Exogenous sodium diethyldithiocarbamate, a Jasmonic acid biosynthesis inhibitor, induced resistance to powdery mildew in wheat

Abstract Jasmonic acid (JA) is an important plant hormone associated with plant–pathogen defense. To study the role of JA in plant–fungal interactions, we applied a JA biosynthesis inhibitor, sodium diethyldithiocarbamate (DIECA), on wheat leaves. Our results showed that application of 10 mM DIECA 0–2 days before inoculation effectively induced resistance to powdery mildew (Bgt) in wheat. Transcriptome analysis identified 364 up‐regulated and 68 down‐regulated differentially expressed genes (DEGs) in DIECA‐treated leaves compared with water‐treated leaves. Gene ontology (GO) enrichment analysis of the DEGs revealed important GO terms and pathways, in particular, response to growth hormones, activity of glutathione metabolism (e.g., glutathione transferase activity), oxalate oxidase, and chitinase activity. Gene annotaion revealed that some pathogenesis‐related (PR) genes, such as PR1.1, PR1, PR10, PR4a, Chitinase 8, beta‐1,3‐glucanase, RPM1, RGA2, and HSP70, were induced by DIECA treatment. DIECA reduced JA and auxin (IAA) levels, while increased brassinosteroid, glutathione, and ROS lesions in wheat leaves, which corroborated with the transcriptional changes. Our results suggest that DIECA can be applied to increase plant immunity and reduce the severity of Bgt disease in wheat fields.

can escape the recognition by some R genes (Dangl, Horvath, & Staskawicz, 2013;Jones & Dangl, 2006). Understanding the molecular basis of plant innate or induced defense responses will enable to find new methods for disease control.
During the long history of co-evolution with pathogens, plants have developed a multifaceted innate immunity system. After the recognition of pathogen invasion, several downstream signaling events are elicited in the plant cell, including influx of Ca 2+ into the cytosol, reactive oxygen species (ROS) accumulation, and transient activation of mitogen-activated protein kinases (MAPK) signaling cascades (Boller & Felix, 2009;Choudhury, Rivero, Blumwald, & Mittler, 2017;Tsuda & Katagiri, 2010). Plant hormones act as immune signals, triggering extensive transcriptional reprogramming, and resulting in an efficient defense response (Bari & Jones, 2009).
JA is involved in the defense against necrotrophic pathogens, preventing plant cell death and inducing defense responses to restrict further pathogen infection (Singh, Singh, Gautam, & Nandi, 2019). Treatment with JA is shown to protect plants against herbivore attack and reduce the severity of infection by necrotrophic fungi (Baldwin, 1998;Thomma, Eggermont, Broekaert, & Cammue, 2000;Zalewski et al., 2019). JA signaling also plays an important role in mediating plant defense against some biotrophic or hemibiotrophic pathogens (De Vleesschauwer, Gheysen, & Höfte, 2013;Yan & Xie, 2015;Zalewski et al., 2019). Exogenous application of MeJA up-regulates some defense genes and results in efficient reduction of disease development (Desmond et al., 2005;Thomma et al., 2000;Wasternack, 2007;Xu et al., 1994). However, contradictory evidences have been published regarding the role of JA in Bgt resistance in wheat. Duan et al., (2014), show that exogenous MeJA significantly enhance Bgt resistance in susceptible wheat varieties, while Xiang et al., (2011), show that MeJA application does not induce resistance to Bgt in wheat. Therefore, the role of JA in plant-fungal interactions is still not clear.
Crosstalk of plant hormones is important for disease resistance, for example, previous reports show an antagonistic relationship between the JA and SA signaling pathways in plant-fungal interactions.
SA can mediate programmed cell death response in plant cells and restrict (hemi) biotrophic pathogens to the infection site, preventing pathogen proliferation (An & Mou, 2011;Nishimura & Dangl, 2010).
Sodium diethyldithiocarbamate (DIECA) has been used as a JA biosynthesis inhibitor in plants and likely inhibits the JA pathway by shunting 13(S)-hydroperoxylinolenic acid to 13-hydroxylinolenic, thereby sharply reducing the precursor pool leading to cyclization and eventual synthesis of JA (Farmer, Caldelari, Pearce, Walker-Simmons, & Ryan, 1994). Application of DIECA has been shown to significantly reduce JA levels in multiple plant species and reduce the expression of some resistance gene, such as TaJRLL1 and PR3 (Hu & Zhong, 2008;Hu, Neill, Cai, & Tang, 2003;Xiang et al., 2011). However, there is no clear and solid report for fungal resistance imposed by different regulated JA levels in wheat. Our results showed that application of DIECA, the inhibitor of JA biosynthesis, could induce resistance to Bgt in wheat, while exogenous MeJA did not. In addition to inhibition of JA after DIECA application, the level of IAA was decreased and brassinosteroid (BR) was increased, and accumulation of glutathione and ROS was observed. These findings corroborated with wide transcriptional regulation induced by DIECA, for example, the up-expression of PR genes and enriched GO terms such as response to growth hormones, activity of glutathione metabolism, oxalate oxidase, and chitinase activity. Moreover, our results suggested that DIECA application can be used to control Bgt in the field.
Arabidopsis lines (Col wild-type and the eds1 and pad4 mutants) were provided by Dr. Zhaorong Hu, China Agriculture University. One-week-old wheat plants were used for both spray and spot inoculations. Wheat plants were inoculated at a density of 100-150 conidia/mm 2 using a blowing machine in a vaccination tower.

