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Vitamin B6 contributes to disease resistance against Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea in Arabidopsis thaliana
Journal of Plant Physiology 175 (2015) 21–25
Contents lists available at ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Short Communications
Vitamin B6 contributes to disease resistance against Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea in Arabidopsis thaliana Yafen Zhang, Xiaoyi Jin, Zhigang Ouyang, Xiaohui Li, Bo Liu, Lei Huang, Yongbo Hong, Huijuan Zhang, Fengming Song, Dayong Li ∗ National Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang 310058, China
a r t i c l e
i n f o
Article history: Received 25 January 2014 Received in revised form 26 May 2014 Accepted 24 June 2014 Available online 18 November 2014 Keywords: Arabidopsis thaliana Vitamin B6 Disease resistance Pseudomonas syringae pv. tomato DC3000 Botrytis cinerea
a b s t r a c t Vitamin B6 (VB6) is an important cofactor for numerous enzymatic reactions and plays an important role in abiotic stress tolerance. However, direct molecular evidence supporting a role for VB6 in plant disease resistance remains lacking. In this study, we explored the possible function of VB6 in disease resistance by analyzing disease phenotypes of Arabidopsis mutants with defects in de novo biosynthetic pathway and salvage pathway of VB6 biosynthesis against Pseudomonas syringae pv. tomato (Pst) DC3000 and Botrytis cinerea. Mutations in AtPDX1.2 and AtPDX1.3 genes involved in the de novo pathway, and in AtSOS4 gene involved in the salvage pathway led to increased levels of diseases caused by Pst DC3000 and B. cinerea. The pdx1.2 and pdx1.3 plants had reduced VB6 contents and showed a further reduction in VB6 contents after infection by Pst DC3000 or B. cinerea. Our preliminary results suggest an important role for VB6 in plant disease resistance against different types of pathogens. © 2014 Elsevier GmbH. All rights reserved.
Introduction Vitamin B6 (VB6), a group of water soluble vitamers including pyridoxal (PL), pyridoxine, pyridoxamine and their phosphorylated derivatives functions as cofactors in many enzymatic reactions (Mittenhuber, 2001; Mooney et al., 2009). De novo biosynthesis of VB6 vitamers requires two protein families, PDX1s and PDX2 (Hill and Spenser, 1996; Dong et al., 2004; Wetzel et al., 2004; TambascoStudart et al., 2005, 2007). In addition, all organisms have a salvage pathway, which functions to convert different VB6 vitamer forms by specific enzymes (Drewke and Leistner, 2001; Mooney et al., 2009). In Arabidopsis thaliana, three PDX1 genes (AtPDX1.1, AtPDX1.2 and AtPDX1.3) and one PDX2 gene, AtPDX2, were identified to be involved in the de novo biosynthetic pathway (Tambasco-Studart et al., 2005, 2007; Wagner et al., 2006); while a pyridoxal kinase (AtSOS4), a PNP/PMP oxidase (AtPDX3) and a pyridoxal reductase (AtPLR1) were found to be required for the salvage pathway (Shi et al., 2002; Lum et al., 2002; González et al., 2007; Sang et al., 2007, 2011; Herrero et al., 2011).
Abbreviations: B., cinereaBotrytis cinerea; dpi, days post-inoculation; PL, pyridoxal; PM, pyridoxamine; PN, pyridoxine; Pst, Pseudomonas syringae pv. tomato; ROS, reactive oxygen species; RT-, PCRreverse transcription-PCR; VB6, vitamin B6. ∗ Corresponding author. Tel.: +86 571 88982271; fax: +86 571 88982271. E-mail address: [email protected] (D. Li). http://dx.doi.org/10.1016/j.jplph.2014.06.023 0176-1617/© 2014 Elsevier GmbH. All rights reserved.
Studies have demonstrated that VB6 has antioxidant activity and plays important roles in regulating cellular antioxidant defense in plants (Vanderschuren et al., 2013). Expression of the genes involved in the de novo biosynthetic pathway was up-regulated under certain abiotic stress conditions (Shi et al., 2002; Denslow et al., 2005, 2007; Titiz et al., 2006). Mutations in AtPDX1 genes led to increased sensitivity to salt, osmotic, and high light stress (Chen and Xiong, 2005; Titiz et al., 2006; Havaux et al., 2009) whereas overexpression of PDX gene resulted in increased tolerance to oxidative stress (Leuendorf et al., 2010; Raschke et al., 2011). The pyridoxal kinase and pyridoxal reductase involved in the salvage pathway were shown to play important roles in abiotic stress response (Shi and Zhu, 2002; González et al., 2007; Herrero et al., 2011; Rueschhoff et al., 2013). It was found that expression of tobacco PDX genes was reduced by infection with an incompatible pathogen and excess VB6 injected into tobacco leaves affected the development of a hypersensitive response and disease caused by different pathogens (Denslow et al., 2005). However, direct molecular evidence supporting a role for VB6 in plant biotic defense remains lacking. In the present study, we examined phenotypes of Arabidopsis mutants with defects in the de novo pathway and the salvage pathway of VB6 biosynthesis against infection by Pseudomonas syringae pv. tomato (Pst) DC3000 and Botrytis cinerea and we found that mutations in AtPDX1.2, AtPDX1.3, and AtSOS4 genes led to increased levels of diseases. Our preliminary results suggest an important role for VB6 in plant disease resistance.
