Overexpression of Nepenthesin Hvnep-1 in Barley Endosperm Reduces Fusarium Head Blight and Mycotoxin Accumulation

agronomy

Article Overexpression of Nepenthesin HvNEP-1 in Barley Endosperm Reduces Fusarium Head Blight and Mycotoxin Accumulation

Zelalem Eshetu Bekalu 1,*, Claus Krogh Madsen 1, Giuseppe Dionisio 1 , Inger Bæksted Holme 1 , Lise Nistrup Jørgensen 2, Inge S. Fomsgaard 2 and Henrik Brinch-Pedersen 1 1 Department of Molecular Biology and Genetics, Research Center Flakkebjerg, Aarhus University, DK-4200 Slagelse, Denmark; [email protected] (C.K.M.); [email protected] (G.D.); [email protected] (I.B.H.); [email protected] (H.B.-P.) 2 Department of Agroecology, Research Center Flakkebjerg, Aarhus University, DK-4200 Slagelse, Denmark; [email protected] (L.N.J.); [email protected] (I.S.F.) * Correspondence: [email protected]

Received: 21 December 2019; Accepted: 29 January 2020; Published: 1 February 2020

Abstract: Fusarium head blight (FHB) causes substantial losses of yield and quality in grains, both in the ﬁeld and in post-harvest storage. To date, adequate natural genetic resistance is not available for the control of FHB. This study reports the cloning and overexpression of a barley (Hordeum vulgare L.) antifungal gene, nepenthesin 1 (HvNEP-1), in the endosperm of barley grains. Transgenic barley lines overexpressing HvNEP-1 substantially reduced FHB severity and disease progression after inoculation with Fusarium graminearum or Fusarium culmorum. The transgenic barley also showed reduced accumulation of the mycotoxin deoxynivalenol (DON) in grain, far below the minimum value allowable for food. Semi-ﬁeld evaluation of four HvNEP-1 transgenic lines revealed substantial reduction of FHB severity and progression as compared with the control H. vulgare cultivar Golden promise (GP) plants. Our study demonstrated the utility of HvNEP-1 for the control of FHB in barley, and possibly other grains such as wheat and maize.

Keywords: Hordeum vulgare; HvNEP-1; endosperm; overexpression; FHB; severity; progression; mycotoxins

1. Introduction Fungal pathogens cause considerable loss of yield and quality in economically important crops. Fusarium head blight (FHB) or scab is one of the major fungal diseases of the Triticeae family in temperate and warm, humid regions of the world [1]. FHB is caused by several Fusarium species, but Fusarium graminearum and Fusarium culmorum are seen as two of the most economically relevant species [1–3]. In the 1990s, an FHB epidemic caused an estimated loss of 2.7 billion USD in the US alone [4]. In 2010, a report on the occurrence of FHB in the US indicated that the disease was on the decline in most states. However, in parts of Ohio, the FHB incidence reached up to 60% [5]. In the same year, the overall impact of the disease in the state of Kansas was valued at 13 million USD. More recently, the state of Kansas experienced an estimated 2.1% yield loss, due to FHB in 2019 [6]. Overall, FHB could lead to >1 billion USD economic losses per year [7]. FHB causes shriveled grains and contamination of grain with mycotoxins, which are harmful to humans and animals if consumed. Mycotoxins produced by Fusarium species belong to the trichothecenes and include deoxynivalenol (DON), nivalenol (NIV), and their derivatives 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), and 4-acetylnivalenol