| Pathogen maintenance and inoculation
Infection types (IT) were classified into six classes in accordance with a previous study with IT 0-4 representing no visible symptoms (0), necrotic flecks (0;), highly resistant (1), resistant (2), susceptible (3), and highly susceptible (4) reactions, respectively (Liu, Sun, Ni, Yang, & McIntosh, 1999). G. cichoracearum strain UCSC1 was maintained by growing it on pad4 mutants. Four-week-old Arabidopsis plants were inoculated using the same methods as those used for inoculation of wheat with strain E09. About 16 plants in each treatment were used for phenotyping, and the representative leaves or plants were used for photograph.

| Coomassie blue staining
For microscopic observations of fungal development, at 24-120 hpi, the Bgt-infected leaves' segments were collected for coomassie blue staining as described by Li et al., (2016). For microscopic observations, leaf segments (5 cm in length) were stored in 50% glycerol and examined under an Olympus BX-43 microscope (Olympus Corporation).
The germination and penetration rates of conidiophores (number of germinated spores and penetrated spores relative to the total number of spores, respectively) were visualized after staining with Coomassie blue. Germinated spores show germinated germ tubes; penetrated spores show developed hyphae and have initiated the formation of young colonies. In each independent experiment, 15-20 leaf segments were observed at 24, 48, 96, and 120 hpi.
Microscopic measurements were used for calculating the mean of germination and penetration rates, using three independent replicated experiments. Statistical significance was determined by paired Student's t test.

| DIECA treatments
DIECA Treatment at seedling stage (1)-DIECA in sterile water containing 0.02% Silwett-L77 (Fisher Scientific, Cat. NC0138454) was used as treatments, while water (0.02% Silwett-L77) was used as control treatment. To select the optimal concentration to be used in the spraying experiment, we applied a preliminary test of DIECA spraying of 0, 0.1, 1, 5, 10, 20, and 30 mM, at seedling stage (when the first leaf was fully expanded). Subsequently, the 5 mM and 10 mM DIECA treatments were selected. DIECA was sprayed onto wheat leaves at seedling stage until the liquid was dripping off the leaves.
Frequency and duration of DIECA treatments-To evaluate the optimal timing of DIECA treatment for effective enhancement of disease resistance, 10 mM DIECA was applied at different time durations, prior to or after powdery mildew inoculation. With continuous powdery mildew inoculation in the greenhouse, plants were sprayed (10 mM DIECA) every 2-6 days.

