Seeing is believing......

One of the objectives for my PhD ( and hopefully a thesis chapter...?) is to completely characterise the  process by which Striga gesnerioides gains entry into the roots of Arabidopsis thaliana. On the outside,  I can first tell if one of the tiny Striga seeds has successfully infected the host root when it begins to swell into a tubercule. In a really good infection, the root can be strung with tiny bobbles, like a chain of knobbly Christmas baubles. But this is a comparatively late stage of the infection process. To understand how the parasite may be suppressing host defences, I need to know at what point it begins to break through the outer root layer, the epidermis, and starts squeezing through the cortical cells towards the central vascular bundle. The key moment is when the parasite connects to the hosts xylem vessels - at which point it can freely withdraw water and nutrients to fuel it's own growth. All of which is very difficult to tell from the outside!

Hence, I have been busy with my scalpel - hacking off sections of infected root to take samples of tissue. I have then been putting these through a long and convoluted protocol to embed these in a material called Technovit - the whole process takes about two weeks and makes the recipe for a wedding cake seem easy by comparison! But this is necessary because Technovit is strong enough to be able to take ultra thin ( 5-10 micro etre ) slices off the sample using the microtome. I have a 'love- hate' relationship with this machine....For my first attempts, I would spend hours and hours on it and yet my sections would look like a jumbled mess under the microscope. Just lately, I have been having more success, learning to bring the blade down in a smooth, rapid motion ( I think of it like a guillotine - if it was my head on the block, how would I like the blade to come down?!). After staining my latest batch of samples, it was exciting to see how they turned out!

The host Arabidopsis root is on the left and the parasite tubercule is on the right, before I reach the point of contact. 

These sections were taken 14 days after I applied the Striga seed to the roots. When I take sections, I slice along a length of root latitudinally. So as I go along the root, all I can see at first is the host root until I reach a tubercule. As the tubercule swells up around the point of entry, initially it doesn't seem to be attached to the root. But as I continue taking sections, the tubercule seems to get closer and closer until I reach the point of contact. At this stage, I can begin to see how much the parasite has disrupted the host root and how extensive its connections to the vascular bundle are.

The point of entry - Striga gesnerioides forces its way in...

Even with these sections however, it can be difficult to tell what is going on ( but you should have seen my first attempts!). Consequently, I am now taking sections from some very exciting Arabidopsis mutants expressing an enzyme called GUS. This means that when I treat the root sections with a certain dye, they will turn green. Because only the host Arabidopsis only expresses GUS and not the parasite, only the host tissue will change colour. So it should be easier to work out which cells belong to Striga and which belong to Arabidopsis. That's the theory anyway - I'll let you know how I get on!

A big jumbled mess....an Arabidopsis root infected with Striga gesnerioides

Can you see the difference? The infected root sample on the right was taken from a GUS-expressing Arabidopsis host, whilst the one on the left comes from a control 'wild-type' host.