Plants have evolved mechanisms to defend themselves against pathogen attack. These defense mechanisms consist of a series of inducible responses (including specific recognition of pathogen invasion, signal transduction and defense gene activation) that result in resistance. Plants responses to pathogen invasion also result in the suppression of various housekeeping activities of the cells, thus diverting the cellular resources to defense responses. Systemic acquired resistance (SAR), an inducible defense response enhanced as a result of initial infection with a necrotising pathogen, lead to long-term resistance in a plant. Differential gene expression of genes related to defense in cultured cotton cells and leaf disks that have been challenged with a purified elicitor from Verticillium dahliae, as well as a chemical inducer of defense responses, DL-b-amino-n-butyric acid, were investigated. The mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) was used to identify differentially expressed genes 5 h after application of either 50 mg mL-1 Verticillium dahliae elicitor or 1 mM DL-b-amino-n-butyric acid to cotton cell suspension cultures and leaf disks. Identified cDNAs up- or down-regulated for this study were classified into seven groups: ‘Transcription factor’, ‘Ubiquitin and Proteasome’, ‘Mitochondria’, ‘Protein kinase/Receptor-like kinase’, ‘Defense/Resistance’, ‘Carbohydrate metabolism/Cell wall’ and ‘Other’. The identified cDNAs up-regulated after Verticillium dahliae elicitor treatment, classified in the ‘Transcription factor’ group, coded for a MYB family transcription factor, zinc finger protein and a RMA1 RING zinc finger protein. The identified cDNA classified in the ‘Mitochondria’ group coded for a cytochrome C oxidase subunit I and II and the cDNA classified in the ‘Protein kinase/Receptor-like kinase’ group coded for a serine/threonine protein kinase. The identified cDNA classified in the ‘Defense/Resistance’ group coded for a disease resistance protein family and the cDNAs classified in the ‘Carbohydrate metabolism/Cell wall’ group coded for a beta-1,4-Nacetylglucosaminyltransferase, a cellulose synthase-like protein, a 3-deoxy-D-manno-octulosonic acid transferase-like protein and a hydroxyproline-rich glycoprotein homolog. In addition, a cDNA classified in the ‘Other’ group, coded for a urea active transporter-like protein. The cDNA identified that was down-regulated after Verticillium dahliae elicitor treatment, classified in the ‘Carbohydrate metabolism/Cell wall’ group, coded for a proline-rich protein family and cDNAs classified in the ‘Other’ group coded for a thioredoxin reductase1 and ‘hookless1’ homologue. Among the identified cDNAs up-regulated after DL-b-amino-n-butyric acid treatment, classified in the ‘Ubiquitin and Proteasome’ group, were a 20S proteasome subunit alpha type 5 and an ubiquitin. The identified cDNA classified in the ‘Mitochondria’ group coded for a NADH dehydrogenase subunit 6, a mitochondrial DNA product. The identified cDNAs classified in the ‘Other’ group coded for an armadillo repeat containing protein and a phosphoinositide-specific phospholipase C. The cDNA identified that was down-regulated after DL-b-amino-n-butyric acid treatment, classified in the ‘Protein kinase/Receptor-like kinase’ group, coded for a casein kinase I like protein. The identified cDNA classified in the ‘Carbohydrate metabolism/Cell wall’ group, coded for a putative glycine rich protein. Also, the identified cDNA classified in the ‘Other’ group, coded for a NADH dehydrogenase subunit F that is coded for by chloroplast DNA. The differential expression of the cDNAs up-regulated after the Verticillium dahliae elicitor treatment was confirmed for seven of the nine cDNA clones with a Reverse Northern dot blot. Also, the differential expression of two cDNAs up-regulated after DL-b-amino-n-butyric acid treatment was confirmed and the induction kinetics was followed with a Reverse Northern dot blot. The mRNAs corresponding to C8B5, the gene encoding an ubiquitin, were detectable after 2.5 h and showed a significant increase in expression up to 7.5 h, after which the expression levels decreased to levels similar to those detected at 2.5 h. The mRNAs corresponding to L4B4, a homologue of an a-type subunit of 20S proteasome, were detectable after 2.5 h with an gradual increase in expression levels up to 7.5 h after which the expression levels decreased to levels similar to those detected at 2.5 h. This study facilitated a better understanding of differential gene regulation during triggering of defense responses in cotton following elicitation with the Verticillium dahliae elicitor and DL-b-aminon- butyric acid. / Prof. I.A. Dubery
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uj/uj:1995 |
| Date | 19 May 2008 |
| Creators | Zwiegelaar, Michele |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Type | Thesis |
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