cc  Welcome to our tenth and final  class of "Frontiers of Life  Science."  Hopefully you will be back for  our spring semester which we're  in the planning for.  Today we have with us Lori  Adams-Phillips.  She's a post-doctoral fellow in  the department of plant  pathology, and her topic is  plant-pathogen interactions.  So with that, Lori, welcome.  >> Thank you.  So I'll just take a few minutes  to introduce myself a little bit  further.  I grew up in Illinois in a town  of 900 people, it's called --  Illinois, so probably many of  you haven't heard of it.  So anyway, I started there, went  to University of Illinois in  Champagne for my bachelors in  crop science, so I have an  agriculture background.  I then went to Texas A&M to get  my PHD in genetics and came here  a few years ago to work in  Dr. Andrew Bent's lab in at the  University of Wisconsin in the  department of plant pathology.  But I have to preface this all  by saying, my training really  isn't in plant pathology, I did  take a couple courses as an  undergraduate, but I've been  learning a lot by being in the  department. 

 And if I'm not able to answer  your questions coming up on the  next few slides, I definitely  know the people that can.  My background is in a lot of  molecular genetics, and I will  get to things like that later  but I wanted to give you just an  overall view of what we do in  the department of plant  pathology.  So, with that, here's a brief  outline of what I'll be talking  about today.  So first, why even bother  studying plant pathology?  I'm going to tell you a little  bit about the department of  plant pathology at UW.  A little bit of plant-pathogen  interactions 101, and then I'm  going to get into a little bit  more of the particulars of my  research and talking about  arabidopsis as a model system.  And then, getting into what I  actually do.  So, starting with, why is plant  pathology important?  It seems pretty obvious, but I  think this sort of sets the  stage for the talk and realizing  how important plant disease is  to all of us.  Essentially all of human food is  derived from photo-synthetic  plants in one way or another,  either we eat it directly or  animals that we eat it.  And plants are prone to  epidemics when grown in  genetically homogeneous  monocultures as we do with all  of our field crops due to  necessity.  Just an example of this, and  this is one of the most famous  of examples of the impact plant  disease can have, comes from the  Irish potato famine in the  1840s. 

 We know now that this was caused  by late blight on potato tubers  and you can see that they're  rotting.  We know now the causal agent of  the potato late blight, which is  call -- and it's an oomycete  pathogen.  Some of the symptoms of potato  late blight include brown leaf  spots, as you can see very  obviously here, and also a very  terrible smell from rotting  potatoes.  And this is a very devastating  disease.  Within days, whole fields can be  completely killed.  And after several successive  years of having this blight hit  the Irish potato farms, the  result was that over  1. 5 million people died, over  2 million emigrated from Ireland  and many died en route.  And even though we don't hear a  whole lot about it too much,  this pathogen still remains a  problem even today.  And in fact this morning I got  an email from a grad student  giving her dissertation talk  about resistance genes to this  pathogen, so there are a lot of  potato farmers here in Wisconsin  and they are affected by this  pathogen.  So part of our work involves  finding resistance to late  blight.  So some of the more current  examples include rice blast,  this is a fungal pathogen of  rice, it's because rice is such  a huge staple for so many in the  world, it's a very devastating  disease and it strikes the seed  head of the --. 

 Let's see if I can point, yep,  there it is.  So the rice seeds are basically  not forming, and it ends up  killing the plant.  It's estimated to kill enough  rice to feed 60 million people.  And interestingly when I was  doing some research for this  topic, I found out that in the  1950s, the US and former Soviet  Union stockpiled rice blast  pathogen for use as a potential  biological weapon.  Of course, they've stopped that  now, but it is an extremely  devastating pathogen and there's  a lot of research going on so  resistance hasn't been found yet  for this pathogen.  Other pathogens include viral  pathogens such as the plum pox  virus, and this is pretty  recent.  In Pennsylvania they found,  they've been having cases of the  plum pox virus and it affects  stone fruit such as peaches, and  it basically reduces the yield  and also fruit quality, so the  fruits just aren't as desirable  to consumers.  But as far as I know the virus  doesn't completely devastate the  tree.  The tree can survive but  basically the marketability of  the fruit is reduced.  And also this one's coming up,  and there's been a lot of talk  about this in our department,  though I don't believe anybody's  working on this one directly. 

 It's called citrus greening,  it's caused by a bacteria, and I  have the name underneath.  This bacteria they believe  originated in China and it's  spread by insects.  There it is.  Yeah, the psyllid insect spreads  the bacteria to the different  trees and they actually found  the insect in Florida in 1998  and then more recently in 2005  found the bacteria strain.  So the two combined can cause  devastating effects to citrus in  that they basically don't ripen  and the fruit is un-marketable.  And this is one of the most  serious threats right now to the  citrus industry.  As you can see right here, this  tree is totally -- and it will  eventually die in a few years.  In other parts of the world, in  Brazil, they've seen up to 70%  reduction in yield and so the  citrus industry is very  concerned with how to monitor  and detect this pathogen and how  to combat it.  And then last but not least  being in the heartland I have to  talk about soybeans.  Soybean -- in particular is a  pathogen that we work on and I  don't work on it directly but in  our lab other people do. 

 This is a nematode that causes  cysts to form in the root, and  this is actually a root nodule  which is different, but then  these tiny white cysts that  form, and those are the  nematodes, and they feed on the  roots, and as you can see this  whole area of the field has been  destroyed by nematode  infestation.  And it's estimated, current  estimates put it at losses up to  $720 million just in the US  alone.  So hopefully I've convinced you  that plant pathology is  important.  And this table, it's a fairly  old table, but it just  illustrates that the loss of  crops due to plant pathogens is  in the billions of dollars every  year.  This is the cost even in 1990,  and you have a waste of land,  fuel, irrigation, and water.  And then last but not least it's  all the pesticides and  fungicides and bactericides that  are applied to try to combat  these diseases.  So now moving on to what we do  in the department of plant  pathology to try to understand  these diseases and help  hopefully combat them someday.  I'll start with a little bit of  history.  I wanted to make a -- in part  because we are coming up on 100  years of being in existence, so  we're coming up on our  centennial celebration of being  a department at University of  Wisconsin.  It kind of started back in the  1890s when a serious disease was  affecting cabbage, up to 95% of  cabbage crops were being lost. 

