Ruiqi Zhang

Pm55, a developmental-stage and tissue-specific powdery mildew resistance gene introgressed from Dasypyrum villosum into common wheat

Key messagePowdery mildew resistance gene Pm55 was physically mapped to chromosome arm 5VS FL 0.60–0.80 of Dasypyrum villosum. Pm55 is present in T5VS·5AL and T5VS·5DL translocations, which should be valuable resources for wheat improvement.AbstractPowdery mildew caused by Blumeria graminis f. sp. tritici is a major wheat disease worldwide. Exploiting novel genes effective against powdery mildew from wild relatives of wheat is a promising strategy for controlling this disease. To identify novel resistance genes for powdery mildew from Dasypyrum villosum, a wild wheat relative, we evaluated a set of Chinese Spring-D. villosum disomic addition and whole-arm translocation lines for reactions to powdery mildew. Based on the evaluation data, we concluded that the D. villosum chromosome 5V controls post-seedling resistance to powdery mildew. Subsequently, three introgression lines were developed and confirmed by molecular and cytogenetic analysis following ionizing radiation of the pollen of a Chinese Spring-D. villosum 5V disomic addition line. A homozygous T5VS·5AL translocation line (NAU421) with good plant vigor and full fertility was further characterized using sequential genomic in situ hybridization, C-banding, and EST-STS marker analysis. A dominant gene permanently named Pm55 was located in chromosome bin 5VS 0.60–0.80 based on the responses to powdery mildew of all wheat-D. villosum 5V introgression lines evaluated at both seeding and adult stages. This study demonstrated that Pm55 conferred growth-stage and tissue-specific dependent resistance; therefore, it provides a novel resistance type for powdery mildew. The T5VS·5AL translocation line with additional softness loci Dina/Dinb of D. villosum provides a possibility of extending the range of grain textures to a super-soft category. Accordingly, this stock is a new source of resistance to powdery mildew and may be useful in both resistance mechanism studies and soft wheat improvement.

Molecular and cytogenetic characterization of a small alien-segment translocation line carrying the softness genes of Haynaldia villosa

The wheat-alien small segment translocation (SAST) lines carrying the beneficial genes from wild species are useful genetic stocks for wheat improvement. In this study, to introduce the grain hardness-related genes of Haynaldia villosa (L.) Schur. into common wheat (Triticum aesitivum L.), the mature female gametes of whole-arm wheat--H. villosa translocation line T5VS·5DL was irradiated by 60CO-γ ray to develop SAST lines involving 5VS. Among the BC2F2 population, six homozygous SAST lines with different fragment sizes of 5VS were identified by GISH, and the exact fragment sizes were further defined using four 5VS-specific markers and four Ha gene-based markers. The results showed that five lines (NAU5VS-1 to NAU5VS-5) carried the softness gene Dina/Dinb of H. villosa, and that NAU5VS-5 had the smallest alien translocation segment, identified to be a 5VS-6AS·6AL terminal translocation. The translocation chromosome 5VS-6AS·6AL was proved to be stably inherited to the successive generations. In the BC3F2 generation, the individuals having the homozygous 5VS-6AS·6AL translocation chromosomes all showed soft grain texture, with an approximately 50% reduction in the SKCS hardness index compared with that of their backcrossing parent. Both the 5VS-6AS·6AL translocation line and the molecular markers developed in this study will be valuable in wheat breeding for soft grain quality improvement.