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Materials and methods Plant materials and growth conditions Seeds for Arabidopsis thaliana wild type Col-0, sos4-1 (At5g37850, CS24930), pdx1.1 (At2g38230, SALK 024245), pdx1.2 (At3g16050, CS872273) and pdx1.3 (At5g01410, SALK 086418) were obtained from Arabidopsis Biological Resource Center. PCRbased genotyping was carried out for SALK 024245, CS872273 and SALK 086418 lines to screen homozygous plants. Gene-specific primers used were as follow: SALK 024245-LP (AAG GCT CAG GTC TTT CTT TGC)/SALK 024245-RP (CTG AGC TTC AAC GAA ATG ACC), SALK 086418-LP (TGA GTC ACA GCC TGA ACA ATG)/SALK 086418RP (AAC AAA ATC CCA CAC TTT CCC) and CS872273-LP (CTT TTT GTT TCC CAA GCT GC)/CS872273-RP (GCT TAG CTT GGT TAA CCG AGG). T-DNA primers LBa1 (TGG TTC ACG TAG TGG GCC ATC G) for SALK 024245 and SALK 086418 lines and LBa3 (TAG CAT CTG AAT TTC ATA ACC AAT CTC GAT ACA C) for CS872273 line were included as suggested on the T-DNA Express Web site (http://www.signal.salk.edu/tdna FAQs.html). Plants homozygous for the PDX1.1, PDX1.2 or PDX1.3 mutation were used for further analysis. The sos4-1 seeds were obtained as a homozygous line deposited in the Arabidopsis Biological Resource Center by Dr. Jian-Kang Zhu (Shi et al., 2002). Seeds were surface sterilized by incubation for 5 min with 70% ethanol, followed by three washings with water. Plants were grown in mixed soil in a growth room under a 10 h light (100 mol s−1 m−2 photons m−2 s−1 of intensity) and 14 h dark cycle at 22 ± 2 ◦ C with 60% relative humidity. Reverse transcription-PCR (RT-PCR) analysis of gene expression Total RNA was extracted from leaf samples using TRIZOL reagent (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized with 500 ng of total RNA using the SuperScript III Kit (Invitrogen, Shanghai, China). One microliter of the first-strand cDNA reaction and 10 pmol of each primer were used for RT-PCR in a total volume of 25 l. PCR conditions were set as 94 ◦ C 30 s, 54–62 ◦ C 30 s and 72 ◦ C 30 s for 25–35 cycles based on the abundance of transcript for each gene, followed by 5 min of final extension at 72 ◦ C. PCR products were electrophoresed on a 1.2% agarose gel. Gene-specific primers used in RT-PCR were as follow: AtPDX1.1-1F, 5 -GTG AGG AGT GTG AAC GGA GC-3 ; AtPDX1.1-1R, 5 -GCA CAA CCA AAT CAT ACG GC-3 ; AtPDX1.2-1F, 5 -GAT GCA GCT AGG TTG TGA TGG-3 ; AtPDX1.2-1R, 5 -TCC ATT GCA TTC TCC AAT CC-3 ; AtPDX1.3-1F, 5 -ATA ATT TCC GGA TCC CGT TC-3 and AtPDX1.3-1R, 5 -CAT CAT CAT CCA TGT TTC GC-3 . An Arabidopsis Actin gene was used as an internal control to normalize the RT-PCR reactions with a pair of gene-specific primers AtActin-1F (5 -GGC GAT GAA GCT CAA TCC AAA CG-3 ) and AtActin1R (5 -GGT CAC GAC CAG CAA GAT CAA GAC G-3 ). The experiments were repeated independently three times. Disease assays Pst DC3000 was grown overnight in King’s B liquid medium containing 25 g/mL rifampicin at 28 ◦ C with shaking. Bacteria were collected, resuspended in 10 mM MgCl2 and adjusted to OD600 = 0.001. Leaves of 4-week-old plants were inoculated by infiltration with bacterial suspension using 1-mL needless syringes and disease symptoms were observed daily. Leaves from 5 independent plants were collected and used for bacterial titering. Leaf punches (6 mm in diameter) were surface sterilized in 70% ethanol for 10 s, homogenized in 500 L 10 mM MgCl2 solution, and plated on King’s B agar plates containing 100 g/mL rifampicin.
Fig. 1. Characterization of T-DNA insertion mutant lines for AtPDX1.1, AtPDX1.2 and AtPDX1.3. (A) Diagrams showing T-DNA locations in the mutant lines. (B) PCR-based genotyping for homozygous plants using combined gene-specific primers and a TDNA primer. Arrows indicate the homozygous plants. (C) Detection of transcripts for AtPDX1.1, AtPDX1.2 and AtPDX1.3 in the obtained pdx1.1, pdx1.2 and pdx.3 mutant plants by RT-PCR analysis with gene specific primers.
B. cinerea was grown on V8 medium (36% V8 juice, 0.2% CaCO3 and 2% agar) at room temperature. Spores were collected and adjusted to 0.5 × 105 spores/mL in 4% maltose and 1% peptone buffer. For detached leaf disease assays, leaves from at least 10 plants were detached and inoculated by dropping 2.5 L of spore suspension onto leaf surface. Alternatively, 4-week-old plants were inoculated by foliar spraying with spore suspension for measurment of VB6 content changes. The inoculated plants or leaves were covered with a transparent plastic film and kept in a growth chamber under similar condition as for plant growth. Lesion diameters were measured 4 days post-inoculation (dpi) and the fungal growth in planta was measured by analyzing transcript level of B. cinerea BcActinA gene using RT-PCR with Arabidopsis Actin as a control (Wang et al., 2009).