DIECA treatment on Bgt-infected detached leaf segments (2)-
Pretreatment included 5 mM and 10 mM DIECA and water, smeared on detached wheat leaf segments placed on agar plates (1% agar, containing 0.05% benzimidazole, SIGMA). One day after pretreatment, the segments were inoculated using the pathogen inoculation method described above.

DIECA treatment in the field (3)-Plants at the adult stage (flow-
ering stage) were sprayed with water, 1 mM DIECA, 5 mM DIECA, or 10 mM DIECA, or were untreated. DIECA or water was sprayed twice (every five days) prior to the outbreak of powdery mildew disease. Two wheat field treatments were used: (a) Artificial field inoculation-in which Xuezao seedlings with Bgt sporulation were transplanted as spreader, in a field containing uninfected Xuezao seedlings. (b) Naturally infected field-in a field located 30-50 m away from the artificial inoculation field.

| MeJA treatments
The water and 0.2, 0.5, and 1 mM MeJA solutions contained 0.02% Silwett-L77 were prepared. MeJA was dissolved in the Silwett-L77 and mixed with water. In the first two days, Xuezao plants were sprayed once a day; at the third day, plants were infected with Bgt E09.

| RNA extraction, CDNA library construction, RNA-SEQ, and data analysis
Leaves for RNA extraction were samples at seedling stage (DIECA treatment 1). The treatments of water or DIECA were applied once a day for 2 days, and on the third day, leaf samples were collected.
Ten leaves of each pot were pooled together, as one biological repetition for RNA extraction. Three biological repetitions were employed for each treatment. Total RNA was extracted using RNA pure Plant Kit (TIANGEN). cDNA Libraries were generated using the NEB Next UltraTM RNA Library Prep Kit for Illumina (NEB) following the manufacturer's recommendations. Paired-end reads were generated on IlluminaHiseq 2500 platform. Sequencing data were analyzed by using BMKCloud (http://en.biocl oud.net/). Adaptor sequences and low-quality sequence reads were removed from the data sets.
Tophat2 tools were used to map the reads to the wheat reference genome (IWGSC RefSeq Annotation v1.0). Only reads with perfect match or one mismatch were further analyzed and annotated based on the reference genome. Gene function was annotated based on the following databases: Nr (NCBI non-redundant protein sequences); Nt (NCBI non-redundant nucleotide sequences); Pfam (Protein family); KOG/COG (Clusters of Orthologous Groups of proteins); Swiss-Prot (A manually annotated and reviewed protein sequence database); KO (KEGG Ortholog database); and GO. Differential expression analysis of the two groups was performed using the DESeq R package (1.10.1). Transcripts with an adjusted p-value ≤ .05 and with |log2 fold change| ≥ 2 found by DESeq were assigned as differentially expressed. GO enrichment analysis of the DEGs was implemented using the GOseq R package based on the Wallenius non-central hyper-geometric distribution (Young, Wakefield, Smyth, & Oshlack, 2010), which can adjust for gene length bias in DEGs. We used KOBAS (Mao, Cai, Olyarchuk, & Wei, 2005) software to test for statistically significant enrichment of DEGs in KEGG pathways.

| Measurements of endogenous fatty acids, glutathione, plant hormones, and DAB staining for ROS
Application of 10 mM DIECA was used once a day for two days (DIECA treatment 1). On the third day, leaf samples were collected for the measurements of endogenous fatty acids, glutathione, and plant hormones. Fatty acid analysis was measured as described by Li et al., (2016), using HP6890 gas chromatograph (Agilent Technologies). Leaf tissues were dried in an oven at 45℃ for 60 hr and then ground into a powder; 200 mg of powder for each sample was placed in a screw capped glass vial. Glutathione and plant hormones, including JA, IAA, SA, BR, gibberellins (GA3 and GA4), dihydrozeatin riboside (DHZR), zeatin riboside (ZR), indolepropionic acid (IPA), and abscisic and acid (ABA), were measured from leaf tissues as described by Cao, Li, Chen, Liu, and Li (2016), and Zhao et al., (2006), with slightly modification by using different internal reference and antibodies. Leaf cell death response was observed at 7 dpi by trypan blue staining as described previously (Koch & Slusarenko, 1990). ROS was estimated using DAB staining solution (0.1 g DAB, 100 ml distilled water, KOH adjusted to pH = 5.8) to stain infected leaves for 8 hr at 28℃ and then 100% ethanol was used to depigment infected leaves for 1 day.