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(4-ANIV) [8]. Fusarium species also produce other mycotoxins such as zearalenone (ZEA), moniliformin, fumonisins, and butenolide. Most of these mycotoxins are associated with fungal virulence and cause toxicosis in humans and other animals [9–11]. Genetic resistance is a highly effective strategy for managing most diseases; however, to date, few wheat (Triticum spp.) and barley accessions with even moderate resistance to FHB have been reported [12]. Resistance to FHB is a quantitative trait that is governed by the combined effects of several quantitative trait loci (QTL), epistasis, and the environment [13]. A major QTL (Fhb1) located on chromosome 3BS and several minor QTL derived from the Chinese cultivar Sumai 3 are the main sources of genetic resistance to FHB in wheat [12,14]. Recently, fine mapping of the Fhb1 locus identified the casual genes associated with Fhb1-mediated resistance in wheat [15–18]. In contrast, sources of FHB resistance are more limited in barley and provide only a modest level of resistance. Due to the polygenic nature of FHB resistance, the development of resistant cultivars with suitable agronomic traits is a challenge [19,20]. Hence, the discovery of antifungal or antitoxin genes in barley would be of considerable interest for the development of FHB resistant cultivars. Aspartic proteases play important roles in biotic and abiotic responses in plants [21,22]. Nepenthesin (EC 3.4.23.12), named after the carnivorous plant Nepenthes, represents a distinct group of aspartic proteases from the A1B subfamily [23]. They have primarily been reported from the pitcher fluid of Nepenthes plants [24,25], and account for the majority of protease activity in the fluid [26]. Purification and characterization of proteases from the pitcher fluid has confirmed that the protease activity was indeed mainly ascribed to nepenthesins [27–29]. Subsequently, cDNA clones of nepenthesins were characterized from Nepenthes alata [30] and N. gracilis [31,32]. Furthermore, nepenthesins were also directly purified and characterized from the pitcher fluid of N. distillatoria [31] and other carnivorous plants [33,34]. Recently, we reported that barley crude protein extracts inactivate microbial phytases via aspartic acid proteinase activity, rather than through classical enzyme inhibition [35]. Activity inhibition-based purification and MS analysis of inhibitory fractions identified HvNEP-1 as a potent inhibitor of fungal phytases [36]. Moreover, the addition of recombinant HvNEP-1 (rHvNEP-1) to the in vitro grown fungus caused a significant reduction of fungal biomass and complete suppression of 15-ADON accumulation [36]. Our recent report presented as a patent described the implication of HvNEP-1 mediated fungal disease resistance in major crop plants [37]. Here, we describe the in vivo characterization of the barley HvNEP-1. Transgenic barley lines overexpressing HvNEP-1 in the endosperm limited FHB disease progression and, substantially, reduced levels of mycotoxin accumulation in the grain. Our findings indicate that HvNEP-1 is an antifungal gene that can be used to control FHB in barley, and possibly other cereals.

2. Materials and Methods

2.1. Plasmid Construction for Plant Transformation For plant transformation, the sequence for the signal peptide lacking HvNEP-1 was PCR amplified from gDNA using primer pairs P1 (see primer information in Table S1). PCR was carried out in a 50 µL reaction mixture containing 100 ng gDNA template (2 µL), 10 µl 5 Herculase II Buffer, 10 µL × 2 mM dNTP mixture, 4 µL primer mixture P1 (10 pmol/µL), 0.5 µL Herculase II DNA polymerase (2 U/µL), 3 µL DMSO, and 20.5 µL MQ. The PCR conditions were a denaturation step at 96 ◦C for 2 min, then, 40 cycles of 96 ◦C for 1 min, 60 ◦C for 20 sec, 72 ◦C for 2.30 min, and a final extension step at 72 ◦C for 2.30 min. Amplicons were detected by electrophoresis in 1% agarose gels stained with ethidium bromide. An endoplasmic reticulum (ER) sorting sequence (SE) KDEL was introduced at the sequence for the C-terminal end of HvNEP-1. The barley HordD signal peptide (HordDsp) was used in place of the HvNEP-1 native signal peptide. The HordD promoter (HordDp) and signal peptide were PCR amplified with primer pairs P2 under the following conditions: initial denaturation at 96 ◦C for 2 min, then 40 cycles of 96 ◦C for 1 min, 60 ◦C for 20 sec, 72 ◦C for 1 min, and a final extension Agronomy 2020, 10, 203 3 of 13

step at 72 ◦C for 2.30 min, and a 4 ◦C hold. The amplicon was fused upstream of the ∆HvNEP-1 and inserted into the barley transformation vector pWBVec8 by In-fusion cloning [38]. In the resulting construct, pWBVec8: HordDp: HordDsp: ∆HvNEP-1: NOS, HvNEP-1 was fused downstream of the HordD promoter and upstream of the Agrobacterium tumefaciens-derived NOS terminator.

