A Genetically Engineered Escherichia coli for Potential Utilization in Fungal Smut Disease Control

Sporisorium scitamineum, the basidiomycetous fungus that causes sugarcane smut and leads to severe losses in sugarcane quantity and quality, undergoes sexual mating to form dikaryotic hyphae capable of invading the host cane. Therefore, suppressing dikaryotic hyphae formation would potentially be an effective way to prevent host infection by the smut fungus, and the following disease symptom developments. The phytohormone methyl jasmonate (MeJA) has been shown to induce plant defenses against insects and microbial pathogens. In this study, we will verify that the exogenous addition of MeJA-suppressed dikaryotic hyphae formation in S. scitamineum and Ustilago maydis under in vitro culture conditions, and the maize smut symptom caused by U. maydis, could be effectively suppressed by MeJA in a pot experiment. We constructed an Escherichia coli-expressing plant JMT gene, encoding a jasmonic acid carboxyl methyl transferase that catalyzes conversion from jasmonic acid (JA) to MeJA. By GC-MS, we will confirm that the transformed E. coli, designated as the pJMT strain, was able to produce MeJA in the presence of JA and S-adenosyl-L-methionine (SAM as methyl donor). Furthermore, the pJMT strain was able to suppress S. scitamineum filamentous growth under in vitro culture conditions. It waits to further optimize JMT expression under field conditions in order to utilize the pJMT strain as a biocontrol agent (BCA) of sugarcane smut disease. Overall, our study provides a potentially novel method for controlling crop fungal diseases by boosting phytohormone biosynthesis.


Introduction
Sporisorium scitamineum is the fungal pathogen that causes sugarcane smut [1]. Diploid teliospores of S. scitamineum are airborne and can survive in harsh environmental conditions. The smut teliospores germinate on sugarcane buds, forming promycelium from which two pairs of haploid sporidia (basidiospores) with opposite mating types, MAT-1 and MAT-2, are generated via a round of meiosis. The sporidia of opposite mating types can recognize each other and fuse, undergoing sexual mating, and afterwards switch from a yeast-like growth style to dikaryotic hyphae, undergoing a so-called dimorphic switch. Dikaryotic hyphae are capable of infecting sugarcane, likely through buds. The smut disease symptoms are characterized by the emergence of a typical structure produced on the apex or side shoots of sugarcane stalks, called "smut whip", which contains teliospores formed from a fusion of two nuclei of the dikaryotic hyphae [2,3].

Total RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)
A RNeasy mini kit (Qiagen (Hilden, Germany), 74104) was used for total RNA extraction. A TURBO DNA-free kit (Invitrogen (Waltham, MA, USA), AM1907) and TransScript first-strand cDNA synthesis supermix (TransGen Biotech (Beijing, China), AT301) were, respectively, used for gDNA removal and first strand cDNA synthesis, following manufacturer's instructions. PowerUp SYBR green master mix (Applied Biosystems (Waltham, MA, USA), A25742) was used for qRT-PCR, with the primers as listed in Table 1, and the reaction was run on a Quant Studio 6 Flex Real-Time PCR system (Applied Biosystems). Relative expression folds were calculated by the 2 −∆∆CT method [22] with ACTIN as an internal control. Statistical analysis was performed by one-way ANOVA and Duncan's multiple analyses.

JMT Gene Induction
Induced expression of JMT gene: The pJMT-carrying strain (hereafter named as pJMT strain) was cultured in 5 mL LB broth containing 50 µg/mL kanamycin (Dingguo, AK177), at 37 • C and shaken at 200 rpm, until OD 600 ≈ 1.0. IPTG (Genview, CI175) was added to the pJMT strain culture to reach the final concentration of 1 mM, and then it was cultured at 28 • C and 200 rpm for another 12 h. The bacterial cells were collected by centrifugation at 1000× g and transferred to 50 mL of LB medium containing 200 µM jasmonic acid (JA, Sigma-aldrich (St. Louis, MO, USA), J2500) and 200 µM SAM (Sigma-aldrich, A4377), and incubated at 28 • C, 200 rpm for 12 h. The E. coli expressing the vector pRSFDUET-1 was used as a negative control.

