Nicotiana benthamiana is a suitable transient system for high-level expression of an active inhibitor of cotton boll weevil α-amylase.

BACKGROUND: Insect resistance in crops represents a main challenge for agriculture. Transgenic approaches based on proteins displaying insect resistance properties are widely used as efficient breeding strategies. To extend the spectrum of targeted pathogens and overtake the development of resistance, molecular evolution strategies have been used on genes encoding these proteins to generate thousands of variants with new or improved functions. The cotton boll weevil (Anthonomus grandis) is one of the major pests of cotton in the Americas. An α-amylase inhibitor (α-AIC3) variant previously developed via molecular evolution strategy showed inhibitory activity against A. grandis α-amylase (AGA). RESULTS: We produced in a few days considerable amounts of α-AIC3 using an optimised transient heterologous expression system in Nicotiana benthamiana. This high α-AIC3 accumulation allowed its structural and functional characterizations. We demonstrated via MALDI-TOF MS/MS technique that the protein was processed as expected. It could inhibit up to 100% of AGA biological activity whereas it did not act on α-amylase of two non-pathogenic insects. These data confirmed that N. benthamiana is a suitable and simple system for high-level production of biologically active α-AIC3. Based on other benefits such as economic, health and environmental that need to be considerate, our data suggested that α-AIC3 could be a very promising candidate for the production of transgenic crops resistant to cotton boll weevil without lethal effect on at least two non-pathogenic insects. CONCLUSIONS: We propose this expression system can be complementary to molecular evolution strategies to identify the most promising variants before starting long-lasting stable transgenic programs.

Trypanosoma vivax infection in sheep: Different patterns of virulence and pathogenicity associated with differentially expressed proteomes.

Cattle trypanosomosis caused by Trypanosoma vivax is a widely distributed disease in Africa and Latin America. It causes significant losses in the livestock industry and is characterized by fluctuating parasitemia, anemia, fever, lethargy, and weight loss. In this study we evaluated the virulence (capacity to multiply inside the host and to modulate the host response) and pathogenicity (ability to produce disease and/or mortality) patterns of two T. vivax strains (TvMT1 and TvLIEM176) in experimentally-infected sheep and determined the proteins differentially expressed in the proteomes of these two strains. Hematological and clinical parameters were monitored in experimentally-infected versus non-infected sheep for 60 days. All the infected animals developed discernable parasitemia at 3 days post-infection (dpi), and the first parasitemia peak was observed at 6 dpi. The maximum average value of parasitemia was 1.3×107 (95% CI, 7.9×105-2×108) parasites/ml in TvLIEM176-infected animals, and 2.5×106 (95% CI, 1.6×105-4×107) parasites/ml in TvMT1-infected ones. Anemia and clinical manifestations were more severe in the animals infected by TvMT1 strain than in those infected by TvLIEM176. In the proteomic analysis, a total of 29 proteins were identified, of which 14 exhibited significant differences in their expression levels between strains. Proteins with higher expression in TvLIEM176 were: alpha tubulin, beta tubulin, arginine kinase, glucose-regulated protein 78, paraflagellar protein 3, and T-complex protein 1 subunit theta. Proteins with higher expression in TvMT1 were: chaperonin HSP60, T-complex protein 1 subunit alpha, heat shock protein 70, pyruvate kinase, glycerol kinase, inosine-5'-monophosphate dehydrogenase, 73kDa paraflagellar rod protein, and vacuolar ATP synthase. There was a difference in the virulence and pathogenicity between the T. vivax strains: TvLIEM176 showed high virulence and moderate pathogenicity, whereas TvMT1 showed low virulence and high pathogenicity. The proteins identified in this study are discussed for their potential involvement in strains' virulence and pathogenicity, to be further defined as biomarkers of severity in T. vivax infections.

Trypanosoma vivax infection in sheep: Different patterns of virulence and pathogenicity associated with differentially expressed proteomes.

Cattle trypanosomosis caused by Trypanosoma vivax is a widely distributed disease in Africa and Latin America. It causes significant losses in the livestock industry and is characterized by fluctuating parasitemia, anemia, fever, lethargy, and weight loss. In this study we evaluated the virulence (capacity to multiply inside the host and to modulate the host response) and pathogenicity (ability to produce disease and/or mortality) patterns of two T. vivax strains (TvMT1 and TvLIEM176) in experimentally-infected sheep and determined the proteins differentially expressed in the proteomes of these two strains. Hematological and clinical parameters were monitored in experimentally-infected versus non-infected sheep for 60 days. All the infected animals developed discernable parasitemia at 3 days post-infection (dpi), and the first parasitemia peak was observed at 6 dpi. The maximum average value of parasitemia was 1.3 × 107 (95% CI, 7.9 × 105-2 × 108) parasites/ml in TvLIEM176-infected animals, and 2.5 × 106 (95% CI, 1.6 × 105-4 × 107) parasites/ml in TvMT1-infected ones. Anemia and clinical manifestations were more severe in the animals infected by TvMT1 strain than in those infected by TvLIEM176. In the proteomic analysis, a total of 29 proteins were identified, of which 14 exhibited significant differences in their expression levels between strains. Proteins with higher expression in TvLIEM176 were: alpha tubulin, beta tubulin, arginine kinase, glucose-regulated protein 78, paraflagellar protein 3, and T-complex protein 1 subunit theta. Proteins with higher expression in TvMT1 were: chaperonin HSP60, T-complex protein 1 subunit alpha, heat shock protein 70, pyruvate kinase, glycerol kinase, inosine-5'-monophosphate dehydrogenase, 73 kDa paraflagellar rod protein, and vacuolar ATP synthase. There was a difference in the virulence and pathogenicity between the T. vivax strains: TvLIEM176 showed high virulence and moderate pathogenicity, whereas TvMT1 showed low virulence and high pathogenicity. The proteins identified in this study are discussed for their potential involvement in strains' virulence and pathogenicity, to be further defined as biomarkers of severity in T. vivax infections.

Optimized transitory ectopic expression of promastigote surface antigen protein in Nicotiana benthamiana, a potential anti-leishmaniasis vaccine candidate.

In recent years, plants have been shown to be an efficient alternative expression system for high-value pharmaceuticals such as vaccines. However, constitutive expression of recombinant protein remains uncertain on their level of production and biological activity. To overcome these problems, transitory expression systems have been developed. Here, a series of experiments were performed to determine the most effective conditions to enhance vaccine antigen transient accumulation in Nicotiana benthamiana leaves using the promastigote surface antigen (PSA) from the parasitic protozoan Leishmania infantum. This protein has been previously identified as the major antigen of a licensed canine anti-leishmaniasis vaccine. The classical prokaryote Escherichia coli biosystem failed in accumulating PSA. Consequently, the standard plant system based on N. benthamiana has been optimized for the production of putatively active PSA. First, the RNA silencing defense mechanism set up by the plant against PSA ectopic expression was abolished by using three viral suppressors acting at different steps of the RNA silencing pathway. Then, we demonstrated that the signal peptide at the N-terminal side of the PSA is required for its accumulation. The PSA ER signaling and retention with the PSA signal peptide and the KDEL motif, respectively were optimized to significantly increase its accumulation. Finally, we demonstrate that the production of recombinant PSA in N. benthamiana leaves allows the conservation of its immunogenic property. These approaches demonstrate that based on these optimizations, plant based systems can be used to effectively produce the biological active PSA protein.

Trypanosoma cruzi: gene expression surveyed by proteomic analysis reveals interaction between different genotypes in mixed in vitro cultures.

