Wheat is an important global economic crop, and is the most widely cultivated plant around the world. Bipolaris sorokiniana is a hemi-biotrophic fungal pathogen that causes spot blotch, root rot and leaf spot diseases in a number of cereals including wheat, barley and other small grain cereals. Genetic engineering is the most economical and least environmentally risky method to controll various diseases. However, identification of molecular mechanisms of pathogen interaction with host is necessary to establish stable resistance in wheat cultivars. Effectors play an essential role in creating or inhibiting hosting resistance, and therefore identifying effectors is important. In this study, we combined bioinformatics approaches with expression analysis during pathogenesis to identify candidate effectors of B. sorokiniana (ND90Pr). For this purpose, small secreted proteins with a size of ≤300 amino acids and rich in cysteine residues were selected. In this study wheat Darab 2 cultivar were inoculated with a spore suspension of B. sorokiniana. RNAs were extracted from the inoculated leaves during 0, 1, 2, 3, 4, 5 and 7 days after inoculation using CTAB-based method and the then cDNA libraries were synthesized. After designing the specific primer pairs, the appropriate annealing temperature of each of them were determined by PCR reaction and expression of four effector candidate genes were eva‎luated by Real-Time PCR reaction. Also, the effect of starvation treatment and carbon and nitrogen restriction on the expression of these genes was investigated. Symptoms of the disease began on the first day after inoculation with leaf paleness and on the second day, chlorosis spots appeared. Necrosis spots appeared on the fifth day after inoculation and on the seventh day after inoculation, all leaf surfaces were burned. The results indicated that in on gene Bs4 containing LysM motif, the highest gene expression level was detected in the third day after inoculation which coincided with leaf chlorosis and appears to play a role in the spread of the disease. Restriction of carbon and nitrogen (B5-N,C) increased the expression of this gene. This gene seems to respond to starvation stress. In the case of Bs6 gene, no expression was detected for this gene in the pathogenicity of B. sorokiniana toward wheat and it is believed that this gene is not involved in pathogenesis. In the case of gene Bs7 and gene Bs9, the highest expression level was detected on the fifth day after inoculation, which coincided with the appearance of leaf necrosis symptoms and may play a role in the pathogenesis process. In the case of gene Bs7 and Bs6, the highest expression level was detected in vitro growth conditions on rich medium, but in gene Bs9, the highest gene expression in carbon-free medium (B5-C) and this gene responded to carbon restriction. In both genes, carbon source deficiency had the highest induction rate of gene expression compared to N and NC treatments. According to the results of gene expression during plant infection, it seems that selected genes have important roles in B. sorokiniana pathogenesis. Key words: Spot Blotch, Gene expression, Plant-microbe interaction, Starvation treatment.

رحيم مهرابي، ابوذر سورني

مجيد طالبي

بدر الدين ابراهيم سيد طباطبايي، بهرام شريف نبي