MeJA or JA Detection
For the extraction of MeJA from the induced pJMT strain cultured in the presence of JA and SAM, the supernatant was collected after centrifuge and mixed with ethyl acetate of equal volume. After phase separation, the ethyl acetate phase was collected and dried by rotary evaporation (EYELA, OSB-2100). Such extracts were dissolved in 1 mL of methanol and filtered through a 0.22-µm microporous filter (Nylon6). For detection of JA from the extracts, high performance liquid chromatography (HPLC) was performed using the following settings: 0.1% formic acid; water = 65/35 (v/v); flow rate = 0.3 mL/min; detection wavelength = 205 nm; detection time = 20 min; and column temperature = 30 • C. The column is Agilent HC-C18(2) 250 × 4.6 mm (5 µm).
MeJA was identified by gas chromatography/mass spectrometry (GC/MS) on an Agilent 7890B/5977B (Agilent Technologies Inc., Santa Clara, CA, USA) series GC system equipped with an Agilent HP-5MS capillary column (30 m × 250 µm × 0.25 µm) in scan mode and split mode. The following temperature program was used: initial temperature of 60 • C (5 min hold), increase to 270 • C at 30 • C/min. Ion source temperature was 220 • C, and transfer line temperature was 270 • C. Electron ionization energy was 70 eV, and m/z = 30~450. MeJA was identified through standard compounds from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) library database.

Pathogenicity Assays
Pot experiment: Maize (Hua Meitian 9; DYQS-16 × TG-9) was grown in pots until the four-leaf stage, under the growth condition set as follows: humidity: 82%; light duration: 12 h. The mixed U. maydis MAT-1 and MAT-2 cells (OD 600 ≈ 1.0, 1:1 in volume), with or without 400 µM MeJA, were injected to the base of maize leaves. Injection with sterile water served as blank control. The injected maize seedlings were allowed to grow under natural conditions for 3 weeks, before examination of disease symptoms and photographing. The pJMT strain's effect on maize smut disease was tested following the same procedure. The pJMT strain was pre-treated with IPTG to induce expression of the JMT gene.
Field experiment: Approximately 290 seedlings of ratoon cane (ROC 22) were used for pJMT treatment, or untreated as control, respectively. The sugarcane cultivar ROC 22 was established as a susceptible cultivar towards smut fungus, as reported [23]. The pathogenicity and biocontrol assay were carried out on an established diseased field (Experimental fields of South China Agricultural University, 23.23 E, 113.63 W), which steadily causes smut disease to the susceptible cane (ROC 22) to a rate above 20%. The tested canes are 3rd year ratoon canes. Liquid-cultured pJMT (pre-treated with IPTG; the concentration of pJMT fermentation product to the soil is approximately 4 × 10 5 cell/cm 2 ) was applied within three days from germination of ratoons after cutting off the aboveground part of the canes, following the established procedure [24].

MeJA Suppresses S. scitamineum Mating/Filamentation
We first tested the effect of MeJA, a phytohormone used by plants against biotic or abiotic stresses [12], on S. scitamineum mating/filamentation, which is a critical step de-termining fungal pathogenicity. The result showed that, with 400-800 µM of MeJA, the filamentation (dikaryotic hyphae growth) of S. scitamineum was completely suppressed, and 200 µM MeJA caused reduced filamentation ( Figure 1A). In contrast, treatment of JA, the precursor of MeJA, did not affect S. scitamineum filamentation even under high concentration (800 µM; Figure 1A). We conclude that MeJA specifically suppressed S. scitamineum dikaryotic hyphae growth.
was applied within three days from germination of ratoons after cutting off the above-ground part of the canes, following the established procedure [24].

