Proteomic analysis of soybean defense response induced by cotton worm (prodenia litura, fabricius) feeding

Proteome Science Proteomic analysis of soybean defense response induced by cotton worm (prodenia litura, fabricius) feeding Rui Fan 0 1 Hui Wang 0 Yongli Wang 0 Deyue Yu 0 0 National Center for Soybean Improvement, National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University , Nanjing 210095 , China 1 College of Chemical and Material Engineering, Quzhou University , Quzhou 324000 , China Background: Cotton worm is one of the main insects of soybean in southern China. Plants may acquire defense mechanisms that confer protection from predation by herbivores. Induced responses can lead to increased resistance against herbivores in many species. This study focuses on searching changed proteins in soybean defense response induced by cotton worm feeding. Results: Ten protein spots that are changed in abundance in response to cotton worm feeding were identified by Two-dimensional gel electrophoresis (2-DE). A total of 11 unique proteins from these spots were identified by MALDI-TOF MS. The mRNA and protein relative expression levels of most changed proteins were up-regulated. These proteins were mainly involved in physiological processes, including active oxygen removal, defense signal transduction, and metabolism regulation. Conclusion: This is the first proteomic analysis of the soybean defense response induced by cotton worm. The differentially expressed proteins could work together to play a major role in the induced defense response. PAL and SAMS were up-regulated at both the protein and mRNA levels. These genes can be strongest candidates for further functional research. Soybean; Induced resistance; 2-DE; qRT-PCR - Background As an important economic crop, soybean provides significant sources of fatty acids, proteins, vitamins, minerals and other nutrients for humans and animals, and it also has nonfood uses, such as in the production of industrial feedstocks and combustible fuels [1,2]. However, insect pests can adversely affect the yield and quality of soybean. Cotton worm is one of the main insect pests of soybean in southern China [3,4]. Therefore, improvement in resistance to cotton worm is one of the main soybean breeding objectives. During the process of evolution, plants have acquired defense mechanisms that confer protection from predation by herbivores. Two modes of resistance to herbivores exist: constitutive resistance, which is expressed independent of an attack; and induced resistance, which is activated only after the plant is attacked or otherwise injured [5]. Compared with constitutive resistance, induced resistance may be more durable and compromises plant fitness less by either decreasing further herbivore damage or increasing plant tolerance to herbivory [6]. Induced responses that lead to increased resistance against herbivores have been reported for over 100 species of plants, such as Arabidopsis [7-9], tobacco [10], tomato [11,12], rice [13], and soybean [14-16]. The resistance responses induced in plants change with different attackers. For example, the level of resistance induced by soybean looper herbivory with subsequent bean leaf beetle feeding was higher than that induced by bean leaf beetle herbivory with subsequent soybean looper feeding [16]. Resistance induced by the soybean looper, Mexican bean beetle and corn earworm in soybean has been reported in many studies [14-17]. However, induction of resistance in soybean by cotton worm has not been reported. Many studies have investigated the induced defense response mechanism of plants. The plant hormones salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are the main players in the regulation of signaling networks involved in induced defense [18-21]. In general, pathogens are more sensitive to SA-dependent responses, whereas herbivorous insects and necrotrophic pathogens are resisted by JA/ET-dependent defenses [22-24]. Ample evidence indicates JA is the main signaling molecule that mediates a plants defense system against herbivores [19,23]. A classic example is the observation that following attack by Manduca sexta larvae, tomato (Solanum lycopersicum) leaves accumulate JA, which results in the activation of genes encoding proteinase inhibitor proteins that inhibit digestive Ser proteinases of herbivorous insects and reduce further insect feeding [8,25]. Induced defense response mechanisms also involve the production of defensive compounds, such as proteinase inhibitors that affect insect feeding [25,26], volatile organic compounds that attract parasitoids and predators of the herbivores that feed on the plant [18,27] and extrafloral nectar that arrests carnivorous arthropods on herbivore-infested plants [28]. Proteomics is a powerful tool for the study of protein dynamics, especially in plant stress responses [29,30]. Analysis of protein profiling is important to understand how genes/proteins are regulated. In the present study, we found that soybean resistance to cotton worm increased after induction of resistance by cotton worm feeding. To investigate which proteins are involved in this induced defense response, we used proteomic approaches to provide an overview of the cotton worm-induced defense response in soybean. Ten protein spots showed differential expression patterns in soybean leaves in response to cotton worm feeding, and 11 proteins were identified. Quantitative RT-PCR (qRT-PCR) analysis indicated differentially expressed proteins also showed changes in mRNA expression level. Results and discussion Induction of soybean defense response by cotton worm feeding To determine whether cotton worm feeding can induce resistance in soybean leaves, we carried out a dual-choice test and force-feeding experiment (Figure 1). Compared with treated leaves, the cotton worm larvae preferred to eat control leaves, where PI < 1 at the four sampling timepoints (Figure 1A). The relative growth rate of cotton worms feeding on control leaves was higher than those feeding on treated leaves except at 6 h (Figure 1B). In the force-feeding experiment, the relative growth rates of cotton worms feeding on control leaves and those feeding on treated leaves were similar. At this timepoint fifth-instar cotton worms were used, which eat greedily and were not sensitive to the food source [31]. In the dual-choice test and force-feeding experiment, the levels of induced resistance were slightly different at the four sampling timepoints, but these results still indicated that cotton worms can induce resistance in soybean. Plants do not exhibit induced resistance to insects unless subjected to a certain degree of damage. In pre-experiments, soybean treated with cotton worm feeding shown induced resistance at a damage level of 20-30% and the levels of resistance were not increased significantly when the damage area increased. The capacity for induced resistance showed no difference also at a damage level between 15-45% in mountain birch [32]. However, when (...truncated)