We have analyzed the comportment in in vitro culture of 2 different genotypes of Trypanosoma cruzi, the agent of Chagas disease, pertaining to 2 major genetic subdivisions (near-clades) of this parasite. One of the stocks was a fast-growing one, highly virulent in mice, while the other one was slow-growing, mildly virulent in mice. The working hypothesis was that mixtures of genotypes interact, a pattern that has been observed by us in empirical experimental studies. Genotype mixtures were followed every 7 days and characterized by the DIGE technology of proteomic analysis. Proteic spots of interest were characterized by the SAMESPOT software. Patterns were compared to those of pure genotypes that were also evaluated every 7 days. One hundred and three spots exhibited changes in time by comparison with T = 0. The major part of these spots (58%) exhibited an under-expression pattern by comparison with the pure genotypes. 32% of the spots were over-expressed; 10% of spots were not different from those of pure genotypes. Interestingly, interaction started a few minutes after the mixtures were performed. We have retained 43 different proteins that clearly exhibited either under- or over-expression. Proteins showing interaction were characterized by mass spectrometry (MALDI-TOF). Close to 50% of them were either tubulins or heat shock proteins. This study confirms that mixed genotypes of T. cruzi interact at the molecular level. This is of great interest because mixtures of genotypes are very frequent in Chagas natural cycles, both in insect vectors and in mammalian hosts, and may play an important role in the transmission and severity of Chagas disease. The methodology proposed here is potentially applicable to any micropathogen, including fungi, bacteria and viruses. It should be of great interest in the case of bacteria, for which the epidemiological and clinical consequences of mixed infections could be underestimated.

Comparative proteomics reveal new HrpX-regulated proteins of Xanthomonas oryzae pv. oryzae.

Pathogenicity of the rice pathogenic bacterium Xanthomonas oryzae pv. oryzae depends on a Hrp (hypersensitive response and pathogenicity) type III secretion system; the expression of which is induced in planta. Expression of the hrp operons is under transcriptional control of two key regulatory proteins, HrpG and HrpX. To identify new proteins that are co-regulated with the type III secretion system, we employed comparative proteomics. Cells of X. oryzae pv. oryzae ectopically expressing hrpX were compared to wild-type cells grown in vitro. Twenty protein spots with different abundances in both samples were identified by 2D-DIGE and LC-MS/MS. Seven spots could be unambiguously identified, corresponding to the HrpB1 protein, two different peptidyl-prolyl cis-trans isomerases, a component of an ATP binding cassette (ABC) transport system, an adenylate kinase, and a secreted protein of unknown function. Interestingly, the isoelectric point of the adenylate kinase was found to be under control of HrpX, most likely due to post-translational modification. Indeed, two glutamate residues of the adenylate kinase were found to be methylated but this modification did not account for the shift in electrophoretic mobility. In summary, we identified new HrpX-regulated proteins of X. oryzae pv. oryzae that might be important for pathogenicity. This article is part of a Special Issue entitled: Trends in microbial proteomics. BIOLOGICAL SIGNIFICANCE: We use 2D-DIGE to compare the proteomes of rice-pathogenic xanthomonads. We identify seven proteins that are co-regulated with the type III secretion system. We find post-translational glutamate methylation of a bacterial adenylate cyclase. The newly identified HrpX-regulated proteins might be important for pathogenicity.

The RYMV-encoded viral suppressor of RNA silencing P1 is a zinc-binding protein with redox-dependent flexibility.

Viral suppressors of RNA interference (VSRs) target host gene silencing pathways, thereby operating important roles in the viral cycle and in host cells, in which they counteract host innate immune responses. However, the molecular mechanisms of VSRs are poorly understood. We provide here biochemical and biophysical features of the dual suppressor/activator VSR P1 protein encoded by the rice yellow mottle virus. In silico analyses of P1 suggested common features with zinc finger proteins and native mass spectrometry unambiguously confirmed that recombinant P1 binds reversibly two zinc atoms, each with a different strength. Additionally, we demonstrate that the reaction of P1 with H2O2 leads to zinc release, disulfide bond formation, and protein oligomerization. A reversible protein modification by redox alterations has only been described for a limited number of zinc finger proteins and has never been reported for VSRs. Those reported here for P1 might be a general feature of Cys-rich VSRs and could be a key regulatory mechanism for the control of RNA silencing.

