An ex vitro hairy root system from petioles of detached soybean leaves for in planta screening of target genes and CRISPR strategies associated with nematode bioassays.

MAIN CONCLUSION: The ex vitro hairy root system from petioles of detached soybean leaves allows the functional validation of genes using classical transgenesis and CRISPR strategies (e.g., sgRNA validation, gene activation) associated with nematode bioassays. Agrobacterium rhizogenes-mediated root transformation has been widely used in soybean for the functional validation of target genes in classical transgenesis and single-guide RNA (sgRNA) in CRISPR-based technologies. Initial data showed that in vitro hairy root induction from soybean cotyledons and hypocotyls were not the most suitable strategies for simultaneous performing genetic studies and nematode bioassays. Therefore, an ex vitro hairy root system was developed for in planta screening of target molecules during soybean parasitism by root-knot nematodes (RKNs). Applying this method, hairy roots were successfully induced by A. rhizogenes from petioles of detached soybean leaves. The soybean GmPR10 and GmGST genes were then constitutively overexpressed in both soybean hairy roots and tobacco plants, showing a reduction in the number of Meloidogyne incognita-induced galls of up to 41% and 39%, respectively. In addition, this system was evaluated for upregulation of the endogenous GmExpA and GmExpLB genes by CRISPR/dCas9, showing high levels of gene activation and reductions in gall number of up to 58.7% and 67.4%, respectively. Furthermore, morphological and histological analyses of the galls were successfully performed. These collective data validate the ex vitro hairy root system for screening target genes, using classical overexpression and CRISPR approaches, directly in soybean in a simple manner and associated with nematode bioassays. This system can also be used in other root pathosystems for analyses of gene function and studies of parasite interactions with plants, as well as for other purposes such as studies of root biology and promoter characterization.

Ectopic expression of an expansin-like B gene from wild Arachis enhances tolerance to both abiotic and biotic stresses.

Plant expansins are structural cell wall-loosening proteins implicated in several developmental processes and responses to environmental constraints and pathogen infection. To date, there is limited information about the biological function of expansins-like B (EXLBs), one of the smallest and less-studied subfamilies of plant expansins. In the present study, we conducted a functional analysis of the wild Arachis AdEXLB8 gene in transgenic tobacco (Nicotiana tabacum) plants to clarify its putative role in mediating defense responses to abiotic and biotic stresses. First, its cell wall localization was confirmed in plants expressing an AdEXLB8:eGFP fusion protein, while nanomechanical assays indicated cell wall reorganization and reassembly due to AdEXLB8 overexpression without compromising the phenotype. We further demonstrated that AdEXLB8 increased tolerance not only to isolated abiotic (drought) and biotic (Sclerotinia sclerotiorum and Meloidogyne incognita) stresses but also to their combination. The jasmonate and abscisic acid signaling pathways were clearly favored in transgenic plants, showing an activated antioxidative defense system. In addition to modifications in the biomechanical properties of the cell wall, we propose that AdEXLB8 overexpression interferes with phytohormone dynamics leading to a defense primed state, which culminates in plant defense responses against isolated and combined abiotic and biotic stresses.

Characterization of raffinose metabolism genes uncovers a wild Arachis galactinol synthase conferring tolerance to abiotic stresses.

Raffinose family oligosaccharides (RFOs) are implicated in plant regulatory mechanisms of abiotic stresses tolerance and, despite their antinutritional proprieties in grain legumes, little information is available about the enzymes involved in RFO metabolism in Fabaceae species. In the present study, the systematic survey of legume proteins belonging to five key enzymes involved in the metabolism of RFOs (galactinol synthase, raffinose synthase, stachyose synthase, alpha-galactosidase, and beta-fructofuranosidase) identified 28 coding-genes in Arachis duranensis and 31 in A. ipaënsis. Their phylogenetic relationships, gene structures, protein domains, and chromosome distribution patterns were also determined. Based on the expression profiling of these genes under water deficit treatments, a galactinol synthase candidate gene (AdGolS3) was identified in A. duranensis. Transgenic Arabidopsis plants overexpressing AdGolS3 exhibited increased levels of raffinose and reduced stress symptoms under drought, osmotic, and salt stresses. Metabolite and expression profiling suggested that AdGolS3 overexpression was associated with fewer metabolic perturbations under drought stress, together with better protection against oxidative damage. Overall, this study enabled the identification of a promising GolS candidate gene for metabolic engineering of sugars to improve abiotic stress tolerance in crops, whilst also contributing to the understanding of RFO metabolism in legume species.