MeJA Suppresses S. scitamineum Mating/Filamentation
We first tested the effect of MeJA, a phytohormone used by plants against biotic or abiotic stresses [12], on S. scitamineum mating/filamentation, which is a critical step determining fungal pathogenicity. The result showed that, with 400-800 µM of MeJA, the filamentation (dikaryotic hyphae growth) of S. scitamineum was completely suppressed, and 200 µM MeJA caused reduced filamentation ( Figure 1A). In contrast, treatment of JA, the precursor of MeJA, did not affect S. scitamineum filamentation even under high concentration (800 µM; Figure 1A). We conclude that MeJA specifically suppressed S. scitamineum dikaryotic hyphae growth.  [19] during sexual mating/filamentous growth, using ZEISS Observer Z1. Arrows denote un-mating sporidia or hyphae. Scale bars = 20 µm. (C) qPCR analysis to assess transcription of a locus gene MFA 1/2 and PRA 1/2, and b locus BE 1/2 and BW 1/2 during S. scitamineum filamentation with or without MeJA treatment. The 1:1 ratio mixed MAT-1 and MAT-2 sporidia was allowed to grow on YePS medium under 28 °C, for 2 d, before total RNA extraction and qRT-PCR analysis. Relative gene expression level was calculated by the 2 −ΔΔCT method [22] with ACTIN as an internal control. Primers used for transcriptional profiling were listed in Table 1. Different letters indicate significant differences, n =  [19] during sexual mating/filamentous growth, using ZEISS Observer Z1. Arrows denote un-mating sporidia or hyphae. Scale bars = 20 µm. (C) qPCR analysis to assess transcription of a locus gene MFA 1/2 and PRA 1/2, and b locus BE 1/2 and BW 1/2 during S. scitamineum filamentation with or without MeJA treatment. The 1:1 ratio mixed MAT-1 and MAT-2 sporidia was allowed to grow on YePS medium under 28 • C, for 2 d, before total RNA extraction and qRT-PCR analysis. Relative gene expression level was calculated by the 2 −∆∆CT method [22] with ACTIN as an internal control. Primers used for transcriptional profiling were listed in Table 1. Different letters indicate significant differences, n = 3. Methanol (20 µL per 40 mL medium) served as solvent control in (A-C). The experiments were repeated three times.
We further assessed whether MeJA affected sexual mating by observing sporidia conjugation between two fluorescent proteins tagged sporidia of opposite mating types, and the following dikaryotic hyphae formation [19]. Under MeJA treatment, we observed abundant sporidia staying as monocyte status, failing to conjugate with the sporidia of the opposite mating type even though they were close to each other ( Figure 1B). In contrast, the untreated or the JA-treated sporidia can form the healthy hyphal, displaying mixed fluorescent signals, suggesting cellular fusion between two sporidia ( Figure 1B). This confirms that MeJA is able to suppress S. scitamineum sexual mating. We further measured the transcriptional levels of a and b locus genes, which control sexual mating and filamentous growth, respectively [25], under different treatment conditions. The results showed that the pheromone genes mfa1/2 and pheromones receptor gene pra1/2 increased their transcription significantly in the presence of MeJA. In contrast, the transcription levels Microorganisms 2023, 11, 1564 6 of 13 of b locus genes controlling filamentous growth and pathogenicity were suppressed by MeJA ( Figure 1C), likely accounting for reduced filamentous growth. Overall, MeJA is a phytohormone with potential in suppressing the pathogenic development of the sugarcane smut fungus.

MeJA Suppresses Maize Smut Disease Symptom in Pot Experiment
As it takes a long time for the systematic infection of S. scitamineum on sugarcane before whip symptoms develop, we instead used maize smut fungus U. maydis to test the effect of MeJA on fungal pathogenicity. U. maydis is a model fungus belonging to basidiomycetes and closely related to S. scitamineum. As it takes long time for typical "whip" symptoms to develop when caused by S. scitamineum infection to the sugarcane, processing a timely application of MeJA would be difficult. Here, we intend to test the potential effect of MeJA on smut disease suppression by using the U. maydis-maize system. First, we tested the effect of MeJA on U. maydis sexual mating and filamentous growth. As is similarly observed in S. scitamineum, MeJA was effective in suppressing U. maydis filamentous growth after sexual mating ( Figure 2A). Overall, MeJA caused reduced dimorphic switch in U. maydis.
We next tested the effect of MeJA on controlling maize smut disease. Approximately 60.87% of U. maydis infected seedlings displayed tumor symptoms on the stems or leaves, and some seedlings were even dead ( Figure 2B,C). MeJA treatment could effectively reduce tumor formation rate, to 41.38% ( Figure 2D). The death rate of U. maydis-infected seedling under MeJA treatment was significantly reduced as compared to un-treated seedlings ( Figure 2D). Therefore, we conclude that MeJA has potential in preventing smut disease caused by fungi.

Construction of Escherichia coli Expressing Plant JMT Gene
We next generated a plasmid for expressing the plant JMT gene (GenBank: AY008434.1), encoding the jasmonic acid carboxyl methyl transferase that catalyzes conversion from JA to MeJA [17]. The JMT gene was driven by a T7 promoter following the lacI repressor; therefore, it is inducible by isopropyl-β-D-thiogalactoside (IPTG) [21]. We transformed this constructed vector into E. coli (hereafter named as pJMT strain) and tested whether it could produce MeJA, by using JA as the precursor and SAM (S-adenosyl-L-methionine) as a methyl donor. Under in vitro culture conditions, IPTG induction led to a significant decrease in JA ( Figure 3A, retention time ≈ 18.5 min) content detected in the supernatant of pJMT strain when compared to what was detected in the supernatant from un-treated pJMT strain ( Figure 3A). This indicates that induced expression of JMT gene catalyzed conversion from JA to MeJA ( Figure 3B). Furthermore, we used GC/MS to detect the presence of MeJA in the supernatant of pJMT strain, with or without IPTG inducion. The results showed that MeJA was detected in IPTG-induced pJMT strain, while not in un-induced strain ( Figure 3C). Overall, the result showed that the induced JMT enzyme was capable of catalyzing conversion from JA to MeJA, likely using SAM as a methyl donor, under in vitro culture conditions. We further tested whether the pJMT strain was able to suppress S. scitamineum filamentous growth by producing MeJA. By either confrontation, or supplying the supernatant of pJMT culture, the dikaryotic hyphae growth of S. scitamineum was significantly suppressed, when the JMT gene was induced by IPTG ( Figure 4A,B). This indicates that the pJMT strain was able to produce and secrete MeJA, and has potential as a biocontrol agent against the sugarcane smut fungus.

