Gel-free/label-free proteomic, photosynthetic, and biochemical analysis of cowpea (Vigna unguiculata [L.] Walp.) resistance against Cowpea severe mosaic virus (CPSMV) Anna Lidia N. Varela a , Setsuko Komatsu b , Xin Wang b , Rodolpho G.G. Silva a , Pedro Filho N. Souza a , Ana Karla M. Lobo a , Ilka M. Vasconcelos a , Joaquim A.G. Silveira a , Jose T.A. Oliveira a, ⁎ a Department of Biochemistry and Molecular Biology, Federal University of Ceara, CE, Brazil b National Institute of Crop Science, National Agriculture and Food Research Organization, Tsukuba 305-8518, Japan abstract article info Article history: Received 6 December 2016 Received in revised form 25 April 2017 Accepted 2 May 2017 Available online 11 May 2017 Cowpea severe mosaic virus (CPSMV) causes significant losses in cowpea (Vigna unguiculata) production. In this present study biochemical, physiological, and proteomic analysis were done to identify pathways and defense proteins that are altered during the incompatible interaction between the cowpea genotype BRS-Marataoã and CPSMV. The leaf protein extracts from mock- (MI) and CPSMV-inoculated plantlets (V) were evaluated at 2 and 6 days post-inoculation (DPI). Data support the assumptions that increases in biochemical (high hydrogen peroxide, antioxidant enzymes, and secondary compounds) and physiological responses (high photosynthesis index and chlorophyll content), confirmed by label-free comparative proteomic approach, in which quantitative changes in proteasome proteins, proteins related to photosynthesis, redox homeostasis, regulation factors/RNA processing proteins were observed may be implicated in the resistance of BRS-Marataoã to CPSMV. This pioneering study provides information for the selection of specific pathways and proteins, altered in this incom- patible relationship, which could be chosen as targets for detailed studies to advance our understanding of the molecular, physiological, and biochemistry basis of the resistance mechanism of cowpea and design approachs to engineer plants that are more productive. Biological significance: This is a pioneering study in which an incompatible relationship between a resistant cowpea and Cowpea severe mosaic virus (CPSMV) was conducted to comparatively evaluate proteomic profiles by Gel-free/label-free methodology and some physiological and biochemical parameters to shed light on how a resistant cowpea cultivar deals with the virus attack. Specific proteins and associated pathways were altered in the cowpea plants challenged with CPSMV and will contribute to our knowledge on the biological process tailored by cowpea in response to CPSMV. © 2017 Elsevier B.V. All rights reserved. Keywords: Cowpea CPSMV, incompatible interaction Biochemistry Physiology Gel free/label-free proteomic 1. Introduction Cowpea [Vigna unguiculata (L.) Walp.] is an important crop that belongs to the family Fabaceae, order Leguminosae, mainly grown in tropical and sub tropical regions in the world for vegetable and grains [1]. Usually cowpea grains contain high protein (20–29%) and starch (56–74%) contents, but low lipid level (0.5–3%) in dry bases and repre- sent a very important source of other several essential nutrients like vitamins and minerals and is devoid of cholesterol [2]. Cowpea is a ro- bust crop, but its production is impaired due to various environmental stresses, including diseases caused by viruses, bacteria, fungi, nema- todes, and insects. Cowpea severe mosaic virus (CPSMV) belongs to the genus Comovirus, family Secoviridae, and has a bipartite linear ssRNA(+) genome composed of RNA-1 and RNA-2 [3]. It is considered a major problem of cowpea in the world, responsible for important losses in the crop production and yield because cowpea plants infected with CPSMV suffer drastic growth reduction and development [4], which depend on the intrinsic susceptibility of each genotype. Typical symptoms in susceptible cowpeas are mosaic, chlorose, yellow patches, foliar distortion, and leaf morphology alterations that lead to reduction of some physiological parameters, primarily photosynthesis [5]. In a compatible interaction, virus-infected plants decline chlorophyll content, light-harvesting complex (LHC), and photosynthesis-related processes (non-photochemical quenching, stomatal conductance, and CO 2 assimilation). These adverse effects on the photosynthetic machin- ery favor the virus propagation and establishment since infected plants diminish the reactive oxygen species (ROS) contents and energy power (ATP and NADPH) production used to respond the viral infection [6,7] . These adverse effects on the photosynthetic machinery favor the virus Journal of Proteomics 163 (2017) 76–91 ⁎ Corresponding author. E-mail address: jtaolive@ufc.br (J.T.A. Oliveira). http://dx.doi.org/10.1016/j.jprot.2017.05.003 1874-3919/© 2017 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Journal of Proteomics journal homepage: www.elsevier.com/locate/jprot