Diuraphis noxia (Russian wheat aphid, RWA) is a major pest on wheat in South Africa and most other wheat growing countries. Being a probing-sucking insect, RWAs insert their stylets into the phloem sieve elements and feed on the phloem sap. This feeding causes necrotic lesions in resistant varieties, or decoloration of leaves and death in susceptible varieties. In an effort to broaden our understanding on the response of the plant to RWA feeding, we synthesized and analyzed expressed sequence tags (ESTs) from suppression subtractive hybridization (SSH) libraries. These libraries were constructed using near isogenic wheat lines susceptible "Tugela" and resistant "TugelaDN" (Dn1) to RWA, as well as accession lines PI137739 (Dn1) and PI294994 (Dn5). Analysis of 200 ESTs from the libraries revealed the involvement of transcripts encoding genes involved in cell maintenance, growth and regulation, plant defense and signaling, photosynthesis and energy production, and of unknown function. A selection of these ESTs, in combination with clones obtained from other sources, were used on a custom array to study the expression profiles of 256 candidate wheat sequences putatively involved in plant defense against RWA. The selected sequences included wheat genomic clones with putative nucleotide binding site (NBS) motifs, rapid amplification of cDNA ends PCR (RACE-PCR), and cDNA clones from RWA induced libraries. Genomic banana and flax clones that were obtained using representative difference analysis (RDA), and suspected to be involved in abiotic stress responses, were also spotted onto the microarray slides. The spotted custom arrays were then hybridized against cDNA isolated from a resistant cultivar "TugelaDN" on 0, 2, 5, and 8 days after infestation, post-labeled with Cy3- or Cy5-fluorescent dyes. The subsequent expression profiling using DNA microarray, RT-PCR, and Northern Blot analysis identified 29 transcripts associated with the feeding response. These transcripts encoded proteins functioning in direct defense and signaling, oxidative burst, cell wall degradation, cell maintenance, photosynthesis, and energy production. Results indicate that plants co-ordinately regulate gene expression when attacked by RWA. It is hypothesized that the NBS-LRR proteins are important in receptor recognition and signaling, which enable the plant to overcome the stresses inflicted by RWA feeding. It is further suggested that the ability to maintain photosynthetic function with resultant energy production is one of the determining factors ensuring the survival of the resistant varieties when coping with the RWA feeding.
Fourteen spring and winter hard red South African wheat (Triticum aestivum L.) cultivars were compared for their regeneration and transient anthocyanin expression efficiencies. Embryonic and non-embryonic callus, as well as plantlets, were obtained from all the cultivars cultured on a modified Murashige and Skoog basal medium supplemented with 5 mg 1. 1 6-Benzylaminopurine. Gamtoos and Tugela On1 were the best cultivars for the development of both roots and shoots. None of the cultivars produced callus or planttets on ML3 medium. Transient anthocyanin expression was obtained in winter, as well as spring, hard red wheat cultivars. Using the nondestructive anthocyanin reporter-gene, it was established that the spring wheat cultivars Palmiet On1, Palmiet On2 and Palmiet On5 were the most suitable cultivars for future transformation studies, determined by the percentage bombarded calli expressing anthocyanin after two to three weeks.Die effektiwiteit met betrekking tot regenerering en kortstondige geenuitdrukking in veertien Suid-Afrikaanse harde-rooi, lente-en winterkoringkultivars (Triticum aestivum L.) is vergelyk. AI die kultivars het embrioniese en nie-embrioniese kallus, sowel as plantjies, op die gewysigde MS medium wat 5 mg 1. 1 SAP bevat, geproduseer.Van al die toepaslike kultivars het Gamtoos en Tugela On1 die beste in terme van stingel-en blaarontwikkeling presteer. Nie een van die kultivars het enige kallus of plante op ML3-medium gevorm nie. Kortstondige antosianienuitdrukking is met harde-rooi winter-en lente-koringcultivars verkry. Deur van die nie-destruktiewe antosianien-verklikkergeen gebruik te maak, asook die persentasie antosianienuitdrukking in kallus, 2 tot 3 weke na bombardering te bereken, is vasgestel dat die lente-koringcultivars, Palmiet On1, Palmiet On2 en Palmiet On5 die mees belowende cultivars vir verdere studies oor transformering blyk te wees.Abbreviations: SAP -6-Senzylaminopurine; CaMV -cauliflower mosaic virus; 2,4-D -2,4-Dichlorophenoxyacetic acid; On -Oiuraphis noxia; ML3 -Murashige and Skoog (1962) basal medium with modifications; MS -Murashige and Skoog (1962) medium; NaOCI-Sodium Hypochlorite; SD -Standard Deviation).
Russian wheat aphid (Diuraphis noxia, Morvilko; RWA) is a major pest on wheat, barley and other triticale in South Africa. Infestation by the RWA results in altered protein expression patterns, which is manifested as differential expression of gene sequences. In the present study, Russian wheat aphid resistant (Tugela DN, Tugela*5/SA2199, Tugela*5/SA463, PI 137739, PI 262660, and PI 294994) and susceptible triticale (Tugela) were infested and cDNA synthesized. A PCR based approach was utilized to amplify the nucleotide binding site conserved region to obtain expressed sequence tags (ESTs) with homology to resistance gene analogs (RGAs). The approach proved highly feasible when the isolation of RGAs is the main objective, since 18% of all obtained ESTs showed significant hits with known RGAs, when translated into their corresponding amino acid sequences and searched against the nonredundant GenBank protein database using the BLASTX algorithm.
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