<TITLE: Rcd5 - A Novel Component in the Regulation of Cell Death
ACADEMIC DOMAIN: natural sciences
DISCIPLINE: biology
EVENT TYPE: seminar presentation
FILE ID: USEMP12C
NOTES: continuation of and continued in USEMD270, seminar also includes presentation USEMP12A

RECORDING DURATION: 17 min 36 sec

RECORDING DATE: 11.4.2007

NUMBER OF PARTICIPANTS: 34

NUMBER OF SPEAKERS: 1

S8: NATIVE-SPEAKER STATUS: German; ACADEMIC ROLE: junior staff; GENDER: male; AGE: 24-30

SS: several simultaneous speakers>


<S8> yeah erm i work in <NAME>'s group and i don't have as fancy pictures <SS> [@@] </SS> [to show as <NAME S2> and as not as pretty flowers as <NAME S4>] we take a plant and we somehow convince it to die <SS> @@ </SS> , okay our basic model system er that we use in the lab is ozone , ozone erm enters the leaf through the stomates and there it erm , it induces the an oxidative burst so it induces the production of (herba) in the oxygen species and also it reacts with some components in the cell wall and to pass a membrane , erm importantly for for my work is that ROS are involved in the cellular signalling and that includes the regulation of programmed cell death , erm the resulting oxidative burst and those of the erm gene expression in response to ozone has similarities to erm pathogen infection and one really important point was it's a really really nice system to study apoplastic reactive oxygen we don't have to manipulate the plants much we don't have to dip them into peculiar solutions we just put them in a chamber and we switch on the ozone we don't have to touch them . okay what is programmed cell death in the shortest version erm you can think of it as a coordinated process erm in which cells actively commit suicide so it's not some effect that crushes up a cell until it can't live anymore but the cell receives a signal and initiates a program that shuts down all required systems until it finally dies , the functions are pretty diverse the functions in development in senescence and also in disease resistance to various pathogens and of course since cells in a living plant die here this needs to be tightly regulated i guess you can imagine what happens if er programmed cell death runs wild then the whole leaf dies or in a more severe case the whole plant dies , and last but not least hormones and also ROS have already been erm described to have pretty important functions in the regulation of this programmed cell death below is just a scheme of what i just erm told you if you imagine this to be cells the first one receives a stimulus and initiates the programmed cell death program and this spreads on and on and on but at one point in a healthy plant the plant can decide okay this has gone enough we limit it here . okay , for my work erm actually two other post-docs in the group <NAME> and <NAME> have been interested in isolating new components in in ROS signalling and while <NAME> did a lot of transcript analyses , er the identified candidates in <NAME> went to ordered lines but <NAME> also ordered one line by accident <SS> @@ </SS> which was not on any area or anything but <NAME> nevertheless had okay we have the seeds let's test it and it had a pretty interesting phenotype for us so er that line is RCD-5 radical-induced cell death number five , erm what it basi- what we knew when we started it erm was that it's erm er similar to erm the stigma-induced stig- er stigma-induced protein one from tobacco and from the sequence analysis we knew that it's er bit about erm 200 amino acid protein it has a signal peptide we have about 30 amino acids and it has the stig-1 domain don't ask what the stig-1 domain does it has been modelled after exactly one protein which is stig-1 but still this comes up when you do when you erm check the protein database er but more interestingly there's there is two erm sistine repeats in there and those are found also in a lo- in a lot of receptor (kinesis) and theoretically there's hypotheses that those might have a function in in ROS signalling but there's not much data shown so er there's a lot of theoretic work on that , the signal peptide erm is supposed to be erm required for extracellular localisation er for receptor pathway the protein has a N glycosylation site and erm this is er the pa- one the second paper actually from the tobacco work where they found that stig-1 is involved in controlling the exudate secretion in the tobacco pistil but when you checked erm over-expression or or antisense class you didn't find phenotypes for it they just they didn't find any any visible phenotypes the only thing th- they saw was the composition of the (xx) , okay erm one more piece of information that we got erm from the former erm post-doc from studies like (kapinsky) in stockholm was when he looked at the RCD-5 promoter that the promoter conf- contains in er according to him pretty prominent position for regulation three elements that they have already been have already described as to be strongly regulated by reactive oxygen species , okay so we just took the knockout plants and erm exposed them to ozone and after six hours ozone exposure you see in colombia well you actually don't see much the plants are just as healthy as before but in RCD-5 you see here that there is erm tissue collapse which erm correlates to cell death , and this you also see when you erm measure cell death via ion leakage so erm ozone has been switched on two hours prior to er point zero at point zero we harvest the plants and put them in water and start to measure the ion leakage for the following hours and we see that in er colombia both ozonated and non-ozonated the ion leakage increases a bit but nothing dramatic happens and the same goes for RCD-5 erm control lines , that have only received clean air not much happens but if you give RCD-5 er ozone the cells die a lot more than wild type plants so this makes us in our group happy we see something die <SS> @@ </SS> you see here this is something we are interested in , erm , if something dies because of ozone because of ROS we of course immediately ask what happens to what what how does it behave in other processes that involve reactive oxygen species and pathogens very often involve ROS so we tested growth of er pseudomonas syringae on RCD-5 plants and the first thing we tried was to take a virulent strain so strain that can dis- can cause disease in normal arabidopsis wild type this also means it grows pretty well in wild type arabidopsis and you can see those bacteria in colombia grow pretty well , in RCD-5 surprisingly this is er the inverted trial here they grow much less so the next question was is this reduction the same that we would see in a erm , in the normal arabidopsis's response to a avirulent pathogen so one that the plant is resistant to and you see here this is in this in sort of the same range , okay RCD-5 is more resistant to to a virulent bacteria and again as for the ozone we could have a look at this in ion leakage we mainly did this because erm the reduction in erm growth of the avirulent strains was the same in , the virulent strain in RCD-5 grew only as much as the avirulent in colombia and when i looked at the plants i s- i thought well maybe they grow a bit faster but you know this is very difficult to see and whenever you take a picture you can show more or less what you want so we decided to measure cell death here , i'm aware that this diagram is very busy but erm what you basically have to have to see here that DC-2000 and DC-2000 AVRV in wild type arabidopsis , the virus strain causes a certain amount of cell death but it's not too much whereas erm the avirulent strain causes much much more so the plants initiate cell death after they face those bacteria , in RCD-5 however this happens much much faster and to a h- and to a higher extent so while in colombia with the avirulent strain you see that this really goes up after about four hours and then it goes higher in RCD-5 this starts much much earlier <P:05> okay so this happens to bacteria er but those bacteria usually like alive cells so when something dies more they can't (go as far) but what happens to a pathogen that actually likes dead tissue , so we checked alternaria brassicicola which is a necrotrophic fungus so this fungus kills cells and then lives on the on the on the dead cells okay the pictures are very bad i said i don't have as nice pictures <SS> @@ </SS> but we quantified the growth here so basically what we did we infected leaves with the with the fungus and after a couple of days harvested the leaves and ran erm quantitative PCR on fungal DNA and also plant DNA and from the ratio we can assess the growth of the fungus so we see that there's a certain amount of growth in colombia we used here another line (xx) which is a erm control in the sense that it's very susceptible to those fungi and here you see that the fungus can grow much much more in (xx) and RCD-5 can grow as much as in (xx) so while these plants are pretty resistant to erm the biotrophic bacteria they are pretty much susceptible to the necrotrophic fungus , so our next step was to cross RCD-5 to different apogenetic backgrounds erm because we wanted to do some analysis on where it's involved in and for some reason that didn't at all work and i tried it <NAME> tried it and we got some of the girls in our lab who are really good at doing crossing and it didn't work so we got a bit suspicious about that and i simply counted erm the viable seeds so i counted both the viable seeds and i also counted embryos and erm well we saw that while you have a certain amount of seeds in colombia in RCD-5 this is approximately half , depending , okay well there is something going on in the (xx) as well which we didn't quite expect because we were interested in the leaf phenotype in in the in the nice cell death that we had there so damn we have a we have have a flawed phenotype now okay and then after erm <NAME> got the idea of well let's let's see what a- what actually happens in the flowers after pollination , and