 is stimulating and I'm sure we will go back home with a more open mind and a more bigger picture of what we do. So as Mateo was saying, this is the leader, the lasers. So as Mateo was saying, I work at ICGB, it's located in Trieste, so it's very similar in mandate and concept to ICTP. We were born in 1987 as a project of the United Nations Industrial Development Organization and we were that project until 1995 and from 1995 we are an independent intergovernmental organization and our mandate is to try and do good science but also remembering that we have a duty to promote biotechnology in developing countries and also doing training, training scientists and promoting this technology so in order to make sure that also developing countries would benefit from this technology, in fact when it was created in the 80s, genetic engineering biotechnology was the revolution so we wanted to make sure that also developing countries would benefit from this new technology. So we are an organization that is composed of three different institutes, one in Trieste here which is also the headquarters and then we call them components, so we're a global research organization so we have a component in New Delhi in India which is actually born at the same time in also 1987 like Trieste and then in 2007 we created a new component in Cape Town South Africa because since it was created in the 80s we have to admit and very happily admit that a lot of countries, developing countries have made incredible progress, countries like India cannot be regarded as developing in terms of biotechnology that now leaders in the field whereas the African content in our view is lagging behind so to give a bit of momentum we created a new institute in 2007 in Cape Town. So we have three components and in each of these components we have research labs, for example in Trieste there are 15, in New Delhi there are 30, in New Delhi is about twice the size of Trieste here, in Trieste there are 200 people in New Delhi there are 400 in Cape Town South Africa where it's actually growing and we hope to bring it up to Trieste standard within a few years, Trieste, sorry Trieste numbers within a few years. So our organization actually belongs to the member states so we have 60 plus member states we have actually almost 80 now and our member states actually own us and we meet once a year to decide which way to go and what projects to do, how to evolve our organization and as you can see here most of all of our member states are from countries with economies in transition. So what we do, we do several things to promote this by technology developing countries apart from having advanced research projects in the three components, we have our specific long terms, we have postdoc fellowships and predoc fellowships which are exclusively reserved to our scientists from our member countries. We organize meeting courses like this one, we organize about 25, we organize them and we sponsor them, okay. We have, we're very proud of this, we have actually our own grant call, once a year we give grants to scientists from member countries so I think that's a very good way, bringing money to scientists, good scientists in these countries that want to do research there and then we also have other programs. So this is just a brief, very brief overview and some of you will come to ICGB on Wednesday, we can get more detail, we have of course updated website and any questions we have you can contact our director general here in Trieste. So I'm an experimental microbiologist and I went to university in the 80s when I was taught that bacteria are unicellular organisms, they're completely different from multicellular organisms, you know we have to forget about multicellularity, you know bacteria can live on its own, likes to live on its own and so on and so forth. So starting, getting interested in quantum sensing in the late 90s I realized that this concept that I was taught at university is probably profoundly wrong. We now believe, at least I believe, that bacteria do not want to live as solitary organisms because probably they're much more vulnerable to stresses, environmental stresses or predators, whereas when they live as communities we're now learning that they can do things that they cannot do, they don't like or they cannot do alone, it's like you know if there's a lot of, also humans, if it's a lot of us we can do incredible things with a lot of us that we cannot do when we're alone, maybe the concept is exactly the same with bacteria. And a lot of scientists have studied their favorite organism and it's shown that through quantum sensing, through the production and detection of small signals, a community of bacteria can do incredible stuff, like they can make a biofilm, they can become virulent, you know it's no point, you know scientists believe, we believe microbiologists, no point attacking a host when there's a few of you, it's better attacking it when there's a lot of you, so synchronization of behavior, if we synchronize our behavior we can be more effective. And this is brought about by the cell density dependent mechanism which we call quorum sensing, the term has been coined in 1994 by P. Greenberg and Cliff Fouquen, Steve Winans, this is a landmark paper which they introduced the term and a lot of work has been done on this and there's a lot of literature on this. And you can find in the literature a lot of signals, you know these signals are being discovered and are continuously being discovered. This is a paper by, from Nottingham Group, which is a review published in 2009, probably this is not complete by now. And I very much believe this is the tip of the iceberg, there's a lot more to be discovered. And you can see here that the message here we have is that they're rather simple molecules. We're going to hear from Ines, she's going to talk about peptides that are produced by gram positive bacteria, we're going to hear several speakers, we'll talk about gram negative bacteria. So if I just start with this, with this class, these almost serine lactones, these are made by gram negative pro bacteria and today, you know, you often read in papers the most common signal molecule, quorum sensing molecule in gram negative bacteria. And I believe it's the most common because it's the easiest to detect actually. It's extremely easy to look for these molecules and to determine the structure. So when we say most common maybe it's a dangerous term, maybe at the moment they're most common, maybe because they are very easy to study. And what you can see here, over 100 species of gram negative pro bacteria have been reported to produce this kind of molecules. And just what I want to say, there's a lot of dialects, so this is the basic structure and you can have the acyl chain that can vary from a 4-carbon to a 20-carbon and a position 3 you can have a ketone or hydroxy or a methylene group. So you have, for example, here is this is the molecule produced by Vibrio fissurei, the first molecule which was discovered and this is a carbon 6 with a ketone, so 3-oxo carbon 6 almost serine lactone. And then you have so many different dialects produced by so many different species, which provides a bit of specificity to the system. It's very, very powerful. So you have a basal level, so bacteria we believe tend to produce them all the time at basal level. So low cell density, we have low concentration. When we have a high cell density of bacteria, what we believe happens is that these almost serine lactones are diffusable. In most cases, we're rather fully diffusable. And the high cell density, so high concentration of almost serine lactones, there is a regulatory protein, sorry, just go back. These are made by one gene, which goes about almost serine lactone synthase, which makes use of common precursor in a living cell, one involved in fatty acid biosynthesis, the other one in a lot of enzyme reactions, takes these two guys, makes the specific AHLs. A high concentration of this high cell density, there is a cognate regulator, which will specifically recognize the AHLs that are being made. This interaction can be very strong. Some cases also reversible. And this leads to then the ability of this looks our family protein to affect gene expression. And it does that by other in a positive way, in a negative way. Again, I'm generalizing, there's a lot of deviations to this thing because there's a lot of studies made on the system. But in general terms, we have, when we have activation, we have this looks our protein, the detector that upon binding to the almost serine lactone, dimerizes, homodimerizes and binds to promoter regions and kicks starts the RNA polymerase to activate transcription. So you can see activating transcription response to cell density. If we have a negative scenario, maybe the dimer can bind to DNA in the promoter region, repressing the ability of the RNA polymerase to bind to the promoter. And this repression is relieved when there is high cell density because the almost serine lactones will bind to the regular and this then will be unable to bind to DNA and repression is relieved. So you can have positive. In most cases, it's positive, but there are beautiful examples also of negative regulation via these kind of signals. So what I actually didn't say when I presented the slide with all the signals and I'm very proud to say that a lot of the work, the foundation work done on this system is actually comes from studying plant-associated bacteria. I've underlined here, these are examples of pathogenic bacteria they use for almost serine lactones and I've underlined here probably, maybe I'm wrong, but probably the best studied species for this form-sensing type system. And you can see here that we have a large portion of these are plant pathogens. And the foundation work done on agrobacterium tumor fashions in Irvinia and Pantoea still are today the leading papers and textbook papers for this system. We're going to hear a lot about pseudomonasurginosa, Pete Greenberg will talk about it, Livia will talk about it. So there'll be talks about pseudomonasurginosa, which is probably maybe the organism that has been mostly studied for this system. So I cannot not mention the work done, the beautiful work done on agrobacterium tumor fashions. This is an incredible organism which has been studied for many reasons. This organism is a pathogen, it causes a tumor, it transfers a piece of DNA from a plasma into a plant cell and has been studied for pathogenic reasons, but also for its use by humans to create genetically modified plants. But anyway, I don't want to go into detail, but I have to mention the beautiful work on the corumnsensing systems, almost like the corumnsensing system, which is involved in the regulation of the conjugation of the plasmid that carries that is important for this disease as well as the copy number of this plasmid. And it's beautiful because how the system is activated through plant signals that are actually produced by the transfer DNA in the plant cell and then there's also the control of the system, the shutting down by lactonases and anti-proteins that block the regulator. So I don't want to go into detail, but this by far I want to say that it is so beautiful and has been studied in major contributions by labs of Steven Ferrand, Steve Wynans, and Clay Foucault, have done beautiful work and it's really a beautiful system to study in all its aspects. And of course, plant beneficial bacteria maybe lag a little bit behind, but a lot of scientists around the world are looking at good guys and we heard at the introduction there's a lot of you studying biological control of plant beneficial bacteria. Also these guys use this kind of corumnsensing for their beneficial interaction with plants. And here it's just by no means exhaustive, but there is a list here of good guys, rhizobium and pseudomonas especially, that use this kind of corumnsensing to regulate plant beneficial interaction and phenotypes related to their symbiotic life with plants. So which also raises the issue that are we going to control, there's a lot of, in my view, controlling corumnsensing can potentially be very useful in agriculture to control plant pathogens, but again we have to be very careful because a lot of beneficial bacteria use the same system to have a beneficial interaction with plants. So going back to this slide of some examples of the signals and you can see here, and I didn't mention that you can see all the plant-associated bacteria, the xanthomonas, the xylellas, the brady rhizobium, so a lot of work is done with people like me and other scientists around the world that are interested in plant-associated bacteria. The other family I want to talk about that is receiving a lot of attention recently is these molecules which is called diffusable signal factors. You know I've been trying to catch up with the reading but you know it's not always easy with all the things we have to do, but these molecules are now widely found in nature and I think we should say that it's because the sensitivity detection methods have improved so much. So for that reason now we are realizing that there's so much more common than we thought, just like the homocerein lactones, right? At first they were confined to xanthomonas and xylella, now we're finding a lot of them and also we're finding a lot of different ones. And again here it's a family of molecules made from you know very similar made from the fatty acid biosynthesis pathway and again here it's just like homocerein lactones, this kind of form-sensing, it has beautiful work being done and again it's basically all plant-associated bacteria. It's also been found in burgled areas and some human opportunistic pathogens now but the fundamental work has been done probably xylella fastidiosa through the you know the work of some Steve Lindow, xanthomonas, the work of several people in India and in the United States have looked at this DSF production and again we're talking about very serious pathogens here. I don't have to say how serious xylella fastidiosa is for the United States in Grapevine and also now in Italy we had a major xylella fastidiosa infection in the southern Italy of olive plants. You know xanthomonas again very very important pathogens they attack a wide range of plants and crops and here you know it's in Brazil they affect citrus and also is extremely important rice pathogen. And in these guys they've done again beautiful work and unlike what occurs in the homocerein lactones we have a classical two component system that is involved in detection of these of these of these diffusible signal factors. So we have a sensor anchored in the periplasmic membrane which can detect the DSF and then and then can initiate a phosphorylate reaction and transfer the signal to the cell. Again beautiful work has shown that there is mechanisms to also keep this quorum sensing obey and this is a topic I think which deserves a lot of attention quorum sensing is very expensive metabolically. A community of cells of bacterial cells that these types of quorum sensing on it's committing a lot of his energy to the event so we need to make sure to be able to shut it off very efficiently and very quickly. And again work here is now beginning to show for example that the sensor component can block the RPFS which is the synthase the protein that synthesizes the diffusible signal factor can be inhibited by the by the sensor when there is a low cell density and high cell density there is a phosphorylate reaction so the sensor will detect the almost the the DSF transfer of the phosphate to a to a sensor which will then affect the concentration of an intracellular messenger which will then free a regulator which will do its job. So again here it takes an extra dimension there is an intracellular signal which is called