Identification and characterization of two foxtail millet varieties in response to S. graminicola infection
Five foxtail millet varieties were evaluated for resistance to downy mildew after inoculation by mixing oospores and seeds in the field and greenhouse. The JG21 variety had the highest susceptibility (disease grade 9) , consistent with the reputation as the worst veriety against the pathogen, with a "grey back" incidence of downy mildew of 52.1% and 60.7% under field and greenhouse conditions, respectively (Table S1 and Fig. S1). The "grey back" incidence rate of the JG40 variety was slightly lower than that of JG21, with a value of 47.6% and 54.4% under field and greenhouse conditions, respectively. The incidence rates of FX3 and JG42 were lower than those of JG21 and JG40. G1 exhibited high resistance (disease grade 1) to S. graminicola, with the "grey back" incidence of downy mildew at 0.00% under both field and greenhouse conditions (Table S1 and Fig. S1). According to the above result, we selected highly susceptible JG21 and highly resistant G1 varieties for subsequent RNA-Seq analysis.
Phenotypic symptoms and transmission electron microscopy observations
The symptom of "grey back" mildew appeared at the seedling stage after S. graminicola infection in the JG21 variety. At the 3-leaf stage (3L), a sparse grey-white mould layer appeared on the back of leaves, and then the white mould layer became denser at the 5-leaf stage (5L), resulting in a large number of sporangiophores and sporangia. At the 7-leaf stage (7L), apart from a large number of mould layers and asexual spores, the leaf colour gradually turned green to yellow-white (Fig. 1A). However, the leaves of the resistant variety G1 did not show obvious symptoms after infection with S. graminicola (Fig. 1A). To clarify the ultrastructural changes in cell morphology in resistant and susceptible varieties, the leaf tissues of resistant and susceptible varieties at 3L, 5L and 7L after inoculation were observed by TEM (Fig. 1B). At 3L, the mesophyll cell and chloroplast double membrane of the susceptible variety JG21 were intact, and there were no markedly abnormal findings. The chloroplast granular layer was clear, and there were fewer starch grains (Fig. 1B-a, b). The mesophyll cells of G1 were arranged in a rectangular shape, and the cell membrane and cell wall were intact and clear; the chloroplasts were spindle-shaped and numerous; the granular layered structure was clear; and starch granule accumulation can be seen in mesophyll cells (Fig. 1B-c, d). At 5L, the leaf ultrastructure of the susceptible variety JG21 changed greatly. The most typical symptom was that the chloroplast was obviously deformed from a spindle to an oval or spherical shape. The number of white and spindle-shaped starch grains increased significantly, covering almost all chloroplast structures (Fig. 1B-e). The internal structure of the chloroplast was damaged, the thylakoids were loosely arranged, the granular layered structure was disordered, the arrangement was irregular, and the number of osmium-philic granules gradually increased (Fig. 1B-f). The cell walls of the resistant variety were relatively complete; the chloroplast granular layer was clear (Fig. 1B-g); there were more starch grains in the chloroplast; and there were papillary protrusions on the inner side of the cell wall (Fig. 1B-h). At 7L, pathogen infection caused severe damage to the chloroplast double-layer membrane of the susceptible variety JG21. Both the membrane structure and the granular layered structure were degraded, and the thylakoid membrane was completely destroyed. The inner and outer chloroplast membranes were obviously degraded and disappeared, the chloroplast was completely disintegrated, and starch grains with gelatinized edges were scattered in the cytoplasmic matrix (Fig. 1B-i). The formation of haustoria and oospores was observed in severely damaged cells (Fig. 1B-j). In the resistant variety G1, the chloroplasts in the cells were partially deformed, and some of the cells became vacuoles without an organelle structure (Fig. 1B-k). In addition, the chloroplast basal lamellae were disordered (Fig. 1B-l).
Physiological changes in the two foxtail millet varieties in response to S. graminicola infection
During their interaction with the host, pathogens can secrete a variety of cell wall degrading enzymes (CWDE), which can destroy the cell wall of the host, lead to necrosis symptoms or accelerate the development of the disease course, and provide nutrients such as sugar sources for the growth and reproduction of pathogenic bacteria [29]. To test this possibility in this study, we measured the activities of seven CWDE enzymes, including Cx, β-G, α-glucanase, β-1,3-glucanase polygalacturonase, PMG and PGTE, which act as virulence factors and elicitors of the host defence response. We determined the activities of CWDEs at 3L, 5L and 7L after infection by S. graminicola (Fig. 2). Expectedly, CX and PGTE activity was significantly higher in the susceptible variety JG21 than in the resistant variety G1 in all infection periods (Fig. 2A, D, F, G). Moreover, the activities of β-G, α-glucanase and PMG were significantly higher in JG21 than in G1 at 5L and 7L, with no considerable differences at 3L between the JG21 and G1 varieties after S. graminicola infection (Fig. 2B, C, E). The activities of cellulose-, hemicellulose- and pectin-degrading enzymes showed obvious differences in both varieties, and higher activities were found in the susceptible variety JG21. It can be speculated that the CWDEs were likely to degrade the host cell wall to obtain enough nutrition for the production of zoosporangia at the important stage of the formation of grey back at 5L.
Transcriptomic response of the resistant and susceptible varieties to S. graminicola
To clarify the molecular mechanism of the interaction between foxtail millet and S. graminicola, a total of 36 RNA samples isolated at 3L, 5L and 7L from ‘G1’ and ‘JG21’ leaves infected by S. graminicola were subjected to RNA-Seq. A total of 328.19 Gb of clean data was obtained after removing low-quality reads. The percentage of Q30 bases was 95.23%, and the average GC percentage was 57.67% (Table S2). After low-quality regions and adapter sequences were removed, an average of 30,498,412.80 bp clean reads were obtained and mapped to the reference genome with an average mapping ratio of 86.70% (Fig. 3). The results showed that the sequence quality of the transcriptome was sufficient for further analysis.
Global transcriptional responses of foxtail millet varieties resistant and susceptible to S. graminicola infection from 3L to 7L were investigated using RNA-Seq. DEGs were identified for S. graminicola-infected varieties relative to uninfected controls using an FDR ≤ 0.01 and a log fold change ≥1.0. A Venn diagram comparing DEGs in inoculated versus uninoculated foxtail millet at the three time points revealed 78, 118, and 321 DEGs in the resistant variety at 3, 5 and 7 L, respectively, among which 51, 88, and 304 DEGs were unique to each time point (Fig. 4A, B, E). In the susceptible variety, 17, 314 and 1105 DEGs were identified at L3, L5 and L7, respectively, among which 16, 311 and 1103 DEGs were unique to each time point (Fig. 4C, D, F). After being infected by the pathogen, the numbers of upregulated and downregulated DEGs increased in both varieties (Fig. 4E, F). We speculated that an increasing number of DEGs were involved in the interaction between foxtail millet and S. graminicola.
In the susceptible variety, the maximum number of DEGs (1211) occurred at 5L relative to the uninfected controls. Smaller numbers of DEGs (1104) were observed at 7L, and the minimum number of DEGs (8) was observed at 3L (Fig. 4C, D). In addition, as shown in Fig. 4 (Fig. 4A, B, C, D), it can be seen that the DEGs were not coexpressed in JG21(0- up, 0-down) or that there was a relatively small number of coexpressed genes (11-up, 3-down) in G1. We speculated that the genes involved in the regulation and the number of genes were different at different stages when the pathogen infected the host.
Common gene ontology enrichment responses to S. graminicola infection between JG21 and G1
To explore the molecular functions that were responsive to S. graminicola infection, we performed gene ontology term enrichment analysis of DEGs. All GO terms were assigned to three groups: biological process (BP), cellular component (CC) and molecular function (MF). The biological process category protein phosphorylation (GO:0006468) is one of the critical processes in plant‒pathogen interactions, which can regulate signal transduction in cells. For example, MAPK was activated, and the expression of defence genes can be induced by the phosphorylation cascade, thus improving plant resistance. The MFs related to serine/threonine kinase activity (GO: 000674), peroxidase activity (GO: 0004601), haem-binding (GO: 0020037) and laccases (GO: 0048046) were important functional groups involved in secondary metabolite production and important defence pathways. Upregulated and downregulated DEGs were associated with the plant defence response. Examples of peroxidase genes, cytochrome P450, laccases, leucine-rich-repeat receptor-like kinases (LRR-LRKs) and serine/threonine-protein kinases (STKs) are shown in Fig. 5. Sixty-eight percent of peroxidase genes were dramatically increased in G1 after S. graminicola infection at the early stage. Similar to peroxidase genes, 84% of the differentially expressed cytochrome P450 genes were upregulated in G1, and 16% of them were decreased at the same stage. The differentially expressed LRR-LRKs (62%) and STKs (42%) were upregulated at 3L and 5L in G1. In addition, three of six laccases (50%) had a much higher expression level in G1 than in JG21 at all infection stages. Overall, pathogen infection induced global gene expression changes in both varieties.
Identification of DEGs in common enrichment KEGG pathways in both varieties
To identify important genes that might be involved in metabolism or biosynthesis critical for downy mildew of foxtail millet resistance in G1, pathway enrichment analyses were performed using KEGG. Only a few defence-associated biosynthetic pathways involving cutin, suberin or wax biosynthesis were enriched in the transcriptome of the resistant varieties, while more pathways, such as glutathione metabolism and phenylalaniene metabolite, were significantly enriched in both resistant and susceptible varieties after inoculation. Comparisons of the expression levels of DEGs in these enriched pathways revealed that most genes were upregulated in the resistant cultivar but downregulated in the susceptible JG21. Among them, glutathione S-transferase genes were greatly upregulated after S. graminicola inoculation in G1 (Fig. 6). In contrast, the differentially regulated glutathione S-transferase genes exhibited distinctive expression patterns in the susceptible variety.
Phenylalanine ammonia-lyases (PALs) are involved in the synthesis of both lignin and phytoalexins to inhibit pathogens from penetrating cell walls [30]. Six PAL genes (Seita.1G240200, Seita.1G240400, Seita.1G240500, Seita.7G240600, Seita.7G168700 and Seita.7G168800) involved in the phenylalanine metabolism pathways were upregulated in G1 and were upregulated in JG21 (Fig. 6). Genes encoding 4-coumarate-CoA ligase (Seita.1G065800, Seita.1G283300 and Seita.6G167900) and trans-cinnamate 4-monooxygenase (Seita.5G361200) were upregulated and involved in lignin biosynthesis through the phenylpropanoid biosynthesis pathway. The defence-related gene PAL was also significantly upregulated in G1, which showed opposite expression patterns in JG21 after S. graminicola induction.
Pathway enrichment analysis revealed that the cutin, suberin and wax biosynthesis pathways were markedly enriched by DEGs. Overall, 11 of the DEGs were significantly differentially expressed between the resistant and susceptible varieties (Fig. 6). All DEGs were downregulated at 3L in both resistant and susceptible varieties. With further pathogen infection, approximately 81.8% (9) of DEGs were rapidly and strongly induced at 5L and 7L in the resistant variety G1. Three CER1 genes (Seita.1G237600, Seita.1G362700 and Seita.7G163300) were also dramatically induced and were involved in cutin and wax synthesis. In particular, the Seita.1G237600 gene showed 274.1- and 119.4-fold expression. Cytochrome 450 86A1/86A4 (Seita.7G197000), as the key enzyme of suberin synthesis, was found to be greatly upregulated, with 45.2- and 70.0-fold expression at 5L and 7L, respectively, in the resistant G1 variety. In contrast, these genes were generally suppressed or not significantly regulated at all infection stages in JG21 (Fig. 6). Divergent patterns of expression in the resistant and susceptible varieties suggest that these genes might be essential for foxtail millet resistance to S. graminicola in the G1 variety.
