Interaction between TaNOX7 and TaCDPK13 Contributes to Plant Fertility and Drought Tolerance by Regulating ROS Production.

Reactive oxygen species (ROS) homeostasis is critical for both physiological processes and stress responses of plants. NADPH oxidases (NOXs) are the key producers of ROS in plants. However, their functions in ROS homeostasis and plant growth regulation in wheat (Triticum aestivum) are little investigated. Here, we cloned and characterized a NOX isoform TaNOX7 in wheat. Overexpression of TaNOX7 in rice led to enhanced root length, ROS production, drought tolerance as well as bigger panicles and higher yield but shorter growth period duration. Further results indicate that TaCDPK13, a member of calcium-dependent protein kinases (CDPKs), can directly interact with TaNOX7 and enhance ROS production in plants. These results demonstrate that TaNOX7 plays crucial roles in wheat development, fertility, and drought tolerance via interaction with TaCDPK13, which may act as an upstream regulator of TaNOX7 to regulate ROS production in wheat.

QTL Mapping and Validation of Adult Plant Resistance to Stripe Rust in Chinese Wheat Landrace Humai 15.

Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a devastating foliar disease that affects common wheat and barley throughout the world. The reasonable deployment of adult plant resistance (APR) wheat varieties is one of the best methods for controlling this disease. Wheat landraces are valuable resources for identifying the genes/QTLs responsible for disease resistance. Humai 15 is a Chinese spring wheat landrace and it has exhibited adequate levels of APR to the prevalent Pst races in field environments for many years. In this study, a population of 177 recombinant inbred lines (RILs) was derived from Humai 15 × Mingxian 169. After screening based on a 90K chip array using 45 RILs and Kompetitive Allelic Specific PCR marker genotyping for the population of RILs, a major effect QTL in Humai 15 was located on the centromere of chromosome 2B, where it accounted for up to 47.2% of the phenotypic variation. Two other minor QTL genes from Humai 15 were located on chromosome arms 3BS and 4BL. The Yr18 gene was identified on chromosome arm 7DS in Mingxian 169.

Screening for simple sequence repeat markers in Puccinia striiformis tritici based on genomic sequence.

Puccinia striiformis f. sp. tritici (Pst) is the obligate biotrophic fungus responsible for stripe rust wheat. In this study, we developed and characterized 20 polymorphic microsatellite markers from the genomic sequence of an isolate of Chinese Pst race CY32. Polymorphism at each simple sequence repeat (SSR) locus was determined using 32 Pst isolates from 7 countries. The number of alleles varied from 2 to 7 across isolates, and the observed and expected heterozygosities ranged from 0.33 to 0.97 (mean 0.62) and 0.23 to 0.73 (mean 0.51), respectively. As expected the genomic SSR markers were more polymorphic than the expressed sequence tag (EST)-SSR markers developed previously. These markers will be more useful for population genetics and molecular genetics studies in Pst.

Isocitrate lyase is required for urediniospore germination of Puccinia striiformis f. sp. tritici.

The PstICL1 gene, which encodes isocitrate lyase, a key enzyme in the glyoxylate cycle, was cloned and characterized in the biotrophic wheat pathogen Puccinia striiformis f. sp. tritici (Pst). Expression analyses of PstICL1 exhibited high levels of transcripts in ungerminated urediniospores, which showed low isocitrate lyase enzyme activity. In planta, PstICL1 expression was continuously down-regulated upon germination. During the later stages of the infection of wheat, the level of PstICL1 expression was extremely low. The function of PstICL1 was identified via mutant complementation. The expression of PstICL1 in Saccharomyces cerevisiae can complement the defects of the △ICL mutant. Using 3-nitropropionate, we observed that inactivation of isocitrate lyase greatly reduced the germination rate of urediniospores, indicating that PstICL1 plays a key role during Pst germination. Furthermore, analysis of lipid bodies revealed that lipid components continuously enter the germ tube from the urediniospore cell during germ tube elongation. Moreover, during this period, the lipid contents continuously decreased, and the total carbohydrates markedly increased, demonstrating that the lipids are being converted into carbohydrates. These results suggest that PstICL1 is required for Pst germination.

