Phytohormonal and Transcriptomic Response of Hulless Barley Leaf in Response to Powdery Mildew Infection

: Powdery mildew (PM) caused by Blumeria graminis (DC.) Golovin ex Speer f. sp. hordei Marchal ( Bgh ) is one of the major yield reducing diseases in hulless barley ( Hordeum vulgare L. var. nudum Hook. f.). Genotypes with contrasting resistance to PM offer unique opportunities to explore the transcriptome in order to understand the expression changes in genes and pathways. In this study, we explored the phytohormone levels and transcriptome of a Bgh susceptible (XL19) and resistant (ZYM1288) hulless barley genotypes at 0, 5, 12, 24, and 36 h post infection (hpi) with Bgh . We found relatively higher levels of abscisic acid, jasmonic acid, salicylic acid, and cytokinins in ZYM1288. The transcriptome analyses identiﬁed 31,354 genes that were enriched in signaling, energy, and defense related pathways. Higher numbers of differentially expressed genes (DEGs) were found in XL19 as compared to ZYM1288 after 5 (3603 vs. 2341) and 12 hpi (3530 vs. 2416). However, after 24 and 36 hpi, the number of DEGs was higher in ZYM1288 as compared to XL19 i.e., 3625 vs. 3034 and 5855 vs. 2725, respectively. Changes in hormone levels drove downstream expression changes in plant-hormone signaling that helped ZYM1288 to perform better under Bgh infection. The expression of DEGs in MAPK-signaling and Toll-like receptor signaling pathways, glucosinolate biosynthesis, glutathione metabolism, brassinosteroid metabolism, and energy related pathways indicated their common roles in defense against PM. Key genes related to PM-resistance were upregulated in the resistant genotype. These genes provide key information towards differences in both genotypes towards resistance to PM. The transcriptomic signatures explored in this study will broaden our understanding towards molecular regulation of resistance to PM in hulless barley.


Introduction
Hulless barley (Hordeum vulgare L. var. nudum Hook. f.) is an important cereal and used in human food, animal feed, and malt products. Particularly, it is an economically important crop for people living in Qingzang highland (including Tibet) [1]. It adapts well in the highlands and can withstand extreme highland conditions, therefore, it has become a principal food of Tibetans [2]. One of the major biotic stresses in barley is the powdery mildew (PM) caused by Blumeria graminis (DC.) Golovin ex Speer f. sp. hordei Marchal (Bgh) and it can cause yield loss up to 25% [3]. It has been reported that PM causes reduction in 1000-kernel weight, crude protein content, and yield in monocots such as winter wheat and barley [4,5].
Bgh has characteristics such as a repertoire of Avr genes, asexual haploid spores, and genetic recombination ability during the growing season, which support its rapid establishment of PM induces changes in phytohormones [21]. The phytohormone analysis showed that at 5 hpi, the abscisic acid (ABA) level increased in XL19 and decreased in ZYM1288 as compared to their respective controls. At this time point, XL19 showed higher ABA levels than ZYM1288. At 12,24, and 36 hpi the ABA levels in XL19 and ZYM1288 were reduced as compared to 0 hpi, where the latter had higher levels as compared to the XL19. (Figure 2i). The auxin levels varied between different time points and showed no trend for indole 3-acetic acid (IAA); the IAA levels increased at 5, and 12 hpi in XL19 and then remained more or less constant at 24 and 36 hpi as compared to 0 hpi. ZYM1288 showed reduced IAA contents in all time points except 24 hpi, where the IAA levels were slightly increased (Figure 2ii). The methylindole-3-acetic acid (ME-IAA) levels in ZYM1288 were lower in all the time points as compared to 0 hpi, while in case of XL19, the ME-IAA levels did not show any trend (Figure 2iii). The indole-3-carboxylic acid (ICA) levels in ZYM1288 were lower than the controls. However, it is to be noted that after 12 and 36 h, then ICA levels were reduced to less than half as compared to the control. In XL19, the ICA contents increased after 5 and 12 hpi, while remained more or less same after 24 and 36 hpi (Figure 2iv). Among all auxins, the Indole-3-carboxaldehyde (ICA-Id) levels showed a decreasing trend in both genotypes with XL19 showed minor reductions while ZYM1288 showed observable reductions and decreased with time ongoing except at 36 hpi ( Figure 2v). The level of dihydrojasmonic acid (H2JA) dropped after the PM infection at all time points, where the susceptible genotype had relatively lower levels when compared with the ZYM1288 (Figure 2vi). The jasmonic acid (JA) levels were lower in ZYM1288 and XL19 after PM infection as compared to their respective controls. However, interestingly, the levels were higher after 12 hpi in XL19. Similar levels of JA were recorded in ZYM1288 after 12 hpi though still lower than 0 hpi (Figure 2vii). Jasmonoyl-L-Isoleucine (JA-ILE) level decreased in ZYM1288. Overall, JA-ILE levels were lower in XL19 except for 12 hpi (Figure 2viii). The methyl Jasmonate (MEJA) levels in XL19 remained more or less constant but in the resistant genotype, the level increased with time until 24 hpi as compared to 0 hpi ( Figure 2ix). Salicylic acid (SA) differed between the genotypes. It was slightly reduced at 5 and 12 hpi then increased at 24 hpi but again reduced at 36 hpi in ZYM1288. In case of XL19, the SA levels were lower after 5, 12, and 24 hpi, while higher after 36 hpi (Figure 2x). It is to be noted that ZYM1288 had higher tZ levels as compared to its control, while in case of XL19, a significant rise was observed at 5 hpi and then the levels decreased unit 24 hpi (but still higher than 0 hpi) and after 36 hpi, the levels were lower than 0 hpi. ZYM1288 had higher trans-zeatin (tZ) levels than XL19 except at 5 hpi ( Figure 2xi).

