Application of Label-Free Shotgun nUPLC–MS^E and 2-DE Approaches in the Study of Botrytis cinerea Mycelium

Abstract

The phytopathogenic fungus Botrytis cinerea infects more than different 200 plant species and causes substantial losses in numerous crops. The B05.10 and T4 wild-type strain genomes have been recently sequenced, becoming a model system for necrotrophic pathogens, as well as opening up new alternatives in functional genomics, such as proteomics. We analyzed B. cinerea mycelium from these two wild-type strains, introducing label-free shotgun nUPLC-MS(E) methodology to complement the 2-DE-MS-based approach. We assessed the label-free nUPLC-MS(E) methodology for protein identification and quantification using five mycelium protein dilutions. A total of 225 and 170 protein species were identified by nUPLC-MS(E) in the B05.10 and T4 strains, respectively. Moreover, 129 protein species were quantified in both strains. Significant differences in protein abundance were found in 15 more abundant and 16 less abundant protein species in the B05.10 strain compared to the T4 strain. Twenty-nine qualitative and 15 significant quantitative differences were found using 2-DE. The label-free nUPLC-MS(E) was a reliable, reproducible and sensitive method for protein identification and quantification to study the B. cinerea mycelial proteome. Results obtained by gel-based and gel-free complementary approaches allow a deeper characterization of this fungus, as well as the identification of potential virulence factors.

Using 7 cm immobilized pH gradient strips to determine levels of clinically relevant proteins in wheat grain extracts

“…However, the complementarity of gel-based and gel-free proteomics approaches was suggested previously (Luque-Garcia et al, 2011; Abdallah et al, 2012). The combination of gel-based and gel-free proteomics was shown to be effective for the analyses of soybean under flooding (Yin et al, 2014), phytopathogenic fungus Botrytis cinerea (Gonzalez-Fernandez et al, 2013), Nicotiana tabacum trichomes (Van Cutsem et al, 2011), the honeybee hemolymph proteome (Bogaerts et al, 2009), or during soybean seed filling (Agrawal et al, 2008). …”

Survival of <i>Escherichia coli</i> under Nutrient-Deprived Conditions: Effect on Cell Envelope Subproteome

“…Membrane protein preparations were obtained according to the method described by Molloy et al [23] with minor variations [24]. Subsequent analysis of these proteins was performed by the Proteomics Core Facility-SGIKER at the University of the Basque Country, using the protocol previously described by Gonzalez-Fernandez et al [25]. Briefly, after protein precipitation by using a 2D Clean-Up Kit (GE Healthcare), the pellet was suspended in RapiGest solution (0.2%) (Waters Corporation) and heated at 85°C for 15 min.…”

“…Peptides were eluted with a linear gradient of acetonitrile (120 min from 3 to 40% and 15 min from 40 to 60% [v/v]). Mass spectra were acquired using a data-independent acquisition mode (MSE) [26] as previously described by Gonzalez-Fernandez et al [25] and processed with ProteinLynx Global SERVER v2.4 Build RC7 (Waters Corporation). Protein identification was carried out using the database search algorithm of the program [27] and the parameters specified by Parada et al [24].…”

Differential accumulation of Xanthomonas campestris pv. campestris proteins during the interaction with the host plant: Contributions of an in vivo system

“…Few studies have analyzed Xanthomonas protein accumulation in the presence of plant extracts and even fewer under in vivo conditions . Recently, high throughput proteomic techniques have been increasingly used to study plant pathogens and have revealed a better view of the changes that occur in protein accumulation . We have previously analyzed the proteome of Xcc during the interaction with Brassica oleracea , using an in vivo system and 2DE, however a low number of proteins could be detected and identified .…”