MicroRNAs involving in cold, wounding and salt stresses in Triticum aestivum L.

MicroRNAs (miRNAs) play critical roles in post-transcriptional regulation and act as important endogenous regulators to various stresses. Cold, wounding and high-salinity are three common environmental stress stimuli influencing crops growth and development. In this study, we identified 31 known miRNAs and 3 novel miRNAs in wheat. Moreover, 19 stress-regulated miRNAs using RT-qPCR data in which the effects of three stresses were surveyed from the known miRNAs. Among them, 16, 12 and 8 miRNAs were regulated under cold, wounding and high-salinity treatments, respectively. Of which 4 miRNAs were highly responsive to cold stress in wheat by northern blot, and 6 wounding-regulated and 3 high-salinity-regulated miRNAs were detected. Meanwhile, miR159, miR393 and miR398 were responsive to multiple stress stimuli. Besides, 2 novel miRNAs were regulated by cold stress. While, the analyses of targets suggested miR159, miR398 and miR6001 could responses to stress conditions in regulation pathways. Taken together, the results of this study suggest that wheat miRNAs may play important roles in response to abiotic stress.

Cloning and molecular characterization of a myosin light chain gene from Puccinia striiformis f. sp. tritici.

The fungus Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust, is an obligate biotrophic basidiomycete. Many studies have found that myosins play important roles during fungal growth and propagation. However, there are few reports on the myosins of Pst. In this study, we cloned and obtained the myosin light chain gene PsMLC1 from Pst and characterized its expression. Furthermore, the function of PsMLC1 was identified by mutant complementation. As a result, we found that expression of PsMLC1 in Schizosaccharomyces pombe mostly complemented the defects of the cdc4 mutant, indicating that PsMLC1 belongs to the myosin light chain family member. Expression studies showed that the transcript levels of PsMLC1 little changed before 24 h post inoculation then was suddenly down-regulated during Pst infection of wheat. By using ML-7, we observed that inactivity of PsMLC1 greatly reduced the germination rate of urediniospores. These results suggest that PsMLC1 is essential for the early stages of Pst infection of wheat but unnecessary for the later stages of infection. This work elucidates the function of the myosins in Pst and may provide some theoretical basis for controlling strip rust.

A conidiation-related gene is highly expressed at the resting urediospore stage in Puccinia striiformis f. sp. tritici.

The production and germination of asexual spores in a diverse group of fungi play a crucial role in their infection cycles. These processes are regulated by a set of genes, namely, conidiation-related genes, involved in the production, morphological characteristics, and differentiation of conidia. In this study, we identified and characterized the PsCon1 gene, which is the first conidiation-related gene identified in Puccinia striiformis f. sp. tritici (Pst). Sequence analysis revealed that PsCON1 has two conserved conidiation-specific protein 6 domains. Single nucleotide polymorphisms and insertion/deletion variations were detected in the coding region of PsCon1 among five Pst races. Quantitative RT-PCR assays revealed that PsCon1 was expressed at the highest level in resting urediospores of Pst, and gradually decreased after germination and infection. However, at 312 hpi, at the stage of forming large amounts of urediospores on leaves, the amount of PsCon1 mRNA was sharply increased but only 0.1-fold that of resting urediospores. Subcellular localization assays indicated PsCon1 heterologously expressed in Fusarium graminearum was located in the cytoplasm of conidia. The results suggest that PsCon1 may play a role in formation or survival of Pst urediospores.

[Characterization and inheritance of resistance to stripe rust in the wheat line Guinong775].