Transcriptomic Responses of XL19 and ZYM1288
The transcriptome of 30 leaf samples (2 genotypes × 5 time points × 3 biological replicates) resulted in 46.25-67.55 million clean reads (average 56.35 million clean reads and 253.59 Gb clean data). The Q30% was ≥93.2% and the average GC content was 56.56%. The clean reads were then mapped to the reference genome [22] and 31,354 genes were obtained (Supplementary Table S1).
The Fragments Per Kilobase of Transcript per Million fragments mapped (FPKM) for XL19 was higher in infected samples as compared to CK. Similarly, the FPKM values in infected ZYM1288 leaves were higher except for 36 hpi. The average Pearson Correlation Coefficient (PCC) for the biological replicates was ~0.79 ( Figure 3a) indicating the reproducibility of the experiment and the reliability of expression data. Higher number of differentially expressed genes (DEGs) was found in XL19 as compared to ZYM1288 after 5 (3603 vs. 2341) and 12 hpi (3530 vs. 2416). However, after 24 and 36 hpi, the number of DEGs was higher in ZYM1288 as compared to XL19 i.e., 3625 vs. 3034 and 5855 vs. 2725, respectively ( Figure 3b). We found 1076 DEGs that were common between all treatment comparisons in both genotypes (Figure 3c). The KEGG pathway enrichment showed that the DEGs were enriched in a large number of pathways related to signaling (plant hormone signaling, MAPK signaling, Toll-like receptor signaling, and plant-pathogen signaling) and energy related pathways (photosynthesis, photosynthesis-antenna proteins, galactose metabolism, starch and sucrose metabolism, nitrogen metabolism, pentose phosphate pathway, citrate cycle, and carbon fixation in photosynthetic organisms). Further, the DEGs were enriched in several defense related pathways such as secondary metabolite

Transcriptomic Responses of XL19 and ZYM1288
The transcriptome of 30 leaf samples (2 genotypes × 5 time points × 3 biological replicates) resulted in 46.25-67.55 million clean reads (average 56.35 million clean reads and 253.59 Gb clean data). The Q30% was ≥93.2% and the average GC content was 56.56%. The clean reads were then mapped to the reference genome [22] and 31,354 genes were obtained (Supplementary Table S1).
The Fragments Per Kilobase of Transcript per Million fragments mapped (FPKM) for XL19 was higher in infected samples as compared to CK. Similarly, the FPKM values in infected ZYM1288 leaves were higher except for 36 hpi. The average Pearson Correlation Coefficient (PCC) for the biological replicates was~0.79 (Figure 3a) indicating the reproducibility of the experiment and the reliability of expression data. Higher number of differentially expressed genes (DEGs) was found in XL19 as compared to ZYM1288 after 5 (3603 vs. 2341) and 12 hpi (3530 vs. 2416). However, after 24 and 36 hpi, the number of DEGs was higher in ZYM1288 as compared to XL19 i.e., 3625 vs. 3034 and 5855 vs. 2725, respectively ( Figure 3b). We found 1076 DEGs that were common between all treatment comparisons in both genotypes (Figure 3c). The KEGG pathway enrichment showed that the DEGs were enriched in a large number of pathways related to signaling (plant hormone signaling, MAPK signaling, Toll-like receptor signaling, and plant-pathogen signaling) and energy related pathways (photosynthesis, photosynthesis-antenna proteins, galactose metabolism, starch and sucrose metabolism, nitrogen metabolism, pentose phosphate pathway, citrate cycle, and carbon fixation in photosynthetic organisms). Further, the DEGs were enriched in several defense related pathways such as secondary metabolite biosynthesis, brassinosteroid metabolism, glutathione metabolism, and glucosinolates biosynthesis (Supplementary Figure S1; Supplementary Table S2).
Agronomy 2021, 11, x 6 of 24 biosynthesis, brassinosteroid metabolism, glutathione metabolism, and glucosinolates biosynthesis (Supplementary Figure S1; Supplementary Table S2).  Powdery mildew infection in other plants such as pumpkin had shown the differential regulation of plant-hormone signaling pathway, which is consistent with our results [23]. Highest number of DEGs were enriched in auxin signaling part of this pathway (Figure 4). The auxin induced proteins (AUXs) were downregulated in ZYM1288 while the auxin transporter protein 1 was upregulated. Additionally, the ARFs or ARF/IAAs showed different regulation patterns, particularly ARF/IAA21, ARF18, three ARF19s, ARF5 and ARF7s were upregulated after 36 h in ZYM1288, while these genes did not differentially  Powdery mildew infection in other plants such as pumpkin had shown the differential regulation of plant-hormone signaling pathway, which is consistent with our results [23]. Highest number of DEGs were enriched in auxin signaling part of this pathway ( Figure 4). The auxin induced proteins (AUXs) were downregulated in ZYM1288 while the auxin transporter protein 1 was upregulated. Additionally, the ARFs or ARF/IAAs showed differ-Agronomy 2021, 11, 1248 7 of 24 ent regulation patterns, particularly ARF/IAA21, ARF18, three ARF19s, ARF5 and ARF7s were upregulated after 36 h in ZYM1288, while these genes did not differentially express at other time points. One ARF/IAA30 was upregulated throughout the four time points in the resistant genotype. Contrastingly, the ARF2 and ARF11s were downregulated in at least one time point in ZYM1288. The GH3.8, and SUARs' expression increased in ZYM1288 after infection as compared to XL19, indicating that XL19 is unable to continue cell enlargement and plant growth ( Figure 4). Contrary to auxins, only six cytokinin signaling related genes i.e., histidine kinases (HKs; HK3, HK4) pseudo histidine-containing phosphotransfer protein 5 (PHP5), two-component response regulations (ORRs; ORR1, ORR3, and ORR26) were differentially expressed. The upregulation of these genes (except HK3) is indicative of higher cytokinin signaling in ZYM1288, which is consistent with tZ levels (Figures 2xi and 4). We found that 14 of 15 DEGs enriched in ABA signaling pathway were upregulated in ZYM1288 as compared to XL19 in at least one time point, which is in agreement with ABA levels ( Figure 2). Together with the higher ABA levels in ZYM1288 leaves, the upregulation of key DEGs benefits the resistant genotype and enhances its resistance against PM, which is consistent with a recent report on barley near-isogenic lines [21]. Similar to this, all the DEGs enriched in gibberellin signaling were upregulated in ZYM1288 except TIF1-like gene. An important observation was that the log2 FC values of these DEGs in XL19/ZYM1288 were higher in all time points. These expression changes indicate that ZYM1288 shows resistance to PM by increased degradation of DELLAs as indicated by increased upregulation of GID2 and GAIs ( Figure 4) [24]. Additionally, the DEGs were enriched in ethylene, brassinosteroid, JA, and SA signaling. It is known that SA, JA, and ethylene enhances defense against pathogens. Thus, the increased accumulation and upregulation of related genes enhances the resistance of ZYM1288 against PM ( Figure 4). b. MAPK signaling pathway and Toll-like receptor signaling pathway.
A previous study on Arabidopsis has shown the involvement of MAPK signaling pathway against PM infection [25]. The KEGG pathway enrichment analysis also indicated that 101 DEGs were enriched in this pathway. Major genes i.e., ANP1, CALM, CAT, CHI-B, EBF1, EIN3, ERECTA, ERFs, FLS2s, MAPKs, MAP2Ks, MAP3Ks, MYC2, PRPs, PP2Cs, PYR/PYLs, WRKY22s, and WRKY33s were differentially expressed between both genotypes at four time points as compared to CK. Most importantly, we noticed that genes involved in early and late response for pathogens i.e., FLS2, MAP2Ks, and PR2C6 were upregulated in ZYM1288 as compared to XL19 ( Figure 5). The PM infection caused the upregulation of genes that are present in pathogen attack signaling and generation of H 2 O 2 related part of MAPK signaling pathway. Defense against PM in ZYM1288 is possibly due to the upregulation of genes such as serine/threonine receptor kinases (ERECTAs), NDPK2, ANP1, and PR2C6 (and PRPs). The genes related to defense response and wounding responses (a part of ethylene and JA signaling of MAPK signaling pathway) i.e., ERF1, ChiB, MYC2, and VIP2 were also upregulated in ZYM1288 as compared to XL19. Taken together, it is clear from the differences in the expression in the XL19 and ZYM1288 that MAPK signaling pathway plays significant role in PM resistance or susceptibility ( Figure 5).
Another important signaling pathway i.e., Toll-like receptor signaling pathway was one of the significantly enriched pathways between XL19 and ZYM1288 hulless barley leaves infected with PM (Supplementary Figure S1). A total of 455 DEGs were enriched in Toll-like receptor signaling pathway. Interestingly, only 9 of 455 genes were annotated as other than interleukin-1 receptor-associated kinases (IRAK1 and IRAK4). Ninety IRAKs were upregulated in ZYM1288 at 5 hpi and at least in one of other time points i.e., 12, 24, and 36 hpi. On the contrary, relatively higher number i.e., 101 IRAKs were downregulated at 5 hpi in ZYM1288. Furthermore, the expression of toll-interacting protein, ubiquitinconjugating enzymes (E2-D, and E2-N), ankyrin, and cathepsin K were upregulated in ZYM1288 at least in one time point. These genes are possibly affecting the increased immunity in ZYM1288 and could be candidates for gene specific characterization studies (Supplementary Table S3).

