Zhensheng Kang

Cytology and ultrastructure of the infection of wheat spikes by Fusarium culmorum

The infection of wheat spikes by Fusarium culmorum, one of the agents responsible for wheat head blight, was examined by light and electron microscopy. Macroconidia of the pathogen germinated 6–12 h after inoculation on all host surfaces they contacted. Developing germ tubes did not infect host tissues immediately, but gave rise to hyphae that grew and branched on host surfaces. Hyphal networks were usually formed 2 d after inoculation (dpi) on the inner surfaces of lemma, glume and palea, but not on the outer surfaces of lemma, glume and rachis. Hyphae on the outer surfaces of lemma and glume often grew over their edges to reach their inner surfaces. Penetration of host tissues occurred by infection hyphae on the inner surfaces of lemma, glume and palea, and on the upper part of the ovary. Occasionally, the pathogen invaded the host tissues through stomatal openings on the inner surface. Thereafter, the pathogen spread downwards to rachilla and rachis node by inter- and intracellular growth from above infected tissues. From the rachis node, hyphae extended downward to rachis and upward to peduncle through vascular bundles and cortical parenchyma tissue. When the pathogen arrived at the rachis 4–5 dpi, hyphae grew upwards and downwards inter- and intracellularly in vascular bundles and cortical parenchyma tissue of the rachis. During colonisation of the wheat spike, a series of alterations occurred in host tissues, including degeneration of host cytoplasm and organelles, collapse of parenchyma cells, disintegration or digestion of host cell walls and appearance of electron-dense coating materials on vessel walls.

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.

High genome heterozygosity and endemic genetic recombination in the wheat stripe rust fungus

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat. Here we report a 110-Mb draft sequence of Pst isolate CY32, obtained using a ‘fosmid-to-fosmid’ strategy, to better understand its race evolution and pathogenesis. The Pst genome is highly heterozygous and contains 25,288 protein-coding genes. Compared with non-obligate fungal pathogens, Pst has a more diverse gene composition and more genes encoding secreted proteins. Re-sequencing analysis indicates significant genetic variation among six isolates collected from different continents. Approximately 35% of SNPs are in the coding sequence regions, and half of them are non-synonymous. High genetic diversity in Pst suggests that sexual reproduction has an important role in the origin of different regional races. Our results show the effectiveness of the ‘fosmid-to-fosmid’ strategy for sequencing dikaryotic genomes and the feasibility of genome analysis to understand race evolution in Pst and other obligate pathogens.

Differential gene expression in incompatible interaction between wheat and stripe rust fungus revealed by cDNA-AFLP and comparison to compatible interaction.

BackgroundStripe rust of wheat, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat worldwide. Due to special features of hexaploid wheat with large and complex genome and difficulties for transformation, and of Pst without sexual reproduction and hard to culture on media, the use of most genetic and molecular techniques in studying genes involved in the wheat-Pst interactions has been largely limited. The objective of this study was to identify transcriptionally regulated genes during an incompatible interaction between wheat and Pst using cDNA-AFLP techniqueResultsA total of 52,992 transcript derived fragments (TDFs) were generated with 64 primer pairs and 2,437 (4.6%) of them displayed altered expression patterns after inoculation with 1,787 up-regulated and 650 down-regulated. We obtained reliable sequences (>100 bp) for 255 selected TDFs, of which 113 (44.3%) had putative functions identified. A large group (17.6%) of these genes shared high homology with genes involved in metabolism and photosynthesis; 13.8% to genes with functions related to disease defense and signal transduction; and those in the remaining groups (12.9%) to genes involved in transcription, transport processes, protein metabolism, and cell structure, respectively. Through comparing TDFs identified in the present study for incompatible interaction and those identified in the previous study for compatible interactions, 161 TDFs were shared by both interactions, 94 were expressed specifically in the incompatible interaction, of which the specificity of 43 selected transcripts were determined using quantitative real-time polymerase chain reaction (qRT-PCR). Based on the analyses of homology to genes known to play a role in defense, signal transduction and protein metabolism, 20 TDFs were chosen and their expression patterns revealed by the cDNA-AFLP technique were confirmed using the qRT-PCR analysis.ConclusionWe uncovered a number of new candidate genes possibly involved in the interactions of wheat and Pst, of which 11 TDFs expressed specifically in the incompatible interaction. Resistance to stripe rust in wheat cv. Suwon11 is executed after penetration has occurred. Moreover, we also found that plant responses in compatible and incompatible interactions are qualitatively similar but quantitatively different soon after stripe rust fungus infection.

Generation and analysis of expression sequence tags from haustoria of the wheat stripe rust fungus Puccinia striiformis f. sp. Tritici

BackgroundStripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat (Triticum aestivum L.) worldwide. In spite of its agricultural importance, the genomics and genetics of the pathogen are poorly characterized. Pst transcripts from urediniospores and germinated urediniospores have been examined previously, but little is known about genes expressed during host infection. Some genes involved in virulence in other rust fungi have been found to be specifically expressed in haustoria. Therefore, the objective of this study was to generate a cDNA library to characterize genes expressed in haustoria of Pst.ResultsA total of 5,126 EST sequences of high quality were generated from haustoria of Pst, from which 287 contigs and 847 singletons were derived. Approximately 10% and 26% of the 1,134 unique sequences were homologous to proteins with known functions and hypothetical proteins, respectively. The remaining 64% of the unique sequences had no significant similarities in GenBank. Fifteen genes were predicted to be proteins secreted from Pst haustoria. Analysis of ten genes, including six secreted protein genes, using quantitative RT-PCR revealed changes in transcript levels in different developmental and infection stages of the pathogen.ConclusionsThe haustorial cDNA library was useful in identifying genes of the stripe rust fungus expressed during the infection process. From the library, we identified 15 genes encoding putative secreted proteins and six genes induced during the infection process. These genes are candidates for further studies to determine their functions in wheat-Pst interactions.

Molecular analysis of common wheat genes encoding three types of cytosolic heat shock protein 90 (Hsp90): functional involvement of cytosolic Hsp90s in the control of wheat seedling growth and disease resistance

Heat shock protein 90 (Hsp90) molecular chaperones play important roles in plant growth and responses to environmental stimuli. However, little is known about the genes encoding Hsp90s in common wheat. Here, we report genetic and functional analysis of the genes specifying cytosolic Hsp90s in this species. Three groups of homoeologous genes (TaHsp90.1, TaHsp90.2 and TaHsp90.3), encoding three types of cytosolic Hsp90, were isolated. The loci containing TaHsp90.1, TaHsp90.2 and TaHsp90.3 genes were assigned to groups 2, 7 and 5 chromosomes, respectively. TaHsp90.1 genes exhibited higher transcript levels in the stamen than in the leaf, root and culm. TaHsp90.2 and TaHsp90.3 genes were more ubiquitously transcribed in the vegetative and reproductive organs examined. Decreasing the expression of TaHsp90.1 genes through virus-induced gene silencing (VIGS) caused pronounced inhibition of wheat seedling growth, whereas the suppression of TaHsp90.2 or TaHsp90.3 genes via VIGS compromised the hypersensitive resistance response of the wheat variety Suwon 11 to stripe rust fungus. Our work represents the first systematic determination of wheat genes encoding cytosolic Hsp90s, and provides useful evidence for the functional involvement of cytosolic Hsp90s in the control of seedling growth and disease resistance in common wheat.

Stage-specific gene expression during urediniospore germination in Puccinia striiformis f. sp tritici

BackgroundPuccinia striiformis f. sp. tritici is an obligate biotrophic pathogen that causes leaf stripe rust on wheat. Although it is critical to understand molecular mechanisms of pathogenesis in the wheat stripe rust fungus for developing novel disease management strategies, little is known about its genome and gene functions due to difficulties in molecular studies with this important pathogen. To identify genes expressed during early infection stages, in this study we constructed a cDNA library with RNA isolated from urediniospores of P. striiformis f. sp. tritici germinated for 10 h.ResultsA total of 4798 ESTs were sequenced from the germinated urediniospore library and assembled into 315 contigs and 803 singletons. About 23.9% and 13.3% of the resulting 1118 unisequences were homologous to functionally characterized proteins and hypothetical proteins, respectively. The rest 62.8% unisequences had no significant homologs in GenBank. Several of these ESTs shared significant homology with known fungal pathogenicity or virulence factors, such as HESP767 of the flax rust and PMK1, GAS1, and GAS2 of the rice blast fungus. We selected six ESTs (Ps28, Ps85, Ps87, Ps259, Ps261, and Ps159) for assaying their expression patterns during urediniospore germination and wheat infection by quantitative real-time PCR. All of them had the highest transcript level in germinated urediniospores and a much less transcript level in un-germinated urediniospores and infected wheat tissues (1–7 dpi). The transcript level of Ps159 increased at later infection stages (6–7 dpi). Our data indicated that these genes were highly expressed in germinated urediniospores and may play important roles in fungal-plant interactions during early infection stages in the wheat stripe rust fungus.ConclusionGenes expressed in germinated urediniospores of P. striiformis f. sp. tritici were identified by EST analysis. Six of them were confirmed by quantitative real-time PCR assays to be highly expressed in germinated urediniospores.

A Secretory Protein of Necrotrophic Fungus Sclerotinia sclerotiorum That Suppresses Host Resistance

SSITL (SS1G_14133) of Sclerotinia sclerotiorum encodes a protein with 302 amino acid residues including a signal peptide, its secretion property was confirmed with immunolocalization and immunofluorescence techniques. SSITL was classified in the integrin alpha N-terminal domain superfamily, and its 3D structure is similar to those of human integrin α4-subunit and a fungal integrin-like protein. When S. sclerotiorum was inoculated to its host, high expression of SSITL was detected during the initial stages of infection (1.5–3.0 hpi). Targeted silencing of SSITL resulted in a significant reduction in virulence; on the other hand, inoculation of SSITL silenced transformant A10 initiated strong and rapid defense response in Arabidopsis, the highest expressions of defense genes PDF1.2 and PR-1 appeared at 3 hpi which was 9 hr earlier than that time when plants were inoculated with the wild-type strain of S. sclerotiorum. Systemic resistance induced by A10 was detected by analysis of the expression of PDF1.2 and PR-1, and confirmed following inoculation with Botrytis cinerea. A10 induced much larger lesions on Arabidopsis mutant ein2 and jar1, and slightly larger lesions on mutant pad4 and NahG in comparison with the wild-type plants. Furthermore, both transient and constitutive expression of SSITL in Arabidopsis suppressed the expression of PDF1.2 and led to be more susceptible to A10 and the wild-type strain of S. sclerotiorum and B. cinerea. Our results suggested that SSITL is an effector possibly and plays significant role in the suppression of jasmonic/ethylene (JA/ET) signal pathway mediated resistance at the early stage of infection.

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.

Identification of expressed genes during compatible interaction between stripe rust ( Puccinia striiformis ) and wheat using a cDNA library

BackgroundWheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat worldwide. To establish compatibility with the host, Pst forms special infection structures to invade the plant with minimal damage to host cells. Although compatible interaction between wheat and Pst has been studied using various approaches, research on molecular mechanisms of the interaction is limited. The aim of this study was to develop an EST database of wheat infected by Pst in order to determine transcription profiles of genes involved in compatible wheat-Pst interaction.ResultsTotal RNA, extracted from susceptible infected wheat leaves harvested at 3, 5 and 8 days post inoculation (dpi), was used to create a cDNA library, from which 5,793 ESTs with high quality were obtained and clustered into 583 contigs and 2,160 singletons to give a set of 2,743 unisequences (GenBank accessions: GR302385 to GR305127). The BLASTx program was used to search for homologous genes of the unisequences in the GenBank non-redundant protein database. Of the 2,743 unisequences, 52.8% (the largest category) were highly homologous to plant genes; 16.3% to fungal genes and 30% of no-hit. The functional classification of all ESTs was established based on the database entry giving the best E-value using the Bevans classification categories. About 50% of the ESTs were significantly homologous to genes encoding proteins with known functions; 20% were similar to genes encoding proteins with unknown functions and 30% did not have significant homology to any sequence in the database. The quantitative real-time PCR (qRT-PCR) analysis determined the transcription profiles and their involvement in the wheat-Pst interaction for seven of the gene.ConclusionThe cDNA library is useful for identifying the functional genes involved in the wheat-Pst compatible interaction, and established a new database for studying Pst pathogenesis genes and wheat defense genes. The transcription patterns of seven genes were confirmed by the qRT-PCR assay to be differentially expressed in wheat-Pst compatible and incompatible interaction.