Identification and Pathogenicity of Fungal Pathogens Causing Black Point in Wheat on the North China Plain.

Fungi associated with black point were isolated from three highly susceptible wheat genotypes in the North China Plain. The 21 isolates represented 11 fungal genera. The most prevalent genera were Alternaria (isolation frequency of 56.7%), Bipolaris (16.1%), and Fusarium (6.0%). The other eight genera were Curvularia, Aspergillus, Cladosporium, Exserohilum, Epicoccum, Nigrospora, Penicillium, and Ulocladium; their isolation frequencies ranged from 0.8 to 4.8%. The pathogenicity of the isolates was individually assessed in the greenhouse by inoculating wheat plants with spore suspensions. Ten of the 21 isolates caused significantly higher incidences of black point than that the controls. These isolates belonged to eight fungal species (A. alternata, B. sorokiniana, B. crotonis, B. cynodontis, C. spicifera, F. equiseti, E. rostratum, and E. sorghinum) based on morphological traits and phylogenetic analysis. The average incidences of black point in the eight fungal species were 32.4, 54.3, 43.0, 41.9, 37.2, 38.8, 50.1, and 34.1%, respectively. B. sorokiniana and A. alternata were determined to be the most important pathogens in the North China Plain based on fungal prevalence and symptom severity. This study is the first to identify E. rostratum as a major pathogen causing black point in wheat.

Proteomic analysis of developing wheat grains infected by powdery mildew (Blumeria graminis f.sp. tritici).

Blumeria graminis f.sp. tritici (Bgt) infection greatly interferes with the normal source-sink relationships and always causes tremendous loss of yield and quality in wheat. To better understand the impact of this pathogen on grain development, proteome characterization during grain development in susceptible wheat cultivar Xinong 979 infected by powdery mildew was investigated by 2-DE and tandem MALDI-TOF/TOF-MS. Identification of 111 differentially expressed protein spots representing 85 unique proteins and six expression patterns showed a chronological description of wheat grain formation. Comparative proteome profiles indicated that 43 protein spots displayed significant abundance change, which is mainly involved in stress/defense responses, primary metabolism, and storage protein. The down-regulation of defense response-related proteins including alpha-purothionin, lactoylglutathione lyase, and alpha-amylase inhibitor CM16 in infected grains compared to control during seed filling might be related to the susceptibility of wheat to Bgt, while the enhanced expression of beta-amylase and glyceraldehyde-3-phosphate dehydrogenase and the down-regulation of ADP glucose pyrophosphorylase in infected grains probably resulted in the negative effects on yield formation. Our data reveal the complex grain metabolism mechanisms and defense responses during compatible interactions of wheat and Bgt, and provide valuable information for further understanding of the underlying molecular processes which can possibly yield novel strategies for breeding resistant cultivars and protection strategies in the field.

Cloning, characterization and expression analysis of the receptor-like kinase gene (RLK) in common wheat.

To investigate whether homologs of wheat (Triticum aestivum L.) LRK10 gene was expressed in powdery mildew-resistant lines after inoculation with Blumeria graminis f. sp. tritici, one degenerate primer for 5'-RACE was designed according to the 6th kinase subdomain of LRK10 and other plant kinases. 5'-RACE was performed with the template cDNA synthesized with RNA extracted from seedling leaves of a powdery mildew-resistant wheat line "99-2439" after inoculation with B. graminis. One 1551 bp cDNA fragment representing a protein kinase gene was obtained (S1125, GenBank accession number: AY584533). Subsequently, a 2255-bp full-length cDNA clone with a complete encoding region (open reading frame, ORF) was obtained by RACE. The clone encodes a polypeptide consisting of 637 amino acid residues. The result of homology search showed that it belongs to a receptor-like kinase gene family in wheat, which was named as wlrk (wheat leaf rust kinase) previously. Similar to LRK10, this protein kinase has five distinct domains: a hydrophobic signal sequence at the amino-terminus, a putative extracellular domain, a transmembrane domain, a highly charged sequence and a serine/threonine kinase domain at the carboxy-terminus, and thus it was named as TaLRK (Triticum aestivum LRK). The expression pattern of TaLRK at transcription level in leaves after B. graminis infection was investigated by semi-quantitative RT-PCR (semi-QRT-PCR), using wheat actin gene as a control. The result showed that the transcription of TaLRK was significantly enhanced by B. graminis infection. The expression pattern of TaLRK in different tissues showed that this new wheat RLK gene was expressed only in green parts of wheat. This study suggests that TaLRK may function in wheat powdery mildew resistance responses.

