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the cellular level using the original property of this virus to infect by itself cellular suspensions ([14]; Brugidou, unpublished data).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).

Research Findings

• In order to confirm the RYMV infection in cellular suspensions, we performed Northern blot analysis of total RNA isolated from non-stress and RYMV-inoculated cellular suspensions harvested at 1 hpi, 2 dpi, 5 dpi, and 7 dpi for both IR64 and Azucena cultivars (Fig. 1). The accumulation of newly synthesized viral RNA was followed by Northern blot analysis using a probe corresponding to RYMV ORF4. No RNA or residual RNA from virus was detected 1 hpi in both IR64 and Azucena cellular suspensions. In contrast, viral RNA (4452 nucleotides) was clearly detected after 2 dpi for both cultivars, and as previously observed in rice leaves, viral subgenomic RNAs were also detected in cellular suspensions for both cultivars.

'Figure 1. Detection of viral RNA. (A) The lanes contain 10 mg of total RNA from nonstressed cellular suspensions and RYMV inoculated cellular suspensions and harvested at 1 hpi, 2 dpi, 5 dpi, and 7 dpi; lanes 1–5, IR64; lanes 6–10, Azucena. Loading of gels is shown by ethidium bromide (EtBr) staining. (B) RNA gel blot confirming RYMV infection in cellular suspensions of IR64 and Azucena.The blot was probed with probe corresponding to RYMV ORF4.'

• Three proteins involved in metabolism revealed a differential profile for IR64 cellular suspensions under RYMV stress: relative abundance of both aldolase C1 (Fig. 2A,No. 44) and phosphoglycerate dehydrogenase (Fig. 2A,No. 13) increased late at RYMV infection, at 5 dpi and 7 dpi, respectively, whereas the relative abundance of 2,3-biphosphoglycerate-independent phosphoglycerate mutase was altered rapidly after RYMV inoculation(Fig. 2A, Nos. 14 and 15). This protein was present on 2-DE gels as doublet spots with a similar molecular mass and pI values. On the contrary, only one enzyme implied in metabolism revealed an altered expression level pattern in Azucena cellular suspensions under RYMV stress: the relative abundance of glyceraldehyde-3-phosphate dehydrogenase (Fig. 2B, No. 65) increased at 7 dpi. This identified protein spot has a molecular mass different to the theoretical mass, suggesting that it corresponded to a fragment of the protein.

'Figure 2. 2-DE map of (A) IR64 cellular suspension and (B) Azucena cellular suspension. The proteins were visualized by Coomassie Brilliant Blue staining. The numbers indicate proteins whose accumulation level changed under RYMV infection.'

• Three metabolic proteins showing a differential expression pattern during RYMV infection in IR64 cells were involved in glycolysis pathway (Table 1).The relative abundance of salt stress-induced protein,pathogenesis-related protein, dehydrin, chaperonins,HSPs, and ethylene-inducible protein have been observed to change during the RYMV infection (Table 1).

'Table 1. Identification of proteins demonstrating relative expression level changes during RYMV infection by MS and database searching'

• Several different patterns of change were observed,including a gradual increase in relative protein abundance occurring during RYMV infection (a total of 10 spots and 16 spots for IR64 and Azucena, respectively), a gradual decrease in relative protein abundance occurring during the RYMV infection (a total of 2 and 5 spots for IR64 and Azucena, respectively), a transient increase in relative protein abundance occurring at one or two time-points followed by a decrease (a total of 16 and 2 spots for IR64 and Azucena, respectively), a transient decrease in relative protein abundance occurring at one or two time points followed by an increase (a total of 12 and 1 spots for IR64 and Azucena, respectively). The overall patterns of differential expression are compared in Fig. 3.

'Figure 3. Number of proteins with deregulated level under RYMV infection (gradual increase or decrease in relative protein abundance, transient increase or decrease in relative protein abundance occurring during RYMV infection).'

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