c. Plant pathogen interaction pathway
One of the key responses in plants to pathogen attack is the activation of plant pathogen interaction pathway. It is a basic resistance pathway but includes the most important transcriptional reprogramming events that help plants to defend themselves against invading pathogens such as PM [26,27]. Between the PM infected XL19 and ZYM1288 hulless barley leaves, 233 DEGs were enriched in this pathway (Supplementary Table S5). Two CDPK genes, which cause the hypersensitive response (HR), (HOVUSG1976600 and HOVUSG1976900) were upregulated in ZYM1288. Similarly, the upregulation of CMLs and CNGC indicates that ZYM1288 senses changes in Ca 2+ concentrations and responds by the stomatal closure, cell wall reinforcement, and HR. Additionally, the upregulation of many FLS2s, MAP3Ks, HSP90kDas, PRPs, and PBS1 indicates that signaling related to the activation of defense related proteins in ZYM1288 is stronger than in XL19. The differential but not consistent regulation of effector triggered immunity (ETI) related genes such as RINs, RPSs, and a large number of RPMs indicate that ETI is involved in defense against PM in both genotypes. Based on the above-mentioned transcriptomic changes, it could be stated that ZYM1288 uses PAMP-triggered immunity (PTI) and ETI related genes to defend itself against the invading PM but both types of immunity mechanisms are active in the tested two genotypes.

c. Plant pathogen interaction pathway
One of the key responses in plants to pathogen attack is the activation of plant pathogen interaction pathway. It is a basic resistance pathway but includes the most important transcriptional reprogramming events that help plants to defend themselves against invading pathogens such as PM [26,27]. Between the PM infected XL19 and ZYM1288 hulless barley leaves, 233 DEGs were enriched in this pathway (Supplementary Table S5). Two CDPK genes, which cause the hypersensitive response (HR), (HOVUSG1976600 and HOVUSG1976900) were upregulated in ZYM1288. Similarly, the upregulation of CMLs and CNGC indicates that ZYM1288 senses changes in Ca 2+ concentrations and responds by the stomatal closure, cell wall reinforcement, and HR. Additionally, the upregulation of many FLS2s, MAP3Ks, HSP90kDas, PRPs, and PBS1 indicates that signaling related to the activation of defense related proteins in ZYM1288 is stronger than in XL19. The differential but not consistent regulation of effector triggered immunity (ETI) related genes such as RINs, RPSs, and a large number of RPMs indicate that ETI is involved in defense against PM in both genotypes. Based on the above-mentioned transcriptomic changes, it could be stated that ZYM1288 uses PAMP-triggered immunity (PTI) and ETI related genes to defend itself against the invading PM but both types of immunity mechanisms are active in the tested two genotypes.

Transcriptional Changes in Energy Related Pathways
Among energy related pathways, the stress effects start from the changes in photosynthetic efficiency in response to modifications in photosynthesis-antenna proteins and photosynthesis pathway [28]. Changes in these pathways are accompanied by other energy and metabolism related pathways including galactose metabolism, starch and sucrose metabolism, nitrogen metabolism, pentose phosphate pathway, citrate cycle, and carbon fixation in photosynthetic organisms [29]. The genes expressed between XL19 and ZYM1288 after the infection of PM were enriched in all the above-mentioned pathways signifying large scale transcriptional changes in energy related pathways. Eight photosystem II (PSII) proteins (PsbW, P680 reaction center D1 and D2 proteins, CP43, oxygen-evolving enhancer 1 and 3, 10 kDa protein, and P700 chla), and four ferredoxin proteins (two ferredoxins and two ferredoxin-NADP+reductases) were differentially regulated. Only two proteins (PsbW and P680 D1) were downregulated in ZYM1288 at 5 hpi indicating that XL19 has reduced photosynthetic activity as compared to ZYM1288. Seven light-harvesting complex proteins (one LhcI and six LhcII proteins) were also differentially expressed. The LhcI protein (chla/b3) and one LhcII (chla/b1) were upregulated in ZYM1288 (Supplementary Table S4). Twenty-seven DEGs were enriched in carbon fixation in photosynthetic organisms pathway (11 up-and 16 downregulated in at least one time point in ZYM1288). One MDH (malate dehydrogenase), one phosphoenolpyruvate carboxylase (PEP), two fructose-bisphosphate aldolases (ALDOA, class I and class II), and one aspartate aminotransferase (AST) were highly downregulated in ZYM1288 as compared to XL19. However, other MDHs were upregulated at all hpi of infection in ZYM1288, suggesting that alternative (or different) key steps are being transcriptionally regulated in response to PM infection in both genotypes. Thirty-three genes were differentially expressed between both genotypes after infection; 17 were downregulated and 16 were upregulated. A higher number of DEGs (108) were enriched in starch and sucrose metabolism. The DEGs that were enriched in the above-mentioned energy related pathways showed different transcription signatures i.e., different genes with similar annotation showed contrasting expression patterns, indicating that complex transcriptomic changes are triggered in response to PM infection and both genotypes vary on how and which step of the pathway is being regulated. For example, 10 beta-fructofuranosidases were differentially regulated at different time points; five were upregulated in ZYM1288 and five were downregulated. Similarly, one MDH was highly downregulated in all time points while other MDHs were up/downregulated in at least one time point in citrate cycle. Nine nitrogen metabolism-related DEGs were found of which only three were upregulated (one carbonic anhydrase, one glutamine synthase, and a MFS transporter) in ZYM1288. The other DEGs in this pathway were upregulated in XL19. These expression changes further support our above statements and suggest that changes in energy metabolism related pathways discussed above are common to both genotypes, thus it could be a general response in hulless barley against PM infection (Supplementary  Table S4).

Transcriptional Changes in Defense Related Pathways
In addition to the plant-pathogen interaction pathway, our analyses indicated the enrichment of other defense related pathways including secondary metabolite biosynthesis, brassinosteroid biosynthesis, glutathione metabolism, and glucosinolate biosynthesis (Figure 6a). Fourteen DEGs related to brassinosteroids biosynthesis were differentially regulated between both genotypes after PM infection. With prolonging infection time, the number of DEGs increased. The steroid 22-alpha-hydroxylase (T9L24.4), two BR6-COX, two CYP724B1s, a BAS1, and one CYP92A6 gene were downregulated in at least one time point except 5 hpi. On the contrary, five CYP724B1s and two CYP92A6s were upregulated in ZYM1288 as compared to XL19. Both CYP724B1 and CYP92A6 are key players in the biosynthesis of brassinosteroids (Figure 6b) [30,31]. The upregulation of these genes suggests higher BR biosynthesis in ZYM1288 thus enhancing resistance against PM [32]. Fifty-nine genes related to glutathione metabolism were expressed between XL19 and ZYM1288 at least at one time point after the PM infection ( Figure 6c); 38 glutathione s-transferases (GSTs), six L-ascorbate peroxidases (APXs), three leucyl aminopeptidases (LAPs), four ribonucleosidediphosphate reductase subunit M1 (RRM1s), two gamma-glutamylcyclotransferase-plant (GGCTs), a glutathione synthase (GSS), an isocitrate dehydrogenase (IDH), a 6-phosphogluconate dehydrogenase (6PGD), and a glutathione reductase (GR). IDH, LAPs, RRM1s, and GSS were downregulated in the resistant genotype however the other genes were upregulated. In particular, we observed that a higher number of GSTs were upregulated in different time points after infection in ZYM1288. Other than the glutathione metabolism, four DEGs (phenylalanine N-monooxygenases, CYP79A2) were enriched in glucosinolate biosynthesis. Three DEGs were highly downregulated in ZYM1288 as compared to XL19 and one was upregulated in all four time points, indicating important role but variable expression pattern. This gene controls the initial steps of conversion of L-phenylalanine to benzyl-glucosinolate, which is known for its vital role in systematic defense against invading pathogens [33]. The upregulation of CYP79A2 in both genotypes indicates that it is a common defense response against PM in hulless barely. Finally, 590 genes enriched in secondary metabolite biosynthesis were differentially expressed between both genotypes after PM infection (Supplementary Table S4). The expression pattern was similar to that of energy related pathways i.e., genes with same annotation were both up-and downregulated in both genotypes indicating a significant role of secondary metabolites in defense against PM in hulless barley ( Figure 6).

Transcriptional Changes in Known PM Related Genes
We also searched the transcriptome of PM infected XL19 and ZYM1288 hulless barley genotypes. Particularly, we searched for DEGs such as Mlo, PEN1, SYP, SNARE, CYP79B2, CYP79B3, WRKY21, SGT1, SKP1, and HSP90, RPM genes, and other resistance related genes i.e., RGAs based on previous reports [15][16][17]. We found that a 1-acyl-sn-glycerol-3-phosphate acyltransferase (PLSC) was highly upregulated in ZYM1288 at all time points, while two ATPbinding cassette genes (subfamily G member 2) were downregulated at 5 hpi in ZYM1288 as compared to XL19. As discussed above, the RPM1s, IRAK1s and IRAK4s showed a variable expression pattern in both genotypes, while HSP90 genes were upregulated in ZYM1288 ( Figure 5; Supplementary Table S3). Similar expression pattern was observed for RPPs, SYP, and STIP1s (stress-induced phosphoprotein 1). Interestingly, we found a DOWNY MILDEW 2-like gene that was downregulated in ZYM1288 as compared to XL19 at 5, 24, and 36 hpi. Three inversins (ankyrin repeat and SOCS box protein 11like) were upregulated in ZYM1288 as compared to XL19. The inorganic pyrophosphatases (iPPase), multidrug resistance proteins (MRPs) showed variable expression pattern in both genotypes. The known resistance genes i.e., Mlas were all upregulated in ZYM1288 as compared to XL19; five upregulated at 5, 12, and 24 hpi, and two upregulated all time points. Three Mlo proteins (HOVUSG2614500, HOVUSG2614900 and HOVUSG5080300) were upregulated in XL19 at all time points after infection. These observations suggest that Mla genes enable ZYM1288 to resist the PM while the XL19 genotype is susceptible due to the increased expression of Mlo genes. A PP2A (serine/threonine-protein phosphatase 2A activator), a PRMC (release factor glutamine methyltransferase), a RRM2 (cold-inducible RNAbinding protein). A gamma-tubulin complex component 6 (TUBGCP6) were also upregulated in ZYM1288 (Figure 7).

qRT-PCR Analysis
The qRT-PCR analysis of seven DEGs in Bgh infected XL19 and ZYM1288 hulless barley genotypes was carried out using Actin gene as an internal control (Figure 8). The relative expression of these seven genes was consistent with that of the FPKM values. These results confirmed that the RNA-seq results are reliable.

Transcriptional Changes in Known PM Related Genes
We also searched the transcriptome of PM infected XL19 and ZYM1288 hulless barley genotypes. Particularly, we searched for DEGs such as Mlo, PEN1, SYP, SNARE, CYP79B2, CYP79B3, WRKY21, SGT1, SKP1, and HSP90, RPM genes, and other resistance related genes i.e., RGAs based on previous reports [15][16][17]. We found that a 1-acyl-sn-glycerol-3phosphate acyltransferase (PLSC) was highly upregulated in ZYM1288 at all time points, while two ATP-binding cassette genes (subfamily G member 2) were downregulated at 5 hpi in ZYM1288 as compared to XL19. As discussed above, the RPM1s, IRAK1s and IRAK4s showed a variable expression pattern in both genotypes, while HSP90 genes were upregulated in ZYM1288 ( Figure 5; Supplementary Table S3). Similar expression pattern was observed for RPPs, SYP, and STIP1s (stress-induced phosphoprotein 1). Interestingly, we found a DOWNY MILDEW 2-like gene that was downregulated in ZYM1288 as compared to XL19 at 5, 24, and 36 hpi. Three inversins (ankyrin repeat and SOCS box protein points. Three Mlo proteins (HOVUSG2614500, HOVUSG2614900 and HOVUSG5080300) were upregulated in XL19 at all time points after infection. These observations suggest that Mla genes enable ZYM1288 to resist the PM while the XL19 genotype is susceptible due to the increased expression of Mlo genes. A PP2A (serine/threonine-protein phosphatase 2A activator), a PRMC (release factor glutamine methyltransferase), a RRM2 (cold-inducible RNA-binding protein). A gamma-tubulin complex component 6 (TUBGCP6) were also upregulated in ZYM1288 (Figure 7).

qRT-PCR Analysis
The qRT-PCR analysis of seven DEGs in Bgh infected XL19 and ZYM1288 hulless barley genotypes was carried out using Actin gene as an internal control (Figure 8). The

Phytohormone Level and Signaling Is Differentially Affected in XL19 and ZYM1288
Auxin is primarily a development related hormone, however, it also plays roles in stress responses in plants. The overlapping expression of auxin responsive genes has been particularly observed in biotic stress responses [34]. The variable levels of auxins and expression patterns of DEGs implicated in auxin signaling propose that auxins are possibly playing role in defense against PM in both genotypes used in this study. The higher expression levels of auxin transporter 1, GH3.8, and SAUR's is possibly one of the main reasons that ZYM1288 leaves stayed green since these proteins influence the cell enlargement and plant growth [35]. In addition to auxin, the increased resistance of ZYM1288 might be due to higher tZ levels. We state this because of the recent reports that cytokinins can play a role as a priming agent and promote pathogen resistance and induce immunity in hosts e.g., barley [21,36]. Increased cytokinin signaling as a result of the upregulation of HKs, PHP5, and ORRs probably played a role in the strong defense response against PM in ZYM1288. Previously, HK5 and PHPs in Arabidopsis have been shown to enhance resistance against fungal pathogens [37,38], while, ORRs mediate interactions between cytokinin and SA in plant immunity [39]. The strong changes in the stress hormones i.e., JA, SA, and ABA are consistent with earlier studies of barley plants (and generally in PM resistant plant species) that showed higher levels of these hormones when infected with PM [21,40]. The increased ABA levels might have triggered the high expression of signaling pathways related DEGs such as ABA-insensitive 5 (ABI5) (Figure 3) that induces the expression of many ABA-responsive genes involved in resistance mechanisms [41]. The increased expression of DEGs related to JA signaling i.e., TIFYs, MYC2s, JAR1, and CLIKE3 and the concertation changes clearly indicates the possibility that ZYM1288 performs better and exhibits resistance to PM, probably due to increased expression of JA signaling related genes and higher JA levels, while XL19 might not. These changes are consistent with the known role of JA signaling in plant resistance to biotrophic pathogens [42]. Finally, the resistance in ZYM1288 might also be due to higher SA levels ( Figure 1) and subsequent activation of many PRP proteins (Figure 3) [43]. Previous studies have also explored such a role of SA against PM in grapevine infected with PM [27]. Taken together,

