Lili Huang

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.

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.

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.

Characterization of a pathogenesis‐related thaumatin‐like protein gene TaPR5 from wheat induced by stripe rust fungus

Pathogenesis-related (PR) proteins, induced in plants in response to various biotic and abiotic stresses, have been assumed to play a role in plant defense system. Proteins of the PR5 family, also named thaumatin-like proteins (TLPs), have been detected in numerous plant species. In this research, a novel PR5 gene, designated as TaPR5, was isolated and characterized from wheat leaves (cv. Suwon 11) infected by the stripe rust pathotype CY23 (incompatible interaction) using the rapid amplification of cDNA ends (RACE). TaPR5 was predicted to encode a protein of 173 amino acids with an estimated molecular mass of 17.6 kDa and a theoretical pI of 4.64. The deduced amino acid sequence of TaPR5 showed a significant sequence similarity with PR5 and TLPs from barley and other plants and contained a putative signal peptide at the amino terminus. Southern blot analysis indicated that TaPR5 is coded by a single-copy gene. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed that TaPR5 transcript is significantly induced and upregulated in the incompatible interaction while in the compatible interaction a relative low level of the transcript was detected. TaPR5 was also induced by phytohormones (SA, JA and ABA) and stress stimuli (wounding, cold temperature and high salinity). Using an assay of onion epidermal cells indicated accumulation of TaPR5 protein in the apoplast. The immunocytochemical method showed that the TaPR5 protein was detected on cell walls of wheat leaves in the incompatible interaction at markedly higher labeling density compared with the compatible interaction.

Wheat TaNPSN SNARE homologues are involved in vesicle-mediated resistance to stripe rust (Puccinia striiformis f. sp. tritici)

Subcellular localisation of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and their ability to form SNARE complexes are critical for determining the specificity of vesicle fusion. NPSN11, a Novel Plant SNARE (NPSN) gene, has been reported to be involved in the delivery of cell wall precursors to the newly formed cell plate during cytokinesis. However, functions of NPSN genes in plant–pathogen interactions are largely unknown. In this study, we cloned and characterized three NPSN genes (TaNPSN11, TaNPSN12, and TaNPSN13) and three plant defence-related SNARE homologues (TaSYP132, TaSNAP34, and TaMEMB12). TaSYP132 showed a highly specific interaction with TaNPSN11 in both yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. We hypothesize that this interaction may indicate a partnership in vesicle trafficking. Expressions of the three TaNPSNs and TaSYP132 were differentially induced in wheat leaves when challenged by Puccinia striiformis f. sp. tritici (Pst). In virus-induced gene silencing (VIGS) assays, resistance of wheat (Triticum aestivum) cultivar Xingzi9104 to the Pst avirulent race CYR23 was reduced by knocking down TaNPSN11, TaNPSN13 and TaSYP132, but not TaNPSN12, implying diversified functions of these wheat SNARE homologues in prevention of Pst infection and hyphal elongation. Immuno-localization results showed that TaNPSN11 or its structural homologues were mainly distributed in vesicle structures near cell membrane toward Pst hypha. Taken together, our data suggests a role of TaNPSN11 in vesicle-mediated resistance to stripe rust.

Optimization of the Fermentation Process of Actinomycete Strain Hhs.015T

Strain Hhs.015T (Saccharothrix yanglingensis sp. nov.), an antagonistic endophytic Saccharothrix actinomycete isolated from roots of cucumber plants, exhibited a broad antimicrobial spectrum in vitro and was active as a biocontrol against plant diseases in field trials. The SSY medium was used for production of antimicrobial metabolites by strain Hhs.015T. However, this medium is too expensive for large-scale production. In this study, an alternative culture medium, based on agricultural waste products (e.g., apple pomace), was optimized. The results showed that the alternative medium contained 15 g apple pomace, 4 g rapeseed meal, 0.1 g KH2PO4, and 0.6 g MgSO4·7H2O in 1 L distilled water. This medium reduced the material costs by 91.5% compared to SSY medium. Response surface methodology (RSM) was used to investigate the influence of environmental variables on production of compounds of antimicrobial metabolites. The optimal conditions achieved were initial pH 7.0, medium volume of 90 mL in 250 mL flasks, rotary speed of 100 rpm, temperature 25°C, and inoculation volume of 15.8%. The antimicrobial activity was increased by 20% by optimizing the environmental parameters. The results obtained allow an efficient production of components with antimicrobial activity by strain Hhs.015T on a large scale at low costs.

Ultrastructural and cytochemical studies on the infection process of Sclerotinia sclerotiorum in oilseed rape

Stem rot, caused by Sclerotinia sclerotiorum (Lib.) de Bary, is one of the major diseases of oilseed rape worldwide. The infection process of S. sclerotiorum in leaves and stems and the alterations of cell wall components in the infected host tissues were examined by electron microscopy and cytochemical labelling techniques. One day after inoculating (dai) leaves and stems with fungal grown agar disks, dense mycelial networks were usually formed on the inoculated tissues. Then, the infection cushions of different size were developed. Mucilage produced by the pathogen covered mycelium and infection cushions. Hyphae forming infection cushions were often flattened and increased in diameter. After removing the infection cushions, the diameters of the numerous pores through which penetration pegs had entered the cuticle of leaves and stems were small and had almost the same diameter. Penetration of leaves and stems occurred 2 and 3 dai, respectively. Small changes in cuticle were observed. After penetration, hyphae of the pathogen extended between the cuticle and epidermal cell walls as well as inside the epidermal cell walls. Then inter- and intra-cellularly spreading hyphae were observed in the hemi- and ultra-thin sections by light- and transmission electron microscopy, 5 dai. Hyphae also colonized xylem and phloem. During colonization marked alterations in the host tissues were detected, including disorganization of cytoplasm, cell organelles, disintegration of cell walls and collapse of host cells. The enzyme- and immunogold-labelling investigations showed obvious degradation of cellulose, xylan and pectin in the host cell walls of infected tissues. The degradation of cell wall components suggests that the pathogen may secrete cell wall degrading enzymes (Cwdes) such as cellulases, xylanases and pectinases during infection and spreading in the oilseed rape tissues.ZusammenfassungDie Weißstängeligkeit, verursacht durch Sclerotinia sclerotiorum (Lib.) de Bary, ist weltweit eine der bedeutendsten Krankheiten an Raps. Der Infektionsprozess an Blättern und Stängeln sowie Veränderungen der Zellwandkomponenten in infizierten Wirtsgeweben wurde mit Hilfe der Elektronen-mikroskopie und cytochemischen Markierungstechniken untersucht. Einen Tag nach Inokulation (dai) der Blätter und Stängel mit pilzbewachsenen Agarscheiben entwickelte sich ein dichtes Myzel auf den inokulierten Geweben. Anschlie-ßend bildeten sich Infektionskissen unterschiedlicher Größe. Das vom Pathogen produzierte Mucigel umgab Hyphen und Infektionskissen. Infektionskissen-bildende Hyphen waren oft abgeflacht und wiesen einen größeren Durchmesser auf. Nach Entfernung der Infektionskissen zeigte sich, dass die zahlreichen Poren, durch die Penetrationshyphen in die Kutikula der Blätter und Stängel eingedrungen waren, einen geringen und einheitlichen Durchmesser aufwiesen. Blätter und Stängel wurden 2 bzw. 3 Tage nach Inokulation penetriert. Geringe Veränderungen an der Kutikula wurden beobachtet. Nach der Penetration breiteten sich die Hyphen zunächst subkutikulär und innerhalb der Epidermiszellwände aus. Die sich anschließend inter- und intrazellulär ausbreiten-den Hyphen wurden 5 dai in Hemi- und Ultradünnschnitten mit dem Licht- und Transmissionselektronenmikroskop untersucht. Die Hyphen besiedelten ebenfalls Xylem und Phloem. In den befallenen Geweben wurden deutliche Veränderungen wie Desorganisation der Zellwände und Kollaps der Wirtszellen nachgewiesen. Die Studien der Enzym- und Immuno-goldmarkierung ergaben deutliche Abbauerscheinungen von Zellulose, Xylan und Pektin in den Zellwänden infizierter Gewebe. Der Abbau der Zellwandkomponenten deutet an, dass S. sclerotiorum imstande ist, Zellwand-abbauende Enzyme wie Zellulasen, Xylanasen und Pektinasen in Geweben der Rapspflanzen auszuscheiden.

Evaluation of endophytic bacterial strains as antagonists of take-all in wheat caused by Gaeumannomyces graminis var. tritici in greenhouse and field

Six endophytic bacterial strains from wheat roots, selected from extensive screening tests, were studied in greenhouse pot experiments against take-all disease of wheat, caused by Gaeumannomyces graminis var. tritici (Ggt). For comparison, the fungicide triadimefon was included. All six bacterial strains and triadimefon significantly reduced take-all in wheat plants between 39.4 and 57.3% compared to the inoculated control. Treatment with the bacterial strains also significantly increased growth parameters of roots (e.g., average length, fresh, and dry weight) in comparison with the inoculated control. One of the endophytic bacterial strains, designated EDR4, which proved to be most consistent in further greenhouse tests, was also examined in field trials against take-all in wheat. In the field experiment 2006/2007, height of wheat plants in Ggt inoculated plots was significantly reduced compared to the non-inoculated treatments. Yield parameters such as kernels per head and thousand kernel weight (TKW) in the inoculated control were lower compared to other treatments. In the inoculated plots, individual treatments with the strain EDR4 increased yield compared to the inoculated control. In the field trial 2007/2008 treatments with strain EDR4 as well as the fungicide triadimefon reduced take-all by 49.6 and 61.9%, respectively, compared to the inoculated control. At harvest, in the inoculated control plant height, seeds per head and TKW were significantly lower compared to the other treatments. Treatments with EDR4 eliminated the detrimental effects of take-all on grain yield to a similar degree as triadimefon application. The strain EDR4 distinctly inhibited growth of Ggt in vitro and was identified as Bacillus subtilis on the basis of its morphology and 16S rRNA sequence analysis as well as physiological and biochemical characteristics. These results indicate that the strain EDR4 has potential in directly controlling take-all disease of wheat.

Endophytic Bacillus subtilis Strain E1R-J Is a Promising Biocontrol Agent for Wheat Powdery Mildew

In this study, the biocontrol efficacies of 14 endophytic bacterial strains were tested against Blumeria graminis f. sp. tritici (Bgt) in pot experiments under greenhouse conditions. Bacillus subtilis strain E1R-j significantly reduced disease index and exhibited the best control (90.97%). When different formulations of E1R-j were sprayed 24 h before Bgt inoculation, fermentation liquid without bacterial cell and crude protein suspension displayed the similar effects; and they reduced disease index more than bacterial cell suspension (109 cfu mL−1) and fermentation liquid without protein. The control effects were not significantly different between 1011 and 109 cfu mL−1 of bacterial cell suspension but were higher than 107 cfu mL−1. Further observations showed that conidial germination and appressorial formation of Bgt were retarded by spraying E1R-j 24 h before Bgt inoculation. Compared with the water check, conidial germination and appressorial formation were decreased by 43.3% and 42.7%, respectively. In the treatment with E1R-j, the number of houstoria significantly reduced and the speed of mycelial extension was slowed down in the wheat leaves. Scanning electron microscopy observation revealed that E1R-j significantly suppressed the conidial germination and caused rupture and deformation of germ tubes. On the surface of wheat leaves, mycelia and conidiophores became shrinking.

Correction: cDNA-AFLP analysis reveals differential gene expression in compatible interaction of wheat challenged with Puccinia striiformis f. sp. tritici

Background: Puccinia striiformis f. sp. tritici is a fungal pathogen causing stripe rust, one of the most important wheat diseases worldwide. The fungus is strictly biotrophic and thus, completely dependent on living host cells for its reproduction, which makes it difficult to study genes of the pathogen. In spite of its economic importance, little is known about the molecular basis of compatible interaction between the pathogen and wheat host. In this study, we identified wheat and P. striiformis genes associated with the infection process by conducting a large-scale transcriptomic analysis using cDNA-AFLP. Results: Of the total 54,912 transcript derived fragments (TDFs) obtained using cDNA-AFLP with 64 primer pairs, 2,306 (4.2%) displayed altered expression patterns after inoculation, of which 966 showed up-regulated and 1,340 downregulated. 186 TDFs produced reliable sequences after sequencing of 208 TDFs selected, of which 74 (40%) had known functions through BLAST searching the GenBank database. Majority of the latter group had predicted gene products involved in energy (13%), signal transduction (5.4%), disease/defence (5.9%) and metabolism (5% of the sequenced TDFs). BLAST searching of the wheat stem rust fungus genome database identified 18 TDFs possibly from the stripe rust pathogen, of which 9 were validated of the pathogen origin using PCR-based assays followed by sequencing confirmation. Of the 186 reliable TDFs, 29 homologous to genes known to play a role in disease/defense, signal transduction or uncharacterized genes were further selected for validation of cDNA-AFLP expression patterns using qRT-PCR analyses. Results confirmed the altered expression patterns of 28 (96.5%) genes revealed by the cDNA-AFLP technique. Conclusion: The results show that cDNA-AFLP is a reliable technique for studying expression patterns of genes involved in the wheat-stripe rust interactions. Genes involved in compatible interactions between wheat and the stripe rust pathogen were identified and their expression patterns were determined. The present study should be helpful in elucidating the molecular basis of the infection process, and identifying genes that can be targeted for inhibiting the growth and reproduction of the pathogen. Moreover, this study can also be used to elucidate the defence responses of the genes that were of plant origin. Published: 30 June 2009 BMC Genomics 2009, 10:289 doi:10.1186/1471-2164-10-289 Received: 26 November 2008 Accepted: 30 June 2009 This article is available from: http://www.biomedcentral.com/1471-2164/10/289