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The effect of potassium phosphite on PR genes expression and the phenylpropanoid pathway in cucumber (Cucumis sativus) plants inoculated with Pseudoperonospora cubensis
Scientia Horticulturae 225 (2017) 366–372
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The effect of potassium phosphite on PR genes expression and the phenylpropanoid pathway in cucumber (Cucumis sativus) plants inoculated with Pseudoperonospora cubensis
MARK
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Moazzameh Ramezania, Fatemeh Rahmania, , Ali Dehestanib a b
Department of Biology, Faculty of Sciences, Urmia University, Urmia, Iran Genetics and Agricultural Biotechnology Institute of Tabarestan & Sari Agricultural Sciences and Natural Resources University, Sari, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords: Potassium phosphite Thaumatin-like protein Ribosome-inactivating protein Defensin Cucumber Pseudoperonospora cubensis
In the present study, the impact of potassium phosphite (KPhi) was investigated at molecular and biochemical levels in response to Pseudoperonospora cubensis infection in cucumber plants. Real-time PCR was employed to explore the differential expression of defense genes against P. cubensis. The highest expression for thaumatin-like protein (TLP), Ribosome-inactivating protein (RIP) and Defensin genes was observed in pre-inoculation plants at 96, 72 and 48 h, respectively. These findings revealed the involvement of these genes in the defense response of cucumber leaves after KPhi treatment and pathogen inoculation. At the biochemical level, more induction in the contents of some end-products of phenylpropanoid pathway, such as phytoalexin, phenolic component, flavonoid and anthocyanin as well as phenylalanine ammonia-lyase (PAL) enzymatic activity was detected in preinoculation plants compared to post-inoculation plants at all-time points. Data suggest that KPhi primes rapid and robust response in plants against infection via activation of defense responses. The negative effects of P. cubensis on cucumber plants could be considerably mitigated by KPhi application before infection.
1. Introduction Pseudoperonospora cubensis is one of the most destructive pathogens in cucumber plants. Researchers have focused on alleviating the damage caused by plant pathogens, and the application of fungicide is one the most effective ways to control infection. Therefore, activating defense responses through the use of chemical and bio-based elicitors should be an alternative method for improving plant tolerance to biotic stresses (Eshraghi et al., 2011). During the interaction between plants and pathogens, plant defense responses exhibit some main features including PR (pathogenesis-related) protein accumulation (Shetty et al., 2009), lignification of cell walls of tissues, and involvement of the phenylpropanoid pathway in plant defense (Somssich and Hahlbrock, 1998). Thaumatin-like protein (TLP) is a type of PR proteins that exhibit antifungal activity and increase plant disease resistance (Wang et al., 2011). Ribosome-inactivating proteins (RIP) are another type of PR proteins that participates in antifungal activity. Plant RIPs with specific modifications of 28 s rRNA inhibit protein synthesis. RIPs have a broad range of antimicrobial activities, including antifungal and antibacterial effects (Hey et al., 1995). PR-12 known as Defensin is another antifungal protein
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Corresponding author. E-mail address: [email protected] (F. Rahmani).
http://dx.doi.org/10.1016/j.scienta.2017.07.022 Received 5 April 2017; Received in revised form 15 July 2017; Accepted 17 July 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
with membrane permeabilization properties (Terras et al., 1992). Plant and pathogen interaction, has received considerable attention due to the synthesis of secondary metabolites such as lignin, phenols and phytoalexin, which add mechanical rigidity and strength to the cell wall and provide barriers to pathogen infection. During these protection processes, key enzymes in the phenylpropanoid pathway, such as phenylalanine ammonia lyase (PAL), play important roles (Bednarek et al., 2005). Among different secondary metabolites, phenolic and flavonoid compounds have protective activities with antioxidant and free radical scavenging properties in plants (Williams et al., 2004). Anthocyanin, as another product of the phenylpropanoid pathway, is usually associated with an increase in PAL activity and has biological roles including protection against many different pathogens, scavenging of free radicals and anti-oxidative activity (Kliebenstein, 2004). In agriculture, potassium phosphite (KPhi) is a fertilizer and activator of natural resistance or systemic resistance (Guest and Bompeix, 1990). A previous study has shown that KPhi is effective against P. cubensis and translocates in both xylem and phloem tissues (Silva et al., 2011). KPhi acts on the pathogen by stimulating host defense responses to inhibit pathogen growth and ultimately, to alter pathogen
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metabolism. KPhi can also induce the accumulation of defense molecules (Eshraghi et al., 2011) such as phytoalexin which is produced as defensive chemical for disease resistance and for overcoming pathogen attack (Holland and O'Keefe, 2010). The objective of this study was to elucidate the role of KPhi in priming defense responses in the P. cubensis–cucumber pathosystem, with an emphasis on the expression of pathogenesis-related genes and various signaling pathways that participate in plant defense. Fig. 1. Representative cucumber plant leaf showing the symptoms caused by P. cubensis pathogen after two weeks of inoculation.
2. Materials and methods 2.1. Plant materials
2.4. RNA extraction and first-strand cDNA synthesis
The study was performed at the laboratories in Genetics and Agricultural Biotechnology Institute of Tabarestan, Sari, Iran. The Isfahan native Cucumis sativus L. seeds were obtained from Pakan Bazr Co., Isfahan, Iran and disinfected in 70% ethanol for 5 min. Seeds were washed by distilled water, dried and sown in pots, filled with sterile soil mix in the greenhouse. The soil had been pre-moistened with distilled water in a growth cabinet (with a 16 h light/8 h dark photoperiod, temperature 24–27 °C and 70% humidity). Experiment was performed on 4 week old seedlings with emergence of secondary true leaves. Plants were divided into 5 groups (each group consisted of 6 pots and each pot contained one plant) and different treatments were performed as follows: 1. The leaves were sprayed with 0 g L−1 KPhi (distilled water) (Control). 2. The leaves were solely treated with 2 g L−1 KPhi three times a day for one day. Four days after KPhi treatment, leaves were harvested at 24, 48, 72 and 96 h (KPhi). 3. The leaves were treated with 2 g L−1 KPhi three times a day for one day. Four days after KPhi application, P. cubensis inoculation was performed and eight days after inoculation, leaves were harvested at 24, 48, 72 and 96 h (Pre- inoculation). 4. Eight days after inoculation of leaves by P. cubensis, leaves were treated with 2 g L−1 KPhi three times a day for one day. Four days later, leaves were harvested at 24, 48, 72 and 96 h (Post-inoculation). 5. The leaves were solely inoculated with P. cubensis and after eight days were harvested at 24, 48, 72 and 96 h (P. cubensis).
