This work was financially supported by the National Key R&#38;D Program of China (2020YFD1001200), the China Agriculture Research System (CARS-11), the specific research fund of The Innovation Platform for Academicians of Hainan Province (YSPTZX202154), the Natural Science Foundation of Hainan Province of China (321RC652), and the Natural Science Foundation of China (No. 31601626).

1. Preparation of black pepper cutting plants for infection
<ol>
	<li>Take a five-node cutting, approximately 40 cm long with a diameter of 0.5 cm, from a healthy and vigorously growing orthotropic branch of black pepper using a disinfected pruning knife or secateurs. Prune the lower three nodes of the plagiotropic branches, with the upper two nodes left with approximately 10 leaves intact.</li>
	<li>Prepare rooting substrate containing soil and animal manure (cow dung or sheep dung) at a ratio of 1:1. Autoclave the rooting substrate at 121 &#176;C for 20 min.</li>
	<li>Insert the cuttings into the rooting substrate at an angle of approximately 50&#176;, with the third node just touching the surface of the substrate and the axillary bud on this node above the substrate. 
	NOTE: The bag used here has the following dimensions: height of 40-60 cm, diameter of 25-30 cm.</li>
	<li>Pour 10-20 L of water over the roots of the plant. Place the cuttings in a greenhouse with 90% shade at a temperature of 25-30 &#176;C for rooting and growth.</li>
</ol>
2. Propagation of Phytophthora capsici (P. capsici)
NOTE: A stock of Phytophthora capsici culture is maintained in the plant protection laboratory of the Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences7.
<ol>
	<li>Brush and clean the potato tubers under running tap water and then cut 200 g of potatoes into cubes of 1 cm3. Place some of the cubes into a beaker containing 800 mL of double-distilled water (ddH2O) and boil for 20 min.</li>
	<li>Filter the broth through double gauze using gravity filtration. Prepare potato dextrose agar (PDA) by adding 20 g of dextrose and 15 g of agar to the filtrate and topping the volume up to 1 L with ddH2O. Autoclave the mixture at 121 &#176;C for 20 min8.</li>
	<li>Pour 20 mL of the sterilized PDA in liquid form into a 9 cm diameter round Petri dish inside a laminar air flow hood. Leave the PDA plates with lids open inside the laminar air flow hood overnight as a means of preventing condensation.</li>
	<li>Use an inoculation loop to pick up mycelia from Phytophthora capsici stock inside a test tube. Place the inoculum with the mycelial side in contact with the PDA in a Petri dish.</li>
</ol>
3. Infection of black pepper
<ol>
	<li>Incubation
	<ol>
		<li>Identify an area 5 cm above the substrate surface and near to the roots on the stem for inoculation.</li>
		<li>Core out a disk of mycelia 0.5 cm in diameter at the growing edge of the Phytophthora capsici culture on PDA in a Petri dish using a stopper borer.</li>
		<li>Damage the stem using a syringe needle and make three holes in a triangular pattern at the selected inoculation area. Cover each hole with a mycelial disk. Position the holes close to each other to ensure the wounded area is fully covered with the mycelial disks.</li>
		<li>Cover the mycelial disks with sterilized moistened cotton pads as a means of preventing drying. Tie the pad onto the stem with a polyethylene strip to maintain the position of the inoculating disks. 
		NOTE: At 8 h after inoculation, the inoculated holes turned black and the lesion extended as time passed. The leaves turned yellow and dropped off, and the inoculated plant died 7-10 days after inoculation. No lesions developed in the control plants. Most of the genes expressed differently following inoculation with Phytophthora capsici in comparison to the control group. Histopathological analysis of the infected tissues demonstrated that Phytophthora capsici colonized in the xylem.</li>
	</ol>
	</li>
	<li>Sample the plant materials of interest and store at -80 &#176;C in liquid nitrogen for use in subsequent studies. 
	NOTE: Liquid nitrogen, plastic bags, marker pens, branch shears, and other materials were prepared prior to the experiments.</li>
	<li>After the specific plant materials are sampled for use, autoclave all the remaining plant materials, left-over Phytophthora capsici culture and culture medium, and all the instruments and labware used in this inoculation work.</li>
</ol>