Proteome of Aedes aegypti in response to infection and coinfection with microsporidian parasites.

Hosts are frequently infected with more than one parasite or pathogen at any one time, but little is known as to how they respond to multiple immune challenges compared to those involving single infections. We investigated the proteome of Aedes aegypti larvae following infection with either Edhazardia aedis or Vavraia culicis, and coinfections involving both. They are both obligate intracellular parasites belonging to the phylum microsporidia and infect natural populations of Ae. aegypti. The results found some proteins only showing modified abundance in response to infections involving E. aedis, while others were only differentially abundant when infections involved V. culicis. Some proteins only responded with modified abundance to the coinfection condition, while others were differentially abundant in response to all three types of infection. As time since infection increased, the response to each of the single parasite infections diverged, while the response to the E. aedis and coinfection treatments converged. Some of the proteins differentially abundant in response to infection were identified. They included two vacuolar ATPases, proteins known to have a role in determining the infection success of intracellular parasites. This result suggests microsporidia could influence the infection success of other intracellular pathogens infecting vector species of mosquito, including viruses, Plasmodium and Wolbachia.

Phylogenetic character mapping of proteomic diversity shows high correlation with subspecific phylogenetic diversity in Trypanosoma cruzi.

We performed a phylogenetic character mapping on 26 stocks of Trypanosoma cruzi, the parasite responsible for Chagas disease, and 2 stocks of the sister taxon T. cruzi marinkellei to test for possible associations between T. cruzi-subspecific phylogenetic diversity and levels of protein expression, as examined by proteomic analysis and mass spectrometry. We observed a high level of correlation (P < 10(-4)) between genetic distance, as established by multilocus enzyme electrophoresis, and proteomic dissimilarities estimated by proteomic Euclidian distances. Several proteins were found to be specifically associated to T. cruzi phylogenetic subdivisions (discrete typing units). This study explores the previously uncharacterized links between infraspecific phylogenetic diversity and gene expression in a human pathogen. It opens the way to searching for new vaccine and drug targets and for identification of specific biomarkers at the subspecific level of pathogens.

RLK7, a leucine-rich repeat receptor-like kinase, is required for proper germination speed and tolerance to oxidative stress in Arabidopsis thaliana.

The leucine-rich repeat class of receptor-like kinase (LRR-RLKs) encoding genes represents the largest family of putative receptor genes in the Arabidopsis thaliana genome. However, very little is known about the range of biological process that they control. We present in this paper the functional characterization of RLK7 that has all the structural features of a receptor-like kinase of the plant-specific LRR type. To this end, we identified and characterized three independent T-DNA insertion mutants, constructed lines carrying truncated versions of this putative receptor, one lacking the cytoplasmic kinase domain (RLK7Δkin) and the other one lacking 14 LRR repeats (RLK7ΔLRR) and generated RLK7 overexpressing lines. We thus provide evidences that RLK7 is involved in the control of germination speed and the tolerance to oxidant stress. First, consistent with the expression kinetics of the RLK7 gene in the seeds, we found that all three mutants showed a delay in germination, whereas the overexpressors, RLK7Δkin and RLK7ΔLRR lines displayed a phenotype of more precocious germination. Second, a non-hypothesis driven proteomic approach revealed that in the seedlings of the three T-DNA insertion lines, four enzymes directly or indirectly involved in reactive oxygen species detoxification, were significantly less abundant. Consistent with this finding, the three mutants were less tolerant than the wild type to a hydrogen peroxide treatment, whereas the overexpressors, RLK7Δkin and RLK7ΔLRR lines presented the opposite phenotype.

Blood-feeding and immunogenic Aedes aegypti saliva proteins.