Southgate that GMP which is linked up to this DSF diffusible signal factor. So just just to to show the complexity of these systems and in this case DSF involving involving a little more complex transmission of the signal and interacting with other components. So the the contribution of plant-associated bacteria to quorum sensing has been fundamental and it's probably going to be also in the future. Why is that? Because I think because plants associated bacteria are so diverse there are so many different now with the new year of microbiomes we are realizing how complex is the microbial life next to a plant and I think the challenges of the future a lot of the studies I mentioned have been from experiments done on a pure culture scenario in your lab you know my favorite organism working you know working on a sterile condition making sure you know another thing we were taught when we joined lab is that make sure your culture is sterile if you get contaminated you ruin all your results actually probably that's completely wrong if we were working with contaminated cultures back then with maybe our results we would get today would be maybe more realistic to what happens in nature because in most cases in nature bacteria do not live as solitary as not solitary excuse me as as pure mono strain scenarios okay there are of course beautiful examples of that but when we look at plant-associated bacteria we need to think about a multi-species scenario and we need to think about the plant so what I'll do in the next 20 minutes is actually talk about my work in my lab hopefully giving some now opening my mind rather than sticking just to the daily problems and giving some messages of microbial community research and the way direction that I think should be going so now I'm very interested in looking at the more realistic scenario of what is all this means to the plant all this communication going on and what about the multi-species effect you know these bacteria live together with so many other bacteria so all the scenarios I've talked about how how does it fit in a multi-species scenario so I've been I've been interested in rice for for for many years and I used rice as my as my model a plant for obvious reasons it's by far the most important crop in the world by far even though it's not grown in the rich countries very much so and I studied back then and I looked a lot of bacteria that live with rice I don't want to go through all this but we looked at good guys and bad guys and a lot of them have quorum sensing and a lot of them and we focused on AHLs almost in lactose because again it was the easiest thing to do at that time very easy to detect but we got really we got really lucky and excited about by the rhizosphere pseudomonas so we had a bunch of strains from India that were isolated from the rhizosphere of rice and we have a bunch of xanthomonas or rhizy pathogens okay that that we knew made dsf but so we got about 25 from the company go buyers send me 25 very pathogenic strains and students in my lab you know Sara, Ferluca and others and here also I had a postdoc Laura Steindler started looking at at do they make homocerein lactose and what to make a very long story short and several years of work the rhizosphere pseudomonas and the pathogen xanthomonas or rhizy actually do not make homocerein lactose in most cases some rhizosphere pseudomonas do okay so rhizosphere pseudomonas in our hands 15 to 20 percent make homocerein lactose and have different systems it's like they've acquired them recently and they have transposals on the side so it's like they've it's been a recent horizontal transfer event but they don't have a core they're no core homocerein lactose systems and the xanthomonas or rhizy basically didn't we we didn't uh detect emotional lactose all this work was done the pre pre genomic year so it was done in 2005 2006 2007 then bingo suddenly within has it happens with genomics suddenly within two or three months there was a genome of xanthomonas or rhizy from group in korea from a group in japan and a group in china and now there are many genomes and same with pseudomonas there were several genomes coming up as well as we were contributing to some of these genomes and what we could see from these rhizosphere good guys and the pathogenic guys is that actually they don't have the gene to make the synth the almost the codes for the synthase that makes homocerein lactose but they have the regulator so they have a luxer family regulator which is very similar to luxer family regulars that bind homocerein lactose as i've shown you and either activate or repress transcription and in fact we we call these luxer solos pits called them luxer orphans we both call them each other actually no problem but but what is now evident from the genomics that these guys are extremely common so it's very common in granite bacteria to have to have bacteria they have these guys devoid of a cognate homocerein lactose synthase so what do they do well they're not very well studied actually and you know works of work of brian hammer has shown that for example e coli is the model organism for having e coli does not make homocerein lactose but it has a luxer solo and what brian has shown that it responds to homocerein lactose so it's like i don't speak