Role of WRKY transcription factors in tolerance of winter wheat 'Triticum aestivum' to aphid 'Sitobion avenae' infestation under different nitrogen regimes

Abstract

The English grain aphid (Sitobin avenae) is one of the most damaging pests of wheat (Triticum aestivum), causing damage both by the abstraction of nutrients and, more importantly, as a vector of viral diseases. Changing agricultural conditions are leading to insect pests becoming a more serious threat to sustainable crop production and therefore understanding the molecular basis of endogenous tolerance to aphid infestation will help mitigate shortfalls in global crop yield. Limiting nitrogen input in wheat increases aphid tolerance, but reduces yield. The present study investigated the response of two commercial winter wheats (Triticum aestivum, Var. Cordiale and Grafton) to biotic (Sitobion avenae) and abiotic (nitrogen) stress. Different growth measurement parameters, including plant height, leaf area, chlorophyll content, NO3 - ion accumulation and relative water content (RWC) were positively correlated with nitrogen level (Chapter 2). Both wheat genotypes exhibited significantly (p < 0.01) greater levels of resistance to aphids at low levels of nitrogen input (2.25 mM) than at intermediate (5.25 mM) or high (7.5 mM) nitrogen, with aphid fecundity reduced from 58 and 61 to 19 and 32 nymphs per adult for Cordiale and Grafton, respectively (Chapter 2). The role of changes in expression of genes for WRKY transcription factors in the stress response was studied in Var. Cordiale over time by RT-qPCR. TaWRKY3 showed large changes in gene expression under aphid and nitrogen stress, suggesting a novel role for this TF in stress (Chapter 3). At 7.5 mM nitrogen maximum expression of TaWRKY3 occurred 6 h after exposure to aphids, returning to basal by 9 h. At 5.25 mM nitrogen, expression occurred earlier and at higher levels than at 7.5 mM nitrogen, but again returned to basal at 9 h. The lowest nitrogen supply resulted in the same rapid onset of gene expression but the magnitude of the response (4-fold) was higher than with high nitrogen. In addition, the response was maintained for a longer period. To investigate the role of WRKY3 in the stress response TILLING lines with mutations to the WRKY3 gene were grown under dual stress. As in the control plants, aphid fecundity on most mutant lines was greater at 7.5 mM than at 2.25 mM nitrogen. However, the mutant 1996 was more resistant than the WT at 7.5 mM nitrogen, and showed no difference between high and low nitrogen, suggesting that WRKY3 may play a role in the link between nitrogen stress and aphid tolerance (Chapter 4). In control plants, concentrations of jasmonic acid (JA) isomers increased as a result of aphid infestation, whereas the concentration of salicylic acid (SA) fell and there was little change in abscisic acid concentration. In the mutant lines, the SA concentration was initially lower than in control plants but increased in 1ine 1996, the concentration of SA was relatively high in line 1171, and the concentration of JA isomers was initially higher than in control plants, increased at 3 h, then decreased (Chapter 4). Protein-DNA interaction assays showed binding of the Wild Type WRKY3 protein to W-box elements (TaPR1-23 flanking sequence, PcPR1- 1 promoter and synthetic) and that the mutation in TILLING line 1996 disrupts binding (Chapter 5). Regulation of PR1 gene expression is important for activation of plant defence responses. The present work suggests that TaWRKY3 may regulate this response through binding to W-box elements in PR1 genes. The data suggest that low nitrogen conditions may prime the defence of wheat against insect attack via a regulatory network of WRKY transcriptions factors. These results provide new knowledge and insight to help inform the effort to produce crops able to be grown under reduced nutrient input.