2.2. Generation of Transgenic Barley Plants The construct was used to transform competent Agrobacterium strain AGL0 using the method already described by An et al. [39]. The bacteria were grown on LB plates containing 100 µg/mL spectinomycin and 25 µg/mL rifampicin for 72 h at 28 ◦C. Positive colonies were identiﬁed by PCR using gene-speciﬁc primers. A positive clone was inoculated into 5 ml MG/L medium (5 g/L mannitol, 1 g/L L-glutamic acid, 0.25 g/L KH PO , 0.1 g/L NaCl, 0.1 g/L MgSO 7H O, 1ng/L biotin, 5 g/L tryptone, 2 4 4· 2 and 2.5 g/L yeast extract) [40], containing 100 µg/mL spectinomycin and 25 µg/mL rifampicin and grown by shaking (150 rpm, 28 ◦C, 48 h). Sterile 15% glycerol stocks were prepared and stored at 80 C. For barley transformation, 200 µL of the glycerol stock was used to inoculate 5 ml MG/L − ◦ medium and grown overnight by shaking (180 rpm, 28 ◦C). The culture was used for transformation of immature barley embryos isolated from barley cultivar Golden promise (GP) following the procedure described by Holme et al. [41] and Bartlett et al. [42].

2.3. PCR Analysis of Transformed Barley Plants After transformation, selection, and regeneration of T0 plants, the gDNA was isolated from young leaves using the procedure described in [43]. PCR was performed to conﬁrm the presence of the hygromycin resistance gene hpt using primer pair P3. Positive transformants were further conﬁrmed with PCR using primer pair P4 designed inside the HordDp and HvNEP-1. The resulting PCR fragment size was 759 bp. Untransformed GP plants used as the negative control were analyzed similarly using both primer pairs.

2.4. Inoculation of Transgenic Barley Lines with Fusarium Two DON chemotype Fusarium isolates, F. graminearum 7775 and F. culmorum 8984, were used to inoculate barley plants. Spore suspensions of both strains were prepared according to Etzerodt et al. [44]. Eight weeks after germination (Zadoks growth stage 60 [45] ), spikes of 10 T0 plants were spray inoculated with a spore suspension (1 105 spores per ml in water containing 0.04% tween 20) (Figure S1a). Brieﬂy, × tillers of the T0 plants were subdivided into three sections. Two of the sections were inoculated with the two Fusarium isolates and the remaining section was sprayed with Milli-Q water (MQ water) (Milli-Q®Direct Water Puriﬁcation System, Merck, Germany) as a control. Two untransformed GP plants at the same stage of development were treated similarly with Fusarium spores and MQ water. Subsequently, the plants were covered with plastic bags for 3 days and transferred into the greenhouse (18 to 21 ◦C and relative humidity 70% to 75%).

2.5. Disease Scoring and Mycotoxin Analysis Spikes of the individual transgenic lines and GP control plants were scored for disease symptoms (FHB severity) at 1, 2, and 3 weeks after inoculation. Percent disease severity was scored as the percentage of infected seeds in a spike. Since the transgene segregated in the T0 plants, the first 3 matured spikes from each transgenic and GP control plants were selected and scored for disease severity. Grains from the selected spikes were used for analysis of mycotoxins and HvNEP-1 expression. Mycotoxin accumulation (DON, NIV, ZEA, T-2, and HT-2) in the seeds of the transgenic lines and control GP plants was quantified in an LC-MS/MS system consisting of an Agilent 1260 infinity chromatographic system (Santa Clara, CA, USA) coupled to an AB Sciex 3200 triple-quadrupole trap mass spectrometer (QTRAP/MS) (AB Sciex, Framingham, USA) according to Etzerodt et al. [46]. Analyst 1.6.3 software (AB Sciex, Framingham, USA) was used to control the LC-MS/MS system. Agronomy 2020, 10, 203 4 of 13

The same numbers of seeds per treatment were used for the analysis of mycotoxins and HvNEP-1 expression. In the Fusarium-treated transgenic and GP control plants, seeds were collected and analyzed separately for F. graminearum (FG) and F. culmorum (FC). For each treatment, 3 seeds per spike (9 seeds per treatment) were collected in liquid nitrogen and dissected longitudinally into equal parts. The seeds were pooled by treatment and one half of each pooled sample was freeze dried, ground, and used for mycotoxin analysis; the other pooled half was used for analysis of HvNEP-1 expression. Similarly, seeds from the non-inoculated control and the GP control plants were collected and analyzed for mycotoxins.

2.6. HvNEP-1 Expression in the Transgenic Barley Lines Total RNA was extracted from the pooled seeds using the method described by Chomczynski and Sacchi [47]. RNA samples were treated with DNase according to the manufacturer’s recommendation (Roche). Reverse transcription of mRNA was performed using Superscript III-RT (Invitrogen) and oligo (dT) 21T-anchor-containing primers. The qPCR was performed in a ﬁnal volume of 12 µL containing 6 µL Power SYBR Green master mix (Applied Biosystems), 1 µL diluted cDNA, 2.4 µL of 1.5 µM primer mix, and 2.6 µL sterile Milli Q water. The samples were set up in 384-well plates and the qPCR was run and detected in an AB7900HT sequence detection system (Applied Biosystems, USA). Gene-speciﬁc qPCR primers were designed according to the available HvNEP-1 sequence. Expression analysis of the seeds from Fusarium (FG, FC) and water (MQ) treated spikes were conducted separately for the transgenic and control plants. The level of Splicing factor 2 (SP2) gene expression was used to normalize HvNEP-1 expression. The qPCR primers are described in the Table S1.

2.7. Field Soil Plot Analysis of Transgenic Barley Lines for Fusarium Resistance

The segregating T1 generation from 4 selected lines (NEP02-01, NEP16, NEP18, and NEP20) and the GP-1 control plants were grown on 5 plots (0.6 m 2.2 m) in a controlled semi-ﬁeld facility × at Aarhus University (Flakkebjerg, 55.321547◦ N) (Figure S1b). Transgene-positive plants from the segregating T1 generation were selected by PCR using hpt primers. The transgenic and non-transgenic control plants (Zadoks stage 60) were spray-inoculated with F. graminearum 7775 spores and covered with nylon mesh for 3 days. Ten transgene-positive plants from the transgenic lines and 53 GP-1 control plants were used to study disease infection rate and progression. The plants were assessed for disease severity on a scale of 1 to 9. In addition, the disease progression (AUDPC) was also examined at 1, 2, and 3 weeks after inoculation.

3. Results

3.1. HvNEP-1-Overexpressing Barley Lines and Assessment of FHB Resistance The overexpression construct was designed to express HvNEP-1 speciﬁcally in the endosperm of barley grains (Figure1a). Immature barley embryos were transformed with the construct using the Agrobacterium strain AGL0. A total of 525 immature embryos were infected with Agrobacterium and 82 T0 plants were regenerated. Among the 82 T0 plants, 24 plants were selected and tested for the insertion of HvNEP-1 by PCR. All tested plants were positive for the transgene. Plants regenerated from 20 out of the 24 lines were transferred into soil and grown in the greenhouse. Ten of the transgenic lines were randomly selected for downstream analysis (Figure1b,c). The lines analyzed by qPCR for seed HvNEP-1 expression showed varying expression of the transgene between the lines. The highest relative expression was seen in line NEP20 (0.4166 ∆Ct) and the lowest was detected in line NEP20-02 (0.0114 ∆Ct) as compared with the reference gene SP2 (Figure1d). Agronomy 2020, 10, 203 5 of 13

Figure 1. Analysis of transgenic barley lines overexpressing HvNEP-1.(a) Gene expression construct used for Agrobacterium-mediated transformation, HordDp: D-hordeum promoter, HordDsp: D-hordeum signal peptide, ∆HvNEP-1: signal peptide lacking HvNEP-1, and NOS terminator; (b) transgenic HvNEP-1-overexpressing Figure 1. Analysis of transgenic barley lines overexpressing HvNEP-1. (a) Gene expression construct used for Agrobacterium-mediated transformation, HordDp: D- barley lines used for downstream studies; (c) PCR analysis of transgenic HvNEP-1-overexpressing barley lines using P5 amplifying fragments inside the HordD hordeum promoter, HordDsp: D-hordeum signal peptide, ΔHvNEP-1: signal peptide lacking HvNEP-1, and NOS terminator; (b) transgenic HvNEP-1-overexpressing promoter and HvNEP-1 gene; and (d) qPCR analysis of transgenic lines with variable expression of the transgene. Values in d are the mean of 3 independent technical barley lines used for downstream studies; (c) PCR analysis of transgenic HvNEP-1-overexpressing barley lines using P5 amplifying fragments inside the HordD promoter replicates and were analyzed by Student’s t-test. Error bars = mean + sd. Signiﬁcant diﬀerences between transgenic and non-transgenic plants are labelled with and HvNEP-1 gene; and (d) qPCR analysis of transgenic lines with variable expression of the transgene. Values in d are the mean of 3 independent technical replicates and asterisks: * p < 0.05, ** p < 0.01, and *** p < 0.001. were analyzed by Student’s t-test. Error bars = mean + sd. Significant differences between transgenic and non-transgenic plants are labelled with asterisks: *p < 0.05, ** p < 0.01, and ***p < 0.001.

