Agroinfiltration methods (FWD)

1, Use the stab culture supplied as a starting material and inoculate 5 mI overnight culture
(28'C for agro):
-5 ml L-broth
-5 µI tetracycline (5 µl/ml final) (Depend on the constructs)
-5 µI kanamycin (50 µl/ml final)  (Depend on the constructs)

2, Use all the 1 ml of the overnight culture to inoculate 50 ml L-broth and grow overnight:
-50 ml L-broth
-50 µI tetracycline (5 µglml final)
-50 µI kanamycin (50 µglml final)
-500µI MES (10 mM final) [optional]
-I0 µI Acetosyringone (20 µM final)

3, Precipitate the bacteria and resuspend the pellet in solution containing:
-10 mM MgCI2
-10 mM MES
-100 µM Acetosyringone

4, Leave on the bench for 2 to 3 hours before agroinfiltration (or overnight).

5, Perform the infiltration with a 2ml syringe. Simply press the syringe (no needle) on the
underside of the leaf, and exert a counter-pressure with your finger on the other side.
Infiltrate the maximum number of leaves, avoid cotyledons.


From David Baulcombe's website

Small RNA Cloning Procedure (FWD)

Introduction

The small RNA cloning procedure is based on adapter ligation. The adapter oligonucleotides are used for priming reverse transcription and for defining the orientation and sequence of the cloned small RNAs. This protocol is isotope free, utilises unmodified small RNAs and is routinely used to characterise miRNAs and siRNAs from various plant tissues. The total nucleic acid (TNA) isolation is adapted from White and Kaper (1989). Our small RNA cloning protocol results from modifications of protocols originally published by the Tuschl, Bartel and Carrington groups (Elbashir et al., 2001; Pfeffer et al., 2003; Lau et al., 2001; Llave et al., 2002) and may be cited as Chappell et al., 2005.

Small RNA cloning protocol

The gel purified small RNAs are ligated directly to a non-phosphorylated 5’-adapter oligonucleotide using T4 RNA ligase. The ligation products are separated from the excess of 5’-adapter on a 15% denaturing polyacrylamide gel and are subsequently ligated to a 5’-phosphorylated 3’-adapter oligonucleotide with a blocked 3’-hydroxyl terminus. The final ligation products are separated from the excess of 3’-adapter and are subjected to reverse transcription and PCR amplification. The gel purified PCR products are digested with EcoRI and NcoI restriction enzymes and subsequently concatamerised using T4 DNA ligase. The concatamers are ligated into an EcoRI-NcoI digested cloning vector and then TOP10 cells are transformed with the recombinant plasmids. Individual colonies are screened for the size of concatamer inserts by PCR and selected PCR fragments are purified and submitted for sequencing. The small RNA sequences are extracted from the sequence manually or automatically using software tools (e.g., Staden Package or software developed in-house).

From :David Baulcombe's website

Books about Arabidopsis

Arabidopsis: A Laboratory Manual

Detef Weigel and Jane Glazebrook

Cold Spring Harbor Lab Press, 2002

A comprehensive,detailed laboratory manual for Arabidopsis including sections on plant growth, genetic analysis, proteomics, and genomics.

The Arabidopsis Book

Chris Somerville and Elliot Meyerowitz

American Society of Plant Biologists.

A web based dynamic compendium of Arabidopsis biology. Contributed chapters written by experts in their fields are available on-line and free of charge as portable document format (PDF) documents.

Arabidopsis : A Practical Approach. (2000)

Zoe Wilson ed.

Oxford University Press, Oxford, UK.

More protocols and methods

Arabidopsis: Annual Plant Reviews, Vol.1. (1998)

Mary Anderson and Jeremy Roberts, eds.

CRC Press, Boca Raton, FL, USA.

Arabidopsis. (1994)

Elliot M. Meyerowitz, Chris R. Somerville, eds.

CSHL Press, New York, USA.

A pretty comprehensive overview of Arabidopsology

Arabidopsis : an Atlas of Morphology and Development. (1993)

John L. Bowman ed.

Springer-Verlag, Berlin & New York.

Images and descriptions of normal and mutant Arabidopsis plants

Methods in Arabidopsis research. (1992)

Csaba Koncz, Nam-Hai Chua, Jeff Schell eds.

Protocols and methods for Arabidopsis researchers

History of Arabidopsis thaliana as a research organism

"Arabidopsis thaliana was discovered by Johannes Thal (hence, thaliana) in the Harz mountains in the sixteenth century, though he called it Pilosella siliquosa (and it has gone through a number of name changes since). The earliest report of a mutant (that I know of) was in 1873 (by A. Braun). F. Laibach first summarized the potential of Arabidopsis thaliana as a model organism for genetics in 1943 - he did some work on it much earlier though, publishing its correct chromosome number in 1907. The first collection of induced mutants was made by Laibach's student E. Reinholz. Her thesis was submitted in 1945, the work published in 1947. Langridge played an important role in establishing the properties and utility of the organism for laboratory studies in the 1950s, as did Rédei and others (such as J.H. van der Veen in the Netherlands, J. Veleminsky in Czechoslovakia and G. Röbbelen in Germany) in the 1960s. One of Rédei's many important contributions was to write scholarly reviews on Arabidopsis, a particularly thorough one is in Bibliographica Genetica vol 20, No. 2, 1970, pp. 1- 151. He wrote a more easily found one in Ann. Rev. Genet. (1975) vol. 9,111-127. Both go through some of the early history of the use of Arabidopsis in the laboratory, though the longer 1970 one has all the details." --from Elliot Meyerowitz, 1998

http://www.arabidopsis.org/portals/education/aboutarabidopsis.jsp#hist

Roger Beachy (Danforth Center) Part 2: Genetic Engineering for Virus Resistance in Plants

