Multiple molecular defense strategies in Brachypodium distachyon surmount Hessian fly (Mayetiola destructor) larvae-induced susceptibility for plant survival

Abstract

The Hessian fly is a destructive pest of wheat causing severe economic damage. Numerous genes and associated biological pathways have been implicated in defense against Hessian fly. However, due to limited genetic resources, compounded with genome complexity, functional analysis of the candidate genes are challenging in wheat. Physically, Brachypodium distachyon (Bd) exhibits nonhost resistance to Hessian fly, and with a small genome size, short life cycle, vast genetic resources and amenability to transformation, it offers an alternate functional genomic model for deciphering plant-Hessian fly interactions. RNA-sequencing was used to reveal thousands of Hessian fly-responsive genes in Bd one, three, and five days after egg hatch. Genes encoding defense proteins, stress-regulating transcription factors, signaling kinases, and secondary metabolites were strongly up-regulated within the first 24 hours of larval feeding indicating an early defense, similar to resistant wheat. Defense was mediated by a hypersensitive response that included necrotic lesions, up-regulated ROS-generating and -scavenging enzymes, and H₂O₂ production. Suppression of cell wall-associated proteins and increased cell permeability in Bd resembled susceptible wheat. Thus, Bd molecular responses shared similarities to both resistant and susceptible wheat, validating its suitability as a model genome for undertaking functional studies of candidate Hessian fly-responsive genes.

Figure 1

Differential expression of Brachypodium genes in response to Hessian fly infestation. Differentially expressed genes (DEGs) in Hessian fly-infested Bd plants were identified over a time-course of 1 (Bd1), 3 (Bd3) and 5 (Bd5) days after egg-hatch (DAH) as compared to the uninfested control plants. (a) Number of statistically significant (>2-fold change, p < 0.05) up-regulated and down-regulated DEGs in Bd samples. Number of genes for each category are indicated (b) Venn diagram depicting shared and uniquely expressed up-regulated (left panel) and down-regulated (right panel) DEGs in Bd1 (orange circle), Bd3 (yellow circle), and Bd5 (blue circle).

Figure 2

Differential expression of defense-related genes in Brachypodium infested with Hessian fly. Number of DEGs encoding several classes of defense-related proteins represented in the Bd transcriptome are indicated in the chart. Heatmaps depict expression profiles of genes encoding defense-related proteins over a time-course of 1 (Bd1), 3 (Bd3), and 5 (Bd5) DAH. All DEGs belonging to one class of defense-related proteins are grouped together within a heatmap. Since all genes represent the Bd Gene-IDs, the prefix “Bradi” has been removed and only the number associated with a particular Gene-ID is given for identification. Green represents up-regulated genes and red represents the down-regulated genes, while genes not differentially expressed at a particular time-point are indicated in black. glp: germin-like proteins, PI: protease inhibitors, PR: pathogenesis-related.

Figure 3

Differential regulation of reactive oxygen species (ROS) metabolism genes in Brachypodium infested with Hessian fly. (a) Bd plants exhibiting dark hypersensitive response (HR)-like necrotic lesions on the main stem. (b) Pie charts showing DEGs encoding ROS-generating (left chart) and -scavenging (right chart) enzymes represented in the Bd transcriptome. (c,d) Heatmaps depicting the expression profiles of enzymes involved in ROS-generation (c) and -scavenging (d) over a time-course of 1 (Bd1), 3 (Bd3), and 5 (Bd5) DAH. Since all genes represent the Bd Gene-IDs, the prefix “Bradi” has been removed and only the number associated with a particular Gene-ID is given for identification. Green represents up-regulated genes and red represents the down-regulated genes, while genes not differentially expressed at a particular time-point are indicated in black. (e) Hessian fly-infested Bd plant (right panel) showing positive (brown coloration) DAB (3,3′-diaminobenzidine) staining at 1 DAH confirming ROS accumulation. Uninfested Bd plants (left panel) were used as controls for the DAB staining. NOX: NADPH-dependent oxidase, AO: amine oxidase, AOX: alternative oxidase, PLA2: phospholipase A2, POX: class III peroxidase, CAT: catalase, APX: ascorbate peroxidase, GPX: glutathione peroxidase, MDAR: dehydroascorbate reductase, TXN: thioredoxin, PRDX: peroxiredoxin, GRX: glutaredoxin, FD: ferrodoxin, GST: glutathione-S-transferase.