| DIECA application induced powdery mildew resistance in wheat
Preliminary test of different DIECA concentrations indicated that plants were more resistant to Bgt with increased concentrations  Microscopic observation showed that germination and penetration rates of Bgt on DIECA-treated leaves were remarkably lower than in untreated or water-treated leaves at 24, 48, 96, and 120 hr post-infection (hpi) (Table S1; Figure 1d). Even after removal of DIECA from the leaves using sterile water before Bgt infection, plants were highly resistant to Bgt ( Figure S2). The specificity of the treatment in four different susceptible wheat cultivars (Chinese Spring, Liaochun 18, Liaochun 10, and Fielder) showed that Bgt resistance was enhanced in all the four wheat cultivars ( Figure S3). Moreover, 5 mM and 10 mM DIECA application induced barley resistance to B. graminis f. sp. hordei (Bgh) ( Figure S4) and also enhanced powdery mildew Golovinomyces cichoracearum UCSC1 resistance in Arabidopsis ( Figure S5).

| Transcriptome analysis of wheat response to DIECA
Resistance to Bgt was observed only when plants were treated by DIECA prior to inoculation. Therefore, we used uninoculated DIECAtreated and water-treated plants for transcriptome analysis by RNA-seq.
A total of 170,850,209 clean reads were obtained, with ≥25,749,518 clean reads in each pool (i.e., one cDNA library prepared from six samples). For each sample, ≥89.47% of the reads had a quality score of Q30 (Table S2). Following assembly, 82.63%-84.26% of the sequences in each library could be mapped to the Chinese Spring wheat genome reference sequence (Tables S3). In total, 118,189 distinct assembled unigenes were annotated after blast searches against several databases (Table S4). Differentially expressed genes (DEGs) between the DIECA and the water-treated libraries were identified using the following criteria: |log 2 fold change| ≥ 2 (p ≤ .05). A total of 432 DEGs were identified, of which 364 were up-regulated in treated plants while 68 were down-regulated (Tables S5 and S6). DEGs identified in different biological replicates were clustered together in a heat map of expression levels, indicating good reproducibility between replicates ( Figure S6).
The highly enriched GO terms of molecular function included glutathione transferase activity, oxalate oxidase activity, and chitinase activity. The highly enriched GO term of biological process included response to growth hormone, lateral root development, and de-etiolation ( Figure 2a). KEGG enrichment analysis showed that the highly enriched pathways were glutathione metabolism, glyoxylate and dicarboxylate metabolism, phenylpropanoid biosynthesis, monoterpenoid biosynthesis, tryptophan metabolism, photosynthesis-antenna proteins, and α-linolenic acid metabolism (Figure 2b). GO and KEGG enrichment analyses showed that pathways most highly enriched in the DEGs were associated with glutathione metabolism and growth hormones (Figure 2).

| Plant hormones
We analyzed the levels of JA and other nine additional plant hormones in the water or DIECA-treated wheat leaves. Our results showed that JA level was decreased by ~88%, in addition, IAA level was decreased by ~65%, and BR level was increased by ~42.8% in the DIECA-treated leaves as compared with the water-treated leaves (Figure 3c, 3d and 3e). No change was observed in other analyzed plant hormones, including SA, GA4, GA3, DHZR, ZR, IPA, and ABA ( Figure S9).
Previous studies showed that SA pathways might synergistically interact with JA or IAA pathways (Patkar et al., 2015;Robert-Seilaniantz, Navarro, Bari, & Jones, 2007;Yuan, Liu, & Lu, 2017); therefore, DIECA was applied on two pad4 and eds1 mutants, which have loss of function of SA-mediated resistance pathways. The results indicated that powdery mildew resistance to UCSC1 was still induced ( Figure S10). This suggests that the induced resistance by DIECA might be independent of the SA pathway.

| DIECA induced cell death and ROS accumulation
We could find visible spontaneous lesions on central parts of some wheat leaves 1 week after 5 mM or 10 mM DIECA application in the

| D ISCUSS I ON
Plant hormones crosstalk mediate complex signal transduction networks, involve in different defense strategies to pathogens (Li, Han, Feng, Yuan, & Huang, 2019). In the current study, we showed that the inhibition of JA biosynthesis by DIECA triggered hormonal alteration and transcriptional reprogramming involved in plant F I G U R E 3 Content of glutathione (a), fatty acids (b), JA (c), IAA (d), and BR (e) levels in water-treated and DIECAtreated Xuezao leaves. Each value is the mean ± SE of three independent biological repetitions. Asterisks indicate a significant difference from the water-treated mock control at p ≤ .05 determined by Student's t test  (Desmond et al., 2005;Thomma et al., 2000;Wasternack, 2007;Xu et al., 1994).
Our results showed that pretreatment with MeJA did not induce effective resistance to Bgt in wheat, while application of JA biosynthesis inhibitor DIECA could induce highly resistance to Bgt in wheat (Figure 1 and Figure S1).
The observed induction of BR in our study corroborates with earlier studies showing that BR plays critical roles in the regulation of plant growth and development as well as responses to biotic and abiotic stress factors (Figure 3e; Peres et al., 2019). BR applications seem to exert an effect on immunity to a wide array of pathogens in different plant species, such as enhancing resistance to Fusarium infection in barely (Ali, Kumar, Khan, & Doohan, 2013) and inducing resistance to powdery mildew in tobacco (Nakashita et al., 2003). Other evidence shows that BR has negative roles in plant resistance in rice, and JAmediated defense can suppress the BR-mediated susceptibility to infection rice black-streaked dwarf virus, suggesting an antagonistic relationship between BR and JA effects in viral defense (He et al., 2017).
The transcriptome analysis results indicated that the most highly enriched GO terms among the DEGs included response to growth hormone ( Figure 2a). Indeed, the observed increase of BR level together with reduction of JA and IAA, after DIECA treatment, might contribute to wheat resistance to Bgt (Figure 3). However, the crosstalk of IAA and JA and BR have been reported to regulate plant F I G U R E 5 DIECA application induced wheat powdery mildew resistance under field conditions. Images of untreated, water-treated, and 1 mM and 5 mM DIECA-treated Xuezao plants in the artificial inoculation field (a) and natural infection field (b) at 5 days after the second application of water or DIECA. 2018, in Beijing resistance and growth (He et al., 2017;Peres et al., 2019;Zhou, Song, & Xue, 2013). Among these three hormones, JA plays an important role in plant growth inhibition (Huang, Liu, Liu, & Song, 2017), and BR is growth-promoting hormone (Lozano-Durán & Zipfel, 2015).
Our result showed that DIECA treatment reduced the growth of Arabidopsis ( Figure S5), and slightly reduction was also observed in wheat ( Figure S2). Since JA was reduced and BR was increased (i.e., both promoting growth), we suggest that the cause of growth reduction might be via the reduction of IAA after DIECA application ( Figure 3). Further studies are needed to determine which of the three hormones (JA, BR, or IAA) is the key regulator, or a synergistic effect of the three hormones is the important factor for inducing Bgt resistance in wheat.
The results of transcriptome analysis indicated that "glutathione transferase activity" was the most highly enriched GO term (e.g., KEGG enrichment analysis; Figure 2b) among the up-regulated DEGs after DIECA application. These genes encoding glutathione transferase and glutathione S-transferase (GST, 2.5.1.18), which function in the glutathione metabolism pathway (Ko00480), were among the up-regulated DEGs after DIECA application ( Figure S7). In some cases, GSTs have been shown to contribute to resistance against powdery mildew (Gullner, Komives, Király, & Schröder, 2018). The GstA1 gene is specifically induced by fungal infection (Mauch & Dudler, 1993). GST is also required for resistance against O. neolycopersici in tomato (Pei et al., 2011).
