Next Article in Journal
HDAC6 and Ovarian Cancer
Next Article in Special Issue
Antioxidant Systems from Pepper (Capsicum annuum L.): Involvement in the Response to Temperature Changes in Ripe Fruits
Previous Article in Journal
The Role of microRNA in Gastric Malignancy
Previous Article in Special Issue
Improved Nutritive Quality and Salt Resistance in Transgenic Maize by Simultaneously Overexpression of a Natural Lysine-Rich Protein Gene, SBgLR, and an ERF Transcription Factor Gene, TSRF1
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 14
Issue 5
10.3390/ijms14059497
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Drought Stress Acclimation Imparts Tolerance to Sclerotinia sclerotiorum and Pseudomonas syringae in Nicotiana benthamiana

by
Venkategowda Ramegowda
1,2,†,‡,
Muthappa Senthil-Kumar
1,‡,
Yasuhiro Ishiga
1,
Amita Kaundal
1,
Makarla Udayakumar
2 and
Kirankumar S. Mysore
1,*
1
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA
2
Department of Crop Physiology, University of Agricultural Science, GKVK, Bangalore 560065, India
*
Author to whom correspondence should be addressed.
†
Present address: Department of Crop, Soil and Environmental Sciences, University of Arkansas, Fayetteville, AR 72701, USA
‡
These authors contributed equally to this work.
Int. J. Mol. Sci. 2013, 14(5), 9497-9513; https://doi.org/10.3390/ijms14059497
Submission received: 12 March 2013 / Revised: 13 April 2013 / Accepted: 22 April 2013 / Published: 2 May 2013
(This article belongs to the Special Issue Abiotic and Biotic Stress Tolerance Mechanisms in Plants)
Browse Figures
Versions Notes

Abstract

:
Acclimation of plants with an abiotic stress can impart tolerance to some biotic stresses. Such a priming response has not been widely studied. In particular, little is known about enhanced defense capacity of drought stress acclimated plants to fungal and bacterial pathogens. Here we show that prior drought acclimation in Nicotiana benthamiana plants imparts tolerance to necrotrophic fungus, Sclerotinia sclerotiorum, and also to hemi-biotrophic bacterial pathogen, Pseudomonas syringae pv. tabaci. S. sclerotiorum inoculation on N. benthamiana plants acclimated with drought stress lead to less disease-induced cell death compared to non-acclimated plants. Furthermore, inoculation of P. syringae pv. tabaci on N. benthamiana plants acclimated to moderate drought stress showed reduced disease symptoms. The levels of reactive oxygen species (ROS) in drought acclimated plants were highly correlated with disease resistance. Further, in planta growth of GFPuv expressing P. syringae pv. tabaci on plants pre-treated with methyl viologen showed complete inhibition of bacterial growth. Taken together, these experimental results suggested a role for ROS generated during drought acclimation in imparting tolerance against S. sclerotiorum and P. syringae pv. tabaci. We speculate that the generation of ROS during drought acclimation primed a defense response in plants that subsequently caused the tolerance against the pathogens tested.

1. Introduction

Apart from thriving under several abiotic stress factors, plants must also defend themselves from pathogen attack [1,2]. When exposed to multiple stress conditions plants activate specific and unique stress responses [3,4]. Plants response to complex biotic and abiotic stress conditions include expression of stress-inducible genes that function in stress tolerance, activation of signaling cascades, production of reactive oxygen species (ROS) and accumulation of hormones such as salicylic acid (SA), ethylene, jasmonic acid (JA) and abscisic acid (ABA) [5–7].
Plant adaptation to any stress situation has been suggested to be mediated by both basal and induced defenses [8,9]. The effectiveness of this basal resistance can be enhanced by pre-exposing plants to specific mild abiotic or biotic stimuli before imposing the full strength of respective stress. For example, prior exposure of plants to cold and drought stress has resulted in more stronger and successful response to the subsequent cold or drought stress, a phenomenon known as acclimation [10,11]. Initial cold acclimation of plants has increased the tolerance to subsequent cold stress in different species [12–15]. Prior exposure of Arabidopsis plants to drought stress or ABA resulted in freezing tolerance in those plants [16]. In wheat, pre-anthesis high-temperature acclimation alleviated damage to the flag leaf caused by post-anthesis heat stress [17]. Initial acclimation of sunflower seedlings to mild temperature provided better tolerance to subsequent high temperature and these heat acclimated seedlings also showed oxidative stress tolerance [18]. Chemical priming of Arabidopsis plants with the non-protein amino acid β-aminobutyric acid (BABA) increased drought and salt stress tolerance through accelerated stress gene expression and stomatal closure mediated by ABA, suggesting the possibility of increasing plant tolerance to abiotic stresses through effective priming of the pre-existing defense pathways [19].
Similarly, prior exposure of plants to biotic stress stimuli resulted in stronger and faster defense response upon subsequent exposure to pathogens, a phenomenon known as priming [20]. Prior pathogen infection or treatments with natural or synthetic compounds like SA, riboflavin, thiamine, menadione, 2,6-dichloroisonicotinic acid (INA), benzothiadiazole (BTH), BABA, or phosphite (Phi) has been shown to prime defense against subsequent pathogen challenge by mostly potentiating SA defense signaling [21–29]. Numerous studies have attempted to understand the components of primed defense. Treatment of Arabidopsis leaves with Phi resulted in enhanced accumulation of H2O2 not only at the site of Phytophthora cinnamomi inoculation but also in cells away from the inoculation [28], indicating the role of ROS in priming the defense response.
In nature, plants are simultaneously exposed to various biotic and abiotic stresses. Plants have mechanisms to survive in such complex environmental conditions [30]. In recent years, there are emerging reports to show that enhanced defense response of abiotic stress acclimated/treated plants to pathogens and vice versa [31]. For example, exposure of Arabidopsis plants to ozone or UV irradiation has induced resistance to virulent phytopathogenic Pseudomonas syringae strains [32] and to tobacco mosaic virus in tobacco [33]. Similarly, infection of various host plants with cucumber mosaic virus improved drought tolerance and also enhanced freezing tolerance in Beta vulgaris [31]. However, very little is known about how prior exposure of plants to abiotic stress improves the capacity of plants to effectively respond to biotic stress.
Upon successful recognition of avirulent pathogens, plants elicit a biphasic ROS accumulation with a rapid but weak transient first phase followed by a massive and prolonged second phase. The second phase increase in ROS, also called oxidative burst, is highly correlated with bacterial disease resistance [34]. Regulation of several abiotic stress signaling pathways is also associated with ROS [35]. Therefore, the generation of ROS has been proposed as a key process that is shared between biotic and abiotic stress responses [35,36]. Rapid production and tight regulation of the steady-state levels of ROS plays a pivotal role in both abiotic stress signaling and disease resistance responses [37,38]. Rapid generation of superoxide (O2−) and accumulation of hydrogen peroxide (H2O2) is a characteristic early feature of cell death or hypersensitive response (HR) following perception of pathogen signals [39–41]. In this study, we used Nicotiana benthamiana to investigate the effect of drought acclimation on subsequent challenge with necrotrophic fungus, Sclerotinia sclerotiorum and hemi-biotrophic bacterial pathogen, P. syringae pv. tabaci. Upon infection, drought acclimated plants showed less disease-induced cell death when compared to control non-acclimated plants. This correlated with the levels of ROS in the drought acclimated plants.

2. Results and Discussion

2.1. Drought Acclimated N. benthamiana Plants Showed Increased ABA and Root-Shoot Ratio

To confirm precise drought stress imposition and acclimation of plants, relative water content (RWC), ABA and root-shoot ratio were measured at the end of stress period. Plants maintained at 100% field capacity (FC) showed nearly 90% RWC, whereas plants at 40% FC reached RWC of 45% (Figure 1a) confirming the cellular water deficiency in the stressed plants. Quantification of ABA in drought stress acclimated N. benthamiana plants showed increased ABA levels (Figure 1b). ABA content is known to increase under drought stress [42]. Reduction in cellular water levels decreases osmotic potential and RWC resulting in closure of stomata to limit water loss through transpiration. Stomatal closure as a result of reduced cellular water levels during drought is mediated by ABA [43]. Consistent with this, N. benthamiana plants undergoing drought acclimation showed reduction in RWC accompanied with increased ABA content as drought level increased. There was also considerable increase in root-shoot ratio in N. benthamiana plants maintained at different FC’s with maximum increase in root growth in plants maintained at 40% FC (Figure 1c). When subjected to drought stress, plants show differential response to root and shoot growth with rapid inhibition of shoot growth and continued root elongation. Root elongation under low water potential is considered to be an adaptation of plants to dry conditions as continued root elongation facilitates water uptake from the soil [44,45]. These physiological and biochemical changes confirms acclimation of N. benthamiana plants subjected to different drought stress levels.

2.2. Drought Acclimation Reduced Sclerotinia sclerotiorum-Induced Cell Death in N. benthamiana

S. sclerotiorum is a broad host range fungus which can cause disease on N. benthamiana [46]. To determine the effect of drought acclimation on S. sclerotiorum-induced cell death, we inoculated drought acclimated N. benthamiana plants, at different FC’s for five days, with actively growing S. sclerotiorum. Drought acclimated N. benthamiana plants showed significantly reduced cell death symptoms caused by the fungus. In contrast, severe cell death was observed in non-acclimated control and plants maintained at 80% FC (Figure 2a,b). It is well known that relative humidity (RH) is an important factor that affects fungal infection and high RH facilitates better infection. Hence a set of plants were also infected with S. sclerotiorum and grown under high RH (80%) to determine whether the observed reduction in cell death was due to the intrinsic capacity of the drought acclimated plants or just mere reduction in RH on the leaf surface. Interestingly, there was no change in cell death or disease symptom development in plants maintained at 40% FC even under high RH when compared to plant maintained at 40% FC with low RH (Figure 2). These results suggest that acclimation to moderate drought stress (40% FC) has enhanced the capacity for effective activation of cellular defense response resulting in improved tolerance to subsequent infection of S. sclerotiorum and the observed tolerance is not due to lack of moist condition. As observed in plant-pathogen interactions, plants are also known to display priming-like reactions to abiotic stresses. It has been reported that biotic and abiotic stresses may interact both positively and negatively either by enhancing the resistance or increasing susceptibility to other stresses [4]. Recent evidences suggest that drought acclimation may enhance resistance to subsequent pathogen infection. In tomato, drought acclimation by three cycles of wilting and recovery enhanced the resistance to the fungus Botrytis cinerea [47]. In Pinus yunnanensis, plants which were exposed to mild water stress during winter showed enhanced resistance to a pathogenic fungus, Leptographium yunnanense [48]. A study in rose showed plant extract from drought stressed leaves controlled the black spot disease caused by Diplocarpon rosae through enhanced expression of pathogenesis-related proteins, peroxidase and accumulation of defense response elicitors [49]. Consistent with these reports, our results also suggest that drought acclimation of N. benthamiana plants might have primed a defense response resulting in enhanced tolerance to subsequent S. sclerotiorum infection.

2.3. Acclimation to Moderate Drought Stress Enhanced Tolerance to Pseudomonas syringae pv. tabaci in N. benthamiana

To examine whether drought acclimation could also improve tolerance to plant-pathogenic bacteria, drought acclimated N. benthamiana plants were syringe (needless) infiltrated with a host pathogen, P. syringae pv. tabaci [50], which causes wildfire disease in tobacco [51]. Disease-induced cell death symptoms were visually scored at 5 days post-inoculation (dpi). More than 65% of the inoculated spots showed complete cell death and nearly 35% of the inoculated spots showed moderate cell death in plants maintained at 100% FC (Figure 3). The trend was very similar in plants maintained at 80% FC with more inoculated spots showing moderate cell death. Strikingly, the inoculated spots of plants maintained in 60% FC showed moderate to mild cell death and none of them showed complete cell death. Interestingly, nearly 30% and 70% of the inoculated spots showed complete cell death in plants maintained at 40% FC and severe drought stress of 20% FC, respectively (Figure 3). Surprisingly, when we quantified the bacterial growth in the inoculated spots of plants maintained in different FC’s, we did not see a significant difference at 3 dpi (data not shown). Disease symptoms induced by bacterial pathogens are not always correlated with bacterial growth as shown earlier [52,53]. From our results we suggest that acclimation of plants to moderate drought stress (60% FC) could limit the P. syringae pv. tabaci-induced disease development in N. benthamiana. At severe drought stress level (20% FC), the disease symptoms-induced by P. syringae pv. tabaci were quite similar to control (100% FC) plants suggesting the importance of moderate drought acclimation in imparting tolerance to bacterial pathogens. Lack of ROS induction and higher levels of ABA could be the reason for increased susceptibility of plants maintained at 20% FC (Figures 1c and 4a,b). Recent reports suggest that ABA plays antagonistic role in biotic stress signaling [30,54,55]. ABA treatment has been shown to antagonize SA, JA and ethylene defense signaling [56,57]. Negative effect of ABA on ROS mediated defense response has also been reported [58]. An ABA deficient tomato mutant plant, with impaired aldehyde oxidase activity which is required in ABA biosynthesis, accumulated H2O2 rapidly following infection by Botrytis cinerea, leading to a stronger defense response and greater resistance to the pathogen compared with the wild-type [58]. The susceptibility of N. benthamiana plants to bacterial pathogen maintained at 20% FC which also accumulated higher levels of ABA could be attributed to antagonistic effect of ABA on SA-mediated defense signaling.

