Comparative TMT Proteomic Analysis Unveils Unique Insights into Helicoverpa armigera (Hübner) Resistance in Cajanus scarabaeoides (L.) Thouars

Abstract

Pigeonpea [Cajanus cajan (L.) Millspaugh] is an economically important legume playing a crucial role in the semi-arid tropics. Pigeonpea is susceptible to Helicoverpa armigera (Hübner), which causes devastating yield losses. This pest is developing resistance to many commercially available insecticides. Therefore, crop wild relatives of pigeonpea, are being considered as potential sources of genes to expand the genetic base of cultivated pigeonpea to improve traits such as host plant resistance to pests and pathogens. Quantitative proteomic analysis was conducted using the tandem mass tag platform to identify differentially abundant proteins between IBS 3471 and ICPL 87 tolerant accession and susceptible variety to H. armigera, respectively. Leaf proteome were analysed at the vegetative and flowering/podding growth stages. H. armigera tolerance in IBS 3471 appeared to be related to enhanced defence responses, such as changes in secondary metabolite precursors, antioxidants, and the phenylpropanoid pathway. The development of larvae fed on an artificial diet with IBS 3471 lyophilised leaves showed similar inhibition with those fed on an artificial diet with quercetin concentrations with 32 mg/25 g of artificial diet. DAB staining (3,3'-diaminobenzidine) revealed a rapid accumulation of reactive oxygen species in IBS 3471. We conclude that IBS 3471 is an ideal candidate for improving the genetic base of cultivated pigeonpea, including traits for host plant resistance.

Keywords: Cajanus cajan; IBS 3471; ICPL 87; TMT; feeding bioassays; phenypropanoid pathway; pigeonpea; quercetin.

Figure 1

Clustering of leaf proteome of IBS 3471 and ICPL 87 displayed as a 3D PCA and heatmap. (A) A 3D Principal Component Analysis (3D PCA) of accumulated proteins of IBS 3471 and ICPL 87. IBS 3471 samples highlighted in a purple circle (126,127N) are at the vegetative stage, blue circle (128C,128N) for the flowering stage, and black circle for the podding stage (127C). For ICPL 87 samples, at vegetative stage are highlighted in a green circle (129N,129C), at the flowering stage in a red circle (131,130C), and a black circle for the podding stage (130N). (B) A heat map showing unsupervised hierarchical clustering of statistically significantly differentially abundant proteins for IBS 3471 and ICPL 87.

Figure 2

Representation of all identified proteins as a volcano plot and statistical summaries of the differentially abundant proteins (DAPs). (A) Volcano plot showing all identified proteins during the vegetative stage. (B) Volcano plot showing all identified proteins during flowering/podding stage. Points above the red horizontal line represent proteins with significantly different abundances (p < 0.05) and the points below the red horizontal line are not significantly different (p < 0.05). All the red point represents proteins with a FC > ±1.5, while all the black points represent proteins with a FC < +1.5. (C) Summary of statistically significantly (p < 0.05) proteins and differentially abundant proteins (DAPs) identified for the two genotypes at the vegetative and flowering/podding growth stage.

Figure 3

GO functional classification of identified proteins and KEGG annotation of differentially abundant proteins (DAPs). (A) PANTHER GO functional classification in terms of biological process, molecular function, and cellular component. (B) KEGG total protein annotation. (C) KEGG metabolism annotation. (D) Biosynthesis of other secondary metabolites annotation.

Figure 3

GO functional classification of identified proteins and KEGG annotation of differentially abundant proteins (DAPs). (A) PANTHER GO functional classification in terms of biological process, molecular function, and cellular component. (B) KEGG total protein annotation. (C) KEGG metabolism annotation. (D) Biosynthesis of other secondary metabolites annotation.

Figure 4

DAB-stained leaf discs to detect the presence of ROS: (A) IBS 3471 DAB-stained leaf discs; (B) ICPL 87 DAB-stained leaf discs; (C) IBS 3471 control leaf discs; (D) ICPL 87 control leaf discs.

Figure 5

Quercetin LCMS chromatogram observed in sample IBS 3471(A), sample ICPL 87 (B), and quercetin standard (C).

Figure 6

Quercetin antibiosis effect on H. armigera larvae. (A) Average larval weight in mg observed on Day 3 after setting up the experiment. (B) Percentage of pupated H. armigera larvae on Day 11 after setting up the experiment. Each bar column represents a different artificial diet larva fed on supplemented with varying concentrations of quercetin (purple bars), lyophilised leaf powder of IBS 3471 (green bar), ICPL 87 (orange bar), and the experimental control, PAD (blue bar). Values labelled with different letters are significantly different at p < 0.01 (Tukey’s HSD test). All data are means ± standard errors (n = 48). PAD: plain artificial diet and QU: quercetin.

Figure 7

Protein accumulation comparisons with qRT-PCR for general phenylpropanoid, quercetin, and monolignol biosynthesis enzymes between IBS 3471 and ICPL 87. Solid arrows denote single biosynthetic steps while the dashed arrows represent multiple biosynthetic steps. Enzymes investigated are in red font and other enzymes in the pathway are black font. Phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate: CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavonoid 3′-hydroxylase (F3′H), flavonol synthase (FLS), cinnamoyl-Coenzyme A reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD). Red shading represent IBS 3471 and green shading is for ICPL 87. Bar graphs represent qRT-PCR expression level and pie chart represent proteomic expression level.