Wheat Avenin-like proteins (TaALP) are atypical storage proteins belonging to the Prolamin superfamily. Previous studies on ALPs have focused on the proteins' positive effects on dough strength, whilst no correlation has been made between TaALPs and the plant immune system. Here, we performed genome-wide characterization of ALP encoding genes in bread wheat. In silico analyses indicated the presence of critical peptides in TaALPs that are active in the plant immune system. Pathogenesis-related nucleotide motifs were also identified in the putative promoter regions of TaALP encoding genes. RT-PCR was performed on TaALP and previously characterised pathogenesis resistance genes in developing wheat caryopses under control and Fusarium graminearum infection conditions, respectively. The results showed 2 that TaALP and NMT genes were upregulated upon F. graminearum inoculation. mRNA insitu hybridization showed that TaALP genes were expressed in the embryo, aleurone and sub-aleurone layer cells. Seven TaALP genes were cloned for the expression of recombinant proteins in Escherichia coli, which displayed significant inhibitory function on F. graminearum under anti-fungal tests. In addition, FHB index association analyses showed that allelic variations of two ALP genes on chromosome 7A were significantly correlated with FHB symptoms. Over-expression of an ALP gene on chromosome 7A showed an enhanced resistance to FHB. Yeast two Hybridization results revealed that ALPs have potential proteases inhibiting effect on metacaspases and beta-glucosidases. A vital infection process related pathogen protein, F. graminearum Beta-glucosidase was found to interact with ALPs.Our study is the first to report a novel function for wheat storage protein in fungal resistance, which greatly advances our understanding of the biological roles of this protein class. The findings in this study is of great significance for future wheat breeding and production.
Preparation of Albumin and Globulin Protein extractsAustralia spring bread wheat varieties Mace and Spitfire were used for albumin and globulin protein extraction. The albumin/globulin proteins were extracted from 100 mg of flour according to the procedure of Dupont et al. (48). Briefly, 100 mg of flour was extracted with 1 mL of 0.3 M NaI, 7.5% 1-propanol (NaI-propanol), and centrifuged at 4500 g for 10 min, After two extractions, the supernatant fractions were pooled in 15 ml tubes, precipitated with four volumes of ice-cold (-20°C) NH 4 Ac-MeOH (0.1 M ammonium acetate in 100% methanol), stored at -20 °C for at least 48 h, and centrifuged as above. The supernatant fluid was transferred into 50 ml tubes and precipitated with four volumes of ice-cold acetone and incubated at -20 °C overnight. Following incubation, the fluid was centrifuged as above to yield albumin/globulin fraction pellets. Determination of Individual ALPs of wheat cv. Mace and Spitfire via RP-HPLC Analysis coupled with SDS-PAGE, MALDI-TOF, and LC/MS RP-HPLC Freeze-dried protein pellets were dissolved in 500 µL 6 M guani...