2.4. Tolerance to Pathogen is Correlated with ROS Levels in Drought Acclimated Plants

We quantified the levels of O2− and H2O2 in plants subjected to drought acclimation at different FC’s. The highest levels of O2− and H2O2 were found in plants maintained at 40% FC. There was 6-fold increase in O2− (Figure 4a) and 2-fold increase in H2O2 fluorescence455 at 40% FC (Figure 4b). At other stress levels (60% and 20% FC), which also showed considerable tolerance against both the pathogens, the O2− levels were higher than non-acclimated control plants. It has been previously proposed that prior exposure of plants to abiotic stress alters pathogen resistance related activities that might result in enhanced resistance to subsequent pathogen challenge [59]. Though the mechanism or components involved in altered activities are not clear, there are evidences suggesting common connection points between biotic and abiotic stresses. Particularly, ROS generation has been suggested to be a central process in mediating the biotic and abiotic stress responses [30,60]. It is well known that the concentration of ROS increases during drought stress conditions [61]. During pathogen infection, plants rapidly generate ROS resulting in oxidative burst that limits pathogen spread through HR or cell death [35]. Our data shows that N. benthamiana plants acclimated at 40% FC drought stress enhanced tolerance to S. sclerotiorum and P. syringae pv. tabaci. The increased tolerance was correlated with higher O2− and H2O2 levels in plants acclimated at 40% FC suggesting that high levels of ROS, very similar to oxidative burst under pathogen infection, might have helped reduce the disease development. It is not clear why the ROS levels decrease at severe stress condition (20% FC). In accordance with the reduced ROS levels, we saw reduced tolerance of N. benthamiana to tested pathogens at 20% FC when compared to moderate stress conditions (40% and 60% FCs).
To verify whether ROS generated in drought acclimated plants can reduce the pathogen infection, we used a ROS inducing chemical, methyl viologen (MV). We monitored the growth of GFPuv expressing P. syringae pv. tabaci pathogen [62] in MV sprayed N. benthamiana leaves. In light exposed plants, MV accepts electrons from photosystem I and transfers them to molecular oxygen generating superoxide in chloroplast [63]. In control plants intense bright green fluorescent spots representing bacterial colonies were observed under long wavelength UV light, while no or a very few green spots were visible on the leaves treated with MV suggesting the role of ROS in inhibiting bacterial growth (Figure 4c). High ROS levels in the plants maintained at 40% FC and complete prevention of bacterial growth in MV sprayed plants suggest that pre-generated ROS in drought acclimated plants might have primed the defense response against necrotrophic fungus, S. sclerotiorum and also to the bacterial pathogen, P. syringae pv. tabaci.

2.5. Drought Acclimation in N. benthamiana Induces Plant Defense Genes

Expression pattern of disease resistance marker genes was analyzed to understand the possible molecular mechanism for the enhanced defense capacity of drought acclimated plants. There was higher induction of PR-5 (pathogenesis-related protein-5) and PDF1.2 (plant defensin 1.2) in drought acclimated plants (Figure 5). The PR-5 gene encodes an osmotin and is induced through many signaling pathways including osmotic stress [64–66]. The PDF1.2 gene encodes a plant defensin, and is induced by pathogen attack both locally and systemically via a JA/ethylene-mediated signaling pathway [67]. Studies have shown that several genes which are part of plant defense response to pathogen attack are also induced by osmotic stress but the role of these proteins in abiotic stress is not fully clear [68,69]. Our results suggest that pre-induction of pathogen defense responsive genes like PR-5 and PDF1.2 in drought acclimated plants might also contributed to the delayed disease development when challenged with S. sclerotiorum and P. syringae pv. tabaci in N. benthamiana.

3. Experimental Section

3.1. Plant Growth Condition

N. benthamiana seeds were germinated on flats filled with soilless potting mixture, Metro-Mix 830, BM7 (SUNGRO Horticulture Distribution, Inc., Bellevue, WA, USA). Three-week-old seedlings were transplanted to 10 cm diameter round pots (one seedling per pot) containing BM7. Plants were watered with fertilizer (20-10-20) containing soluble trace element mix (The Scotts Co., Marysville, OH, USA). The light intensity maintained in the greenhouse was 600 μE/m2/s with RH of 65%. The day/night cycles for temperature and photoperiod were 22 ± 2 °C/19 ± 2 °C and 14 h day/10 h night, respectively.

3.2. Drought Stress Imposition

Five-week-old plants were subjected to drought stress following gravimetric method of stress imposition. Soil moisture content at 100% field capacity (FC) was determined using the formula; Soil moisture content at 100% FC = [(SW − DW)/DW] × 100; Where SW-saturated soil weight taken after flooding the soil followed by overnight gravitational drainage; DW-oven dry soil weight. For stress treatments, soil water status was gradually brought down to specific FC (80%, 60%, 40% and 20%) by weighing the pots daily at a fixed time of the day [70]. Set of plants which reached specific FC early were maintained at that level by replenishing the lost water through evapotranspiration until the last set reaching 20% FC. Further, plants were maintained continuously at respective FC’s for five more days for acclimation (Figure 1a). Drought acclimated plants were then used for stress effect quantification and pathogen experiments. During the pathogen infection, plants were maintained at specified FC. A set of plants at 100% FC were used as non-acclimated controls.

3.3. RWC and Root-Shoot Ratio Measurement

RWC was determined following the protocol described by Flower and Ludlow, 1986 [71]. Briefly, after determining the fresh weight (FW), samples were immediately hydrated, by floating on de-ionized water in a closed Petri dish, to full turgidity for 4 h under normal room light and temperature and turgid weight (TW) was obtained. Samples are then oven dried at 50 °C until they reach constant weight to determine dry weight (DW). RWC was calculated using the formula:
RWC  ( % ) = [ ( FW - DW ) / ( TW - DW ) ] × 100
To estimate the root-shoot ratio, the aboveground and belowground parts were harvested separately and oven dried at 50 °C until they reach constant weight. The aboveground weight was considered as shoot and the below ground weight as root and the root-shoot ratio was calculated using following formula; Root-shoot ratio = Root dry weight/shoot dry weight.

3.4. ABA Quantification

Leaf tissue was ground to fine powder in liquid nitrogen and ABA was extracted in extraction buffer, containing 80% methanol with 100 mg/L butylated hydroxytoluene and 500 mg/L citric acid, in darkness for 16 h with constant shaking at 4 °C. The mixture was centrifuged at 5000 rpm for 5 min and the supernatant was diluted 10-fold with TBS buffer (50 mM Tris, 1 mM MgCl2, 150 mM NaCl, pH 7.8). The ABA concentration was quantified using the Phytodetek competitive ELISA kit (Agdia, Elkhardt, IN, USA) following the manufacturer’s instructions. A standard curve of different ABA dilutions [(+) cis/trans ABA; Sigma Aldrich, St. Louis, MO, USA)] was constructed to calculate the sample ABA concentrations.

3.5. Pseudomonas syringae pv. tabaci Inoculation and Scoring the Extent of Disease-Induced Cell Death

P. syringae pv. tabaci was grown in King’s B medium at 30 °C supplemented with rifampicin (25 μg/mL). Bacterial cells were harvested by centrifugation of overnight grown culture at 5000 rpm for 10 min, washed twice and re-suspended to the desired concentration (0.00002 OD) in sterile water. The bacterial suspension was used to inoculate leaves of drought acclimated N. benthamiana plants at respective FC’s using a needless syringe [50]. Plants were maintained continuously under drought for three more days and then re-watered. The disease-induced cell death symptom was scored at five days post-inoculation (dpi).

3.6. Sclerotinia sclerotiorum Inoculum Preparation and Plant Inoculation

S. sclerotiorum culture was initially grown on PDA (Difco, Sparks, MD, USA) medium and agar plugs (5 mm diameter) from actively growing regions were used as inoculum. Leaves of N. benthamiana plants maintained at different FC’s were inoculated with agar plugs and maintained at 65% RH. For increased RH, another set of plants inoculated with S. sclerotiorum were maintained at 80% RH chamber. All plants were kept at 22 ± 2 °C on a 14 h photoperiod at light intensity of 200 μE/m2/s. The disease symptoms were recorded at 5 dpi.

3.7. Quantification of ROS in Drought Acclimated Plants

Leaf tissue from drought acclimated plants, at a specified FC for 5 days, was used for quantifying O2− and H2O2. Superoxide content was quantified by incubating leaf disks in 1 mL of K-phosphate buffer (20 mM, pH 6.0) containing 500 μM XTT (Polyscience Europe, Eppelheim, Germany) in darkness at 25 °C on a shaker. The increase in absorbance (A470) in the incubation medium was measured using spectrophotometer [72]. For determining H2O2 levels, leaf disks were pre-incubated for 30 min in 3 mL of K-phosphate buffer (20 mM, pH 6.0) to remove pre-formed H2O2 and were then incubated in 3 mL of the same buffer containing 5 μM scopoletin [7-hydroxy-6-methoxy-2H-1-benzopyran-2-one (Sigma Aldrich, St. Louis, MO, USA)] [73] and 3 μg/mL horseradish peroxidase in darkness at 25 °C on a shaker. The decrease in fluorescence (excitation: 346 nm, emission: 455 nm) in the incubation medium was measured using reagent blanks as reference.

3.8. Inoculation and Monitoring Bacterial Growth in Methyl Viologen Pre-Treated Plants

Initially a set of four-week-old N. benthamiana plants were sprayed with 5 μM MV. Six-hours later, plants were inoculated with P. syringae pv. tabaci expressing GFPuv [63] by vacuum infiltration. The inoculated plants were kept in greenhouse at 22 ± 2 °C. Two-days after inoculation, bacterial growth was visualized in leaves directly under UV light in a dark room. The photographs were taken using regular digital camera under UV light.

3.9. Real-Time Quantitative RT-PCR (qRT-PCR) Analysis

Total RNA was extracted from 100 mg of leaf tissue collected at the end of drought acclimation period using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. Two microgram of DNase treated total RNA was used for cDNA synthesis using SuperScript™ III First-Strand Synthesis System (Invitrogen™ life technologies, Grand Island, NY, USA). Equal dilution of cDNA was used as a template for qRT-PCR analysis using gene-specific primers (Supplementary Table S1) and the SYBR® Green (Sigma Aldrich, St. Louis, MO, USA). The PCR reactions were run on Bio-Rad Thermal Cycler (Bio-Rad Life Science Research, Hercules, CA, USA) using the following program: 95 °C for 2 min, 95 °C for 15 s, 55 °C for 1 min for 40 cycles. The NbActin was used as internal control to normalize gene expression levels. Quantification of the relative changes in gene expression was performed using the 2−ΔΔCT method [74].

4. Conclusion

The results from our study indicate that drought acclimation of N. benthamiana plants enhance tolerance to S. sclerotiorum and P. syringae pv. tabaci. The increase in ROS levels in drought acclimated plants could have primed the defense response in plants to subsequent pathogen infection very similar to second phase of oxidative burst in plant-pathogen interaction. Our study further strengthens the concept that ROS could be the central process connecting abiotic and biotic stress responses.

Supplementary Information

ijms-14-09497-s001.pdf

Acknowledgments

This project was funded by The Samuel Roberts Noble Foundation. VR thanks The Wood Whelan Research Fellowships of International Union of Biochemistry and Molecular Biology (WWF-IUBMB) and The Kirkhouse Trust, UK for travel and research support at KSM lab. Authors thank Janie Gallaway for excellent plant care.

Conflict of Interest

The authors declare no conflict of interest.

References

  1. Wang, W.; Vinocur, B.; Altman, A. Plant responses to drought, salinity and extreme temperatures: Towards genetic engineering for stress tolerance. Planta 2003, 218, 1–14. [Google Scholar]
  2. Hammond-Kosack, K.E.; Jones, J.D.G. Response to Plant Pethogens. In Biochemistry and Molecular Biology of Plants; Buchannan, B., Gruissem, W., Jones, R.L., Eds.; Society of Plant Physiologists: Rockville, MD, USA, 2000; pp. 1102–1157. [Google Scholar]
  3. Rizhsky, L.; Liang, H.; Shuman, J.; Shulaev, V.; Davletova, S.; Mittler, R. When defense pathways collide. The response of Arabidopsis to a combination of drought and heat stress. Plant Physiol 2004, 134, 1683–1696. [Google Scholar]
  4. Atkinson, N.J.; Urwin, P.E. The interaction of plant biotic and abiotic stresses: From genes to the field. J. Exp. Bot 2012, 63, 3523–3543. [Google Scholar]
  5. Yamaguchi-Shinozaki, K.; Shinozaki, K. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu. Rev. Plant. Biol 2006, 57, 781–803. [Google Scholar]
  6. Bostock, R.M. Signal crosstalk and induced resistance: Straddling the line between cost and benefit. Annu. Rev. Phytopathol 2005, 43, 545–580. [Google Scholar]
  7. Lorenzo, O.; Solano, R. Molecular players regulating the jasmonate signalling network. Curr. Opin. Plant Biol 2005, 8, 532–540. [Google Scholar]
  8. Bray, E.A.; Bailey-Serres, J.; Weretilnyk, E. Responses to Abiotic Stresses; American Society of Plant Physiologists: Rockville, MD, USA, 2000; pp. 1158–1203. [Google Scholar]
  9. Hasegawa, P.M.; Bressan, R.A.; Zhu, J.K.; Bohnert, H.J. Plant cellular and molecular responses to high salinity. Annu. Rev. Plant Physiol. Plant Mol. Biol 2000, 51, 463–499. [Google Scholar]
  10. Lang, V.; Palva, E.T. The expression of a rab-related gene, rab18, is induced by abscisic acid during the cold acclimation process of Arabidopsis thaliana (L.) Heynh. Plant Mol. Biol 1992, 20, 951–962. [Google Scholar]
  11. Knight, H.; Brandt, S.; Knight, M.R. A history of stress alters drought calcium signalling pathways in Arabidopsis. Plant J 1998, 16, 681–687. [Google Scholar]
  12. Bridger, G.M.; Yang, W.; Falk, D.E.; McKersie, B.D. Cold acclimation increases tolerance of activated oxygen in winter cereals. J. Plant Physiol 1994, 144, 235–240. [Google Scholar]
  13. Gilmour, S.J.; Hajela, R.K.; Thomashow, M.F. Cold Acclimation in Arabidopsis thaliana. Plant Physiol 1988, 87, 745–750. [Google Scholar]
  14. Jennings, P.; Saltveit, M.E. Temperature effects on imbibition and germination of cucumber (Cucumis sativus) seeds. J. Am. Soc. Hortic. Sci 1994, 119, 464–467. [Google Scholar]
  15. Prasad, T.K.; Anderson, M.D.; Martin, B.A.; Stewart, C.R. Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxide. Plant Cell 1994, 6, 65–74. [Google Scholar]
  16. Mantyla, E.; Lang, V.; Palva, E.T. Role of abscisic acid in drought-induced freezing tolerance, cold acclimation, and accumulation of LT178 and RAB18 proteins in Arabidopsis thaliana. Plant Physiol 1995, 107, 141–148. [Google Scholar]
  17. Wang, X.; Cai, J.; Jiang, D.; Liu, F.; Dai, T.; Cao, W. Pre-anthesis high-temperature acclimation alleviates damage to the flag leaf caused by post-anthesis heat stress in wheat. J. Plant Physiol 2011, 168, 585–593. [Google Scholar]
  18. Senthil-Kumar, M.; Srikanthbabu, V.; Mohan Raju, B.; Ganeshkumar; Shivaprakash, N.; Udayakumar, M. Screening of inbred lines to develop a thermotolerant sunflower hybrid using the temperature induction response (TIR) technique: A novel approach by exploiting residual variability. J. Exp. Bot 2003, 54, 2569–2578. [Google Scholar]
  19. Jakab, G.; Ton, J.; Flors, V.; Zimmerli, L.; Metraux, J.P.; Mauch-Mani, B. Enhancing Arabidopsis salt and drought stress tolerance by chemical priming for its abscisic acid responses. Plant Physiol 2005, 139, 267–274. [Google Scholar]
  20. Conrath, U.; Pieterse, C.M.; Mauch-Mani, B. Priming in plant-pathogen interactions. Trends Plant Sci 2002, 7, 210–216. [Google Scholar]
  21. Kauss, H.; Krause, K.; Jeblick, W. Methyl jasmonate conditions parsley suspension cells for increased elicitation of phenylpropanoid defense responses. Biochem. Biophys. Res. Commun 1992, 189, 304–308. [Google Scholar]
  22. Kauss, H.; Jeblick, W. Pretreatment of parsley suspension cultures with salicylic acid enhances spontaneous and elicited production of H2O2. Plant Physiol 1995, 108, 1171–1178. [Google Scholar]
  23. Katz, V.A.; Thulke, O.U.; Conrath, U. A benzothiadiazole primes parsley cells for augmented elicitation of defense responses. Plant Physiol 1998, 117, 1333–1339. [Google Scholar]
  24. Zimmerli, L.; Jakab, G.; Metraux, J.P.; Mauch-Mani, B. Potentiation of pathogen-specific defense mechanisms in Arabidopsis by beta-aminobutyric acid. Proc. Natl. Acad. Sci. USA 2000, 97, 12920–12925. [Google Scholar]
  25. Ahn, I.P.; Kim, S.; Lee, Y.H.; Suh, S.C. Vitamin B1-induced priming is dependent on hydrogen peroxide and the NPR1 gene in Arabidopsis. Plant Physiol 2007, 143, 838–848. [Google Scholar]
  26. Borges, A.A.; Dobon, A.; Exposito-Rodriguez, M.; Jimenez-Arias, D.; Borges-Perez, A.; Casanas-Sanchez, V.; Perez, J.A.; Luis, J.C.; Tornero, P. Molecular analysis of menadione-induced resistance against biotic stress in Arabidopsis. Plant Biotechnol. J 2009, 7, 744–762. [Google Scholar]
  27. Zhang, S.; Yang, X.; Sun, M.; Sun, F.; Deng, S.; Dong, H. Riboflavin-induced priming for pathogen defense in Arabidopsis thaliana. J. Integr. Plant Biol 2009, 51, 167–174. [Google Scholar]
  28. Eshraghi, L.; Anderson, J.; Aryamanesh, N.; Shearer, B.; McComb, J.; Hardy, G.E.S.; O’Brien, P.A. Phosphite primed defence responses and enhanced expression of defence genes in Arabidopsis thaliana infected with Phytophthora cinnamomi. Plant Pathol 2011, 60, 1086–1095. [Google Scholar]
  29. Massoud, K.; Barchietto, T.; Le Rudulier, T.; Pallandre, L.; Didierlaurent, L.; Garmier, M.; Ambard-Bretteville, F.; Seng, J.M.; Saindrenan, P. Dissecting phosphite-induced priming in Arabidopsis infected with Hyaloperonospora arabidopsidis. Plant Physiol 2012, 159, 286–298. [Google Scholar]
  30. Fujita, M.; Fujita, Y.; Noutoshi, Y.; Takahashi, F.; Narusaka, Y.; Yamaguchi-Shinozaki, K.; Shinozaki, K. Crosstalk between abiotic and biotic stress responses: A current view from the points of convergence in the stress signaling networks. Curr. Opin. Plant Biol 2006, 9, 436–442. [Google Scholar]
  31. Xu, P.; Chen, F.; Mannas, J.P.; Feldman, T.; Sumner, L.W.; Roossinck, M.J. Virus infection improves drought tolerance. New Phytol 2008, 180, 911–921. [Google Scholar]
  32. Sharma, Y.K.; Leon, J.; Raskin, I.; Davis, K.R. Ozone-induced responses in Arabidopsis thaliana: The role of salicylic acid in the accumulation of defense-related transcripts and induced resistance. Proc. Natl. Acad. Sci. USA 1996, 93, 5099–5104. [Google Scholar]
  33. Yalpani, N.; Enyedi, A.J.; Leon, J.; Raskin, I. UV light and ozone stimulate accumulation of salicylic acid, pathogenesis-related protein and virus resistance in tobacco. Planta 1994, 193, 372–376. [Google Scholar]
  34. Lamb, C.; Dixon, R.A. The oxidative burst in plant disease resistance. Annu. Rev. Plant Physiol. Plant Mol. Biol 1997, 48, 251–275. [Google Scholar]
  35. Apel, K.; Hirt, H. Reactive oxygen species: Metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol 2004, 55, 373–399. [Google Scholar]
