Dataset Information

Transcriptional profiling of root-knot nematode induced feeding sites in cowpea (Vigna unguiculata L. Walp.) using a soybean genome array.

ABSTRACT:

Background

The locus Rk confers resistance against several species of root-knot nematodes (Meloidogyne spp., RKN) in cowpea (Vigna unguiculata). Based on histological and reactive oxygen species (ROS) profiles, Rk confers a delayed but strong resistance mechanism without a hypersensitive reaction-mediated cell death process, which allows nematode development but blocks reproduction.

Results

Responses to M. incognita infection in roots of resistant genotype CB46 and a susceptible near-isogenic line (null-Rk) were investigated using a soybean Affymetrix GeneChip expression array at 3 and 9 days post-inoculation (dpi). At 9 dpi 552 genes were differentially expressed in incompatible interactions (infected resistant tissue compared with non-infected resistant tissue) and 1,060 genes were differentially expressed in compatible interactions (infected susceptible tissue compared with non-infected susceptible tissue). At 3 dpi the differentially expressed genes were 746 for the incompatible and 623 for the compatible interactions. When expression between infected resistant and susceptible genotypes was compared, 638 and 197 genes were differentially expressed at 9 and 3 dpi, respectively.

Conclusions

In comparing the differentially expressed genes in response to nematode infection, a greater number and proportion of genes were down-regulated in the resistant than in the susceptible genotype, whereas more genes were up-regulated in the susceptible than in the resistant genotype. Gene ontology based functional categorization revealed that the typical defense response was partially suppressed in resistant roots, even at 9 dpi, allowing nematode juvenile development. Differences in ROS concentrations, induction of toxins and other defense related genes seem to play a role in this unique resistance mechanism.

SUBMITTER: Das S

PROVIDER: S-EPMC2996976 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In this study we have looked at the transcriptome profile of both incompatible and compatible cowpea-RKN interaction for two different time points using the Affymetrix soybean GeneChip. This is the first study of this kind in cowpea-RKN interaction. This study provides a broad insight into the Rk-mediated resistance in cowpea and creates an excellent dataset of potential candidate genes involved in both nematode resistance and parasitism, which can be further tested for their role in this biological process using functional genomics approaches. our results have shown that the root-knot nematode resistant pathway is still partially suppressed at 9 days post inoculation in resistant cowpea root. There is indication that subtle variation of ROS concentration, induction of toxins and other defense related genes play a role in this unique resistance mechanism. Further functional analysis of these differentially expressed genes will help us to understand this intriguing plant-nematode interaction in a more precise manner. Experiment Overall Design: Seeds of CB46 (resistant) and null-Rk (susceptible) cowpea genotypes were surface sterilized using 10% (v/v) bleach solution and were planted in growth pouches. Plants were grown under controlled environmental conditions of 26.7Â°C Â± 0.5Â°C constant temperature and daily light/dark cycles of 16/8 hours. This temperature was used because it lies within the optimum temperature range of 26 â 28 Â°C for development and reproduction of M. incognita on cowpea in growth pouches. Each pouch was inoculated with 3000 J2 in 5 ml of deionized water, 12 days after planting (dap). Equal number of pouches was mock inoculated with 5 ml of deionized water to use as control. Nematode infected root tissue was excised using a sterile scalpel at 3 days post inoculation (dpi) and 9 dpi respectively under a magnifying glass and flash frozen immediately in liquid nitrogen. Approximately equal amount of root tissue was also excised and flash frozen from the control plants. The harvested tissue was stored in -80Â°C until RNA isolation. For 9 dpi samples 3 biological replicates were used for each treatment. So total number of soybean GeneChips used for 9dpi samples was 12. For 3 dpi samples two biological replicates were used for each treatment and a total of 8 GeneChips were used. Expression signals were first analyzed in Microarray Suite 5.0 software (MAS 5, Affymetrix Inc.) to determine the âpresentâ probe set list. The detection algorithm uses probe pair intensities to generate a detection p-value and assign a âpresentâ, âmarginalâ, or âabsentâ call. Each probe pair in a probe set has a potential vote in determining whether the measured transcript is or is not âpresentâ. The vote is described by the discrimination score (R). R is calculated for each probe pair and compared to a predefined threshold, Tau. Probe pairs with R higher than Tau vote âpresentâ and the voting result is summarized as a p-value. The greater the number of discrimination scores (R) that are above Tau, the smaller the p-value and the more likely the given transcript is truly present in the sample. Only probe sets with a âpresentâ call in all three replicates of at least one treatment was considered to be âexpressedâ.

Project description:BackgroundCowpea is the most important food grain legume in Sub-Saharan Africa. However, no study has so far assessed rhizobial biodiversity and/or nodule functioning in relation to strain IGS types at the continent level. In this study, 9 cowpea genotypes were planted in field experiments in Botswana, South Africa and Ghana with the aim of i) trapping indigenous cowpea root-nodule bacteria (cowpea "rhizobia") in the 3 countries for isolation, molecular characterisation using PCR-RFLP analysis, and sequencing of the 16S - 23S rDNA IGS gene, ii) quantifying N-fixed in the cowpea genotypes using the 15N natural abundance technique, and iii) relating the levels of nodule functioning (i.e. N-fixed) to the IGS types found inside nodules.ResultsField measurements of N2 fixation revealed significant differences in plant growth, delta15N values, %Ndfa and amounts of N-fixed between and among the 9 cowpea genotypes in Ghana and South Africa. Following DNA analysis of 270 nodules from the 9 genotypes, 18 strain IGS types were found. Relating nodule function to the 18 IGS types revealed significant differences in IGS type N2-fixing efficiencies. Sequencing the 16S - 23S rDNA gene also revealed 4 clusters, with cluster 2 forming a distinct group that may be a new Bradyrhizobium species. Taken together, our data indicated greater biodiversity of cowpea bradyrhizobia in South Africa relative to Botswana and Ghana.ConclusionsWe have shown that cowpea is strongly dependant on N2 fixation for its N nutrition in both South Africa and Ghana. Strain IGS type symbiotic efficiency was assessed for the first time in this study, and a positive correlation was discernible where there was sole nodule occupancy. The differences in IGS type diversity and symbiotic efficiency probably accounts for the genotype x environment interaction that makes it difficult to select superior genotypes for use across Africa. The root-nodule bacteria nodulating cowpea in this study all belonged to the genus Bradyrhizobium. Some strains from Southern Africa were phylogenetically very distinct, suggesting a new Bradyrhizobium species.