Bacillus subtilis CBR05 induces Vitamin B6 biosynthesis in tomato through the de novo pathway in contributing disease resistance against Xanthomonas campestris pv. vesicatoria

Expression profiling for genes involved in Vitamin B6 (VitB6) biosynthesis was undertaken to delineate the involvement of de novo and salvage pathway induced by Bacillus subtilis CBR05 against, Xanthomonas campestris pv. vesicatoria in tomato. Pyridoxine biosynthesis (PDX) genes such as PDX1.2 and PDX1.3, were found to be overexpressed significantly at 72 hpi in B. subtilis and pyridoxine inoculated plants. Most significant upregulation was observed in the transcript profile of PDX1.3, which showed more than 12- fold increase in expression. Unfortunately, salt sensitive overlay4 (SOS4) profiling showed irregular expression which corroborates that SOS4 role in VitB6 biosynthesis needs further studies for deciphering a clear notion about their role in tomato. Antioxidant enzymes i.e., superoxide dismutase, catalase, polyphenol oxidase, and peroxidase activities clearly demonstrate escalation till 48 hpi and gets reduced in 72 hpi. Pot trials also confirm that B. subtilis compared to pyridoxine supplementation alone show plant disease resistance and elongated roots. The present study confirms that B. subtilis, as a versatile agent in eliciting induced systemic resistance regulated by de novo pathway as a model for plant defense against X. campestris pv. vesicatoria substantiated by VitB6 biosynthesis. Nevertheless, the study is preliminary and needs further evidence for affirming this phenomenon.

vitB6 oxidase (PDX3) 8,9 . Recently, a pyridoxal reductase has been characterized in Arabidopsis thaliana, which is essential for conversion of PL into PN 10 .
Studies showed that abiotic stress response comprises of pyridoxal kinase and pyridoxal reductase enzymes which play a significant role mediated by salvage pathway [8][9][10][11] . Previous studies also showed that abiotic stress regulates up-regulation of de novo biosynthetic pathway genes [12][13][14][15][16][17] . A. thaliana (AtPDX1) gene mutation renders the plants for the high rate of sensitivity to stress conditions like high light, salt and stress. Nevertheless, PDX gene overexpression provides escalated tolerance in combating oxidative stress 16,17 . The gene for pyridoxal kinase, SOS4 which is a key factor in the salvage pathway was alienated to sequential response to salinity and osmotic stress [8][9][10][11] .
Moreover, previous studies clearly depict salicylic acid (SA), methyl jasmonate (JA), and ethylene (ET) act as the chemical counterparts in inducing plant defense response abating oxidative stress through overexpression of PDX transcript in Nicotiana tabacum and Hevea brasiliensis 18,19 . The studies lucidly show that the hypersensitive response (HR) in tobacco leaves infected with Pseudomonas syringae pv. phaseolicola was affected with excess VitB6 content, rather P. syringae pv. tabaci infection resulted in augmented disease severity 18 . However, direct molecular evidence supporting a role for VitB6 in biocontrol agent, Bacillus subtilis inoculated plants and their plant biotic defense remains lacking. The present study was focused to analyze the expression profiling of de novo (PDX1.2, PDX1. 3,PDX2), and salvage pathway (SOS4) genes to demarcate involvement of de novo and salvage pathway in VitB6 biosynthesis by B. subtilis CBR05 upon challenge emancipated by Xanthomonas campestris pv. vesicatoria (XCV) in tomato.
Among plant diseases encountered worldwide, bacterial spot disease, caused by XCV pose a serious threat owing to complex pathogen variability which affects economically important crops production [20][21][22][23][24][25] . Bacterial diversity poses additional threats to biocontrol efficacy of various agents of antagonistic control and inadequacy of effective strategies to gain disease resistance and efficient abatement through chemical control 26 . Besides, restrictions on the use of chemical pesticides due to concerns about their impact on the environment and human health are increasing rapidly 27 . Biological control of disease using microbial antagonists is an eco-friendly alternative to chemical pesticide and is being studied extensively on several different plant diseases [28][29][30][31][32] . Strains of the Gram-positive bacterium, B. subtilis have been regarded to protect plants opposing fungal and bacterial pathogens. B. subtilis has been regarded as a versatile bacterium possessing plant growth promotion along with enhanced crop protection mediated by ISR (induced systemic resistance) 30,31,33 . Elicitation of ISR by these strains has demonstrated importance in greenhouse or field trials [34][35][36][37][38] . Mode of action by which B. subtilis instilling broad-spectrum antagonistic activity against various phytopathogens is attributed to the ability to produce spores, antibiotic production, lytic enzymes and capacity to resists adverse environments [39][40][41] . Thus, it is clearly evident that Bacillus sp. is an essential regulator of ISR.
B. subtilis is also a predominant endophytic bacterium which marks it as an efficient biocontrol agent against vascular pathogens 28,42 . Endophyte colonization triggers the reprogramming of the host action, favoring secondary metabolism and inducing changes in the plant development 43 . For example, B. subtilis strain confers protection to melon plants encountering the cucurbit powdery mildew by activating SA and JA dependent defense response 44 . Moreover, they produce the volatile compound such as acetoin, which triggered the ISR. In A. thaliana, B. subtilis UMAF6639 restricts pathogen dissemination and disease progression in its aerial parts through ET-and SA-dependent and JA-independent response 45 . Cyclic lipopeptides such as iturins and fengycins from B. subtilis acting as potent antibiotics in the biocontrol of the tomato wilt disease caused by the phytopathogenic bacterium Ralstonia solanacearum 46 . A change in a JA receptor resulted in the sufficient signal transduction, fertility and defense conflicting insects, in addition to resistance in opposing P. syringae pathogenic strains 47 . Hence, the recent identification of host target modification could be a promising approach to protect plants from pathogen attack. Thus B. subtilis acts a significant antagonist in field conditions both in the external environment and endophytic colonization thereby contributing to ISR in eliciting a host defense response for increased productivity and devoid of loss due to phytopathogens, VitB6 possess antioxidant activity and can modulate plant defense by regulating antioxidant status in plants 48 . However, collaborating VitB6 biosynthesis with biocontrol, disease resistance, ISR, stress alleviation, and tolerance is still absent with regard to B. subtilis CBR05 against XCV. In the present study, we examined B. subtilis CBR05 induced VitB6 biosynthetic genes in tomato confronted with XCV. Earlier up to the literature collected it was positively corroborated this is the first report involving B. subtilis CBR05 induced expression of VitB6 biosynthetic genes in tomato against XCV apart from biocontrol proficiency, ISR and plant improvement based on growth characters. We affirm that the present study could be rationalized for similar diseases not only in tomato but can be extended to other crops belonging to the Solanaceae family. The study would be a launch pad in further characterization and involvement of crosstalk between the gene regulation of de novo and salvage pathway in ISR mediated by B. subtilis. VitB6 contents in the plants without XCV inoculation and with XCV were examined to assess B. subtilis-induced expression of the VitB6 biosynthetic genes. VitB6 contents in pathogen-inoculated plants were lower than that in the mock-inoculated plants, showing reduction of 14, 6, and 15% for XCV, XCV + B. subtilis, and XCV + pyridoxine, respectively, at 24 hpi, as compared to those of mock-inoculated plants. These results, in consort with the fact that the transcript abundance of VitB6 gene in tomato after infection by XCV, show that reduced biosynthesis and hence reduced VitB6 content. It might be a natural defense response of plants to pathogenic infection as a part of defense mechanism. However, the VitB6 contents in the XCV + pyridoxine + B. subtilis inoculated plants, showing increase of 12, 93 and 76% at 24, 48 and 72 hpi, respectively, compared to that of mock-inoculated plants (Fig. 2). The result clearly shows that Bacillus inoculation has roles in VitB6 biosynthesis in all intervals indicating a way for profiling the specific genes. Differential regulation of the VitB6 genes could also involve a crosstalk between de novo and salvage pathway genes. Hence, further resistance mechanisms could positively associate with antioxidant profiling for establishing prominent disease resistance strategies.  www.nature.com/scientificreports www.nature.com/scientificreports/ XCV effect on superoxide dismutase (SOD) and catalase (CAT) activity. As shown in Fig. 3a, B.

VitB6 biosynthetic genes expression and changes in
subtilis + XCV inoculated plants and XCV + pyridoxine inoculated plants had a significant increase in SOD activity at 24 hpi as compared to mock-inoculated plants (115 and 57%, respectively) and XCV inoculated plants (111 and 54%, respectively). We also found significant increase in SOD activity in the XCV + pyridoxine + B. subtilis inoculated plants, compared them with those in mock-inoculated pants (82%) and XCV inoculated plants (54%), at 24 hpi followed by decrease in SOD activity at 48-72 hpi (Supplementary Table S1). However, the activity of CAT in tomato plants increased rapidly after pathogen inoculation. CAT activity was significantly higher in the XCV + B. subtilis, XCV + pyridoxine and XCV + pyridoxine + B. subtilis by 44, 34 and 34%, respectively at 24 hpi, compared to those in XCV inocualted plants (Fig. 3b). We did not find any significant difference in the SOD and CAT activity at 48 hpi both in B. subtilis and X. campestris inoculated plants. Reduced levels of SOD and CAT activity was observed in XCV and pyridoxine inoculated plants at  hpi.

XCV effect on polyphenol oxidase (PPO) and peroxidase (POD) activity. Patterns of increase in
PPO activity in the leaves during 24-72 hpi are illustrated in Fig. 3c. In, B. subtilis + XCV inoculated plants had a noteworthy increase in PPO activity at 48 hpi as compared to mock-inoculated controls (120%), XCV alone (4%), and XCV + pyridoxine (42%) inoculated plants. However, the PPO activity was significantly higher in the XCV + pyridoxine + B. subtilis inoculated plants, showing an increase of 18% compared with that in the XCV inoculated plants at 48 hpi. As shown in Fig. 3d, the treatment groups had a significant increase in POD production as compared to mock inoculated plants at 24 and 48 hpi. In, B. subtilis inoculated plants had a significant increase in POD activity at 24-72 hpi as compared to XCV, pyridoxine, and mock-inoculated controls. Induction of POD activity in the leaves was observed during 24 hpi and followed by a decline at 48-72 hpi. At 48 hpi, XCV inoculated plants showing an increase of 78% total POD activity than the mock-inoculated healthy control. Pot trials also corroborate that B. subtilis compared to pyridoxine supplementation alone show biocontrol activities on the leaf (Fig. 4). Figure 5 shows that root length of B. subtilis and pyridoxine inoculated plants were greatly increased by co-inoculation. Among inoculation, XCV + pyridoxine + B. subtilis (23.5 cm), followed by B. subtilis + XCV (16.5 cm), XCV + pyridoxine (16.0 cm) and mock-inoculated plants (14.0 cm) showed an increase in root length. Whereas, XC inoculated plants showed decreased in root length (9.0 cm).

Discussion
VitB6 is a collective term for a group of six interconvertible compounds 1,7 . It is most notable for its contribution to amino acid, carbohydrate and fatty acid metabolism where it serves as a cofactor for enzymes 48 . Despite these facts, the effect of VitB6 on plant development, the mechanisms controlling its biosynthesis and stress tolerance are still poorly understood. Reactive oxygen species (ROS) quenching and antioxidant properties by VitB6 have been an extensively studied phenomenon. VitB6 vitamers have been affirmed as significant singlet oxygen quenchers in vitro 49 . Nevertheless, well documented in A. thaliana, it needs to be assessed for its definitive role 50 . Further, PDX1.2 serves its role in sustaining VitB6 under critical conditions and stabilizes PDX1s under abiotic stress 51 . The literature available for most of the crop plants shows the manifestation of both de novo and salvage pathways in VitB6 genes for enhanced disease resistance upon phytopathogens infection. Recently, Zhang et al. 52 proved that Botrytis cinerea-infected tomato plants show the involvement of the de novo vitB6 biosynthetic pathway and not salvage pathway by affirming SlPDX1.2 and SlPDX1.3 genes but not SlSOS4 through gene silencing studies based on disease severity. Also, Ralstonia solani RsolPDX1, RsolPDX2, and RsolPLR were the three principal factors involved in the VitB6 pathway in R. solani AG3 53 . Most of the literature pertaining to VitB6 biosynthesis addresses de novo pathway mostly but salvage pathway at rare circumstances, interestingly lack of introns were denoted for plant-pathogen induced response for synthesis 18 . Hence, we could not clearly find a gap in abridging salvage pathway in oxidative stress-induced plant defense and antioxidant properties. The present study further adds novel results for the environmentally useful bacteria in eliciting a plethora of optimal outcomes. A www.nature.com/scientificreports www.nature.com/scientificreports/ non-native DXP-dependent VitB6 pathway in B. subtilis for the production of pyridoxine was shown to be present earlier 54 . Till date, the role of B. subtilis for the production of pyridoxine or involvement in VitB6 biosynthesis have not been classically addressed. It has been made possible through altered mechanisms like metabolic engineering strategies 54 . The present study provides molecular insights into the cumulative plant growth promotion, ISR and de novo pathway involvement of VitB6 biosynthesis.
Transcript profiling indicates overexpression of all four VitB6 biosynthetic genes was increased under stress; whereas, differential expression patterns were observed. PDX1.2, and PDX1.3, were found to be overexpressed significantly after 72 hpi with pyridoxine and B. subtilis in XCV inoculated plants showing that optimal synthesis of VitB6 could be possibly corroborated to the involvement of de novo pathway in tomato after XCV. Most significant upregulation was observed in the transcript profile of PDX1.3, which showed more than 12-fold increase in expression at 72 hpi. We also found significant up regulation of PDX2 in XCV + B. subtilis and XCV + pyridoxine + B. subtilis inoculated plants, which were 3.054-and 3.44-fold, respectively, at 48 hpi. From our results, we can clearly infer that SOS4 expression has not been significant. Increased expression of PDX1.3, PDX1.3, and PDX2 levels in our study proves that B. subtilis inoculated plants has roles in balancing the VitB6 biosynthesis. The previous study also showed that in A. thaliana, PDX2 balancing B6 vitamer levels 55 . This possibly explains that de novo pathway predominates over the salvage pathway having mechanisms as a bypass. The absence of a salvage pathway may be due to a demarcated pathway as that of the non-native pathway. In a similar study by Torky 56 , it clearly shows that VitB6 primes defense response and disease resistance in Capsicum annum upon Tobacco Mosaic Virus (TMV) infection. We also hypothesize that B. subtilis plays similar roles in the emancipation of cohesive response as that of TMV. In addition, VitB6 contents in XCV inoculated plants slightly decreased compared to those in the mock-inoculated plants. Moreover, a slight increase in the VitB6 content in mock-inoculated tomato leaves may be responsive to stress or wounding caused by mock infiltration. These results indicate that expression of VitB6 biosynthetic genes in tomato down-regulated after infection with XCV and hence reduced VitB6 content might be a natural response of plants to pathogenic infection as a part of defense mechanism 18 .
Previous studies showed thatVitB6 vitamers can act as antioxidant in plants and may act as an important modulator of redox status during pathogen defense response 12,18,49 . When compared with those in the control plants, increased disease level and increased accumulation of SOD after infection with XCV was observed. The increase www.nature.com/scientificreports www.nature.com/scientificreports/ of SOD activity accelerated enzymatic conversion of the superoxide anion to H 2 O 2 and led to further accumulation of H 2 O 2 in the XCV inoculated plants. Hence, increased accumulation of H 2 O 2 will lead to production of CAT and POD. We also found that activity of PPO was induced in XCV inoculated plants compared to those in the mock-inoculated control plants. Moreover, antioxidant activity was higher in B. subtilis inoculated compared to those of other treatments. Previous studies, the showed that enhanced activities of antioxidant enzymes in plant tissues are positively associated with ISR and plant disease suppression 30,37,41 . Moreover, antioxidant activity in mock-inoculated healthy control was consistently present in all the test plants. The enzyme activity in control plants increased over time after inoculation showing native antioxidant enzyme presence.
Further, tomato plants infiltrated with XCV showed increased disease symptoms compared to those infiltrated with biocontrol agent, B. subtilis CBR05. However, co-inoculation with B. subtilis showed increased plant growth and decreased disease severity. At the same time supplementation of VitB6 vitamers like pyridoxine show comparatively low levels in the VitB6 synthesis and plant growth properties. Our results showed that the pyridoxine treated plants appeared in yellow color. Thus, the changed coloration may result from changed pigmentation 12 . Moreover, leaves infiltrated with pyridoxine and XCV increased the severity of chlorosis and necrosis associated with disease. Further inoculation of pyridoxine with B. subtilis colonizes root in plants and expresses root parameters. These results suggest that a possible role of pyridoxine in promoting root cell division and elongation. In our study, it clearly shows the above phenomena wherein, B. subtilis CBR05 extensively promote root growth in length and altered health in tomato roots through induced systemic resistance 57 . Further, active principles in B. subtilis have been attributed to surfactin in wheat as an elicitor 58 . With this view, active principles of B. subtilis could possibly act as a launch pad for the development of novel elicitors from our pilot study. Interestingly it has been observed that B. subtilis remarkably show similar patterns of 100 mM pyridoxine supplementation. Our proposition stating B. subtilis to be adaptable tool invoke several questions for the application in field conditions. Hence, pot culture experiments guarantee the application modalities in plants. The omnipresence of B. www.nature.com/scientificreports www.nature.com/scientificreports/ subtilis owing to several competencies in suppressing plant pathogens has been extensively studied [39][40][41] . B. subtilis has been well documented for usage against Fusarium wilt, Sclerotina sclerotiorum, Pythium, Phytophthora, Rhizoctonia, Septoria, and Verticillium in tomato 59 . This was further characterized at the molecular level showing a distinct lineage for the B. subtilis with copious prominent roles in plant enhancement mechanisms. XCV infection in Chinese cabbage has been studied for plant growth promotion and ISR which indicate Bacillus sp. of predominance 60 . Nevertheless, Xanthomonas abatement along with numerous molecular dissection mechanisms and VitB6 biosynthesis is lacking. Our study makes this versatile B. subtilis to be utilized as a broad spectrum agriculturally important microorganism. The present study is a holistic approach to address the B. subtilis eluding XCV with multiple molecular insights.
Current research focuses on assorted approach in interpreting real-time expression patterns of B. subtilis CBR05 induced VitB6 biosynthetic genes against XCV in tomato. The advancement in identifying B. subtilis CBR05 is the inherent objective of the present study. PDX1.2, PDX1.3, and PDX2 are the overexpressed VitB6 biosynthetic genes denoting involvement of de novo pathway in VitB6 biosynthesis induced by B. subtilis CBR05 against XCV. However, genes involved in salvage pathway have been not regulated as like de novo pathway genes. This is the first report that B. subtilis induces expression of VitB6 biosynthesis and can be a versatile bacterium in multifarious roles in tomato plant upgraded productivity. The present study can be rationalized for any crop and VitB6 biosynthesis.

Materials and Methods
Microorganism and culture conditions. In the present study, Xanthomonas campestris pv. vesicatoria (XCV) KACC11154 obtained from Korean Agricultural Culture Collection (KACC), South Korea, was used as the phytopathogen. B. subtilis CBR05 used in this study were obtained from Prof. Se Chul Chun lab, used as a biocontrol agent. Bacterial cells were subcultured in tryptic soy broth. They were transferred to tryptic soy agar and incubated at 30 °C for 24 h. The bacterial cells were harvested and resuspended in 10 mM MgCl 2. B. subtilis CBR05 viable population was adjusted to 10 8 CFU/ml for further studies.
Plant materials and growth conditions. We obtained seeds of tomato from Korea seed resource center.
They were surface sterilized with 70% ethanol for 5 min, finally rinsed three times with sterile distilled water. Tomato seedlings were transferred into plastic pots after four weeks. The experiment was carried out in a randomized complete block design with three plants as replicates in each of the following treatments: (i) Mock (4% maltose and 1% peptone solution) (ii) XCV alone (iii) XCV + B. subtilis (iv) XCV + 100 mM pyridoxine (v) XCV + B. subtilis + 100 mM pyridoxine. Pots were incubated in a growth room. Three pots were maintained per treatment, each with a single plant and arranged in a completely randomized design with three replications.
Total RNA isolation and cDNA synthesis. Leaf samples were collected from tomato plants. Total RNA was isolated from frozen tomato tissue using the RNeasy Plant Mini Kit (Qiagen, Germany), according to manufacturer's instructions. Genomic DNA contamination from RNA samples was removed by DNAse treatment. Formaldehyde agarose gel electrophoresis was used for the verification of the integrity of total RNA. We determined the purity and concentration of the total RNA by spectrophotometry (NanoDrop ND-1000 Spectrophotometer; Celbio, Italy). RNA samples were reverse transcribed into cDNA using First-strand cDNA synthesis kit (Bioneer, Korea).
Quantitative PCR (qPCR). The synthesized cDNA was used as a template for real-time PCR reactions using the instrument CFX96 ™ Real-Time System (Bio-Rad, Hercules, CA, USA). The sequences of primers used in this study for RT-qPCR analysis were listed in Supplementary Table S2. Each reaction (25 μL) contained 12.5 μL of AccuPower ® 2x Greenstar qPCR Master Mix (Bioneer, Korea), forward and reverse primer (0.4 μM), 5 μL of diluted cDNA sample and nuclease-free water. The qPCR reactions were carried out using the following PCR cycling conditions: 95 °C for 10 min (1 cycle), the 20 s at 95 °C followed by annealing and extension at 60 °C for 45 s (40 cycles). The actin gene was used as the internal reference for normalization. The expression level of the VitB6 gene transcripts was calculated using CFX Manager Software (Bio-Rad). All samples were analyzed with three independent total RNA samples.
Measurement of VitB6 content. VitB6 contents were determined using a bioassay with Yeast strain (Saccharomyces cerevisiae ATCC9080), auxotrophic for VitB6. Leaf samples were collected from each experiment and leaf extracts were prepared with a protocol as described earlier 18 . Cells were grown in Pyridoxine Y medium for 12 h at 30 °C, 220 rpm. Overnight culture was washed two times with Pyridoxine Y medium for removal of residual pyridoxine. One milliliter of leaf extract was added to tubes containing 5 × 10 8 yeast cells in 5 ml of Pyridoxine Y medium. Assay tubes were incubated for 16 h, at 30 C, 220 rpm. Yeast growth was measured by spectrophotometry at 540 nm. Pyridoxine was used as a standard.
Determination of antioxidant enzymes. Determination of SOD activity. SOD was determined using the SOD activity kit (Enzo Life Sciences Inc., USA), according to manufacturer's instructions. The enzyme extract (25 μl) was added to 150 μl of Master Mix. The reaction was started by adding 25 μl of 1× xanthine solution to all the wells. The well without enzyme solution (added 25 μl of the 1× SOD buffer) were considered as a control. The total SOD activity was measured using a microtiter plate reader, optical density (OD) at 450 nm. The specific activity was expressed as units per milligram of protein (Units mg −1 protein).
Determination of CAT activity. The catalase fluorometric detection kit (Enzo-Catalog # ADI-907-027) used to detect CAT activity by measuring the amount of H 2 O 2, according to the manufacturer's protocol. The reaction