<ol>
	<li>Gordo, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+sequencing+of+black+pepper+root+transcriptome.">High-throughput sequencing of black pepper root transcriptome.</a> BMC Plant Biology. 12 (1), (2012).</li><li>Leonian, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+and+fruit+blight+of+Peppers+caused+by+Phytophthora+capsici+sp.+Nov.">Stem and fruit blight of Peppers caused by Phytophthora capsici sp. Nov.</a> Phytopathology. 12 (9), 401-408 (1922).</li><li>Ding, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priming+maize+resistance+by+its+neighbors:+Activating+1,4-benzoxazine-3-ones+synthesis+and+defense+gene+expression+to+alleviate+leaf+disease.">Priming maize resistance by its neighbors: Activating 1,4-benzoxazine-3-ones synthesis and defense gene expression to alleviate leaf disease.</a> Frontiers in Plant Science. 6, 830 (2015).</li><li>Fonseca, C. E. L., Vianda, D. R., Hansen, J. L., Pell, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+among+forage+quality+traits,+vigor,+and+disease+resistance+in+alfalfa.">Associations among forage quality traits, vigor, and disease resistance in alfalfa.</a> Crop Science. 39 (5), 1271-1276 (1999).</li><li>Altier, N. A., Thies, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+resistance+to+Pythium+seedling+disease+in+Alfalfa+using+a+culture+plate+method.">Identification of resistance to Pythium seedling disease in Alfalfa using a culture plate method.</a> Plant Disease. 79 (4), 341-345 (1995).</li><li>Pratt, R. G., Rowe, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+simplified+leaf+inoculation+procedures+for+identification+of+quantitative+resistance+to+Sclerotinia+trifoliorum+in+Alfalfa+seedling.">Evaluation of simplified leaf inoculation procedures for identification of quantitative resistance to Sclerotinia trifoliorum in Alfalfa seedling.</a> Plant Disease. 82 (10), 1161-1164 (1998).</li><li>Hao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+transcriptome+sequencing+of+black+pepper+(Piper+nigrum+L.)+and+an+analysis+of+genes+involved+in+phenylpropanoid+metabolism+in+response+to+Phytophthora+capsici.">De novo transcriptome sequencing of black pepper (Piper nigrum L.) and an analysis of genes involved in phenylpropanoid metabolism in response to Phytophthora capsici.</a> BMC Genomics. 17 (1), 1-14 (2016).</li><li>Dong, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+inoculation+and+classification+of+maize+ear+rot+caused+by+Fusarium+verticillioides.">Field inoculation and classification of maize ear rot caused by Fusarium verticillioides.</a> Bio-protocol. 8 (23), 3099 (2018).</li><li>English, J. T., Laday, M., Bakonyi, J., Schoelz, J. E., &#201;rsek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+molecular+characterization+of+species+hybrids+derived+from+induced+fusion+of+zoospores+of+Phytophthora+capsica+and+Phytophthora+nicotianae.">Phenotypic and molecular characterization of species hybrids derived from induced fusion of zoospores of Phytophthora capsica and Phytophthora nicotianae.</a> Mycological Research. 103 (8), 1003-1008 (1999).</li><li>Chatterjee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant+activity+of+some+phenolic+constituents+from+green+pepper+(Piper+nigrum+L.)+and+fresh+nutmeg+mace+(Myristica+fragrans).">Antioxidant activity of some phenolic constituents from green pepper (Piper nigrum L.) and fresh nutmeg mace (Myristica fragrans).</a> Food Chemistry. 101 (2), 515-523 (2007).</li><li>Pfender, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+sporangia+and+release+of+zoospores+by+Phytophthora+megasperma+in+soil.">Production of sporangia and release of zoospores by Phytophthora megasperma in soil.</a> Phytopathology. 67 (5), 657-663 (1977).</li><li>Nagila, A., Schutte, B. J., Sanogo, S., Idowu, O. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chile+pepper+sensitivity+to+mustard+seed+meal+applied+after+crop+emergence.">Chile pepper sensitivity to mustard seed meal applied after crop emergence.</a> HortScience. 56 (2), 1-7 (2021).</li><li>Lamour, K. 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In this study, the basal head was pinpricked to damage and provide an effective inoculation system in the black pepper plant. Mycelial pellets then covered the three holes to retain moisture and enable the pathogen to infect the plant well. Following inoculation, the leaves turned yellow and dropped off and the inoculated plants died. No lesions developed in the control plants. Most genes expressed differently following inoculation with Phytophthora capsici in comparison to the control group. Fungal diseases are responsible for structural and physiological disorders in a significant number of crops, leading to decreased productivity and economic losses for their producers. Structural studies employing histological techniques on the mode of penetration and colonization of plant tissues by fungi provide a detailed indication of the interactions between the pathogen and the plant tissue. These studies have revealed important aspects to help understand the monocycle of diseases. Histopathological analysis of infected tissues demonstrated Phytophthora capsici colonized in the xylem. This method provides a better means for solving the instability that is caused by traditional inoculation methods including soil drench or root dipping. An effective inoculation and precise sampling system for Phytophthora capsici on black pepper plants are essential for studying this plant-pathogen interaction. It also provides a promising means for studying the mode of action between plants and other soil-borne plant pathogens in agricultural precision breeding.
At the same time, this protocol represents a more efficient way of providing reference for the incubation of woody vine. In previous studies, pathogens were inoculated by root dipping with spore suspensions cultured in V8 medium9. It takes 7 days for the spore suspension to be ready, whereas the use of PDA to culture Phytophthora capsici takes just 5 days. The PDA plate was sealed using permeable surgical tape as a means of avoiding contamination from other bacteria and fungi. The cultures were kept at room temperature. The method used in this study can save more time and be performed more rapidly. Black pepper is a woody vine with many sugars and phenols10, and the zoospores produced by Phytophthora capsici generally occur in soils, making it difficult to infect black pepper vines and cause infection instability in the root11. This protocol provides better results, enabling a strong interaction between vine plants and soil-borne pathogens. The detection of the dynamic process between plants and pathogens is visible and convenient. 
The irrigating root method is fast and time-saving, but a problem remains unsolved for black pepper. Phytophthora capsici is a soil-borne pathogen that generally infects plant roots via sporangia and zoospores12. In nature, the sporangia is able to spread via rain and irrigation. Once the zoospores become attached to the plant surface, the germ tubes may quickly develop and penetrate the plant tissue, which results in infection13,14. This can cause uncertainty that choosing hyphae as an infection source will be similar to the spore suspension. The method used in this study starts with pinpricking the basal head of the black pepper plant to damage it. The damaged area is then covered with Phytophthora capsici and moisture is retained, ensuring that the pathogen can infect the plant well. This method is better at solving the instability caused by traditional inoculation methods including soil drench or root dipping. It is also a promising method for studying the mode of action between plants and other soil-borne plant pathogens in agricultural precision breeding.

