Recently, the CRISPR/Cas9 system has been widely used to precisely edit plant genomes. Due to the difficulty in Agrobacterium-mediated genetic transformation of wheat, the reported applications in CRISPR/Cas9 system were all based on the biolistic transformation. In the present study, we efficiently applied targeted mutagenesis in common wheat (Triticum aestivum L.) protoplasts and transgenic T0 plants using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Seven target sites in three genes (Pinb, waxy and DA1) were selected to construct individual expression vectors. The activities of the sgRNAs were evaluated by transforming the constructed vectors into wheat protoplasts. Mutations in the targets were detected by Illumina sequencing. Genome editing, including insertions or deletions at the target sites, was found in the wheat protoplast cells. The highest mutation efficiency was 6.8% in the DA1 gene. The CRISPR/Cas9 binary vector targeting the DA1 gene was then transformed into common wheat plants by Agrobacterium tumefaciens-mediated transformation, resulting in efficient target gene editing in the T0 generation. Thirteen mutant lines were generated, and the mutation efficiency was 54.17%. Mutations were found in the A and B genomes of the transgenic plants but not in the D genome. In addition, off-target mutations were not detected in regions that were highly homologous to the sgRNA sequences. Our results showed that our Agrobacterium-mediated CRISPR/Cas9 system can be used for targeted mutations and facilitated wheat genetic improvement.

Biotic stresses do damage to the growth and development of plants, and yield losses for some crops. Confronted with microbial infections, plants have evolved multiple defense mechanisms, which play important roles in the never-ending molecular arms race of plant–pathogen interactions. The complicated defense systems include pathogen-associated molecular patterns (PAMP) triggered immunity (PTI), effector triggered immunity (ETI), and the exosome-mediated cross-kingdom RNA interference (CKRI) system. Furthermore, plants have evolved a classical regulation system mediated by miRNAs to regulate these defense genes. Most of the genes/small RNAs or their regulators that involve in the defense pathways can have very rapid evolutionary rates in the longitudinal and horizontal co-evolution with pathogens. According to these internal defense mechanisms, some strategies such as molecular switch for the disease resistance genes, host-induced gene silencing (HIGS), and the new generation of RNA-based fungicides, have been developed to control multiple plant diseases. These broadly applicable new strategies by transgene or spraying ds/sRNA may lead to reduced application of pesticides and improved crop yield.

The CRISPR/Cas9 system has been successfully used in hexaploid wheat. Although it has been reported that the induced mutations can be passed to the next generation, gene editing and transmission patterns in later generations still need to be studied. In this study, we demonstrated that the CRISPR/Cas9 system could achieve efficient mutagenesis in five wheat genes via Agrobacterium-mediated transformation of an sgRNA targeting the D genome, an sgRNA targeting both the A and B homologues and three tri-genome guides targeting the editing of all three homologues. High mutation rates and putative homozygous or biallelic mutations were observed in the T0 plants. The targeted mutations could be stably inherited by the next generation, and the editing efficiency of each mutant line increased significantly across generations. The editing types and inheritance of targeted mutagenesis were similar, which were not related to the targeted subgenome number. The presence of Cas9/sgRNA could cause new mutations in subsequent generations, while mutated lines without Cas9/sgRNA could retain the mutation type. Additionally, off-target mutations were not found in sequences that were highly homologous to the selected sgRNA sequences. Overall, the results suggested that CRISPR/Cas9-induced gene editing via Agrobacterium-mediated transformation plays important roles in wheat genome engineering.

