An improved method for RNA extraction from urediniospores of and wheat leaves infected by an obligate fungal pathogen, Puccinia striiformis f. sp. tritici

Programmed cell death (PCD) is a physiological process to remove redundant or harmful cells, for the development of multicellular organisms, or for restricting the spread of pathogens (hypersensitive response). Metacaspases are cysteine-dependent proteases which play an essential role in PCD. Triticum aestivum metacaspase 4 (TaMCA4) is a type II metacaspase gene cloned from ‘Suwon11’ wheat, with typical structural features such as peptidase C14 caspase domain and a long linker sequence between the two subunits. Transient expression of TaMCA4 in tobacco leaves failed to induce PCD directly but enhanced cell death triggered by a mouse Bax gene or a candidate effector gene from Puccinia striiformis f. sp. tritici. Enhancement of PCD was also observed in wheat leaves co-bombarded with TaMCA4. When challenged with the avirulent race of P. striiformis f. sp. tritici, the expression level of TaMCA4 in wheat leaves was sharply upregulated, whereas the transcript level was not significantly induced by the virulent race. Moreover, knocking down TaMCA4 expression by virus-induced gene silencing enhanced the susceptibility of Suwon11 to the avirulent race of P. striiformis f. sp. tritici and reduced the necrotic area at infection sites.

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Distribution of Puccinia striiformis f. sp. tritici races and virulence in wheat growing regions of Kenya from 1970 TO 2014

Plant Disease ◽

10.1094/pdis-11-20-2341-re ◽

2021 ◽

Author(s):

Mercy Wamalwa ◽

Ruth Wanyera ◽

Julian Rodriguez-Algaba ◽

Lesley Boyd ◽

James Owuoche ◽

...

Keyword(s):

Stripe Rust ◽

Fungal Pathogen ◽

Puccinia Striiformis ◽

Yellow Rust ◽

Stripe Rust Resistance ◽

Scar Markers ◽

Seedling Resistance ◽

Sequence Characterized Amplified Region ◽

Triticum Spp ◽

Virulence Diversity

Stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici (Pst), is a major threat to wheat (Triticum spp.) production worldwide. The objective of this study was to determine the virulence of Pst races prevalent in the main wheat growing regions of Kenya, which includes Mt. Kenya, Eastern Kenya, and the Rift Valley (Central, Southern, and Northern Rift). Fifty Pst isolates collected from 1970 to 1992 and from 2009 to 2014 were virulence phenotyped using stripe rust differential sets, and 45 isolates were genotyped with sequence characterized amplified region (SCAR) markers to differentiate among the isolates and identify aggressive strains PstS1 and PstS2. Virulence corresponding to stripe rust resistance genes Yr1, Yr2, Yr3, Yr6, Yr7, Yr8, Yr9, Yr17, Yr25, Yr27 and the seedling resistance in genotype Avocet S were detected. Ten races were detected in the Pst samples obtained from 1970 to 1992, and three additional races were detected from 2009 to 2014, with a single race being detected in both periods. The SCAR markers detected both Pst1 and Pst2 strains in the collection. Increasing Pst virulence was found in the Kenyan Pst population, and that diverse Pst race groups dominated different wheat growing regions. Moreover, recent Pst races in east Africa indicated possible migration of some race groups into Kenya from other regions. This study is important in understanding Pst evolution and virulence diversity and useful in breeding wheat cultivars with effective resistance to stripe rust. Keywords: pathogenicity, Puccinia f. sp. tritici stripe (yellow) rust, Triticum aestivum

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Application of Double-Strand RNAs Targeting Chitin Synthase, Glucan Synthase, and Protein Kinase Reduces Fusarium graminearum Spreading in Wheat

Frontiers in Microbiology ◽

10.3389/fmicb.2021.660976 ◽

2021 ◽

Vol 12 ◽

Author(s):

Peng Yang ◽

Shu-Yuan Yi ◽

Jun-Na Nian ◽

Qing-Song Yuan ◽

Wei-Jie He ◽

...

Keyword(s):

Protein Kinase ◽

Fusarium Graminearum ◽

Fungal Pathogen ◽

Chitin Synthase ◽

Mycelium Growth ◽

Rnai Construct ◽

Glucan Synthase ◽

Kinase C ◽

Wheat Leaves

Controlling the devastating fungal pathogen Fusarium graminearum (Fg) is a challenge due to inadequate resistance in nature. Here, we report on the identification of RNAi molecules and their applications for controlling Fg in wheat through silencing chitin synthase 7 (Chs7), glucan synthase (Gls) and protein kinase C (Pkc). From transgenic Fg strains four RNAi constructs from Chs7 (Chs7RNAi−1, −2, −3, and −4), three RNAi constructs from Gls (GlsRNAi−2, −3, and −6), and one RNAi construct from Pkc (PkcRNAi−5) were identified that displayed effective silencing effects on mycelium growth in medium and pathogenicity in wheat spikes. Transcript levels of Chs7, Gls and Pkc were markedly reduced in those strains. Double-strand RNAs (dsRNAs) of three selected RNAi constructs (Chs7RNAi-4, GlsRNAi-6 and PkcRNA-5) strongly inhibited mycelium growth in vitro. Spray of those dsRNAs on detached wheat leaves significantly reduced lesion sizes; the independent dsRNAs showed comparable effects on lesions with combination of two or three dsRNAs. Expression of three targets Chs7, Gls, and Pkc was substantially down-regulated in Fg-infected wheat leaves. Further application of dsRNAs on wheat spikes in greenhouse significantly reduced infected spikelets. The identified RNAi constructs may be directly used for spray-induced gene silencing and stable expression in plants to control Fusarium pathogens in agriculture.

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Mutations in wheat leading to enhanced resistance to the fungal pathogen of yellow rust

Plant Protection Science ◽

10.17221/10324-pps ◽

2002 ◽

Vol 38 (SI 1 - 6th Conf EFPP 2002) ◽

pp. S73-S75

Author(s):

L.A. Boyd ◽

J.A. Howie ◽

T. Worland ◽

R. Stratford ◽

P.H. Smith

Keyword(s):

Fungal Pathogen ◽

Puccinia Striiformis ◽

Yellow Rust ◽

Field Resistance ◽

Deletion Mutants ◽

Molecular Characterisation ◽

Wheat Line ◽

Enhanced Resistance ◽

Negative Effect ◽

Mutant Lines

The isolation and study of plant resistance genes is revealing a story more complicated than the gene-for-gene hypothesis originally implied. The story of resistance is complicated even further by the discovery of genes that appear to have a negative effect on resistance. Early studies in the wheat line Hobbit ‘sib’ identified a number of chromosomes that reduced the level of field resistance to the fungal pathogen Puccinia striiformis f.sp. tritici, the causal agent of yellow rust on wheat. From a series of deletion mutants generated in Hobbit ‘sib’ a number of mutant lines were selected that gave enhanced resistance to yellow rust. The phenotypic, genetic and molecular characterisation of some of these mutants is presented.

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An improved method for RNA extraction from common bean seeds and validation of reference genes for qPCR

Cropp Breeding and Applied Biotechnology ◽

10.1590/1984-70332017v17n2a22 ◽

2017 ◽

Vol 17 (2) ◽

pp. 150-158 ◽

Cited By ~ 7

Author(s):

Wendell Jacinto Pereira ◽

Priscila Zaczuk Bassinello ◽

Claudio Brondani ◽

Rosana Pereira Vianello

Keyword(s):

Common Bean ◽

Reference Genes ◽

Rna Extraction ◽

Improved Method

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Development of SCAR Markers and an SYBR Green Assay to Detect Puccinia striiformis f. sp. tritici in Infected Wheat Leaves

Plant Disease ◽

10.1094/pdis-06-15-0693-re ◽

2016 ◽

Vol 100 (9) ◽

pp. 1840-1847 ◽

Cited By ~ 8

Author(s):

L. Gao ◽

H. X. Yu ◽

X. H. Kang ◽

H. M. Shen ◽

C. Li ◽

...

Keyword(s):

Detection Limit ◽

Puccinia Striiformis ◽

Pathogenic Fungus ◽

Sybr Green ◽

Post Inoculation ◽

Scar Markers ◽

Sequence Characterized Amplified Region ◽

Rust Pathogen ◽

Pcr Method ◽

Wheat Leaves

Stripe rust, caused by the pathogenic fungus Puccinia striiformis f. sp. tritici, is an important disease of wheat worldwide. A rapid and reliable detection of the pathogen in latent infected wheat leaves is useful for accurate and early forecast of outbreaks and timely application of fungicides for managing the disease. Using the previously reported primer pair Bt2a/Bt2b, a 362-bp amplicon was obtained from P. striiformis f. sp. tritici and a 486-bp amplicon was obtained from both P. triticina (the leaf rust pathogen) and P. graminis f. sp. tritici (the stem rust pathogen). Based on the sequence of the 362-bp fragment, two pairs of sequence characterized amplified region (SCAR) primers were designed. PSTF117/PSTR363 produced a 274-bp amplicon and TF114/TR323 produced a 180-bp amplicon from P. striiformis f. sp. tritici, whereas they did not produce any amplicon from P. triticina, P. graminis f. sp. tritici, or any other wheat-infecting fungi. The detection limit of PSTF117/PSTR363 was 1 pg/µl and TF114/TR323 was 100 fg/µl. Both SCAR markers could be detected in wheat leaves 9 h post inoculation. An SYBR Green RT-PCR method was also developed to detect P. striiformis f. sp. tritici in infected leaves with the detection limit of 1.0 fg DNA from asymptomatic leaf samples of 6 h after inoculation. These methods should be useful for rapid diagnosis and accurate detection of P. striiformis f. sp. tritici in infected wheat leaves for timely control of the disease.

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Application of Near-Infrared Spectroscopy to Quantitatively Determine Relative Content of Puccnia striiformis f. sp. tritici DNA in Wheat Leaves in Incubation Period

Journal of Spectroscopy ◽

10.1155/2017/9740295 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12 ◽

Cited By ~ 3

Author(s):

Yaqiong Zhao ◽

Yilin Gu ◽

Feng Qin ◽

Xiaolong Li ◽

Zhanhong Ma ◽

...

Keyword(s):

Infrared Spectroscopy ◽

Incubation Period ◽

Stripe Rust ◽

Near Infrared Spectroscopy ◽

Near Infrared ◽

Puccinia Striiformis ◽

Quantitative Detection ◽

Support Vector ◽

Infected Leaf ◽

Wheat Leaves

Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a devastating wheat disease worldwide. Potential application of near-infrared spectroscopy (NIRS) in detection of pathogen amounts in latently Pst-infected wheat leaves was investigated for disease prediction and control. A total of 300 near-infrared spectra were acquired from the Pst-infected leaf samples in an incubation period, and relative contents of Pst DNA in the samples were obtained using duplex TaqMan real-time PCR arrays. Determination models of the relative contents of Pst DNA in the samples were built using quantitative partial least squares (QPLS), support vector regression (SVR), and a method integrated with QPLS and SVR. The results showed that the kQPLS-SVR model built with a ratio of training set to testing set equal to 3 : 1 based on the original spectra, when the number of the randomly selected wavelength points was 700, the number of principal components was 8, and the number of the built QPLS models was 5, was the best. The results indicated that quantitative detection of Pst DNA in leaves in the incubation period could be implemented using NIRS. A novel method for determination of latent infection levels of Pst and early detection of stripe rust was provided.

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Genetic resistance to yellow rust infection of the wheat ear is controlled by genes controlling foliar resistance and flowering time

10.1101/2021.04.27.441654 ◽

2021 ◽

Author(s):

Laura Bouvet ◽

Simon Berry ◽

Paul Fenwick ◽

Sarah Holdgate ◽

Ian J Mackay ◽

...

Keyword(s):

Triticum Aestivum ◽

Genetic Analysis ◽

Flowering Time ◽

Genetic Control ◽

Puccinia Striiformis ◽

Yellow Rust ◽

Genetic Resistance ◽

Founder Population ◽

Foliar Resistance ◽

Wheat Leaves

Yellow rust (YR), or stripe rust, is a fungal infection of wheat (Triticum aestivum L.) caused by the pathogen Puccinia striiformis f. sp. tritici (Pst). While much research has focused on YR infection of wheat leaves, we are not aware of reports investigating the genetic control of YR resistance in other wheat structures, such as the ears. Here we use an eight-founder population to undertake genetic analysis of glume YR infection in wheat ears. Five quantitative trait loci (QTL) were identified, each explaining between 3.4% and 6.8% of the phenotypic variation. Of these, three (QYrg.niab-2D.1, QYrg.niab-4D.1 and QYrg.niab-5A.1) co-located with QTL for leaf YR resistance previously identified in the same population. Additional leaf YR resistance QTL previously identified in the population were not detected as controlling glume resistance, with the remaining two glume YR QTL linked to genetic loci controlling flowering time. The first, QYrg.niab-2D. 1, mapped to the major flowering time locus Photoperiod-D1 (Ppd-D1), with the early-flowering allele from the founder Soissons conferring reduced glume YR resistance. The second, QYrg.niab-4A.1, was identified in one trial only, and was located close to a flowering time QTL. This indicates earlier flowering results in increased glume YR susceptibility, likely due to exposure of tissues during environmental conditions more favourable for Pst infection. Collectively, our results provide first insights into the genetic control of YR resistance in glumes, ontrolled by subsets of QTL for leaf YR resistance and flowering time. This work provides specific genetic targets for the control of YR resistance in both the leaves and the glumes, and may be especially relevant in Pst-prone agricultural environments where earlier flowering is favoured.

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RT-LAMP has high accuracy for detecting SARS-CoV-2 in saliva and naso/oropharyngeal swabs from asymptomatic and symptomatic individuals

10.1101/2021.06.28.21259398 ◽

2021 ◽

Author(s):

Stephen P Kidd ◽

Dan Burns ◽

Bryony Armson ◽

Andrew D Beggs ◽

Emma L. A Howson ◽

...

Keyword(s):

Preparation Method ◽

Rna Extraction ◽

Improved Method ◽

Diagnostic Specificity ◽

Sample Preparation Method ◽

Viral Loads ◽

Healthcare Settings ◽

Frequent Interval ◽

Oropharyngeal Swab ◽

Asymptomatic Individuals

Previous studies have described RT-LAMP methodology for the rapid detection of SARS-CoV-2 in nasopharyngeal/oropharyngeal swab and saliva samples. Here we describe the validation of an improved simple sample preparation method for Direct SARS-CoV-2 RT-LAMP, removing the need for RNA extraction, using 559 swabs and 86,760 saliva samples from asymptomatic and symptomatic individuals across multiple healthcare settings. Using this improved method we report a diagnostic sensitivity (DSe) of 70.35% (95% CI 63.48-76.60%) on swabs and 84.62% (79.50-88.88%) on saliva, with diagnostic specificity (DSp) 100% (98.98-100.00%) on swabs and 100% (99.72-100.00%) on saliva when compared to RT-qPCR. Analysing samples with RT-qPCR ORF1ab CT values of <25 and <33 (high and medium-high viral loads, respectively), we found DSe of 100% (96.34-100%) and 77.78% (70.99-83.62%) for swabs, and 99.01% (94.61-99.97%) and 87.32% (80.71-92.31%) for saliva. We also describe RNA RT-LAMP (on extracted RNA) performed on 12,619 swabs and 12,521 saliva samples to provide updated performance data with DSe and DSp of 95.98% (92.74-98.06%) and 99.99% (99.95-100%) for swabs, and 80.65% (73.54-86.54%) and 99.99% (99.95-100%) for saliva, respectively. We also report on daily samples collected from one individual from symptom onset where both Direct and RNA RT-LAMP detected SARS-CoV-2 in saliva collected on all six days where symptoms were recorded, with RNA RT-LAMP detecting SARS-CoV-2 for an additional further day. The findings from these studies demonstrate that RT-LAMP testing of swabs and saliva is potentially applicable to a variety of use-cases, including frequent, interval-based testing of saliva from asymptomatic individuals via Direct RT-LAMP that may be missed using symptomatic testing alone.

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