er <NAME> and me embarked on a on a project which we by now refer to as the fun stuff which involves hand pollinating flowers and after that staining it and what we saw that er in the arabidopsis flower in the papillae on the stigma erm in colombia you don't see much er superoxide but in RCD-5 when it's pollinated you see here erm a lot of blue which indicates that there's a lot of superoxide produced and since this is a very small picture we have it here so best example is here here you have a papilla and here you have a pollen tube where the (both) er have a pollen grain where the tube is going in and here you get a lot of blue staining indicating that there is a lot of superoxide produced here , erm just the fact that there is superoxide there doesn't mean much so we checked on on if papillae in RCD-5 are (er) are more or less alive than in wild type and for that we did trypan blue staining , er this is one of the fir- er some of the first pictures i made for this so erm i'm currently have plans erm have have erm samples which i which i have to go to microscopy with but what we saw in those first samples that there is actually not much er cell death in colombia when you pollinate it but if you focused to the RCD-5 flowers you get a lot of examples where you have a pollen tube and er er a pollen grain and pollen tube going to stigma and you get this dark blue staining down here meaning that there should be a higher amount of cell death in the flowers there which would actually explain our diff- differences in the crossing . and erm of course we have to check if actually of the some of the pollinated of our hand-pollinated flowers pollen tubes at all grow in we have to do aniline blue staining and basically you just erm , what you see here is pollen tubes going down in the flower and this works so we now have to look into more detail what happens in those flowers , but now we have ready plants over-expressing RCD-5 with different tags and here's a (xx) (portfolio) (extractor) where you can see in the transgenic lines here you have er actually two bands of protein which you al- or which we always get while you have nothing in the other in the control lines and you also have (xx) expression lines but this is a picture from onion where we're trying to erm , to now resolve the localisation of RCD-5 it is erm predicted to be apoplastic and from tobacco there is data that it's apoplastic this is pictures which i just did erm on the weekend so we now are thinking about good controls to check apoplastic localisation and if anyone has any suggestions for that i'm mo- you're most welcome to tell me i'd be really happy to hear something about that , okay so the RCD-5 knockout plant show a leaf phenotype we looked at the transcript levels but er initial data show- erm suggests , erm that while the the the transcript levels are low okay i'm sorry there is a mistake here that the transcript levels are high in flowers but er they are low in leaves but still we have a leaf phenotype so our but our data also suggests that the RCD-5 is not so strong in regulating the transcript level but just from er recent western blots we got the idea that actually protein might be regulated by a stability and or by a post translational modification so by doing more and more experiments on the cell death erm , level we sort of get the idea where we think about if RCD-5 could be part of a cellular surveillance machinery that constantly monitors in no matter what what level it it it monitors for the presence of ROS and erm it has actually been recently shown by the end of last year that there's a lot of ROS and also n- er (N-O) available in the flowers so this would go well with the idea that high levels are of RCD-5 are required in arabidopsis flowers to just monitor there what goes on and keep telling the cells don't die this is okay guys so don't die . and this was er the model we initially before we had erm our preliminary data for expression on programmed cell death on the negative regulation so there here again you have the cells and they get a stimulus and upon the stimulus we er cell death is initiated ROS and other signals go from cell to cell and erm spread the cell death other signals prec- would precede the cell death front and er induced up-regulation of negative regulators and at a certain level when there's enough of those regulators present the lesion would be contained cell death would be stopped well we thought that RCD-5 could be one of those we don't have the final proof yet but the data suggests that it's not that easy would have been too nice but we still think that this model in a general sense d- erm could be true we just might look of RCD-5 at er a- at a different aspect in this regulation , okay er our ox stress group <NAME> being the leader of the pack er <NAME> is here and <NAME> who is not in the picture have initiated the study and erm now i took over most of the project but <NAME> still contributes really a lot to that yeah thank you very much </S8>
<APPLAUSE>
