Who dented the car?

Shock and horror when I came out of work tonight to see a big dent in the front left corner of Regine's BMW which she has lent to us for the long weekend. I knew I had not hit anything so was quite surprised it had potentially been swiped by another car at work. But closer inspection once I got back to the Guest showed there was rust in the middle of the dent. Whoo! What a relief. Today has been very cold with a little rain so very different to our first two and half weeks of sunny spring weather. Tomorrow is a public holiday in Germany as it is 1 May. Having a car for the weekend means we will be able to explore a little further afield than we have managed on our bikes.

Yesterday I had stimulating discussions with post doc Lay Sun and PhD student Xiaowei. Both are working on effectors. Lay Sun reminded me of how difficult it is to prove an effector is being transferred to the cytosol of the plant cell. One method they use which is very helpful is to soak the leaves in NaCl which causes the plant cells to plasmolyse around the hyphae making it easier to see if a reporter such as GFP is just in the hyphae or also in the surrounding space. The discussion with Xiaowei had a similar theme. Checking for secretion of an effector out of the fungus is reasonably straight forward but showing uptake into the plant cell is more difficult. Having a 3D structure of Cmu1, which he presented at Asilomar, is allowing him to use a rational design approach to analysing the function of the protein.

Today I reviewed the 9th manuscript of the 10 I have to manage for the August issue of Current Opinion in Plant Biology. Just one more referee report needed before all have gone back to authors for revision. This is the first time I have been in an editorial role for a journal. Quite a cumbersome portal to get familiar with but I have now got the hang of it. Of course I have learnt a lot reading these 10 manuscripts. Hopefully dealing with the revisions will be more straightforward.

I enjoyed catching up with Katrin this morning and Kimberly late afternoon by Skype.

A productive day of manuscript writing

Today I completed Matt Nicholson's manuscript on the cloning and functional analysis of two indole-diterpene gene clusters from Penicillium. This work was started in 2006 so its rather sobering to look back at how rapidly the technology has changed - who would make a cosmid library these days when you can sequence the whole genome. However, we did use second generation Solexa technology in 2010 to sequence the cosmids. What will now make publishing the paper difficult is a recent publication from a group at Hokkaido University who in a tour de force have reconstituted  penitrem biosynthesis in Aspergillus oryzae.

Yesterday I had very enjoyable discussions with two PhD students. Mariana has successfully developed the CRISPR/Cas9 technology for U. maydis.  They have some very nice tools in U. maydis including a  good selectable marker (cbx = carboxin) and an autonomously replicating vector. Once the selectable marker is removed the cells rapidly lose the plasmid and mutagenesis by CRISPR/Cas9 is shut down. They also have a nice selection system as the transition from yeast to filamentous growth generates a fuzzy phenotype on charcoal. CRISPR/Cas9 mutagenesis is a potentially powerful tool for silencing multiple genes such as those that comprise a gene family.

My second meeting was with Philipp who is studying an effector protein called Stp1 from effector cluster 10 of U. maydis. He has identified a putative plant interactor of this protein using Y2H and confirmed by co-IP. He is also making use of comparative genomics. We are fortunate to have such a good collection of fungal genome sequences from other Clavicipitaceae to inform our studies by comparative genomics - thanks to Chris Schardl.

Today there was a symposium for prospective students who want to study at the MPI for PhD. Each of them had to do a presentation of their MSc work. An interesting but highly selective way of recruiting students.

Weekend in Frankfurt

Christine and I spent the weekend in Frankfurt which is just one hour by train from Marburg. On Saturday we visited the Goethe Museum which is a rebuild of the 4 storey house where Johann Goethe lived. I was intrigued by the large water heaters in every room fed by water heated from fireplaces on each of the landings. Someone was kept busy keeping all those fires going. Today we visited the Staedel Museum which is one of the best art museums in Germany. It is currently hosting a Monet exhibition so it was very busy. I do like the design of the most recent downstairs extension which makes use of glass portals to let in natural light from outside - but some of the art was a little puzzling such as the single black rectangular painting. Being Sunday we felt we should do what the Germans do and treat ourselves to a piece of cake - see below. Now we are catching up on the news of the devastating earthquake in Nepal. A very scary time for everyone there but especially the climbers at Everest Base camp and those at Camp 1 above the Kumbu icefall as they are trapped there until a new route is established through the icefall.