In the second part of the lecture, Beachy explains how different biotechnology strategies can be used to produce crops resistant to specific viral infections

Roger Beachy (Danforth Center) Part 2: Genetic Engineering for Virus Resistance in Plants

In the second part of the lecture, Beachy explains how different biotechnology strategies can be used to produce crops resistant to specific viral infections. 

Roger Beachy (Danforth Center) Part 1: Biology of Plant Virus Infection

This seminar describes the cell and molecular biology of plant virus infection. The first lecture will discuss how virus replication centers are set up in plants and how viruses use host cell mechanisms to facilitate cell to cell movement and eventual pathogenesis.

CASSAVA Brown Streak Disease part 2

CASSAVA Brown Streak virus

A deadly cassava virus-Cassava brown streak virus

A deadly cassava virus

International Potato Center (CIP)/Inoculacion/evaluacion de clones/progenies/papa-virus PVX/PVY/PLRV

Evaluation of Selected Clones for extreme resistance to PVY and PVX viruses - 4/5 to 2009

Evaluation of Selected Clones for extreme resistance to PVY and PVX viruses - 3/5 to 2009

Evaluation of Selected Clones for extreme resistance to PVY and PVX viruses - 2/5 to 2009

Evaluation of Selected Clones for extreme resistance to PVY and PVX viruses - 1/5 to 2009

Inoculacion/evaluacion de clones/progenies/papa-virus PVX/PVY/PLRV

Evaluacion de Clones Selectos para resistencia extrema a los virus PVY y PVX - 4/5 - 2009

International Potato Center (CIP)

Top 10 plant viruses in molecular plant pathology- from MOLECULAR PLANT PATHOLOGY

Top 10 plant viruses in molecular plant pathology

1. KAREN-BETH G. SCHOLTHOF
2.  , 
3. SCOTT ADKINS
4.  ,
5. HENRYK CZOSNEK
6.  , 
7. PETER PALUKAITIS
8.  ,
9. EMMANUEL JACQUOT
10.  , 
11. THOMAS HOHN
12.  ,
13. BARBARA HOHN
14.  , 
15. KEITH SAUNDERS
16.  , 
17. THIERRY CANDRESSE
18.  , 
19. PAUL AHLQUIST
20.  , 
21. CYNTHIA HEMENWAY
22.  , 
23. GARY D. FOSTER ^,*

http://onlinelibrary.wiley.com/doi/10.1111/j.1364-3703.2011.00752.x/abstract

Many scientists, if not all, feel that their particular plant virus should appear in any list of the most important plant viruses. However, to our knowledge, no such list exists. The aim of this review was to survey all plant virologists with an association with Molecular Plant Pathology and ask them to nominate which plant viruses they would place in a 'Top 10' based on scientific/economic importance. The survey generated more than 250 votes from the international community, and allowed the generation of a Top 10 plant virus list for Molecular Plant Pathology. The Top 10 list includes, in rank order, (1) Tobacco mosaic virus, (2) Tomato spotted wilt virus, (3) Tomato yellow leaf curl virus, (4) Cucumber mosaic virus, (5) Potato virus Y, (6) Cauliflower mosaic virus, (7) African cassava mosaic virus, (8) Plum pox virus, (9) Bromemosaic virus and (10) Potato virus X, with honourable mentions for viruses just missing out on the Top 10, including Citrus tristeza virus, Barley yellow dwarf virus, Potato leafroll virus and Tomato bushy stunt virus. This review article presents a short review on each virus of the Top 10 list and its importance, with the intent of initiating discussion and debate amongst the plant virology community, as well as laying down a benchmark, as it will be interesting to see in future years how perceptions change and which viruses enter and leave the Top 10.

Preparing Cassava for Mingling into Posho-Video from Youtube

Preparing Cassava for Mingling into Posho

I have studied cassava viruses for two years,unfortunately, I haven't seen the plant in the field. It is interesting they prepared food from cassava in the following video.

Video from Youtube

Cassava Brown Streak Virus

Cassava Brown Streak Virus-one big problem in East African

Before We thought there is only one Cassava brown streak virus, the origin of the virus in highland region was from lowland.Now studies showed that there are actually two distinct viruses:Cassava brown streak virus and Uganda Cassava brown streak virus. The complete genome of two virus are available couple of years ago.

This is one interesting virus I have been studied, I got many surprised from this virus, If time allow, I would like do more in future.