Utilization of E. coli pJMT Strain in Controlling Smut Diseases
We next tested the effect of pJMT strain in controlling smut diseases, caused by U. maydis on maize or by S. scitamineum on sugarcane. As sugarcane smut caused by S. scitamineum takes a longer time (usually 3-6 months) to develop the typical "whip" symptom, we firstly tested the potential disease control capacity of pJMT on maize seedlings grown in pots. The results showed that U. maydis infection caused tumors formed on the stem of maize seedlings, either treated with pJMT strain or the control strain carrying vector only ( Figure 5). The disease incidence rate seems to show no obvious difference between pJMT or the control strain-treated plants ( Figure 5). Therefore, the pJMT strain did not seem to be effective in suppressing tumor formation on the maize stems or leaves.  we used GC/MS to detect the presence of MeJA in the supernatant of pJMT strain, with or without IPTG inducion. The results showed that MeJA was detected in IPTG-induced pJMT strain, while not in un-induced strain ( Figure 3C). Overall, the result showed that the induced JMT enzyme was capable of catalyzing conversion from JA to MeJA, likely using SAM as a methyl donor, under in vitro culture conditions. We further tested whether the pJMT strain was able to suppress S. scitamineum filamentous growth by producing MeJA. By either confrontation, or supplying the supernatant of pJMT culture, the dikaryotic hyphae growth of S. scitamineum was significantly suppressed, when the JMT gene was induced by IPTG ( Figure 4A,B). This indicates that the pJMT strain was able to produce and secrete MeJA, and has potential as a biocontrol agent against the sugarcane smut fungus. Under laboratory conditions, pJMT strain showed a significant inhibitory effect on the dimorphic switch of S. scitamineum ( Figure 4B). We assumed that it could be a potential biocontrol agent for protecting sugarcane from smut disease. A field experiment was conducted to evaluate the control ability of pJMT strain against sugarcane smut. Approximately 290 sugarcanes from 90 clumps were grown from ratoons in an established diseased field and used for untreated control and pJMT treatment plots, respectively. Liquid-cultured pJMT (pre-treated with IPTG; the concentration of pJMT fermentation product to the soil is approximately 4 × 10 5 cell/cm 2 ) were applied within three days from the germination of ratoons. The disease canes grew short and slim, and the whips appeared from the trunks of a very early stage, in either untreated or pJMT-treated plots ( Figure 6). There was no significant difference in the disease-occurring rate between the untreated control plot (20.6% diseased seedlings out of a total 294 seedlings) and the pJMT strain treatment (18.6% diseased out of a total of 294 seedlings).
In summary, the engineered E. coli pJMT strain-which can catalyze a JA-to-MeJA conversion-displayed a mild suppression effect on maize smut in the pot experiment and had no obvious effect on sugarcane smut under field conditions. This strain needs further optimization before it can be applied as a biocontrol agent against smut diseases.

Utilization of E. coli pJMT Strain in Controlling Smut Diseases
We next tested the effect of pJMT strain in controlling smut diseases, caused by U. maydis on maize or by S. scitamineum on sugarcane. As sugarcane smut caused by S. scitamineum takes a longer time (usually 3-6 months) to develop the typical "whip" symptom, we firstly tested the potential disease control capacity of pJMT on maize seedlings grown in pots. The results showed that U. maydis infection caused tumors formed on the stem of maize seedlings, either treated with pJMT strain or the control strain carrying vector only ( Figure 5). The disease incidence rate seems to show no obvious difference between pJMT or the control strain-treated plants ( Figure 5). Therefore, the pJMT strain did not seem to be effective in suppressing tumor formation on the maize stems or leaves. Under laboratory conditions, pJMT strain showed a significant inhibitory effect on the dimorphic switch of S. scitamineum ( Figure 4B). We assumed that it could be a potential biocontrol agent for protecting sugarcane from smut disease. A field experiment was conducted to evaluate the control ability of pJMT strain against sugarcane smut. Approximately 290 sugarcanes from 90 clumps were grown from ratoons in an established diseased field and used for untreated control and pJMT treatment plots, respectively. Liquid-cultured pJMT (pre-treated with IPTG; the concentration of pJMT fermentation product to the soil is approximately 4 × 10 5 cell/cm 2 ) were applied within three days from the germination of ratoons. The disease canes grew short and slim, and the whips were used for testing smut con trol capacity of pJMT strain. Three plots containing approximately 290 seedlings, out of 90 clump were, respectively, set as control or pJMT treatment plots. The pJMT culture was pre-treated wit IPTG before being applied to the ratoons within 3 days after cutting the above-ground parts. Dis ease symptoms, including typical symptom and obvious growth defects of the infected seedling were evaluated at three months post growth of the ratoons. A seedling with typical black whi and slim stem symptoms is displayed in left panel. The disease occurring rate based on the num ber of black whips is quantified and presented in the right panel. A significant difference (* p 0.0165) in the number of diseased seedlings in comparison between the pJMT treatment and un treated (control) plot was determined by a two-tailed Student's t-test.

