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Wheat dwarf virus (WDV, genus Mastrevirus, family Geminiviridae) is one of the causal agents of wheat viral disease, which severely impacts wheat production in most wheat-growing regions in the world. Currently, there is little information about natural resistance against WDV in common wheat germplasms. CRISPR/Cas9 technology is being utilized to manufacture transgenic plants resistant to different diseases. In the present study, we used the CRISPR/Cas9 system targeting overlapping regions of coat protein (CP) and movement protein (MP) (referred to as CP/MP) or large intergenic region (LIR) in the wheat variety 'Fielder' to develop resistance against WDV. WDV-inoculated T1 progenies expressing Cas9 and sgRNA for CP/MP and LIR showed complete resistance against WDV and no accumulation of viral DNA compared with control plants. Mutation analysis revealed that the CP/MP and LIR targeting sites have small indels in the corresponding Cas9-positive plants. Additionally, virus inhibition and indel mutations occurred in T2 homozygous lines. Together, our work gives efficient results of the engineering of CRISPR/Cas9-mediated WDV resistance in common wheat plants, and the specific sgRNAs identified in this study can be extended to utilize the CRISPR/Cas9 system to confer resistance to WDV in other cereal crops such as barley, oats, and rye. [ABSTRACT FROM AUTHOR]

Summary: Barley yellow dwarf viruses (BYDVs) cause widespread damage to global cereal crops. Here we report a novel strategy for elevating resistance to BYDV infection. The 17K protein, a potent virulence factor conserved in BYDVs, interacted with barley IMP‐α1 and ‐α2 proteins that are nuclear transport receptors. Consistently, a nuclear localization signal was predicted in 17K, which was found essential for 17K to be transported into the nucleus and to interact with IMP‐α1 and ‐α2. Reducing HvIMP‐α1 and ‐α2 expression by gene silencing attenuated BYDV‐elicited dwarfism, accompanied by a lowered nuclear accumulation of 17K. Among the eight common wheat CRISPR mutants with two to four TaIMP‐α1 and ‐α2 genes mutated, the triple mutant α1aaBBDD/α2AAbbdd and the tetra‐mutant α1aabbdd/α2AAbbDD displayed strong BYDV resistance without negative effects on plant growth under field conditions. The BYDV resistance exhibited by α1aaBBDD/α2AAbbdd and α1aabbdd/α2AAbbDD was correlated with decreased nuclear accumulation of 17K and lowered viral proliferation in infected plants. Our work uncovers the function of host IMP‐α proteins in BYDV pathogenesis and generates the germplasm valuable for breeding BYDV‐resistant wheat. Appropriate reduction of IMP‐α gene expression may be broadly useful for enhancing antiviral resistance in agricultural crops and other economically important organisms. [ABSTRACT FROM AUTHOR]

Summary: The LRK10‐like receptor kinases (LRK10L‐RLKs) are ubiquitously present in higher plants, but knowledge of their expression and function is still limited. Here, we report expression and functional analysis of TtdLRK10L‐1, a typical LRK10L‐RLK in durum wheat (Triticum turgidum L. ssp. durum). The introns of TtdLRK10L‐1 contained multiple kinds of predicted cis‐elements. To investigate the potential effect of these cis‐elements on TtdLRK10L‐1 expression and function, two types of transgenic wheat lines were prepared, which expressed a GFP‐tagged TtdLRK10L‐1 protein (TtdLRK10L‐1:GFP) from the cDNA or genomic DNA (gDNA) sequence of TtdLRK10L‐1 under the native promoter. TtdLRK10L‐1:GFP expression was up‐regulated by the powdery mildew pathogen Blumeria graminis f. sp. tritici (Bgt) in both types of transgenic plants, with the scale of the elevation being much stronger in the gDNA lines. Both types of transgenic plants exhibited enhanced resistance to Bgt infection relative to wild type control. Notably, the Bgt defence activated in the gDNA lines was significantly stronger than that in the cDNA lines. Further analysis revealed that a putative MYB transcription factor binding site (MYB‐BS, CAGTTA) located in TtdLRK10L‐1 intron I was critical for the efficient expression and function of TtdLRK10L‐1 in Bgt defence. This MYB‐BS could also increase the activity of a superpromoter widely used in ectopic gene expression studies in plants. Together, our results deepen the understanding of the expression and functional characteristics of LRK10L‐RLKs. TtdLRK10L‐1 is likely useful for further dissecting the molecular processes underlying wheat defence against Bgt and for developing Bgt resistant wheat crops. [ABSTRACT FROM AUTHOR]

Summary: Gliadins are a major component of wheat seed proteins. However, the complex homoeologous Gli‐2 loci (Gli‐A2, ‐B2 and ‐D2) that encode the α‐gliadins in commercial wheat are still poorly understood. Here we analyzed the Gli‐D2 locus of Xiaoyan 81 (Xy81), a winter wheat cultivar. A total of 421.091 kb of the Gli‐D2 sequence was assembled from sequencing multiple bacterial artificial clones, and 10 α‐gliadin genes were annotated. Comparative genomic analysis showed that Xy81 carried only eight of the α‐gliadin genes of the D genome donor Aegilops tauschii, with two of them each experiencing a tandem duplication. A mutant line lacking Gli‐D2 (DLGliD2) consistently exhibited better breadmaking quality and dough functionalities than its progenitor Xy81, but without penalties in other agronomic traits. It also had an elevated lysine content in the grains. Transcriptome analysis verified the lack of Gli‐D2 α‐gliadin gene expression in DLGliD2. Furthermore, the transcript and protein levels of protein disulfide isomerase were both upregulated in DLGliD2 grains. Consistent with this finding, DLGliD2 had increased disulfide content in the flour. Our work sheds light on the structure and function of Gli‐D2 in commercial wheat, and suggests that the removal of Gli‐D2 and the gliadins specified by it is likely to be useful for simultaneously enhancing the end‐use and health‐related traits of common wheat. Because gliadins and homologous proteins are widely present in grass species, the strategy and information reported here may be broadly useful for improving the quality traits of diverse cereal crops. [ABSTRACT FROM AUTHOR]