Two functional CC-NBS-LRR proteins from rye chromosome 6RS confer differential age-related powdery mildew resistance to wheat.

Rye (Secale cereale), a valuable relative of wheat, contains abundant powdery mildew resistance (Pm) genes. Using physical mapping, transcriptome sequencing, barley stripe mosaic virus-induced gene silencing, ethyl methane sulfonate mutagenesis, and stable transformation, we isolated and validated two coiled-coil, nucleotide-binding site and leucine-rich repeat (CC-NBS-LRR) alleles, PmTR1 and PmTR3, located on rye chromosome 6RS from different triticale lines. PmTR1 confers age-related resistance starting from the three-leaf stage, whereas its allele, PmTR3, confers typical all-stage resistance, which may be associated with their differential gene expression patterns. Overexpression in Nicotiana benthamiana showed that the CC, CC-NBS, and CC-LRR fragments of PMTR1 induce cell death, whereas in PMTR3 the CC and full-length fragments perform this function. Luciferase complementation imaging and pull-down assays revealed distinct interaction activities between the CC and NBS fragments. Our study elucidates two novel rye-derived Pm genes and their derivative germplasm resources and provides novel insights into the mechanism of age-related resistance, which can aid the improvement of resistance against wheat powdery mildew.

Development of novel wheat-rye 6RS small fragment translocation lines with powdery mildew resistance and physical mapping of the resistance gene PmW6RS.

KEY MESSAGE: Novel wheat-rye 6RS small fragment translocation lines with powdery mildew resistance were developed, and the resistance gene PmW6RS was physically mapped onto 6RS-0.58-0.66-bin corresponding to 18.38 Mb in Weining rye. Rye (Secale cereale L., RR) contains valuable genes for wheat improvement. However, most of the rye resistance genes have not been successfully used in wheat cultivars. Identification of new rye resistance genes and transfer of these genes to wheat by developing small fragment translocation lines will make these genes more usable for wheat breeding. In this study, a broad-spectrum powdery mildew resistance gene PmW6RS was localized on rye chromosome arm 6RS using a new set of wheat-rye disomic and telosomic addition lines. To further study and use PmW6RS, 164 wheat-rye 6RS translocation lines were developed by 60Coγ-ray irradiation. Seedling and adult stage powdery mildew resistance analysis showed that 106 of the translocation lines were resistant. A physical map of 6RS was constructed using the 6RS translocation and deletion lines, and PmW6RS was localized in the 6RS-0.58-0.66-bin, flanked by markers X6RS-3 and X6RS-10 corresponding to the physical interval of 50.23-68.61 Mb in Weining rye genome. A total of 23 resistance-related genes were annotated. Nine markers co-segregate with the 6RS-0.58-0.66-bin, which can be used to rapidly trace the 6RS fragment carrying PmW6RS. Small fragment translocation lines with powdery mildew resistance were backcrossed with wheat cultivars, and 39 agronomically acceptable homozygous 6RS small fragment translocation lines were obtained. In conclusion, this study not only provides novel gene source and germplasms for wheat resistance breeding, but also laid a solid foundation for cloning of PmW6RS.

Genome-wide identification and functional characterization of wheat Brassinazole-resistant transcription factors in response to abiotic stresses and stripe rust infection.

Brassinazole-resistant (BZR) transcription factors (TFs) are key players in brassinolides (BRs) signaling pathway, which is widely involved in regulating plant growth and development, as well as in plant responding to a variety stresses. Despite their critical roles, little is known about BZR TFs in wheat. In this study, we performed genome-wide analysis of BZR gene family from wheat genome, and 20 TaBZRs were identified. Based on the phylogenetic relationships of TaBZR and BZRs from rice and Arabidopsis, all BZR genes were clustered into four groups. The intron-exon structural patterns and conserved protein motifs of TaBZRs showed high group specificity. TaBZR5, 7, and 9 were significantly induced after salt, drought treatment, and stripe rust infection. However, TaBZR16, which was significantly upregulated under NaCl application, was not expressed during wheat-stripe rust fungus interaction. These results indicated that BZR genes in wheat play different roles in response to various stresses. The results of this study will lay a foundation for further in-depth functional studies of TaBZRs and will provide information for the breeding and genetic improvement of wheat against drought and salt stresses.

Development and Identification of a Wheat-Rye Breeding Line for Harmonious Improvement Between Powdery Mildew Resistance and High Yield Potential.

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is a devastating disease that seriously threatens wheat yield and quality. To control this disease, host resistance is the preferred measure. However, wheat breeding is a complex process with elusive exchange and recombination of the traits from their parents. Increased resistance often leads to a decline in other key traits, such as yield and quality. Developing breakthrough germplasms with harmonious powdery mildew resistance and other key breeding traits is attractive in wheat breeding. In this study, we developed an ideal wheat breeding line AL46 that pyramided its hexaploid triticale parent-derived desirable yield traits and its wheat parent-derived powdery mildew resistance gene Pm2. Sequential genomic in situ hybridization (GISH), multicolor GISH, multicolor fluorescence in situ hybridization, and molecular marker analyses revealed that AL46 was a wheat-rye T1RS·1BL translocation line. Genetic analysis combined with function marker detection and sequence alignment were used to confirm that AL46 carried the Pm2 gene. Then, we evaluated the powdery mildew resistance and comprehensive traits of AL46, and just as we designed, AL46 showed harmonious powdery mildew resistance with some key breeding traits. This study not only developed an ideal wheat germplasm resource but also provided a successful example for pyramiding breeding, which could be a promising direction for wheat improvement in the future.

Identification of a Wheat Powdery Mildew Dominant Resistance Gene in the Pm5 Locus for High-Throughput Marker-Assisted Selection.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), poses a severe threat to wheat yield and quality worldwide. Rapid identification and the accurate transference of effective resistance genes are important to the development of resistant cultivars and the sustainable control of this disease. In the present study, the wheat line AL11 exhibited high levels of resistance to powdery mildew at both the seedling and adult plant stages. Genetic analysis of the AL11 × 'Shixin 733' mapping population revealed that its resistance was controlled by a single dominant gene, tentatively designated PmAL11. Using bulked segregant RNA-Seq and molecular marker analysis, PmAL11 was mapped to the Pm5 locus on chromosome 7B where it cosegregated with the functional marker Pm5e-KASP. Sequence alignment analysis revealed that the Pm5e-homologous sequence in AL11 was identical to the reported recessive gene Pm5e in wheat landrace 'Fuzhuang 30'. It appears that PmAL11 was most probably Pm5e, but it was mediated by a dominant inheritance pattern, so it should provide a valuable resistance resource for both genetic study and wheat breeding. To efficiently use and trace PmAL11 in breeding, a new kompetitive allele-specific PCR marker AL11-K2488 that cosegregated with this gene was developed and confirmed to be applicable in the different wheat backgrounds, thus promoting its use in the marker-assisted selection of PmAL11.

Identification of the major QTL QPm.cas-7D for adult plant resistance to wheat powdery mildew.

Developing effective and durable host plant resistance is crucial for controlling powdery mildew, a devastating disease caused by Blumeria graminis f. sp. tritici (Bgt). In the present study, we dissected the genetic basis of the adult plant resistance to powdery mildew using a recombinant inbred line (RIL) composed of 176 F9 RILs population derived from a cross between PuBing 3228 (P3228) and susceptible cultivar Gao 8901. P3228 exhibits stable adult-plant resistance to powdery mildew in the field over consecutive years. We identified two QTLs on chromosomes 7DS (QPm.cas-7D) and 1AL (QPm.cas-1A) contributed by P3228, and one QTL on 3DS (QPm.cas-3D) contributed by Gao 8901, which could explain 65.44%, 3.45%, and 2.18% of the phenotypic variances, respectively. By analyzing the annotated genes in the 1.168 Mb physical interval of the major QTL QPm.cas-7D, we locked a previously cloned adult-plant resistance gene Pm38 that was most probably the candidate gene of QPm.cas-7D. Sequence alignment analysis revealed that the candidate gene of QPm.cas-7D in P3228 was identical to the reported Pm38 sequence. Two haplotypes QPm-7D-R and QPm-7D-S were identified in the whole Pm38 genomic regions between P3228 and Gao 8901. To apply QPm.cas-7D in wheat breeding, we developed a kompetitive allele-specific PCR (KASP) marker Kasp5249 that is closely linked with these haplotypes. It is worth mentioning that the QPm-7D-R haplotype significantly decreased TKW and underwent negative selection for higher yields in China wheat breeding. In this study, we identified a major QTL QPm.cas-7D and revealed the relationship between its resistance and yield, which could be beneficial for further applications in wheat disease resistance and high-yield breeding.

Molecular Cytogenetic Identification of a New Wheat-Rye 6R Addition Line and Physical Localization of Its Powdery Mildew Resistance Gene.

Rye (Secale cereale L.), a naturally cross-pollinating relative of wheat, is a tertiary gene donor and of substantial value in wheat improvement. Wheat powdery mildew is caused by Blumeria graminis f. sp. tritici (Bgt), which seriously affects yield and quality worldwide. Identifying and transferring new, effective resistance genes against powdery mildew from rye is important for wheat breeding. The current study developed a wheat-rye line YT2 resistant to powdery mildew by crossing, backcrossing, and self-pollination for multiple generations between octoploid triticale 09R2-100 and common wheat cultivar Shixin 616. YT2 was confirmed to be a 6R disomic addition and T1RS⋅1BL translocation line by genomic in situ hybridization (GISH), multicolor fluorescence in situ hybridization (mc-FISH), multicolor-GISH (mc-GISH), and molecular marker analyses. Disease responses to different Bgt isolates and genetic analysis showed that the powdery mildew resistance gene of YT2 was derived from the rye chromosome 6R of 09R2-100, which differed from the previously reported Pm genes from rye including Pm20 on 6RL. Resistance phenotype of different translocation lines and deletion lines derived from YT2 combined with newly developed 6RL-specific markers analysis suggested that the powdery mildew resistance gene of YT2 was localized to the region in chromosome 6RL: 890.09-967.51 Mb and flanked by markers XM189 and X4M19, corresponding to the reference genome of Weining rye. Therefore, YT2 could be used as a promising bridging parent for wheat disease resistance improvement.

A Novel Wheat-Rye 2R (2D) Disomic Substitution Line Pyramids Two Types of Resistance to Powdery Mildew.

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is a devastating disease of wheat that seriously affects yield and quality worldwide. Because of the extensive growth of wheat cultivars with homogeneous genetic background, exploring novel resistant resources from wheat relatives has become important for increasing the genetic diversity of wheat. Rye (Secale cereale) is a wheat relative possessing abundant resistance genes because of its high variation. Wheat line AL69, resistant to powdery mildew, was developed by crossing, backcrossing, and self-pollination for multiple generations between hexaploid triticale Zhongsi 237 and common wheat cultivar Zimai 17. Through genomic in situ hybridization (GISH) and multicolor fluorescence in situ hybridization (FISH), nondenaturing FISH, multicolor GISH, and selection with specific molecular markers, AL69 was determined to be a wheat-rye 2R (2D) disomic substitution line. Testing with different B. graminis f. sp. tritici isolates and genetic analysis showed that the all-stage resistance (also called seedling resistance) of AL69 was conferred by the cataloged powdery mildew resistance gene Pm4b derived from Zimai 17, and its adult-plant resistance was derived from the alien chromosome 2R of Zhongsi 237, which was found to be different from the previously reported rye-derived Pm genes, including Pm7 on 2RL. In addition, AL69 showed improved spike number per plant, spike length, fertile spikelet number per spike, kernel number per spike, and grain yield per plant compared with its wheat parent Zimai 17. An elite line S251 combining powdery mildew resistance with excellent agronomic performance was selected from the progenies of AL69 and wheat cultivar Jimai 22. Therefore, AL69 has two types of resistance genes to powdery mildew and improved agronomic traits through pyramiding and thus can be used as a promising genetic stock for wheat breeding.

Nontraditional Luminescent Molecular Aggregates Encapsulated by Wormlike Silica Nanoparticles for Latent Fingerprint Detection.

The phenomenon of nontraditional luminescence has attracted wide attention and curiosity of researchers due to its inexplicable photoluminescence paradigm without aromatic or extended π-systems. The present work puts forward a neotype of a light-emitting nitrogenous small molecule, namely, N-stearoyl-hydroxyproline (L-C16-Hyp), which could emit weak light in aggregation states through the restriction mechanism of intramolecular motion, exhibiting properties comparable to those of AIEgens. Using these molecular aggregates as anionic surfactant micelles to incorporate within the silica matrix, we prepared fluorescent nanoparticles (FL-NPs) by a one-pot method for expedient visualization of latent fingerprints (LFPs). The FL-NPs exhibit an excitation range from 335 to 365 nm, resulting in nontraditional luminescence observed between 410 and 440 nm. The enhanced luminescent FL-NPs may derive from the collective entities or assemblies of restricted L-C16-Hyp, which can be reasonably explicated by an effect termed as cluster-triggered emission (CTE). Theoretical calculations demonstrated that this luminescence pattern belongs to partial charge transfer, which is mainly attributed to the close interaction between the tertiary amino and adjacent carboxyl in the L-C16-Hyp structure. Moreover, some merits of FL-NPs, such as wormlike nanomorphology, stable photophysical properties, low toxicity, great adhesion to multiple substrates, easy to get raw material, an inexpensive, simple process, and rapid detection without any further modification or assistance, provide the feasibility of efficacious LFP detection. Overall, this study will provide insights into the design and application of luminescent materials with unconventional groups.

qPCR and loop mediated isothermal amplification for rapid detection of Ustilago tritici.