Discussion
Sugarcane smut caused by Sporisorium scitamineum is considered as the most seriou and widespread disease of sugarcane [2], severely affecting sugarcane yields and qual ty, and thus causes significant economic loss [26][27][28]. The disease control methods fo sugarcane smut include physical control, fungicide control, and disease-resistant breed ing [29][30][31]. The application of fungicides is not only expensive, but also leads to seriou environmental pollution problems and drug-resistance to the pathogens with increasin fungicide doses in pursuit of the desired control effects. Although disease-resistan breeding is the most effective method to control sugarcane smut in agricultural practic es, the breeding process of disease-resistant varieties is difficult and time-consumin [32,33], and ever-evolving pathogens cause the loss of resistance of disease-resistant va rieties [34]. At present, a highly efficient, sustainable, and environmentally friendly con trol strategy for sugarcane smut is still lacking, and is in urgent need.
Phytohormones not only regulate plant growth, but also precipitate/regulate plan immune responses to pathogens. Auxin regulates plant cell division, enlargement, an differentiation [35]. Exogenous addition of auxin increases the tolerance of Medicag truncatula to Macrophomina phaseolina infection [36], indicating a positive regulation o plant immunity. In Arabidopsis thaliana, the exogenous addition of auxin affects the pro liferation of Pseudomonous syringae [37], but does not affect Fusarium oxysporum [38]. Sa icylic acid (SA) and jasmonic acid (JA) are important defense hormones. Necrotrophi pathogens can activate JA-related defense responses [39]. SA plays an important role i defending against infection by biotrophic and hemibiotroph pathogens [40]. Moreove as a plant hormone, SA can interact with a variety of plant hormone-related signalin pathways to activate the immune response and disease resistance of plants [41]. The ap plication of MeJA to roots or foliar tissues can enhance transcriptional expression level  (23.23 E, 113.63 W) were used for testing smut control capacity of pJMT strain. Three plots containing approximately 290 seedlings, out of 90 clumps, were, respectively, set as control or pJMT treatment plots. The pJMT culture was pre-treated with IPTG before being applied to the ratoons within 3 days after cutting the above-ground parts. Disease symptoms, including typical symptom and obvious growth defects of the infected seedlings, were evaluated at three months post growth of the ratoons. A seedling with typical black whip and slim stem symptoms is displayed in left panel. The disease occurring rate based on the number of black whips is quantified and presented in the right panel. A significant difference (* p = 0.0165) in the number of diseased seedlings in comparison between the pJMT treatment and untreated (control) plot was determined by a two-tailed Student's t-test.