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Fig. 2. Enhanced levels of disease caused by Pst DC3000 in the pdx1.2, pdx1.3 and sos4 plants. (A) Representatives of disease symptom on leaves of wild type and mutant plants. Four-week-old plants were infiltrated with bacterial suspension (OD600 = 0.001) of Pst DC3000 and photos were taken 5 days after inoculation. (B) Bacterial titers in inoculated leaves of the wild-type and mutant plants. Leaf samples were collected at 0, 2 and 5 d after inoculation and bacterial titers in colony forming units (CFU)/cm2 leaf area was measured. Data presented in (B) are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level.
Measurement of VB6 contents VB6 contents were analyzed by a bioassay-based measurement with a yeast strain (ATCC9080) auxotrophic for VB6. Leaf samples were collected from at least 5 individual plants before and after pathogen inoculation and leaf extracts were prepared with a protocol as described previously (Denslow et al., 2005). One milliliter of leaf extract was added to 5 mL of pyridoxol Y medium inoculated with 5 × 106 yeast cells. Yeast growth was measured at 17 h and 24 h with a spectrophotometer at OD540 . A standard curve was made using the same protocol. Total VB6 in leaf extracts was determined based on comparison to the standard curve. Results and discussion The Arabidopsis T-DNA insertion lines with mutations in AtPDX1.1, AtPDX1.2 and AtPDX1.3, three genes involved in the de novo biosynthetic pathway (Tambasco-Studart et al., 2005), and AtSOS4, a pyridoxal kinase gene involved in the salvage pathway (Shi et al., 2002), were used to explore the possible role of VB6 in plant disease resistance. The T-DNA insertions were located in promoter regions of the AtPDX1.1 and AtPDX1.2 genes and in coding region of the AtPDX1.3 gene, respectively (Fig. 1A) and homozygous mutant plants were identified by genotyping with gene-specific primers flanking the insertion sites (Fig. 1B). RT-PCR analysis revealed that no transcripts of AtPDX1.1, AtPDX1.2 and
AtPDX1.3 were detected in the pdx1.1, pdx1.2 and pdx1.3 mutant plants, respectively (Fig. 1C). These data indicate that the pdx1.1, pdx1.2 and pdx1.3 are null mutants of the AtPDX1.1, AtPDX1.2 and AtPDX1.3 genes, respectively. The sos4-1 mutant harbors a point mutation in the AtSOS4 gene (Shi et al., 2002). These homozygous mutant lines were used for the further experiments as described below. We first examined and compared the levels of disease caused by a virulent strain of Pst DC3000 on the wild type Col-0 and the mutant plants. At 5 dpi, chlorotic lesions were observed in the inoculated leaves of all plant genotypes but these chlorotic lesions were much extensive and larger in the inoculated leaves of the pdx1.2, pdx1.3 and sos4 plants than that in the wild type and the pdx1.1 plants (Fig. 1A). The chlorotic lesions and bacterial populations in the inoculated leaves of the wild type and pdx1.1 plants were comparable in all experiments (Fig. 1A), indicating that PDX1.1 might have no role in disease resistance against Pst DC3000. At 2 dpi, the bacterial population in the inoculated leaves of the pdx1.2 plants showed a 10-fold higher over that in the wild type plants, but no significant difference in bacterial populations was observed among the wild type, pdx1.3 and sos4 plants (Fig. 1B). At 5 dpi, the bacterial populations in inoculated leaves of the pdx1.2, pdx1.3 and sos4 plants were 2.04 × 109 , 8.35 × 108 and 4.34 × 108 cfu/cm2 , respectively, resulting in a significant increase in bacterial growth relative to that in the wild-type plants (7.93 × 107 cfu/cm2 )(Fig. 1B). These results indicate that mutations in AtPDX1.2, AtPDX1.3 and AtSOS4
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Fig. 3. Increased level of disease caused by B. cinerea in the pdx1.2, pdx1.3 and sos4 plants. (A and B) Representatives of disease symptom and lesion sizes on leaves of wild type and mutant plants. Four-week-old plants were inoculated by dropping a 2.5 L spore suspension (1 × 105 spores/mL) onto leaves and photos were taken 4 days after inoculation. Data presented in (B) are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level. (C) In planta growth of B. cinerea in wild type and mutant plants after infection. Fungal growth was measured by analyzing expression level of B. cinerea BcActinA gene using RT-PCR with Arabidopsis Actin mRNA as a control. The experiments were repeated three times with similar results.
