IC4R010-Proteomic-2004-14730683

Project Title

• Proteome analysis of cultivar-specific deregulations of Oryza sativa indica and O. sativa japonica cellular suspensions undergoing Rice yellow mottle virus infection

The Background of This Project

• Despite several studies investigating the effects of virus infection, no global research on proteins controlling viral disease was reported. Moreover, proteomic approaches to analyze virus-responsive proteins in both susceptible and partially resistant hosts have not yet been reported.
• In this study, the researchers propose to investigate protein deregulations and their assignment to early/late and host compatibility, using the Rice yellow mottle virus (RYMV)-rice interaction as a model. The RYMV, a member of the genus of Sobemovirus, was first reported in the ’70s [9]. RYMV is endemic to Africa [10, 11], and is considered as very destructive for rice production. With only four open reading frames, this virus could be considered as a model for studying genetics and genomics resistance [12]. The genetic basis of RYMV partial resistance has been studied using a doubled haploid population (IR64 (O. sative indica; highly susceptible to RYMV) 6 Azucena (O. sativa japonica; partly resistant to RYMV)) [13]. Genomic approaches of “RYMV-responsive genes” analysis tend to show that the interactions between RYMV and host are highly complex in both susceptible and partially resistant rice cultivars. Using ESTs and cDNA-amplified fragment length polymorphism (AFLP) analysis, we have already demonstrated in planta that RYMV stress involves changes in photosynthesis and metabolism (VentelonDebout et al., in press).
• In this study, the researchers focus our work on protein deregulation at the cellular level using the original property of this virus to infect by itself cellular suspensions ([14]; Brugidou, unpublished data).

Plant Culture & Treatment

• Cells from two cultivars were used: IR64 (O. sativa indica)and Azucena (O. sativa japonica). IR64 is a high-yielding cultivar developed at the International Rice Research Institute (IRRI) while Azucena is a traditional upland cultivar from the Philippines. For both cultivars, 1.5 g of cellular suspensions was grown in 30 mL of an appropriate medium [23]. The medium was changed every week, for one month, to obtain mainly synchronized cells. Inoculation was performed 3 days after changing the medium, by addition of 100 mg of RYMV. For both cultivars, RYMV inoculated cellular suspensions were harvested at different time points (1 hpi (hour post inoculation), 2 dpi (days after inoculation), 5 dpi, and 7 dpi). At each time point,samples were harvested in duplicates, washed twice in medium, and pooled. In parallel, the noninfected cellular suspensions were harvested at 1 hpi, 5 dpi, and 7 dpi, and used as control.

Protein Extraction and 2-D PAGE

• Aliquots (1 g) of synchronized cell samples were ground to a fine powder in liquid nitrogen. Protein extraction and precipitation was performed in 10% v/v TCA in acetone with 0.07% v/v b-mercaptoethanol at 2207C for 1 h, followed by centrifugation for 10 min at 47C, 50 0006g. The pellets were washed twice with cold acetone containing 0.07% b-mercaptoethanol and centrifuged for 10 min at 47C, 50 0006g, and the pellets dried. Proteins were solubilized in 500 mL of lysis buffer (9 M urea, 4% w/v CHAPS,0.5% v/v Triton X-100, 0.5% IPG buffer, 3 mM tributylphosphine (TBP)). Finally, the sample was centrifuged 10 min at 207C, 60 0006g. Concentration of the supernatant was determined using protein assay (2D-Quant kit; Amersham, UK).

• At each time point, 2-DE was performed for two replications of both cultivars. All experiments of each replication were performed at the same time. Cell proteins (1 mg per sample) were loaded onto a IPG gel strip (240 mm, pH 4–7; Amersham). Isoelectric focusing (IEF) was conducted by using IPGphor (Amersham) system. IPG strips were rehydrated for 9 h prior to electrophoresis. IEF conditions were: 50 V for 10 h, 300 V for 10 min, 300–8000 V gradient for 3 h, and 8000 V for 13 h. The IPG gel strips were placed for 20 min in 15 mL of equilibration buffer (6 Murea, 0.375 M Tris-HCl, 30% v/v glycerol, 2% SDS w/v, 3 mM TBP). The second dimension was run on vertical 12% polyacrylamide-SDS gels. Gels were stained with colloidal Coomassie Brilliant Blue. The gels were scanned and spot intensities were analyzed using the software Image Master-2D (Amersham). A Student’s t-test was performed to evaluate the level of significance of any quantitative change in the level of analyzed proteins between control and each replicate at each time-point after RYMV inoculation. A change in protein abundance was considered significant if the difference between control and any time-point achieved the p , 0.05 significance level (t-test).

Labs working on this Project

• IRD, Institut de Recherche pour le Développement,Montpellier, France
• Laboratoire de Spectrométrie de Masse Bio-Organique,Strasbourg, France

Corresponding Author

• Marjolaine Ventelon-Debout:ventelon@mpl.ird.fr & Christophe Brugidou:brugidou@mpl.ird.fr