Exploring different types of resistance genes and deploying diversity resistance genes among different wheat producing regions are important strategy to control stripe rust epidemic in large scale. The resistance spectra of the wheat line Guinong 775 at seedling stage was tested in greenhouse with ten isolates of Puccinia triticina West. f. sp. tritici; and the inheritance of resistance was investigate with F(2:3) and BC(1) populations derived from a cross between Guinong 775 and a susceptible cultivar Avocet (S), inoculated with isolates CYR32 and CH42, which poses a notable threat to current wheat varieties carrying Yr26 resistance gene. The results showed that Guinong 775 was immune or nearly immune to all the tested races; however, the resistant germplasm 92R137, Chuanmai 42, Guinong 22, and Yr24/6*Avocet (S) were susceptible to CH42. The resistance of Guinong 775 was conferred by a single dominant gene to CYR32 and another single dominant gene to CH42, and these genes proved to be different.

Cloning and characterization of the actin gene from Puccinia striiformis f. sp. tritici.

The fungus Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust, is an obligate biotrophic basidiomycete. Urediniospores are the most common spore type involved in the epidemiology of this disease. Tip growth of germ tubes of germinated urediniospores is a key step during infection of wheat, but few studies have investigated it so far. Recent research has found that actin is closely associated with hyphal tip growth. In this study, we have cloned and obtained the full-length actin cDNA from P. striiformis f. sp. tritici and characterized its expression. Furthermore, actin filament (F-actin) patterns were visualized microscopically during germ tube formation. The most conspicuous actin-containing structures were actin patches. They were mainly concentrated near the hyphal tip and scattered throughout the cortex. By using cytochalasin B, we observed that depolymerization of F-actin greatly reduced the germination rate of urediniospores and disrupted the transport of vesicles to the germ tube tip, indicating that F-actin played a key role in the tip growth of P. striiformis f. sp. tritici. This work helps us to understand the tip growth mechanism of P. striiformis f. sp. tritici, and may provide a theoretical framework for designing novel pesticides.

Analysis of differential transcriptional profiling in wheat infected by Blumeria graminis f. sp. tritici using GeneChip.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a devastating disease of wheat. The use of wheat cultivars resistant to powdery mildew provides an effective, economical, and environmentally friendly method to control the disease. Previously, we identified a dominant resistance gene, temporarily named Pmhym, from the wheat cultivar Hongyoumai. In order to screen differential transcripts related to Pmhym-mediated resistance, four F3 homozygous resistant and four susceptible progenies derived from the Hongyoumai/Yumai13 cross were selected to construct two different pools, respectively, representing an incompatible and compatible interaction with Bgt. Pre-inoculated control and the pathogen-inoculated treatments at 24 h post inoculation (hpi) were used. Three groups of differential genes were categorized from three comparisons as pre- and post-induced, respectively, in two interactions, and post-induced between incompatible and compatible interaction. It was found that salicylic acid (SA), jasmonate (JA), and ethylene (ET) signaling-related genes were differentially expressed, thus suggesting that they are involved in the defensive response against Bgt infection. In compatible interactions, the genes involved in the abscisic acid (ABA) signaling pathway might be inhibitory to the above-mentioned three pathways, resulting in a susceptible reaction. Genes involved in disease/defense, signal transduction, and reactive oxygen species (ROS) metabolism were up-regulated in incompatible interactions, implying a role in resistant response. The results of qRT-PCR analysis on several candidate genes were consistent in their expression patterns as revealed by microarray analysis. The differential expression analyses in the present study are good candidates for further elucidation of wheat defensive response to powdery mildew.

Saccharothrix yanglingensis sp. nov., an antagonistic endophytic actinomycete isolated from cucumber plant.