The secondary true leaves (100 mg fresh weight) were frozen in liquid nitrogen and stored at −80 °C. TRIzol reagent (Invitrogen, USA) was used for RNA extraction according to the manufacturer’s instructions. The RNA concentration was determined by measuring the absorbance at 260 nm, and its intensity was visualized on 1% agarose gel. Approximately 2 μg of total RNA was further treated with DNaseI and used for first-strand cDNA synthesis using oligo (dT) primers, 10 mM dNTPs, and reverse transcriptase according to the manufacturer’s instructions (Fermentas). 2.5. Real-time PCR conditions Aliquots of the cDNA were used as template for real-time PCR analysis with SYBR Green Real-time PCR Master Mix (Thermo Scientific). For RT-PCR analysis, gene-specific primers were designed using Bio Edit 7.0.9.0 and Oligo Explorer V1.4 Software (Table 1). Actin was used as an internal control. For real-time PCR, the following program was used: 3 min at 95 °C, denaturation at 95 °C for 25 s, annealing at 60 °C for 20 s and extension at 72 °C for 25 s for 40 cycles. To check the amplified product, a melting curve analysis was performed. To determine the relative gene induction levels, the ΔCT method was used (Livak and Schmittgen, 2001). 2.6. Determination of phytoalexin
2.2. KPhi stock solution preparation Phytoalexin were extracted according to the modified method of Andreu et al. (2001). Leaves (1 g fresh weight) were mixed by chloroform/acetic acid/methanol (50:5:45 v/v/v, 10 mL) using a homogenizer. The homogenate was kept overnight at room temperature and was then filtered. Chloroform and 0.2 M acetic acid were added in equal volumes to the container. The mixture was shaken, and two layers were separated. The chloroform layer containing phytoalexin was evaporated to dryness. The dried sample was resolved in 1 mL hexane and 2 mL sulfuric acid, and then agitated and centrifuged at 1000 rpm for 30 min. After 20 min, the absorbance of the red layer was recorded at 500 nm using a spectrophotometer (Biochrom WPA Biowave II). Phytoalexin content was calculated as μg g−1 FW.
A stock solution of filter-sterilized KPhi (pH 5, adjusted with KOH) was freshly prepared from phosphorous acid (AppliChem, Darmstadt, Germany). KPhi was applied to the plants as a foliar spray at the concentration of 2 g L−1 (Mofidnakhaei et al., 2016). 2.3. P. cubensis culture and plant inoculation P. cubensis spores were aseptically produced on freshly grown cucumber leaves using the method described by Waffa (2002) in Sari Agriculture University. The spore density was determined using a bright line haemocytometer and adjusted to a concentration of 4.5 × 105 spores mL−1 using sterile distilled water. P. cubensis inoculation was carried out as follows: leaves were sprayed by P. cubensis (with concentration of 4.5 × 105 mL−1 under controlled condition) three times a day for two days. Then, transferred to a growth cabinet with a 10 h photoperiod at 27 ± 2 °C to allow lesions to develop. Disease symptoms were observed as macroscopically visible necrotic spots. First, necrotic yellow spots appeared on the upper leaf surfaces, spreading from the marginal area to the central leaf surface followed by a change from yellow spots to a dried grayish leaf. The inoculated leaf underwent defoliation and the entire plant eventually became necrotic (Fig. 1). The samples were collected in aluminum foils and stored at −80 °C for molecular and biochemical assessments at different time points (24, 48, 72 and 96 h), as mentioned specifically for each assessment.
2.7. PAL activity analysis Preparation of PAL enzyme extract: to prepare the crude enzyme extract, 0.1 g leaf tissue was homogenized in 15 mL of 0.05 M phosphate buffer (phosphate buffer, pH = 7). The homogenate was centrifuged at 10,000 g for 15 min. The supernatant was used for further analysis. The extraction was conducted at 4 °C. PAL enzymatic activity was measured essentially according to the modified method of Hahlbrock and Grisebach (1979). PAL activity measurements were based on the formation of cinnamic acid at 290 nm. Approximately 0.1 mL PAL enzyme extract was mixed with 0.3 mL L-phenylalanine (50 mM). Phosphate buffer (0.05 M) was added to the reaction mixture (3 mL). After the reaction mixture was incubated at 30 °C for 15 min, 367
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Table 1 Sequences of the gene-specific primer pairs used in real time experiment. Gene
Accession number
Forward primer
Reverse primer
RIP TLP Defensin Actin
NC_026660.1 XM_004149913.2 XM_004151139 XM_004135239.2
5′-CTCAAGACTTCGTCGGTGTCAA-3′ 5′-ATGAAGGCAAAGGCTGGTGTGA-3′ 5′-GAGGCGAGGGTATGCGAATC-3′ 5′-GATTCTGGTGATGGTGTGAGTC-3′
5′-CGCCAGAGTTCACTAGCCTA-3′ 5′-AGGGTGACATAGATGATCCCAG-3′ 5′-GCAGCGACGGCAAATC-3′ 5′-TCGGCAGTGGTGGTGAACAT-3′
Fig. 2. The relative transcription levels of A) TLP, B) RIP, and C) Defensin in control, KPhi, pre-inoculation, post-inoculation and P. cubensis plants at 24, 48, 72 and 96 h. Data represent the mean ± SD, n = 3; level of significance: P < 0.05.
centrifuged. The absorbance of the samples was measured at 765 nm using a spectrophotometer. The total phenolic content was calculated by comparison with a standard curve of gallic acid and results were expressed as mg of gallic acid equivalents per g FW. Finally, the total phenolic content was calculated as the mg g−1 FW.
the absorbance was recorded at 290 nm using a spectrophotometer (Biochrom WPA Biowave II). For product determination, a standard curve of cinnamic acid was created. PAL activity was calculated as U mg−1 protein. 2.8. Extract preparation to determine the total phenolic and flavonoid contents
2.10. Total flavonoids To determine the total flavonoid content, the aluminum chloride calorimetric method was performed, and quercetin was used to generate the standard calibration curve. To prepare the quercetin solution, 5.0 mg quercetin was dissolved in 1.0 mL methanol. Next, 0.6 mL standard quercetin solution was separately mixed with 0.6 mL of 2% aluminum chloride. The solution was incubated for 60 min at room temperature. The absorbance was measured at 420 nm using a spectrophotometer. The total flavonoid content was calculated as mg g−1 FW (Zhishen et al., 1999).
Dried plant materials (10 mg) were extracted with 75 mL (95%) ethanol at 4 °C for 10 min. The solvent was evaporated at 40 °C under a reduced pressure chamber. The evaporated extract was used for further analysis. 2.9. Total phenolic content The total phenolic contents of the extracts were determined using folin and ciocalteu reagents according to Marinova et al. (2005). Fresh leaf sample (0.2 g) was mixed with 0.6 mL of water and 0.2 mL of folinciocalteu reagent (previously diluted with water (1:1, v/v). After 5 min, 1 mL of saturated sodium carbonate solution was added to the mixture, and the volume of the reaction was adjusted to 3 mL with distilled water. The reaction solution was kept in the dark for 30 min and then
2.11. Extraction of anthocyanin pigment For the determination of anthocyanin pigment content, the extract was prepared according to Fuleki and Francis (1968). Cucumber leaves 368
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plants at 48 h (0.95 U mg−1 protein) while the lowest in control plants (0.54 U mg−1 protein) followed by P. cubensis (0.56 U mg−1 protein) at 24 h. The results indicate induction of PAL activity by KPhi treatment. However, more activity was always evident in the pre-inoculation compared to the post-inoculation plants at the same time point (Fig. 4). The phenolic content showed a gradual increase during the experimental period under all treatments. The highest phenolic content was observed in pre-inoculation plants (120.85 mg g−1 FW) at 96 h and P.cubensis plants exhibited the lowest content (81. 80 mg g−1 FW) at 24 h. The pre-inoculation plants displayed more phenolic content than post-inoculation plants at different time points (Fig. 5A). The highest production of flavonoids was detected in KPhi (0.49 mg g −1 FW) at 96 h which was followed by KPhi and pre-inoculation plants (0.48 mg g −1 FW) at 72 h. The lowest flavonoid content was recorded in the control plants (0.34 mg g −1 FW) at 24 h. However, KPhi introduced greater effect on flavonoid content in preinoculated than post-inoculation plants during the experimental period (Fig. 5B). The maximum amount of anthocyanin was detected in KPhi plants (0.86 mg g−1 FW) at 96 h, and the lowest observed in control plants (0.43 mg g−1 FW) at 24 h. The results support a greater effectiveness of pre-inoculation than post-inoculation in induction of anthocyanin production at all-time points (Fig. 5C).
were homogenized with an ethanol solution acidified with 1.5 M HCl (85:15 v/v), pH 1.0, and the absorbance was read at 520 nm using a spectrophotometer. Anthocyanin content was calculated as mg g−1 FW. 2.12. Statistical analysis The experiments were arranged as factorial based on completely randomized design (CRD). Analysis of variance and significance of differences among means were performed by two-way ANOVA and LSD using SPSS software (version 23, SPSS, Chicago, IL). Means were compared at a significance level of P < 0.05. Experiments were performed in three biological replicates. 3. Results 3.1. Molecular assays Differential expression of TLP was clearly evident among the treatments (Fig. 2A). The highest level of transcription was observed at 96 h in pre-inoculation plants. KPhi and post-inoculation plants showed the lowest transcription level at 24 h. A comparison between pre and post-inoculation plants showed an enhanced effectiveness of KPhi treatment in pre-inoculation than post-inoculation plants at 72 and 96 h. No significant difference was observed between control and P. cubensis-inoculated plants during the entire experiment. The transcription of RIP gene was significantly induced in KPhi, preinoculation and post-inoculation plants at 72 h (Fig. 2B) with pre-inoculation plants exhibiting the highest transcription. This induction was followed by a sharp decrease in pre-inoculation and post-inoculation plants at 96 h. The control plants showed the lowest transcription of the RIP gene during the experiment. However, pre-inoculation plants always accumulated more transcripts than post-inoculation plants except at 24 h. The expression of Defensin gene revealed a peak of transcription in KPhi (20 fold), pre (19 fold) and post-inoculation (17 fold) plants at 48 h, compared to the P.cubensis plants at this time (Fig. 2C). In contrast, neither the control nor the P. cubensis plants showed any significant difference in expression during the experimental period. A comparison between pre and post-inoculation plants showed higher accumulation of Defensin mRNA in pre-inoculation compared to postinoculation plants at different harvesting times (Fig. 2C).
4. Discussion Plants respond to pathogen attack or elicitor treatments by activating a wide variety of protective mechanisms designed to prevent pathogen replication and spreading (Eshraghi et al., 2011). There are numerous reports concerning the protective effects of KPhi against pathogen infection in a range of crops (Daniel and Guest, 2006; Dharani et al., 2014; Eshraghi et al., 2011; Guest and Bompeix, 1990; Machinandiarena et al., 2012). The increase in TLP expression in pre-inoculation plants revealed a positive effect of KPhi on the expression of TLP resistance gene against P. cubensis. TLP was transcriptionally up-regulated in response to P. cubensis in pre-inoculation leaves and peaked after 72 h, showing higher activity at the later stages of infection. However, TLP is also related to SAR (systemic acquire resistance) or inhibition of hyphal growth (Uknes et al., 1992). The increase in TLP in response to KPhi may be a consequence of its antifungal activity when applied before infection as well as its ability to reduce esterase secretion by pathogens (Eshraghi et al., 2011). The up- regulation of TLP in KPhi plants demonstrate that an elicitor, such as KPhi, can prime the expression of TLP in the absence of pathogen. These results suggest that TLP is an antifungal protein that may be more effective against fungal invasion and spread. The fungal penetration through plant cell walls and cell membranes is an important pathway for suppression of the plant defense system (He et al., 2016). The maximum RIP expression was observed during the late stage of the experiment in pre and post-inoculation plants. Late expression of RIP is due to its role in the inhibition of fungal growth (Shabir, 2010).
3.2. Biochemical assays The highest amount of phytoalexin was observed in pre-inoculation (0.048 μg g−1 FW) plants at 96 h while the lowest was determined in P. cubensis (0.028 μg g−1 FW) plants at 24 h. The phytoalexin content appeared with gradual increase during the experimental period under all treatments. However, the higher content was always detected in the pre-inoculation than post-inoculation plants at the same time point (Fig. 3). The highest PAL enzymatic activity was detected in pre-inoculation
Fig. 3. The phytoalexin content in control, KPhi, pre-inoculation, postinoculation and P. cubensis plants at 24, 48, 72 and 96 h. Data represent the mean ± SD, n = 3; level of significance: P < 0.05.
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Fig. 4. A comparison of PAL enzyme activity in control, KPhi, pre-inoculation, post-inoculation and P. cubensis plants at 24, 48, 72 and 96 h. Data represent the mean ± SD, n = 3; level of significance: P < 0.05.