genes resulted in increased bacterial growth and enhanced disease development, indicating the requirement of AtPDX1.2, AtPDX1.3 and AtSOS4 for resistance against Pst DC3000. We next examined and compared the levels of disease caused by B. cinerea, a necrotrophic fungus causing gray mold disease (Mengiste, 2012), and of in planta fungal growth on the wild type Col-0 and the mutant plants. In our experiments, B. cinerea-caused lesions on detached leaves from the wild type and pdx1.1 plants were similar; however, disease lesions on detached leaves form the pdx1.2, pdx1.3 and sos4 plants were significantly larger than that of the wild type plants (Fig. 2A), showing an increase of 55–75% over that of the wild type plants (Fig. 2B). Analysis of the transcript for the B. cinerea BcActinA gene as an indicative of the fungal growth rate in planta (Benito et al., 1998) confirmed that the pdx1.2, pdx1.3 and sos4 plants were more susceptible to Botrytis infection than the wild type plants. At 1 and 2 dpi, growth of B. cinerea in leaf tissues of the pdx1.2, pdx1.3 and sos4 plants was much higher than that in the wild type plants (Fig. 2C). These data indicate that mutations in AtPDX1.2, AtPDX1.3 and AtSOS4 genes also resulted in increased susceptibility to B. cinerea. We further analyzed the changes of VB6 contents in the wild type, pdx1.2 and pdx1.3 plants before and after inoculation with Pst DC3000 or B. cinerea using the bioassay-based measurement (Denslow et al., 2005). Without pathogen inoculation, the VB6 content in the wild type plants was approximately 1.3 g/g fresh weight (FW)(Fig. 4), which is comparable to the VB6 contents in Col-0 plants determined by HPLC (Leuendorf et al., 2010; Tambasco-Studart et al., 2005; Ristilä et al., 2011). The VB6 contents in the pdx1.2 and pdx1.3 plants were lower than that in the wild type plants, showing reduction of 30% for the pdx1.2 plants and 40% for the pdx1.3 plants as compared to that of the wild type plants (Fig. 4). This is in agreement with a previous observation that the pdx1.3 plants grown in soil or sterile culture medium contained reduced
VB6 contents (Titiz et al., 2006). After infection by Pst DC3000 or B. cinerea, the VB6 contents in the wild type plants were slightly reduced (Fig. 4); however, the VB6 contents in the pdx1.2 and pdx1.3 plants showed a further reduction as compared with those in the wild type plants (Fig. 4). These results, along with the fact that the expression of PDX1 gene in tobacco was down-regulated after infection by an incompatible pathogen P. syringae pv. phaseolicola (Denslow et al., 2005), indicate that reduced biosynthesis and hence reduced VB6 content might be a natural response of plants to pathogenic infection as a part of defense mechanism. VB6 has been shown to be an antioxidant capable of scavenging reactive oxygen species (ROS) such as singlet oxygen and superoxide anion, and hence play important roles in regulating cellular antioxidant defense (Bilski et al., 2000; Chen and Xiong, 2005; Titiz et al., 2006; Denslow et al., 2007; Havaux et al., 2009). ROS are required differentially for defense response against biotrophic/hemibiotrophic pathogens like Pst DC3000 as one of early signaling events and for development of disease caused by necrotrophic fungi such as B. cinerea. Recently, we found that silencing of tomato SlPDX1.2 and SlPDX1.3 genes resulted in increased disease severity caused by B. cinerea, reduced contents of VB6 and decreased cellular antioxidant capacity (Zhang et al., 2014). Therefore, these results further confirm that the function of VB6 in plant defense response is associated with its antioxidant activity as an antioxidant and modulator of ROS (Denslow et al., 2005). However, the relationship between VB6 and ROS in response to pathogen infection becomes an open question to be further elucidated (Fig. 4). Results from experiments with the pdx1.2 and pdx1.3 plants indicate a significant correlation between reduction in VB6 contents and the increased levels of diseases caused by Pst DC3000 and B. cinerea. However, this is not the case for the sos4 plants, which contain higher levels of total VB6 content (González et al., 2007) but showed increased levels of diseases caused by Pst DC3000
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Fig. 4. Reduced VB6 contents in pdx1.2 and pdx1.3 plants before and after infection with Pst DC3000 or B. cinerea. Four-week-old plants were inoculated by spraying with with bacterial suspension (OD600 = 0.001) of Pst DC3000 (A) or spore suspension (1 × 105 spores/mL) of B. cinerea (B) or and leaf samples were collected at 0 and 24 h after inoculation. Total VB6 content was measured by yeast bioassay. Data presented in are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level.
and B. cinerea. Such contradictory was also found in the pdx1.3 and sos4 plants that showed similar abiotic stress responses but had opposite contents of VB6 (Chen and Xiong, 2005; Titiz et al., 2006; Havaux et al., 2009). It is reasonable that the reduced contents of VB6 in pdx1.2 and pdx1.3 plants led to decreased capacity of antioxidant activity, resulting in increased susceptibility to pathogens (Figs. 2 and 3). However, the salvage pathway, in which the SOS4 acts as a key enzyme, functions to convert different VB6 vitamers between each other (McCormick and Chen, 1999). Although the sos4 plants contain higher levels of total VB6 content (González et al., 2007), the content of active VB6 forms might be not changed or even reduced. Further detailed analysis the contents of different VB6 forms in sos4 plants by HPLC will be helpful to clarify this point. Alternatively, it was shown recently that, despite differing VB6 content in whole leaf tissue, both the pdx1.3 and sos4 mutants share a common deficiency in VB6 in chloroplast (Rueschhoff et al., 2013). VB6 deficiency in chloroplasts in the pdx1.3 and sos4 plants can be one of the causes that lead to some of similar phenotypes in response to abiotic stresses and pathogenic infection. Interestingly, silencing of SlSOS4 did not affect the response to tomato plants to infection of B. cinerea (Zhang et al., 2014). Thus, it is possible that the functions of SOS4 and even the salvage pathway of VB6 vary among different plants in response to pathogen infection. Nevertheless, our preliminary data presented in this study provide direct molecular evidence suggesting an important role for VB6 in plant disease resistance against pathogens. However, the mechanism for the function of VB6 in plant defense response needs to be further investigated. Acknowledgements This work was supported by the National Basic Research Program of China (2009CB119005), the National Key Technology R & D Program of China (2011BAD12B04), the Ph.D. Program Foundation for Young Teachers of Ministry of Education of China (no. 20100101120079) and the Scientific Research Fund of Department of Education of Zhejiang Province (no. Y200909712), the National Natural Science Foundation of China (Nos. 31101422).