Figure 4

Differential regulation of Brachypodium genes involved in the biosynthesis of secondary metabolites in response to Hessian fly larval attack. Schematic representation of secondary metabolites produced in plants via Shikimate, MVA (mevalonic acid), and MEP (methylerythritol phosphate) pathways. The pathways are localized to the chloroplast and the cytosol. The DEGs involved in the production of secondary metabolites are displayed. Green arrows denote up-regulated and red arrows denote down-regulated genes. Heatmaps depict the expression profiles of the DEGs over a time-course of 1 (Bd1), 3 (Bd3), and 5 (Bd5) DAH, with all genes producing one type of secondary metabolite clustered together. Since all genes represent the Bd Gene-IDs, the prefix “Bradi” has been removed and only the number associated with a particular Gene-ID is given for identification. On the heatmaps, green represents up-regulated genes and red represents down-regulated genes, while genes not differentially expressed at a particular time-point are inducated in black. AS: anthranilate synthase, TS: tryptophan synthase, IGPS: indole-3-glycerol phosphate synthase, ADT: arogenate dehydratase, CM: chorismate mutase, PAL: phenylalanine ammonia lyase, HCT: hydroxycinnamoyl-CoA shikimate/ quinate hydroxycinnamoyl transferase, C4H: cinnamate-4-hydroxylase, STS: stilbene synthase, CHS: chalcone synthase, FLS: flavonol synthase, DHR: dihydroflavonol 4-reductase, CCR: cinnamoyl CoA reductase, CAD: cinnamyl alcohol dehydrogenase, DXS: deoxyxylulose-5-phosphate synthase, GGPS: geranylgeranyl pyrophosphate synthase, FDPS: farnesyl diphosphate synthase, TPS: terpene synthase, TPC: terpenoid cyclase, DMAPP/IPP: dimethylallyl pyrophosphate/isopentenyl diphosphate, FPP: farnesyl pyrophosphate, GPP: geranylgeranyl pyrophosphate.

Figure 5

Differential expression of genes encoding protein kinases in Brachypodium infested with Hessian fly larvae. Number of DEGs encoding various types of protein kinases represented in the transcriptome of Hessian fly-infested Bd plants are indicated in the chart. Heatmaps depict expression profiles of DEGs encoding protein kinases over a time-course of 1 (Bd1), 3 (Bd3), and 5 (Bd5) DAH. All DEGs belonging to one type of protein kinase are clustered together within the heatmap. Since all genes represent the Bd Gene-IDs, the prefix “Bradi” has been removed and only the number associated with a particular Gene-ID is given for identification. Green represents up-regulated genes and red represents the down-regulated genes, while genes not differentially expressed at a particular time-point are indicated in black. CRK: cysteine-rich receptor-like kinase, STK: serine threonine protein kinase, TPK: tyrosine protein kinase, MAPK: mitogen-activated protein kinase, CDK: cyclin-dependent kinase, CDPK: calcium-dependent protein kinase, WAK: wall-associated kinase, S-LPK: s-locus lectin protein kinase, L-LPK: conA lectin-like kinase, LPK: lectin protein kinase, PK: protein kinase, RLK: receptor kinase/receptor-like kinase, LRRK: leucine rich repeat kinase.

Figure 6

Cell wall metabolism in Brachypodium infested with Hessian fly larvae. Diagram showing list of genes involved in cell wall-related metabolism. Upward arrows indicate up-regulation and downward arrows indicate down-regulation of most members of a particular gene within the Hessian fly-infested Bd transcriptome. Asterisk represents a gene family with up- and down-regulated members. Heatmaps depict expression profiles of DEGs encoding cell wall-associated proteins and polysaccharides (cellulose, hemicellulose and pectins). The DEGs are grouped within a heatmap based on their associated function. Since all genes represent the Bd Gene-IDs, the prefix “Bradi” has been removed and only the number associated with a particular Gene-ID is given for identification. Green and red represent up-regulated and down-regulated genes, respectively, while genes not differentially expressed at a particular time-point are indicated in black. Neutral red stained Bd represents change in plant cell permeability in the main stem harboring the Hessian fly larvae at 4 DAH (left panel). Uninfested control Bd plants were pin pricked and stained with neutral red (right panel) to distinguish staining caused by larval feeding from that caused by physical damage. PRP: proline-rich protein, HPRG: hydroxyproline-rich glycoprotein, AGP: arabinogalactan protein, PGIP: polygalacturonase inhibiting protein, CWII: cell wall invertase inhibitor, GRP: glycine-rich protein, CesA: cellulose synthase, Csl: cellulose synthase-like, COBRA: glycosyl-phosphatidyl inositol-anchored protein, SuSy: sucrose synthase, PME: pectin methylesterase, PAE: pectin acetylesterase, PLL: pectin-lyase like, XTH: xyloglucan endotransglucosylase/hydrolase, β-gal: beta-galactosidase, β-xyl: beta-D-xylosidase, β-glu: beta-glucosidase, α-glu: alpha-glucosidase, GH: glycosylhydrolase, GT: glycosyltransferase, EXP: expansin.

Figure 7

Model showing the major pathways involved during Brachypodium-Hessian fly interactions. Damage to cell wall and membrane by Hessian fly larval feeding triggers the production of a hypersensitive response resulting in the production of ROS such as H₂O₂ through the action of NADPH oxidase. Multiple defense strategies are mounted simultaneously. Jasmonic acid (JA)-responsive genes are induced leading to up-regulation of PR proteins. Various transcription factors (TF) such as WRKY trigger defense response genes such as different kinases, lectins, and protease inhibitors, while a number of photosynthesis and cell wall-associated genes are repressed resulting in delayed or suppressed cell wall fortification. Increased cytokinins (CK) induce secondary metabolite formation that may directly affect the survivability of the larvae. Some defense-responsive heat shock proteins (HSPs) play a role in resistance to the larvae, while some small HSPs induce susceptibility. WAK2: wall-associated kinase 2, S-LPK: s-locus protein kinase, L-LPK: conA lectin-like kinase, LPK: lectin protein kinase, LRRK: leucine rich repeat kinase, glp: germin-like protein, CAT: catalase, PRDX: peroxiredoxin, TXN: thioredoxin, GST: glutathione-S-transferase, TS: tryptophan synthase, PAL: phenylalanine ammonia lyase.