Bezanilla Lab
Moss Methods
The moss Physcomitrella patens is a powerful model system to study plant gene function. Unique among plants, moss performs efficient homologous recombination enabling precise genetic manipulation, including gene deletions and allele replacements. With a completely sequenced genome, facile genetic alteration, and a relatively simple developmental pattern, moss provides an unparalleled tool for dissecting plant gene function. Moss is simple to propagate and transform, and requires little space to maintain. This lab manual contains information about propagation, transformation, and DNA and protein isolation.


 

 


Moss Propagation

Moss should be propagated weekly. A detailed explanation can be found in the moss manual.

Protoplasting
By removing the cell walls from moss tissue, it is possible to generate a suspension of single cells called protoplasts. Moss protoplasts can be transformed with DNA and then regenerated into whole plants. For details on this procedure, please refer to the moss manual.

         
Growth Assay
Moss protoplasts given the proper growth conditions will within a few days regenerate into a whole plant. Thousands of plants can be regenerated. Taking pictures of the plants, it is possible to get measurements of overall plant size and shape.
RNAi Assay
Moss performs RNAi-induced gene silencing. Double stranded RNA can be generated by transformation of DNA constructs containing inverted repeats of the sequence targeted for gene silencing. My laboratory uses a robust RNAi assay that enables rapid identification of gene function.
Complementation Assay
Complementation studies provide validation of loss-of-function studies and mechanistic insights into gene function. By targeting untranslated regions of sequence, RNAi-induced phenotypes can be rescued by expression of a construct containing only the open reading frame.
   

 

 

 

 

Plasmids
General disclaimer: plasmid sequences are not always perfect, when we know of a discrepancy, we will try and note it.
Expression plasmids
For expression of your favorite protein using the maize ubiquitin promoter. Can be used for transient overexpression or for generating a stable line. All vectors have moss sequences for homologous recombination based targeting. When exogenous sequences are inserted into these loci, there is no affect on growth or development. Cloning into these vectors is mediated by Gateway recombination based cloning from Invitrogen.

pTHUBIgate – hygromycin resistant in plants, targets the Pp108 locus

pTKUBIgate – kanamycin resistant in plants, targets a redundant copy of the ARPC2 gene
pTZUBIgate – zeocin resistant in plants, targets a redundant copy of the ARPC3 gene
RNAi vectors (for details refer to moss manual)
For expression of inverted repeat constructs to generate double stranded RNA
pUGi – control RNAi vector, hygromycin resistance in plants
pUGGi – destination vector for cloning fragments in inverted repeats using LR clonase, hygromycin resistance in plants
pZUGi – control RNAi vector, zeocin resistance in plants
pZUGGi – destination vector for cloning fragments in inverted repeats using LR clonase, zeocin resistance
2-fragment recombination (for details refer to moss manual)
For generating epitope tagged versions of your favorite protein.
mEGFP-L1R5 – element 1 for N-terminal tagging
mEGFP-L5L2 – element 2 for C-terminal tagging
mCherry-L1R5 – element 1 for N-terminal tagging
mCherry-L5L2 – element 2 for C-terminal tagging
3XFLAG-L5L2 – element 2 for C-terminal tagging

Destination Vectors:
Any gateway-enabled destination vector (for example pTHUBIgate, pTKUBIgate, or pTZUBIgate).

   
3-fragment recombination for generating knock out constructs (refer to moss manual)
element 2 resistance cassettes:
nos_Lox_Hygro_Lox-R4R3
nos_Lox_Zeo_Lox-R4R3
nos_Lox_Kan_Lox-R4R3
destination vector:
pGEM-Gate link to map and sequence
4-Fragment recombination for generating knock in constructs (refer to moss manual)
element 2 for C-terminal tagging gene at locus:
mEGFP-L5L4
3XmEGFP-L5L4
mCherry-L5L4
element 3 resistance cassettes are the same as the vectors above used for generating knock outs

destination vector is the same as the vector above used for generating knock outs

Moss Lines

NLS4

Golgi

Vacuole


kanamycin resistant line expressing GFP:GUS localized to the nucleus

hygromycin resistant line expressing YFP targeted to the Golgi

kanamycin resistant line expressing Vam3-mEGFP (mEGFP targeted to the tonoplast membrane)

ER

Microtubules

Actin



hygromycin resistant line expressing mEGFP targeted to the endoplasmic reticulum.

kanamycin resistant line expressing mEGFP-tubulin.

hygromycin resistant line expressing Lifeact-mEGFP


Peroxisome

hygromycin resistant line expressing mEGFP targeted to the peroxisome

Resources

Moss Resources

The Physcomitrella patens genome website
http://www.phytozome.net/physcomitrella

Moss resources available from Germany
http://www.cosmoss.org

Moss resources available from Japan
http://moss.nibb.ac.jp

Moss Research in the U.S.:

Axtell Lab
http://axtell-lab-psu.weebly.com/

McDaniel Lab
http://mcdaniellab.wordpress.com/

Quatrano Lab
http://www.biology.wustl.edu/faculty/quatrano/index.php

Estelle Lab: Mike's moss resources
http://labs.biology.ucsd.edu/estelle/moss2.html

Theg Lab
http://www-plb.ucdavis.edu/labs/Theg

Vidali Lab
http://users.wpi.edu/~lvidali/

 

Generally Helpful Biology Resources

The National Center for Biotechnology Information
http://www.ncbi.nlm.nih.gov

European Bioinformatics Institute
http://www.ebi.ac.uk

Has a great clustalw server for multiple sequence analysis
http://www.ebi.ac.uk/Tools/clustalw2/index.html

Expasy – excellent bioinformatics tools
http://www.expasy.ch

Coiled-coil prediction software, COILS
http://www.ch.embnet.org/software/COILS_form.html

Coiled-coil prediction software, Paircoil2
http://groups.csail.mit.edu/cb/paircoil2

Molecular Toolkit from Colorado State
http://www.vivo.colostate.edu/molkit/index.html

Kyte Doolittle Hydrophobicity Plots
http://gcat.davidson.edu/rakarnik/KD.html

Great free software for DNA manipulation for Macs
http://serialbasics.free.fr/Serial_Cloner.html

Great free software for sequence chromatograms
http://mekentosj.com/science/4peaks

MultiSite gateway technology from Invitrogen
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning/GatewayC-Misc/MultiSite-Gateway-Pro-Kits.html