1. Software dependencies and installation
<ol>
	<li>Install software dependencies from their home site or using conda.
	<ol>
		<li>Download and install Perl, if it is not already installed, from its home site (https://www.perl.org/get.html). 
		NOTE: Represented results were predicted using Perl v5.32.0.</li>
		<li>Download Blast+, an alignment program, from its home site (https://www.ncbi.nlm.nih.gov/books/NBK279671/) as an executable and as source code. 
		NOTE: Represented results were predicted using the BLAST 2.6.0+.</li>
		<li>Install precompiled package of RNAfold from https://www.tbi.univie.ac.at/RNA/.</li>
		<li>Alternatively, install these softwares using the following conda: i)&#160;conda install -c bioconda blast; ii) conda install -c bioconda viennarna.</li>
	</ol>
	</li>
</ol>
2. The mirMachine setup and testing
<ol>
	<li>Download the latest version of the mirMachine scripts and the mirMachine submission script from GitHub, https://github.com/hbusra/mirMachine.git, and then set the scripts path into the&#160;PATH.</li>
	<li>Use the test data provided at the GitHub to make sure that the mirMachine along with all its dependencies have been downloaded correctly.</li>
	<li>Run the mirMachine on the test data shown below. 
	bash mirMachine_submit.sh -f iwgsc_v2_chr5A.fasta -i mature_high_conf_v22_1.fa.filtered.fasta -n 10 
	NOTE: Set the -n option to 10 as the test data contains only one chromosome of the wheat genome. At defaults, the -n option is set to 20.</li>
	<li>Control the hairpins.tbl.out.tbl output files for the predicted mature miRNAs, their predicted precursors, and their locations on the chromosomes.</li>
	<li>Check the log files for the program outputs and warnings.</li>
</ol>
3. Homology-based miRNA identification
<ol>
	<li>Run the mirMachine using the bash script shown below: 
	bash mirMachine_submit.sh -f $genome_file -i $input_file -m $mismatches -n $number_of_hits</li>
	<li>Check the predicted miRNAs. Find the output file named $input_file.results.tbl.hairpins.tbl.out.tbl for the predicted miRNAs. Find the output file named $input_file.results.tbl.hairpins.fsa for the pre-miRNA FASTA sequences. Find the output file named $input_file.results.tbl.hairpins.log for the hairpin log file.</li>
</ol>
4. Novel miRNA identification
<ol>
	<li>Preprocess the sRNA-seq FASTQ files into proper FASTA format. Trim adaptors if needed. Do not trim low-quality reads; instead, remove them. Remove reads containing N. Convert the FASTQ file into FASTA file ($input_file).</li>
	<li>Run the mirMachine using the bash script shown below. 
	bash mirMachine_submit.sh -f $genome_file -i $input_file -n $number_of_hits -sRNAseq -lmax $lmax -lmin $lmin -rpm $rpm 
	NOTE: $mismatches was set to 0 for sRNA-seq based predictions.</li>
	<li>Check the predicted miRNAs. Find the output file named $input_file.results.tbl.hairpins.tbl.out.tbl for the predicted miRNAs. Find the output file named $input_file.results.tbl.hairpins.fsa for the pre-miRNA FASTA sequences. Find the output file named $input_file.results.tbl.hairpins.log for the hairpin log file.</li>
</ol>
5. Advance parameters
NOTE: The defaults are defined for all the parameters except for the genome file and the input miRNA file.
<ol>
	<li>Set the -db option to a blast database to skip the building reference database within the pipeline.</li>
	<li>Set the -m option to the number of mismatches allowed. 
	NOTE: At defaults, -m option was set to 1 for homology-based predictions and 0 for the sRNA-seq-based predictions.</li>
	<li>Set the -n to the number of hits to eliminate after alignment (default to 20). Change this based on the species.</li>
	<li>Use the -long to assess the secondary structures for the suspect list.</li>
	<li>Use the -s to activate the novel miRNA prediction based on sRNA-seq data.</li>
	<li>Set the -lmax option to the maximum length of the sRNA-seq reads to include in the screening.</li>
	<li>Set the -lmax option to the minimum length of the sRNA-seq reads to include in the screening.</li>
	<li>Use the -rpm option to set the Reads Per Million (RPM) threshold. 
	NOTE: For advanced parameters like the length of pri-miRNAs/pre-miRNAs, experienced users are encouraged to modify the scripts for their research of interest. Additionally, if the users intend to skip some steps or prefer to use modified outputs, the submission script can be modified by simply adding # at the beginning of the lines to skip those lines.</li>
</ol>

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Our miRNA pipeline, SUmir, has been used for the identification of many plant miRNAs for the last decade. Here, we developed a new, fully automated, and freely available miRNA identification and annotation pipeline, mirMachine. Furthermore, a number of miRNA identification pipelines including, but not limited to the previous pipeline, were dependent on UNAfold software21, which became a commercial software over time, although once being freely available. This new and fully automated mirMachine is ...

Advances in next generation sequencing technologies have widened the understanding of RNA structures and regulatory elements, revealing functionally important non-coding RNAs (ncRNAs). Among different types of ncRNAs, microRNAs (miRNAs) constitute a fundamental regulatory class of small RNAs with a length between 19 and 24 nucleotides in plants1,2. Since the discovery of the first miRNA in the nematode Caenorhabditis elegans3, the presence and the functions of miRNAs have been studied extensively in animal and plant genomes as well4,5,6. miRNAs function by targeting mRNAs for cleavage or translational repression7. Accumulating evidence has also shown that miRNAs are involved in a wide range of biological processes in plants including growth and development8, self-biogenesis9, and several biotic and abiotic stress responses10.
In plants, miRNAs are initially processed from long primary transcripts called pri-miRNAs11. These pri-miRNAs generated by RNA polymerase II inside the nucleus are long transcripts forming an imperfect fold-back structure12. The pri-miRNAs later undergo a cleavage process to produce endogenous single-stranded (ss) hairpin precursors of miRNAs called pre-miRNAs11. The pre-miRNA forms a hairpin-like structure wherein a single strand folds into a double-stranded structure to excise an miRNA duplex (miRNA/miRNA*)13. Dicer-like protein cuts both strands of the miRNA/miRNA* duplex, leaving 2-nucleotide 3&#39;-overhangs14,15. The miRNA duplex is methylated inside the nucleus, which protects the 3&#39;-end of the miRNA from degradation and uridylation activity16,17. A helicase unwinds the methylated miRNA duplex after export and exposes the mature miRNA to the RNA-induced silencing complex (RISC) in the cytosol18. One strand of the duplex is mature miRNA incorporated into RISC , whereas the other strand, miRNA*, is degraded. The miRNA-RISC complex binds to the target sequence leading to either mRNA degradation in case of full complementarity or translational repression in case of partial complementarity13.
Based on the expression and biogenesis features, guidelines for miRNA annotation have been described15,19. With the defined guidelines, Lucas and Budak developed the SUmir pipeline to perform a homology-based in silico miRNA identification in plants9. The SUmir pipeline was composed of two scripts: SUmirFind and SUmirFold. SUmirFind performs similarity searches against known miRNA datasets through National Center for Biotechnology Information (NCBI) Basic Local Alignment Search tool (BLAST) screening with modified parameters to include hits with only 2 or fewer mismatches and to avoid bias towards shorter hits (blastn-short -ungapped -penalty -1 -reward 1). SUmirFold evaluates the secondary structure of the putative miRNA sequences from BLAST20 results using UNAfold21. SUmirFold differentiates miRNAs from small interfering RNAs by the identification of the characteristics of hairpin structure. Moreover, it differentiates miRNAs from other ssRNAs such as tRNA and rRNA by the parameters, minimum fold energy index &#62; 0.67 and GC content of 24-71%. This pipeline has been recently updated by adding two additional steps to (i) increase sensitivity, (ii) increase annotation accuracy, and (iii) provide genomic distribution of the predicted miRNA genes22. Given the high conservation of plant miRNA sequences23, this pipeline was originally designed for homology-based miRNA prediction. Novel miRNAs, however, could not be accurately identified with this bioinformatics analysis as it heavily relied on sequence conservation of miRNAs between closely related species.
This paper presents a new and fully automated miRNA pipeline, mirMachine that 1) can identify known and novel miRNAs more accurately (for example, the pipeline now uses sRNA-seq-based novel miRNA predictions as well as homology-based miRNA identification) and 2) is fully automated and freely available. The outputs have also included the genomic distributions of the predicted miRNAs. mirMachine was tested for both homology-based and sRNA-seq-based predictions in wheat and Arabidopsis genomes. Although initially released as free software, UNAfold became a commercial software in the last decade. With this upgrade, the secondary structure prediction tool was switched from UNAfold to RNAfold so that mirMachine can be freely available. Users can now execute a short submission script to run the fully automated mirMachine pipeline (examples are provided at https://github.com/hbusra/mirMachine.git).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>https://www.ncbi.nlm.nih.gov/books/NBK279671/</td>
			<td></td>
			<td></td>
			<td>Blast+</td>
		</tr>
		<tr>
			<td>https://github.com/hbusra/mirMachine.git</td>
			<td></td>
			<td></td>
			<td>mirMachine submission script</td>
		</tr>
		<tr>
			<td>https://www.perl.org/get.html</td>
			<td></td>
			<td></td>
			<td>Perl</td>
		</tr>
		<tr>
			<td>https://www.tbi.univie.ac.at/RNA/</td>
			<td></td>
			<td></td>
			<td>RNAfold</td>
		</tr>
		<tr>
			<td>Arabidopsis TAIR10</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triticum aestivum (wheat, IWGSC RefSeq v2)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