 And the cabbage growers  contacted the university for  help.  And they sent out Dr. Russell  and he discovered that there was  a bacterial agent involved in  these cabbage crop losses.  And we now know that that  bacteria is called xanthomonoas  campestris and it causes black  rot disease of cabbage.  And further studies went on to  find resistant varieties of this  cabbage or change the  cultivation practices to help  combat that pathogen.  Dr. Russell then became dean in  1907 in the college of  agriculture at UW.  And he recognized the importance  of having a department of plant  pathology and he appointed  Professor Jones, right here, as  the first department chairman in  1910.  And thus the department of plant  pathology was born from the  needs of growers.  And this has always been an  important aspect that our  department keeps in mind, that  the needs of the growers are  important.  And here just to show you, this  is Russell labs, this is where  our department is housed.  This is a beautiful picture of  Allen Gardens behind, but on the  other side is Babcock dairy, and  we love to take breaks once in a  while and have ice cream and  think about our science for a  while and it's a nice location. 

 Just a few more things about our  department.  We study a number of  economically relevant crops such  as potato, tomato, apple, corn,  fruit crops, even forest trees.  There are a number of diseases  and there's many more than this,  but I just put a few; soft rot,  bacterial wilt, apple scab,  brown stem rot of soybean,  Aflatoxin, which is produced by  the fungus Aspergillus, potato  early dying, and the pine cancre  disease.  And so our department has  several functions.  First of all, we do both basic  and applied research in disease  epidemiology, genetics, disease,  physiology, and microbial  ecology.  WE also educate undergraduates  and graduates in plant pathology  and plant-microbe interactions  and then again, this is an  important component of our  department, is to provide  extension and outreach to foster  understanding of plant diseases  and enhance plant health, food  safety, and profitable and  sustainable agriculture.  So there's very close ties with,  for example, the potato growers  association, the soybean  association of Wisconsin, and  other such organizations.  And this is an interesting piece  of information that might be  useful to everybody.  The university and our  department has the plant disease  diagnostic clinic, and you can  actually drop off a sample from  your garden if you're concerned  about a specific pathogen, maybe  it's a tree that you don't want  to lose, and you can bring in a  sample, and I think it's $15,  depending on what they do, and  they can diagnose what disease  you have.  And those specimens are also  useful because we often get an  email, "Oh, there's a neat  specimen downstairs, you want to  come and check it out?"  So it's also an educational  tool as well. 

 So just wanted to give you an  overview of our department and  now, just going to switch gears  a little bit and this is a  rather long section, but I just  wanted to introduce you to some  basic and fundamental concepts  of plant-pathogen interactions  and also what is the current  state of knowledge in this area  right now.  So, many of you, if you've had  any plant pathology classes,  this is the sort of classic  example, or it's fundamental to  plant pathology, plant disease  research is understanding the  disease triangle.  You need a conducive  environment, you need a  susceptible plant, and a  virulent pathogen and then you  get disease.  Another way to look at this  triangle, and it says sort of  the same thing but you can  visualize it differently, it's  sort of at the intersection of  having the proper weather  conditions, pre- and  post-infection, having a plant  with a susceptible genotype, and  also how are they planted.  Are they planted extremely close  together in monoculture, things  like that.  Also, what about the pathogen?  What is the pathogen?  This is the genotype, how much  of the pathogen -- is the plant  receiving?  And at the intersection of all  that is plant disease.  So, I alluded to this at the  very beginning.  What kinds of pathogens are  plants exposed to?  And it's amazing when you start  to think about all the different  kinds of pathogens that are out  there.  So I talked about the bacterial  pathogens, in particular the  example of citrus greening.  Other pathogens, and this is a  very tiny example, but some that  we've actually studied in our  lab are pseudomonas syringae,  which causes leaf speck in  tomato, and xanthomonas  campestris, and I showed that  slide at the beginning too. 

 That causes black rot in  cabbage.  We also have the oomycete  pathogens.  Oomycetes used to be categorized  as fungi, but they are now their  own little separate class of  pathogen.  Those, I gave the example of  potato late blight, that was --,  also hyaloperonospora parasitica  causes downy mildew.  Fungal pathogens, and I gave the  example of rice blast, that was  -- but also other fungal  pathogens include eryslphe, that  causes mildew, and alternaria  which causes early blight.  Yeah, yeah!  >> How close are these  particularly to bacterial  pathogens in humans?  >> They're very close, actually.  So pseudomonas is a human  pathogen and so, there's not a  whole lot of difference, in  fact, you can actually get  pseudomonas-- that'll cause some  symptoms in plants as well.  So it can actually cross over  and be both human and plant  pathogens.  Others, pseudomonas syringae  is not a human pathogen.  >> (Inaudible)  >> Sometimes, so  yeah, for that example, you can  have pseudomonas --, but   pseudomonas -- is definitely  not, hopefully not, I work with  pseudomonas syringae, and it is  definitely not a human pathogen. 

 And it is interesting and I'm  not entirely sure if we know  what makes the difference, what  makes it virulent in human  versus animal, but certainly the  pathogens themselves, based at  the molecular level, are very  similar to each other.  So, what makes them be a human  pathogen versus a plant pathogen  is an interesting topic.  So we also have viruses, and I  mentioned plum pox virus.  There's also cauliflower mosaic  virus and turnip crinkle virus.  And last but not least we also  have to think about nematodes  and aphids.  And I gave the example of  soybean cyst nematodes, there's  also root knot nematode and  other nematodes that are  devastating to plants.  And also aphids can transmit  diseases as well.  Yeah?  >> They seem to be one-specific,  like potato or tomato.  >> Well, yeah.  It depends on the path a lot. 

 There's specific, so this one,  -- or tomato, there's different  pathobars of these strains, and  those can be causing--, because  you can get -- which causes  disease on soybeans.  So within the species there's  different pathobars that cause  different diseases on different  plants.  And on this, even though I say  tomato, for example, -- can  cause this disease on other  plants like tobacco, and I'll be  talking about -- later, so it  doesn't necessarily only  correspond to that one plant  species.  So, how do pathogens get inside  the plants?  And there's a few main routes of  entry.  One of them include the  somates, so these are important  structures in transferration,  and also carbon dioxide gas  exchange.  But when those stomates are open  they provide great openings for  pathogens.  Also, feeding by insects, you  can get -- damage, so pathogens  can enter that way.  And also the weather plays a  huge role in pathogen entry, and  so you can get even just rain  splash is enough to get  pathogens to where they need to  be on a leaf, but more extreme  weather such as hail damage can  cause wounding, a lot of  wounding.  Any time you wound a plant you  leave it open to pathogen  invasion.  So, what do they do?  What do the pathogens do?  There are two main classes of  pathogens. 