Glutathione has also been reported to be an important molecule for plant-pathogen resistance. The γ-glutamylcysteine synthetase mutant pad2-1 contains only about 22% of the wild-type amount of glutathione and is highly susceptible to oomycete pathogen Phytophthora brassicae; feeding mutant plants glutathione can restore the glutathione level and resistance to the pathogen (Parisy et al., 2007). In addition to glutathione generation, we have identified ROS accumulation in wheat leaves in DIECA-treated leaves ( Figure 4c and d). ROS have been postulated to be an integral part of the plant defense response, acting as local and systemic signal molecules that are involved in the activation of antimicrobial defenses (Waszczak, Carmody, & Kangasjärvi, 2018). Furthermore, transcriptome analysis indicates that DIECA induced genes encoding peroxidase 1, peroxidase 2, and peroxidase 3, which have been reported to play a significant role in generating H 2 O 2 during the plant defense response and in conferring resistance to a wide range of pathogens (Bindschedler et al., 2006). In plant-pathogen interactions, ROS participate in a coordinated way in regulating the hypersensitive response (Bellin, Asai, Delledonne, & Yoshioka, 2013). Thus, ROS and glutathione accumulation, identified in the current study, may be an important part of DIECA-induced resistance mechanism to Bgt in wheat.
The downstream genes of pathways in plant disease resistance may be involved in these biological processes, especially inducing the expression of some PR genes, including PR1, PR2, chitinase (PR3, PR8, and PR11), peroxidase (PR9), and oxalate oxidase (PR15 and PR 16). Most of those PR genes can be used as potential candidate genes for improvement of the pathogen resistance of wheat and barley (Wang et al., 2018). For example, PR genes encoding hydrolytic enzymes chitinases and β-1,3-glucanases are very important in plants for invading pathogen, and overexpression of some PR genes improve the resistance to pathogens in plants (Ali et al., 2018;Ebrahim, Usha, & Singh, 2011). It suggested that those DIECA-induced PR genes, such as encoding PR1.1, PR1, PR10, PR4a, chitinase 8, beta-1,3-glucanase, and beta-glucosidase 31, might contribute to resistance to powdery mildew in wheat, and those PR genes could be used as candidate genes for improving wheat resistance (Table S5).
From agricultural point of view, our results clearly showed that spraying with DIECA 0-2 days prior to Bgt inoculation induced effective resistance in wheat. Our results also show that DIECA did not have a direct toxic effect on the growth of Bgt hyphae ( Figure 1b). Furthermore, when DIECA-pretreated wheat leaves were washed before Bgt infection, leaves were still highly resistant ( Figure S2). The results of our two-year field experiments showed that DIECA application in wheat fields could significantly reduce the severity of powdery mildew disease ( Figure 5). Unlike in the greenhouse experiments where 10 mM DIECA was the appropriate concentration for inducing resistance, 1 or 5 mM DIECA could effectively be used to control Bgt in field during heading date of wheat.

| CON CLUDING REMARK S
By implementing integral metabolic, transcriptomic and plant pathology methods, we show here that inhibition of JA biosynthesis led to alteration in IAA and BR which triggered the accumulation of ROS and glutathione and up-regulation of pathogenesis-related genes. Eventually inducing defense responses to Bgt in Wheat. Our results indicate that spraying with DIECA can be used to control Bgt in the field.

ACK N OWLED G M ENTS
We are grateful to Professors Zhaorong Hu, Xiayu Duan, and Qianhua Shen for kindly providing plant and fungus materials.
We are grateful to Dr. Gen Xu and Dr. Hui Fang for the analysis of fatty acid content. We are grateful to Dr. Dong Li and Prof. Baomin

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

AUTH O R CO NTR I B UTI O N S
CX, QS, YL, and LQ conceived the project. YL and LQ performed most of the experiments. QZ, XZ, HL, and XC provided help for experiments. YL wrote the manuscript. LQ, TK, and CX improved and revised this manuscript.