Early plant defense-related responses triggered by a novel glycoprotein from Streptomycin

A novel glycoprotein from Streptomyces sp. ZX01, GP-1, has high activity against tobacco mosaic virus (TMV). With small molecular weight and water-soluble characteristics, GP-1 has tremendous commercial value. Though previous study indicated a plant immunity-inducing effect, the antiviral mechanism of GP-1 is still unclear. In this study, early plant defense-related responses, such as Ca2+ transient peak, callose apposition, oxidative burst, hypersensitive response (HR), programmed cell death (PCD), NO rising and stomatal closure, were thoroughly investigated and we studied the mechanism of how GP-1 can induce virus resistance in Nicotiana benthamiana in additional detail. Results showed that GP-1 could induce Ca2+cyt rapidly both in tobacco leaves and suspension cells, followed by ROS and NO elevation. Similar with typical pathogen-associated molecular patterns (PAMPs), GP-1 induces callose deposition and stomatal closure to form defense barriers for pathogen invasion. The expression of defense-related genes further supported these phenomena. The gene with CAM5 was obtained by RNA sequencing in Nicotiana benthamiana. movement, photosynthesis, hormone response, nuclear enzyme system and gene expression. More and more studies have confirmed that the CaM signaling pathway plays

2 Abstract Background A novel glycoprotein from Streptomyces sp. ZX01, GP-1, has high activity against tobacco mosaic virus (TMV). With small molecular weight and water-soluble characteristics, GP-1 has tremendous commercial value. Though previous study indicated a plant immunityinducing effect, the antiviral mechanism of GP-1 is still unclear.

Results
In this study, early plant defense-related responses, such as Ca2+ transient peak, callose apposition, oxidative burst, hypersensitive response (HR), programmed cell death (PCD), NO rising and stomatal closure, were thoroughly investigated and we studied the mechanism of how GP-1 can induce virus resistance in Nicotiana benthamiana in additional detail. Results showed that GP-1 could induce Ca2+cyt rapidly both in tobacco leaves and suspension cells, followed by ROS and NO elevation. Similar with typical pathogenassociated molecular patterns (PAMPs), GP-1 induces callose deposition and stomatal closure to form defense barriers for pathogen invasion. The expression of defense-related genes further supported these phenomena. The gene with CAM5 was obtained by RNA sequencing in Nicotiana benthamiana.

Conclusions
Through analyses of expression of SA, JA and CAM5, it was shown that GP-1 enhanced the virus resistance of tobacco by improving expression of CAM5 and the SA and JA content.