mirmachine: a one-stop shop for plant mirna annotation

Of different types of noncoding RNAs, microRNAs (miRNAs) have arguably been in the spotlight over the last decade. As post-transcriptional regulators of gene expression, miRNAs play key roles in various cellular pathways, including both development and response to a/biotic stress, such as drought and diseases. Having high-quality reference genome sequences enabled identification and annotation of miRNAs in several plant species, where miRNA sequences are highly conserved. As computational miRNA identification and annotation processes are mostly error-prone processes, homology-based predictions increase prediction accuracy. We developed and have improved the miRNA annotation pipeline, SUmir, in the last decade, which has been used for several plant genomes since then. 
This study presents a fully automated, new miRNA pipeline, mirMachine (miRNA Machine), by (i) adding an additional filtering step on the secondary structure predictions, (ii) making it fully automated, and (iii) introducing new options to predict either known miRNA based on homology or novel miRNAs based on small RNA sequencing reads using the previous pipeline. The new miRNA pipeline, mirMachine, was tested using The Arabidopsis Information Resource, TAIR10, release of the Arabidopsis genome and the International Wheat Genome Sequencing Consortium (IWGSC) wheat reference genome v2.

The miRNA pipeline, mirMachine, described above was applied to the test data for the fast evaluation of the performance of the pipeline. Only the high-confidence plant miRNAs deposited at miRBase v22.1 were screened against the chromosome 5A of IWGSC wheat RefSeq genome v224. mirMachine_find returned 312 hits for the nonredundant list of 189 high-confidence miRNAs with a maximum of 1 mismatch allowed (Table 1). mirMachine_fold classified 49 of them as putative miRNAs depending on ...

Watch this Scientific Journal Video about mirMachine: A One-Stop Shop for Plant miRNA Annotation at JoVE.com

mirMachine: A One-Stop Shop for Plant miRNA Annotation

Herein, we present a new and fully automated miRNA pipeline, mirMachine that 1) can identify known and novel miRNAs more accurately and 2) is fully automated and freely available. Users can now execute a short submission script to run the fully automated mirMachine pipeline.

Of different types of noncoding RNAs, microRNAs (miRNAs) have arguably been in the spotlight over the last decade. As post-transcriptional regulators of ...

mirmachine-a-one-stop-shop-for-plant-mirna-annotation

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2.4K Views.  Crop Improvement and Genetics Research Unit, CA, USA. Herein, we present a new and fully automated miRNA pipeline, mirMachine that 1) can identify known and novel miRNAs more accurately and 2) is fully automated and freely available. Users can now execute a short submission script to run the fully automated mirMachine pipeline.

Video: mirMachine: A One-Stop Shop for Plant miRNA Annotation

Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, Brussel sprouts etc. Rearing of the insects takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Pieris rapae. One for the larvae and the other to contain the adults, the butterflies. In order to investigate the role of plant hormones and toxic plant chemicals in resistance to this insect pest, we demonstrate two experiments. First, determination of the role of jasmonic acid (JA - a plant hormone often indicated in resistance to insects) in resistance to the chewing insect Pieris rapae. Caterpillar growth can be compared on wild-type and mutant plants impaired in production of JA. This experiment is considered "No Choice", because larvae are forced to subsist on a single plant which synthesizes or is deficient in JA. Second, we demonstrate an experiment that investigates the role of glucosinolates, which are used as oviposition (egg-laying) signals. Here, we use WT and mutant Arabidopsis impaired in glucosinolate production in a "Choice" experiment in which female butterflies are allowed to choose to lay their eggs on plants of either genotype. This video demonstrates the experimental setup for both assays as well as representative results.

Plant resistance to chewing insect herbivores can be tested in several ways. Here, we demonstrate how to set-up a choice and a no-choice experiment with the model plant Arabidopsis thaliana to identify resistance against the pest species Pieris rapae.

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which ...

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

A &#946;-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

A &#946;-glucuronidase GUS Based Cell Death Assay

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Annotation of Plant Gene Function via Combined Genomics, Metabolomics and Informatics

Given the ever expanding number of model plant species for which complete genome sequences are available and the abundance of bio-resources such as knockout mutants, wild accessions and advanced breeding populations, there is a rising burden for gene functional annotation. In this protocol, annotation of plant gene function using combined co-expression gene analysis, metabolomics and informatics is provided (Figure 1). This approach is based on the theory of using target genes of known function to allow the identification of non-annotated genes likely to be involved in a certain metabolic process, with the identification of target compounds via metabolomics. Strategies are put forward for applying this information on populations generated by both forward and reverse genetics approaches in spite of none of these are effortless. By corollary this approach can also be used as an approach to characterise unknown peaks representing new or specific secondary metabolites in the limited tissues, plant species or stress treatment, which is currently the important trial to understanding plant metabolism.

Combination of genomics, co-expression gene analysis and the identification of target compounds via metabolism give gene functional annotation.

Given the ever expanding number of model plant species for which complete genome sequences are available and the abundance of bio-resources such as ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

mirMachine: A One-Stop Shop for Plant miRNA Annotation

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Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

A β-glucuronidase (GUS) Based Cell Death Assay

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Annotation of Plant Gene Function via Combined Genomics, Metabolomics and Informatics

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on ¹⁵N Metabolic Labeling and Quantitative Mass Spectrometry

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants