Project description:Screening cDNA clones from SSH library by hybridization with cDNA used to construct the library. Treated samples were drought-stressed cowpea plants, and control samples were cowpea plants subjected to a standard watering regime. cDNA clones from forward and reverse libraries are spotted on the same array, but data from each library were analysed separately after normalization using SSHscreen software (http://microarray.up.ac.za/SSHscreen ) to calculate Enrichment Ratio 3 (ER3) and Enrichment Ratio 2 (ER2) values for each clone. ER3 is a measure of differential expression, and was determined using a set of hybridizations with unsubtracted treated (UT) and unsubtracted control (UC) cDNA. ER2 is a measure of the relative abundance of a clone's transcript in the original tester sample, relative to other transcripts in the sample prior to the SSH process. ER2 for the forward library was determined using a set of hybridizations with subtracted treated (ST) and unsubtracted treated (UT) cDNA. ER2 for the reverse library is determined using a set of hybridizations with subtracted control (SC) and unsubtracted control (UC) cDNA.
Project description:Screening cDNA clones from SSH library by hybridization with cDNA used to construct the library. Treated samples were drought-stressed cowpea plants, and control samples were cowpea plants subjected to a standard watering regime. cDNA clones from forward and reverse libraries are spotted on the same array, but data from each library were analysed separately after normalization using SSHscreen software (http://microarray.up.ac.za/SSHscreen ) to calculate Enrichment Ratio 3 (ER3) and Enrichment Ratio 2 (ER2) values for each clone. ER3 is a measure of differential expression, and was determined using a set of hybridizations with unsubtracted treated (UT) and unsubtracted control (UC) cDNA. ER2 is a measure of the relative abundance of a clone's transcript in the original tester sample, relative to other transcripts in the sample prior to the SSH process. ER2 for the forward library was determined using a set of hybridizations with subtracted treated (ST) and unsubtracted treated (UT) cDNA. ER2 for the reverse library is determined using a set of hybridizations with subtracted control (SC) and unsubtracted control (UC) cDNA. Direct comparison of UT and UC cDNAs to calculate ER3 values for F and R library, including dye swaps and replicate arrays. Direct comparison of ST and UT cDNAs to calculate ER2 values for F library, including dye swaps and replicate arrays. Direct comparison of SC and UC cDNAs to calculate ER2 values for R library, including dye swaps and replicate arrays.
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.
Project description:Background. Cowpea, Vigna unguiculata L. Walp., is one of the most important food and forage legumes in the semi-arid tropics. While most cowpea accessions are susceptible to the root parasitic weed Striga gesnerioides, several cultivars have been identified that show race-specific resistance. Cowpea cultivar B301 contains the RSG3-301 gene for resistance to S. gesnerioides race SG3, but is susceptible to race SG4z. When challenged by SG3, roots of cultivar B301 develop a strong resistance response characterized by a hypersensitive reaction and cell death at the site of parasite attachment. In contrast, no visible response occurs in B301 roots parasitized by SG4z. Results. Gene expression in the roots of the cowpea cultivar B301 during compatible (susceptible) and incompatible (resistant) interactions with S. gesnerioides races SG4z and SG3, respectively, were investigated at 6 and 13 days post-inoculation (dpi), in the early and late stages of the resistance response using a Nimblegen custom design cowpea microarray. A total of 111 genes were differentially expressed in B301 roots at 6 dpi; this number increased to 2102 genes at 13 dpi. At 13 dpi, a total of 1944 genes were differentially expressed during compatible (susceptible) interactions of B301 with SG4z . Genes and pathways involved in signal transduction, programmed cell death and apoptosis, and defense response to biotic and abiotic stress were differentially expressed in the early resistance response; at the later time point, enrichment was primarily for defense-related gene expression, and genes encoding components of lignifications and secondary wall formation. In compatible interactions (B301 – SG4z), multiple defense pathways were repressed, including those involved in lignin biosynthesis and secondary cell wall modifications, while cellular transport processes for nitrogen and sulfur were increased. Conclusion. Distinct changes in global gene expression profiles occur in host roots following successful and unsuccessful attempted parasitism by Striga. Induction of specific defense related genes and pathways defines components of a unique resistance mechanism. Some genes and pathways up-regulated in the host resistance response to SG3 are repressed in the susceptible interactions, suggesting that the parasite is targeting specific components of the host’s defense. These results add to our understanding of plant-parasite interactions and the evolution of resistance to parasitic weeds.
Project description:Background. Cowpea, Vigna unguiculata L. Walp., is one of the most important food and forage legumes in the semi-arid tropics. While most cowpea accessions are susceptible to the root parasitic weed Striga gesnerioides, several cultivars have been identified that show race-specific resistance. Cowpea cultivar B301 contains the RSG3-301 gene for resistance to S. gesnerioides race SG3, but is susceptible to race SG4z. When challenged by SG3, roots of cultivar B301 develop a strong resistance response characterized by a hypersensitive reaction and cell death at the site of parasite attachment. In contrast, no visible response occurs in B301 roots parasitized by SG4z. Results. Gene expression in the roots of the cowpea cultivar B301 during compatible (susceptible) and incompatible (resistant) interactions with S. gesnerioides races SG4z and SG3, respectively, were investigated at 6 and 13 days post-inoculation (dpi), in the early and late stages of the resistance response using a Nimblegen custom design cowpea microarray. A total of 111 genes were differentially expressed in B301 roots at 6 dpi; this number increased to 2102 genes at 13 dpi. At 13 dpi, a total of 1944 genes were differentially expressed during compatible (susceptible) interactions of B301 with SG4z . Genes and pathways involved in signal transduction, programmed cell death and apoptosis, and defense response to biotic and abiotic stress were differentially expressed in the early resistance response; at the later time point, enrichment was primarily for defense-related gene expression, and genes encoding components of lignifications and secondary wall formation. In compatible interactions (B301 M-bM-^@M-^S SG4z), multiple defense pathways were repressed, including those involved in lignin biosynthesis and secondary cell wall modifications, while cellular transport processes for nitrogen and sulfur were increased. Conclusion. Distinct changes in global gene expression profiles occur in host roots following successful and unsuccessful attempted parasitism by Striga. Induction of specific defense related genes and pathways defines components of a unique resistance mechanism. Some genes and pathways up-regulated in the host resistance response to SG3 are repressed in the susceptible interactions, suggesting that the parasite is targeting specific components of the hostM-bM-^@M-^Ys defense. These results add to our understanding of plant-parasite interactions and the evolution of resistance to parasitic weeds. A Nimblegen custom design cowpea microarray investigating gene expression in the roots of the cowpea cultivar B301 during compatible (susceptible) and incompatible (resistant) interactions with S. gesnerioides races SG4z and SG3, respectively, at 6 and 13 days post-inoculation (dpi), in the early and late stages of the resistance response.