 Biotropes need living tissue to  survive, where necrotrophic  pathogens actually feed on the  dead tissue of the plant.  And how do they do that?  They deplete photosynthates and  carbohydrates, so they use up  all the plant's reserves.  They divert metabolism, cause  lesions and blemishes which make  our fruits and crops  undesirable.  But most importantly they can  cause necrosis and blockage of  key tissues which is where you  get plant death.  And the way that they do that is  they can plug up the vasculature  or the veins, the main network  where the plants are getting  their nutrients.  They rot the roots, so that's a  very bad thing to happen.  Stems, again, along with the  vasculature, rots leaves,  fruits, flowers.  They don't undergo  photosynthesis as well.  But how do they do that?  The pathogens are very  sneakative, lots of tools in  their toolbox.  They have lots of toxins,  enzymes that can degrade plant  cell walls, enzymes such as  pectinases. 

 They secrete slime, some  pathogens do that, not all,  that's known as extracellular  polymeric substance or EPS, and  this is the kind of slime that  ends up plugging up the  vasculature.  And I'm gonna talk about this in  a little bit more detail because  this is an area that we're  interested in, that they can  suppress host defenses with high  specificity.  And again, I'm gonna get into  that later.  So this brings up the question,  do plants have an immune system?  And looking at this field, this  is a field that has been  inoculated.  These are test plants, like  soybean, and they've been  inoculated with phytophthora,  causes root rot, and these are  basically different genotypes,  but they've been inoculated  with the same pathogen.  And you can see, I mean, these  guys are doing great, doing very  well, kind of brings up the  question, how are they defending  themselves?  And thinking about, what is an  immune system?  We think about it in humans as a  specialized group of cells that  are deployed to kill or limit  the spread of an invading  pathogen, but what if we think  about it as any system to limit  pathogens?  We don't often think about  plants having an immune system.  I mean, they don't have  circulating cells, so this isn't  happening, but plant cells have  preformed and inducible defenses  and I'm going to talk about what  that means in the next slide, so  that's an important defense  mechanism.  And I'm also gonna briefly touch  on, there's parallels with  innate immune systems in animals  and in plants.  So, in animals, in ourselves, we  have innate immune systems and  adaptive immune systems, and it  turns out that plants also have  an innate immune system that has  similarities to ours.  So, just talking a little bit  about these preformed versus  active defense responses, some  of the ones that are already in  place, they don't need to be  induced, when the pathogen  completely attacks the plant. 

 First of all, we've got some  barriers.  There's a cuticle layer, an  epidermis, there's a cell wall  that's pretty hard to get into.  But if they manage to get into  that, there's a number of  chemical inhibitors such as --  and these are all different  chemicals, that act as  anti-fungal, anti-bacterial  proteins, insecticides that are  there in place when the pathogen  first encounters the plant.  There's also a whole other  arsenal that's induced by  pathogen attack.  And that includes reinforcement  of the cell wall, and I'm going  to be talking about --, but  other reinforcements are -- and  other cell wall proteins.  And then there's this whole  arsenal of pathogenesis  proteins, these phytoalexins,  pathogenosis-related proteins,  --, glucinase, reactive oxygen  species, so hydrogen peroxide,  these are all again chemicals  that can inhibit pathogens and  that are induced.  And then last but not least,  there's the hypersensitive  response, which is a type of  programmed cell that can actually  stop the pathogen in its path.  It's a way that the plant  sacrifices a few cells to stop  the pathogen from spreading.  So, we're gonna talk about those  kind of things in a little bit  more detail.  So, we know that the plant has  these defenses in place. 

 How do they recognize the  pathogen in the first place?  How do they know that they're  there?  And this is a saga that goes on  for several parts, so you'll  have to bear with me.  How do they recognize pathogens?  One of the first things that  they can do is detect what are  called PAMPS, and PAMPS stands  for pathogen-associated  molecular patterns.  And all that is something  that is widely conserved among  pathogen molecules, and I've  shown here a bacterium.  And I'm pretty much going to  stick with bacteria for the most  part, I work with bacterial  pathogens, so I'll stick with  that.  But these PAMPS also can come  from fungal pathogens as well,  so chitin is a big part of a  fungus, it's a part of the  structure of a fungus.  Also, double-stranded RNA from  viruses.  So these are very conserved  pathogen molecules.  I'll talk next about flagellin,  these are the structures that  help bacteria swim, and not all  bacteria have flagellin by the  way, but quite a few of them do.  But stray or degraded flagellin  can elicit innate immune  responses in both plants and  animals.  So we actually have a system in  place as well to detect  flagellin, those structures that  all the bacteria to be mobile  and to swim around. 

 And it induces an innate immune  response in humans.  So this is just a cartoon  showing, we have a receptor  called toll-like receptor, that  sits at the surface of the cell,  and when a bacterial flagellin  is detected it can activate  certain genes and signaling to  active immediate inflammatory  responses.  And this is, again, different  than an adaptive immune system  which has antibodies and  recognition and things like  that.  But this is sort of an immediate  response to pathogen detection.  Similarly in plants, they also  can recognize bacterial  flagellin, it's a different type  of receptor, a different name, a  little bit different structure.  But it does the same thing, it  activates signaling in the  plant cell to induce defenses.  This is just another summary  slide of what I just showed you.  There's the pathogen being  recognized by a receptor so the  flagellins are binding, and now  we're activating MAPKKK, these  aren't really that important  what they are, they're just  genes downstream that get  activated.  That turn on transcription of  genes and trigger immune  responses, they give a very low  level, basal level of  resistance.  So it's kind of another low  level of resistance. 

 However, we have, the pathogens  can quickly outsmart this and  they counterattack.  So, what they can do is, this is  again a bacteria.  They can inject effector  proteins, so these are actually  proteins that are secreted into  the cell directly, this is an  apparatus, a type-3 secretion  apparatus.  These are found in both human  pathogens and plant pathogens,  and it's the same way in humans  as well.  The bacteria can inject these  effector proteins into a plant  cell and they actually target  specific genes and specific  proteins in plants to block  plant defense responses.  So this ends up becoming a very  virulent bacteria, it can cause  disease.  This also occurs, this type of  delivery of virulence proteins,  also occurs in fungi as well.  So this is a plant cell on the  outside, and this is a  haustorium.  The fungus has penetrated into  the plant cell, has formed a  haustorium, and it's not yet  clear how they get transported,  but they do get transported.  These are, again, effector  proteins that get transferred  into the plant cytoplasm, that  can block the trigger of host  defense responses. 