Proteomics unravels new candidate genes for Meloidogyne resistance in wild Arachis.

Arachis stenosperma is a wild peanut relative exclusive to South America that harbors high levels of resistance against several pathogens, including the peanut root-knot nematode (RKN) Meloidogyne arenaria. In this study, a proteomic survey of A. stenosperma-M. arenaria interaction using 2-DE and LC-MS/MS identified approximately 1400 proteins, out of which 222 were differentially abundant (DAPs) when RKN inoculated root samples were compared to the control. Most of these DAPs were assigned to functional categories related to plant responses to pathogens including stress, glycolysis, redox and tricarboxylic acid cycle. The comparison between the transcriptome (RNA-Seq) and proteome expression changes, showed that almost 55% of these DAPs encode genes with a similar expression trend to their protein counterparts. Most of these genes were induced during RKN infection and some were related to plant defense, such as MLP-like protein 34 (MLP34), cinnamoyl-CoA reductase 1 (CCR1), enolase (ENO), alcohol dehydrogenase (ADH) and eukaryotic translation initiation factor 5A (eIF5A). The overexpression of AsMLP34 in Agrobacterium rhizogenes transgenic roots in a susceptible peanut cultivar showed a reduction in the number of M. arenaria galls and egg masses, indicating that AsMLP34 is a promising candidate gene to be exploited in breeding programs for RKN control in peanut. SIGNIFICANCE: The use of an integrated approach to compare plant-nematode transcriptional and translational data enabled the identification of a new gene, AsMLP34, for Meloidogyne resistance.

Comparative proteomics and gene expression analysis in Arachis duranensis reveal stress response proteins associated to drought tolerance.

Peanut wild relatives (Arachis spp.) have high genetic diversity and are important sources of resistance to biotic and abiotic stresses. In this study, proteins were analyzed in root tissues of A. duranensis submitted to a progressive water deficit in soil and the differential abundance was compared to transcript expression profiles obtained by RNA-seq and qRT-PCR. Using a 2-DE approach, a total of 31 proteins were identified, most of which were associated with stress response and drought perception. These comprised a chitinase-2 (unique to stressed condition), an MLP-like protein, a glycine-rich protein DOT1-like, a maturase K and heat shock-related proteins (HSP70 - an isoform unique to the control, and HSP17.3). Other proteins unique to the control condition comprised a transcription initiation factor IIF subunit alpha-like protein, a SRPBCC ligand-binding domain superfamily protein, an Adenine phosphoribosyl transferase, a Leo1-like protein, a Cobalamine-independent methionine synthase and a Transmembrane emp24 domain-containing protein p24delta9-like. Correlation of mRNA expression and corresponding protein abundance was observed for 15 of the identified proteins, with genes encoding the majority of proteins (14) negatively regulated in stressed roots. Proteins identified in this study offer potential for the genetic improvement of cultivated peanut for drought tolerance. SIGNIFICANCE: The comparison of protein abundance and corresponding transcript expression levels (RNA-seq and qRT-PCR) revealed that 15 of the identified proteins showed similar expression behavior, with the majority (14 proteins) negatively regulated in stressed roots. Chitinase-2 (Cht2) was the only protein with an upregulation behavior in all approaches. These proteins appear to play an important role in drought tolerance in A. duranensis and may be further explored in peanut genetic breeding programs.

Early responses to dehydration in contrasting wild Arachis species.