Background
Tobacco mosaic virus (TMV), a notorious plant pathogen, has a wide host range of over 885 plant species in 65 families, and causes serious economic losses worldwide (Yonghui Ge, et al., 2012). Besides the practical benefits related to controlling TMV and treating infected commercial hosts, TMV represents a paradigm in virology and represents a very difficult challenge for the researcher. Though conventional techniques of eradication from the soil have been tested over the last century, few virus-curative agrochemicals are available in the market (Andrea, L., et al., 2017). Induction of plant resistance, either achieved by chemicals (systemic acquired resistance, SAR) or by rhizobacteria (induced systemic resistance, ISR) is a possible and/or complementary alternative to manage virus infections in crops (Faoro, F., & Gozzo, F., 2015).
Some artificially synthesized chemicals and natural chemicals or proteins have been shown to have SAR inducing effects. The antiviral agent ningnanmycin (NNM) lead to curative rates of 30-60% (Ouyang, G., et al., 2008), however, the results mainly refer to lab tests and more data on open-field applications are needed (Andrea, L., et al.,2017). BTH (trademarks: Bion TM in Europe, Actigard TM in USA), is a functional analogue of salicylate. It was the first commercial product to induce an artificial type of SAR against systemic virus infection, e.g. TMV in tobacco Xanthi "nn" (Friedrich, L., et al., 1996).
Microbial elicitors of plant defence are active small molecules produced during the interaction between a pathogen and its host, and include oligosaccharides, glycoproteins, glycopeptides, proteins, polypeptides, lipids, and other cellular metabolites. These inducers are recognized by pattern-recognition receptors (PRRs) on the surfaces of plant cells and trigger plant defense responses, resulting in systemic resistance (Dewen, Q. et al.,2017). Chitosans are natural, non-toxic and inexpensive products obtained by deacetylation of chitin from the exoskeleton of crustacean and other arthropods. The capacity of inducing resistance against viral diseases, such as bean common mosaic virus (BCMV), bean yellow mosaic virus (BYMV), alfalfa mosaic virus (AMV), peanut stunt virus (PSV) and TMV, by chitosans is long known. Unfortunately, the use of chitosans in crop protection is still hampered by some limitations, such as unstable activity and water 4 insolubility (Faoro, F., & Gozzo, F., 2015). Plant growth promoting rhizobacteria (PGPR), as well as photoprotein-induced systemic resistance, is the other two worthwhile strategies that could be developed to curb virus infection and spread (Prasad, V., et al., 2014).
Additionally, Jacalin-type lectin, RTM1 (RESTRICTED TEV MOVEMENT 1) from Arabidopsis, two other lectin-like proteins isolated from Cyamopsis tetragonoloba and Dioicin 2, a typical ribosome inactivating protein (RIP) from Phytolacca dioica, showed strong antiviral activity. However, the type of resistance they induced does not depend on HR or SAR signaling, and the molecular weights of these proteins hindered the industrial production (Faoro, F., & Gozzo, F., 2015).
GP-1, a glycoprotein from Streptomyces sp. ZX01 with high activity against TMV, was previously isolated in our lab, which has a small molecular weight of 8479 Da and a watersolubility easing its production (Zhang, G., et al., 2015). The protective efficacy (87.58%) of GP-1 was significantly higher than the curative efficacy (13.44%) on Nicotiana tabacum indicating a plant immunity-inducing effect. Further experiments proved that GP-1 induced the SAR, in the form of increasing of superoxide dismutase (SOD), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) activity, decreasing of malondialdehyde (MDA) content and strongly expressing of pathogenesis-related proteins (PRs) in tobacco plant (Zhang, G., et al., 2016). The anti-virus mechanism of polysaccharides, chitosan as an example, has been thoroughly studied, which involves Ca 2+ transient peak, callose apposition, oxidative burst, hypersensitive response (HR), programmed cell death (PCD), NO rising and stomatal closure (Srivastava, N., et al., 2009;Wang, W., et al., 2008).
However, limited information can be found for glycoproteins. In this study, the early plant defense responses were examined using GP-1 to elucidate the immunity-inducing mechanism of glycoproteins.

HR and PCD induced by GP-1
The induction of necrosis by GP-1 was determined by observing the development of a necrotic spot at the site of injection of 50 μl of GP-1 (100 μg/mL) into tobacco leaves. A transparent spot was visible in the infiltrated area at 4-8 h after inoculation, and the necrotic lesion became obvious at 12-24 h post-treatment (Fig. 1a). Evans blue staining showed an obvious cell death after GP-1 treatment (Fig. 1b). Serial dilution of the elicitor showed that GP-1 induced PCD at a concentration as low as 50 μg/mL. Additionally, GP-1 can quickly induce PCD in a time-and concentration-dependent manner (Fig. 1c).
Altogether, GP-1 may activated receptors on the plasma membrane and induced HR and PCD.

Induction of ROS production
Hydrogen peroxide polymerized by 3, 3'-diaminobenzidine (DAB), which forms a dark redbrown precipitate, was detected, and the sites of H 2 O 2 accumulation. A strong signal was obviously observed microscopically in tobacco leaves after 2 h after treatment with GP-1 (100 μg/mL) (Fig. 2a). ROS production induced by GP-1 was also visualized through DCFH-DA in tobacco suspension cells (Fig. 2b). The quantification of H 2 O 2 in tobacco leaves and cell suspension and compared to a negative control was further conducted. GP-1 treatment caused a rapid increase in H 2 O 2 in leaves, which reached a maximum at about 3 min, followed by a gradual decrease to a level similar to that of the negative control in 10 minutes (Fig. 2c). Similar phenomenon of H 2 O 2 production was observed for tobacco suspension cells, with the peak of H 2 O 2 production appearing 30 min after GP-1 treatment ( Fig. 2d).
Effects of GP-1 on [Ca 2+ ] cyt elevation 6 It was observed that, the resting [Ca 2+ ] cyt was not changing in control cells. GP-1 treatment in tobacco cells induced an elevation of [Ca 2+ ] cyt at about 10s. [Ca 2+ ] cyt peak was observed after about 200s and a periodic increase was observed 200s-1200s later (Fig. 3). The addition of Ca 2+ chelator EGTA suppressed the [Ca 2+ ] cyt increase induced by GP-1, indicating that [Ca 2+ ] cyt elevations depended on the Ca 2+ influx from the extracellular medium. These data provided evidence for the involvement of Ca 2+ in the GP-1 mediated signaling.
Elevation of NO levels and stomatal closure induced by GP-1 GP-1 induces the appearance of cells showing bright fluorescence due to NO accumulation (Fig. 4a) and NO production showed a dose-dependent manner (Fig. 4b). Application of 100 μg/mL GP-1 resulted a rapid increase of NO in both tobacco suspension cells (Fig. 4c) and leaves (Fig. 4d).
As GP-1 may induce the accumulation of NO, it may also have a role in stomatal closure.
In this study, GP-1 could induce stomatal closure in tobacco, as is the case with ABA. The effect of GP-1 on promotion of stomatal closure was significant (P < 0.05) at concentrations above 50 μg/mL. Application of 50 μg/mL, 100 μg/mL, 200 μg/mL GP-1 reduced stomatal apertures by 23.3%, 38.9%, and 49.6%, respectively, and showed a does-dependent manner ( To further confirm the results of transcriptome sequencing, the transcript of several genes that involved in immune reactions were analyzed by qRT-PCR (Fig. 7). HSR203J, STR319, SGT, LOX, WIPK, CalS, and CaM5, that are involved in HR establishment, SA or JA synthesis, callose deposition, and Ca 2+ signal pathway, were selectively picked out for examination. Results showed that all the examined genes were significantly up-regulated by GP-1 treatment (Fig. 7).

Discussion
Biological active molecules, such as oligosaccharides, polypeptides, and lipids can be recognized by receptors on the surfaces of plant cells and trigger plant defense responses, resulting in systemic resistance (Dewen, Q. et al., 2017). Among these 8 inducers, chitosan has been well investigated and the anti-virus mechanism of chitosan has been studied thoroughly (Pichyangkura, R. & S. Chadchawan, 2015;Hadwiger, L.A., 2013). A glycoprotein, GP-1, with high activity against TMV from Streptomyces sp. ZX01 was isolated in our lab (Zhang, G., et al., 2016). To further elucidate the immunityinducing mechanism of GP-1, some early plant defense responses were examined in this study.
A burst in oxidative metabolism that leads to the accumulation of superoxide (O 2-) and H 2 O 2 is considered a significant early event in the plant defense system and has been proposed as key factors in the control of both developmentally and environmentally induced PCD . The rapid increase of H 2 O 2 in both leaves and tobacco suspension cells treated by GP-1 (Fig. 2) proved a plant defense system was activated.
The production of ROS by NADPH oxidases are mainly controlled by Ca 2+ via direct binding to EF-hand motifs and phosphorylation by Ca 2+ -dependent protein kinases (Kadota, Y., K. Shirasu, & C. Zipfel, 2015). What's more, Ca 2+ influx can also led to rapid production of NO and activation of mitogen-activated protein kinases (MAPKs) (Ma, W., et al., 2008). In this study, GP-1 was proved to be involved in Ca 2+ mediated signaling (Fig. 3) and NO production ( Fig. 4) Plant hormones as well as microbial elicitors, such as chitosan ( Srivastava, N., et al.,2009), can modulate stomata, during which NO is a key element among the signaling elements leading to stomatal closure (Agurla, S., G. Gayatri, & A.S. Raghavendra, 2014).
Stomatal closure restricts the entry of pathogens into leaves and forms a part of plant defense response (Agurla, S., G. Gayatri, & A.S. Raghavendra, 2014). In this study, the increased NO by GP-1 treatment indeed led to stomatal closure (Fig. 5).
Upon pathogen infection, callose is deposited in cell wall appositions, called Papillae, at the sites of attack to form effective chemical and physical defense barriers for pathogen invasion . Purified pathogen-associated molecular patterns (PAMPs), including flg22, elf18 and chitosan, have been shown to induce callose deposits in leaves or cotyledons of Arabidopsis, which has emerged as an indicator of plant immune responses (Wu, S., L. Shan, & P. He, 2014). As expected, GP-1 induced callose deposition like elicitors mentioned above (Fig. 6). HSR203J plays a functional role in the establishment of the HR. The temporal and spatial patterns of HSR203J activation in leaves and roots inoculated with Pseudomonas solanacearum indicate that the HSR203J promoter exhibits a rapid (3 to 6 h postinoculation) and high level of induction only in plant cells inoculated with the HR-inducing bacterial isolate (Pontier, D., et al., 2014). In this study, the rapid induction of HSR203J by GP-1 was also observed, supporting the establishment of HR and PCD by GP-1. As the promoter of HSR203J does not respond to various stress conditions and is only very weakly induced during compatible interactions (Pontier, D., et al., 2014), GP-1 may have similar function with incompatible pathogenic bacterium. STR319, as another marker for the HR (Keller, H., et al.,1998), participates in sesquiterpenoid biosynthesis and is also induced by GP-1 rapidly with a similar pattern with HSR203J.
The expression of SGT (UDP-Glc: SA glucosyltransferase), which converts SA to a conjugated and stable form (Chen, Z., et al., 2009) (Kobayashi, M., et al., 2010). Though GP-1 could enhance the biosynthesis pathways of both JA and SA, WIPK expression was still induced by GP-1 treatment (Fig. 7). This result supported a JAdependent signal pathway was also involved in the plant protection by GP-1. This hypothesis was further confirmed by the increased contents of SA and JA after GP-1 treatment (Fig. 8).
As callose deposition was observed after GP-1 treatment, the expression of callose synthase (CalS) was further monitored. The results showed that GP-1 induced the expression of CalS with a similar pattern with that of HSR203J (Fig. 7), further proving the callose deposition effects of GP-1.
Its' well known that CaM involves in many cellular processes, such as, enzyme activity regulation, sexual regulation, cell division and differentiation, cytoskeleton and cell movement, photosynthesis, hormone response, nuclear enzyme system and gene

Conclusion
This study explained the mechanism of the GP-1-induced resistance of tobacco to the TMV.
Substantial GP-1 can be obtained by fermentation of Streptomyces sp. ZX01. This method is cost effective with simple extraction techniques and without subjection to external environmental conditions. In addition, GP-1 is highly active, safe for humans and animals, and is inexpensive to prepare. Therefore, this glycoprotein possesses significant potential for commercial agricultural applications.

12
The purification of the glycoprotein GP-1 was realised following the modified method of Zhang et al (2015). In short, the supernatant of Streptomyces sp. ZX01 culture was filtered using a 10 kDa ultrafiltration membrane. The filtrate was purified by using DEAE-52 column (10 × 2 cm). The 0.1 M NaCl eluent was collected, and then applied to a Sephadex G-75 gelfiltration column (1.2 cm × 100 cm), and eluted with deionized water at a flow rate of 0.3 mL/min. Total glycoproteins identitied and collected by HPLC (Ailgent 1260, USA) with TSK-GEL G2000SW XL column (7.8 × 300 mm, 5 μm). The molecular weight of GP-1 approximately is 8.5 kDa. GP-1 were sterilized by filtration through a Millipore filter (0.22 μm).

Cells culture and treatments
Tobacco suspension cells (N. tabacum var. samsun NN) (The cells obtained from Academy of Agricultural Sciences) were routinely propagated and cultured as described previously (Wang, W., et al., 2008). For the experiments, cells were reinoculated (1% w/v inoculum) during their exponential growth phase. At the fourth day of culture, GP-1 was added to the medium.

Evans blue staining and quantification
To observe the dying cells in GP-1 induced tobacco leaves, Evans blue staining was performed as described (Xing, F., et al., 2013). Detached leaves were submerged in Evans blue solution (0.25%, w/v) for 5 h. Then the leaves were boiled in 95% ethanol for 15 min to remove the chlorophyll completely for observation and photos taking. The blue precipitates were solubilized with 1% (w/v) SDS in 50% (v/v) methanol at 50 °C for 20 min and quantified by measuring the absorbance at 600 nm.

In vivo Detection of H 2 O 2 and ROS
The in vivo detection of H 2 O 2 was carried out using 3,3'-diaminobenzidine (DAB) according to Thordal-Christensen et al. (2010). DAB polymerizes locally as soon as it comes into 13 contact with H 2 O 2 in the presence of peroxidase, giving a reddish-brown polymer. DAB is taken up by living plant tissue and can be used to show H 2 O 2 production when peroxidase activity is present (Thordal-Christensen, H., et al., 2010). The leaves from tobacco were cut, placed in 1 mg/mL DAB-HCl, pH 3.8 (Sigma, MO, USA; #D-8001) and incubated in the growth chamber for 8 h prior to sampling. When sampling occurred later than 8 h after treatment, the leaves were cut and placed in water at the time of inoculation. At specific time-points after treatment the DAB reactions were examined in leaves cleared in boiling ethanol (96%) for 10 min. The samples were stored and examined in 96% ethanol. H 2 O 2 is visualized as a reddish-brown coloration.
When applied to intact cells, the nonionic, nonpolar DCFH-DA crosses cell membranes and is hydrolyzed enzymatically by intracellular esterases to nonfluorescent DCFH. In the presence of ROS, DCFH is oxidized to highly fluorescent dichlorofluorescein (DCF). Therefore, the intracellular DCF fluorescence can be used as an index to quantify the overall ROS in cells (Wang, H. & J.A. Joseph, 1999). Five μM (final concentration) DCFH-DA solubilized in ethanol were added to the 1 mL tobacco cell suspension cultures and incubated on a shaker at room temperature in the dark for 1 h and then rinsed twice with fresh suspension buffer to wash off excessive fluorophore probe. GP-1 (100 μg/mL) was added to the cells and incubated in microplate for 30 min before imaging with 488 nm excitation and 525 nm emission filters.

Determination of H 2 O 2
Hydrogen peroxide was extracted from plant tissues as described by Patterson et al. (1984). Fresh leaves or suspension cells (0.5 g) were homogenized in cold acetone in 1 mL acetone. Titanium reagent (20% TiCl 2 in HCl) was added to a known volume of extract supernatant to give a Ti (IV) concentration of 2%. The Ti-H 2 O 2 complex, together with unreacted Ti, was then precipitated by adding 0.2 mL 17 M ammonia solution for each 1 mL of extract. The precipitate was washed five times with acetone by resuspension, drained, and dissolved in 2 N H 2 SO 4 (3 mL). The absorbance of the solution was measured at 410 nm against blanks which had been prepared similarly but without plant tissue.
Measurement of nitric oxide (NO) and cytoplasmic Ca 2+ ([Ca 2+ ] cyt ) NO accumulation was determined using the fluorophore probe DAF-FMDA as described previously (Foresi, N., et al., 2010). Briefly, the tobacco suspension cells were incubated with 5 μM DAF-FMDA for 1 h in the dark at 25℃ on a rotary shaker (120 rpm) and then rinsed twice with fresh suspension buffer to wash off excessive fluorophore probe. Cells were then transferred into 96-well plates (100 μL of cells per well), and treated with GP-1 in the dark. NO production was measured using a 96-well Gemini EM Fluorescence Microplate Reader with 495 nm excitation and 515 nm emission filters. Fluorescence was expressed as relative fluorescence units. [Ca 2+ ] cyt accumulation were determined by corresponding fluorescent probe Fluo-3AM (5 μM) using the same method with NO except that the [Ca 2+ ] cyt experiment was operated at 37℃, the excitation and emission for Ca 2+ were 506 and 526-nm. For each treatment, measurements of NO and Ca 2+ production over time were performed on the same batch of cells.

Stomatal closure in epidermal strips
Stomatal bioassays were carried out essentially as described by Srivastava et al. (Srivastava, N., et al., 2009) Table 1. The reaction mixture was incubated for 30 s at 95°C, and for 40 cycles of 10 s at 95°C and 30 s at 56°C. Each assay included three technical and two biological replicates. The relative gene expression was quantified by using 2 -∆∆Ct method (Livak, K.J. & T.D. Schmittgen, 2001). At each time point, the relative expression of a gene from the treatment was compared against that from the control.

Library construction and RNA-seq analysis
Our previous study found that the expression level of most resistant genes reached the highest value at 11h after GP-1 treatment. Therefore, in this study, the transcriptome was measured in N. benthamiana that were treated with GP-1 for 11 h. N. benthamiana was treated with 100 μg mL -1 GP-1 and water, respectively. Five N. benthamiana leaves were mixed as a biological repeat, and repeated three times in each group. Total RNA was extracted using the TRIzol reagent. Library construction was performed according to instructions of NEBNext® Ultra TM RNA Library Prep Kit for RNA Illumina and sequenced on Illumina Hiseq TM 2500/Miseq TM sequencer. Raw reads in FASTQ format were first processed through in-house Perl scripts. Clean data were obtained by removing low-quality reads and reads containing ploy-N from raw data (Bolger et al., 2014). Clean reads were mapping to the reference sequence (transcript Homo_sapiens. GRCh38.p10) by Bowtie 2 v2.1.0 (Langmead et al., 2012), and the expression levels were analyzed by samtools v0.1.19 ). Next the DEG seq v1.20.0 (Wang et al., 2010)

Determination of salicylic acid and jasmonic acid
After treated with 100 µg mL − 1 of GP-1 by spraying, tobacco leaves were used for SA extraction and quantified by HPLC as described by Marianne (2002). The contents of jasmonic acid in leaves were measured by gas chromatograph according to the method described by Lan et al. (2004) and Deng et al.(2009)    Stomatal aperture was reduced in diameter after GP-1 or ABA treatment for 3 h.

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to download.