Mosquito-transmitted pathogens pass through the insect's midgut (MG) and salivary gland (SG). What occurs in these organs in response to a blood meal is poorly understood, but identifying the physiological differences between sugar-fed and blood-fed (BF) mosquitoes could shed light on factors important in pathogens transmission. We compared differential protein expression in the MGs and SGs of female Aedes aegypti mosquitoes after a sugar- or blood-based diet. No difference was observed in the MG protein expression levels but certain SG proteins were highly expressed only in BF mosquitoes. In sugar-fed mosquitoes, housekeeping proteins were highly expressed (especially those related to energy metabolism) and actin was up-regulated. The immunofluorescence assay shows that there is no disruption of the SG cytoskeletal after the blood meal. We have generated for the first time the 2-DE profiles of immunogenic Ae. aegypti SG BF-related proteins. These new data could contribute to the understanding of the physiological processes that appear during the blood meal.

Identification of proteomic changes during human liver development by 2D-DIGE and mass spectrometry.

BACKGROUND/AIMS: The aim of this study was to identify human liver proteins that are associated with different stages of liver development. METHODS: We collected liver samples from 14 fetuses between 14 and 41 weeks of development, one child and four adults. Proteins which exhibited consistent and significant variations during development by two-dimensional differential in gel electrophoresis (2D-DIGE) were subjected to peptide mass fingerprint analysis by MALDI-TOF mass spectrometry. Real-time PCR analysis confirmed, at the transcriptional level, the data obtained by the proteomic approach. RESULTS: Among a total of 80 protein spots showing differential expression, we identified 42 different proteins or polypeptide chains, of which 26 were upregulated and 16 downregulated in developing in comparison to adult liver. These proteins could be classified in specific groups according to their function. By comparing their temporal expression profiles, we identified protein groups that were associated with different developmental stages of human fetal liver and suggest that the changes in protein expression observed during the 20- to 36-week time window play a pivotal role in liver development. CONCLUSIONS: The identification of these proteins may represent good markers of human liver and stem cells differentiation.

Identification of total and differentially expressed excreted-secreted proteins from Trypanosoma congolense strains exhibiting different virulence and pathogenicity.

Animal trypanosomosis is a major constraint to livestock productivity in the tropics and has a significant impact on the life of millions of people globally (mainly in Africa, South America and south-east Asia). In Africa, the disease in livestock is caused mainly by Trypanosoma congolense, Trypanosoma vivax, Trypanosoma evansi and Trypanosoma brucei brucei. The extracellular position of trypanosomes in the bloodstream of their host requires consideration of both the parasite and its naturally excreted-secreted factors (secretome) in the course of pathophysiological processes. We therefore developed and standardised a method to produce purified proteomes and secretomes of African trypanosomes. In this study, two strains of T. congolense exhibiting opposite properties of both virulence and pathogenicity were further investigated through their secretome expression and its involvement in host-parasite interactions. We used a combined proteomic approach (one-dimensional SDS-PAGE and two-dimensional differential in-gel electrophoresis coupled to mass spectrometry) to characterise the whole and differentially expressed protein contents of secretomes. The molecular identification of differentially expressed trypanosome molecules and their correlation with either the virulence process or pathogenicity are discussed with regard to their potential as new diagnostic or therapeutic tools against animal trypanosomosis.

"Pathogeno-proteomics".

Many scientists working on pathogens (viruses, bacteria, fungi, parasites) are betting heavily on data generated by longitudinal genomic-transcriptomic-proteomic studies to explain biochemical host-vector-pathogen interactions and thus to contribute to disease control. Availability of genome sequences of various organisms, from viruses to complex metazoans, led to the discovery of the functions of the genes themselves. The postgenomic era stimulated the development of proteomic and bioinformatics tools to identify the locations, functions, and interactions of the gene products in tissues and/or cells of living organisms. Because of the diversity of available methods and the level of integration they promote, proteomics tools are potentially able to resolve interesting issues specific not only to host-vector-pathogen interactions in cell immunobiology, but also to ecology and evolution, population biology, and adaptive processes. These new analytical tools, as all new tools, contain pitfalls directly related to experimental design, statistical treatment, and protein identification. Nevertheless, they offer the potency of building large protein-protein interaction networks for in silico analysis of novel biological entities named "interactomes," a way of modeling host-vector-pathogen interactions to define new interference strategies.