Black pepper (Piper nigrum L.) is a woody climber and one of the most important spice crops. It is known as the "King of Spices"1 and is cultivated in over 40 countries and regions throughout Asia, Africa, and Latin America. Phytophthora root rot is the most devastating disease of black pepper, and is caused by the oomycete Phytophthora capsici. This pathogen also infects cucurbits, eggplants, chili peppers, and tomatoes2,3. With black pepper, an entire crop can sometimes be decimated by this disease. The expansion of pepper planting areas is restricted as a result of the unavailability of resistant varieties, which has significantly hindered the development of the Chinese black pepper industry. An effective inoculation and a precise sampling system for Phytophthora capsici on black pepper plants are essential for studying this plant-pathogen interaction.
The identification and screening of resistance in germplasm resources is the basic requirement for researching the pathogenicity of the pathogen and the breeding and utilization of resistant varieties. A widely used approach is to use a variety of identification methods based on plant species and pathogen groups. Current identification methods include population identification, individual identification, organ identification, tissue identification, cell identification, biochemical identification, and molecular identification, which have been developed in recent years4,5. There has been success in these areas, but there are also many problems. No matter which method is chosen, the basic requirements of plant resistance identification are consistent, including clear objectives, reliable results, and methods that are simple, rapid, and easy to standardize. This principle must also be followed in the identification of black pepper resistance.
In natural field conditions, the identification of disease resistance can be influenced by many environmental factors. Therefore, it was proposed that detached leaves and irrigated roots be used in the laboratory to identify disease resistance. Young leaves from healthy plants were inoculated in vitro in the laboratory, and the diseased leaves area was measured by inoculating the pathogen in order to identify the disease resistance of plants6. However, in vitro leaf inoculation can only be used for general resistance identification and not for molecular interaction studies. Despite this, the disease-resistant status often presents in the irrigated root inoculation, causing uncertainty in the follow-up study of molecular breeding for disease resistance. Therefore, fast and simple indoor detection methods are essential. This study aims to provide a method for resistance identification in the laboratory.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar powder</td>
			<td>Solarbio</td>
			<td>A8190</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean bench</td>
			<td>Haier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Xilong Scientific</td>
			<td>15700501</td>
			<td></td>
		</tr>
		<tr>
			<td>High temperature sterilizing oven</td>
			<td>Zaelway</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish plates</td>
			<td>Biosharp</td>
			<td>BS-90-D</td>
			<td></td>
		</tr>
	</tbody>
</table>

an effective inoculation method for phytophthora capsici on black pepper plants

Piper nigrum L. (black pepper) is a typical woody vine that is an economically important spice crop across the world. Black pepper production is significantly impacted by root rot disease caused by Phytophthora capsici, which has seriously influenced the industry development as a "choke point" problem. However, the molecular genetic mechanism of resistance in black pepper is unclear, leading to slow progress in the development of new black pepper varieties. An effective inoculation and precise sampling system for Phytophthora capsici on black pepper plants is essential for studying this plant-pathogen interaction. The main aim of this study is to demonstrate a detailed methodology where the basal head of black pepper is inoculated with Phytophthora capsici, while also providing a reference for the inoculation of woody vine plants. The basal head of the black pepper plant was pinpricked to damage it, and mycelial pellets covered the three holes to retain the moisture so the pathogen could infect the plant well. This method provides a better way of solving the instability that is caused by traditional inoculation methods including soil drench or root dipping. It also provides a promising means for studying the mode of action between plants and other soil-borne plant pathogens in agricultural precision breeding.

Figure 1 shows the symptoms of black pepper leaves following P. capsici inoculation. Figure 2 shows the symptoms of black pepper stems following P. capsici inoculation. The pathogen infected the black pepper at the basal stem; symptoms including leaf yellowing, wilting appearing, xylem browning, and vessel blacking gradually appearing.&#160;Figure 3 shows most of the genes expressed differently after inoculation with Phytophthora capsici compared with the control group. Figure 4 has demonstrated Phytophthora capsici colonized in the xylem by the histopathological analysis of infected tissues.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63002/63002fig01.jpg" /> 
Figure 1: The symptoms of black pepper leaves following P. capsici inoculation7. CK:&#160;control group; Inoculated: after inoculation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63002/63002fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63002/63002fig02.jpg" /> 
Figure 2: The symptom of black pepper stems following P. capsici inoculation7. Inoculated: after inoculation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63002/63002fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63002/63002fig03.jpg" /> 
Figure 3: Detailed expression profiles of the genes in the black pepper roots. Error bars in the figures denote the standard error of expression levels from three biological replications. CK-8, CK-12, CK-24, CK-48, 8, 12, 24, and 48 on the x-axis refer to 8, 12, 24, and 48 h on the control and 8, 12, 24, and 48 h, respectively, following inoculation with P. capsici. The y-axis represents the relative expression level compared with ubiquitin. Each column represents the mean value plus SD (standard deviation) from three biological replicates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63002/63002fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63002/63002fig04.jpg" /> 
Figure 4: Histopathological analysis of infected tissues. The comparison between toluidine blue O staining alone (left column) and cotton blue and safranin O double-staining (right column) (20X). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63002/63002fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants at JoVE.com

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

Pinpricking the basal head of the black pepper plant is a brief and time-saving method to damage it. Here, we provided detailed steps with a video for infecting black pepper plants.

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

Piper nigrum L. (black pepper) is a typical woody vine that is an economically important spice crop across the world. Black pepper production is ...

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Nanyang Technological University

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Genetics

3.2K Views.  Chinese Academy of Tropical Agricultural Science (CATAS). Pinpricking the basal head of the black pepper plant is a brief and time-saving method to damage it. Here, we provided detailed steps with a video for infecting black pepper plants.

Video: An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

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Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

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Single Molecule Analysis of Laser Localized Psoralen Adducts

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Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

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Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

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When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in regulating gene expression at the post-transcriptional level. Sequencing sRNA libraries by Next Generation Sequencing (NGS) methods has been widely employed to identify and analyze miRNA transcriptomes in the last decade, resulting in a rapid increase of miRNA discovery. However, two major challenges arise in plant miRNA annotation due to increasing depth of sequenced sRNA libraries as well as the size and complexity of plant genomes. First, many other types of sRNAs, in particular, short interfering RNAs (siRNAs) from sRNA libraries, are erroneously annotated as miRNAs by many computational tools. Second, it becomes an extremely time-consuming process for analyzing miRNA transcriptomes in plant species with large and complex genomes. To overcome these challenges, we recently upgraded miRDeep-P (a popular tool for miRNA transcriptome analyses) to miRDeep-P2 (miRDP2 for short) by employing a new filtering strategy, overhauling the scoring algorithm and incorporating newly updated plant miRNA annotation criteria. We tested miRDP2 against sequenced sRNA populations in five representative plants with increasing genomic complexity, including Arabidopsis, rice, tomato, maize and wheat. The results indicate that miRDP2 processed these tasks with very high efficiency. In addition, miRDP2 outperformed other prediction tools regarding sensitivity and accuracy. Taken together, our results demonstrate miRDP2 as a fast and accurate tool for analyzing plant miRNA transcriptomes, therefore a useful tool in helping the community better annotate miRNAs in plants.

A bioinformatics pipeline, namely miRDeep-P2 (miRDP2 for short), with updated plant miRNA criteria and an overhauled algorithm, could accurately and efficiently analyze microRNA transcriptomes in plants, especially for species with complex and large genomes.

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite cells. Satellite cells are characterized by the expression of the transcription factor Pax7 and their location underneath the basal lamina in the resting skeletal muscle. Upon injury, satellite cells get activated, undergo self-renewal or differentiation to either form new myofibers or to fuse with damaged ones. The functionality of satellite cells in vivo can be investigated using a cardiotoxin based injury model of skeletal muscle. To study the function of one gene during the regeneration of skeletal muscle, transgenic mouse models are mostly used. Here, we present an alternative method to transgenic mice, to investigate the gene function in satellite cells during regeneration, e.g., in cases where transgenic mice are not available. We combine the cardiotoxin mediated injury of a specific skeletal muscle with the injection of a self-delivering siRNA into the regenerating muscle which is then taken up by satellite cells among other cells. Thereby, we provide a method to analyze gene function in satellite cells during regeneration under physiological conditions without the need for transgenic mice.

We describe an in vivo method to investigate the functional loss of a specific gene in satellite cells by using a combination of cardiotoxin mediated injury of skeletal muscle and the injection of a self-delivering siRNA.

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite ...

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

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Chapters in this video

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

Single Molecule Analysis of Laser Localized Psoralen Adducts

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo