Proteomic analysis by iTRAQ-PRM provides integrated insight into mechanisms of resistance in pepper to Bemisia tabaci (Gennadius)

Abstract

Background: The Bemisia tabaci is a major leaf feeding insect pest to pepper (Capsicum annuum), causing serious damage to pepper growth and yield. It is particularly important to study the mechanism of pepper resistance to B. tabaci, and to breed and promote the varieties of pepper resistant to B. tabaci. However, very limited molecular mechanism is available about how plants perceive and defend themselves from the destructive pest. Proteome technologies have provided an idea method for studying plant physiological processes in response to B. tabaci.

Results: Here, a highly resistant genotype and a highly susceptible genotype were exposed to B. tabaci feeding for 48 h to explore the defense mechanisms of pepper resistance to B. tabaci. The proteomic differences between both genotypes were compared using isobaric tag for relative and absolute quantification (iTRAQ). The quantitative data were validated by parallel reaction monitoring (PRM). The results showed that 37 differential abundance proteins (DAPs) were identified in the RG (resistant genotype), while 17 DAPs were identified in the SG (susceptible genotype) at 48 h after B. tabaci feeding. 77 DAPs were identified when comparing RG with SG without feeding. The DAP functions were determined for the classification of the pathways, mainly involved in redox regulation, stress response, protein metabolism, lipid metabolism and carbon metabolism. Some candidate DAPs are closely related to B. tabaci resistance such as annexin D4-like (ANN4), calreticulin-3 (CRT3), heme-binding protein 2-like (HBP1), acidic endochitinase pcht28-like (PR3) and lipoxygenase 2 (LOX2).

Conclusions: Taken together, this study indicates complex resistance-related events in B. tabaci interaction, provides novel insights into the molecular mechanism underlying the response of plant to B. tabaci, and identifies some candidate proteins against B. tabaci attack.

Keywords: Bemisia tabaci; PRM; Pepper; Proteome; Resistance; iTRAQ.

Fig. 1

Strategy for analysis of protein expression in pepper leaves by 4-plex isobaric tagging. SG, susceptible genotype; RG, resistant genotype; SB, susceptible genotype infested with B. tabaci; RB, resistant genotype infested with B. tabaci; SC, SB control; RC, RB control

Fig. 2

Symptom of different pepper genotypes, a susceptible genotype (SG) and a resistant genotype (RG) exposed to B. tabaci feeding. a Representative images of the SG following B. tabaci infestation. b Representative images of the RG following B. tabaci infestation. Left, the plant phenotype; Right, the mature B. tabaci populations on single leaf from either the SG or RG. Plants were infested with B. tabaci in the greenhouse for 72 h. c Anti-selectivity of different pepper varieties on B. tabaci

Fig. 3

Identification and analysis of the proteome by iTRAQ a Distribution of the number of peptides. The X axis represents the scope of the number of unique peptides, and the Y axis represents the number of proteins and corresponding cumulative percent. b Distribution of the proteins’sequence coverage. The pie chart displays the proportion of the number of the different proteins within the scope of coverage in the total protein amount. c Venn diagram of unique and shared proteins of three batches

Fig. 4

Venn diagram of differential abundance proteins (DAPs). a Venn diagram; b The number of up-regulated and down-regulated DAPs

Fig. 5

Gene ontology (GO) analysis of all differential abundance proteins (DAPs)

Fig. 6

Relative expression levels of selected proteins measured by PRM in the RB-RC, SB-SC and RC-SC. RB-RC represents protein level changes in the resistant genotype after B. tabaci infestation; SB-SC represents protein level changes in the susceptible genotype after B. tabaci infestation; RC-SC represents protein level changes in the resistant/susceptible genotype under control conditions. The protein samples for PRM were exacted from peppers treated with B. tabaci for 48 h

Fig. 7

Real-time PCR analysis of genes encoding the selected proteins in RB-RC, SB-SC and RC-SC. RB-RC represents protein level changes in the resistant genotype after B. tabaci infestation; SB-SC represents protein level changes in the susceptible genotype after B. tabaci infestation; RC-SC represents protein level changes in the resistant/susceptible genotype under control conditions. The expression levels of CAT, SOD, HBP1, LOX2, PLD and PR3 were quantified relative to the value obtained from control samples (B. tabaci-free plants). The column means the relative expression level of genes and the line means the trend value of iTRAQ