Molecular response of wheat to Bipolaris sorokiniana under nitrogen stress

Abstract

Spot blotch caused by Bipolaris sorokiniana (Cochliobolus sativus) is a serious disease of wheat grown in warm climates. The impact of plant diseases is influenced by plant nutrition, and it has previously been reported that nutrient stress affects spot blotch severity. The investigation of gene expression in response to stress is an excellent approach to crop improvement. The interaction between nitrogen supply, spot blotch disease severity and the regulation of WRKY TF defence gene expression in Iraqi commercial wheat and Brachypodium was investigated. Nitrogen levels significantly affected the growth of wheat cultivars Rashid (susceptible to spot blotch) and Latifia (resistant to spot blotch) and susceptibility to B. sorokiniana in a dose-dependent manner under growth room conditions. Levels of the internal transcribed spacer of B. sorokiniana ribosomal RNA, measured with three primer pairs, increased with increasing pathogen load. Plant growth with reduced nitrogen (0.75 mM, 3.75 mM) resulted in the up-regulation of all seven TaWRKY genes investigated and in the upregulation of PR1 in both Rashid and Latifia compared to when grown under optimal (7.5 mM) conditions; in contrast, the RuBisCO large subunit (rbcL) gene was down-regulated. Similar trends were observed in Brachypodium for the corresponding WRKY TF genes. The response to infection in the more resistant cultivar Latifia was earlier than in susceptible Rashid, with up-regulation of WRKY TF genes occurring by 24 hours post infection (hpi) compared to 72 h for Rashid. Measurement of chitin content using fluorescence microscopy not only showed the ability to detect spot blotch in the early stages during the biotrophic phase but also demonstrated that it would produce an absolute measure of the fungal load, thus providing a reliable method to measure the development of disease and spot blotch resistance. For all three fungal genes transcription was >50% in the infected susceptible Rashid compared to infected resistant Latifia after 72 hpi. TILLING lines (with mutations in TaWRKY19 and TaWRKY68a) showed different development patterns and defence responses to B. sorokiniana compared to the wild-type (Cadenza cultivar). Microscopic analysis of the chitin content of the W68aQ245 mutant showed a greater visualisation of the pathogen abundance and spread correlating with high levels of damage in the infected leaves under nitrogen stress in a dose-dependent manner (from 0.75 mM to 3.75 mM and 7.5 mM), in contrast to the wild-type. Interestingly, in older plants (25 days old) pre-inoculation with Micromonospora luteifusca showed an enhanced defence response in Rashid and Cordaile, which was significantly greater than in Latifia following infection by B. sorokiniana as verified by disease severity, number of spores and chitin content. Surprisingly expression of WRKY TFs was significantly higher in Latifia and Cordiale than Rashid in response to spot blotch infection in the presence of Micromonospora. The rbcL gene showed significant down-regulation of expression post spot blotch challenge in both the presence and absence of the Micromonospora inoculation in Rashid. However, in Latifia and Cordiale, rbcL was only downregulated in the absence of the bacterium post spot blotch challenge. This study shows that there is a complex pattern of changes in the expression of genes encoding WRKY transcription factors in response to nitrate stress and infection by B. sorokiniana. There remains a great deal of potential in the use of WRKY TFs to better understand how wheat responds to nitrogen stress and B. sorokiniana infection, and how the two might be linked. Continued investigation into the function of WRKY TFs will provide a basis via which we can understand, interpret and potentially manipulate this link in a way that ensures wheat production is improved and ongoing global food security increased.