Wild peanut relatives (Arachis spp.) are genetically diverse and were selected throughout evolution to a range of environments constituting, therefore, an important source of allelic diversity for abiotic stress tolerance. In particular, A. duranensis and A. stenosperma, the parents of the reference Arachis A-genome genetic map, show contrasting transpiration behavior under limited water conditions. This study aimed to build a comprehensive gene expression profile of these two wild species under dehydration stress caused by the withdrawal of hydroponic nutrient solution. For this purpose, roots of both genotypes were collected at seven time-points during the early stages of dehydration and used to construct cDNA paired-end libraries. Physiological analyses indicated initial differences in gas exchange parameters between the drought-tolerant genotype of A. duranensis and the drought-sensitive genotype of A. stenosperma. High-quality Illumina reads were mapped against the A. duranensis reference genome and resulted in the identification of 1,235 and 799 Differentially Expressed Genes (DEGs) that responded to the stress treatment in roots of A. duranensis and A. stenosperma, respectively. Further analysis, including functional annotation and identification of biological pathways represented by these DEGs confirmed the distinct gene expression behavior of the two contrasting Arachis species genotypes under dehydration stress. Some species-exclusive and common DEGs were then selected for qRT-PCR analysis, which corroborated the in silico expression profiling. These included genes coding for regulators and effectors involved in drought tolerance responses, such as activation of osmosensing molecular cascades, control of hormone and osmolyte content, and protection of macromolecules. This dataset of transcripts induced during the dehydration process in two wild Arachis genotypes constitute new tools for the understanding of the distinct gene regulation processes in these closely related species but with contrasting drought responsiveness. In addition, our findings provide insights into the nature of drought tolerance in wild germoplasm, which might be explored as novel sources of diversity and useful wild alleles to develop climate-resilient crop varieties.

The genome structure of Arachis hypogaea (Linnaeus, 1753) and an induced Arachis allotetraploid revealed by molecular cytogenetics.

Peanut, Arachis hypogaea (Linnaeus, 1753) is an allotetraploid cultivated plant with two subgenomes derived from the hybridization between two diploid wild species, A. duranensis (Krapovickas & W. C. Gregory, 1994) and A. ipaensis (Krapovickas & W. C. Gregory, 1994), followed by spontaneous chromosomal duplication. To understand genome changes following polyploidy, the chromosomes of A. hypogaea, IpaDur1, an induced allotetraploid (A. ipaensis × A. duranensis)4x and the diploid progenitor species were cytogenetically compared. The karyotypes of the allotetraploids share the number and general morphology of chromosomes; DAPI+ bands pattern and number of 5S rDNA loci. However, one 5S rDNA locus presents a heteromorphic FISH signal in both allotetraploids, relative to corresponding progenitor. Whilst for A. hypogaea the number of 45S rDNA loci was equivalent to the sum of those present in the diploid species, in IpaDur1, two loci have not been detected. Overall distribution of repetitive DNA sequences was similar in both allotetraploids, although A. hypogaea had additional CMA3+ bands and few slight differences in the LTR-retrotransposons distribution compared to IpaDur1. GISH showed that the chromosomes of both allotetraploids had preferential hybridization to their corresponding diploid genomes. Nevertheless, at least one pair of IpaDur1 chromosomes had a clear mosaic hybridization pattern indicating recombination between the subgenomes, clear evidence that the genome of IpaDur1 shows some instability comparing to the genome of A. hypogaea that shows no mosaic of subgenomes, although both allotetraploids derive from the same progenitor species. For some reasons, the chromosome structure of A. hypogaea is inherently more stable, or, it has been at least, partially stabilized through genetic changes and selection.

Phenotypic effects of allotetraploidization of wild Arachis and their implications for peanut domestication.

PREMISE OF THE STUDY: Several species of Arachis have been cultivated for their edible seeds, historically and to the present day. The diploid species that have a history of cultivation show relatively small signatures of domestication. In contrast, the tetraploid species A. hypogaea evolved into highly domesticated forms and became a major world crop, the cultivated peanut. It seems likely that allotetraploidization (hybridity and/or tetraploidization) in some way enhanced attractiveness for cultivation. Here we investigate this using six different hybridization and tetraploidization events, from distinct Arachis diploid species, including one event derived from the same wild species that originated peanut. METHODS: Twenty-six anatomical, morphological, and physiological traits were examined in the induced allotetraploid plants and compared with their wild diploid parents. KEY RESULTS: Nineteen traits were transgressive (showed strong response to hybridization and chromosome duplication): allotetraploids had larger leaves, stomata and epidermal cells than did their diploid parents. In addition, allotetraploids produced more photosynthetic pigments. These traits have the same trend across the different hybrid combinations, suggesting that the changes are more likely due to ploidy rather than hybridity. In contrast, seed dimensions and seed mass did not significantly change in response to hybridization or tetraploidization. CONCLUSIONS: We suggest that the original allotetraploid that gave rise to cultivated peanut may have been attractive because of an increase in plant size, different transpiration characteristics, higher photosynthetic capacity, or other characteristics, but contrary to accepted knowledge, increased seed size was unlikely to have been important in the initial domestication.

Reference Gene Selection for qPCR Analysis in Tomato-Bipartite Begomovirus Interaction and Validation in Additional Tomato-Virus Pathosystems.

Quantitative Polymerase Chain Reaction (qPCR) is currently the most sensitive technique used for absolute and relative quantification of a target gene transcript, requiring the use of appropriated reference genes for data normalization. To accurately estimate the relative expression of target tomato (Solanum lycopersicum L.) genes responsive to several virus species in reverse transcription qPCR analysis, the identification of reliable reference genes is mandatory. In the present study, ten reference genes were analyzed across a set of eight samples: two tomato contrasting genotypes ('Santa Clara', susceptible, and its near-isogenic line 'LAM 157', resistant); subjected to two treatments (inoculation with Tomato chlorotic mottle virus (ToCMoV) and its mock-inoculated control) and in two distinct times after inoculation (early and late). Reference genes stability was estimated by three statistical programs (geNorm, NormFinder and BestKeeper). To validate the results over broader experimental conditions, a set of ten samples, corresponding to additional three tomato-virus pathosystems that included tospovirus, crinivirus and tymovirus + tobamovirus, was analyzed together with the tomato-ToCMoV pathosystem dataset, using the same algorithms. Taking into account the combined analyses of the ranking order outputs from the three algorithms, TIP41 and EF1 were identified as the most stable genes for tomato-ToCMoV pathosystem, and TIP41 and EXP for the four pathosystems together, and selected to be used as reference in the forthcoming expression qPCR analysis of target genes in experimental conditions involving the aforementioned tomato-virus pathosystems.

Cowpea-Meloidogyne incognita interaction: Root proteomic analysis during early stages of nematode infection.

Cowpea (Vigna unguiculata L. Walp) is an important legume species well adapted to low fertility soils and prolonged drought periods. One of the main problems that cause severe yield losses in cowpea is the root-knot nematode Meloidogyne incognita. The aim of this work was to analyze the differential expression of proteins in the contrasting cultivars of cowpea CE 31 (highly resistant) and CE 109 (slightly resistant) during early stages of M. incognita infection. Cowpea roots were collected at 3, 6, and 9 days after inoculation and used for protein extraction and 2-DE analysis. From a total of 59 differential spots, 37 proteins were identified, mostly involved in plant defense, such as spermidine synthase, patatin, proteasome component, and nitrile-specifier protein. A follow-up study was performed by quantitative RT-PCR analysis of nine selected proteins and the results revealed a very similar upregulation trend between the protein expression profiles and the corresponding transcripts. This study also identified ACT and GAPDH as a good combination of reference genes for quantitative RT-PCR analysis of the pathosystem cowpea/nematode. Additionally, an interactome analysis showed three major pathways affected by nematode infection: proteasome endopeptidase complex, oxidative phosphorylation, and flavonoid biosynthesis. Taken together, the results obtained by proteome, transcriptome, and interactome approaches suggest that oxidative stress, ubiquitination, and glucosinolate degradation may be part of cowpea CE 31 resistance mechanisms in response to nematode infection.