Background: PhasiRNAs (phased secondary siRNAs) play important regulatory roles in the development processes and biotic or abiotic stresses in plants. Some of phasiRNAs involve in the reproductive development in grasses, which include two categories, 21-nt (nucleotide) and 24-nt phasiRNAs. They are triggered by miR2118 and miR2275 respectively, in premeiotic and meiotic anthers of rice, maize and other grass species. Wheat (Triticum aestivum) with three closely related subgenomes (subA, subB and subD), is a model of allopolyploid in plants. Knowledge about the role of phasiRNAs in the inflorescence development of wheat is absent until now, and the evolution ofPHASloci in polyploid plants is also unavailable.Results: Using 261 small RNA expression datasets from various tissues, a batch ofPHAS(phasiRNA precursors) loci were identified in the young spike of wheat, most of which were regulated by miR2118 and miR2275 in their target site regions. Dissection ofPHASand their trigger miRNAs among the diploid (AA and DD), tetraploid (AABB) and hexaploid (AABBDD) genomes ofTriticumindicated that distribution ofPHASloci were dominant randomly in local chromosomes, while miR2118 was dominant only in the subB genome. The diversity ofPHASloci in the three subgenomes of wheat and their progenitor genomes (AA, DD and AABB) suggested that they originated or diverged at least before the occurrence of the tetraploid AABB genome. The positive correlation between thePHASloci or the trigger miRNAs and the ploidy of genome indicated the expansion of genome was the major drive force for the increase ofPHASloci and their trigger miRNAs inTriticum. In addition, the expression profiles of thePHAStranscripts suggested they responded to abiotic stresses such as cold stress in wheat.Conclusions: Altogether, non-coding phasiRNAs are conserved transcriptional regulators that display quick plasticity inTriticumgenome. They may be involved in reproductive development and abiotic stress in wheat. It could be referred to molecular research on male reproductive development inTriticum.

Disease resistance genes encoding proteins with nucleotide binding sites and Leucine-Rich Repeat (NB-LRR) domains include many members involved in the effector-triggered immunity pathway in plants. The transcript levels of these defense genes are negatively regulated by diverse microRNAs (miRNAs) in angiosperms and gymnosperms. In wheat, using small RNA expression datasets and degradome datasets, we identified five miRNA families targeting NB-LRR defense genes in monocots, some of which arose in the Triticeae species era. These miRNAs regulate different types of NB-LRR genes, most of them with coil-coiled domains, and trigger the generation of secondary small interfering RNAs (siRNA) as a phased pattern in the target site regions. In addition to acting in response to biotic stresses, they are also responsive to abiotic stresses such as heat, drought, salt, and light stress. Their copy number and expression variation in Triticeae suggest a rapid birth and death frequency. Altogether, non-conserved miRNAs as conserved transcriptional regulators in gymnosperms and angiosperms regulating the disease resistance genes displayed quick plasticity including the variations of sequences, gene copy number, functions, and expression level, which accompanied with NB-LRR genes may be tune-regulated to plants in natural environments with various biotic and abiotic stresses.

Plant cell walls are dynamic structural matrices that provide mechanical support while regulating growth, transport, environmental sensing, and defense. Their properties emerge from the coordinated biosynthesis, deposition, assembly, and remodeling of cellulose, hemicelluloses, pectins, lignin, and specialized wall polymers, yet how wall architecture is coupled to integrity surveillance and whole-plant physiology remains incompletely understood. Here, we synthesize current knowledge of plant cell wall classification, major polymer biosynthesis and assembly, cell wall integrity signaling, and physiological functions. We compare primary, secondary, and specialized walls and discuss how differences in their composition and organization confer distinct mechanical and functional properties. We then examine how wall-derived oligosaccharides, apoplastic peptides, receptor-like kinases, Ca²⁺ signaling, reactive oxygen species, cytoskeletal dynamics, and hormonal crosstalk translate wall perturbations into adaptive responses. Viewed in an integrated physiological context, wall composition, architecture, and remodeling shape anisotropic growth, morphogenesis, tissue repair, xylem hydraulics, ion homeostasis, abiotic stress adaptation, and pathogen defense. Collectively, current evidence supports a view of the cell wall as an active regulatory interface rather than a passive scaffold. Finally, we highlight unresolved questions concerning mechanochemical sensing, spatial heterogeneity, and growth–defense trade-offs, and discuss how advances in spatial omics, live-cell imaging, biomechanics, genome editing, and computational modeling may help resolve how wall properties are regulated across cells, tissues, and environmental contexts.