Genome-wide transcriptomic and proteomic analyses of bollworm-infested developing cotton bolls revealed the genes and pathways involved in the insect pest defence mechanism

Abstract

Cotton bollworm, Helicoverpa armigera, is a major insect pest that feeds on cotton bolls causing extensive damage leading to crop and productivity loss. In spite of such a major impact, cotton plant response to bollworm infection is yet to be witnessed. In this context, we have studied the genome-wide response of cotton bolls infested with bollworm using transcriptomic and proteomic approaches. Further, we have validated this data using semi-quantitative real-time PCR. Comparative analyses have revealed that 39% of the transcriptome and 35% of the proteome were differentially regulated during bollworm infestation. Around 36% of significantly regulated transcripts and 45% of differentially expressed proteins were found to be involved in signalling followed by redox regulation. Further analysis showed that defence-related stress hormones and their lipid precursors, transcription factors, signalling molecules, etc. were stimulated, whereas the growth-related counterparts were suppressed during bollworm infestation. Around 26% of the significantly up-regulated proteins were defence molecules, while >50% of the significantly down-regulated were related to photosynthesis and growth. Interestingly, the biosynthesis genes for synergistically regulated jasmonate, ethylene and suppressors of the antagonistic factor salicylate were found to be up-regulated, suggesting a choice among stress-responsive phytohormone regulation. Manual curation of the enzymes and TFs highlighted the components of retrograde signalling pathways. Our data suggest that a selective regulatory mechanism directs the reallocation of metabolic resources favouring defence over growth under bollworm infestation and these insights could be exploited to develop bollworm-resistant cotton varieties.

Keywords: Gossypium hirsutum; Helicoverpa armigera; biotic stress response; defence mechanism; proteome; transcriptome.

Figure 1

Bollworm infested biotic stress induction in cotton bolls, G. hirsutum L. cv. Bikaneri Narma. (a) Method of biotic stress induction in cotton bolls under field conditions. (b) Boll developmental stages of control (CN) and bollworm infected tissues (BS) used in the current study (0, 2, 5 and 10 dpa/days post anthesis). (c) Schematic overview of proteome and transcriptome data generation and analyses workflow. (d) Number of differentially expressed transcripts (DETs) during boll development stages under BS as compared to their respective stages of CN. (e) Cluster analysis showing the differentially expressed transcripts related to biotic stress. (f) Number of differentially expressed proteins (DEPs) during boll development stages under BS as compared to their respective stages of CN.

Figure 2

Diagrammatic view of the up‐ and down‐regulated transcripts (a, b) and proteins (c, d) among the boll developmental stages under bollworm infestation in comparison with their respective controls. Gene Ontology‐based annotation and classification of the differentially expressed proteins into the (e) biological process, (f) cellular component and (g) molecular function categories.

Figure 3

Representative Coomassie stained 2D PAGE proteome profile of Control, CN (a) and Boll worm infested, BS (b) cotton bolls. Annotated 2D spots corresponding to the differentially expressed proteins identified using MALDI TOF/TOF. Detailed list of identified proteins are tabulated in Table S14. (c) 2D Spot profile of representative proteins showing differential expression under bollworm infestation in comparison with their respective control bolls during boll developmental stages (0, 2, 5 dpa). 2D PAGE proteome profile of infested and control bolls during developmental stages are presented in Figure S1.

Figure 4

Differentially expressed transcription factors (a) and phytohormone (b) under bollworm infestation as compared to their respective control. Numbers 1–4 represents different stages; (1)—0 dpa, (2)—2 dpa, (3)—5 dpa and (4)—10 dpa. Cluster analysis performed using log₂‐transformed fold change values showing the differentially expressed transcripts related to transcription factors (c) and phytohormones (d) at boll developmental stages. Putative transcription factors and phytohormones at each stage are presented in Tables S2 and S3.

Figure 5

Heat map view of the cluster analysis depicting the expression pattern of differentially expressed transcripts (DETs) (a) and differentially expressed proteins (DEPs) (b). Hierarchical cluster analyses of DETs (fold change ±3) and DEPs (fold change ±1.5) under biotic stress as compared to their respective control samples during fibre development stages (0, 2, 5 and 10 dpa). List of Affymetrix cotton probe set IDs, and fold change for transcripts present in each cluster are presented in Table S16. List of Spot IDs, protein accessions and fold change for proteins are presented in Table S17. The hierarchical clustering was performed using complete linkage method with Euclidean distance based on fold change data compared to control samples using Cluster 3.0.

Figure 6

Putative model depicting the regulation of molecular events in cotton bolls subjected to bollworm infestation. Genes related to the redox regulation—peroxidase (PX), super oxide dismutase (SODs), metabolic process—trehalose phosphate synthase (TPS), trehalose phosphate phosphatase (TPP), galactinol synthase (GolS), signalling cascades—calcium‐dependent protein kinase (CDPK), calcium‐binding proteins (CBPs), mitogen‐activated protein kinase (MAPK), enhanced disease resistance 1 (EDR1), phytohormone synthesis—lipoxygenase (LOX2, LOX3), allene oxide synthase (AOS), allene oxide cyclase (AOC), 12‐oxo‐phytodienoic acid reductase (OPR3), S‐adenosylmethionine synthetase (SAM), 1‐aminocyclopropane‐1‐carboxylate (ACC) synthase/oxidase, isochorismatase hydralase (ICH), isochorismate pyruvate lyase (IPL), 3‐oxo‐5‐alpha‐steroid 4‐dehydrogenase (DET 2), 24‐sterol C‐methyltransferase (SMT2‐2), transcription factors—ethylene insensitive 3(EIN3), heat‐shock transcription factor (HSf‐1), multiprotein bridging factor 1c (MBF1c), retrograde signalling—alternative oxidase (AOX), pentatricopeptide repeat containing protein (GUN1), defence—pathogenesis‐related (PR) protein, photosynthesis—photosystem (PS I, PS II), cytochrome complex (Cyt), light harvesting complex (LHC), hydroxy methyl bilane synthase (HMBS) and growth—carbohydrate active enzymes (CAZymes) are annotated along with their expression pattern. Upward pointing arrow indicates up‐regulation and downward pointing arrow indicates down‐regulation of respective genes. The overall pattern suggests the selective regulation of signalling cascades favouring defence over growth in bollworm‐infested cotton bolls.