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Contents lists available at ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Short Communications
Vitamin B6 contributes to disease resistance against Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea in Arabidopsis thaliana Yafen Zhang, Xiaoyi Jin, Zhigang Ouyang, Xiaohui Li, Bo Liu, Lei Huang, Yongbo Hong, Huijuan Zhang, Fengming Song, Dayong Li ∗ National Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang 310058, China
a r t i c l e
i n f o
Article history: Received 25 January 2014 Received in revised form 26 May 2014 Accepted 24 June 2014 Available online 18 November 2014 Keywords: Arabidopsis thaliana Vitamin B6 Disease resistance Pseudomonas syringae pv. tomato DC3000 Botrytis cinerea
a b s t r a c t Vitamin B6 (VB6) is an important cofactor for numerous enzymatic reactions and plays an important role in abiotic stress tolerance. However, direct molecular evidence supporting a role for VB6 in plant disease resistance remains lacking. In this study, we explored the possible function of VB6 in disease resistance by analyzing disease phenotypes of Arabidopsis mutants with defects in de novo biosynthetic pathway and salvage pathway of VB6 biosynthesis against Pseudomonas syringae pv. tomato (Pst) DC3000 and Botrytis cinerea. Mutations in AtPDX1.2 and AtPDX1.3 genes involved in the de novo pathway, and in AtSOS4 gene involved in the salvage pathway led to increased levels of diseases caused by Pst DC3000 and B. cinerea. The pdx1.2 and pdx1.3 plants had reduced VB6 contents and showed a further reduction in VB6 contents after infection by Pst DC3000 or B. cinerea. Our preliminary results suggest an important role for VB6 in plant disease resistance against different types of pathogens. © 2014 Elsevier GmbH. All rights reserved.
Introduction Vitamin B6 (VB6), a group of water soluble vitamers including pyridoxal (PL), pyridoxine, pyridoxamine and their phosphorylated derivatives functions as cofactors in many enzymatic reactions (Mittenhuber, 2001; Mooney et al., 2009). De novo biosynthesis of VB6 vitamers requires two protein families, PDX1s and PDX2 (Hill and Spenser, 1996; Dong et al., 2004; Wetzel et al., 2004; TambascoStudart et al., 2005, 2007). In addition, all organisms have a salvage pathway, which functions to convert different VB6 vitamer forms by specific enzymes (Drewke and Leistner, 2001; Mooney et al., 2009). In Arabidopsis thaliana, three PDX1 genes (AtPDX1.1, AtPDX1.2 and AtPDX1.3) and one PDX2 gene, AtPDX2, were identified to be involved in the de novo biosynthetic pathway (Tambasco-Studart et al., 2005, 2007; Wagner et al., 2006); while a pyridoxal kinase (AtSOS4), a PNP/PMP oxidase (AtPDX3) and a pyridoxal reductase (AtPLR1) were found to be required for the salvage pathway (Shi et al., 2002; Lum et al., 2002; González et al., 2007; Sang et al., 2007, 2011; Herrero et al., 2011).
Abbreviations: B., cinereaBotrytis cinerea; dpi, days post-inoculation; PL, pyridoxal; PM, pyridoxamine; PN, pyridoxine; Pst, Pseudomonas syringae pv. tomato; ROS, reactive oxygen species; RT-, PCRreverse transcription-PCR; VB6, vitamin B6. ∗ Corresponding author. Tel.: +86 571 88982271; fax: +86 571 88982271. E-mail address: [email protected] (D. Li). http://dx.doi.org/10.1016/j.jplph.2014.06.023 0176-1617/© 2014 Elsevier GmbH. All rights reserved.
Studies have demonstrated that VB6 has antioxidant activity and plays important roles in regulating cellular antioxidant defense in plants (Vanderschuren et al., 2013). Expression of the genes involved in the de novo biosynthetic pathway was up-regulated under certain abiotic stress conditions (Shi et al., 2002; Denslow et al., 2005, 2007; Titiz et al., 2006). Mutations in AtPDX1 genes led to increased sensitivity to salt, osmotic, and high light stress (Chen and Xiong, 2005; Titiz et al., 2006; Havaux et al., 2009) whereas overexpression of PDX gene resulted in increased tolerance to oxidative stress (Leuendorf et al., 2010; Raschke et al., 2011). The pyridoxal kinase and pyridoxal reductase involved in the salvage pathway were shown to play important roles in abiotic stress response (Shi and Zhu, 2002; González et al., 2007; Herrero et al., 2011; Rueschhoff et al., 2013). It was found that expression of tobacco PDX genes was reduced by infection with an incompatible pathogen and excess VB6 injected into tobacco leaves affected the development of a hypersensitive response and disease caused by different pathogens (Denslow et al., 2005). However, direct molecular evidence supporting a role for VB6 in plant biotic defense remains lacking. In the present study, we examined phenotypes of Arabidopsis mutants with defects in the de novo pathway and the salvage pathway of VB6 biosynthesis against infection by Pseudomonas syringae pv. tomato (Pst) DC3000 and Botrytis cinerea and we found that mutations in AtPDX1.2, AtPDX1.3, and AtSOS4 genes led to increased levels of diseases. Our preliminary results suggest an important role for VB6 in plant disease resistance.
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Materials and methods Plant materials and growth conditions Seeds for Arabidopsis thaliana wild type Col-0, sos4-1 (At5g37850, CS24930), pdx1.1 (At2g38230, SALK 024245), pdx1.2 (At3g16050, CS872273) and pdx1.3 (At5g01410, SALK 086418) were obtained from Arabidopsis Biological Resource Center. PCRbased genotyping was carried out for SALK 024245, CS872273 and SALK 086418 lines to screen homozygous plants. Gene-specific primers used were as follow: SALK 024245-LP (AAG GCT CAG GTC TTT CTT TGC)/SALK 024245-RP (CTG AGC TTC AAC GAA ATG ACC), SALK 086418-LP (TGA GTC ACA GCC TGA ACA ATG)/SALK 086418RP (AAC AAA ATC CCA CAC TTT CCC) and CS872273-LP (CTT TTT GTT TCC CAA GCT GC)/CS872273-RP (GCT TAG CTT GGT TAA CCG AGG). T-DNA primers LBa1 (TGG TTC ACG TAG TGG GCC ATC G) for SALK 024245 and SALK 086418 lines and LBa3 (TAG CAT CTG AAT TTC ATA ACC AAT CTC GAT ACA C) for CS872273 line were included as suggested on the T-DNA Express Web site (http://www.signal.salk.edu/tdna FAQs.html). Plants homozygous for the PDX1.1, PDX1.2 or PDX1.3 mutation were used for further analysis. The sos4-1 seeds were obtained as a homozygous line deposited in the Arabidopsis Biological Resource Center by Dr. Jian-Kang Zhu (Shi et al., 2002). Seeds were surface sterilized by incubation for 5 min with 70% ethanol, followed by three washings with water. Plants were grown in mixed soil in a growth room under a 10 h light (100 mol s−1 m−2 photons m−2 s−1 of intensity) and 14 h dark cycle at 22 ± 2 ◦ C with 60% relative humidity. Reverse transcription-PCR (RT-PCR) analysis of gene expression Total RNA was extracted from leaf samples using TRIZOL reagent (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized with 500 ng of total RNA using the SuperScript III Kit (Invitrogen, Shanghai, China). One microliter of the first-strand cDNA reaction and 10 pmol of each primer were used for RT-PCR in a total volume of 25 l. PCR conditions were set as 94 ◦ C 30 s, 54–62 ◦ C 30 s and 72 ◦ C 30 s for 25–35 cycles based on the abundance of transcript for each gene, followed by 5 min of final extension at 72 ◦ C. PCR products were electrophoresed on a 1.2% agarose gel. Gene-specific primers used in RT-PCR were as follow: AtPDX1.1-1F, 5 -GTG AGG AGT GTG AAC GGA GC-3 ; AtPDX1.1-1R, 5 -GCA CAA CCA AAT CAT ACG GC-3 ; AtPDX1.2-1F, 5 -GAT GCA GCT AGG TTG TGA TGG-3 ; AtPDX1.2-1R, 5 -TCC ATT GCA TTC TCC AAT CC-3 ; AtPDX1.3-1F, 5 -ATA ATT TCC GGA TCC CGT TC-3 and AtPDX1.3-1R, 5 -CAT CAT CAT CCA TGT TTC GC-3 . An Arabidopsis Actin gene was used as an internal control to normalize the RT-PCR reactions with a pair of gene-specific primers AtActin-1F (5 -GGC GAT GAA GCT CAA TCC AAA CG-3 ) and AtActin1R (5 -GGT CAC GAC CAG CAA GAT CAA GAC G-3 ). The experiments were repeated independently three times. Disease assays Pst DC3000 was grown overnight in King’s B liquid medium containing 25 g/mL rifampicin at 28 ◦ C with shaking. Bacteria were collected, resuspended in 10 mM MgCl2 and adjusted to OD600 = 0.001. Leaves of 4-week-old plants were inoculated by infiltration with bacterial suspension using 1-mL needless syringes and disease symptoms were observed daily. Leaves from 5 independent plants were collected and used for bacterial titering. Leaf punches (6 mm in diameter) were surface sterilized in 70% ethanol for 10 s, homogenized in 500 L 10 mM MgCl2 solution, and plated on King’s B agar plates containing 100 g/mL rifampicin.
Fig. 1. Characterization of T-DNA insertion mutant lines for AtPDX1.1, AtPDX1.2 and AtPDX1.3. (A) Diagrams showing T-DNA locations in the mutant lines. (B) PCR-based genotyping for homozygous plants using combined gene-specific primers and a TDNA primer. Arrows indicate the homozygous plants. (C) Detection of transcripts for AtPDX1.1, AtPDX1.2 and AtPDX1.3 in the obtained pdx1.1, pdx1.2 and pdx.3 mutant plants by RT-PCR analysis with gene specific primers.
B. cinerea was grown on V8 medium (36% V8 juice, 0.2% CaCO3 and 2% agar) at room temperature. Spores were collected and adjusted to 0.5 × 105 spores/mL in 4% maltose and 1% peptone buffer. For detached leaf disease assays, leaves from at least 10 plants were detached and inoculated by dropping 2.5 L of spore suspension onto leaf surface. Alternatively, 4-week-old plants were inoculated by foliar spraying with spore suspension for measurment of VB6 content changes. The inoculated plants or leaves were covered with a transparent plastic film and kept in a growth chamber under similar condition as for plant growth. Lesion diameters were measured 4 days post-inoculation (dpi) and the fungal growth in planta was measured by analyzing transcript level of B. cinerea BcActinA gene using RT-PCR with Arabidopsis Actin as a control (Wang et al., 2009).