 And furthermore, this happens in  nematodes as well, so all these  different classes of pathogens.  This is a figure of a nematode  that's feeding on a plant cell,  and up here in the gland, I  guess, are these effector  proteins, and they're secreted  through this stylet and the  feeding tube and get injected  into the plant cell.  And those again have activities  that can block plant defenses.  So this is a common mechanism  that lots of plant pathogens use  to elicit virulence, or that's  how they block those different  responses.  So in this diagram that sort of  sums this up, we have, this is a  bacteria, we're back to  bacteria.  We have bacteria secreting  through a secretion apparatus,  these effector proteins that  block some of these pathways,  and so you no longer get  activation of the PAMP-triggered  immune responses.  Thus, now your plant is  susceptible.  But the story isn't over yet.  The plants recognize the  pathogen invasion in some cases.  So, in this scenario, we have a  bacteria, again with this whole  repertoire. 

 I should have mentioned this  before, these effector proteins,  it can vary from pathogen to  pathogen, but there can be up to  forty to fifty types of effector  proteins found and that are  secreted into plant cells.  Sometimes those effector  proteins when they're secreted  are actually recognized by  plants and that's because they  have evolved what are called  resistance genes, R genes, and  they recognize specific effector  proteins, and those are termed  avirulent proteins.  This is a lot of lingo that's  been around for years and years  and so we would call, they're  still effector proteins, but now  that they're recognized by the  plant we call them a-virulence  proteins.  And once the plant has  recognized this protein, it's  recognized this AVR protein from  the bacteria, it can now trigger    appropriate defense responses,  and actually elicit extremely  strong responses, even stronger  than those elicited just by --  and the immune system alone.  So, just one quick slide about  what are plant r-genes, what are  they actually?  There can be hundreds of them in  plants, several hundred, let's  just say for example, in tomato,  there could be up to several  hundred of these.  Most of them are cloned to date  encode NBS-LRR proteins.  It's just an acronym it stands  for nucleocite binding site  loose --.  That's not really that  important, except to say that  they're all very similar in  structure, so here's an example  of different classes of r-genes,  and it's just a diagram showing  this would be the LRR, this  little red ribbon.  A lot of them have a very  similar structure, and there's a  LRR here and here.  And that LRR, the name can be  important in recognizing either  directly or indirectly the  presence of those avr molecules. 

 It's really not known exactly  how they directly interact, so  there's still a lot of work to  be done on how exactly does the  r-gene figure out that the avr,  what's that interaction?  And so there's still a lot of  research to be done.  And regardless, and they knew  this for years, before we ever  knew the molecular structure or  what the protein was,  regardless, r-avr interactions  elicit a strong plant defense  response.  And I did talk about, I showed  this slide before, but what I'm  talking about when I say a  strong response is that  hypersensitive response, the one  that's going to sacrifice a few  cells to stop the spread of the  pathogen.  And I want to just talk about it  a little bit further because  it's an interesting phenomenon.  So, this is a slide taken from  some experiments, I believe they  were done in our lab.  I didn't personally do them, but  this is --, and I'm going to  show you all about -- in a  little bit.  But, it's interesting, one of  these leaves has been  inoculated with a virulent  pathogen that contains effector  proteins that are not recognized  by the plant, and the other one  has been inoculated with an  avirulent pathogen that contains  a protein that is recognized by  the plant, and it will elicit a  hypersensitive response.  When you look at it you would  say, well, this one looks pretty  sad, I would say this one is  definitely losing out.  It's very susceptible, because  you can see that the plant cells  are dying.  But when you look back that the  -- and look at the results over  time, these leaves were  inoculated with a high titer of  bacteria and were absorbed after  24 hours. 

 And by high titer I mean, like  one times ten to the eighth  colony-forming units, and that's  like 100 million bacteria.  Whereas these were inoculated  at a low titer, so maybe  something like 10,000 bacteria  and that's over five days.  You can see that when we  inoculate at a high level, the  avirulent strains, this happens  to be --, the plants recognizing  this avr gene, inducing this  hypersensitive response.  If we inoculate the plants with  a lower titer and look over five  days, you can see that the  virulent pathogen is actually  the one causing disease.  You've got --, you've got black  spots, it actually forms black  spots on the leaves whereas the  avirulent strain is now  resistant.  So what's going on there?  Well, we've got actually here  with the high titer, we can see  what we call macroscopic cell  def.  and we use this when we're  trying to study how does the  hypersensitive response occur.  And we want to be able to just  look at the plants and see them.  But with such a low titer you  can look at microscopically,  these are cells that have been  stained with -- which stains for  cell death.  And you can see that just a few  individual cells are dying. 

 That's enough to restrict the  growth of pathogen, and thus  it'll be resistant.  It causes the plant to be  resistant.  This resistance, the  hypersensitive response also  comes into play against fungal  pathogens as well, and this  happens to be the fungal  pathogen that causes downy  mildew.  So these are again, these would  be plants that have been  inoculated with downy mildew  that the plant does not, or DOES  recognize, excuse me.  And this is a strain of downy  mildew that the plant does not  recognize.  And so what the stain is  actually staining for is the  fungal growth, and you can see  that here the plant immediately  stopped it and contained it.  And in this interaction it did  not.  And again, this is due to the  activation of that cell death to  stop the growth of the pathogen.  So another big question is,  after the plant recognizes this,  so it's figured out that the  bacteria is secreting an  effector protein and can  recognize that, how does it turn  on defense related genes?  This is a huge big black box.  And suffice it to say, it's  complicated. 

 So we, there's lots of research  going on.  And in fact this review is from  2003, so if you can imagine that  even more has been added to the  picture, and I'm not even going  to attempt to take you through  it, but I just wanted to mention  that it's very, very complicated  and there's a lot of genes going  on.  At the top, these are all those  different r-genes, if you  recognize the ndllr.  These are all kind of resistance  genes, and downstream of that,  there's all kinds of genes.  They all have functions that are  sometimes separate and sometimes  they interact with each other.  So, the story is very  complicated and that's about all  I wanted to say to that regard,  but it's not so simple that it's  like a linear pathway and one  gene gets activated and the next  gene and the next gene.  Turns out, lots of genes talk to  each other, they interact,  there's actually molecules,  there's cells, there's hormones,  ethylenes, there's -- acid,  there's nitric oxide, there's  all kinds of compounds as well  as proteins that are activated.  So we've got a lot ahead of us  to do.  So just again, to summarize up  that last section, the  r-proteins from the plants, the  resistance genes can actually  recognize some effectors that  are secreted by bacteria, and  when that occurs, when this  recognition of the effector  occurs, then you can get  effector-triggered immune  responses, such as the  hypersensitive response and  that's a great, a good response  to have, that's what's going to  lead to plant resistance.  But the saga continues, it keeps  going, so pathogens can evolve  to escape detection. 

 You can see, this will go back  and forth and back and forth,  and in fact it does, it just  goes round and round.  And that's why we have  susceptibility and resistance  and it can change and you can  get plants that once were  resistant, and no longer are  resistant.  For this reason, now the  bacteria can evolve to either  lose the effector that was once  recognized by the plant, or it  can be modified so that the  plant can no longer detect it.  So now the bacteria is secreting  effectors, but the one that the  plant was recognizing is  missing.  And now the pathogen can block  defense responses and we get  susceptibility.  But through breeding and  cross-pollination, things like  that, you can now plant, the  story can go back and forth,  plants can get new r-genes to  detect those new effectors and  back and forth.  And so this brings us up to sort  of the current, the latest  central dogma of plant-pathogen  interactions.  This was published a couple  years ago and it's widely talked  about in our field, so you can,  this is the current state of  what we think is going on.  But you have the plants, plants  can detect these  pathogen-associated molecular  patterns such as flagellin, but  that only elicits a modest level  of resistance.  Pathogen effectors can, in turn,  target, and turn off that immune  response, they can target those  proteins that are important in  plant defense and block it,  resulting in susceptibility. 

 However, plants can evolve  r-genes to recognize these  effector proteins, triggering  resistance, but then the  pathogen effectors can be lost  and new ones can be gained, and  you now get susceptibility, but  the plant can turn around and  r-genes that recognize it.  So you go back and forth, back  and forth.  But this helps to explain why  some plants that were once  resistance are no longer  resistant because the pathogen  has changed slightly.  Or once a pathogen that was  virulent has lost virulence.  So that's our current model.  >> Does that type of thing  happen more in plants than in  animals?  >> Well, that's an interesting  question.  I would say, just due to the  last, what we're doing with the  vast numbers that we have of  crops, and thinking about, in  terms of generations, but we can  produce in one season.  If we've ever though about  humans, a plant can produce a  hundred seeds and then we plant  those, and I think we've  accelerated this process.  But it's an interesting  question, whether it occurs in  animals.  >> Have you come up with any  good penicillin shots that would  work for plants?  >> For plants, well, in terms of  traditional breeding, and I  actually have a little section  at the very end about biotech  and things like that, in terms  of plant breeding you can go out  into wild varieties. 

 That's kind of the biggest thing  right now, is to find wild  relatives so, what do I mean by  that?  Soybean has been the most  domesticated for so many years,  and you can go back to where  some of the wild relatives are  in China and try to look for  sources of resistance there, and  cross those in.  And try to maybe look for new  sources of r-genes to cross  into, and try to combat that.  And actually, the -- defense  today on late blight.  She went back to potato, the  potato originated in South  America, she found some wild  relatives there of potato and  crossed those into potato  varieties that we use now.  And found some resistance as  well.  So you can kind of go back to  wild relatives to get  resistance.  Okay.  So, anyway, does anybody else  have any questions on that, I'm  going to move on, switch gears a  little bit and tell you about  arabidopsis.  Yeah.  >> Do pathogens die out when  they don't have a host, do they  just naturally die out?  And do things like modern --  farming perpetuate these  pathogens?  >> Well, those are all good  questions. 

 AS far as dying out, I would say  that that is probably the case,  based on selection that, if  they're no longer thriving then  that population will eventually  die out.  But probably, a new one that  evolves to become more virulent  is going to take over.  So it's not that the whole  strain would die out, it might  evolve to become a slightly  different, if that makes sense,  but the original strain probably  would die out.  As far as no-till, that's--  >> (Inaudible)  >> Yes, and I know  they're trying to at least  rotate those, the idea I guess  with no-till was not to disturb,  there's also the whole natural  ecosystem too in the soil that  exists.  So, I'm being mean to all  pathogens, all microbes, because  there are so many beneficial  microbes as well.  I think the idea with no-till is  to help keep some of those  around sort of as natural  combatants so there's perfectly  fine strains of microbes out  there that actually help the  plant, so I don't know if that  answers that question or not,  but there's definitely  differences in farming  practices.  A lot of that, too, is to help  keep the soil less compact and  things like that.  So I just thought I would talk a  little bit about arabidopsis.  It's, I'm guessing, it's a  plant, I don't know how many of  you have heard of it.  Because in the elevator all the  time at work, everybody's  saying, what is that plant?  So not many people know what it  is. 

 But it turns out that it's  actually a huge model system,  not only for plant-pathogen  research but for, it's a model  system for just plant biology  research.  So there's about, maybe eleven  or twelve labs on campus that  use it as a system, as a model  to send lots of plant pathogens.  Just to tell you a little bit  more about arabidopsis.  I think it, I'll say a little  bit about the history.  It's been around in the lab  since about 1960 or so and it  was developed, I'd say in the  last twenty years, to be a model  system, and the reason that it  was popular is that it's very  tiny.  And these are all huge, blown-up  pictures.  It's a member of the mustard  family, of the Brassicaccae  family, so in the Brassicaccae  you have your cabbage, your  radish, broccoli, cauliflower,  turnip.  And so a lot of the times we  like to argue that it's an  economically relevant crop.  Actually it's not, it's  considered more of a weed, but  it does, it's very closely  related to cabbage and radish  and those.  I put this picture in here just  to illustrate. 

 It shows the seeds down here,  they're actually quite large,  and I thought, that's funny  because they are tiny.  And this shows it even better.  The seeds are extremely small,  these little tiny dots.  So it's good and it's bad,  they're very prolific.  What you'll get, and this is a  seedpod right here, let me just  back up.  In fact, here it is more  closely.  Inside, this is called a --,  inside one of these -- there's  upwards of thirty seeds inside  the pod and so you can get lots  of seeds from a plant.  There's a flower.  They're self-pollinated.  There's two stages of growth,  this is a tiny little seedling. 

 They have kind of the rosette  stage and then they produce  these stalks with these flowers,  and let's see.  Oh, and they're found all over  the world, there's actually,  over 750 natural accessions that  have been collected from around  the world.  It's probably a bit difficult to  see.  But this is the US here, so  there's quite a bit found in the  US and quite a large part of  them found in Europe as well.  So they occur all over the  place, but they really are  considered more of a weed.  So this brings up the question,  why study it?  Why not study a species of  agronomic importance?  And I did ask that question a  lot.  Some of the advantages of using  a model system, and I'll be  showing these in the next few  slides is that they're easy to  genetically manipulate.  And we're talking about basic  research, not applied research.  So they're easy to manipulate,  they're easy to transform with  genes, we can make transgenics,  there's a large information  base.  Again, one of the reasons it was  used and brought to -- attention  is its rapid life cycle. 