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The transgenic lines were assessed for FHB resistance and mycotoxin accumulation at the 85 to 87 growth stage (in the Zadoks scale, [45]). Disease severity of the 10 T0 lines was compared to two untransformed control GP plants. FHB severity was scored as the percentage of infected seeds in a spike after one, two, and three weeks after inoculation. The transgenic lines showed a substantial reduction in FHB severity as compared with the control plants. The mean percent of infection in the transgenic plants varied from 3.41% to 23.08%, whereas the mean percent of infection in the control GP plants ranged from 31.88% to 50% for both F. graminearum and F. culmorum strains (Figure2a). The progression of FHB in the spikes of the transgenic lines and control GP plants was assessed for the ﬁrst three weeks after inoculation, and the area under the disease progress curve (AUDPC) was calculated (Figure2b). The mean AUDPC of FHB progress was higher in the control GP plants than thatAgronomy in the 2019 transgenic, 9, x FOR PEER lines. REVIEW 2 of 13

Figure 2. Transgenic barley lines overexpressing HvNEP-1 showed reduced severity of Fusarium Figure 2. Transgenic barley lines overexpressing HvNEP-1 showed reduced severity of Fusarium head blight (FHB) in the greenhouse. (a) FHB disease severity scoring 3 weeks after inoculation with head blight (FHB) in the greenhouse. (a) FHB disease severity scoring 3 weeks after inoculation with F. graminearum (FG) and F. culmorum (FC) spores and the water control Milli-Q water (MQ). Values are F. graminearum (FG) and F. culmorum (FC) spores and the water control Milli-Q water (MQ). Values the mean of 3 independent technical replicates. Error bars = mean + sd. Letters on the top of the are the mean of 3 independent technical replicates. Error bars = mean + sd. Letters on the top of the column bars indicate statistically significant differences (p = 0.05, least significant difference (LSD) test); column bars indicate statistically significant differences (p = 0.05, least significant difference (LSD) (b) area under the disease progress curve (AUDPC) analysis of FHB disease progression evaluated for test); (b) area under the disease progress curve (AUDPC) analysis of FHB disease progression 3 weeks after inoculation. Bars refer to the median AUDPC, and the minimum and maximum values evaluated for 3 weeks after inoculation. Bars refer to the median AUDPC, and the minimum and are indicated with error bars. maximum values are indicated with error bars.

3.3. Semi-Field Analysis of the HvNEP-1 Transgenic Lines To test the reliability of HvNEP-1-mediated resistance to FHB, four Fusarium-infected transgenic lines and control GP plants were grown in a field plot covered with an insect-proof net (Figure S1b). Ten transgene-positive plants from each transgenic line were compared to 53 untransformed GP-1 plants. Line NEP20 showed the lowest disease infection rate and progression as compared with the other transgenic lines (NEP02-01, NEP16 and NEP18) and the untransformed control plant GP-1 (Figure 3a). Three weeks after infection, line NEP20 had an average infection score of 1.9, whereas the control GP plants were scored at 4.15 (on a one to nine scale) (Figure 3b). AUDPC was calculated at one, two, and three weeks after inoculation. After three weeks, the AUDPC was significantly higher in the GP control plants (35%) than in the transgenic NEP20 plants (24.5%) (Figure 3c).