Transcriptome Profiling of Wild Arachis from Water-Limited Environments Uncovers Drought Tolerance Candidate Genes.

Peanut (Arachis hypogaea L.) is an important legume cultivated mostly in drought-prone areas where its productivity can be limited by water scarcity. The development of more drought-tolerant varieties is, therefore, a priority for peanut breeding programs worldwide. In contrast to cultivated peanut, wild relatives have a broader genetic diversity and constitute a rich source of resistance/tolerance alleles to biotic and abiotic stresses. The present study takes advantage of this diversity to identify drought-responsive genes by analyzing the expression profile of two wild species, Arachis duranensis and Arachis magna (AA and BB genomes, respectively), in response to progressive water deficit in soil. Data analysis from leaves and roots of A. duranensis (454 sequencing) and A. magna (suppression subtractive hybridization (SSH)) stressed and control complementary DNA (cDNA) libraries revealed several differentially expressed genes in silico, and 44 of them were selected for further validation by quantitative RT-PCR (qRT-PCR). This allowed the identification of drought-responsive candidate genes, such as Expansin, Nitrilase, NAC, and bZIP transcription factors, displaying significant levels of differential expression during stress imposition in both species. This is the first report on identification of differentially expressed genes under drought stress and recovery in wild Arachis species. The generated transcriptome data, besides being a valuable resource for gene discovery, will allow the characterization of new alleles and development of molecular markers associated with drought responses in peanut. These together constitute important tools for the peanut breeding program and also contribute to a better comprehension of gene modulation in response to water deficit and rehydration.

Analysis of the leaf transcriptome of Musa acuminata during interaction with Mycosphaerella musicola: gene assembly, annotation and marker development.

BACKGROUND: Although banana (Musa sp.) is an important edible crop, contributing towards poverty alleviation and food security, limited transcriptome datasets are available for use in accelerated molecular-based breeding in this genus. 454 GS-FLX Titanium technology was employed to determine the sequence of gene transcripts in genotypes of Musa acuminata ssp. burmannicoides Calcutta 4 and M. acuminata subgroup Cavendish cv. Grande Naine, contrasting in resistance to the fungal pathogen Mycosphaerella musicola, causal organism of Sigatoka leaf spot disease. To enrich for transcripts under biotic stress responses, full length-enriched cDNA libraries were prepared from whole plant leaf materials, both uninfected and artificially challenged with pathogen conidiospores. RESULTS: The study generated 846,762 high quality sequence reads, with an average length of 334 bp and totalling 283 Mbp. De novo assembly generated 36,384 and 35,269 unigene sequences for M. acuminata Calcutta 4 and Cavendish Grande Naine, respectively. A total of 64.4% of the unigenes were annotated through Basic Local Alignment Search Tool (BLAST) similarity analyses against public databases.Assembled sequences were functionally mapped to Gene Ontology (GO) terms, with unigene functions covering a diverse range of molecular functions, biological processes and cellular components. Genes from a number of defense-related pathways were observed in transcripts from each cDNA library. Over 99% of contig unigenes mapped to exon regions in the reference M. acuminata DH Pahang whole genome sequence. A total of 4068 genic-SSR loci were identified in Calcutta 4 and 4095 in Cavendish Grande Naine. A subset of 95 potential defense-related gene-derived simple sequence repeat (SSR) loci were validated for specific amplification and polymorphism across M. acuminata accessions. Fourteen loci were polymorphic, with alleles per polymorphic locus ranging from 3 to 8 and polymorphism information content ranging from 0.34 to 0.82. CONCLUSIONS: A large set of unigenes were characterized in this study for both M. acuminata Calcutta 4 and Cavendish Grande Naine, increasing the number of public domain Musa ESTs. This transcriptome is an invaluable resource for furthering our understanding of biological processes elicited during biotic stresses in Musa. Gene-based markers will facilitate molecular breeding strategies, forming the basis of genetic linkage mapping and analysis of quantitative trait loci.