Fig. 5. A comparison of A) phenol, B) flavonoid and C) anthocyanin in control, KPhi, pre-inoculation, post-inoculation and P. cubensis plants at 24, 48, 72 and 96 h. Data represent the mean ± SD, n = 3; level of significance: P < 0.05.
enhances secretion, which could lead to a more rapid elicitation of plant defense responses and thereby contribute to the enhanced resistance of KPhi plants. However, in this study, the enhanced expression of PR genes following treatment with KPhi in the absence of P. cubensis (KPhi plants), especially TLP and Defensin, cannot be explained by an enhanced release of elicitors by the pathogen. Defense-related genes can encode a variety of proteins, including enzymes that participate in secondary metabolism, PR-proteins and regulatory proteins that regulate the expression of other defense-related genes (Bengtsson et al., 2014). The activation of PR genes can trigger the phenylpropanoid pathway, which leads to phytoalexin, phenolic, flavonoid and anthocyanin accumulations (El-kereamy et al., 2011). For example, TLP from Camellia sinensis is associated with activation of the phenylpropanoid pathway in potato (Acharya et al., 2013). The consistent results were obtained in gene expression and biochemical assays in KPhi and pre-inoculation leaves, revealing a relationship between them. Overally, it can be concluded that PR proteins have an active second line of defense that contributes indirectly to other
RIP displayed greater induction in pre and post-inoculation than in KPhi plants due to the antifungal role of RIP. This result is in agreement with findings showing that KPhi can generate resistance through the accumulation of hydrogen peroxide and RIP expression in Arabidopsis (Eshraghi et al., 2011) and potato (Machinandiarena et al., 2012). The increased Defensin mRNA level, after KPhi treatment and pathogen inoculation, could be attributed to its role in protection via activation of pathogen defense-related genes. Jiang et al. (2012) reported that overexpression of a broccoli Defensin gene led to enhanced resistance to downy mildew. Defensin, as an antifungal gene, can protect plants in different ways, such as by interacting with the cell membrane and disrupting the pathogen membrane integrity, leading to cell death, or stimulating root development and consequently, improving plant pathogen resistance (Gomez-Vasquez et al., 2004). KPhi may indirectly affect the induction of defense responses through its effect on the pathogen. A previous study has shown that KPhi can lead to pathogen growth inhibition by causing morphological changes in the hyphal wall (Wilkinson et al., 2001). This phenomenon 370
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Author contribution statement
defense regulatory mechanisms such as the phenylpropanoid and phytoalexin pathways (El-kereamy et al., 2011). The contents of phytoalexin in KPhi, pre and post-inoculation leaves were higher than control and P. cubensis leaves. A silicon-mediated accumulation of phytoalexins has been reported in cucumber plants in response to powdery mildew disease through enhancing antifungal activity in the infected leaves (Fawe et al., 1998). This antifungal activity was attributable to the presence of low-molecular-weight phytoalexins. It has been suggested that KPhi acts primarily on pathogens by altering fungal cells, which can force fungi to behave as an incompatible isolate leading to the development of resistance in host plants (Gottstein and Kuc, 1989). In this study, minimum phytoalexin content was detected in P. cubensis leaves in comparison to the other treatments. From a biological perspective, the multiplicity of active compounds may confer a distinct advantage to the plant. Previous studies have shown that fungi can develop the ability to detoxify phytoalexin (VanEtten et al., 1995). Stimulation of PAL activity by a chemical inducer or invasive pathogen demonstrated that the PAL enzyme is involved in carbon flow from phenylalanine to the metabolism of phytoalexin, phenolic, flavonoid, and anthocyanin contents. PAL genes are transcriptionally activated after microbial infection or treatment of plant cells with microbial elicitors (Bednarek et al., 2005). The phenolic and flavonoid components were higher under KPhi related treatments. The accumulation of phenolic compounds in leaves may be due to the inhibition of catalase activity, which in turn induces PAL gene expression and the synthesis of phenolic compounds (Vermerris and Nicholson, 2006). Phenol metabolism is normally activated in plants in response to pathogens, due not only to the mechanical roles played by phenolics in cell walls but also to their anti-fungal properties (Harborne, 1991). However, total phenols have long been considered important defense-related compounds that are naturally present at high levels in resistant varieties of many crops (Gogoi et al., 2001). It can be concluded that activation of PAL by KPhi resulted in an increase in phenolic and flavonoid contents as an end products of the pathway, creating a physical barrier against pathogen invasion. Based on their anti-pathogen activity, flavonoids can inactivate pathogen adhesion and cell envelope transport proteins (Naoumkina et al., 2010). The anthocyanin content displayed more induction in KPhi and preinoculation plants. KPhi stimulates the accumulation of anthocyanin by activating genes involved in the synthesis of anthocyanin (Giberti et al., 2012). The KPhi application on strawberry activated plant defense mechanisms by producing a higher concentration of anthocyanin (Estrada-Ortiz et al., 2013). The increased production of end-products of the phenylpropanoid pathway is known to be important in plant defense which is linked to the resistance properties of elicitors such as KPhi (Daayf et al., 2000).
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5. Conclusions KPhi primes plants for a rapid and intense response to infection involving heightened activation of a range of defense responses. In addition to its priming activity, KPhi also induces some features of the defense response, such as the expression of PR defense genes and phenylpropanoid pathway suggesting that KPhi facilitates the recruitment of a broad array of defense responses. A comparison of pre-inoculation and post-inoculation plants showed that application of KPhi before pathogen inoculation was more effective. This observation suggests that the pre-activation of genes activates rapid defense responses prior to P. cubensis inoculation, enhancing plant resistance to P. cubensis. This research enriches understanding of the best time for induction to promote protection against pathogen infection. The results suggest that the negative effects of P. cubensis on infected cucumber plants could be considerably mitigated by foliar spray with KPhi before infection. 371
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Overexpression of a thaumatin-like protein gene from Vitis amurensis improves downy mildew resistance in Vitis vinifera grapevine. Protoplasma. http://dx.doi.org/10.1007/ s00709-016-1047-y. Hey, T.D., Hartley, M., Walsh, T.A., 1995. Maize ribosome-inactivating protein (b-32). Homologs in related species, effects on maize ribosomes, and modulation of activity by pro-peptide deletions. Plant Physiol. 107, 1323–1332. http://dx.doi.org/10.1104/ pp.107.4.1323. Holland, K.W., O'Keefe, S.F., 2010. Recent application of Peanut phytoalexins. Recent Pat. Food Nutr. Agric. 22, 221–231. Jiang, M., He, C., Miao, L., Zhang, Y., 2012. Overexpression of a broccoli Defensin gene BoDFN enhances downy mildew resistance. J. Integr. Agr. 11, 1137–1144. http://dx. doi.org/10.1016/S2095-3119(12)60107-5. Kliebenstein, D.J., 2004. Secondary metabolites and plant/environment interactions: a view through Arabidopsis thaliana tinged glasses. Plant Cell Environ. 27, 675–684. http://dx.doi.org/10.1111/j.1365-3040.2004.01180.x. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C (T)). Methods 25, 402–408. http://dx. doi.org/10.1006/meth.2001.1262. Machinandiarena, M.F., Lobato, M.C., Feldman, M.L., Daleo, G.R., Andreu, A.B., 2012. Potassium phosphite primes defense responses in potato against Phytophthora infestans. J. Plant Physiol. 169, 1417–1424. http://dx.doi.org/10.1007/s10658-0119741-2. Marinova, D., Ribarov, F., Atanassova, M., 2005. Total phenolics and total flavonoids in Bolgarian fruits and vegetables. J. Chem. Technol. Metall. 40, 255–260. Mofidnakhaei, M., Abdossi, V., Dehestani, A., Pirdashti, H., Babaeizad, V., 2016. Potassium phosphite affects growth, antioxidant enzymes activity and alleviates disease damage in cucumber plants inoculated with Pythium ultimum. Arch. Phytopathology Plant Protect 49, 207–221. http://dx.doi.org/10.1080/03235408. 2016.1180924. Naoumkina, M.A., Zhao, Q., Gallego-Giraldo, L., Dai, X., Zhao, P.X., Dixon, R.A., 2010. Genome-wide analysis of phenylpropanoid defence pathways. Mol. Plant Pathol. 11, 829–846. http://dx.doi.org/10.1111/j.1364-3703.2010.00648.x. Shabir, H.W., 2010. Inducing fungus-resistance into plants through biotechnology. Not. Sci. Biol. 2, 14–21. http://dx.doi.org/10.15835/nsb.2.2.4594. Shetty, N.P., Jensen, J.D., Knudsen, A., Finnie, C., Geshi, N., Blennow, A., Collinge, D.B., Jørgensen, H.J., 2009. Effects of β-1, 3-Glucan from Septoria tritici on structural