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3.2. Mycotoxin Analysis of the HvNEP-1-Overexpressing Lines The mycotoxins levels were analyzed in the grains of Fusarium-infected and water-treated transgenic (T0) and non-transformed GP plants three weeks after inoculation (Table1). Fusarium-infected seeds were sorted into seeds with FHB symptoms (FC/FG, FHB+) and seeds with no FHB symptoms (FC/FG, FHB–). In FHB- seeds from transgenic plants, mycotoxin levels were below detection level in all samples, except from some NIV accumulation in NEP02-01 (FC, 77 µg/kg DM and FG, 124 µg/kg DM) and NEP06 (FG, 171 µg/kg DM). Water-treated control plants showed no sign of infections and mycotoxin accumulation. Similarly, water-treated transgenic plants did not develop infections, although a minor NIV level (163 µg/kg DM) was seen in line NEP16. In the FHB+ non-transgenic plants, both F. culmorum and F. graminearum caused a strong accumulation of DON, and also a strong accumulation of NIV (except for GP-2) and ZEA (except for GP-1). The highest levels of DON were seen in F. culmorum-infected (FC, FHB+) GP-1 control seeds. In the transgenic plants infected with F. culmorum (FC, FHB+), DON levels were substantially reduced as compared with the F. culmorum-infected (FC, FHB+) non-transgenic GP plants. For nine out of the 10 transgenic plants, DON levels were below the detection level. For the NEP20 line, in which DON levels were detected, we measured 152 µg DON/kg DW, which was still far below the DON levels in the controls. For the F. graminearum-infected (FG, FHB+) seeds of transgenic plants, DON levels were below the detection level in six out of the 10 lines. The NEP04 line had a DON content just above the detection level, but NEP19 and NEP20 had high levels of DON (8170 and 40,073 µg/kg DW, respectively). ZEA levels were also strongly reduced in the transgenic lines. Only line NEP20 had a detectable ZEA level (256 µg/kg DW). In contrast, high levels of NIV were still seen in the transgenic lines. For some lines (NEP02-01, NEP06, and NEP20 for F. culmorum-infected (FC, FHB+) seeds and NEP02-01, NEP05, and NEP06 for F. graminearum-infected (FG, FHB+) seeds), NIV levels were higher than in the untransformed GP plants. In the non-transgenic FHB– plants, higher levels of DON were detected in both F. culmorum and F. graminearum infected plants as compared with transgenic FHB– plants.

3.3. Semi-Field Analysis of the HvNEP-1 Transgenic Lines To test the reliability of HvNEP-1-mediated resistance to FHB, four Fusarium-infected transgenic lines and control GP plants were grown in a ﬁeld plot covered with an insect-proof net (Figure S1b). Ten transgene-positive plants from each transgenic line were compared to 53 untransformed GP-1 plants. Line NEP20 showed the lowest disease infection rate and progression as compared with the other transgenic lines (NEP02-01, NEP16 and NEP18) and the untransformed control plant GP-1 (Figure3a). Three weeks after infection, line NEP20 had an average infection score of 1.9, whereas the control GP plants were scored at 4.15 (on a one to nine scale) (Figure3b). AUDPC was calculated at one, two, and three weeks after inoculation. After three weeks, the AUDPC was signiﬁcantly higher in the GP control plants (35%) than in the transgenic NEP20 plants (24.5%) (Figure3c). Agronomy 2020, 10, 203 8 of 13

Table 1. Mycotoxin analyses of transgenic and control GP plants treated with F. graminearum and F. culmorum spores or water.

HvNEP-1 Transgenic Lines and GP Controls NEP02-01 NEP02-02 NEP04 NEP05 NEP06 NEP16 NEP18 NEP19 NEP20 NEP20-02 GP-1 GP-2 15938 794 1627 1231 9198 925 1257 <50 25043 462 7264 77 NIV FHB+ <50 <50 <50 <50 52 <50 <50 <50 152 <50 37091 2324 DON <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 186 55 ZEA FC 77 <50 <50 55 <50 <50 <50 <50 <50 <50 <50 <50 NIV FHB– <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 142 855 DON

<5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 15 ZEA kg DW) /

35510 6243 1543 16377 29727 597 97 <50 53 2399 13050 <50 NIV g µ FHB+ 5295 <50 56 <50 <50 <50 <50 8170 40073 <50 6022 1664 DON <5 <5 <5 <5 <5 <5 <5 <5 256 <5 <5 38 ZEA FG 124 <50 <50 <50 171 <50 <50 <50 <50 <50 <50 <50 NIV FHB– <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 569 240 DON

<5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 ZEA Mycotoxins ( Samples used for analysis <50 52 <50 <50 <50 163 <50 <50 <50 <50 <50 <50 NIV MQ <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 DON <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 ZEA One half of 9 grains (3 per spike) collected from 3 spikes per treatment in a plant were pooled together for mycotoxin analysis. GP-1 and GP-2 stands for the two Golden promise cultivar plants used as controls. The FC and FG denote for spikes infected with spores of F. culmorum (FC) and F. graminearum (FG) fungal species, respectively. FHB+ and FHB– stand for grains that show and do not show FHB symptoms, respectively. MQ stands for grains treated with water. The levels of deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEA) mycotoxins were measured. Detectable limits for DON, NIV, and ZEA are >50 µg, >50 µg, and >5 µg per kg of DW, respectively. Agronomy 2020, 10, 203 9 of 13 Agronomy 2019, 9, x FOR PEER REVIEW 3 of 13

Figure 3. Semi-field analysis of transgenic barley lines overexpressing HvNEP-1 against FHB infection. Figure 3. Semi-field analysis of transgenic barley lines overexpressing HvNEP-1 against FHB (a) Semi-field evaluation of Fusarium-infected four T1 transgenic lines (NEP02-01, NEP16, NEP18, andinfection. NEP20) (a) and Semi-field control evaluation Golden promise of Fusarium (GP) plants-infected in thefour semi-field T1 transgenic plots lines 3 weeks (NEP02-01, after infection; NEP16, (bNEP18,) Fusarium andinfection NEP20) leveland control of T1 transgenic Golden promise and control (GP) GP plants plants in scored the semi-field on a scale plots of 1–9. 3 weeks Values after are theinfection; mean of (b 10) Fusarium and 53 biological infection replicateslevel of T1 for transgenic transgenic and (NEP02-01, control GP NEP16, plants NEP18, scored andon a NEP20) scale of and1–9. GPValues plants, are respectively. the mean of Letters 10 and on 53 the biological top of the replic columnates barsfor transgenic indicate statistically (NEP02-01, significant NEP16, NEP18, differences and (pNEP20)= 0.05, and LSD GP test); plants, (c) AUDPC respectively. for the semi-field-grownLetters on the top T1 of transgenic the column lines bars and indicate control statistically GP plants. Errorsignificant bars = differencesmean + sem. (p Bars= 0.05, refer LSD to test); the median (c) AUDPC AUDPC, for andthe semi-field-grown the minimum and T1 maximum transgenic values lines areand indicated control GP with plants. error Error bars. bars = mean + sem. Bars refer to the median AUDPC, and the minimum and maximum values are indicated with error bars. 4. Discussion 4. DiscussionFusarium head blight is a devastating disease of barley and other crops worldwide. ResistantFusarium cultivars headcould blight reduce is a devastating damage from disease FHB. of However,barley and so other far, crops only limitedworldwide. resources Resistant of resistancecultivars could genes reduce are available. damage In from the FHB. current However, study, the so antifungalfar, only limited potential resources of barley of resistanceHvNEP-1 geneswas investigatedare available. by In transgenic the current overexpression study, the antifungal in the developing potential barleyof barley endosperm. HvNEP-1 The was ten investigated investigated by Ttransgenic0 barley plants overexpression had variable in levelsthe developing of HvNEP-1 barleyexpression, endosperm. but no The noticeable ten investigated phenotypic T0 barley differences plants werehad variable observed levels between of HvNEP-1 the transgenic expression, and controlbut no noticeable GP plants, phenotypic even in the differences highest expressing were observed lines (Figurebetween1b). the Studies transgenic of Fusarium and control-infected GP plants, plants even showed in the that highest overexpression expressing of linesHvNEP-1 (Figurein 1b). transgenic Studies barleyof Fusarium improved-infected resistance plants toshowed FHB boththat overexpression by limiting primary of HvNEP-1 infection in (Type-Itransgenic resistance) barley improved and the spreadresistance of theto FHB disease both (AUDPC) by limiting (Type-II primary resistance) infection (Type-I as compared resistance) with and the the untransformed spread of the controldisease plants(AUDPC) [48]. (Type-II Limiting infectionresistance) and as spread compared of disease with isth important,e untransformed particularly, control from plants a yield [48]. perspective. Limiting However,infection accumulationand spread of of diseasemycotoxins is important, is another unfortunateparticularly, eff ectfrom of Fusariuma yield perspective.infections. The However, current studyaccumulation demonstrated of mycotoxins that, endosperm-specific is another unfortunate overexpression effect of of FusariumHvNEP-1 infections.reduced the The accumulation current study of mycotoxinsdemonstrated in the that, infected endosperm-specific grains significantly overexpression (Type-IV resistance) of HvNEP-1 [49]. In reduced contrast tothe the accumulation non-transgenic of controlmycotoxins plants in infected the infected with F.grains culmorum significantly, in which (T theype-IV level resistance) of DON was [49]. high, In thecontrast accumulation to the non- of DONtransgenic in the control infected plants transgenic infected grains with was F. veryculmorum low (below, in which the detectionthe level levelof DON in nine was out high, of thethe tenaccumulation lines). This of was DON in agreementin the infected with transgenic our previous grainsin vitro was experiments,very low (below where the the detection accumulation level in ofnine 15-ADON out of the was ten reduced lines). This in cultures was in withagreement rHvNEP-1 with our [36]. previous For the F.in graminearum-vitro experiments,infected where plants, the sevenaccumulation transgenic of lines15-ADON had DON was levelsreduced below in cultures the detection with rHvNEP-1 level. The [36]. lower For mycotoxin the F. graminearum- formation ininfected the transgenic plants, seedsseven was transgenic accompanied lines byhad decreased DON levels disease below progression the detection in the transgeniclevel. The plantslower mycotoxin formation in the transgenic seeds was accompanied by decreased disease progression in