An endophytic actinomycete strain, designated Hhs.015(T), was isolated from roots of cucumber seedlings. The endophytic isolate was identified by means of a polyphasic taxonomic approach. On the basis of 16S rRNA gene sequence similarities, strain Hhs.015(T) was closely related to members of the genus Saccharothrix. DNA-DNA hybridization with the four closest relatives, Saccharothrix longispora NRRL B-16116(T), Saccharothrix xinjiangensis NRRL B-24321(T), Saccharothrix autraliensis CGMCC 4.1355(T) and Saccharothrix espanaensis CGMCC 4.1714(T), gave similarity values of 33.8, 28.2, 44.1 and 29.5%, respectively, which indicated that strain Hhs.015(T) represents a novel species of the genus Saccharothrix. This is consistent with the morphological, physiological and chemotaxonomic data. As a whole, these results suggest that strain Hhs.015(T) represents a novel Saccharothrix species. The name Saccharothrix yanglingensis sp. nov. is proposed, with the type strain Hhs.015(T) (=CGMCC 4.5627(T) = KCTC 19722(T)).

Characterization of a wheat HSP70 gene and its expression in response to stripe rust infection and abiotic stresses.

Members of the family of 70-kD heat shock proteins (HSP70 s) play various stress-protective roles in plants. In this study, a wheat HSP70 gene was isolated from a suppression subtractive hybridization (SSH) cDNA library of wheat leaves infected by Puccinia striiformis f. sp. tritici. The gene, that was designated as TaHSC70, was predicted to encode a protein of 690 amino acids, with a molecular mass of 73.54 KDa and a pI of 5.01. Further analysis revealed the presence of a conserved signature that is characteristic for HSP70s and phylogenetic analysis demonstrated that TaHSC70 is a homolog of chloroplast HSP70s. TaHSC70 mRNA was present in leaves of both green and etiolated wheat seedlings and in stems and roots. The transcript level in roots was approximately threefold less than in leaves but light-dark treatment did not charge TaHSC70 expression. Following heat shock of wheat seedlings at 40°C, TaHSC70 expression increased in leaves of etiolated seedlings but remained stable at the same level in green seedlings. In addition, TaHSC70 was differentially expressed during an incompatible and compatible interaction with wheat-stripe rust, and there was a transient increase in expression upon treatment with methyl jasmonate (MeJA) treatment. Salicylic acid (SA), ethylene (ET) and abscisic acid (ABA) treatments had no influence on TaHSC70 expression. These results suggest that TaHSC70 plays a role in stress-related responses, and in defense responses elicited by infection with stripe rust fungus and does so via a JA-dependent signal transduction pathway.

Characterization of a novel wheat NAC transcription factor gene involved in defense response against stripe rust pathogen infection and abiotic stresses.

Proteins encoded by the NAC gene family constitute one of the largest plant-specific transcription factors, which have been identified to play many important roles in both abiotic and biotic stress adaptation, as well as in plant development regulation. In the current paper, a full-length cDNA sequence of a novel wheat NAC gene, designated as TaNAC4, was isolated using in silico cloning and the reverse transcription PCR (RT-PCR) methods. TaNAC4 sharing high homology with rice OsNAC4 gene was predicted to encode a protein of 308 amino acid residues, which contained a plant-specific NAC domain in the N-terminus. Transient expression analysis indicated that the deduced TaNAC4 protein was localized in the nucleus of onion epidemical cells. Yeast one-hybrid assay revealed that the C-terminal region of the TaNAC4 protein had transcriptional activity. The expression of TaNAC4 was largely higher in the wheat seedling roots, than that in leaves and stems. TaNAC4 transcript in wheat leaves was induced by the infection of strip rust pathogen, and also by exogenous applied methyl jasmonate (MeJA), ABA and ethylene. However, salicylic acid (SA) had no obvious effect on TaNAC4 expression. Environmental stimuli, including high salinity, wounding, and low-temperature also induced TaNAC4 expression. These results indicate that this novel TaNAC4 gene functions as a transcriptional activator involved in wheat response to biotic and abiotic stresses.

[Research progress in molecular ecology of Puccinia striiformis f. sp. tritici].

Stripe rust caused by Puccinia striiformis f. sp. tritici Westend is one of the most important epidemic diseases of wheat in the world. To understand the population evolution of this rust fungus will facilitate the control of this disease and the breeding of rust-resistant wheat. In the past decades, DNA molecular markers have been applied to investigate the population genetics of wheat stripe rust fungus, and rapid progress has brought out in the research of P. striiformis molecular ecology. In this paper, the main research progress in the molecular ecology of stripe rust was reviewed, and the limitations and trends of related research in China were discussed.