A survey of genes involved in Arachis stenosperma resistance to Meloidogyne arenaria race 1.

Root-knot nematodes constitute a constraint for important crops, including peanut (Arachis hypogaea L.). Resistance to Meloidogyne arenaria has been identified in the peanut wild relative Arachis stenosperma Krapov. & W. C. Greg., in which the induction of feeding sites by the nematode was inhibited by an early hypersensitive response (HR). Here, the transcription expression profiles of 19 genes selected from Arachis species were analysed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), during the early phases of an A. stenosperma-M. arenaria interaction. Sixteen genes were significantly differentially expressed in infected and non-infected roots, in at least one of the time points analysed: 3, 6, and 9 days after inoculation. These genes are involved in the HR and production of secondary metabolites related to pathogen defence. Seven genes encoding a resistance protein MG13, a helix-loop helix protein, an ubiquitin protein ligase, a patatin-like protein, a catalase, a DUF538 protein, and a resveratrol synthase, were differentially expressed in all time points analysed. Transcripts of two genes had their spatial and temporal distributions analysed by in situ hybridisation that validated qRT-PCR data. The identification of candidate resistance genes involved in wild peanut resistance to Meloidogyne can provide additional resources for peanut breeding and transgenic approaches.

Global transcriptome analysis of two wild relatives of peanut under drought and fungi infection.

BACKGROUND: Cultivated peanut (Arachis hypogaea) is one of the most widely grown grain legumes in the world, being valued for its high protein and unsaturated oil contents. Worldwide, the major constraints to peanut production are drought and fungal diseases. Wild Arachis species, which are exclusively South American in origin, have high genetic diversity and have been selected during evolution in a range of environments and biotic stresses, constituting a rich source of allele diversity. Arachis stenosperma harbors resistances to a number of pests, including fungal diseases, whilst A. duranensis has shown improved tolerance to water limited stress. In this study, these species were used for the creation of an extensive databank of wild Arachis transcripts under stress which will constitute a rich source for gene discovery and molecular markers development. RESULTS: Transcriptome analysis of cDNA collections from A. stenosperma challenged with Cercosporidium personatum (Berk. and M.A. Curtis) Deighton, and A. duranensis submitted to gradual water limited stress was conducted using 454 GS FLX Titanium generating a total of 7.4 x 10(5) raw sequence reads covering 211 Mbp of both genomes. High quality reads were assembled to 7,723 contigs for A. stenosperma and 12,792 for A. duranensis and functional annotation indicated that 95% of the contigs in both species could be appointed to GO annotation categories. A number of transcription factors families and defense related genes were identified in both species. Additionally, the expression of five A. stenosperma Resistance Gene Analogs (RGAs) and four retrotransposon (FIDEL-related) sequences were analyzed by qRT-PCR. This data set was used to design a total of 2,325 EST-SSRs, of which a subset of 584 amplified in both species and 214 were shown to be polymorphic using ePCR. CONCLUSIONS: This study comprises one of the largest unigene dataset for wild Arachis species and will help to elucidate genes involved in responses to biological processes such as fungal diseases and water limited stress. Moreover, it will also facilitate basic and applied research on the genetics of peanut through the development of new molecular markers and the study of adaptive variation across the genus.

Characterization of WRKY co-regulatory networks in rice and Arabidopsis.

