Wheat powdery mildew resistance gene Pm13 encodes a mixed lineage kinase domain-like protein.

Wheat powdery mildew is one of the most destructive diseases threatening global wheat production. The wild relatives of wheat constitute rich sources of diversity for powdery mildew resistance. Here, we report the map-based cloning of the powdery mildew resistance gene Pm13 from the wild wheat species Aegilops longissima. Pm13 encodes a mixed lineage kinase domain-like (MLKL) protein that contains an N-terminal-domain of MLKL (MLKL_NTD) domain in its N-terminus and a C-terminal serine/threonine kinase (STK) domain. The resistance function of Pm13 is validated by mutagenesis, gene silencing, transgenic assay, and allelic association analyses. The development of introgression lines with significantly reduced chromosome segments of Ae. longissima encompassing Pm13 enables widespread deployment of this gene into wheat cultivars. The cloning of Pm13 may provide valuable insights into the molecular mechanisms underlying Pm13-mediated powdery mildew resistance and highlight the important roles of kinase fusion proteins (KFPs) in wheat immunity.

Phylogenetic analysis of wall associated kinase genes in Triticum and characterization of TaWAK7.

Wall-associated kinases (WAKs), a group of receptor-like kinases (RLKs), have been found to play important roles in defending against pathogens and in various developmental processes. However, the importance of this family in wheat remains largely unknown. Wheat powdery mildew is caused by Blumeria graminis f. sp. tritici (Bgt) which initiates infection on the cell surface and forms haustoria inside the cell, therefore, the defense to Bgt involves extracellular and subsequently intracellular signals. In this study, WAKs were identified genome-wide and phylogenetically analyzed, then a transmembrane WAK gene putatively participated in pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) and effector-triggered immunity (ETI) to Bgt was functionally and evolutionarily investigated. In total, 1,193 WAKs were identified from wheat and its Gramineae relatives. Phylogenetic analysis indicated that WAKs expanded through tandem duplication or segment duplication. TaWAK7, from chromosome 2A, was identified as a Bgt-inducible gene both in susceptible and resistant materials but showed distinct responsive patterns. Functional analysis showed that TaWAK7 was involved in both the basal and resistance (R)-gene mediated resistances. The specific gene structures and protein characteristics of TaWAK7 together with its orthologs were characterized both in subgenomes of Triticum and in the A genome of multiple wheat accessions, which revealed that TaWAK7 orthologs underwent complex evolution with frequent gene fusion and domain deletion. In addition, three cytoplasmic proteins interacting with TaWAK7 were indicated by yeast-two-hybrid and BiFC assays. Binding of TaWAK7 with these proteins could change the subcellular localization of TaWAK7 from the plasma membrane to the cytoplasm. This study provides a better understanding of the evolution of WAKs at the genomic level and TaWAK7 at the gene level, and provides useful clues for further investigation of how WAKs transmit the extracellular signals to the cytoplasm to activate defense responses.

[Nutrition diagnosis and fertilization of Pinus armandii forest in the Qinling Mountains, China]. / ç§¦å²­åŽå±±æ¾è¥å »è¯Šæ–­ä¸Žæž—åœ°æ–½è‚¥.

We examined the contents of total nitrogen (TN) and total phosphorus (TP) in needle, litter, and soil of Pinus armandii forest on the southern slope of the Qinling Mountains in Shaanxi, China. We conducted nutrition diagnosis of P. armandii forest, analyzed nutrient limitation pattern, the fertilization types and amount to increase growth, to provide support for precision fertilization and scientific cultivation of big timber of P. armandii. The results showed that the proportion of big timber (DBH>26 cm) and individuals with the potential to grow into big timber (18 cm

Development of Novel Wheat-Aegilops longissima 3S1 Translocations Conferring Powdery Mildew Resistance and Specific Molecular Markers for Chromosome 3S1.