 So within six to eight weeks of  planting the seed, you can  harvest the seed.  And that's a huge jump from,  say, corn or soybean or tomato.  I used to work in soy actually,  that was my grad work, and it  would take at least three or  four months to get from seed to  seed.  So you can see that for your  experiments.  So, research is always, how can  we do it faster?  There's prolific seed production  and they're easy to cultivate in  a small space.  And I wanted to bring one in,  but just, they're -- big.  They're probably this big, and  they're probably this tall from  when they got stalks with seeds  on them.  So if you can imagine, you can  pack a ton of these things into  a small space until you can work  with them in the lab.  And they're easy to transform  using agrobacteria--.  Have you talked about plant  transgenics or anything?  I'm not going to have time to  talk about it, but the way that  you make a transgenic plant is,  instead of traditionally just  breeding in a gene that you want  the plant to have, you can  actually transfer it with a  bacteria. 

 You can put the gene you want on  a piece of DNA and put into this  strain of bacteria, it has to be  this strain, this is a very  special strain of agrobacteria.  It actually causes -- in corn,  that's where it originally was  found.  But it turns out, this strain of  bacteria can actually inject or  transfer its DNA into the plant,  or into its DNA and that's how  you can make a transgenic plant.  And it turns out this process,  I'm going to get to that process  in a minute.  Let me go over a few more of the  fun cool facts about  arabidopsis.  It's got a small genome, it was  sequenced in 2000, I think it  was the first plant genome that  was sequenced.  Now rice is also sequenced.  But it was fully sequenced so we  know, the whole DNA, these are  the five chromosomes.  There's a large number of lines,  mutant lines.  Lots of resources. 

 There's a website that's devoted  to housing all of this  information.  And just as an example, there's  one gene, this is an r-gene  called --, and it confers  resistance to --.  So if we want to know about --,  we go to the website, we type it  in, and we can find out all  kinds of things.  We can look for markers that can  detect the gene, we look for  mutants.  I know all this stuff is just  numbers, but it represents all  kinds of information that you  can find.  If you wanted to make a mutation  of that gene, you can find out  what the sequence of the gene  is, anyway, you can find out all  kinds of information from this  database.  It's all found online.  >> Do scientists believe they  know what all the chromosomes  contain and what all that  material is like?  >> The problem is that just  having the sequence alone  doesn't mean anything.  The human genome was sequenced  and that's great and at first it  seemed like, we're going to know  everything, we've got the  sequence.  But that's just a lot of As, Ts,  Cs, and Gs. 

 So you've got to go in there and  figure out what do all those  genes encode for?  What do they do?  How do they interact?  And so having the sequence alone  is definitely useful but it  certainly doesn't tell you  everything.  >> Do you have a way to delete?  >> To delete the genes?  Not a good way.  This is the t-DNA, again this is  from the agrobacterium, I wish I  had brought a picture of that,  but again the t-DNA, it can  insert a piece of DNA into the  locus that you're interested in,  so it can disrupt function that  way, by inserting a big piece of  DNA in the open reading frame,  it can block function.  And that's how we have all these  collections, and almost, the  goal is to get a mutation in  almost every single gene.  And they're getting there, I  can't tell you how many -  there's 2500 genes in  arabidopsis and I thought they  had, maybe half of them.  So then of course you've got to  go in and look at them and say,  well what does it do?  And do all these tests.  But certainly, we don't really  have deletions, it's more of an  insertion to disrupt function.  And then of course there's  naturally occurring -- and things  like that.  So I just want to briefly  mention the transformation  because this is kind of  interesting.  Also, the method that we use was  really developed in our lab, so  Andrew Bent really defined this  method and published it and it's  been widely used and everybody  uses it, so I just thought I  would mention it really quick. 

 It's basically revolutionized  the work with arabidopsis.  No tissue culture, because to  make a transgenic the  old-fashioned way, you have  start a tissue culture, you make  callous, and then you induce the  plant to sheet and induce the  plant to root.  So this doesn't involve any of  that, minimal effort, and you  can generate thousands of  transformants.  So how do we do it?  Here's some pictures.  There's the arabidopsis, they're  starting to flower and that's an  important thing, the bacteria  attack basically the flowers  there.  So you wait till they're  flowering, we dip them in a  solution of bacteria, this is  agrobacterium, containing a  plasma, it's a circular piece of  DNA, you insert the trait that  you want the plant to express.  Put that on the plasma, then you  dip the plants in the bacteria,  and the bacteria actually invade  the stem cells and the ovary  tissue basically in the flower  and they integrate into the  plant genome.  You wait till this seeds some,  you take the seeds, plate them  on a plate that has a selectable  marker, that's something I  forgot to mention that on this  plasmade, you have something  such as -- or some kind of  antibiotic so you can determine  which plants carry your gene of  interest and which ones don't.  The green ones are obviously  living and those would be the  ones that are transgenic,  whereas the rest are dying;  plants can't live on -- plates.  So that's sort of in a nutshell  how you can make a transformant  of arabidopsis. 

 And it's really one of the only  plants that you can do that,  that we know of.  I think they're trying to work  on that in other plants but  nobody's really had any success.  The fact that you can just dip  it in the solution and let it  grow, and you're done.  Again, it takes months, with  tomato, it would take at least  three months to get a  transformant from tissue  culture, so it's a lot quicker.  And the beauty of that is that  you can over express a gene, you  can try to knock out a gene that  way, you can do all kinds of  things.  And then in terms of disease  resistance research, pseudomonas  we talked about this a little  bit, it does affect arabidopsis  so we can use it in the lab to  study plant-pathogen  interaction.  Normally pseudomonas causes  bacterial speck on tomato but it  also affects arabidopsis and  causes -- and the dark spots.  Just a little background if  you're interested.  Pseudomonas is an epiphytic, it  lives on the surface of the  leaves, and quite happily  actually.  Then it can enter through leaf  somates, grow in the  intracellular spaces, so in the  spaces between the -- cells, and  when it's ready it can become  virulent on arabidopsis and  cause lesions and things like  that. 