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The effect of potassium phosphite on PR genes expression and the phenylpropanoid pathway in cucumber (Cucumis sativus) plants inoculated with Pseudoperonospora cubensis
MARK
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Moazzameh Ramezania, Fatemeh Rahmania, , Ali Dehestanib a b
Department of Biology, Faculty of Sciences, Urmia University, Urmia, Iran Genetics and Agricultural Biotechnology Institute of Tabarestan & Sari Agricultural Sciences and Natural Resources University, Sari, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords: Potassium phosphite Thaumatin-like protein Ribosome-inactivating protein Defensin Cucumber Pseudoperonospora cubensis
In the present study, the impact of potassium phosphite (KPhi) was investigated at molecular and biochemical levels in response to Pseudoperonospora cubensis infection in cucumber plants. Real-time PCR was employed to explore the differential expression of defense genes against P. cubensis. The highest expression for thaumatin-like protein (TLP), Ribosome-inactivating protein (RIP) and Defensin genes was observed in pre-inoculation plants at 96, 72 and 48 h, respectively. These findings revealed the involvement of these genes in the defense response of cucumber leaves after KPhi treatment and pathogen inoculation. At the biochemical level, more induction in the contents of some end-products of phenylpropanoid pathway, such as phytoalexin, phenolic component, flavonoid and anthocyanin as well as phenylalanine ammonia-lyase (PAL) enzymatic activity was detected in preinoculation plants compared to post-inoculation plants at all-time points. Data suggest that KPhi primes rapid and robust response in plants against infection via activation of defense responses. The negative effects of P. cubensis on cucumber plants could be considerably mitigated by KPhi application before infection.
1. Introduction Pseudoperonospora cubensis is one of the most destructive pathogens in cucumber plants. Researchers have focused on alleviating the damage caused by plant pathogens, and the application of fungicide is one the most effective ways to control infection. Therefore, activating defense responses through the use of chemical and bio-based elicitors should be an alternative method for improving plant tolerance to biotic stresses (Eshraghi et al., 2011). During the interaction between plants and pathogens, plant defense responses exhibit some main features including PR (pathogenesis-related) protein accumulation (Shetty et al., 2009), lignification of cell walls of tissues, and involvement of the phenylpropanoid pathway in plant defense (Somssich and Hahlbrock, 1998). Thaumatin-like protein (TLP) is a type of PR proteins that exhibit antifungal activity and increase plant disease resistance (Wang et al., 2011). Ribosome-inactivating proteins (RIP) are another type of PR proteins that participates in antifungal activity. Plant RIPs with specific modifications of 28 s rRNA inhibit protein synthesis. RIPs have a broad range of antimicrobial activities, including antifungal and antibacterial effects (Hey et al., 1995). PR-12 known as Defensin is another antifungal protein
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Corresponding author. E-mail address: [email protected] (F. Rahmani).
http://dx.doi.org/10.1016/j.scienta.2017.07.022 Received 5 April 2017; Received in revised form 15 July 2017; Accepted 17 July 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
with membrane permeabilization properties (Terras et al., 1992). Plant and pathogen interaction, has received considerable attention due to the synthesis of secondary metabolites such as lignin, phenols and phytoalexin, which add mechanical rigidity and strength to the cell wall and provide barriers to pathogen infection. During these protection processes, key enzymes in the phenylpropanoid pathway, such as phenylalanine ammonia lyase (PAL), play important roles (Bednarek et al., 2005). Among different secondary metabolites, phenolic and flavonoid compounds have protective activities with antioxidant and free radical scavenging properties in plants (Williams et al., 2004). Anthocyanin, as another product of the phenylpropanoid pathway, is usually associated with an increase in PAL activity and has biological roles including protection against many different pathogens, scavenging of free radicals and anti-oxidative activity (Kliebenstein, 2004). In agriculture, potassium phosphite (KPhi) is a fertilizer and activator of natural resistance or systemic resistance (Guest and Bompeix, 1990). A previous study has shown that KPhi is effective against P. cubensis and translocates in both xylem and phloem tissues (Silva et al., 2011). KPhi acts on the pathogen by stimulating host defense responses to inhibit pathogen growth and ultimately, to alter pathogen
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metabolism. KPhi can also induce the accumulation of defense molecules (Eshraghi et al., 2011) such as phytoalexin which is produced as defensive chemical for disease resistance and for overcoming pathogen attack (Holland and O'Keefe, 2010). The objective of this study was to elucidate the role of KPhi in priming defense responses in the P. cubensis–cucumber pathosystem, with an emphasis on the expression of pathogenesis-related genes and various signaling pathways that participate in plant defense. Fig. 1. Representative cucumber plant leaf showing the symptoms caused by P. cubensis pathogen after two weeks of inoculation.
2. Materials and methods 2.1. Plant materials
2.4. RNA extraction and first-strand cDNA synthesis
The study was performed at the laboratories in Genetics and Agricultural Biotechnology Institute of Tabarestan, Sari, Iran. The Isfahan native Cucumis sativus L. seeds were obtained from Pakan Bazr Co., Isfahan, Iran and disinfected in 70% ethanol for 5 min. Seeds were washed by distilled water, dried and sown in pots, filled with sterile soil mix in the greenhouse. The soil had been pre-moistened with distilled water in a growth cabinet (with a 16 h light/8 h dark photoperiod, temperature 24–27 °C and 70% humidity). Experiment was performed on 4 week old seedlings with emergence of secondary true leaves. Plants were divided into 5 groups (each group consisted of 6 pots and each pot contained one plant) and different treatments were performed as follows: 1. The leaves were sprayed with 0 g L−1 KPhi (distilled water) (Control). 2. The leaves were solely treated with 2 g L−1 KPhi three times a day for one day. Four days after KPhi treatment, leaves were harvested at 24, 48, 72 and 96 h (KPhi). 3. The leaves were treated with 2 g L−1 KPhi three times a day for one day. Four days after KPhi application, P. cubensis inoculation was performed and eight days after inoculation, leaves were harvested at 24, 48, 72 and 96 h (Pre- inoculation). 4. Eight days after inoculation of leaves by P. cubensis, leaves were treated with 2 g L−1 KPhi three times a day for one day. Four days later, leaves were harvested at 24, 48, 72 and 96 h (Post-inoculation). 5. The leaves were solely inoculated with P. cubensis and after eight days were harvested at 24, 48, 72 and 96 h (P. cubensis).
The secondary true leaves (100 mg fresh weight) were frozen in liquid nitrogen and stored at −80 °C. TRIzol reagent (Invitrogen, USA) was used for RNA extraction according to the manufacturer’s instructions. The RNA concentration was determined by measuring the absorbance at 260 nm, and its intensity was visualized on 1% agarose gel. Approximately 2 μg of total RNA was further treated with DNaseI and used for first-strand cDNA synthesis using oligo (dT) primers, 10 mM dNTPs, and reverse transcriptase according to the manufacturer’s instructions (Fermentas). 2.5. Real-time PCR conditions Aliquots of the cDNA were used as template for real-time PCR analysis with SYBR Green Real-time PCR Master Mix (Thermo Scientific). For RT-PCR analysis, gene-specific primers were designed using Bio Edit 7.0.9.0 and Oligo Explorer V1.4 Software (Table 1). Actin was used as an internal control. For real-time PCR, the following program was used: 3 min at 95 °C, denaturation at 95 °C for 25 s, annealing at 60 °C for 20 s and extension at 72 °C for 25 s for 40 cycles. To check the amplified product, a melting curve analysis was performed. To determine the relative gene induction levels, the ΔCT method was used (Livak and Schmittgen, 2001). 2.6. Determination of phytoalexin
2.2. KPhi stock solution preparation Phytoalexin were extracted according to the modified method of Andreu et al. (2001). Leaves (1 g fresh weight) were mixed by chloroform/acetic acid/methanol (50:5:45 v/v/v, 10 mL) using a homogenizer. The homogenate was kept overnight at room temperature and was then filtered. Chloroform and 0.2 M acetic acid were added in equal volumes to the container. The mixture was shaken, and two layers were separated. The chloroform layer containing phytoalexin was evaporated to dryness. The dried sample was resolved in 1 mL hexane and 2 mL sulfuric acid, and then agitated and centrifuged at 1000 rpm for 30 min. After 20 min, the absorbance of the red layer was recorded at 500 nm using a spectrophotometer (Biochrom WPA Biowave II). Phytoalexin content was calculated as μg g−1 FW.
A stock solution of filter-sterilized KPhi (pH 5, adjusted with KOH) was freshly prepared from phosphorous acid (AppliChem, Darmstadt, Germany). KPhi was applied to the plants as a foliar spray at the concentration of 2 g L−1 (Mofidnakhaei et al., 2016). 2.3. P. cubensis culture and plant inoculation P. cubensis spores were aseptically produced on freshly grown cucumber leaves using the method described by Waffa (2002) in Sari Agriculture University. The spore density was determined using a bright line haemocytometer and adjusted to a concentration of 4.5 × 105 spores mL−1 using sterile distilled water. P. cubensis inoculation was carried out as follows: leaves were sprayed by P. cubensis (with concentration of 4.5 × 105 mL−1 under controlled condition) three times a day for two days. Then, transferred to a growth cabinet with a 10 h photoperiod at 27 ± 2 °C to allow lesions to develop. Disease symptoms were observed as macroscopically visible necrotic spots. First, necrotic yellow spots appeared on the upper leaf surfaces, spreading from the marginal area to the central leaf surface followed by a change from yellow spots to a dried grayish leaf. The inoculated leaf underwent defoliation and the entire plant eventually became necrotic (Fig. 1). The samples were collected in aluminum foils and stored at −80 °C for molecular and biochemical assessments at different time points (24, 48, 72 and 96 h), as mentioned specifically for each assessment.
2.7. PAL activity analysis Preparation of PAL enzyme extract: to prepare the crude enzyme extract, 0.1 g leaf tissue was homogenized in 15 mL of 0.05 M phosphate buffer (phosphate buffer, pH = 7). The homogenate was centrifuged at 10,000 g for 15 min. The supernatant was used for further analysis. The extraction was conducted at 4 °C. PAL enzymatic activity was measured essentially according to the modified method of Hahlbrock and Grisebach (1979). PAL activity measurements were based on the formation of cinnamic acid at 290 nm. Approximately 0.1 mL PAL enzyme extract was mixed with 0.3 mL L-phenylalanine (50 mM). Phosphate buffer (0.05 M) was added to the reaction mixture (3 mL). After the reaction mixture was incubated at 30 °C for 15 min, 367
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Table 1 Sequences of the gene-specific primer pairs used in real time experiment. Gene
Accession number
Forward primer
Reverse primer
RIP TLP Defensin Actin
NC_026660.1 XM_004149913.2 XM_004151139 XM_004135239.2
5′-CTCAAGACTTCGTCGGTGTCAA-3′ 5′-ATGAAGGCAAAGGCTGGTGTGA-3′ 5′-GAGGCGAGGGTATGCGAATC-3′ 5′-GATTCTGGTGATGGTGTGAGTC-3′
5′-CGCCAGAGTTCACTAGCCTA-3′ 5′-AGGGTGACATAGATGATCCCAG-3′ 5′-GCAGCGACGGCAAATC-3′ 5′-TCGGCAGTGGTGGTGAACAT-3′
Fig. 2. The relative transcription levels of A) TLP, B) RIP, and C) Defensin in control, KPhi, pre-inoculation, post-inoculation and P. cubensis plants at 24, 48, 72 and 96 h. Data represent the mean ± SD, n = 3; level of significance: P < 0.05.
centrifuged. The absorbance of the samples was measured at 765 nm using a spectrophotometer. The total phenolic content was calculated by comparison with a standard curve of gallic acid and results were expressed as mg of gallic acid equivalents per g FW. Finally, the total phenolic content was calculated as the mg g−1 FW.
the absorbance was recorded at 290 nm using a spectrophotometer (Biochrom WPA Biowave II). For product determination, a standard curve of cinnamic acid was created. PAL activity was calculated as U mg−1 protein. 2.8. Extract preparation to determine the total phenolic and flavonoid contents
2.10. Total flavonoids To determine the total flavonoid content, the aluminum chloride calorimetric method was performed, and quercetin was used to generate the standard calibration curve. To prepare the quercetin solution, 5.0 mg quercetin was dissolved in 1.0 mL methanol. Next, 0.6 mL standard quercetin solution was separately mixed with 0.6 mL of 2% aluminum chloride. The solution was incubated for 60 min at room temperature. The absorbance was measured at 420 nm using a spectrophotometer. The total flavonoid content was calculated as mg g−1 FW (Zhishen et al., 1999).
Dried plant materials (10 mg) were extracted with 75 mL (95%) ethanol at 4 °C for 10 min. The solvent was evaporated at 40 °C under a reduced pressure chamber. The evaporated extract was used for further analysis. 2.9. Total phenolic content The total phenolic contents of the extracts were determined using folin and ciocalteu reagents according to Marinova et al. (2005). Fresh leaf sample (0.2 g) was mixed with 0.6 mL of water and 0.2 mL of folinciocalteu reagent (previously diluted with water (1:1, v/v). After 5 min, 1 mL of saturated sodium carbonate solution was added to the mixture, and the volume of the reaction was adjusted to 3 mL with distilled water. The reaction solution was kept in the dark for 30 min and then
2.11. Extraction of anthocyanin pigment For the determination of anthocyanin pigment content, the extract was prepared according to Fuleki and Francis (1968). Cucumber leaves 368
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plants at 48 h (0.95 U mg−1 protein) while the lowest in control plants (0.54 U mg−1 protein) followed by P. cubensis (0.56 U mg−1 protein) at 24 h. The results indicate induction of PAL activity by KPhi treatment. However, more activity was always evident in the pre-inoculation compared to the post-inoculation plants at the same time point (Fig. 4). The phenolic content showed a gradual increase during the experimental period under all treatments. The highest phenolic content was observed in pre-inoculation plants (120.85 mg g−1 FW) at 96 h and P.cubensis plants exhibited the lowest content (81. 80 mg g−1 FW) at 24 h. The pre-inoculation plants displayed more phenolic content than post-inoculation plants at different time points (Fig. 5A). The highest production of flavonoids was detected in KPhi (0.49 mg g −1 FW) at 96 h which was followed by KPhi and pre-inoculation plants (0.48 mg g −1 FW) at 72 h. The lowest flavonoid content was recorded in the control plants (0.34 mg g −1 FW) at 24 h. However, KPhi introduced greater effect on flavonoid content in preinoculated than post-inoculation plants during the experimental period (Fig. 5B). The maximum amount of anthocyanin was detected in KPhi plants (0.86 mg g−1 FW) at 96 h, and the lowest observed in control plants (0.43 mg g−1 FW) at 24 h. The results support a greater effectiveness of pre-inoculation than post-inoculation in induction of anthocyanin production at all-time points (Fig. 5C).
were homogenized with an ethanol solution acidified with 1.5 M HCl (85:15 v/v), pH 1.0, and the absorbance was read at 520 nm using a spectrophotometer. Anthocyanin content was calculated as mg g−1 FW. 2.12. Statistical analysis The experiments were arranged as factorial based on completely randomized design (CRD). Analysis of variance and significance of differences among means were performed by two-way ANOVA and LSD using SPSS software (version 23, SPSS, Chicago, IL). Means were compared at a significance level of P < 0.05. Experiments were performed in three biological replicates. 3. Results 3.1. Molecular assays Differential expression of TLP was clearly evident among the treatments (Fig. 2A). The highest level of transcription was observed at 96 h in pre-inoculation plants. KPhi and post-inoculation plants showed the lowest transcription level at 24 h. A comparison between pre and post-inoculation plants showed an enhanced effectiveness of KPhi treatment in pre-inoculation than post-inoculation plants at 72 and 96 h. No significant difference was observed between control and P. cubensis-inoculated plants during the entire experiment. The transcription of RIP gene was significantly induced in KPhi, preinoculation and post-inoculation plants at 72 h (Fig. 2B) with pre-inoculation plants exhibiting the highest transcription. This induction was followed by a sharp decrease in pre-inoculation and post-inoculation plants at 96 h. The control plants showed the lowest transcription of the RIP gene during the experiment. However, pre-inoculation plants always accumulated more transcripts than post-inoculation plants except at 24 h. The expression of Defensin gene revealed a peak of transcription in KPhi (20 fold), pre (19 fold) and post-inoculation (17 fold) plants at 48 h, compared to the P.cubensis plants at this time (Fig. 2C). In contrast, neither the control nor the P. cubensis plants showed any significant difference in expression during the experimental period. A comparison between pre and post-inoculation plants showed higher accumulation of Defensin mRNA in pre-inoculation compared to postinoculation plants at different harvesting times (Fig. 2C).
4. Discussion Plants respond to pathogen attack or elicitor treatments by activating a wide variety of protective mechanisms designed to prevent pathogen replication and spreading (Eshraghi et al., 2011). There are numerous reports concerning the protective effects of KPhi against pathogen infection in a range of crops (Daniel and Guest, 2006; Dharani et al., 2014; Eshraghi et al., 2011; Guest and Bompeix, 1990; Machinandiarena et al., 2012). The increase in TLP expression in pre-inoculation plants revealed a positive effect of KPhi on the expression of TLP resistance gene against P. cubensis. TLP was transcriptionally up-regulated in response to P. cubensis in pre-inoculation leaves and peaked after 72 h, showing higher activity at the later stages of infection. However, TLP is also related to SAR (systemic acquire resistance) or inhibition of hyphal growth (Uknes et al., 1992). The increase in TLP in response to KPhi may be a consequence of its antifungal activity when applied before infection as well as its ability to reduce esterase secretion by pathogens (Eshraghi et al., 2011). The up- regulation of TLP in KPhi plants demonstrate that an elicitor, such as KPhi, can prime the expression of TLP in the absence of pathogen. These results suggest that TLP is an antifungal protein that may be more effective against fungal invasion and spread. The fungal penetration through plant cell walls and cell membranes is an important pathway for suppression of the plant defense system (He et al., 2016). The maximum RIP expression was observed during the late stage of the experiment in pre and post-inoculation plants. Late expression of RIP is due to its role in the inhibition of fungal growth (Shabir, 2010).
3.2. Biochemical assays The highest amount of phytoalexin was observed in pre-inoculation (0.048 μg g−1 FW) plants at 96 h while the lowest was determined in P. cubensis (0.028 μg g−1 FW) plants at 24 h. The phytoalexin content appeared with gradual increase during the experimental period under all treatments. However, the higher content was always detected in the pre-inoculation than post-inoculation plants at the same time point (Fig. 3). The highest PAL enzymatic activity was detected in pre-inoculation
Fig. 3. The phytoalexin content in control, KPhi, pre-inoculation, postinoculation and P. cubensis plants at 24, 48, 72 and 96 h. Data represent the mean ± SD, n = 3; level of significance: P < 0.05.
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Fig. 4. A comparison of PAL enzyme activity in control, KPhi, pre-inoculation, post-inoculation and P. cubensis plants at 24, 48, 72 and 96 h. Data represent the mean ± SD, n = 3; level of significance: P < 0.05.