  36. Torres, M.A.; Dangl, J.L. Functions of the respiratory burst oxidase in biotic interactions, abiotic stress and development. Curr. Opin. Plant Biol 2005, 8, 397–403. [Google Scholar]
  37. Laloi, C.; Apel, K.; Danon, A. Reactive oxygen signalling: The latest news. Curr. Opin. Plant Biol 2004, 7, 323–328. [Google Scholar]
  38. Guan, L.M.; Zhao, J.; Scandalios, J.G. Cis-elements and trans-factors that regulate expression of the maize Cat1 antioxidant gene in response to ABA and osmotic stress: H2O2 is the likely intermediary signaling molecule for the response. Plant J 2000, 22, 87–95. [Google Scholar]
  39. Doke, N. Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthora infestans and to the hyphal wall components. Physiol. Plant Pathol 1983, 23, 345–357. [Google Scholar]
  40. Auh, C.K.; Murphy, T.M. Plasma membrane redox enzyme is involved in the synthesis of O2− and H2O2 by phytophthora elicitor-stimulated rose cells. Plant Physiol 1995, 107, 1241–1247. [Google Scholar]
  41. Grant, M.; Brown, I.; Adams, S.; Knight, M.; Ainslie, A.; Mansfield, J. The RPM1 plant disease resistance gene facilitates a rapid and sustained increase in cytosolic calcium that is necessary for the oxidative burst and hypersensitive cell death. Plant J 2000, 23, 441–450. [Google Scholar]
  42. Zhang, A.; Jiang, M.; Zhang, J.; Tan, M.; Hu, X. Mitogen-activated protein kinase is involved in abscisic acid-induced antioxidant defense and acts downstream of reactive oxygen species production in leaves of maize plants. Plant Physiol 2006, 141, 475–487. [Google Scholar]
  43. Davies, W.J.; Zhang, J.H. Root signals and the regulation of growth and development of plants in drying soil. Annu. Rev. Plant Physiol. Plant Mol. Biol 1991, 42, 55–76. [Google Scholar]
  44. Westgate, M.E.; Boyer, J.S. Osmotic adjustment and the inhibition of leaf, root, stem and silk growth at low water potentials in maize. Planta 1985, 164, 540–549. [Google Scholar]
  45. Sharp, R.E.; Davies, W.J. Regulation of growth and development of plants growing with a restricted supply of water. In Plants Under Stress; Jones, H.G., Flowers, T.J., Jones, M.B., Eds.; Cambridge University Press: Cambridge, UK, 1989; pp. 71–93. [Google Scholar]
  46. Williams, B.; Kabbage, M.; Kim, H.J.; Britt, R.; Dickman, M.B. Tipping the balance: Sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment. PLoS Pathog 2011, 7, e1002107. [Google Scholar]
  47. Achuo, E.A.; Prinsen, E.; Höfte, M. Influence of drought, salt stress and abscisic acid on the resistance of tomato to Botrytis cinerea and Oidium neolycopersici. Plant Pathol 2006, 55, 178–186. [Google Scholar]
  48. Salle, A.; Ye, H.; Yart, A.; Lieutier, F. Seasonal water stress and the resistance of Pinus yunnanensis to a bark-beetle-associated fungus. Tree Physiol 2008, 28, 679–687. [Google Scholar]
  49. Gachomo, E.W.; Kotchoni, S.O. Extract from drought-stress leaves enhances disease resistance through induction of pathogenesis related proteins and accumulation of reactive molecules. Biotechnology 2008, 7, 273–279. [Google Scholar]
  50. Rojas, C.M.; Senthil-Kumar, M.; Wang, K.; Ryu, C.M.; Kaundal, A.; Mysore, K.S. Glycolate oxidase modulates reactive oxygen species-mediated signal transduction during nonhost resistance in Nicotiana benthamiana and Arabidopsis. Plant Cell 2012, 24, 336–352. [Google Scholar]
  51. Taguchi, F.; Suzuki, T.; Inagaki, Y.; Toyoda, K.; Shiraishi, T.; Ichinose, Y. The siderophore pyoverdine of Pseudomonas Syringae pv. tabaci 6605 is an intrinsic virulence factor in host tobacco infection. J. Bacteriol 2010, 192, 117–126. [Google Scholar]
  52. Wang, K.; Uppalapati, S.R.; Zhu, X.; Dinesh-Kumar, S.P.; Mysore, K.S. SGT1 positively regulates the process of plant cell death during both compatible and incompatible plant-pathogen interactions. Mol. Plant Pathol 2010, 11, 597–611. [Google Scholar]
  53. Uppalapati, S.R.; Ishiga, Y.; Ryu, C.M.; Ishiga, T.; Wang, K.; Noël, L.D.; Parker, J.E.; Mysore, K.S. SGT1 contributes to coronatine signaling and Pseudomonas syringae pv. tomato disease symptom development in tomato and Arabidopsis. New Phytol 2011, 189, 83–93. [Google Scholar]
  54. Asselbergh, B.; De Vleesschauwer, D.; Hofte, M. Global switches and fine-tuning-ABA modulates plant pathogen defense. Mol. Plant Microbe Interact 2008, 21, 709–719. [Google Scholar]
  55. Yasuda, M.; Ishikawa, A.; Jikumaru, Y.; Seki, M.; Umezawa, T.; Asami, T.; Maruyama-Nakashita, A.; Kudo, T.; Shinozaki, K.; Yoshida, S.; et al. Antagonistic interaction between systemic acquired resistance and the abscisic acid-mediated abiotic stress response in Arabidopsis. Plant Cell 2008, 20, 1678–1692. [Google Scholar]
  56. Kusajima, M.; Yasuda, M.; Kawashima, A.; Nojiri, H.; Yamane, H.; Nakajima, M.; Akutsu, K.; Nakashita, H. Suppressive effect of abscisic acid on systemic acquired resistance in tobacco plants. J. Gen. Plant Pathol 2010, 76, 161–167. [Google Scholar]
  57. Anderson, J.P.; Badruzsaufari, E.; Schenk, P.M.; Manners, J.M.; Desmond, O.J.; Ehlert, C.; Maclean, D.J.; Ebert, P.R.; Kazan, K. Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis. Plant Cell 2004, 16, 3460–3479. [Google Scholar]
  58. Asselbergh, B.; Curvers, K.; Franca, S.C.; Audenaert, K.; Vuylsteke, M.; Van Breusegem, F.; Hofte, M. Resistance to Botrytis cinerea in sitiens, an abscisic acid-deficient tomato mutant, involves timely production of hydrogen peroxide and cell wall modifications in the epidermis. Plant Physiol 2007, 144, 1863–1877. [Google Scholar]
  59. Ayres, P.G. The interaction between environmental stress injury and biotic disease physiology. Annu. Rev. Phytopathol 1984, 22, 53–75. [Google Scholar]
  60. Ton, J.; Flors, V.; Mauch-Mani, B. The multifaceted role of ABA in disease resistance. Trends Plant Sci 2009, 14, 310–317. [Google Scholar]
  61. Miller, G.; Suzuki, N.; Ciftci-Yilmaz, S.; Mittler, R. Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant Cell Environ 2010, 33, 453–467. [Google Scholar]
  62. Wang, K.; Kang, L.; Anand, A.; Lazarovits, G.; Mysore, K.S. Monitoring in planta bacterial infection at both cellular and whole-plant levels using the green fluorescent protein variant GFPuv. New Phytol 2007, 174, 212–223. [Google Scholar]
  63. Mehler, A.H. Studies on reactions of illuminated chloroplasts. I. Mechanism of the reduction of oxygen and other Hill reagents. Arch. Biochem. Biophys 1951, 33, 65–77. [Google Scholar]
  64. Martin, G.B. Functional analysis of plant disease resistance genes and their downstream effectors. Curr. Opin. Plant Biol 1999, 2, 273–279. [Google Scholar]
  65. Glazebrook, J. Genes controlling expression of defense responses in Arabidopsis—2001 status. Curr. Opin. Plant Biol 2001, 4, 301–308. [Google Scholar]
  66. Hammond-Kosack, K.E.; Jones, J.D. Resistance gene-dependent plant defense responses. Plant Cell 1996, 8, 1773–1791. [Google Scholar]
  67. Penninckx, I.A.; Eggermont, K.; Terras, F.R.; Thomma, B.P.; De Samblanx, G.W.; Buchala, A.; Métraux, J.P.; Manners, J.M.; Broekaert, W.F. Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway. Plant Cell 1996, 8, 2309–2323. [Google Scholar]
  68. Zhu, B.; Chen, T.H.; Li, P.H. Expression of three osmotin-like protein genes in response to osmotic stress and fungal infection in potato. Plant Mol. Biol 1995, 28, 17–26. [Google Scholar]
