Plant C2 domain proteins play important roles in diverse cellular processes including growth, development, and membrane targeting, as well as in abiotic and biotic stress adaptations by sensing intracellular Ca 2+ signals. In this study, we isolated a novel C2 domain protein gene, TaERG3, from wheat infected by Puccinia striiformis f. sp. tritici. TaERG3 was predicted to encode a 144 amino acid protein with molecular mass of 15.68 kD and isoelectric point of 3.93. Analysis of the deduced amino acid sequence of TaERG3 using InterProScan revealed the presence of an N-terminal calciumdependent phospholipid-binding module (C2 domain, 5 to 103). Transient expression analysis showed that the TaERG3 protein was predominately and uniformly localized in the plasmalemma and nucleus of onion epidermal cells. Quantitative real-time PCR analyses indicated that TaERG3 transcript was differentially induced in both incompatible and compatible interactions, as well as by applied abscisic acid (ABA) and CaCl 2 . However, the significant transcript changes induced by methyl jasmonate, ethylene, and salicylic acid treatments were not as dramatic as those induced by ABA. TaERG3 was also up-regulated by environmental stimuli including low temperature and high salinity. These results imply that TaERG3 might be involved in wheat defence responses against stripe rust and abiotic stresses in an ABA-dependent signalling pathway.

A novel gene induced during hypersensitive reaction (HIR) in wheat was identified using in silico cloning and designated as TaHIR2. The TaHIR2 gene was deduced to encode a 284-amino acid protein, whose molecular mass and isoelectric point (pI) were 31.05 kD and 5.18, respectively. Amino acid sequence analysis demonstrated the presence of stomatins, prohibitin, flotillins, HflK/C (SPFH) domain and prohibitin homologue for the TaHIR2 protein. Phylogenetic analysis of 13 HIR genes from different monocots indicated that TaHIR2 was highly homologous to HvHIR2. Transient expression analysis using particle-mediated bombardment showed that the TaHIR2 fusion protein was located in the onion epidermal cells. Quantitative RT-PCR analyses revealed that TaHIR2 transcripts were significantly accumulated in adult wheat leaves with maximum induction at 18 h post inoculation with the stripe rust, whereas slightly up-regulation could also be observed in the compatible reaction at the seedling stage. These results suggest that TaHIR2 may play an active role in wheat defense against stripe rust.

Autophagy-related 8 (ATG8) protein has been reported to be involved in plant’s innate immune response, but it is not clear whether such genes play a similar role in cereal crops against obligate biotrophic fungal pathogens. Here, we reported an ATG8 gene from wheat (Triticum aestivum), designated TaATG8j. This gene has three copies located in chromosomes 2AS, 2BS, and 2DS. The transcriptions of all three copies were upregulated in plants of the wheat cultivar Suwon 11, inoculated with an avirulent race (CYR23) of Puccinia striiformis f. sp. tritici (Pst), the causal fungal pathogen of stripe rust. The transient expression of TaATG8j in Nicotiana benthamiana showed that TaATG8j proteins were distributed throughout the cytoplasm, but mainly in the nucleus and plasma membrane. The overexpression of TaATG8j in N. benthamiana slightly delayed the cell death caused by the mouse apoptotic protein BAX (BCL2-associated X protein). However, the expression of TaATG8j in yeast (Schizosaccharomyces pombe) induced cell death. The virus-induced gene silencing of all TaATG8j copies rendered Suwon 11 susceptible to the avirulent Pst race CYR23, accompanied by an increased fungal biomass and a decreased necrotic area per infection site. These results indicate that TaATG8j contributes to wheat resistance against stripe rust fungus by regulating cell death, providing information for the understanding of the mechanisms of wheat resistance to the stripe rust pathogen.

Plant cell surface-localized receptor-like kinases (RLKs) recognize invading pathogens and transduce the immune signals inside host cells, subsequently triggering immune responses to fight off pathogen invasion. Nonetheless, our understanding of the role of RLKs in wheat resistance to the biotrophic fungus Puccinia striiformis f. sp. tritici (Pst) remains limited. During the differentially expressed genes in Pst infected wheat leaves, a Leucine-repeat receptor-like kinase (LRR-RLK) gene TaBIR1 was significantly upregulated in the incompatible wheat-Pst interaction. qRT-PCR verified that TaBIR1 is induced at the early infection stage of Pst. The transient expression of TaBIR1-GFP protein in N. bentamiana cells and wheat mesophyll protoplasts revealed its plasma membrane location. The knockdown of TaBIR1 expression by VIGS (virus induced gene silencing) declined wheat resistance to stripe rust, resulting in reduced reactive oxygen species (ROS) production, callose deposition, and transcripts of pathogenesis-related genes TaPR1 and TaPR2, along with increased Pst infection area. Ectopic overexpression of TaBIR1 in N. benthamiana triggered constitutive immune responses with significant cell death, callose accumulation, and ROS production. Moreover, TaBIR1 triggered immunity is dependent on NbBAK1, the silencing of which significantly attenuated the defense response triggered by TaBIR1. TaBIR1 interacted with the NbBAK1 homologues in wheat, co-receptor TaSERK2 and TaSERK5, the transient expression of which could restore the impaired defense due to NbBAK1 silencing. Taken together, TaBIR1 is a cell surface RLK that contributes to wheat stripe rust resistance, probably as a positive regulator of plant immunity in a BAK1-dependent manner.

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