BACKGROUND: The WRKY transcription factor gene family has a very ancient origin and has undergone extensive duplications in the plant kingdom. Several studies have pointed out their involvement in a range of biological processes, revealing that a large number of WRKY genes are transcriptionally regulated under conditions of biotic and/or abiotic stress. To investigate the existence of WRKY co-regulatory networks in plants, a whole gene family WRKYs expression study was carried out in rice (Oryza sativa). This analysis was extended to Arabidopsis thaliana taking advantage of an extensive repository of gene expression data. RESULTS: The presented results suggested that 24 members of the rice WRKY gene family (22% of the total) were differentially-regulated in response to at least one of the stress conditions tested. We defined the existence of nine OsWRKY gene clusters comprising both phylogenetically related and unrelated genes that were significantly co-expressed, suggesting that specific sets of WRKY genes might act in co-regulatory networks. This hypothesis was tested by Pearson Correlation Coefficient analysis of the Arabidopsis WRKY gene family in a large set of Affymetrix microarray experiments. AtWRKYs were found to belong to two main co-regulatory networks (COR-A, COR-B) and two smaller ones (COR-C and COR-D), all including genes belonging to distinct phylogenetic groups. The COR-A network contained several AtWRKY genes known to be involved mostly in response to pathogens, whose physical and/or genetic interaction was experimentally proven. We also showed that specific co-regulatory networks were conserved between the two model species by identifying Arabidopsis orthologs of the co-expressed OsWRKY genes. CONCLUSION: In this work we identified sets of co-expressed WRKY genes in both rice and Arabidopsis that are functionally likely to cooperate in the same signal transduction pathways. We propose that, making use of data from co-regulatory networks, it is possible to highlight novel clusters of plant genes contributing to the same biological processes or signal transduction pathways. Our approach will contribute to unveil gene cooperation pathways not yet identified by classical genetic analyses. This information will open new routes contributing to the dissection of WRKY signal transduction pathways in plants.

Identification of drought-responsive genes in roots of upland rice (Oryza sativa L).

BACKGROUND: Rice (Oryza sativa L.) germplasm represents an extraordinary source of genes that control traits of agronomic importance such as drought tolerance. This diversity is the basis for the development of new cultivars better adapted to water restriction conditions, in particular for upland rice, which is grown under rainfall. The analyses of subtractive cDNA libraries and differential protein expression of drought tolerant and susceptible genotypes can contribute to the understanding of the genetic control of water use efficiency in rice. RESULTS: Two subtractive libraries were constructed using cDNA of drought susceptible and tolerant genotypes submitted to stress against cDNA of well-watered plants. In silico analysis revealed 463 reads, which were grouped into 282 clusters. Several genes expressed exclusively in the tolerant or susceptible genotypes were identified. Additionally, proteome analysis of roots from stressed plants was performed and 22 proteins putatively associated to drought tolerance were identified by mass spectrometry. CONCLUSION: Several genes and proteins involved in drought-response, as well as genes with no described homologs were identified. Genes exclusively expressed in the tolerant genotype were, in general, related to maintenance of turgor and cell integrity. In contrast, in the susceptible genotype, expression of genes involved in protection against cell damage was not detected. Several protein families identified in the proteomic analysis were not detected in the cDNA analysis. There is an indication that the mechanisms of susceptibility to drought in upland rice are similar to those of lowland varieties.

Plant-pathogen interactions: what is proteomics telling us?

Over the years, several studies have been performed to analyse plant-pathogen interactions. Recently, functional genomic strategies, including proteomics and transcriptomics, have contributed to the effort of defining gene and protein function and expression profiles. Using these 'omic' approaches, pathogenicity- and defence-related genes and proteins expressed during phytopathogen infections have been identified and enormous datasets have been accumulated. However, the understanding of molecular plant-pathogen interactions is still an intriguing area of investigation. Proteomics has dramatically evolved in the pursuit of large-scale functional assignment of candidate proteins and, by using this approach, several proteins expressed during phytopathogenic interactions have been identified. In this review, we highlight the proteins expressed during plant-virus, plant-bacterium, plant-fungus and plant-nematode interactions reported in proteomic studies, and discuss these findings considering the advantages and limitations of current proteomic tools.