis, Pyrus communis, and Malus pumila because the PGIPs of these plants may have different evolutionary scenarios.
Considering the structure of PGIP from Phaseolus vulgaris (De Matteo et al., 2006), we found that the Nterminal regions of the VtPGIP contained disulfide bridges and the C-terminal regions contain disulphide bonds (Figure 4).The N-terminal regions of the VtPGIP consist of an α-helix residue and a short β-strand that forms Hbonds with the residues of sheet B1.VtPGIP structure, which contains seven α-helix motifs, were analyzed using Swiss-Pdb Viewer 3.7.In the VtPGIP structure, the Cterminal region comprises the last two 3 10 -helices, the last strand of sheet B2, and a short loop (Figure 4).

Analysis of the VtPGIP promoter
The promoter sequence of VtPGIP, with a length of 1650 bp (Figure 5), was obtained (JF832390) and the promoter sequence was analyzed (Figure 5 and Table 1a,b).The promoter of the VtPGIP gene contained cis-acting elements involved in the responses to ABA (ABRE), SA (TCA-element), and fungal elicitor responsive element (Box-W1).The promoter also contains transcription factor binding sites, such as Nkx2-5 and c-Rel.

Analysis of the subcellular distribution of the VtPGIP protein
The control proteins were uniformly distributed in the cells (Figure 6A, 6B, and 6C).The VtPGIP-EGFP fusion proteins were predominately located in the plant cell wall (Figure 6D, 6E, 6F).The signal peptide sequence of VtPGIP was analyzed online and the result indicated that the sequences should have accordant distribution located in the plant cell wall.

Effect of the treatments on VtPGIP gene expression
Studies on plant defense signaling have revealed that plants adopt a network of signal transduction pathways via different kinds of signaling molecules.Gene products were verified after the respective PCR products were sequenced.VtPGIP expression was analyzed in the treated leaves and then compared with that in the untreated control leaves.The result confirmed that VtPGIP expression was induced after ABA and SA treatment (Figures 7A and 7B).The highest transcription level was found at 2 h after SA treatment compared with the control group (0 h), and the highest VtPGIP expression was increased 57.68 times compared with that of the control group (Figure 7B).For the ABA treatment, the highest VtPGIP expression was found at 6 h, and this expression was 10 times greater than that of the control group (Figure 7A).The results confirmed that the VtPGIP gene was strongly induced by ABA treatment and consisted of the promoter sequence containing cis-acting elements involved in the responses to ABA and SA.
The relative VtPGIP expressions from the leaves inoculated with four different pathogens were monitored.The results showed that VtPGIP expression from the leaves infected with fungi was higher than that in the control group (Figure 8).After the plants were infected with B. cinerea, VtPGIP expression increased 100 times higher than that of the control group.Similarly, after C. diplodiella vaccination, VtPGIP expression relative to that of the control group was also approximately 100 times higher.After E. cichoracearum and G. cigulata were inoculated, VtPGIP expression increased approximately 10 times compared with that of the control group.These results suggested that VtPGIP was involved in plant disease resistance.

DISCUSSION
The structural data and the close homology with other PGIPs confirmed that the sequence was PGIP.VtPGIP was previously isolated and showed a high degree of similarity to PGIP genes from other plants.For the amino acid sequence, the species of the same genus were classified into the same group, but E. grandis, P. communis, and M. pumila may indicate that PGIPs have various evolutionary histories.In all of the PGIPs studied, the LRR domain is conserved, which reveals that proteinprotein interactions are involved in immune functions and  recognition of non-self molecules in plants (Jones, 2001).
The VtPGIP protein may inhibit PG activity because the sequence "function-sites" of LRR did not change during evolutionary development.
The three-dimensional structure of the bean PGIP has been investigated (Penninck et al., 1996), and results showed that the two extended B sheets in the LRR region are highly conserved; these B sheets are also involved in the interactions with pathogen PGs (De Matteo et al., 2003).The consensus LRR domain of the VtPGIP showed similarities to the LRR regions of other PGIPs and PGIP-like plant proteins.The three-dimensional structures of VtPGIP were also analyzed (Figure 4).In the structural view, VtPGIP showed numerous sites similar to P. vulgris PGIP (PvPGIP); hence, it may also contain PG-binding sites.The sequences of the amino acid residue of VtPGIP were analyzed and four cysteine residues were identified at the flanks of the LRR regions within the N-and C-terminal domains; this result is consistent with that of the PGIP of bean (De Matteo et al., 2006).The results suggested that the type of N-terminal extension affects substrate specificity and may determine the functional diversity of these enzymes.The N-and Cterminal cysteine-rich regions then form disulphide bridges, which are considered to be important in maintaining and providing additional stability of the secondary and tertiary structures.In the functional view, glycosylation possibly provides higher stability and increased protease resistance.This characteristic has been observed in many fungal PGs and may be crucial for their enzymatic activity.The six N-linked glycosylation sites were found in the LRR domains in VtPGIP (Figure 2).The positions and numbers of the N-linked glycosylation sites were not highly conserved among plant PGIPs (Mattei et al., 2001).
The subcellular localization of the transiently expressed PGIP protein was determined.The result showed that VtPGIP was localized in the plant cell wall, and this finding is consistent with that in previous studies on the localization of many other PGIPs (De Lorenzo et al., 2001).The plant cell wall provides structural stability (Shanmugam, 2005) and functions as the first barrier to counteract pathogens; PGs are also the first enzymes to be secreted when pathogens encounter plant cell walls (De Lorenzo and Ferrari, 2002).PGIPs are located in the plant cell wall and limit fungal invasion by counteracting the action of PGs.
Plants may have evolved mechanisms to respond to pathogens and other stress-related molecules (Ramonell et al., 2002).PGIPs may have an important function in the overall resistance to biotic and abiotic stresses (Li et al., 2003).The characteristics of PGIP promoter sequence was analyzed using promoter prediction software and transcription factor prediction software because the regulation of gene expression only covers a small part of the non-coding region; experimental studies are often difficult to conduct without the specific software.The promoter sequences were analyzed online to detect the presence of putative cis-acting regulatory elements in the promoter regions of VtPGIP (Table 1a,b).Several elements, including ABRE, Box-4, G-box, W1-box, HSE, TC-rich repeats, and TCA-element, were detected.VtPGIP expression was regulated by ABA, SA, and fungi.Such hormones and pathogens functioned as positive regulators of VtPGIP expression in the present experiment.In addition, several elements containing a binding site for Nkx2-5 and c-Rel transcription factors were  The structure of the promoter was analysed according to three different online software programs.Bioinformatics software was used to analyse the promoter.Some transcription factors and their positions were obtained in the TF Search, but the positions of unknown or new transcription factors could not be analysed; The promoter of the VtPGIP gene contain cis-acting elements involved in the responses to abscisic acid (ABRE), salicylic acid (TCA-element) and fungal elicitor responsive element (Box-W1) and it contain some transcription factor binding sites, such as Nkx2-5;c-Rel.The structure of the promoter was analysed according to three different online software programs.Bioinformatics software was used to analyse the promoter.Some transcription factors and their positions were obtained in the TF Search, but the positions of unknown or new transcription factors could not be analysed; The promoter of the VtPGIP gene contain cis-acting elements involved in the responses to abscisic acid(ABRE), salicylic acid(TCA-element) and fungal elicitor responsive element(Box-W1).and it contain some transcription factor binding sites, such as Nkx2-5;c-Rel.7B).VtPGIP expression in the untreated control leaves was analyzed and positively confirmed the relative expression of VtPGIP after ABA and SA treatment.On the basis of signaling molecules, plant defense genes were activated and regulated by different signal transduction pathways.SA, a product of the phenylpropanoid pathway, is involved in the expression of localized hypersensitivity reactions and systemic acquired resistance (Raskin, 1992).Studies have shown that SA has a critical function in the defense signaling pathway.In many plant species, SA levels increase as PR gene expression and disease resistance are activated (Johnson et al., 2003).The phytohormone ABA is involved in plant abiotic stress response and regulation of various biotic stress responses.Enhanced resistance against necrotrophic pathogens was demonstrated;  The results of this study suggested that the PGIP gene may be involved in SA and ABA-regulated defence responses, as the expression of VtPGIP strongly increased after treatment.They showed the maximum expression level of induction of VtPGIP was observed at 6 h and 2 h by ABA and SA treatments, respectively.Then expression levels of the VtPGIP transcripts decreased.After a longer period of time, it would return to basal levels of expression.
this procedure was based on primed callose accumulation controlled by an ABA-dependent defense pathway (Ton and Mauch-Mani, 2004).Plants secrete different defense proteins to protect themselves from pathogen invasion.These kinds of defense proteins are also induced by various signal molecules, such as SA and ABA.Thus, many important families of defense proteins are expressed and regulated by different signal molecules via different transduction pathways.Mechanical wounding or damage possibly occurs in the infection site where defense-related genes may be activated against pathogens (Cheong et al., 2002).Microarray studies have demonstrated that mechanical wounding and insect feeding account for distinct and overlapping sets of gene activation (Reymond et al., 2000).PGIPs are regulated during development and after wounding and pathogen infection or treatments with elicitors, SA, and cold temperature (De Lorenzo et al., 2001;Ferrari et al., 2003;Li et al., 2003).
PGIPs are also induced in many plant tissues under various environmental conditions, and PGIPs have been isolated in many plants.Other signaling molecules or defense response activators can induce PGIP gene expression (Ferrari et al., 2003).In the present study, VtPGIP gene expression was regulated by SA and ABA signal transduction pathways.
proteins.It was found that this protein was localized in the plant cell wall.The importance of PGIPs in plant defense has been elucidated by a series of studies.For instance, the overexpression of the PGIP gene in Arabidopsis reduces symptoms and colonization by B. cinerea (Ferrari et al., 2003).In the present study, the VtPGIP expression level from pathogen-infected leaves (C.diplodiella, E. cichoracearumr, G. cigulata, and B. cinerea) was higher than that of the control leaves.This phenomenon is more evident in B. cinerea and C. diplodiella than in other species.
In conclusion, PGIP has an important function in plant disease resistance.The PGIP gene expression levels were enhanced in response to applied SA and ABA.This result may be used as a basis to increase plant resistance to pathogen as induced by SA or ABA treatment in V. thunbergii.Hence, appropriate plant hormones should be applied in resistant engineering of other plants.PGIP expression in treated plants is possibly upregulated compared with untreated plants.As a result, plants may exhibit greater resistance to pathogens after treatments with appropriate plant hormones.

Figure 3 .
Figure 3.The signal peptides were identified using Signal P, and the Signal P-NN.The most likely cleavage site is between positions 27 and 28: SLS-ER.

Figure 4 .
Figure 4. Ribbon representation of the crystal structure of the VtPGIP.As with the ribbon representation of the crystal structure of PGIP2 from Phaseolus vulgaris (De Matteo et al., 2006), the figure showed that the VtPGIP had a righthanded superhelical fold typical of LRR proteins.The fold of the VtPGIP consists of a central LRR domain flanked by the Nand C-terminal cysteine-rich regions and, also like the crystal structure of PGIP2 from Phaseolus vulgaris, the secondary structure of the LRR motif showed seven α-helices located on the convex side of the VtPGIP protein.Meanwhile, the majority of the LRR proteins had two B sheets connected with the helices on the convex side by loops or b-turns were analysed by Swiss-Pdb Viewer 3.7, parallel to the B2 sheet.B2 is distorted of the twisted shape of the molecule and the variable length of the β-strands (Hegedus et al., 2008).From this figure, it showed the VtPGIP had twenty-two B -sheets.Sheets B1 and B2 are xLx and xxLxLxx regions, respectively, and the helix is the xLt/sGx region.

Figure 5 .
Figure 5.The promoter sequence analysed by Plantcare, it showed that it had ABRE (cisacting element involved in the abscisic acid responsiveness), Box-4, G-box, W1-box (fungal elicitor responsive element), HSE, TC-rich repeats and other transcription factorbinding sites on the positive strand.A TCA-element (cis-acting element involved in salicylic acid responsiveness) was on the negative strand.The expression of PGIP might be correlated with abscisic acid, salicylic acid, light, the fungal elicitor, heat stress and defence and stress responsiveness.

Figure 6
Figure 6 Subcellular localization of VtPGIP in onion epidermal cells.The onion epidermal cells were transformed with p35S:VtPGIP-EGFP(D-F).The expression and subcellular distribution of the proteins were examined under a fluorescent microscope (C, F) and a light microscope (B, E) and then merged (A, D).The data shown were representative of three independent experiments.EGFP: Enhanced green fluorescent proteins.It was found that this protein was localized in the plant cell wall.

Figure 7 .
Figure 7. Time course of the changes in mRNA levels of PGIP gene in leaves after ABA (A) and SA (B) treatments.The leaves were harvested 0, 2, 6, 16, 24, 48, and 72 h after the ABA (A) and SA (B) treatments.Analysis was performed by real-time RT-PCR (see Materials and methods).The results of this study suggested that the PGIP gene may be involved in SA and ABA-regulated defence responses, as the expression of VtPGIP strongly increased after treatment.They showed the maximum expression level of induction of VtPGIP was observed at 6 h and 2 h by ABA and SA treatments, respectively.Then expression levels of the VtPGIP transcripts decreased.After a longer period of time, it would return to basal levels of expression.

Fig. 7 Figure 8 .
Fig. 7 Time course of the changes in mRNA levels of PGIP gene in leaves after ABA (a) and SA (b) treatm leaves were harvested 0, 2, 6, 16, 24, 48, ).Hence, plants have evolved PGIPs with different recognition capabilities to counteract numerous PGs secreted by pathogenic fungi.Arabidopsis thaliana possesses two PGIP genes, which function in response to Botrytis cinerea infection; however, Arabidopsis PGIPs cannot inhibit PGs produced by F. moniliforme and A. niger; by comparison, Arabidopsis PGIPs can inhibit PGs produced by Colletotrichum gloesporioides, Stenocarpella maydis, and B. cinerea

Table 1a .
The structure of the promoter according to promoter prediction software.

Table 1b .
Structure of the promoter according to transcription factor prediction software.