Fig. 5. A comparison of A) phenol, B) flavonoid and C) anthocyanin in control, KPhi, pre-inoculation, post-inoculation and P. cubensis plants at 24, 48, 72 and 96 h. Data represent the mean ± SD, n = 3; level of significance: P < 0.05.
enhances secretion, which could lead to a more rapid elicitation of plant defense responses and thereby contribute to the enhanced resistance of KPhi plants. However, in this study, the enhanced expression of PR genes following treatment with KPhi in the absence of P. cubensis (KPhi plants), especially TLP and Defensin, cannot be explained by an enhanced release of elicitors by the pathogen. Defense-related genes can encode a variety of proteins, including enzymes that participate in secondary metabolism, PR-proteins and regulatory proteins that regulate the expression of other defense-related genes (Bengtsson et al., 2014). The activation of PR genes can trigger the phenylpropanoid pathway, which leads to phytoalexin, phenolic, flavonoid and anthocyanin accumulations (El-kereamy et al., 2011). For example, TLP from Camellia sinensis is associated with activation of the phenylpropanoid pathway in potato (Acharya et al., 2013). The consistent results were obtained in gene expression and biochemical assays in KPhi and pre-inoculation leaves, revealing a relationship between them. Overally, it can be concluded that PR proteins have an active second line of defense that contributes indirectly to other
RIP displayed greater induction in pre and post-inoculation than in KPhi plants due to the antifungal role of RIP. This result is in agreement with findings showing that KPhi can generate resistance through the accumulation of hydrogen peroxide and RIP expression in Arabidopsis (Eshraghi et al., 2011) and potato (Machinandiarena et al., 2012). The increased Defensin mRNA level, after KPhi treatment and pathogen inoculation, could be attributed to its role in protection via activation of pathogen defense-related genes. Jiang et al. (2012) reported that overexpression of a broccoli Defensin gene led to enhanced resistance to downy mildew. Defensin, as an antifungal gene, can protect plants in different ways, such as by interacting with the cell membrane and disrupting the pathogen membrane integrity, leading to cell death, or stimulating root development and consequently, improving plant pathogen resistance (Gomez-Vasquez et al., 2004). KPhi may indirectly affect the induction of defense responses through its effect on the pathogen. A previous study has shown that KPhi can lead to pathogen growth inhibition by causing morphological changes in the hyphal wall (Wilkinson et al., 2001). This phenomenon 370
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Author contribution statement
defense regulatory mechanisms such as the phenylpropanoid and phytoalexin pathways (El-kereamy et al., 2011). The contents of phytoalexin in KPhi, pre and post-inoculation leaves were higher than control and P. cubensis leaves. A silicon-mediated accumulation of phytoalexins has been reported in cucumber plants in response to powdery mildew disease through enhancing antifungal activity in the infected leaves (Fawe et al., 1998). This antifungal activity was attributable to the presence of low-molecular-weight phytoalexins. It has been suggested that KPhi acts primarily on pathogens by altering fungal cells, which can force fungi to behave as an incompatible isolate leading to the development of resistance in host plants (Gottstein and Kuc, 1989). In this study, minimum phytoalexin content was detected in P. cubensis leaves in comparison to the other treatments. From a biological perspective, the multiplicity of active compounds may confer a distinct advantage to the plant. Previous studies have shown that fungi can develop the ability to detoxify phytoalexin (VanEtten et al., 1995). Stimulation of PAL activity by a chemical inducer or invasive pathogen demonstrated that the PAL enzyme is involved in carbon flow from phenylalanine to the metabolism of phytoalexin, phenolic, flavonoid, and anthocyanin contents. PAL genes are transcriptionally activated after microbial infection or treatment of plant cells with microbial elicitors (Bednarek et al., 2005). The phenolic and flavonoid components were higher under KPhi related treatments. The accumulation of phenolic compounds in leaves may be due to the inhibition of catalase activity, which in turn induces PAL gene expression and the synthesis of phenolic compounds (Vermerris and Nicholson, 2006). Phenol metabolism is normally activated in plants in response to pathogens, due not only to the mechanical roles played by phenolics in cell walls but also to their anti-fungal properties (Harborne, 1991). However, total phenols have long been considered important defense-related compounds that are naturally present at high levels in resistant varieties of many crops (Gogoi et al., 2001). It can be concluded that activation of PAL by KPhi resulted in an increase in phenolic and flavonoid contents as an end products of the pathway, creating a physical barrier against pathogen invasion. Based on their anti-pathogen activity, flavonoids can inactivate pathogen adhesion and cell envelope transport proteins (Naoumkina et al., 2010). The anthocyanin content displayed more induction in KPhi and preinoculation plants. KPhi stimulates the accumulation of anthocyanin by activating genes involved in the synthesis of anthocyanin (Giberti et al., 2012). The KPhi application on strawberry activated plant defense mechanisms by producing a higher concentration of anthocyanin (Estrada-Ortiz et al., 2013). The increased production of end-products of the phenylpropanoid pathway is known to be important in plant defense which is linked to the resistance properties of elicitors such as KPhi (Daayf et al., 2000).
AD designed the research; MR conducted the research and analyzed the data; MR, AD and FR wrote the paper; FR and AD had primary responsibility for the final content. All authors have read and approved the final manuscript. Acknowledgement This research was financially supported by by Urmia University and Genetics and Agricultural Biotechnology Institute of Tabarestan. References Acharya, K., Pal, A.K., Gulati, A., Kumar, S., Singh, A.K., Ahuja, P.S., 2013. Overexpression of Camellia sinensis thaumatin-like protein, CsTLP in potato confers enhanced resistance to Macrophomina phaseolina and Phytophthora infestans infection. Mol. Biotechnol. 54, 609–622. http://dx.doi.org/10.1007/s12033-012-9603-y. Andreu, A., Oliva, C.R., Distel, S., Daleo, G.R., 2001. Production of phytoalexins, glycoalkaloids and phenolics in leaves and tubers of potato cultivars with different degrees of field resistance after infection with Phytophthora infestans. 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5. Conclusions KPhi primes plants for a rapid and intense response to infection involving heightened activation of a range of defense responses. In addition to its priming activity, KPhi also induces some features of the defense response, such as the expression of PR defense genes and phenylpropanoid pathway suggesting that KPhi facilitates the recruitment of a broad array of defense responses. A comparison of pre-inoculation and post-inoculation plants showed that application of KPhi before pathogen inoculation was more effective. This observation suggests that the pre-activation of genes activates rapid defense responses prior to P. cubensis inoculation, enhancing plant resistance to P. cubensis. This research enriches understanding of the best time for induction to promote protection against pathogen infection. The results suggest that the negative effects of P. cubensis on infected cucumber plants could be considerably mitigated by foliar spray with KPhi before infection. 371
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