Powdery mildew of wheat, caused by Blumeria graminis f. sp. tritici, is a destructive disease of common wheat. Cultivation of resistant varieties is the most cost-effective disease management strategy. Previous studies reported that chromosome 3Sl#2 present in Chinese Spring (CS)-Aegilops longissima 3Sl#2(3B) disomic substitution line TA3575 conferred resistance to powdery mildew. In this study, we further located the powdery mildew resistance gene(s) to the short arm of chromosome 3Sl#2 (3Sl#2S) by evaluating for B. graminis f. sp. tritici resistance of newly developed CS-Ae. longissima 3Sl#2 translocation lines. Meanwhile, TA7545, a previously designated CS-Ae. longissima 3Sl#3 disomic addition line, was reidentified as an isochromosome 3Sl#3S addition line and evaluated to confer resistance to powdery mildew, thus locating the resistance gene(s) to the short arm of chromosome 3Sl#3 (3Sl#3S). Based on transcriptome sequences of TA3575, 10 novel chromosome 3SlS-specific markers were developed, of which 5 could be used to distinguish between 3Sl#2S and 3Sl#3S derived from Ae. longissima accessions TL20 and TA1910 (TAM4) and the remaining 5 could identify both 3Sl#2S and 3Sl#3S. Also, CL897, one of five markers specific to both 3Sl#2S and 3Sl#3S, could be used to detect Pm13 located at chromosome 3Sl#1S from Ae. longissima accession TL01 in diverse wheat genetic backgrounds. The powdery mildew resistance genes on chromosomes 3Sl#2S and 3Sl#3S, the CS-Ae. longissima 3Sl#2 translocation lines, and the 3SlS-specific markers developed in this study will facilitate the transfer of B. graminis f. sp. tritici resistance genes into common wheat and provide new germplasm resources for powdery mildew resistance breeding.

Genome-wide impacts of alien chromatin introgression on wheat gene transcriptions.

Agronomic characteristics and tolerance to biotic and abiotic stresses in hexaploid wheat can be drastically improved through wheat-alien introgression. However, the transcriptional level interactions of introduced alien genes in the wheat genetic background is rarely investigated. In this study, we report the genome-wide impacts of introgressed chromosomes derived from Ae. longissima on gene transcriptions of the wheat landrace Chinese Spring. RNA-seq analyses demonstrated 5.37% and 4.30% of the genes were significantly differentially expressed (DEGs) in CS-Ae. longissima disomic 3Sl#2(3B) substitution line TA3575 and disomic 6Sl#3 addition line TA7548, respectively when compared to CS. In addition, 561 DEGs, including 413 up-regulated and 148 down-regulated or not transcribed genes, were simultaneously impacted by introgressed chromosomes 3Sl#2 and 6Sl#3, which accounts for 41.25% of the DEGs in TA3575 and 38.79% in TA7548. Seventeen DEGs, annotated as R genes, were shared by both introgression lines carrying chromosomes 3Sl#2 and 6Sl#3, which confer resistance to powdery mildew. This study will benefit the understanding of the wheat gene responses as result of alien gene(s) or chromosome intogression and the plant defense response initiated by powdery mildew resistance genes in chromosomes 3Sl#2 and 6Sl#3.

A spontaneous wheat-Aegilops longissima translocation carrying Pm66 confers resistance to powdery mildew.

KEY MESSAGE: A spontaneous Robertsonian T4SlS·4BL translocation chromosome carrying Pm66 for powdery mildew resistance was discovered and confirmed by RNA-seq, molecular marker, and in situ hybridization analyses. Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a severe disease of bread wheat worldwide. Discovery and utilization of resistance genes to powdery mildew from wild relatives of wheat have played important roles in wheat improvement. Aegilops longissima, one of the S-genome diploid wild relatives of wheat, is a valuable source of disease and pest resistance for wheat. Chromosome 4Sl from Ae. longissima confers moderate resistance to powdery mildew. In this study, we conducted RNA-seq on a putative Chinese Spring (CS)-Ae. longissima 4Sl(4B) disomic substitution line (TA3465) to develop 4Sl-specific markers to assist the transfer of a Bgt resistance gene from 4Sl by induced homoeologous recombination. A pairwise comparison of genes between CS and TA3465 demonstrated that a number of genes on chromosome 4BS in CS were not expressed in TA3465. Analysis of 4B- and 4Sl-specific molecular markers showed that 4BS and 4SlL were both missing in TA3465, whereas 4BL and 4SlS were present. Further characterization by genomic and fluorescent in situ hybridization confirmed that TA3465 carried a spontaneous Robertsonian T4SlS·4BL translocation. Powdery mildew tests showed that TA3465 was resistant to 10 of 16 Bgt isolates collected from different regions of China, whereas CS was susceptible to all those Bgt isolates. The powdery mildew resistance gene(s) in TA3465 was further mapped to the short arm of 4Sl and designated as Pm66.

Physical Mapping of Pm57, a Powdery Mildew Resistance Gene Derived from Aegilops searsii.

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is one of many severe diseases that threaten bread wheat (Triticum aestivum L.) yield and quality worldwide. The discovery and deployment of powdery mildew resistance genes (Pm) can prevent this disease epidemic in wheat. In a previous study, we transferred the powdery mildew resistance gene Pm57 from Aegilops searsii into common wheat and cytogenetically mapped the gene in a chromosome region with the fraction length (FL) 0.75-0.87, which represents 12% segment of the long arm of chromosome 2Ss#1. In this study, we performed RNA-seq using RNA extracted from leaf samples of three infected and mock-infected wheat-Ae. searsii 2Ss#1 introgression lines at 0, 12, 24, and 48 h after inoculation with Bgt isolates. Then we designed 79 molecular markers based on transcriptome sequences and physically mapped them to Ae. searsii chromosome 2Ss#1- in seven intervals. We used these markers to identify 46 wheat-Ae. searsii 2Ss#1 recombinants induced by ph1b, a deletion mutant of pairing homologous (Ph) genes. After analyzing the 46 ph1b-induced 2Ss#1L recombinants in the region where Pm57 is located with different Bgt-responses, we physically mapped Pm57 gene on the long arm of 2Ss#1 in a 5.13 Mb genomic region, which was flanked by markers X67593 (773.72 Mb) and X62492 (778.85 Mb). By comparative synteny analysis of the corresponding region on chromosome 2B in Chinese Spring (T. aestivum L.) with other model species, we identified ten genes that are putative plant defense-related (R) genes which includes six coiled-coil nucleotide-binding site-leucine-rich repeat (CNL), three nucleotide-binding site-leucine-rich repeat (NL) and a leucine-rich receptor-like repeat (RLP) encoding proteins. This study will lay a foundation for cloning of Pm57, and benefit the understanding of interactions between resistance genes of wheat and powdery mildew pathogens.

Discovery of powdery mildew resistance gene candidates from Aegilops biuncialis chromosome 2Mb based on transcriptome sequencing.

Powdery mildew is one of the most widespread diseases of wheat. The development and deployment of resistant varieties are one of the most economical and effective methods to manage this disease. Our previous study showed that the gene(s) at 2Mb in Chinese Spring (CS)-Aegilops biuncialis 2Mb disomic addition line TA7733 conferred a high level of resistance to powdery mildew of wheat. In this study, resistance spectrum of TA7733 was assayed by using 15 Blumeria graminis f. sp. tritici (Bgt) isolates prevalent in different regions of China. The result indicated that TA7733 was highly resistant to all tested Bgt isolates and the gene(s) on chromosome 2Mb conferred broad-spectrum resistance to powdery mildew. In order to characterize mechanism of powdery mildew resistance by identifying candidates R-genes derived from Ae. biuncialis chromosome 2Mb and develop 2Mb-specific molecular markers, we performed RNA-seq analysis on TA7733 and CS. In total we identified 7,278 unigenes that showed specific expression in TA7733 pre and post Bgt-infection when compared to CS. Of these 7,278 unigenes, 295 were annotated as putative resistance (R) genes. Comparatively analysis of R-gene sequences from TA7733 and CS and integration CS Ref Seq v1.0 were used to develop R-gene specific primers. Of 295 R-genes we identified 53 R-genes were specific to 2Mb and could be involved in powdery mildew resistance. Functional annotation of majority of the 53 R-genes encoded nucleotide binding leucine rich repeat (NLR) protein. The broad-spectrum resistance to powdery mildew in TA7733 and availability of 2Mb-derived putative candidate R-gene specific molecular markers identified in this study will lay foundations for transferring powdery mildew resistance from 2Mb to common wheat by inducing CS-Ae. biuncialis homoeologous recombination. Our study also provides useful candidates for further isolation and cloning of powdery mildew resistance gene(s) from Ae. biuncialis chromosome 2Mb.

Physical Mapping of Stem Rust Resistance Gene Sr52 from Dasypyrum villosum Based on ph1b-Induced Homoeologous Recombination.

Wheat stem rust caused by Puccinia graminis f. sp. tritici (Pgt) had been a devastating foliar disease worldwide during the 20th century. With the emergence of Ug99 races, which are virulent to most stem rust resistance genes deployed in wheat varieties and advanced lines, stem rust has once again become a disease threatening global wheat production. Sr52, derived from Dasypyrum villosum and mapped to the long arm of 6V#3, is one of the few effective genes against Ug99 races. In this study, the wheat-D. villosum Robertsonian translocation T6AS·6V#3L, the only stock carrying Sr52 released to experimental and breeding programs so far, was crossed with a CS ph1b mutant to induce recombinants with shortened 6V#3L chromosome segments locating Sr52. Six independent homozygous recombinants with different segment sizes and breakpoints were developed and characterized using in situ hybridization and molecular markers analyses. Stem rust resistance evaluation showed that only three terminal recombinants (1381, 1380, and 1392) containing 8%, 22%, and 30% of the distal segment of 6V#3L, respectively, were resistant to stem rust. Thus, the gene Sr52 was mapped into 6V#3L bin FL 0.92-1.00. In addition, three molecular markers in the Sr52-located interval of 6V#3L were confirmed to be diagnostic markers for selection of Sr52 introgressed into common wheat. The newly developed small segment translocation lines with Sr52 and the identified molecular markers closely linked to Sr52 will be valuable for wheat disease breeding.

Applying foliar stoichiometric traits of plants to determine fertilization for a mixed pine-oak stand in the Qinling Mountains, China.

BACKGROUND: The Chinese Natural Forest Protection program has been conducted nationwide and has achieved resounding success. However, timber importation has increased; therefore, producing more domestic timber is critical to meet the demand for raw materials. Fertilization is one of the most effective silviculture practices used to improve tree and stand growth. However, determining the appropriate type and amount of elements is necessary for effective fertilization of big timber in different forest types and environmental conditions. Stoichiometric theory provides the criteria to assess nutrient limitation in plants and offers important insight into fertilizer requirements of forested ecosystems. METHODS: Nitrogen (N) and phosphorus (P) concentrations in plants' leaves, mineral soil, and litter were investigated in a mixed pine-oak stand. RESULTS: The big timber rate for Pinus tabuliformis, Pinus armandii and Quercus aliena var. acutesserata is 57.71%, 22.79% and 2.78% of current existing individuals respectively. Foliar N and P concentrations were 9.08 and 0.88 mg g-1, respectively. The N:P in the plants was 10.30. N concentration and N:P in mineral soil decreased from 0-30 cm soil depth. For litter, N and P concentrations were 16.89 and 1.51 mg g-1, respectively, and N:P was 11.51. Concentrations of N and P in mineral soil and litter did not significantly affect plants'leaf concentrations. Similar result was also obtained between litter and mineral soil concentrations. Nitrogen storage in mineral soil was significantly correlated with foliar N:P in the plants. DISCUSSION: Foliar N:P of dominant tree species and the plants, and foliar N concentration in Pinus tabuliformis and P. armandii, and foliar P concentration of P. armandii in the mixed pine-oak stand was lower than that in Chinese and other terrestrial plants. Foliar nutrients in the plants were not affected by soil nutrients. According to the criteria of nutrient limitation for plants, growth of dominant tree species was N limited; therefore, 1.49 t ha-1 pure N should be added to forest land to as fertilizer.

[Construction of the eukaryotic expression vector with IL-2 gene and VP2 gene of PPV and research on immunogenicity].

To construct gene vaccine of PPV and to investigate the effects of interleukin 2 (IL-2) as an adjuvant on immune responses in mouse, the recombinant expression plasmid of pCIneo-IL2-VP2 was constructed and transfected into PK-15 cells by lipofectamine, the expressed product was detected by immunofluore assay. To study the immune effects of DNA vaccine in vitro and in vivo, mice were used as the animal model. The recombinant plasmid pCIneo-IL2-VP2, the control plasmid pCI-neo and the PPV live vaccine were immunized by intramuscular injection. Anti-PPV antibodies were measured by ELISA, lymphocyte proliferation activity was detected using MTT method, and the specific killing activities of CTL were assayed too. The results show that the immunized mice produced PPV antibody after one week, and reached to highest after four weeks. Compared with the control group, the pCIneo-IL2-VP2 immunized group produced significant differences in the antibody titers, the lymphocyte proliferation activity and the specific killing activities of CTL. The pCIneo-IL2-VP2 induced humoral and cellular immunity responses similarly to that the live vaccine induced. These results manifested that the PPV DNA vaccine successfully induced humoral and cellular immunity response in mice with the IL-2 gene as an adjuvant.