Our previous study indicated that glycerol application induced resistance to powdery mildew (Bgt) in wheat by regulating two important signal molecules, glycerol-3-phosphate (G3P) and oleic acid (OA18:1). Transcriptome analysis of wheat leaves treated by glycerol and inoculated with Bgt was performed to identify the activated immune response pathways. We identified a set of differentially expressed transcripts (e.g., TaGLI1, TaACT1, and TaSSI2) involved in glycerol and fatty acid metabolism that were upregulated in response to Bgt infection and might contribute to G3P and OA18:1 accumulation. Gene Ontology (GO) enrichment analysis revealed GO terms induced by glycerol, such as response to jasmonic acid (JA), defense response to bacterium, lipid oxidation, and growth. In addition, glycerol application induced genes (e.g., LOX, AOS, and OPRs) involved in the metabolism pathway of linolenic and alpha-linolenic acid, which are precursor molecules of JA biosynthesis. Glycerol induced JA and salicylic acid (SA) levels, while glycerol reduced the auxin (IAA) level in wheat. Glycerol treatment also induced pathogenesis related (PR) genes, including PR-1, PR-3, PR-10, callose synthase, PRMS, RPM1, peroxidase, HSP70, HSP90, etc. These results indicate that glycerol treatment regulates fatty acid metabolism and hormones cross-talk and induces the expression of PR genes that together contribute to Bgt resistance in wheat.

Starch and prolamin composition and content are important indexes for determining the processing and nutritional quality of wheat (Triticum aestivum L.) grains. Several transcription factors (TFs) regulate gene expression during starch and protein biosynthesis in wheat. Storage protein activator (TaSPA), a member of the basic leucine zipper (bZIP) family, has been reported to activate glutenin genes and is correlated to starch synthesis related genes. In this study, we generated TaSPA-B overexpressing (OE) transgenic wheat lines. Compared with wild-type (WT) plants, the starch content was slightly reduced and starch granules exhibited a more polarized distribution in the TaSPA-B OE lines. Moreover, glutenin and ω- gliadin contents were significantly reduced, with lower expression levels of related genes (e.g., By15, Dx2, and ω-1,2 gliadin gene). RNA-seq analysis identified 2023 differentially expressed genes (DEGs). The low expression of some DEGs (e.g., SUSase, ADPase, Pho1, Waxy, SBE, SSI, and SS II a) might explain the reduction of starch contents. Some TFs involved in glutenin and starch synthesis might be regulated by TaSPA-B, for example, TaPBF was reduced in TaSPA-B OE-3 lines. In addition, dual-luciferase reporter assay indicated that both TaSPA-B and TaPBF could transactivate the promoter of ω-1,2 gliadin gene. These results suggest that TaSPA-B regulates a complex gene network and plays an important role in starch and protein biosynthesis in wheat.

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.

Reactive oxygen species (ROS)- and hypersensitive response (HR)-mediated cell death have long been known to play critical roles in plant immunity to pathogens. Wheat powdery mildew caused by Blumeria graminis f. sp. tritici ( Bgt) is a destructive wheat pathogen. Here, we report a quantitative analysis of the proportion of infected cells with local apoplastic ROS (apoROS) versus intracellular ROS (intraROS) accumulation in various wheat accessions that carry different disease resistance genes ( R genes) at a series of time points postinfection. The proportion of apoROS accumulation was 70 to 80% of the infected wheat cells detected in both compatible and incompatible host−pathogen interactions. However, intensive intraROS accumulation followed by localized cell death responses was detected in 11 to 15% of the infected wheat cells, mainly in wheat lines that carried nucleotide-binding leucine-rich repeat R genes (e.g., Pm3F, Pm41, TdPm60, MIIW72, and Pm69). The lines that carry unconventional R genes, Pm24 ( Wheat Tandem Kinase 3) and pm42 (a recessive R gene), showed fewer intraROS responses, whereas 11% of Pm24 line-infected epidermis cells still showed HR cell death, suggesting that different resistance pathways are activated there. Here, we also demonstrated that ROS could not act as a strong systemic signal for inducing high resistance to Bgt in wheat, although it induced the expression of pathogenesis-related genes. These results provide new insights into the contribution of intraROS and localized cell death to immune responses against wheat powdery mildew.

Powdery mildew, caused by the fungus Blumeria graminis f. sp. tritici (Bgt), has limited wheat yields in many major wheat-production areas across the world. Introducing resistance genes from wild relatives into cultivated wheat can enrich the genetic resources for disease resistance breeding. The powdery mildew resistance gene Pm60 was first identified in diploid wild wheat Triticum urartu (T. urartu). In this study, we used durum as a ‘bridge’ approach to transfer Pm60 and Pm60b into hexaploid common wheat. Synthetic hexaploid wheat (SHW, AABBA^uA^u), developed by crossing T. urartu (A^uA^u) with durum (AABB), was used for crossing and backcrossing with common wheat. The Pm60 alleles were tracked by molecular markers and the resistance to powdery mildew. From BC₁F₁ backcross populations, eight recombinant types were identified based on five Pm60-flanking markers, which indicated different sizes of the introgressed chromosome segments from T. urartu. Moreover, we have selected two resistance-harboring introgression lines with high self-fertility, which could be easily used in wheat breeding system. Our results showed that the durum was an excellent ‘bridge’ for introducing the target gene from diploid T. urartu into the hexaploid cultivated wheat. Moreover, these introgression lines could be deployed in wheat resistance breeding programs, together with the assistance of the molecular markers for Pm60 alleles.