Transcriptome Analysis for Abnormal Spike Development of the Wheat Mutant dms.

BACKGROUND: Wheat (Triticum aestivum L.) spike development is the foundation for grain yield. We obtained a novel wheat mutant, dms, characterized as dwarf, multi-pistil and sterility. Although the genetic changes are not clear, the heredity of traits suggests that a recessive gene locus controls the two traits of multi-pistil and sterility in self-pollinating populations of the medium plants (M), such that the dwarf genotype (D) and tall genotype (T) in the progeny of the mutant are ideal lines for studies regarding wheat spike development. The objective of this study was to explore the molecular basis for spike abnormalities of dwarf genotype. RESULTS: Four unigene libraries were assembled by sequencing the mRNAs of the super-bulked differentiating spikes and stem tips of the D and T plants. Using integrative analysis, we identified 419 genes highly expressed in spikes, including nine typical homeotic genes of the MADS-box family and the genes TaAP2, TaFL and TaDL. We also identified 143 genes that were significantly different between young spikes of T and D, and 26 genes that were putatively involved in spike differentiation. The result showed that the expression levels of TaAP1-2, TaAP2, and other genes involved in the majority of biological processes such as transcription, translation, cell division, photosynthesis, carbohydrate transport and metabolism, and energy production and conversion were significantly lower in D than in T. CONCLUSIONS: We identified a set of genes related to wheat floral organ differentiation, including typical homeotic genes. Our results showed that the major causal factors resulting in the spike abnormalities of dms were the lower expression homeotic genes, hormonal imbalance, repressed biological processes, and deficiency of construction materials and energy. We performed a series of studies on the homeotic genes, however the other three causal factors for spike abnormal phenotype of dms need further study.

[Analysis of the functions of wheat resistance-related genes by a transient expression system].

Transient expression system was used to analyze the functions of three resistance- related genes: TaTBL, TaPK1 and TaTST. Target genes were constructed into plant expression vectors and transformed into leaf epidermal cells of a powdery mildew-susceptible wheat variety by gene gun. GUS gene was co-transformed with target gene to mark the transformed cells. After transformation, leaf surface was inoculated with powdery mildew conidiospores. Forty eight hours after inoculation, penetration of the fungus and formation of haustoria in transformed cells were observed to evaluate the effects of the target gene's products on the invasion of powdery mildew. The results implied that all these three genes, when transiently expressed in leaf epidermal cells of susceptible wheat variety, could partly inhibit the penetration of conidiospores and formation of haustoria, and to some extent increase the resistance of cells to powdery mildew.

Cloning, characterization and expression of wheat EDR1 (enhanced disease resistance) gene.

To investigate if there is an EDR1 pathway in wheat (Triticum aestivum L.), a pair of degenerate primers was designed according to the cDNAs of Arabidopsis thaliana EDR1 gene and its homologs were used to isolate EDR1 gene homologs from wheat. RT-PCR was conducted on the cDNA template synthesized with RNA of wheat leaves. A 627-bp cDNA fragment representing an EDR1 gene (named as TaEDR1) was isolated (GenBank accession number: AY743662). Subsequently, the 3050-bp full-length cDNA sequence of TaEDR1, which encodes a polypeptide consisting of 959 amino acid residues, was obtained by RACE technique. The amino acid sequence of TaEDR1 and that of barley (Hordeum vulgare) EDR1 (signed as HvEDR1) show 92% identity. There is a highly conserved catalytic domain of serine/threonine protein kinases in the C-terminus of TaEDR1. Because this protein has a putative nuclear localization motif, it probably functions in the nucleus. This study provides the first molecular biological evidence of the presence of an EDR1 homolog in common wheat. The transcription pattern of TaEDR1 was investigated in leaves after inoculation with Blumeria graminis (DC.) E.O. Speer f. sp. tritici Em. Marchal (Bgt) through semi-quantitative RT-PCR (semi-QRT-PCR). The result showed that the transcribing of TaEDR1 was enhanced by Bgt. The expression pattern of the TaEDR1 gene in different tissues showed that it expressed in leaves, stems, spikes and roots. This study suggests that the TaEDR1, a MAP kinase kinase kinase, may function in wheat defense responses.