Discussion
Sugarcane smut caused by Sporisorium scitamineum is considered as the most serious and widespread disease of sugarcane [2], severely affecting sugarcane yields and quality, and thus causes significant economic loss [26][27][28]. The disease control methods for sugarcane smut include physical control, fungicide control, and disease-resistant breeding [29][30][31]. The application of fungicides is not only expensive, but also leads to serious environmental pollution problems and drug-resistance to the pathogens with increasing fungicide doses in pursuit of the desired control effects. Although disease-resistant breeding is the most effective method to control sugarcane smut in agricultural practices, the breeding process of disease-resistant varieties is difficult and time-consuming [32,33], and ever-evolving pathogens cause the loss of resistance of disease-resistant varieties [34]. At present, a highly efficient, sustainable, and environmentally friendly control strategy for sugarcane smut is still lacking, and is in urgent need.
Phytohormones not only regulate plant growth, but also precipitate/regulate plant immune responses to pathogens. Auxin regulates plant cell division, enlargement, and differentiation [35]. Exogenous addition of auxin increases the tolerance of Medicago truncatula to Macrophomina phaseolina infection [36], indicating a positive regulation of plant immunity. In Arabidopsis thaliana, the exogenous addition of auxin affects the proliferation of Pseudomonous syringae [37], but does not affect Fusarium oxysporum [38]. Salicylic acid (SA) and jasmonic acid (JA) are important defense hormones. Necrotrophic pathogens can activate JA-related defense responses [39]. SA plays an important role in defending against infection by biotrophic and hemibiotroph pathogens [40]. Moreover, as a plant hormone, SA can interact with a variety of plant hormone-related signaling pathways to activate the immune response and disease resistance of plants [41]. The application of MeJA to roots or foliar tissues can enhance transcriptional expression levels of resistance genes in sugarcane towards various classes of soil-borne pests and pathogens [42]. MeJA increases plant resistance by regulating antioxidant defense systems in sugarcane seedlings [43], Panax ginseng C.A. Mey. roots [44], and Glycine max L. leaves [45]. Furthermore, foliar application of MeJA could improve the grape and wine quality [46].
In this study, we found that the phytohormone MeJA could inhibit the dimorphic transition, a critical step in pathogenic development, of smut fungi S. scitamineum and U. maydis (Figures 1 and 2A). The a and b locus play an important role in sexual mating and hypha formation [3,5,24]. The qRT-PCR results of the coding genes showed that MeJA inhibits S. scitamineum sexual mating while causing a significant increase in the transcriptional levels of the mating type genes, pheromone genes mfa1/2, and pheromones receptor gene pra1/2. We infer that suppressed transcription of b locus genes is more important for regulating filamentation, which is inhibited by MeJA. Up-regulation of the a locus genes does not guarantee promoted sexual mating, as the encoded pheromone peptides need post-translational modification [8] for activation, and the pheromone receptors Pra1/2 need to recognize the activated pheromone peptide from the opposite mating-type [47]. In addition, we noticed a limited difference in the transcription of the a and b locus genes in presence of MeJA, which would almost completely suppress mating/filamentous growth. We hypothesize that in addition to suppression of a and b locus gene transcription, MeJA may target other pathways critical to the filamentation of S. scitamineum, which were not identified in this study.
Furthermore, we found that the exogenous addition of MeJA suppressed disease symptoms (tumors formation) caused by a U. maydis infection in maize seedlings, under the pot experiment ( Figure 2B). The pJMT strain, heterogeneously expressing the plant JMT gene, was able to catalyze MeJA production using JA as a precursor ( Figure 3). Although, under in vitro culture conditions, the formation of S. scitamineum dikaryotic hyphae could be effectively inhibited by confrontation or by supplying the supernatant of the pJMT culture (Figure 4), suggesting that such a MeJA-producing pJMT strain could be potentially used as a biocontrol agent against smut diseases caused by the fungal pathogens. However, direct application of this pJMT strain to the infected maize or sugarcane seedlings, in pot experiments or in field experiments, did not show an obvious disease control ability ( Figures 5 and 6). When performing the pot and field experiments, we pre-treated the pJMT culture with IPTG to induce JMT expression. A possible reason for the failure of the pJMT strain in controlling smut diseases may be the fact that the symptom development process in the field is long and complicated. In the future, we would like to further modify this strain by using a strong, constitutive, and native promoter of E. coli to drive the JMT gene. Alternatively, a pathogen-inducible promoter would be screened and tested.
In conclusion, our results demonstrate that the phytohormone MeJA could suppress a dimorphic switch, which was established as a key step of pathogenicity for smut fungi (S. scitamineum and U. maydis). We further constructed a pJMT strain by expressing the plant's JMT gene, and verified that the engineered strain could catalyze a conversion from JA to MeJA. At present, this pJMT construct showed no capacity in suppressing smut disease either in maize (pot condition) or sugarcane (field condition). However, the idea that a bacteria strain expressing a JMT gene could utilize a plant-source JA to facilitate MeJA production was shown to suppress the dikaryotic hyphae growth of smut fungi in this study. Future attempts will be conducted to monitor and enhance the colonization ability and/or strain stability of the pJMT strain, with the aim to develop it as a useful tool in the prevention and management of smut diseases.