Genes related to hormonal pathways in response to downy mildew challenge
Phytohormones such as SA, JA, and ET are known to play major roles in regulating plant defence responses against various pathogens [31]. A total of 11 DEGs were identified as being involved in hormone signal transduction pathways, which involve auxin (IAA), salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA) and gibberellic acid (GA), all of which play an important role in downy mildew resistance (Fig. 7). Five genes (3 SAUR71, 1 GH3 auxin-responsive promoter and 1 GH3.1) were upregulated and significantly induced in the resistant variety G1, except Seita.5G434800, which was also downregulated in JG21. PR1 is the marker gene of SA and JA signal transduction. In this study, two PR-1 genes, Seita.2G124900 and Seita.2G124800, were significantly induced and upregulated in G1 but downregulated in JG21. Moreover, DEGs of TGAL8 and NPR5 showed opposite expression trends in the resistant G1 and susceptible JG21, respectively (Fig. 7). In addition, PYL4 (Seita.3G207900), which is associated with ABA metabolism, was not significantly differentially expressed in either variety (Fig. 7). Our finding that defence-related genes, including PR and auxin-responsive genes, showed opposite expression patterns between G1 and JG21 after S. graminicola infection indicates that these genes play essential roles in downy mildew resistance in G1.
Identification of DEGs in specific enrichment of starch and sucrose metabolism and the fatty acid elongation pathway in the susceptible variety JG21
Plant pathogen infection leads to the deposition of starch molecules in the chloroplast and the accumulation of sucrose in infected plant cells [32, 33]. We investigated the expression pattern of transcripts related to starch and sucrose metabolism under S. graminicola infection (Fig. 8). The expression of most genes related to sugar metabolism was also enhanced in the JG21 leaves (Fig. 8). Notably, beta-amylase (BMY) and sucrose-catabolism genes (SUSY) showed the highest expression levels at 5 L following S. graminicola attack in JG21, and these genes can promote the formation of glucose and fructose. Moreover, the expression of sucrose phosphate synthase (SPS), β-fructofuranosidase, which plays key roles in the sucrose synthesis process, also only markedly increased and was upregulated at 5L in JG21. Consistently, seven enzymes, glucan endo-1,3-β-glucosidase, β-glucosidase (β-G), pectinesterase (PE), sucrose 1-fructosyltransferase (1-SST), fructokinase-2 (FRK), UTP-glucose-1-phosphate uridylyltransferase and beta-D-xylosidase 4, involved in cellulose hydrolysis and sucrose accumulation were upregulated in JG21 at 5L, which was the opposite expression pattern reported in G1 in response to S. graminicola infection. We speculate that pathogen attack hijacked plant sucrose transporters, increased sugar outflow from infected sites, and eventually led to pathogen growth and plant defence at 5L.
The primary metabolism included elongation of fatty acids. Genes associated with fatty acid elongation, including nine 3-ketoacyl-CoA synthase, 4 very-long-chain 3-oxoacyl-CoA reductase and 1 very-long-chain (3R)-3-hydroxy acyl-CoA dehydratase, were also generally upregulated at 5L in the susceptible variety. The gene expression level of these DEGs was lower than that of 5L infected by S. graminicola (Fig. 8). In contrast, the expression of the DEGs was slightly induced and upregulated in 3L and then gradually decreased until 7L in the resistant variety (Fig. 8). Fatty acid elongation-related genes were suppressed by the pathogen in the 'G1' accession but somehow induced in the 'JG21' variety, consistent with the concept that S. graminicola is likely to be a fatty acid auxotroph that requires lipids from the host.
Validation of transcriptomics data by RT‒qPCR
To verify the differentially expressed genes based on RNA-Seq data, nine DEGs potentially involved in the defence against S. graminicola and representing different expression patterns in both the resistant and susceptible strains were selected for qRT‒PCR analysis. These genes included Seita.5G053000 (peroxidase), Seita.9G193900 (P450 84A1), Seita.7G168700 (PAL), Seita.3G270200 (β-D-Glucosidase), Seita.6G093400 (4-coumarate CoA ligase), Seita.7G234000 (cinnamyl alcohol dehydrogenase), Seita.2G366600 (photosystem I chlorophyll-a), Seita.2G328100 (multicopper oxidase) and Seita.1G253500 (receptor protein kinase). The relative expression values of all the tested genes were calculated using the constitutively expressed Actin gene. The results also showed a linear correlation between RNA-Seq and qRT‒PCR expression values for R2 (correlation coefficient of 0.71). The expression profiles of all tested genes resulting from qRT‒PCR were in line with our RNA-Seq data analysis results (Fig. 9).