Loose smut of wheat caused by the basidiomycete fungus Ustilago tritici, a seed-borne disease, is difficult to control because of the expanse of wheat planting area and difficulty in pathogen detection. In this study, real-time fluorescence quantitative PCR (qPCR) and loop-mediated isothermal amplification (LAMP) assays are used to rapidly amplify the DNA of U. tritici. Five pairs of primers for qPCR and two series primers for LAMP were designed. Primarily, the specificity of the primer was assessed by using genomic DNA of U. tritici, Fusarium graminearum, Blumeria graminis, Rhizoctonia cerealis, Puccinia striiformis, Bipolaris sorokiniana, and Alternaria solani as templates. Further, the amplification systems were optimized. Finally, the sensitivity of qPCR and LAMP assays were evaluated. The results showed that the primer Y-430 F/R, Y-307 F/R, Y-755 F/R, and Y-139 F/R for qPCR and primers L-139 and L-988 for LAMP could be used for U. tritici detection. In the sensitivity test, the detection limit of qPCR assay was identified as 10 pg µL-1 of genomic DNA, the detection limit for LAMP assay was 100 fg µL-1. We successfully performed qPCR and LAMP assays on wheat loose smut wheat samples. This paper establishes two methods for U. tritici detection, which can be used for diagnosis of wheat loose smut in the laboratory and in the field.

Protective effect of 4-Methoxy benzyl alcohol on the neurovascular unit after cerebral ischemia reperfusion injury.

OBJECTIVE: Cerebral ischemia reperfusion injury (CIRI) is a major cause of ischemic stroke (IS) deterioration. Considering the intricate mechanism of the pathological process of CIRI, most drugs only work on one target. The neurovascular unit (NVU) puts forward the concept of neuroprotection from nerve protection to global stabilization. The NVU plays an important role in maintaining the brain microenvironment. This would promote neuronal survival and overall neurological recovery, which would likely lead to the reduction of mortality rate. Previous studies have shown that 4-methoxy benzyl alcohol (4-MA) ameliorated neurological score and cerebral infarct volume and reduced the concentration of Evans blue (EB) in brain tissue. In this research, we investigated the effects of 4-MA on NVU microenvironment improvement in rats impaired by middle cerebral artery occlusion/reperfusion (MCAO/R). METHODS: First, we established a rat model of middle cerebral artery occlusion (MCAO) so as to use Western blot analysis, immunofluorescence and transmission electron microscopy (TEM) evaluating the NVU's protection of 4-MA. Then we established a primary cortical neuron model of oxygen glucose deprivation and re-oxygenation (OGD/R) with the objective of identifying whether 4-MA exhibited anti-oxidant and anti-apoptotic effects on neurons. RESULTS: NVU ultra structural changes were improved by 4-MA. Immunofluorescence and western blot showed that 4-MA protected NVUs through enhancement of the expression of the symbolic neuronal proteins Microtubule Associated Protein-2(MAP-2), and attenuation of protein expression of Asy symbolic protein Glial Fibrillary Acidic Protein(GFAP). Furthermore, in the OGD/R model of I/R injury in vitro, 4-MA significantly increased Superoxide dismutase(SOD), Nitric Oxide(NO), B-cell lymphoma-2(Bcl-2), decreased Bcl-2-Associated X(Bax) and increased Bcl-2/Bax. CONCLUSION: 4-MA can play the role of anti-ischemic stroke drug by ameliorating the microenvironment of NVUs while its neuroprotective effects will contribute towards the inhibition of the antioxidant and anti-apoptotic activities.

Neuroprotective effect of ethyl acetate extract from gastrodia elata against transient focal cerebral ischemia in rats induced by middle cerebral artery occlusion.

OBJECTIVE: To investigate the protective effect of neuroprotection against transient focal cerebral ischemia of the extract from Tianma (Rhizoma Gastrodiae) and the possible mechanisms underlying the action. METHODS: Cerebral ischemia-reperfusion injury was induced through middle cerebral artery occlusion (MCAO). Adult male Sprague-Dawley rats were randomly divided into four groups: sham-operated, ischemia-reperfusion model, 102.6 mg/kg extract treated and 11.4 mg/kg extract treated groups. The extract was prepared from gastrodia elata with ethyl acetate. The effect of the extract tested on rat neurological deficits and Cerebral index, cerebral infarct volume, brain injury, terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and B-cell lymphoma-2 (Bcl-2) positive cells. RESULTS: The extract was able to reduce neurological scores, cerebral index and cerebral infarction rate. The brain injury was also relieved by the extract. The results of immunofluorescence staining analysis indicated that the extract increased the expression of Bcl-2 and reduced TUNEL-positive cells significantly in the extract treated groups. CONCLUSION: These results suggested that the extract relieved ischemic injury induced by transient focal cerebral ischemia in rats, and this neuroprotective effect might be partially due to the attenuated apoptosis pathway.