 So that's the pathogen that we  use, we and many others.  This is the  arabidopsis-pseudomonas syringae  interaction, is probably the  best, well-studied system for  plant-pathogen interactions in  basic research labs.  So us and many other labs.  There'll be national meetings  with 300 to 500 people, all  studying the same thing so  everybody uses it as a model.  And pseudomonas has been  sequenced as well.  So arabidopsis responds to  bacterial flagellin, if you'll  remember at the beginning I was  talking about how flagellin can  induce innate immune responses,  so that's something that we're  really interested in at the lab.  And what you can actually do is  plate arabidopsis seeds, you can  plate them on augur plates, grow  them for a few days and then  pick them and plate them on  these 24-wall plates that have  liquid in them.  And you can grow, they actually  grow quite happily in liquid.  And you can add flagellin in the  form of a peptide, just a tiny  little peptide that you can  dissolve in water and put into  plates and then you start to see  these growing in culture, and  then do assays over time, you  can look at -- production,  callous deposition, -- addition,  within minutes, hours, and days.  And I'm going to go through what  all of these assays are when I  get to my research so I didn't  want to go through them in  detail but just to say that  arabidopsis does respond to  bacterial flagellin. 

 So we can use it as a system to  study the plant innate immune  response.  So now I'm transitioning from  what is arabidopsis, why do we  use it, and its importance in  our system to my post-doctoral  research, in a nutshell.  I tried to make it a little bit  succinct, and I guess at this  point, were there any questions  on arabidopsis?  >> You submerge that whole plant  when you--?  >> Yeah, you dip it all the way,  you know this is kind of an  interesting, Let me back up.  Because what we do is, we grow  the plants in --, and we put the  seeds on over that so when you  dip it the soil doesn't fall out  but we basically dip it all the  way up to the base of the  rosette, we're basically trying  to target the entire plant but  mostly we're just tying to get  the flowers, because that's what  the bacterium are mostly tied  with.  At this stage there will be a  lot of stems coming up here that  you don't want to miss either.  So, all this is bacteria and  a little bit of sugar because  the bacteria like sugar, it  makes them happy and grow.  And the whole process takes  maybe a half an hour if you're  fast.  All right, so, yeah?  >> (Inaudible)  >> A certain what? Acidity?  >> Tonicity.  >> Oh.  Well, the only other thing we  add to it is we add a surfactant  that helps the bacteria hold on  to the leaves but other than  that it's water, sucrose,  surfactant, and bacteria. 

 And there's no, we don't worry  about any sort of pH or anything  like that.  Nope.  The one thing my advisor did  was, when they published his  paper, because they'd already  known you could do this with  arabidopsis, what he did was  basically break it all down.  Because it used to be that you'd  put in salts, all these  vitamins, all these things, and  it was thought that you needed  all that.  And it turns out you can strip  all that away and it's as simple  as a 5% sucrose solution, and a  little bit of a surfactant, and  that's it.  So they did some research to  show that none of those other  things really made a big  difference with transferational  rates.  So this is where it gets pretty  molecular, investigating the  role of poly (ADP-ribosylation)  in plant-pathogen interactions.  And I'm going to take you  through, what is poly  ADP-ribosylation, because I get  that all the time, nobody's  heard of it.  And even in my field because  actually it's very very new to  plant research.  Only several people have  published on it in the entire  world so it's pretty new. 

 So this is just, what is poly  ADP-ribosylation.  I'm just going to go over the  mechanism real quick, and I  think you've been through  genetics and things like that,  this is just a picture of the  central diagram, the DNA goes to  the RNA, gets translated into  proteins.  I put this on here just so you  could visualize it, what are we  talking about?  We're talking about a  modification, a post-translation  modification of the protein.  So you can kind of forget about  all of this and just think about  the protein, because there's a  lot that happens after the RNA  has been translated and it forms  a protein there's a tremendous  amount that can happen  afterwards.  There's a huge number of  modifications that happen, and  there's actually up to 300 of  these that have been documented.  And there's different kinds of  chemical groups that can get  attached, so you've got  phosphorylation, we've go  modifications such as  acetylation, mehtylation, these  are all chemical modifications  but each of those modifications  can kind of tell the protein to  do something different.  And just as another example, and  it's not so important, this  slide was basically to  illustrate that once a protein's  made, it's not going to finish,  there's still a huge amount that  gets done to the protein, we  call it "decorated" there's all  these different modifications  that make it do different  things.  So this is just to illustrate  the importance of  post-translational  modifications.  If we think about the human  genome, it's been sequenced, we  know that there are 30,000  genes, but they predicted,    through -- and other computer  approaches and other sampling,  they figured out that there's  almost 1. 8 million  types of protein variants.  So if you think about, it's not  just one gene one protein, you  have all this happening  afterwards. 

 And the post-translational  modifications can account for a  huge portion of protein  variation so this whole scene  gets very complicated.  But I just wanted to illustrate  how important a  post-translational modification  is because poly ADP-ribosylation  is a post-translational  modification.  So how does poly  ADP-ribosylation modify  proteins?  So what is this protein  modification doing?  If we just step back in simple  terms, where does it all begin?  It starts with NADs, -- I didn't  promise there'd be no chemistry,  there's a little chemistry, but  it's a source of ADP ribose, so  the ADP ribose molecule's here.  Just to reiterate, NAD is an  enzyme found in all cells  involved in redox reactions so  it's definitely abundant, it's a  very important molecule, but it  does serve as the source of the  ADP ribose.  The process of ADP-ribosylation  is actually very simple.  There's two enzymes that are  involved.  One's called poly ADP ribose and  this enzyme has many functions.  It can take from NAD it can take  the ADP ribose and attach it to  a receptor protein and modify  the function of that protein by  putting on this ADP ribose  group.  It can also catalyze the  elongation of many of these ADP  ribose groups, so you've got  this protein and parts coming  along here attaching ribose  after ribose all the way up to  200 ADP ribose, so this molecule  is extremely negatively charged.  And it alters its function. 

 The only other enzyme involved  in this process is PARG, which  is poly ADP ribose  glycohydrolase, and a  glycolhydrolase just means that  it creates the ribose-ribose  bond.  So PARG comes along, creates the  bond, and releases the ADP  ribose, so you've got -- that  puts on the ADP ribose and PARG  that takes it off.  So it's two enzymes and they  work, they kind of counteract  each other.  So there's many impacts that  this modification can have on  cells.  I mentioned that in the  beginning, the source of ADP  ribose is from NAD.  So you can deplete NAD pools  which is in effect a lot of  redox reactions so you've got  effects there, and you can,  actually the whole ADP ribose  itself can signal and I'm not  going to get into that, but  there's things that the ADP  ribose polymer can do.  And then you can also modify the  protein, it's post-translational  modification, it can activate or  inactivate a protein.  So that's the things it can do.  And this is interesting from  just a biological standpoint, a  lot of this research is being  done -- systems for at least 50  or 60 years there's been a lot  of research done on this.  And it's been shown to be  important in lots of things,  apoptosis, DNA repair, gene  expression, cell cycle  regulation, chromatin  remodeling, probably a lot of  the things that you've been  hearing about this year. 