Two members of TaRLK family confer powdery mildew resistance in common wheat

BackgroundPowdery mildew, caused by Blumeria graminearum f.sp. tritici (Bgt), is one of the most severe fungal diseases of wheat. The exploration and utilization of new gene resources is the most effective approach for the powdery mildew control.ResultsWe report the cloning and functional analysis of two wheat LRR-RLKs from T. aestivum c.v. Prins- T. timopheevii introgression line IGV1-465, named TaRLK1 and TaRLK2, which play positive roles in regulating powdery mildew resistance in wheat. The two LRR-RLKs contain an ORF of 3,045 nucleotides, encoding a peptide of 1014 amino acids, with seven amino acids difference. Their predicted proteins possess a signal peptide, several LRRs, a trans-membrane domain, and a Ser/Thr protein kinase domain. In response to Bgt infection, the TaRLK1/2 expression is up-regulated in a developmental-stage-dependent manner. Single-cell transient over-expression and gene-silencing assays indicate that both genes positively regulate the resistance to mixed Bgt inoculums. Transgenic lines over-expressing TaRLK1 or TaRLK2 in a moderate powdery mildew susceptible wheat variety Yangmai 158 led to significantly enhanced powdery mildew resistance. Exogenous applied salicylic acid (SA) or hydrogen peroxide (H2O2) induced the expression of both genes, and H2O2 had a higher accumulation at the Bgt penetration sites in RLK over-expression transgenic plants, suggesting a possible involvement of SA and altered ROS homeostasis in the defense response to Bgt infection. The two LRR-RLKs are located in the long arm of wheat chromosome 2B, in which the powdery mildew resistance gene Pm6 is located, but in different regions.ConclusionsTwo members of TaRLK family were cloned from IGV1-465. TaRLK1 and TaRLK2 contribute to powdery mildew resistance of wheat, providing new resistance gene resources for wheat breeding.

Characterization of a Putative New Semi-Dominant Reduced Height Gene, Rht_NM9, in Wheat (Triticum aestivum L.).

Plant height is an important agronomic trait in cereal crops, and can affect both plant architecture and grain yield. New dwarfing genes are required for improving the genetic diversity of wheat. In this study, a novel dwarf mutant, NM9, was created by treating seeds of the wheat variety NAU9918 with ethyl methanesulfonate (EMS). NM9 showed obvious phenotypic changes, which were distinct from those caused by other dwarfing genes, especially the reduced plant height, increased effective tiller number, and elongated spike and grain length. The reduced plant height in NM9 was attributable to a semi-dominant dwarfing gene Rht_NM9, which was flanked by two closely linked SNP markers, SNP34 and SNP41, covering an 8.86-Mb region on the chromosome arm 2AS. The results of gibberellic acid (GA) sensitivity evaluation, comparative genomics analysis and allelism test indicated that Rht_NM9 was neither allelic to Rht7 and Rht21 nor homoeoallelic to Rht8, so Rht_NM9 was proposed to be a new dwarfing locus on the homoeologous group 2 chromosomes of wheat. Rht_NM9 has a negative effect on plant height and positive effects on effective tiller number and grain size, thus, Rht_NM9 could be used for elucidating the mechanisms underlying plant architecture and grain development.

Overexpression of ERF1-V from Haynaldia villosa Can Enhance the Resistance of Wheat to Powdery Mildew and Increase the Tolerance to Salt and Drought Stresses

The APETALA 2/Ethylene-responsive element binding factor (AP2/ERF) transcription factor gene family is widely involved in the biotic and abiotic stress regulation. Haynaldia villosa (VV, 2n = 14), a wild species of wheat, is a potential gene pool for wheat improvement. H. villosa confers high resistance to several wheat diseases and high tolerance to some abiotic stress. In this study, ERF1-V, an ethylene-responsive element-binding factor gene of the AP2/ERF transcription factor gene family from wild H. villosa, was cloned and characterized. Sequence and phylogenetic analysis showed that ERF1-V is a deduced B2 type ERF gene. ERF1-V was first identified as a Blumeria graminis f. sp. tritici (Bgt) up-regulated gene, and later found to be induced by drought, salt and cold stresses. In responses to hormones, ERF1-V was up-regulated by ethylene and abscisic acid, but down-regulated by salicylic acid and jasmonic acid. Over expression of ERF1-V in wheat could improve resistance to powdery mildew, salt and drought stress. Chlorophyll content, malondialdehyde content, superoxide dismutase and peroxidase activity were significantly differences between the recipient Yangmai158 and the transgenic plants following salt treatment. Furthermore, the expression levels of some stress responsive genes were differences after drought or salt treatments. Although ERF1-V was activated by the constitutive promoter, the agronomic traits, including flowering time, plant height, effective tiller number, spikelet number per spike and grain size, did not changed significantly. ERF1-V is a valuable gene for wheat improvement by genetic engineering.

