Rutin-Mediated Priming of Plant Resistance to Three Bacterial Pathogens Initiating the Early SA Signal Pathway

Flavonoids are ubiquitous in the plant kingdom and have many diverse functions, including UV protection, auxin transport inhibition, allelopathy, flower coloring and insect resistance. Here we show that rutin, a proud member of the flavonoid family, could be functional as an activator to improve plant disease resistances. Three plant species pretreated with 2 mM rutin were found to enhance resistance to Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Pseudomonas syringae pv. tomato strain DC3000 in rice, tobacco and Arabidopsis thaliana respectively. While they were normally propagated on the cultural medium supplemented with 2 mM rutin for those pathogenic bacteria. The enhanced resistance was associated with primed expression of several pathogenesis-related genes. We also demonstrated that the rutin-mediated priming resistance was attenuated in npr1, eds1, eds5, pad4-1, ndr1 mutants, and NahG transgenic Arabidopsis plant, while not in either snc1-11, ein2-5 or jar1 mutants. We concluded that the rutin-priming defense signal was modulated by the salicylic acid (SA)-dependent pathway from an early stage upstream of NDR1 and EDS1.


Introduction
Flavonoids belong to an important class of secondary metabolites in plants, which can be divided into several subgroups by the diversity of chemical radical groups [1]. They exhibit broad biological functions including defense (antibacterial activity), UV protection, auxin transport inhibition, allelopathy, energy transfer, control of respiration and photosynthesis and flower coloring in plant [2]. Rutin is one of the huge families of flavoniods which was broadly distributed in fruits, vegetables and other plant food sources [3,4]. Even in tobacco leaves, the content of rutin is approximately reached to 80 μg g -1 fresh weight [5]. Rutin has anti-inflammatory and strong antioxidant properties too. It was reported to attach to metal ions and phase. Cells were collected by centrifugation and resuspended in distilled water to form a gradient concentration of 10 6 , 10 7 and 10 8 CFU ml -1 (equal to approximate 0.2 OD). The bacterial suspensions were grown on PSA solid medium containing 0 to 4 mM rutin which purchased from Sangon Biotech (Shanghai, CN), at 28°C for 24 h.
Pathogenic bacteria Ralstonia solanacearum SD (Isolated from Shandong Province, China at the year of 2011) and Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) were cultured as described above and the medium were replaced with Nutrient Agar (NA) medium and King's B (KB) medium, respectively. All the pathogens were only studied in the lab and greenhouse. There has no specific permissions were required for these locations/activities.

Plant material and pathogen inoculation
Rice Mudanjiang 8 (Oryzae sativa cv. japonica) plants were grown in the greenhouse at 28°C, 70% relative humidity and with a 12 h photoperiod. Each of five plants at booting stage were sprayed with a solution containing different concentrations of rutin diluted in distilled water, which was supplemented with 0.02% Tween 20. The control plants were sprayed with 0.02% Tween 20 only. The plants were inoculated with PXO99 (Philippine race 6) by the leaf clipping method after three days of pre-spraying with rutin, as described previously [24]. The disease was scored by measuring the lesion length at 7 and 14 days after inoculation. The results show average values of triple experiments.
N. benthamiana were grown in the greenhouse under a 16 h light/8 h dark cycle at 25°C, with 70% relative humidity. Eight-week-old plants were sprayed with different concentrations of rutin diluted in distilled water containing with 0.02% Tween 20. The control plants were sprayed with 0.02% Tween 20. The plants were inoculated with 10 8 CFU ml -1 of R. solanacearum SD through hypodermic injection with a syringe after three days of treatment [25]. Different growth stages of R. solanacearum SD were detected after inoculation to draw the growth curve. Bacteria in leaves was counted by determining the CFU of 1 g leaves (fresh weight) either pretreated or untreated with rutin on NA medium [20]. At least three plants for each time point were inoculated through leaf injection with the bacterial suspension. The same experiment was repeated in triplicate at the greenhouse.

RNA extraction and qRT-PCR
Total RNA was isolated from 100 mg plant tissue with TRI reagent according to the manufacturer's instructions (T9424, Sigma-Aldrich, USA). 0.5 μg RNA was used for first-strand cDNA synthesis using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, CN). Quantitative PCR was performed with SYBR 1 Premix Ex Taq™ (Tli RNaseH Plus) (Takara, Dalian, CN) on the IQ5 Real-Time PCR System (Bio-Rad, USA). The following PCR program from the reference was used [24]: 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 55°C for 15 s, and 72°C for 30 s. A heat dissociation curve (55-95°C) following the final cycle of the PCR was checked to test the specificity of the PCR amplification. OsActin of rice, NbEF1α of tobacco and AtActin2 of Arabidopsis were used as internal control to standardize the results. We used NCBI database to get the gene sequences and Primer Premier 5 to design the primers. For each gene, qRT-PCR assays were repeated at least twice with triplicates runs. Relative expression levels were measured using the 2 -⊿⊿Ct analysis method. The sequence of each primer for all detected genes is listed in Table 1.

Data treatment
All experiments were performed in three replicates with similar results. Each replicate contained at least three plants. The quantitative data were performed by Student's t test (two-tail t

Results
Rutin has limited antibacterial action to all tested bacterial pathogens at 2 mM or lower concentration To evaluate the effects of rutin against bacterial pathogens, four strains represented as three bacteria species were growth on cultural medium plus with different concentration of rutin. Among four strains, PXO99 and RH3 were belong to typical strain of Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), they were shown no clear growth inhibition on PSA contains either 0.5 mM, 1.0 mM or 2mM rutin as well as R. Solanacearum SD and Pst DC3000 (Fig 1). The growth inhibition of all four pathogens was only observed with 4 mM rutin supplemented in PSA when their inoculum titrations were lower as 10 6 CFU ml -1 . Compared with rutin, the quercetin demonstrated a better growth inhibition capacity to all four tested pathogens (S1 Fig). These results were indicative of limited antibacterial ability of rutin for tested bacteria.

Rutin promoted resistance against Ralstonia solanacearum in Nicotiana benthamiana
Previous studies described that AtMYB12-overexpressing tobacco was resistant against R. solanacearum SD as well as enriched rutin. To test whether rutin could directly activated the plant resistance, in this study, we investigated the effect of rutin on the defense response against R. solanacearum SD in N. benthamiana. Most leaves in the control group showed water-soaked symptoms, wilted post three days inoculation, as shown in Fig 2a. However, the wilted symptoms were attenuated in N. benthamiana leaves when it was pretreated with rutin from 1 mM to 4 mM. The stronger attenuation disease symptoms were observed to associate with rutin concentration on inoculation plants (Fig 2a). In addition, rutin hardly inhibit bacteria growth at a concentration of 2 mM in the cultural medium (Fig 1). Therefore, this concentration was chosen for subsequent experiments. The bacterial growth curve indicated that pre-sprayed rutin could remarkably protect N. benthamiana from R. solanacearum SD infection at a concentration of 2 mM (Fig 2b). Compared to pretreatment with 2 mM rutin, more than 4.82 folds of bacteria had been evaluated on control plant at 48 hpi (Fig 2b). Additionally, in spite of less antibacterial ability than quercetin, pretreated with 2 mM rutin presented better resistance to R. solanacearum SD (S2a Fig).  We also analyzed the transcription level of PR genes: NbPR1a, NbNOA1 (nitric oxide-associated 1) which is related to NO production and to defense responses [27], and NbrbohB (respiratory burst oxidase homologs B) which is related in active oxygen species generation [28]. Without inoculation of R. Solanacearum SD, the transcription level of NbPR1a was slightly upregulated one day after spraying with rutin and turn to down-regulation at 3 dpi compared with spraying with water in N. benthamiana (Fig 2c). So we selected three days as the interval time between spraying rutin and R. solanacearum SD inoculation to balance the weak activation defense caused by spraying rutin. We observed there was a more rapid and strong increased expression levels of PR genes, including NbPR1a, NbNOA1 and NbrbohB in rutinpretreated plants than in control plants when R. solanacearum SD was inoculated (Fig 2d). The transcript levels reached their maximum value at 6 hpi for NbNOA1 (7.26-fold highter than the control) and NbrbohB (3.33-fold highter than the control), and 24 hpi for NbPR1a (2.44-fold highter than the control) in rutin-pretreated leaves, respectively. These results suggested that rutin primed the expressing activation of several PR genes in challenged N. benthamiana.
Suppressed the proliferation of Xanthomonas oryzae pv. oryzae by prespaying rutin on rice To test whether rutin could enhance resistance against bacterial pathogen in other host, we have evaluated the efficacy of rutin against PXO99 which caused bacterial blight disease in rice. The plants were inoculated with PXO99 three days later after sprayed with different concentrations of rutin as 1 mM, 2mM and 4 mM respectively. The lesion length of rice leaves was measured post 14 days inoculation. It was averaged to 14.32 ± 3.75 cm for control plant which pre-spraying with 0.02% Tween 20 only. And the average lesion length was reduced to 9.76 ± 2.65 cm, 7.79 ± 2.19 cm and 6.94±0.57 cm for pretreatment with 1 mM, 2mM and 4 mM of rutin respectively (Fig 3a). Statistical data also suggested that the lesions caused by PXO99 were suppressed in rutin-pretreated Mudanjiang 8 (Fig 3a). As 2 mM rutin inhibited little or nothing to PXO99 in vitro, and it has dramatically reduced the lesion length in rice after pre-spraying 2 mM rutin (Fig 1), therefore, we chose 2 mM rutin for subsequent experiments. Capture our attentions, compared with the control, the lesion length was dramatically reduced for rutin pre-spraying rice leaves since 7 d-post-inoculation (Fig 3b). Interestingly, the reductive lesion length was almost similar with each other between rutin-and quercetin-pretreated leaves at 14 dpi (S2b Fig). To investigate whether the pre-spraying 2 mM rutin affected the proliferation of PXO99 in rice leaves or not, we conducted a growth curve experiments in rice. Compared to spray 0.02% Tween 20 only, the number of colonies from spraying rutin leaves has no clear difference post 2 days inoculation, while it was regarding to 5.01 times and 26.92 times reduction at 4 dpi and 6 dpi, respectively (Fig 3c). These results were suggested that pretreated with rutin could enhance rice to against PXO99.
Because enhanced plant disease resistance is usually related to the expression of PR genes, to elucidate the rutin-mediated resistance, the expression pattern of several PR genes was investigated in rice. The results demonstrated that all six PR genes included PR-1a, PR-1b, PR-10, phenylalanine ammonia lyase (PAL), peroxidase (POX) and LOX genes were up-regulated expression after inoculation with PXO99 both for pre-spraying with 0.02% Tween 20 and 2 mM rutin. But the expression of chloramphenicol acetyl transferase (CAT) was not significant changed post rutin treatment compared to the control. (Fig 3d). The expression levels of PR- 1a, PR-1b PR-10 and POX genes were all reached their maximum levels at the 12 h post inoculation, and were approximately 4.98-, 5.05-, 3.39-fold and 4.23-fold higher than the control, respectively. The maximum transcription level of the PAL gene was obtained at 24 h post inoculation. The transcription of LOX was also induced more highly in treated plants compared to the control plants after inoculation. It was reached high values at the 12 h and 48 h time points after the initiation of inoculation which was approximately 5.71-and 8.23-folds higher than the control plants, respectively.

Enhanced the resistance against to Pst DC3000 in Arabidopsis thaliana
In addition to the above mentioned rice-Xoo and N. benthamiana-R. solanacearum investigation, we also tested the function of rutin in Arabidopsis thaliana. The results demonstrated that rutin also protected susceptible Arabidopsis ecotype Columbia-0 (Col-0) against the virulent Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000). After inoculation with Pst DC3000, typical wilting and chlorotic symptoms was observed on the leaves without pre-spraying with rutin at 3 dpi. However, attenuated disease symptoms were observed on Arabidopsis leaves pre-sprayed with 1, 2 and 4 mM rutin (Fig 4a). The proliferation of Pst DC3000 was indicated that the growth had been inhibited in leaves which were pretreated wtih 2 mM rutin (Fig 4b). More than 111.78 folds of Pst DC3000 had been identified in control leaves.
To understand the mechanisms involved in rutin-mediated resistance in Arabidopsis, we analyzed the expression patterns of four PR genes including AtPR1, AtPR2, AtPR5, and AtPAL, which are involved in defense responses to pathogen attack (Fig 4c). Similar with our observation in N. benthamiana-R. solanacearum and rice-Xoo interactions, four PR genes was shown more rapidly and stronger expressing activation in rutin-pretreated plants than in control plants after inoculation with Pst DC3000 (Fig 4c). These results suggested that rutin had the function of primed resistance in a broadly range of host, including Arabidopsis.

Rutin-mediated priming is dependent on the SA signal pathway in Arabidopsis
Plant hormones were known as the signals of plant defense. To explore the resistance signal transduction pathway mediated by rutin, a set of Arabidopsis mutants were used for investigation which was involved in SA, JA and ethylene (ET) dependent pathway. The NahG transgenic plant abolishes the accumulation of SA, and the Arabidopsis mutant npr1 was a typical mutant of SA-dependent pathway. jar1-1 and ein2-5 were typical mutants of JA-and ET-dependent pathway. If the rutin-mediated priming defense is dependent on one of them, the inhibition growth of Pst DC3000 by pretreatment with 2 mM rutin will be attenuated. The results demonstrated that it was still able to inhibit the growth of Pst DC3000 in jar1-1 and in ein2-5, while not in npr1-1 and NahG plants (Fig 5). It was indicated that the rutin-mediated plant resistance is dependent on the SA signal pathway in Arabidopsis and independent on the JA and ET pathways.

Rutin-mediated signaling initiated from upstream of NDR1, PAD4 and EDS1
To obtain more details about the signals of rutin-mediated resistance, we had investigated the growth of Pst DC3000 in several other mutants involved in SA signaling pathway, including with snc1-11, pad4-1, ndr1, eds5 and eds1 [29]. SNC1 is encoded an interleukin-1 receptor-like nucleotide-binding site leucine-rich repeat type of resistance (R)-like gene residing in the RPP5 gene cluster which possibly mediates race-specific disease resistance [30][31][32]. EDS1 and PAD4 belong to two lipase-like proteins [33], NDR1 is a putative membrane-binding protein [34] and EDS5 is an MATE-like SA transporter which pumped the SA from the chloroplast to the cytoplasm [35]. These proteins belong to three upstream components responsible for the transduction of SA signals and for downstream pathways triggered by the R protein. Except in snc1-11, the inhibition growth of Pst DC3000 by pretreatment with 2 mM rutin had been attenuated in pad4-1, ndr1, eds 5 and eds1 (Fig 6). These results were further suggested that the rutinmediated resistance was dependent on the SA signal pathway, which was initiated upstream of NDR1, PAD4 and EDS1.

Discussion
Rutin, classified as a polyphenolic substance, had also shown to exhibit bactericidal and fungicidal activity in vitro assay. The antibacterial activity of rutin was reported to specific bacteria species, such as Xanthomonas campestris, Agrobacterium tumefaciens, Xylella fastidiosa etc [19,20]. The possible mechanism of action is presumably as follows: first, such polyphenolic substances most likely disrupt the cell wall and the cell membrane integrity of microbial cells, which leads to the release of intracellular components and causes the electron transfer at the membrane, the repression of nucleotide synthesis and ATP activity, thereby inhibiting the growth of microorganisms [36]; Second, rutin excessively scavenges the reactive oxygen species of microbes, leading to a reduction in the normal physiological function of reactive oxygen [37]. But rutin were effective in inhabiting bacteria causing the plant disease at relative high minimum inhibitory activity (MIC) which means to weaker bactericidal activity than other phenolic compounds [19,20]. In this study, we have measured the inhibition efficiency of rutin against four plant bacterial pathogens, including R. solanacearum SD, Xanthomonas oryzae pv. oryzae (PXO99), Xanthomonas oryzae pv. oryzicola (RH3) and Pst DC3000. The results demonstrated that rutin was only functional in very high concentration over 4 mM (Fig 1). Our other study demonstrated that the AtMYB12-overexpressing tobacco had approximately enriched the averaged concentration of rutin as 1.43 mM in fresh weight. It was also enhanced resistance against R. solanacearum (Li et al., unpublished data). Together, the conclusions of this work were consistent with previous studies that rutin has demonstrated weak antibacterial activity to against three additional species of plant gram-negative bacterial pathogens.
In vitro assays, 2 mM rutin hardly inhibit the growth of R. solanacearum SD, PXO99 and Pst DC3000 in medium. Causing we hardly quantify the concentration of rutin for intercellular space, we couldn't completely eliminate the direct inhibition caused by the antibacterial agent. However, spraying 2 mM of rutin dramatically reduced the growth of those bacteria in each host plant, which implied that other resistance mechanisms had been triggered (Figs 2 and 3). Notably, the foliar application of 2 mM rutin almost rarely affected the expression of the SAresponsive PR1a gene on N. benthamiana (Fig 2c). This was indicated that rutin couldn't directly activate the basal plant defense. Interestingly, when challenged with a pathogen, the plants pre-spraying rutin show a faster and stronger expression of PR1a than control as well as other PR genes (Figs 2d, 3d and 4c). The delay of occurrence resistance was indicated that rutin promotes disease resistance by a priming mechanism. In addition, exogenous application of rutin simultaneously enhanced the expression of genes which involved into SA, reactive oxygen species and nitric oxide signal pathway (Figs 2 and 3), indicating the ownstream signaling activated by rutin was complex.
Many chemicals or plant metabolic components have also been reported to induce or prime plant defense responses that are dependent on the SA signal transduction pathway. However, Fig 6. Analysis of rutin-primed resistance in Arabidopsis mutants. a-f represent as Wild type, eds5, snc1-11, pad4-1, eds1, and ndr1 respectively. The growth rate of Pseudomonas syringae pv tomato strain DC3000 was measured. In total, samples from control and treated plants were measured at 0, 3 days post inoculation. The data were collected from 10 plants. The data showed representative experiments that were repeated three times. The values are means ±SE. The asterisks denote significant differences (t -test, P < 0.01).
doi:10.1371/journal.pone.0146910.g006 most of these studies primarily focused on the characterization of the effects of these components using NahG and npr1 mutants [12,13,15], except for azelaic acid which has been analyzed to induce plant defense responses dependent on NDR1 and PAD4, which are two importance components involved in the upstream signals of SA [14]. Rutin-stimulated plant resistance was compromised in many defective SA pathway mutants, confirming that SA signaling was required for rutin-primed disease resistance (Figs 5 and 6). Our data have also identified that NDR1, PAD4 and EDS1 were required for rutin-primed plant defense (Fig 6). This result implied that rutin-primed plant resistance might slightly differ from other plant activators. NDR1 and EDS1 mediated the signal downstream from the major subsets of R proteins, including the CC-NBS-LRR type and the TIR-NBS-LRR type, and they represent an important node acting upstream of SA in PTI [38,39]. Interestingly, we have determined that snc1-11 mutant did not affect the rutin-primed resistance, given the possibility that rutin may specifically affect the other R proteins or downstream components of the SNC1 and other R protein.
Based on these results, the possible work model for rutin-primed defense is described (Fig 7), which temporally suggests that the resistance signal is initiated upstream from NDR1, PAD4 and EDS1, followed by activating SA signal transduction. Even though we did not decipher the beginning signals or the targeted receptor of rutin in plants, nevertheless, this study it still offers a new research insight into this newly characterized plant activator.
Flavonoids play a critical role in preventing human diseases and have been evolved as a protective mechanism for different plants. In this study, we also found that rutin as a component of flavonoids which could involve into plant immunity with a broad range of host. Together with the quercetin [22], it is feasible suggestion of a conserve mechanism for priming the plant immunity with other components of flavonoids. As rutin was functional at a relative high concentration and economical cost, it is formidable to use directly as a purify bactericide. However, there has increasingly growing of reports that the high content of rutin could be regulated synthesis and accumulated in plant by several transcriptional factors, including with AtMYB11, AtMYB12 and AtMYB111 [23,[40][41][42]. It was provided opportunity to promote the use of rutin by reducing the economic cost in future. Additionally, AtMYB12-expression tobacco was also reported to be resistant to insects, such as aphid, whitefly, Spodoptera litura and Helicoverpa armigera, by the high-level accumulation of rutin [23,40]. And our previous study showed that the flavonol enriched AtMYB12-expression tobacco enhanced the resistance against pathogens, such as R. solanacearum, Colletotrichum nicotianae Averna and Alternaria alternata. The priming resistance identified with rutin would be helpful to understand the resistance generated by AtMYB12-expression tobacco. It was opens the opportunity to make the daily nutrient and biosafety bactericide with overcapacity simultaneously by transgenic method.