 So it's been shown in -- to be  important there.  But even in humans it's been  shown to have a role in injury  after heart attack, stroke,  heart failure, liver and kidney  failure and arthritis.  And actually it's a huge  pharmaceutical industry right  now to develop pharmaceuticals  that turn off these PARG  proteins, and even in cancer  research.  And I just saw recently they  actually got some inhibitors of  this process, they've developed  pharmaceuticals that are now in  clinical trials.  So it's really important in  humans, in lots of cellular  processes.  But we don't know anything about  it in plants.  And I don't have a real good  segue, because you're probably  wondering, what does this have  to do with plant pathology.  But we've figured out from a lot  of gene expression studies that  the genes involved in PARG and  PARP are important, they're  up-regulated when plants respond  to pathogens.  So that's why I started getting  interested in -- and plant  defense.  Because again the expression of  these genes was increased in  plants responding to pathogen  attacks. 

 That's sort of my premise, but I  found out that this is really  important in -- research as  well.  So this is also part, I think, I  got some funding from the  National Institute of Health to  actually do some of this  research in plants, and I think  in part because of its role in  human processes.  So the next few slides I'm just  going to show you a little bit  of data that I have regarding  poly ADP-ribosylation in  plant-pathogen interactions.  So just going to show you, just  briefly, that we've done a  number of times.  We've got ways to detect poly  ADP ribose, so that polymer, you  can detect it using an antibody.  And we've found that in plants  that are infected with an  avirulent strain of pseudomonas  you see an increase in the  levels of that polymer,  indicating that poly  ADP-ribosylation is occurring in  response to infection.  What we're trying to do is  determine what proteins are poly  ADP-ribosylated?  So there's an increase in poly  ADP ribose, what are the  proteins that are being  ribosylated?  We don't know, so we have a grad  student working on that and  she's working on it.  So that's just one of the  questions we're interested in.  And the next three or four  slides just going to finish up.  I'm going to show you a little  bit of work I'm doing with PARP  inhibitors. 

 And as I alluded to before, it's  a multi-billion dollar industry,  there's lots of trials, they're  trying to use PARP inhibitors to  treat Alzheimer's, protect  against heart attack, stroke,  and chemotherapy.  There's a lot of evidence in  mouse studies and cell-culture  studies that these can be  important to be able to block  PARP activity.  Apparently this process is  induced in response to stress.  So the inhibitors that I use are  aminobenzamide and  methoxybenzamide.  It doesn't matter what they are,  just to give you the names.  But they are shown to be potent  inhibitors of PARP activity and  we can use it to conditionally  turn it off.  So instead of just knocking out  PARP genes, we can add a little  bit of inhibitor and turn them  off whenever we want to.  So I've been using these  inhibitors in a lot of my plant  studies, in particular using,  trying to investigate  arabidopsis innate immune  responses.  And in PARP because it's an acid  where I can add flagellin and  inhibitor to the wells of these,  it's all liquid culture, and  then I can test for all these  different responses and see if I  inhibit PARP activity, if I  block poly ADP ribosylation, can  I disrupt or enhance innate  immune responses in plants?  And that's what the last three  slides are going to be about.  One of the assays that we've  looked at is looking at the  production of ROS production, so  things like hydrogen peroxide  which we know accumulate. 

 And we have an assay that can  detect luminescence in the  presence of --.  So when we add flagellin to a  seedling looking at it over  time, we see a huge increase in  ROS when you compare it to the  untreated, which is way down  here.  So there's basically no ROS.  When we add inhibitor and  flagellin, there doesn't seem to  be a difference.  It doesn't seem to enhance it,  it doesn't seem to decrease it.  I've repeated it several times,  there's really no difference.  And inhibitor alone doesn't do  anything either.  But when I look at downstream  callous deposition, this is one  of those classic responses, that  when you treat plants with  flagellin you get callous  forming, and this is an enhancer  of cell wall and it's thought to  protect the plant against  further invasion, so it's  basically strengthening the cell  wall.  When you treat it with flagellin  you see these little deposits  occurring.  That's the callous. 

 But when I treat with inhibitor  and then flagellin I don't get  any callous deposition, so it's  blocking that response.  And when I let the seedlings  grow longer, for several days,  on those plates and then I weigh  them, what you normally see, and  let's just focus on this things,  which is Col, it's Columbia,  it's the ecotype that we use, it  represents a wild-type plants.  And fls2 is actually a mutant  plant that can't respond to  flagellin.  So looking at here you've got,  you weigh the seedlings you've  got lots, they're 30 milligrams  or whatever untreated.  You treat them with flagellin,  that's that flg22, you see a  significant decrease in seedling  weight and that's the classic  response that's called seedling  growth inhibition, it's the  classic response to flagellin.  Fls2 plants don't respond to it  because they can't receive  flagellin.  I add the inhibitor the plants  don't really like it much but  they're okay.  But when I add inhibitor and  flagellin they get really,  really tiny.  So what does that mean?  It just means that we know that  it seems like by blocking PARP  we've uncovered the fact that  poly ADP-ribosylation can play a  role in plant defense response  to pathogens.  In particular looking at the  innate immune response. 

 We see that some responses  aren't blocked.  I didn't mention gene response.  These responses aren't blocked,  but callous deposition as well  as lignin is blocked and  furthermore the plants actually,  with seedling growth inhibition  get even smaller.  So there's something going on,  poly ADP-ribosylation is doing  something, which is more than we  knew before.  So now we're interested in what  proteins are poly  ADP-ribosylated?  Why does PARP inhibition block  callous deposition?  What is happening?  We really don't know, we just  discovered the phenomenon.  So we'll develop some genetic  screens and do some micro  analysis, which means we're  going to look at all the genes  that express the whole lot of  them.  At the same time, in response  inhibitor -- analysis, we're  also going to look at poly ADP  ribosylation in other  plant-pathogen interactions.  I'm in the process of finding a  job and I hope to take some of  these projects with me when I do  find one.  So it's a lot of unanswered  questions and hoping to develop  sort of a ten, fifteen-year  research plan so I can answer  these questions because it's  definitely not going to be  answered overnight.  We've got lots of work ahead of  us. 

 So with that I would just  acknowledge my lab members,  first Andrew Bent, who is the  PI, the -- of lab and Amy who's  the grad student on the project,  and the other post-doc students  on the lab.  And I put this here, if you have  any questions afterwards I can  give you my email address too,  because I was thinking with some  of the plant pathology questions  I may or may not be able to  answer, but I'll try to the best  of my ability, but you can still  feel free to email me.  And I can get back to you as  well.  So, that's it.