but i can listen okay in the case in our case we were we were obviously trying to follow that route probably it could be that these guys don't have the ability to produce but they have the ability to listen probably of hls produced by neighbors there were also reports in early 2000 about uh regarding homocerein lactone mimics so that plants produce compounds that would activate homocerein lactone biosensors so maybe you know there's still today that's believed that plants produce compounds that can interfere or talk to these luxer proteins so we were following these two scenarios and to make a very long story short the work of two phc students in one postdoc okay sara ferruga and one gonzales worked as phc students and sujata supermoney as a postdoc the basic we basically showed that in the benefit good guys and in the in the good rhizosphere pseudomonas and the bad guys these two luxer proteins have evolved away have evolved away the ability to buy homocerein lactose but they're blinding they're binding to a plant compound uh and when they do that they are they're active they're very important for biocontrol in collaboration with with christoph keel in university of los ang using cucumber and a funger and uh and uh pithy and uh my city pithy mesamolo we shown that this is very important for biocontrol and exanthomonas arrives it's important for virulence so we've done a lot of genetics microarray and mutants and blah blah to show this but the message i want to just give you here is that we found these this kind of the color subfamily of luxer proteins okay so when i say biocontrol is is if we for example in this case what we did the experiment if we inoculate our cucumber plants with this guy they would keep the pathogen away and the mechanism of that is other they produce antimicrobial compounds and they also induce the immunity of the plant so controlling biologically a pathogen with another with in this case with a bacteria so the message i want to give here is is that uh we found this family let's hold this subfamily luxer proteins which we call solos uh very very common in bacteria and this is by no this is a you know three four year old uh tree that we constructed about this family and you can see here all these guys are plant associated we have the rhizobium we have the pseudomonas syringes we have the rhodobactyls agribacterium exanthomonas so we published that is we think this is a very widespread communication system with uh with the plant the message here this is one and i think might be this is one of many that we still have to discover these guys that live next to plants communicate with each other but also evolve the ability to communicate with the plant and this is very important in terms of microbiome because in my view in applications if we understand these these communication systems then we can maybe we can engineer we can we can uh tailor the plant for a good beneficial recruitment of its microbiome especially in the root you know the root uh the root the soil closest to the root is called the rhizosphere and this is often paralleled with the gut the human gut because the this this area of the plant uh just to tell you the plant releases 15 percent of the carbon and nitrogen that it has into the rhizosphere and it does so we believe for a good recruitment of the microbiome so if we understand this kind of communication then we can maybe tailor the plant for a good recruitment and then we can then move on to the next generation of agriculture so we can start reducing the use of chemicals and we start making healthier healthier plants through a good microbiome recruitment now the work this probably some of you are asking what is the signal and p Greenberg is uh is uh continuing with this is very interested in this and he's made important contribution and he's and he's beginning to to think that it could be a peptide because uh peptide transporters is involved and also peptidase is involved but again it will be so fantastic if you can determine what this uh what this family of compounds is because then we can move on maybe to the next part which is engineering plants or to control pathogens and recruit good guys so that's the message I want to give with respect to to to that uh again I think that in terms of this kind of communication which affects the microbiome we are so behind there's beautiful beautiful communication between plants bacteria when we talk about rhizobium when we talk about agro bacteria but that's it we need to move on to the other guys that constitute the majority of the microbiome of the plants we have to understand in my view have to understand how how they communicate with the plant and how they create communities by communicating with the plant and there's a lot of work to be done there I think okay the last 10 minutes is is um is is the other part that interests us here in Trieste is the multi-species aspect you know and as we know from a lot of microbiome studies especially beneficial guys or even pathogens they live in in in very complex communities so how do they do how do they do their job and how do they communicate so I I was I was lucky that I accidentally bumped into this microorganism because Taha Hosni is a PhD student from Morocco that was working in Perugia he contacted me