Cloning, mapping and protein expression of wheat thaumatin protein gene (TaTLP1).

To understand wheat powdery mildew resistance mechanism, reverse-transcription polymerase chain reaction (RT-PCR) and cDNA library screening were performed to isolate the full-length cDNA of wheat thaumatin protein gene from wheat-Haynaldia villosa 6VS/6AL translocation line. The putative amino acid sequence of this gene consists of 173 amino acid residues, and is an acid polypeptide. It was highly homologous to thaumatin proteins isolated from other plants, so it is designated as TaTLP1 (GenBank accession number: AF384146). Northern blot analysis of TaTLP1 showed that the transcription difference obviously existed between resistant wheat-Haynaldia villosa 6VS/6AL translocation line and susceptible "Yangmai 5". The result of western blot showed that the protein expression product of TaTLP1 gene in wheat seedling leaf was a soluble cytoplasm protein, whose expression was induced by fungus Erysiph graminis and apparently related to disease resistance of the 6VS/6AL translocation line. Southern blot indicated that the TaTLP1 gene had 1-2 copies in wheat genome, and had been localized on the specific region of 7B and 7D chromosomes in wheat.

Molecular cloning, characterization and mapping of a rhodanese like gene in wheat.

To isolate genes related to resistance to Erysiphe graminis (Blumeria graminis) DC. f. sp. tritici in wheat (Triticum aestivum L.), differential display analysis was conducted for mRNA extracted from seedlings of a wheat-Haynaldia villosa 6VS/6AL translocation line 92R137 that contains a powdery mildew resistance gene Pm21. A full-length cDNA sequence named TaTST (Triticum aestivum thiosulfate sulfurtransferase) homologous to the thiosulfate sulfurtransferase (rhodanese) in Datisca glomerata was isolated. Northern blot showed that the expression of TaTST was enhanced after infection with Erysiphe graminis. TaTST was mapped on the short arm of 6B chromosomes of wheat through Southern blot and GSP-PCR using Chinese Spring nullisomic/tetrasomic lines and ditelosomic lines. There is a homologue of TaTST on 6VS too.

Transgenic barley with overexpressed PTrx increases aluminum resistance in roots during germination.

A transgenic barley line (LSY-11-1-1) with overexpressed Phalaris coerulescens thioredoxin gene (PTrx) was employed to measure the growth, protein oxidation, cell viability, and antioxidase activity in barley roots during germination on the presence of 2 mmol/L AlCl(3) on filter paper. The results show that (1) compared with the non-transgenic barley, LSY-11-1-1 had enhanced root growth, although both were seriously inhibited after AlCl(3) treatment; (2) the degree of protein oxidation and loss of cell viability in roots of LSY-11-1-1 were much less than those in roots of non-transgenic barley, as reflected by lower contents of protein carbonyl and Evans blue uptakes in LSY-11-1-1; (3) activities of catalase (CAT), glutathione peroxidase (GPX), ascorbate peroxidase (APX), and glutathione reductase (GR) in LSY-11-1-1 root tips were generally higher than those in non-transgenic barley root tips, although these antioxidase activities gave a rise to different degrees in both LSY-11-1-1 and non-transgenic barley under aluminum stress. These results indicate that overexpressing PTrx could efficiently protect barley roots from oxidative injury by increasing antioxidase activity, thereby quenching ROS caused by AlCl(3) during germination. These properties raise the possibility that transgenic barley with overexpressed PTrx may be used to reduce the aluminum toxicity in acid soils.