Agronomy 2020, 10, 203 10 of 13 as compared with the non-transgenic controls. Contamination of food and feed with mycotoxins is a worldwide problem. Although acute mycotoxicosis caused by high doses is rare in animals and humans, exposure to Fusarium mycotoxins can alter the human and animal susceptibility to infectious diseases by affecting the intestinal health and the innate and adaptive immune system [50]. The current study demonstrates that Fusarium resistance in barley induced by HvNEP-1 can facilitate a protection against mycotoxins. Furthermore, the reliability of HvNEP-1 as an antifungal gene was examined under semi-field conditions where plants were grown in soil plots covered by an insect net (Figure3a, Figure S1b). The result from the semi-field experiment was consistent with the greenhouse study where the overexpression of HvNEP-1 strongly suppressed FHB disease severity and progression (Figure3b,c). On the basis of the semi-field analysis, the expression level of HvNEP-1 in line NEP20 was sufficient to reduce the infection rate and disease progression. The current study describes a new resistance mechanism against FHB in barley. HvNEP-1 is demonstrated to have a strong potential as an FHB resistance gene and for reducing mycotoxins in infected seed. The transgenic approach to manage FHB used here can potentially minimize fungicide use, post-harvest sorting of infected grains, and combat mycotoxin-associated health risks. The use of azole fungicides for Fusarium control has aroused growing concern over the last years. Fungicides such as tebuconazole induces triazole-resistance in Aspergillus fumigatus [51]. At the same time, broad-spectrum triazoles are the primary drugs in the treatment of patients with prophylaxis of aspergillosis [52]. Fusarium resistant plants require less fungicide, and thereby alleviate the formation of triazole-resistant microorganisms in the environment. Since the HvNEP-1 gene was isolated from barley, it can reduce safety concerns and could potentially also be used in cis- and intra-genesis approaches [41]. Similarly, NEP-based strategies can be developed in other species where NEP candidates are available. Further studies are essential to understand the exact mechanism of HvNEP-1-based resistance, as well as to show if variation in HvNEP-1 levels is available in the natural germplasm and the extent to which these variants have the potential to protect the crop against FHB.

5. Conclusions The results of this study provide insights into the novel HvNEP-1 based Fusarium resistance mechanism in barley that limits primary infection, the spread of the disease, and synthesis of mycotoxins in the grains. Utilization of HvNEP-1 based Fusarium resistance in our crops could help to reduce the use of azole fungicides in agriculture. Future studies would show to what extent HvNEP-1 based Fusarium resistance can be utilized in other crops.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4395/10/2/203/s1. Author Contributions: Z.E.B. performed most of the experiments, data analysis and manuscript preparation; C.K.M. and G.D. participated in the HvNEP-1 cloning and review of the manuscript; I.B.H. was involved in Agrobacterium-mediated transformation of HvNEP-1 in barley; I.S.F. analysis of transgenic grains for mycotoxin accumulation; L.N.J. participated in the inoculation and scoring of transgenic plants with Fusarium; H.B.-P. developed the study concept, designed the experiment and manuscript preparation. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors acknowledge Lis Bagnkop Holte, Ole Bråd Hansen, and Kirsten Heinrichson for technical assistance in the lab and greenhouse. Conﬂicts of Interest: The authors declare no competing ﬁnancial interests.

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