  69. Ingram, J.; Bartels, D. The molecular basis of dehydration tolerance in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol 1996, 47, 377–403. [Google Scholar]
  70. Ramegowda, V.; Senthil-Kumar, M.; Nataraja, K.N.; Reddy, M.K.; Mysore, K.S.; Udayakumar, M. Expression of a finger millet transcription factor, EcNAC1, in tobacco confers abiotic stress-tolerance. PLoS One 2012, 7, e40397. [Google Scholar]
  71. Flower, D.J.; Ludlow, M.M. Contribution of osmotic adjustment to the dehydration tolerance of water-stressed pigeonpea (Cajanus cajan (L.) millsp.) leaves. Plant Cell Environ 1986, 9, 33–40. [Google Scholar]
  72. Sutherland, M.W.; Learmonth, B.A. The tetrazolium dyes MTS and XTT provide new quantitative assays for superoxide and superoxide dismutase. Free Radic. Res 1997, 27, 283–289. [Google Scholar]
  73. Schopfer, P.; Plachy, C.; Frahry, G. Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin, and abscisic acid. Plant Physiol 2001, 125, 1591–1602. [Google Scholar]
  74. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods 2001, 25, 402–408. [Google Scholar]
Ijms 14 09497f1 1024
Figure 1. Drought acclimation of N. benthamiana plants. Drought stress was imposed on five-week-old plants following gravimetric approach. After all sets of plants reached specific FC’s, they were continued to be maintained at that level for five more days for acclimation (a). Stress levels were confirmed by stress-induced changes in relative water content (RWC) (b); ABA (c) and root-shoot ratio (d) at the end of acclimation period. Each bars represents standard error (n = 6). The one-way analysis of variance (ANOVA) was performed (p = 0.05), and letters above the data points indicate the significance and data points with the same letters are not significantly different.
Figure 1. Drought acclimation of N. benthamiana plants. Drought stress was imposed on five-week-old plants following gravimetric approach. After all sets of plants reached specific FC’s, they were continued to be maintained at that level for five more days for acclimation (a). Stress levels were confirmed by stress-induced changes in relative water content (RWC) (b); ABA (c) and root-shoot ratio (d) at the end of acclimation period. Each bars represents standard error (n = 6). The one-way analysis of variance (ANOVA) was performed (p = 0.05), and letters above the data points indicate the significance and data points with the same letters are not significantly different.
Ijms 14 09497f1
Ijms 14 09497f2 1024
Figure 2. Sclerotinia sclerotiorum-induced cell death on N. benthamiana plants. Leaves of control and drought acclimated plants were inoculated with potato dextrose agar (PDA) plugs with actively growing S. sclerotiorum cultures. Photographs were taken at 4 dpi (a) and necrotic area was visually scored 0 (no cell death) to 4 (severe cell death) and expressed as percent of each score (b). Five independent plants were analyzed for each treatment (each with 30 inoculation spots). Experiments were repeated twice with reproducible results.
Figure 2. Sclerotinia sclerotiorum-induced cell death on N. benthamiana plants. Leaves of control and drought acclimated plants were inoculated with potato dextrose agar (PDA) plugs with actively growing S. sclerotiorum cultures. Photographs were taken at 4 dpi (a) and necrotic area was visually scored 0 (no cell death) to 4 (severe cell death) and expressed as percent of each score (b). Five independent plants were analyzed for each treatment (each with 30 inoculation spots). Experiments were repeated twice with reproducible results.
Ijms 14 09497f2
Ijms 14 09497f3 1024
Figure 3. Disease-induced cell death in N. benthamina plants inoculated with Pseudomonas syringae pv. tabaci. Leaves of control and drought acclimated plants were inoculated with P. syringae pv. tabaci and pathogen-induced cell death was visually scored 0 (no cell death) to 5 (100% cell death at the inoculated spot) at 5 dpi. Three independent plants were analyzed for each treatment (each plant with 5 inoculation spots). Experiments were repeated twice with reproducible results.
Figure 3. Disease-induced cell death in N. benthamina plants inoculated with Pseudomonas syringae pv. tabaci. Leaves of control and drought acclimated plants were inoculated with P. syringae pv. tabaci and pathogen-induced cell death was visually scored 0 (no cell death) to 5 (100% cell death at the inoculated spot) at 5 dpi. Three independent plants were analyzed for each treatment (each plant with 5 inoculation spots). Experiments were repeated twice with reproducible results.
Ijms 14 09497f3
Ijms 14 09497f4a 1024Ijms 14 09497f4b 1024
Figure 4. ROS levels in drought acclimated N. benthamiana plants. The ROS contents were measured in plants maintained at different FC’s at the end of stress period. The O2− (a) and H2O2 (b) were quantified by XTT and scopoletin assay, respectively. Each bars represent the standard error (n = 6). The one-way analysis of variance (ANOVA) was performed (p = 0.05), and letters above the data points indicate the significance and data points with the same letters are not significantly different. (c) Visualization of bacterial growth in methyl viologen (MV) treated N. benthamiana leaves infected with Pseudomonas syringae pv. tabaci. Plants were sprayed with ROS inducer, MV, followed by infection with GFPuv expressing P. syringae pv. tabaci. Photographs were taken under UV light at 4 dpi.
Figure 4. ROS levels in drought acclimated N. benthamiana plants. The ROS contents were measured in plants maintained at different FC’s at the end of stress period. The O2− (a) and H2O2 (b) were quantified by XTT and scopoletin assay, respectively. Each bars represent the standard error (n = 6). The one-way analysis of variance (ANOVA) was performed (p = 0.05), and letters above the data points indicate the significance and data points with the same letters are not significantly different. (c) Visualization of bacterial growth in methyl viologen (MV) treated N. benthamiana leaves infected with Pseudomonas syringae pv. tabaci. Plants were sprayed with ROS inducer, MV, followed by infection with GFPuv expressing P. syringae pv. tabaci. Photographs were taken under UV light at 4 dpi.
Ijms 14 09497f4aIjms 14 09497f4b
Ijms 14 09497f5 1024
Figure 5. Increased expression of plant defense genes in drought acclimated plants. The relative mRNA expression levels of PR-5 (a) and PDF1.2 (b) were quantified by qRT-PCR in drought acclimated and control plants (100% FC). The fold change values were calculated using the 2−ΔΔCT method and represented as changes in mRNA levels relative to 100% FC. NbActin was used as an internal control to normalize gene expression levels. The data are averages of two biologically independent experiments each consisting of three technical replicates.
Figure 5. Increased expression of plant defense genes in drought acclimated plants. The relative mRNA expression levels of PR-5 (a) and PDF1.2 (b) were quantified by qRT-PCR in drought acclimated and control plants (100% FC). The fold change values were calculated using the 2−ΔΔCT method and represented as changes in mRNA levels relative to 100% FC. NbActin was used as an internal control to normalize gene expression levels. The data are averages of two biologically independent experiments each consisting of three technical replicates.
Ijms 14 09497f5