Secretome of animal trypanosomes.

Animal trypanosomosis is one of the most severe constraints to agricultural development in sub-Saharan Africa and is also an important disease of livestock in Latin America and Asia. The causative agents are various species of protozoan parasites belonging to the genus Trypanosoma, among which T. congolense and T. evansi are the major pathogenic species. The extracellular position of trypanosomes obliges us to consider both the parasite and its excreted/secreted factors in the course of the physiopathologic process. The advent of proteomics led us to propose a comparative approach of the proteome (i.e., the whole parasite content) and the secretome (i.e., naturally excreted/secreted molecules) of T. congolense and T. evansi with particular attention to common and specific molecules between strains of differing virulence and pathogenicity. The molecular identification of differentially expressed trypanosome molecules correlated with either the virulence process or the pathogenicity will provide new potential molecular targets for improved field diagnosis and chemotherapy of animal trypanosomosis.

Malaria Plasmodium agent induces alteration in the head proteome of their Anopheles mosquito host.

Despite increasing evidence of behavioural manipulation of their vectors by pathogens, the underlying mechanisms causing infected vectors to act in ways that benefit pathogen transmission remain enigmatic in most cases. Here, 2-D DIGE coupled with MS were employed to analyse and compare the head proteome of mosquitoes (Anopheles gambiae sensu stricto (Giles)) infected with the malarial parasite (Plasmodium berghei) with that of uninfected mosquitoes. This approach detected altered levels of 12 protein spots in the head of mosquitoes infected with sporozoites. These proteins were subsequently identified using MS and functionally classified as belonging to metabolic, synaptic, molecular chaperone, signalling, and cytoskeletal groups. Our results indicate an altered energy metabolism in the head of sporozoite-infected mosquitoes. Some of the up-/down-regulated proteins identified, such as synapse-associated protein, 14-3-3 protein and calmodulin, have previously been shown to play critical roles in the CNS of both invertebrates and vertebrates. Furthermore, a heat shock response (HSP 20) and a variation of cytoarchitecture (tropomyosins) have been shown. Discovery of these proteins sheds light on potential molecular mechanisms that underlie behavioural modifications and offers new insights into the study of intimate interactions between Plasmodium and its Anopheles vector.

The pitfalls of proteomics experiments without the correct use of bioinformatics tools.

The elucidation of the entire genomic sequence of various organisms, from viruses to complex metazoans, most recently man, is undoubtedly the greatest triumph of molecular biology since the discovery of the DNA double helix. Over the past two decades, the focus of molecular biology has gradually moved from genomes to proteomes, the intention being to discover the functions of the genes themselves. The postgenomic era stimulated the development of new techniques (e.g. 2-DE and MS) and bioinformatics tools to identify the functions, reactions, interactions and location of the gene products in tissues and/or cells of living organisms. Both 2-DE and MS have been very successfully employed to identify proteins involved in biological phenomena (e.g. immunity, cancer, host-parasite interactions, etc.), although recently, several papers have emphasised the pitfalls of 2-DE experiments, especially in relation to experimental design, poor statistical treatment and the high rate of 'false positive' results with regard to protein identification. In the light of these perceived problems, we review the advantages and misuses of bioinformatics tools - from realisation of 2-DE gels to the identification of candidate protein spots - and suggest some useful avenues to improve the quality of 2-DE experiments. In addition, we present key steps which, in our view, need to be to taken into consideration during such analyses. Lastly, we present novel biological entities named 'interactomes', and the bioinformatics tools developed to analyse the large protein-protein interaction networks they form, along with several new perspectives of the field.