SYNMIKRO symposium

Today I attended the 2015 SYNMIKRO symposium in the beautiful Alte Aula (see pictures below) in the old part of Philipps Universitat. SYNMIKRO is a Marburg consortium for Synthetic Biology. The theme of the symposium today was biosensors and regulatory circuits. What an amazing collection of talks with many different examples of biosensor use including light regulated biosensors that modulate glucose and lipid levels in mammalian cells and in turn activate genes for restoring homeostasis, to biosensors to monitor chemicals such as arsenic in the environment. The arsenic biosensor has been trialled in Bangladesh where there are something like 50 million wells for drinking water that need to be monitored. There was another great talk from a scientist at SANOFI on the use of S. cerevisiae as a cell factory for synthesising the antimalarial compound artemisin done in collaboration with various groups around the world on a no profit/no loss basis and funded by the Gates foundation - no less than about 8 genes had to be introduced into yeast. But now they are producing tons of the product. They have also metabolically engineered yeast to make the anti-inflammatory, hydrocortisone - some very sophisticated gene engineering. One little snippet I enjoyed was the use of some cells engineered with a biosensor to detect oil spills in the ocean but when they got to sea to do their tests they realised the Dutch government had no regulations on the use of GM microbes on ships - never mind the impact of the oil! The opening talk was almost science fiction where they were working on 'mind control of gene expression' using a wireless powered optogenetic implant that responded to electrical signals. On the more frivolous side a blue light sensor to activate NO synthesis to replace the blue pill!

Week 1

Its now two weeks since we arrived in Marbury which is a pretty city snuggled in the Lahn valley. We are staying in an apartment at the University Guest house, which is on the Eastern side of the valley. There are other university/institute visitors and post docs staying here. Its quite basic but fulfills all our needs as its warm, has a good hot shower and is clean. Downside is the poor internet service. It has been a good time to arrive in Marburg as it has just started to warm up after a prolonged grey/bleak winter. When we arrived last week the trees were all quite bare but within a few days all the buds have burst so all the trees are now green with leaf. It also coincided with the start of the spring/summer semester for students so the town is busy with students. On the warm days they can be found having BBQs and drinking beer on the banks of the Lahn – students are the same everywhere! It is a 25 min walk up through the forest to the Max Planck University and science departments associated with Philipps University. I think most of the arts and commerce faculties are located downtown. This walk is a pleasant way to start the day.

The Institute itself has wonderful facilities. I have a large office to myself, which is good for reading and writing. This last week I attended an Institute Seminar on bacterial motility, a Department seminar from post doc Pierre Grognet, a journal club and then on Friday the weekly round table lab meeting involving short verbal presentations by the different groups on their results for the last week – no powerpoint but muffins and coffee. Last week I met with post docs Pierre Grognet and Libera Lo Presti.

Pierre is investigating how Um (Ustilago maydis) senses the host leaf surface during infection. Previous work showed that Um senses both hydrophobicity and hydroxy fatty acids (cutin monomers/oxylipins). Sensing of hydrophobicity is through Msb2, a mucin, and Sho1, with transduction of the signal through the pheromone MAP kinase pathway. However, nothing is known about the sensing of HFAs. He gave a talk on this work in the volatiles session at Asilomar (see concurrent sessions and poster #556), which some of you may have attended.

Libera is developing the biotin tag assay for effectors and gave a talk on this work in the Effector Biology session at Asilomar. The strategy is to tag the effector with a Biotag (small peptide) that serves as a substrate for BirA, the bacterial biotin ligase. If the effector is translocated into a plant cell transgenic for the expression of BirA then it will become biotinylated and can be picked up after IP and Mass Spec. However, as elegant as this assay sounds its much more difficult than you would imagine – which highlights once again the challenges on unambiguously demonstrating effector transport into the plant cell cytoplasm. The gold standard remains TEM of immunogold labeled cells using antibody to the effector. A useful control is to use the split GFP system as described in Nature Biotechnology (2005) 23: 102-107. This relies on tagging the effector with a very short N-terminal component of GFP (C11), which can bind to the longer C-terminal component (C10), as in BiFC, to form a functional GFP.  For the system to work well it is important there is no damage to the host tissue through stress or other means as this will lead to leakage of the proteins from one compartment into the next. 

On Thursday Christine and I bought bicycles from a bike tour company downtown so we are now mobile! But we are living up a very steep hill so still need to find a better way down into the valley. Yesterday we tried them out with a 25 km ride down the Lahn and through some lovely little villages including Argenstein, Roth, Niederwalgern, Niederweimar, Gisselberg then back to Marburg after which we had very sore backsides. Last evening we went to hear the Marburg symphony orchestra play some Mozart in Elisabethkirch, a beautiful 13^th century Gothic church that holds the tomb of St Elizabeth of Hungary. It is one of the landmarks of the city, together with Landgraves castle (Landgrafenschloss), the old university and the town hall (Rathus), which has some beautiful old paintings.

On Sunday we walked the Behring trail a 5 km walk that celebrates the life of Emil Behring who received the Nobel Prize in 1901 for the development of vaccines for diphtheria and tetanus. There is a magnificent Mausoleum in the forest where he is buried. The manufacturing site today is a series of at least 20 buildings nestled in a valley within the forest an opening to the outskirts of Marburg.