Curricular Resources
Host-parasite Phylogeny Workbench Investigations
This lab focuses on constructing molecular phylogenies of Xanthomonas
phytopathogens and the hosts that they target.
Objectives:
Use the Biology Workbench to construct phylogenies of Xanthomonas and its
plant hosts.
Compare host and parasite trees to each other and assess the role of
cospeciation in the history of the groups.
[The following workbench protocol is adapted from the West Nile Virus
workspace (author: Stacey Kiser)]
Warning: The Biology Workbench pages are research-level web sites. As
students we will use default settings for most of the software. You will also have
to be patient and scroll many times to find the control buttons. Ask for assistance
when you need help or get stuck.
Step 1: Starting your own session
Go to the Biology Workbench and select the Enter the Biology Workbench link to
begin a session.
Select the Set up a free account link and follow the instructions to set up your
own free account. You are now able to work on your problem (or future sequence
problems) from any internet computer. Saved sequences and alignments will be
saved on the supercomputer site at San Diego State University for some time
(months or years).
Record your account name _____________________
The most basic structure of the site asks you to choose a specific set of Tools,
which offers you a specific set of commands. Scroll down the information screen
that comes up after you log in. Near the bottom you will find the Tool Bar.
Choose Session Tools to begin a new session for your lab group.
To start a new session, select the Start New Session option in the choice window
and hit the Run button. It will ask you to type in a name for your session. Enter
something that identifies your group and the exercise, then hit the Start New
Session button. You will then return to the above web page. Your new session
name should be highlighted (instead of the Default Session example above). If it
is not highlighted, scroll down through the list of session names and click on the
grey button next to your session name to highlight it.
Your group name is ___________________________
You all can access your group's session from any internet connection again by
simply logging in to my account and selecting your group's session name at this
web page. Select the Resume Session choice then Run to get back into your
previous session.
Step 2: Obtaining DNA Sequences
Make sure that the radio button next to your new session name is selected, then
click on the Alignment Tools button. Run. Now click the Add New Aligned
Sequence button. Run.
Download from Bedrock the nucleic acid file (lrp_DNA_align) data file to your
desktop.
Browse and upload the file lrp_DNA_align. This file contains previously collected
lrp sequence data for various Xanthomonas taxa and two outgroups
(Pseudomonas and Ralstonia). Select this alignment.
A summary table of these data (Xanthomonas_taxa_table) is available below.
Step 3: Viewing and interpreting the alignment.
Select BOXSHADE – Color-Coded Plots of Pre-Aligned Sequences. Run. Keep
default settings (for now…you can play later). Click Submit.
Do all of the sequences have the same number of nucleotides?
Where do you notice missing data (dashes)? Do you think all are
insertions/deletions?
Note where differences tend to occur? Many changes occur at every third
position. Why?
Click Return when you are done examining the sequences.
Step 4: Producing a Phylogeny
Click DRAWGRAM – Draw Rooted Phylogenetic Tree From Alignment. Run. Use
default settings on the page that appears. Click Submit. Scroll down to visualize
your phylogenetic tree.
Note: The rooting is somewhat arbitrary in this tree, but necessary for easy
visualization.
Questions:
1. Are all Xanthomonas axonopodis strains closely related to each other?
2. Are the strains that infect citrus all found together?
3. Do you see evidence of host shifting?
4. Species that include the name vesicatoria all infect tomatoes. What do you
notice about their distribution in the phylogeny.
POTENTIAL INVESTIGATIONS
Choosing useful genes
Many genes could potentially be chosen to address Xanthomonas
interrelationships—the leucine-responsive regulatory protein [lrp] is only one of
them. Lrp, a global metabolic regulator protein, is not directly involved in
Xanthomonas pathogenicity (see Cubero & Graham, 2004), and hopefully acts
as a neutral marker that has simply kept a record of the evolution of the group.
Goncalves & Rosato (2002) sequenced the 16s-23s rDNA intergenic spacer
regions (its1, its2, its3) of 21 Xanthomonas, The taxa they chose are not identical
to those of Cubero & Graham (2004). See the West Nile Virus problem space
(author: Stacey Kiser) steps 2 through 4 to learn how to use Biology Workbench
to find DNA sequences that have not been gathered for you. You will of course
have to use different search terms to find these data.
Choosing a locus believed to be involved in pathogenicity (avr, pth, rpf, etc.)
would be very interesting, but could produce spurious results—taxa could
resemble each other as a result of common selective pressures.
Host-parasite phylogeny congruence
Should host and parasite trees have anything in common? Should they agree
with each other in terms of their branching structures (see the Host-parasite
figure below)? An alignment of angiosperm matK sequences (matK_DNA_align)
is available below for your analysis.
You can search to find other loci that would be better to address Xanthomonasangiosperm host-parasite coevolution? Do closely-related strains of
Xanthomonas appear to shift hosts frequently? It may be helpful to review
material on Xanthomonas taxonomy (http://grove.ufl.edu/~jbjones).
DNA sequences vs. proteins
DNA sequences can potentially possess more information that protein sequence.
Why do you think this might be so? Hint: Can you unambiguously reconstruct a
DNA sequence if you know the protein sequence to which it corresponds?
Protein sequences, on the other hand, are the functional units (e.g. enzymes,
transcription factors, receptors) upon which selection can act.
Are phylogenetic trees method sensitive?
Different methods exist for inferring the history of a lineage
(http://evolution.genetics.washington.edu/phylip/software.html). Do alternate
methods reconstruct the same sets of nested relationships?
Citations
Cubero, J. and J.H. Graham. 2004. The leucine-responsive regulatory protein
(lrp) gene for characterization of the relationship among Xanthomonas species.
Int. J. Syst. Evol. Microbiology 54: 429-437.
Goncalves, E.R. and Y.B. Rosato. 2002. Phylogenetic analysis of Xanthomonas
species based upon 16s-23s rDNA intergenic spacer sequences. Int. J. Syst.
Evol. Microbiology 52: 355-361.
Additional resources
Xanthobase – X. oryzae pv. oryzae database – http://microbe.dna.affrc.go.jp
Xanthomonas taxonomy lecture – http://grove.ufl.edu/~jbjones
Joe Felsenstein’s list of phylogeny programs http://evolution.genetics.washington.edu/phylip/software.html
Florida map set
This is a set of five maps of the peninsula portion of Florida, showing climate,
citrus leaf miner, citrus production, citrus canker and 1900 foot eradication zones.
Consider asking students to make transparencies of these maps so that
they can be overlaid one on the next.
What patterns of relationships can be discerned among these variables?
American Phytopathological Society Lesson on Citrus Canker
Tim R. Gottwald, USDA, ARS, Orlando, Florida
James H. Graham, University of Florida, Lake Alfred, Florida
http://www.apsnet.org/education/LessonsPlantPath/CitrusCanker/default.htm
This material is informational, but might be useful as a resource in a lab on hostpathogen relations, as the basis of a brochure for consumers of citrus nursery
stocks, etc.