Proteome analysis of plant-virus interactome: comprehensive data for virus multiplication inside their hosts.

Known host-parasite molecular interactions are widespread among parasite families, but these interactions have to be particularly large considering that viruses generally encode few proteins. Although some particular virus-host interactions are well described, no global study has yet shown multiple and simultaneous interactions in a host-parasite biological system. To prove that these multiple interactions occur in biological conditions, the complexes formed by a plant virus (rice yellow mottle virus) and the proteins of its natural host (rice) were extracted and purified from infected tissue sample. Remarkably mass spectrometry permitted the identification of a large number of proteins from the complexes that are involved in different functions not encoded by the virus but probably essential for its biological life cycle. This recruiting of proteins was strongly confirmed by the repetition of experiments using different pairs of virus-host and the use of high salt concentration to extract the complexes. We mainly identified proteins involved in plant defense, metabolism, translation, and protein synthesis and some proteins involved in transport. This study demonstrates that viruses are able to recruit many proteins from their hosts to ensure their development. Among different pairs of virus-host, similar protein functions were identified suggesting a particular importance of these proteins for viruses. The identification of particular paralog proteins among multigenic families suggests the high specificity of the recruiting for some protein functions.

Multigenic families and proteomics: extended protein characterization as a tool for paralog gene identification.

In classical proteomic studies, the searches in protein databases lead mostly to the identification of protein functions by homology due to the non-exhaustiveness of the protein databases. The quality of the identification depends on the studied organism, its complexity and its representation in the protein databases. Nevertheless, this basic function identification is insufficient for certain applications namely for the development of RNA-based gene-silencing strategies, commonly termed RNA interference (RNAi) in animals and post-transcriptional gene silencing (PTGS) in plants, that require an unambiguous identification of the targeted gene sequence. A PTGS strategy was considered in the study of the infection of Oryza sativa by the Rice Yellow Mottle Virus (RYMV). It is suspected that the RYMV recruits host proteins after its entry into plant cells to form a complex facilitating virus multiplication and spreading. The protein partners of this complex were identified by a classical proteomic approach, nano liquid chromatography tandem mass spectrometry. Among the identified proteins, several were retained for a PTGS strategy. Nevertheless most of the protein candidates appear to be members of multigenic families for which all paralog genes are not present in protein databases. Thus the identification of the real expressed paralog gene with classical protein database searches is impossible. Consequently, as the genome contains all genes and thus all paralog genes, a whole genome search strategy was developed to determine the specific expressed paralog gene. With this approach, the identification of peptides matching only a single gene, called discriminant peptides, allows definitive proof of the expression of this identified gene. This strategy has several requirements: (i) a genome completely sequenced and accessible; (ii) high protein sequence coverage. In the present work, through three examples, we report and validate for the first time a genome database search strategy to specifically identify paralog genes belonging to multigenic families expressed under specific conditions.

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

We have used two-dimensional gel electrophoresis with mass spectrometry analysis to study the temporal patterns of protein expression during RYMV (Rice yellow mottle virus) infection in rice cells of two cultivars: IR64, Oryza sativa indica, susceptible, and Azucena, O. sativa japonica, partially resistant to RYMV. Proteomic analysis of nonstressed and RYMV inoculated cells showed statistically significant changes in the relative levels of 40 IR64 proteins and 24 Azucena proteins. Protein identification using mass spectrometry was attempted for all the differentially regulated proteins. This global analysis detected 32 hypothetical "new" proteins. Nineteen differentially regulated proteins were identified for IR64 cultivar, while 13 were identified for Azucena cultivar, including proteins in three functional categories: metabolism, stress-related proteins, and translation. These data revealed that a number of proteins regulated by abiotic stress response pathway were activated by RYMV in both cultivars (such as salt-induced protein, heat shock proteins (HSPs), superoxide dismutase (SOD), and others have functions consistent with the susceptibility or partially resistance trait (such as dehydrin, proteins involved in glycolysis pathway).