to come to my lab to to to look at quorum sensing this in this organism and this organism is a pathogen of olive okay it's not a serious pathogen can be controlled quite easily if if good practice is is is employed by the farmer these guys these guys live on on the surface of on the aerial surfaces and there is a damage it will get in and it will cause a tumor cause hyperplasia by producing hormones this guy produces two or three plant hormones don't think it's like agribacterium there's no transfer of DNA to to to the chromosome of plants it just does that it just does a by gene expression in the bacteria so we Taha came to the lab and we showed that this guy makes homocernolactones we found the system classical system a synthase and a regulator we inactivated the genes and we could then if inoculated one-year-old olive plants and you can see that after 60 days we have a good olive not being produced and when we infect with a wild tap with when we when we don't have the signal we have a much smaller olive not we don't have a regulator we have a smaller olive not you know it's it's you know it was not too it was nice but so many examples of plant pathogen and bacteria they use this kind of system to regulate violence well it got exciting afterwards when when he also said to me said to me I isolate a lot of bacteria from the olive notes you know I take the olive not from nature and I isolate but yeah of course I pick up the pathogen mostly but there's other guys in the olive not and one of the guys that that he that he regularly finds is this Irina Toletana which was actually classified in 2004 in Spain as a Irina Toletana which this is a harmless or even beneficial epiphyde epiphyde and endophyte so epiphyde lives on the surface endophyte lives inside plants I'll come to endophytes in the last few minutes of my talk so this harmless or beneficial bacteria is found abundantly found in the in the olive notes so I thought hmm so here we have a pathogen that takes a lot of his energy and is involved to create a disease why does it create disease because he wants to grow and wants to eat and wants to colonize and wants to take over the world and yet inside the olive not it takes other guys in are they cheats are they just there but you know how how is it how is the community coordinated is it are they cooperating are they competing and I'm not a social microbiologist but obviously this attracted me attracted the attention so we started working with this guy as well and what we what we shown I don't have the slide is that it has almost certain lactones it makes almost certain lactones excuse me and it makes a structure the same almost like tone as as as the pathogen exactly the same one is a ca 3 ox almost like so we made mutants of this guy of this corns system in this serbinia and then we did co-infections so here again this is an experiment when we took 20 year old olive plants this is done in Roberto Bono Euro lab in University of Perugia so one year old olive plants and he infects with the with the wild type and then we make co-infections with the wild type and the harmless end of alvinia and we the first thing we saw is that when we do that we have bigger knots okay so when we have the pathogen we have a good knot but whenever the pathogen and they were really with bigger knots so that was a clear indication that these two guys like each other they work better together we then did co-infections with the wild type together excuse me with the wild sorry with the pathogen not making the almost like to so the the mutant of the synthase of the pathogen which I already showed you in the previous slide the all the the knot is reduced considerably because it uses quorum sensing for virulence okay and then when we co-infect that that mutant together with the wild type or vena tuletana which I told you makes the same almost like tone we restore the olive knot formation all right right so so the the mutant of the pathogen is is let's say the lactone is is provided by the beneficial or harmless co-resonant of the knot okay and when we do the same experiment with the with the virulence vena tuletana regulatory mutant when we co-infect the regulatory mutant with the virulence vena tuletana we don't see this because of course the pathogen does not have the regulator to detect almost an lactone I hope it's clear so it was I think this work was was very nice and I that clearly showed I mean this these not take 60 days to form so this is a very it might be very stable cooperation interaction and what is it very important is that when we look at cell numbers see a fuse of the pathogen and of their pseudomonas and their vena if we have if we look at the open circle the open circles or the open triangles these are single inoculations and this is measuring see a fuse over the 60 day period of the olive knot formation if we do single inoculation obviously we see the pathogen the not forms and the pathogen number increases obviously and when we just infect the virulence alone it's not a pathogen we just infected the number go down dramatically when we put them together the two wild types we can see and we go look at the numbers the pathogen goes up significantly so the pathogen can grow better when the virulence present and look at dramatically the