Fighting between googlefight and waste of time

Have you heard about the googlefight? Have you used it to choose the better words?
when I attended one writing course, the teacher has recommended us to use google fight to the which word are more popular. Today I just see one interesting fight between googlefight and waste of time. the later one is the winner, does it mean something? :-)

Difference between hypothetical proteins and predicted protein

what is the difference between hypothetical proteins and predicted protein?

and what is the similarity between them ? Is it like this:

The biological result showed the protein function as other protein, but no other result, we call it hypothetical protein, if we have the sequences we could predict it using the bioinformatic methods, we call it predicted protein. Is it right or wrong？

Homeobox

A homeobox is a DNA sequence found within genes that are involved in the regulation of patterns of anatomical development (morphogenesis) in animals, fungi and plants. Homeobox genes encode transcription factors that typically switch on cascades of other genes. The homeodomain binds DNA in a sequence-specific manner. However, the specificity of a single homeodomain protein is usually not enough to recognize only its desired target genes. Most of the time, homeodomain proteins act in the promoter region of their target genes as complexes with other transcription factors. Such complexes have a much higher target specificity than a single homeodomain protein. Homeodomains are encoded both by genes of the Hox gene clusters and by other genes throughout the genome. The homeobox domain was first identified in a number of drosophila homeotic and segmentation proteins, but is now known to be well-conserved in many other animals, including vertebrates.[4][5][6] Hox genes encode homeodomain-containing transcriptional regulators that operate differential genetic programs along the anterior-posterior axis of animal bodies.[7] The domain binds DNA through a helix-turn-helix (HTH) structure. The HTH motif is characterised by two alpha-helices, which make intimate contacts with the DNA and are joined by a short turn. The second helix binds to DNA via a number of hydrogen bonds and hydrophobic interactions, which occur between specific side chains and the exposed bases and thymine methyl groups within the major groove of the DNA.[6] The first helix helps to stabilise the structure. The motif is very similar in sequence and structure in a wide range of DNA-binding proteins (e.g., cro and repressor proteins, homeotic proteins, etc.). One of the principal differences between HTH motifs in these different proteins arises from the stereo-chemical requirement for glycine in the turn which is needed to avoid steric interference of the beta-carbon with the main chain: for cro and repressor proteins the glycine appears to be mandatory, while for many of the homeotic and other DNA-binding proteins the requirement is relaxed. from:http://en.wikipedia.org/wiki/Homeobox

How to determine the real protein structure

I think the real protein structure still is a difficult question in Nowadays. Although there are already quite a lot of 3D structrues in the database, they are mostly from crystallized protein using different methods. Most of them are not the structure when they are functional.there are also quite a lot of prediction software/web server, however, most of them are based on the database....

CELL Transcriptional Control of Gene Expression by MicroRNAs

Just listened the author's presentation, It is very good one, clear and interesting. the followsing is one paper he published on CELL in 2010,which is just small part of his talk.

During the discussion, there are some questions concerning the talk.

1, the specificity of the methylation,
2, the protein level changes whihc didn't mentioned in the talks and published papers.

► The moss DICER-LIKE1a (PpDCL1a) protein is required for miRNA biogenesis

► The related PpDCL1b protein is required for target cleavage but not miRNA biogenesis

► In PpDCL1b mutants, genes encoding miRNA targets are silenced by DNA methylation

► This epigenetic gene silencing is initiated by high miRNA to target RNA ratios

Summary

MicroRNAs (miRNAs) control gene expression in animals and plants. Like another class of small RNAs, siRNAs, they affect gene expression posttranscriptionally. While siRNAs in addition act in transcriptional gene silencing, a role of miRNAs in transcriptional regulation has been less clear. We show here that in moss Physcomitrella patens mutants without a DICER-LIKE1b gene, maturation of miRNAs is normal but cleavage of target RNAs is abolished and levels of these transcripts are drastically reduced. These mutants accumulate miRNA:target-RNA duplexes and show hypermethylation of the genes encoding target RNAs, leading to gene silencing. This pathway occurs also in the wild-type upon hormone treatment. We propose that initiation of epigenetic silencing by DNA methylation depends on the ratio of the miRNA and its target RNA.

http://www.cell.com/abstract/S0092-8674(09)01570-0

hypothetical proteins

Genes are either (i) homologous to genes of unknown function, and are typically referred to as “conserved hypothetical” genes, or (ii) do not have any known homologs termed“hypothetical” or “non characterized” or “unknown” because it is unclear whether they encode actual proteins. Since it is often unclear whether they encode actual proteins, the latter genes are commonly referred to as“hypothetical”, “uncharacterized”, or “unknown” proteins.

approaches developed for predicting protein function:
sequence similarity
phylogenetic profiles
protein-protein interactions
protein complexes
gene expression profiles

Classical way to infer function is based on sequence similarity using sequence database searching programs such as FASTA and PSI-BLAST.

Lack of sequence similarity in the database to the protein of interest creates difficulties for functional predictions. However, examples of dissimilar function for similar proteins are also available.

Approaches to predict protein fucntion by in silico methods are discussed HERE

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1891709/