Phytohormone Level and Signaling Is Differentially Affected in XL19 and ZYM1288
Auxin is primarily a development related hormone, however, it also plays roles in stress responses in plants. The overlapping expression of auxin responsive genes has been particularly observed in biotic stress responses [34]. The variable levels of auxins and expression patterns of DEGs implicated in auxin signaling propose that auxins are possibly playing role in defense against PM in both genotypes used in this study. The higher expression levels of auxin transporter 1, GH3.8, and SAUR's is possibly one of the main reasons that ZYM1288 leaves stayed green since these proteins influence the cell enlargement and plant growth [35]. In addition to auxin, the increased resistance of ZYM1288 might be due to higher tZ levels. We state this because of the recent reports that cytokinins can play a role as a priming agent and promote pathogen resistance and induce immunity in hosts e.g., barley [21,36]. Increased cytokinin signaling as a result of the upregulation of HKs, PHP5, and ORRs probably played a role in the strong defense response against PM in ZYM1288. Previously, HK5 and PHPs in Arabidopsis have been shown to enhance resistance against fungal pathogens [37,38], while, ORRs mediate interactions between cytokinin and SA in plant immunity [39]. The strong changes in the stress hormones i.e., JA, SA, and ABA are consistent with earlier studies of barley plants (and generally in PM resistant plant species) that showed higher levels of these hormones when infected with PM [21,40]. The increased ABA levels might have triggered the high expression of signaling pathways related DEGs such as ABA-insensitive 5 (ABI5) (Figure 3) that induces the expression of many ABA-responsive genes involved in resistance mechanisms [41]. The increased expression of DEGs related to JA signaling i.e., TIFYs, MYC2s, JAR1, and CLIKE3 and the concertation changes clearly indicates the possibility that ZYM1288 performs better and exhibits resistance to PM, probably due to increased expression of JA signaling related genes and higher JA levels, while XL19 might not. These changes are consistent with the known role of JA signaling in plant resistance to biotrophic pathogens [42]. Finally, the resistance in ZYM1288 might also be due to higher SA levels ( Figure 1) and subsequent activation of many PRP proteins (Figure 3) [43]. Previous studies have also explored such a role of SA against PM in grapevine infected with PM [27]. Taken together, ZYM1288 shows resistance to PM possibly due to increased levels of defense related phytohormones and cytokinin and subsequent expression changes in the related signaling genes.

MAPK Signaling Pathway and Toll-Like Receptor Signaling Pathways Are Activated in Response to PM in Hulless Barley
MAPKs are evolutionarily conserved proteins in plants and are required for the activation of plant immunity [44]. MAPK signaling is one of the earliest signaling events after the plant has sensed the pathogen attack. The regulation of more than a hundred DEGs in this pathway strongly indicates the possibility that PM attack is sensed and signals are sent to downstream pathways for the activation or inactivation of genes/proteins [45] (Figure 4). Upon infection with PM, the differential expression of FLS2s in both genotypes suggests that FLS2-mediated defense response might be active; relatively higher FLS2s in ZYM1288 are possibly enabling its better resistance to PM [46]. We propose this since a similar expression trend of WRKY33s was noted in ZYM1288 ( Figure 4). The higher expression of MAPKs (and MAP2Ks and MAP3Ks) during early infection hours in ZYM1288 is quite relatable to their downstream presence of pathogen sensors/receptors [44]. Furthermore, the differential regulation of NDPKs, ANP1, PRPs, MAPK, MAP2K, and MAP3Ks in both genotypes indicate the possibility that that hulless barley, after PM attack, prepares for cell death and/or H 2 O 2 production, accumulation of reactive oxygen species, and activates defense related proteins [47]. We discussed in the above section that JA might also be playing role in defense responses against PM in ZYM1288. In this regard, the increased expression of CHI-Bs, ERF1s, and VIP2 (along with MYC2s) in ZYM1288 is possibly generating wounding as well as defense responses through the cross-talk between ethylene and JA signaling [48]. Taken together, it can be suggested that the MAPK signaling pathway is one of the defense strategies that is general to hulless barley genotypes to send downstream signals for defense responses. Relatively higher expression of key genes in MAPK signaling pathway might be enabling ZYM1288 to show higher resistance to PM. Further, the results showed that triggering toll-like receptor kinase signaling pathway related genes (especially a higher number of IRAK1s and IRAK4s) is possibly a common response to PM in hulless barley. This proposition is based on the observation of variable expression of these genes in both studied genotypes (Supplementary Table S2). It is known that these receptor like kinases act as a bridge between PM signals and intracellular regulatory machinery in plants [49].

PM Infection Modulates Changes in Energy Related Pathways in Hulless Barley
Powdery mildew infection significantly affects the photosynthesis and subsequent energy related processes [50]. One of the reasons for the increasing leaf yellowing with the time in XL19 is possibly due to the reduced expression of lhc1 (HOVUSG0250900) as compared to ZYM1288, meaning that the capture and delivery of the excitation energy to PSI in XL19 was reduced [51]. Further, the downregulation of relatively higher number of photosynthesis associated proteins (photosystem II (PSII) proteins and ferredoxin proteins) in XL19 reduced its photosynthetic potential that possibly affected downstream energy metabolism related pathways [52,53]. The slight yellowing noticed in ZYM1288 at latter time points (36 hpi) is possibly because of the downregulation of ferredoxin-NADP+ reductase, P680 D1, and a PSII 10 kDa protein (Supplementary Table S3). Thus, PM affects photosynthesis and photosynthesis-antenna proteins in hulless barley regardless of the genetic background but the resistant genotype showed higher expression of related genes, particularly at early infection stages [54]. PM also affected the downstream pathways i.e., carbon fixation in photosynthetic organisms, nitrogen metabolism, citrate cycle, and starch and sucrose metabolism in both genotypes. These changes are consistent with the findings that PM infection significantly impaired the tricarboxylic acid cycle, electron transfer capacity, photochemical efficiency in rubber tree [55]. These transcriptomic signatures are complex and should be explored with a specific focus on their role in defense against PM in barley. Previously, it is known that PM infection significantly alters the CO 2 fixation and light utilization in sugar beet leaves [56]. Possibly, the PM is affecting hulless barley leaves similarly, since both genotypes exhibited differential regulation of the key enzymes (MDH, PEP, ALDOAs, AS, and RBCSs) [57][58][59]. Such changes at biochemical level have also been reported in rubber tree [55].

PM Triggers Changes in Defense-Associated Pathways in Hulless Barley
The plant-pathogen interaction pathway is the major pathway associated with defense mechanisms in plants is [60]. CDPKs are present in cytoplasm which (along with CNGCs, CMLs, NOS, and RBOs) execute PTI and develop the HR and reinforce cell wall (and stomatal closure) [61]. In addition to these roles, the CKPKs in barley have been shown to antagonistically control the entry of PM into the host cell [62]. Therefore, the differential expression of CDPKs in both genotypes indicated that hulless barley uses HR to PM infection. However, the upregulation of multiple genes in these key stages of HR in ZYM1288 like CMLs, CNGC, and NOS enabled this genotype for better resistance against invading PM ( Figure 5). Further, HR is also driven by ETI in which RINs, RPMs, and RPSs effect SGT1, HSP90, and RAR1 [63]. The effector triggered genes, i.e., RINs, RPMs, and RPSs, were differentially expressed in both genotypes, indicating that hulless barley also relies on these genes for resistance against PM. However, upregulation of HSP90 in ZYM1288 could be another reason for enhanced resistance to PM.
Apart from the PTI and ETI, several other pathways such as brassinosteroid biosynthesis, glutathione metabolism, and glucosinolate metabolism are essential defense strategies in plants [64][65][66]. The higher expression of T9L24.44, BR6-COX1, CYP724B1s, BAS1, and CYP92A6s in ZYM1288 must have increased the brassinosteroid levels that led to resistance to PM. This is supported by the known function of these genes in brassinosteroid biosynthesis and the fact that brassinosteroids defend plants against broad range of diseases, particularly against the virulent PM Odium sp. in tobacco [67]. Furthermore, the upregulation of two BRI1s in ZYM1288 is also in agreement with the results of an earlier study that brassinosteroid led increased resistance in uzu barley lines [67]. In addition to brassinosteroids, studies in other plants have highlighted the role of glutathione in plant signaling and ultimately defense responses against biotic stresses [68]. The differential expression of DEGs related to this pathway in the leaf transcriptome of XL19 and ZYM1288 suggests that PM infection triggers changes in glutathione metabolism in hulless barley. Particularly, the upregulation of GSTs is consistent with the previous findings that PM infection induced higher transcripts of GstA1 gene in barley and it was suggested that it mainly prevents plant cell disruption and death by PM [69]. However, other studies identified that GST activity can be variable in resistant and susceptible genotypes, which is consistent with our results [70,71]. Apart from these two pathways, the large-scale transcriptomic changes in secondary metabolite biosynthesis indicates that secondary metabolites may also play important roles in resistance to PM. This in agreement with the transcriptome level studies in wheat, barley, grapevine, Vitis pseudoreticulata, Fragaria vesca, pumpkin, and melon [19,20,[72][73][74]. Secondary metabolites play diverse roles in plant innate immunity and the differential regulation of this pathway in both hulless barley genotypes is indicative of diverse role of this pathway against resistance to PM (Supplementary Table S3).

XL19 and ZYM1288 Differ in the Expression of Known PM Resistance Loci/Genes
In barley, the control of Bgh is associated with many resistance-related loci. The comparative transcriptome of both genotypes suggested the presence of both Mlo and Mla loci. The resistance of ZYM1288, based on leaf transcriptome, is possibly due to the downregulation of the Mlo genes and the upregulation of Mla genes. This is quite understandable since the knockdown of Mlo genes in grapevine reduces the susceptibility to PM, thus, the relative upregulation of these genes in XL19 made this genotype susceptible. Contrastingly, the Mla locus in barley gives isolate-specific immunity to PM [75], thus the ZYM1288 is a PM resistant genotype. Apart from these loci, it is known that SNARE-dependent antimicrobial secretion pathway limits the PM penetration [76], including different SNARE proteins (including SYPs) [77]. The upregulation of two SYPs (syntaxin 1B/2/3) in ZYM1288 suggested their role in limiting the Bgh penetration. On the other hand, in XL19, its down-regulation as compared to ZYM1288 is possibly due to the fact that syntaxin-dependent exocytosis is required for mlo-based defense as observed previously [78,79]. Another known regulator of barley defense against PM is the SKP1, that controls the abundance of PM susceptibility factor (RACB) [80]. This gene was active in both genotypes since its differential expression was modulated contrastingly in XL19 as well as ZYM1288 (Figure 7). Apart from the possible roles of these genes in resistance to PM, the resistance in ZYM1288 can also be explained by the high upregulation of PLSC gene (Figure 7) since a recent study reported glycerol-mediated PM resistance due to increased expressed on glycerol 3-phosphate [81]. The upregulation of inversins also increased PM resistance in ZYM1288 as soybean transgenic lines containing ankyrin-repeat containing gene exhibited enhanced resistance to fungus (Fusarium virguiforme) [81].

Conclusions
In this study, we compared the phytohormone levels and the transcriptome of two hulless barley genotypes with contrasting resistance to PM. ZYM1288 genotype was resistant to PM due to increased levels of defense related phytohormones i.e., ABA, SA, and JA. These increased levels in ZYM1288 triggered downstream phytohormone signaling cascade as noticed by the upregulation of genes such as ABI5, TIFYs, MYC2s, JAR1, and CLIKE3, and PRP. The JA signaling as a part of MAPK signaling pathway possibly induced wounding and defense responses. The PM infection triggered the MAPK signaling pathway, toll-like receptor kinase signaling, and plant-pathogen interaction pathways. Infection with Bgh modulated changes in the important energy related pathways. Genes related to both ETI and PTI were expressed in both ZYM1288 and XL19, however, increased expression of major genes in ZYM1288 possibly governed resistance to PM. Expression of brassinosteroid biosynthesis related genes was increased in ZYM1288, while the glutathione, glucosinolate, and secondary metabolite biosynthesis pathways showed variable expression patterns at different key steps of each pathway. The susceptibility of XL19 is indicated by the higher expression of Mlo genes, lower expression of Mla genes as compared to ZYM1288. The upregulation of Mla, syntaxin, and PLSC together with the regulation of brassinosteroid pathway, energy related pathways, signaling pathways, and plant-pathogen interaction pathway gave higher Bgh resistance potential to ZYM1288. The transcriptomic signatures identified in ZYM1288 are an important basis for future breeding of hulless barley for increased resistance against PM.

Plant Materials, Growth Conditions, and Inoculation
Two hulless barley genotypes whose contrasting resistance levels to PM (Blumeria graminis) f. sp. Hordei (Bgh) i.e., susceptible variety Xila 19 (XL19) and resistant variety ZYM1288 were identified in a separate study at the Agricultural Research Institute of the Academy of Agricultural and Animal Husbandry of the Tibet Autonomous Region, China. Seeds were grown in plastic flowerpots (8 cm diameter) in a greenhouse at 22 • C temperature and a photoperiod of 12 h. The pathogen infected samples were collected from a local field and maintained on susceptible variety XL19 in the greenhouse. Humidity, temperature, and photoperiod for pathogen infected samples were maintained at 70%, 22/18 • C, and 14 h light/10 h darkness, respectively. When both XL19 and ZYM1288 seedlings reached two-leaf and one heart stage, they were infected with the Bgh as reported earlier [2], and the leaf samples were harvested at four time points i.e., 5, 12, 24, and 36 h, and control (CK, 0 h; the non-infected plants were considered as 0 h post infection (hpi)). The Bgh isolate was maintained on a susceptible hulless barley cultivar Z13 at Agricultural Research Institute of the Academy of Agricultural and Animal Husbandry of the Tibet Autonomous Region, China. Three biological replicates were collected for each time point and variety for phytohormone and transcriptome analyses.

Phytohormone Detection
Phytohormones were detected by MetWare (http://www.metware.cn/, accessed on 2 February 2020) based on the AB Sciex QTRAP 6500 LC-MS/MS platform. Briefly, 50 mg fresh weight of leaves per replicate for each variety were ground into powder in liquid nitrogen and extracted with methanol/water/formic acid (V/V/V, 15:4:1). The extracts were then dried by evaporating under nitrogen gas stream, followed by the addition of 80% methanol (V/V), and filtration through a 0.22 µm PFTE membrane filter. The filtrate was then subjected to LC-MS/S analyses as described previously [9,82].

Transcriptome Sequencing, Data Analyses, Differential Gene Expression, and Enrichment Analyses
Total RNA was extracted from each biological replicate of XL19 and ZYM1288 and after checking its purity, it was quantified and integrity was tested. The cDNA was synthesized, libraries were prepared, and sequenced on Illumina HiSeq platform (Illumina Inc., San Diego, CA, USA) at MetWare (http://www.metware.cn/, accessed on 2 February 2020). The methods for cDNA synthesis, library construction, and sequencing were adopted from previous reports [83].
FastQC was used for quality assessing and obtaining clean reads followed by checking the GC content distribution (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 2 February 2020). The clean reads were stitched in Trinity [84] and compared to the reference genome [22,85] using HISAT2 [86] and the proportion of generated sequence reads that were aligned to the reference genome as calculated as total mapped reads/fragments.
The gene expression was quantified as Fragments Per Kilobase of transcript per Million Fragments Mapped (FPKM) [87]. The Pearson Correlation Coefficient (PCC) was estimated between the expression of biological replicates [83]. Differentially expressed genes (DEGs) between XL19 and ZYM1288 treatments were identified by using DESeq2 [88] by normalizing the read counts in featureCounts [89]. We used fold change (FC) ≥ 1 [90] and FDR correction set at p < 0.05 as screening conditions. Venn diagrams of DEGs were prepared in InteractiVenn [91].
The DEGs were annotated in different databases such as Kyoto Encyclopedia of Genes and Genomes (KEGG) [92], Gene Ontology (GO) [93], Clusters of Orthologous Groups (COG) [94], PfAM, Swissprot [95], Eukaryotic Orthologous Groups of proteins (KOG) [96] databases. To determine the gene expression, we compared the sequenced reads with the unigene library in Bowtie [97], and Fragments Per Kilobase of transcript per Million Fragments Mapped (FPKM) was estimated in combination with featureCounts (1.6.1) [89]. KEGG pathway enrichment of DEGs was done by using KOBAS2.0 and FDR correction (p < 0.05) was used to reduce false positive prediction [98]. The DEGs for specific pathway were filtered based on annotations and enrichment analyses and heatmaps were prepared in TBtools [99].

qRT-PCR Analysis of Selected DEGs
The expression of seven genes that were differentially expressed in response to Bgh infection in XL19 and ZYM was quantified by QRT-PCR to check the reliability of the RNA-Seq results. The primers used for analysis are listed in Table 1. GAPDH gene [100] was used as an internal control. The analysis was performed using applied Biosystems StepOnePlus TM Real-Time System (Applied Biosystems, Waltham, MS, USA). SYBR ® Select Master Mix (2X) was used for reactions. The reaction conditions were 90 • C for 10 min followed by 40 cycles of denaturation, annealing, and extension. Each reaction included denaturation at 90 • C for 10 s, annealing at 60 • C for 60 s, and extension at 72 • C for 30 s. The relative expression of each gene was calculated using 2 (− CT) method. The values of the relative expression were used to calculate mean and standard deviation in Microsoft Excel 2019 ® . Furthermore, we also determined the correlation (R 2 ) between the RT-PCR expression data and RNA-seq data in R.