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Fig. 2. Enhanced levels of disease caused by Pst DC3000 in the pdx1.2, pdx1.3 and sos4 plants. (A) Representatives of disease symptom on leaves of wild type and mutant plants. Four-week-old plants were infiltrated with bacterial suspension (OD600 = 0.001) of Pst DC3000 and photos were taken 5 days after inoculation. (B) Bacterial titers in inoculated leaves of the wild-type and mutant plants. Leaf samples were collected at 0, 2 and 5 d after inoculation and bacterial titers in colony forming units (CFU)/cm2 leaf area was measured. Data presented in (B) are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level.
Measurement of VB6 contents VB6 contents were analyzed by a bioassay-based measurement with a yeast strain (ATCC9080) auxotrophic for VB6. Leaf samples were collected from at least 5 individual plants before and after pathogen inoculation and leaf extracts were prepared with a protocol as described previously (Denslow et al., 2005). One milliliter of leaf extract was added to 5 mL of pyridoxol Y medium inoculated with 5 × 106 yeast cells. Yeast growth was measured at 17 h and 24 h with a spectrophotometer at OD540 . A standard curve was made using the same protocol. Total VB6 in leaf extracts was determined based on comparison to the standard curve. Results and discussion The Arabidopsis T-DNA insertion lines with mutations in AtPDX1.1, AtPDX1.2 and AtPDX1.3, three genes involved in the de novo biosynthetic pathway (Tambasco-Studart et al., 2005), and AtSOS4, a pyridoxal kinase gene involved in the salvage pathway (Shi et al., 2002), were used to explore the possible role of VB6 in plant disease resistance. The T-DNA insertions were located in promoter regions of the AtPDX1.1 and AtPDX1.2 genes and in coding region of the AtPDX1.3 gene, respectively (Fig. 1A) and homozygous mutant plants were identified by genotyping with gene-specific primers flanking the insertion sites (Fig. 1B). RT-PCR analysis revealed that no transcripts of AtPDX1.1, AtPDX1.2 and
AtPDX1.3 were detected in the pdx1.1, pdx1.2 and pdx1.3 mutant plants, respectively (Fig. 1C). These data indicate that the pdx1.1, pdx1.2 and pdx1.3 are null mutants of the AtPDX1.1, AtPDX1.2 and AtPDX1.3 genes, respectively. The sos4-1 mutant harbors a point mutation in the AtSOS4 gene (Shi et al., 2002). These homozygous mutant lines were used for the further experiments as described below. We first examined and compared the levels of disease caused by a virulent strain of Pst DC3000 on the wild type Col-0 and the mutant plants. At 5 dpi, chlorotic lesions were observed in the inoculated leaves of all plant genotypes but these chlorotic lesions were much extensive and larger in the inoculated leaves of the pdx1.2, pdx1.3 and sos4 plants than that in the wild type and the pdx1.1 plants (Fig. 1A). The chlorotic lesions and bacterial populations in the inoculated leaves of the wild type and pdx1.1 plants were comparable in all experiments (Fig. 1A), indicating that PDX1.1 might have no role in disease resistance against Pst DC3000. At 2 dpi, the bacterial population in the inoculated leaves of the pdx1.2 plants showed a 10-fold higher over that in the wild type plants, but no significant difference in bacterial populations was observed among the wild type, pdx1.3 and sos4 plants (Fig. 1B). At 5 dpi, the bacterial populations in inoculated leaves of the pdx1.2, pdx1.3 and sos4 plants were 2.04 × 109 , 8.35 × 108 and 4.34 × 108 cfu/cm2 , respectively, resulting in a significant increase in bacterial growth relative to that in the wild-type plants (7.93 × 107 cfu/cm2 )(Fig. 1B). These results indicate that mutations in AtPDX1.2, AtPDX1.3 and AtSOS4
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Fig. 3. Increased level of disease caused by B. cinerea in the pdx1.2, pdx1.3 and sos4 plants. (A and B) Representatives of disease symptom and lesion sizes on leaves of wild type and mutant plants. Four-week-old plants were inoculated by dropping a 2.5 L spore suspension (1 × 105 spores/mL) onto leaves and photos were taken 4 days after inoculation. Data presented in (B) are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level. (C) In planta growth of B. cinerea in wild type and mutant plants after infection. Fungal growth was measured by analyzing expression level of B. cinerea BcActinA gene using RT-PCR with Arabidopsis Actin mRNA as a control. The experiments were repeated three times with similar results.
genes resulted in increased bacterial growth and enhanced disease development, indicating the requirement of AtPDX1.2, AtPDX1.3 and AtSOS4 for resistance against Pst DC3000. We next examined and compared the levels of disease caused by B. cinerea, a necrotrophic fungus causing gray mold disease (Mengiste, 2012), and of in planta fungal growth on the wild type Col-0 and the mutant plants. In our experiments, B. cinerea-caused lesions on detached leaves from the wild type and pdx1.1 plants were similar; however, disease lesions on detached leaves form the pdx1.2, pdx1.3 and sos4 plants were significantly larger than that of the wild type plants (Fig. 2A), showing an increase of 55–75% over that of the wild type plants (Fig. 2B). Analysis of the transcript for the B. cinerea BcActinA gene as an indicative of the fungal growth rate in planta (Benito et al., 1998) confirmed that the pdx1.2, pdx1.3 and sos4 plants were more susceptible to Botrytis infection than the wild type plants. At 1 and 2 dpi, growth of B. cinerea in leaf tissues of the pdx1.2, pdx1.3 and sos4 plants was much higher than that in the wild type plants (Fig. 2C). These data indicate that mutations in AtPDX1.2, AtPDX1.3 and AtSOS4 genes also resulted in increased susceptibility to B. cinerea. We further analyzed the changes of VB6 contents in the wild type, pdx1.2 and pdx1.3 plants before and after inoculation with Pst DC3000 or B. cinerea using the bioassay-based measurement (Denslow et al., 2005). Without pathogen inoculation, the VB6 content in the wild type plants was approximately 1.3 g/g fresh weight (FW)(Fig. 4), which is comparable to the VB6 contents in Col-0 plants determined by HPLC (Leuendorf et al., 2010; Tambasco-Studart et al., 2005; Ristilä et al., 2011). The VB6 contents in the pdx1.2 and pdx1.3 plants were lower than that in the wild type plants, showing reduction of 30% for the pdx1.2 plants and 40% for the pdx1.3 plants as compared to that of the wild type plants (Fig. 4). This is in agreement with a previous observation that the pdx1.3 plants grown in soil or sterile culture medium contained reduced
VB6 contents (Titiz et al., 2006). After infection by Pst DC3000 or B. cinerea, the VB6 contents in the wild type plants were slightly reduced (Fig. 4); however, the VB6 contents in the pdx1.2 and pdx1.3 plants showed a further reduction as compared with those in the wild type plants (Fig. 4). These results, along with the fact that the expression of PDX1 gene in tobacco was down-regulated after infection by an incompatible pathogen P. syringae pv. phaseolicola (Denslow et al., 2005), indicate that reduced biosynthesis and hence reduced VB6 content might be a natural response of plants to pathogenic infection as a part of defense mechanism. VB6 has been shown to be an antioxidant capable of scavenging reactive oxygen species (ROS) such as singlet oxygen and superoxide anion, and hence play important roles in regulating cellular antioxidant defense (Bilski et al., 2000; Chen and Xiong, 2005; Titiz et al., 2006; Denslow et al., 2007; Havaux et al., 2009). ROS are required differentially for defense response against biotrophic/hemibiotrophic pathogens like Pst DC3000 as one of early signaling events and for development of disease caused by necrotrophic fungi such as B. cinerea. Recently, we found that silencing of tomato SlPDX1.2 and SlPDX1.3 genes resulted in increased disease severity caused by B. cinerea, reduced contents of VB6 and decreased cellular antioxidant capacity (Zhang et al., 2014). Therefore, these results further confirm that the function of VB6 in plant defense response is associated with its antioxidant activity as an antioxidant and modulator of ROS (Denslow et al., 2005). However, the relationship between VB6 and ROS in response to pathogen infection becomes an open question to be further elucidated (Fig. 4). Results from experiments with the pdx1.2 and pdx1.3 plants indicate a significant correlation between reduction in VB6 contents and the increased levels of diseases caused by Pst DC3000 and B. cinerea. However, this is not the case for the sos4 plants, which contain higher levels of total VB6 content (González et al., 2007) but showed increased levels of diseases caused by Pst DC3000
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Fig. 4. Reduced VB6 contents in pdx1.2 and pdx1.3 plants before and after infection with Pst DC3000 or B. cinerea. Four-week-old plants were inoculated by spraying with with bacterial suspension (OD600 = 0.001) of Pst DC3000 (A) or spore suspension (1 × 105 spores/mL) of B. cinerea (B) or and leaf samples were collected at 0 and 24 h after inoculation. Total VB6 content was measured by yeast bioassay. Data presented in are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level.
and B. cinerea. Such contradictory was also found in the pdx1.3 and sos4 plants that showed similar abiotic stress responses but had opposite contents of VB6 (Chen and Xiong, 2005; Titiz et al., 2006; Havaux et al., 2009). It is reasonable that the reduced contents of VB6 in pdx1.2 and pdx1.3 plants led to decreased capacity of antioxidant activity, resulting in increased susceptibility to pathogens (Figs. 2 and 3). However, the salvage pathway, in which the SOS4 acts as a key enzyme, functions to convert different VB6 vitamers between each other (McCormick and Chen, 1999). Although the sos4 plants contain higher levels of total VB6 content (González et al., 2007), the content of active VB6 forms might be not changed or even reduced. Further detailed analysis the contents of different VB6 forms in sos4 plants by HPLC will be helpful to clarify this point. Alternatively, it was shown recently that, despite differing VB6 content in whole leaf tissue, both the pdx1.3 and sos4 mutants share a common deficiency in VB6 in chloroplast (Rueschhoff et al., 2013). VB6 deficiency in chloroplasts in the pdx1.3 and sos4 plants can be one of the causes that lead to some of similar phenotypes in response to abiotic stresses and pathogenic infection. Interestingly, silencing of SlSOS4 did not affect the response to tomato plants to infection of B. cinerea (Zhang et al., 2014). Thus, it is possible that the functions of SOS4 and even the salvage pathway of VB6 vary among different plants in response to pathogen infection. Nevertheless, our preliminary data presented in this study provide direct molecular evidence suggesting an important role for VB6 in plant disease resistance against pathogens. However, the mechanism for the function of VB6 in plant defense response needs to be further investigated. Acknowledgements This work was supported by the National Basic Research Program of China (2009CB119005), the National Key Technology R & D Program of China (2011BAD12B04), the Ph.D. Program Foundation for Young Teachers of Ministry of Education of China (no. 20100101120079) and the Scientific Research Fund of Department of Education of Zhejiang Province (no. Y200909712), the National Natural Science Foundation of China (Nos. 31101422).
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