Share and Cite

MDPI and ACS Style

Ramegowda, V.; Senthil-Kumar, M.; Ishiga, Y.; Kaundal, A.; Udayakumar, M.; Mysore, K.S. Drought Stress Acclimation Imparts Tolerance to Sclerotinia sclerotiorum and Pseudomonas syringae in Nicotiana benthamiana. Int. J. Mol. Sci. 2013, 14, 9497-9513. https://doi.org/10.3390/ijms14059497

AMA Style

Ramegowda V, Senthil-Kumar M, Ishiga Y, Kaundal A, Udayakumar M, Mysore KS. Drought Stress Acclimation Imparts Tolerance to Sclerotinia sclerotiorum and Pseudomonas syringae in Nicotiana benthamiana. International Journal of Molecular Sciences. 2013; 14(5):9497-9513. https://doi.org/10.3390/ijms14059497

Chicago/Turabian Style

Ramegowda, Venkategowda, Muthappa Senthil-Kumar, Yasuhiro Ishiga, Amita Kaundal, Makarla Udayakumar, and Kirankumar S. Mysore. 2013. "Drought Stress Acclimation Imparts Tolerance to Sclerotinia sclerotiorum and Pseudomonas syringae in Nicotiana benthamiana" International Journal of Molecular Sciences 14, no. 5: 9497-9513. https://doi.org/10.3390/ijms14059497

Article Metrics

Back to TopTop