Cloning of a putative hypersensitive induced reaction gene from wheat infected by stripe rust fungus.

The hypersensitive response (HR) is one of the most efficient forms of plant defense against biotrophic pathogens and results in localized cell death and the formation of necrotic lesions. In this study, a novel putative hypersensitive induced reaction (HIR) gene from wheat leaves infected by incompatible stripe rust pathogen CY23, designated as Ta-hir1, was identified by using rapid amplification of cDNA ends (RACE). Ta-hir1 encodes 284 amino acids, with a predicted molecular mass of 31.31 KDa. A phylogenetic analysis showed that Ta-hir1 was highly homologous to Hv-hir1 from barley at both cDNA and deduced amino-acid levels. Amino-acid sequence analysis of the wheat HIR protein indicated the presence of the SPFH (Stomatins, Prohibitins, Flotillins and HflK/C) protein domain typical for stomatins which served as a negative regulator of univalent cation permeability, especially for potassium. The expression profile of the Ta-hir1 transcript detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time polymerase chain reaction (real time-PCR), respectively, showed that the highest expression occurred 48 h post inoculation (hpi), which is consistent with our previous histopathology observations during the stripe rust fungus-wheat incompatible reaction.

[Systemic resistance induced by biocontrol agents in plants and its biochemical and cytological mechanisms].

Microbial biocontrol agents (BCAs) are generally used for controlling plant diseases via antagonistic mechanisms including competition, antibiosis, parasitism, and cross-protection. Some BCAs can even promote plant growth, and provide induced systemic resistance (ISR), i. e., induce the plants to have resistance against pathogens including phytopathogenic fungi, bacteria and virus, and in some cases, pest insects and nematodes. ISR is characterized by non-specific, wide spectrum and systemic. It is phenotypically similar to the systemic acquired resistance (SAR) induced by the infection of pathogens, and with the same efficiency but without hypersensitive response (HR) and visible symptoms in plant as SAR, which is helpful to open a new way to develop and improve safer and environmentally friendly strategies for plant protection. In this paper, the research advances on ISR mediated by biocontrol fungi and bacteria, elicitors or determinants, and signaling transduction pathways were summarized, with more emphasis on the biochemical and cytological mechanisms of host plant defense reaction induced by free-living and endophytic BCAs. The potential application of ISR in biocontrol of plant diseases was also discussed.

[Screen, identification and optimized fermentation condition of an actinomycete strain against pathogenic fungus Fulvia fulva].

An actinomycete strain xjy was isolated from the soil of cotton field in Xinjiang against pathogenic fungus Fulvia fulva. The growth of 23 plant pathogens and 6 bacteria were strongly inhibitded by strain xjy on PDA plate. Antimicrobial spectrum of fermentation filtrate of strain xjy is extensive and selective to different pathogens. The morphology, cultural characteristics, physiological and biochemical properties, chemotaxonomy and 16S rDNA sequences of this strain were studied. The strain showed faint yellow vegetative mycelium, spiral spore-bearing filaments and column spores with smooth surface. No pigment was produced in culture. The cell wall type I and sugar type C showed the strain with streptomyces character. A phylogenetic tree was constructed by comparing with the published 16S rDNA sequences of the related species and showed 99.6% identity of nucleotide sequence of 16S rDNA with Streptomyces lavendulae. From the polyphasic taxonomical view, the strain xjy falls into Streptomyces lavendulae. The optimum fermentation condition of strain xjy for producing the most effective ferment filtrate were cultured in 2% soybean flour, 2% glucose, 0.8% NaCl, 0.2% CaCO3, 0.32% (NH4)2 SO4, the initial pH of 7.0, at 28 degrees C and shaked at 180r/mim for 6d. These results are valuable to strain application, antibiotics purification and its industrialization.