A disulphide isomerase gene (PDI-V) from Haynaldia villosa contributes to powdery mildew resistance in common wheat.

In this study, we report the contribution of a PDI-like gene from wheat wild relative Haynaldia villosa in combating powdery mildew. PDI-V protein contains two conserved thioredoxin (TRX) active domains (a and a′) and an inactive domain (b). PDI-V interacted with E3 ligase CMPG1-V protein, which is a positive regulator of powdery mildew response. PDI-V was mono-ubiquitinated by CMPG1-V without degradation being detected. PDI-V was located on H. villosa chromosome 5V and encoded for a protein located in the endoplasmic reticulum. Bgt infection in leaves of H. villosa induced PDI-V expression. Virus induced gene silencing of PDIs in a T. durum-H. villosa amphiploid compromised the resistance. Single cell transient over-expression of PDI-V or a truncated version containing the active TXR domain a decreased the haustorial index in moderately susceptible wheat cultivar Yangmai 158. Stable transgenic lines over-expressing PDI-V in Yangmai 158 displayed improved powdery mildew resistance at both the seedling and adult stages. By contrast over-expression of point-mutated PDI-VC57A did not increase the level of resistance in Yangmai 158. The above results indicate a pivotal role of PDI-V in powdery mildew resistance and showed that conserved TRX domain a is critical for its function.

Cereal cyst nematode resistance gene CreV effective against Heterodera filipjevi transferred from chromosome 6VL of Dasypyrum villosum to bread wheat

Cereal cyst nematodes (CCN) are a global economic problem for cereal production. Heterodera filipjevi is one of the most commonly identified and widespread CCN species found in many wheat production regions of the world. Transferring novel genes for resistance to H. filipjevi from wild relatives of wheat is a promising strategy for protection of wheat crops. A set of wheat–Dasypyrum villosum chromosome addition lines, T6V#4S·6AL translocation lines and their donor parental lines were tested for their response to the nematode. D. villosum and wheat–D. villosum disomic addition line DA6V#4 were resistant. As T6V#4S·6AL translocation lines were susceptible, resistance was presumed to be located on chromosome 6V#4L. The objective of this study was to produce and characterize wheat–6V#4L translocations and confirm the chromosome location of the resistance. Introgression lines T6V#4L·6AS, T6V#4L-4BL·4BS and DT6V#4L were developed and subjected to molecular cytogenetic analysis. These and four additional wheat–6V#4 introgression lines were tested for response to H. filipjevi in the greenhouse. The results indicated that introgression lines DA6V#4, T6V#4L·6AS, T6V#4L-4BL·4BS, T6V#4L·6V#4S-7BS and DT6VL#4 had higher levels of H. filipjevi resistance than their recurrent parent. However, Del6V#4L-1 and translocation line T6V#4S·6AL were equally susceptible to wheat cv. Chinese Spring. The CCN resistance gene, temporarily named CreV, was therefore physically mapped to chromosome arm 6V#4L FL 0.80–1.00. Translocation chromosomes T6V#4L·6AS transferred to a modern wheat cv. Aikang 58 with its co-dominant molecular markers could be utilized as a novel germplasm for CCN resistance breeding in wheat.

Agronomic characterization and genetic analysis of the supernumerary spikelet in tetraploid wheat (Triticum turgidum L.)

Abstract The supernumerary spikelets (SS) characters of tetraploid wheat (Triticum turgidum L.) resulting in more spikelets and kernels per spike, thus enhancing sink capacity may contribute to potential wheat yield improvement. In order to investigate the effect of different SS types on agronomic characters and understand the genetic base of SS phenotype in tetraploid wheat, near isogenic lines (NILs), bh-50 with normal spikelets (NS), bh-51 with four-rowed spikelets (FRS), bh-52 with short-ramified spikelets (SRS), and bh-53 with long-ramified spikelets (LRS) in a Triticum durum cv. ZY1286 genetic background were developed by continuous backcrossing. Agronomic characters showed that the SS phenotype lines, bh-51, bh-52 and bh-53 have significant increase in the number of spikelets and grains per spike compared with the NS phenotype line bh-50 (P

TaNAC6s are involved in the basal and broad-spectrum resistance to powdery mildew in wheat

NACs are important transcriptional factors involved in growth and development as well as responses to abiotic and biotic stresses in plants. In this study, TaNAC6 was identified as a differentially expressed gene between two lines with broad-spectrum resistance to powdery mildew, NAU9918 and OEStpk-V, and their corresponding susceptible isogenic lines, SM-1 and Yangmai158, after Bgt inoculation by transcriptome analysis. Then, three homoeologous genes of TaNAC6 were cloned and named as TaNAC6-A, TaNAC6-B and TaNAC6-D, respectively. Each member of TaNAC6s was subcellular localized to the nucleus and displayed the transcriptional activation activity. However, the responses of them to pathogens and phytohormones were different. Transient overexpression of each TaNAC6 reduced the haustorium index of Yangmai158, and stable transformation of TaNAC6-A enhanced its resistance against Bgt, implying that TaNAC6s play important roles in basal resistance. Silencing of TaNAC6s compromised the resistance of OEStpk-V and NAU9918 suggesting that TaNAC6s play positive roles in the broad-spectrum resistance against Bgt. TaNAC6s might be induced by JA and then feedback regulate the JA pathway leading to improved resistance to Bgt. The role of TaNAC6s and their orthologous genes HvNAC6 and ATAF1 in the powdery mildew resistance implied these NAC6 genes share a common signal pathway across species.

A malectin-like/leucine-rich repeat receptor protein kinase gene, RLK-V, regulates powdery mildew resistance in wheat: A malectin-like/LRR receptor protein kinase gene

Pattern recognition receptors (PRRs) can trigger plant immunity through the recognition of pathogen-associated molecular patterns. In this study, we report that a malectin-like/leucine-rich repeat receptor protein kinase gene, RLK-V, from Haynaldia villosa putatively acts as a PRR to positively regulate powdery mildew resistance caused by Blumeria graminis f. sp. tritici (Bgt) in wheat. RLK-V has two alternatively spliced transcripts corresponding to an intact RLK-V1.1 and a truncated RLK-V1.2 caused by intron retention. Expression analysis showed that both transcripts could be up-regulated by Bgt in resistant materials, whereas the functional RLK-V1.1 was expressed only after Bgt inoculation. Promoter activity assays indicated that RLK-V could respond to Bgt even in susceptible wheat. Silencing of RLK-V in Pm21-carrying resistant materials resulted in compromised resistance to Bgt. In addition, over-expression of RLK-V1.1 in Pm21-lacking susceptible Yangmai158 and SM-1 by single-cell transient expression and stable transformation in Yangmai158 could improve powdery mildew resistance. We propose that RLK-V regulates basal resistance to powdery mildew, which is also required for broad-spectrum resistance mediated by the Pm21 gene. Over-expression of RLK-V1.1 could trigger cell death in Nicotiana benthamiana, and RLK-V1.1 transgenic wheat accumulated more reactive oxygen species and displayed a stronger hypersensitive response than did the recipient, which led to enhanced Bgt resistance. However, constitutive activation of RLK-V1.1 resulted in the abnormal growth of transgenic plants.