virulence vena now can grow when the pathogen is there so so we then Daniel Passos a PhD student from Brazil spent time in my lab he was then he then went to Cairo Ramos lab in University of Malaya which is probably the best scientist that looks at the virulence mechanism of these guys and he has a beautiful micro plant system quick system that he that he has to infect small plants of olives and we and so we marked the the pathogen green constitutive green and and and and there being a constitutive red and he could show very clearly that these two guys co-localizing the olive knot through the period a lot many days and they're very happy together the red and the green in bed with each other and and are very happy living together so what do we do now we'll we did several things and we're not making good progress actually so I'm very discouraged and I don't you know and we hope to pick it up again and pick up some positive results so we've done the the quorum sensing regular on in both systems and we have all the genes that are regulated by the by the in the pathogen and in their vineyard by quorum sensing we've done some phenotypic microarray to see what kind of metabolism is is going on in the pathogen in the wildtide and in the pathogen and in the and in their vineyard and we see them when we put them together when we call when we make a phenotypic microarray of them together we we we have better better metabolism you know we've used the bilog a lot of bilog plates for that in collaboration with Stefan Mokali in Florence we did that he has computer programs to detect this this kind of this kind of these bilog plates and so so so also what we've done was shown here is along along the side along the talk of Daniel even though we're not we're not very good in bioinformatics but Daniel was actually spent a lot of time it was actually he actually looked at the metabolism of the single in the Irvine alone and in the and in the pathogen alone and then in both together we seem to increase metabolic potential obviously we have more genes together we the pan genome of both together can has more metabolic potential so together with the these three pieces of data the the quorum sensing regulon which we know it's important for virulence and and they share the signals as i've shown you the phenotypic microarray and this in silico data we're working on a set of compounds that are present in the plant and might be involved in what Daniel mentioned in a in a in a in a better lie in a better metabolic profile when these two guys are together both benefiting from each other in in the metabolism of the in the metabolism of these compounds this is where we are so here you know the signals i want to give these guys to share hs signals from our right from our um regular data quorum sensing regular metabolic pathways metabolic complementary in my view can play a very important role and and i think this is a model to study could be a very nice model to study this kind of metabolic interaction and signaling between a pathogen and uh and a beneficial or harmless uh so again the concept of pathobium now we we we have this word in the literature pathobium so the you know the uh we have to think of the pathogen coming to the plant uh meeting a lot of other guys and maybe in some cases the guys that it meets actually have evolved to to collaborate and join up to do a better job of being in in this case increasing the ability to make to make the olive knot and to cause disease and to and to proliferate finally i i want to finish up because again here i think there's a i think there's an important message in terms of community we we've been interested in in my lab now for for a few years on this aspect of beneficial using bacteria to to fortify the plant to improve the microbiome of the plant so we can decrease the use of chemicals in in agriculture you know most it is believing that 99 percent of additives in agriculture at the moment are chemical we just cannot afford to go like that so there's a big interest worldwide by private companies and also labs to bring this down and i read uh in my reading i realized that they want to break it down by at least 90 by 2020 and the biggest promise as an alternative is the microbial inoculants right so we are we are trying to to get into this field and we're using endophytes and endophytes are in most cases recruited from the soil and there are bacteria that can get into the plant mechanism of entry is still rather unclear but they can get into the roots and someone can actually migrate up and live in intercellular spaces inside the plant and in many cases they're beneficial they can produce hormones they fix nitrogen they keep pathogens away okay so we had a we had a big project on rice well big we had a grant for italian standards rather a big grant in rice and we we are italy is the biggest rice producer in europe okay so so we we went to italians eat rice once a week which is in terms of asia it's a joke but this is what we do and i uh in general family will make results once a week so we went out there in the italian areas where they grow rice and we had a we went out to isolate endophytes i don't want to spend too much time how we did it but we we took plants which are live which are going submerged or non-submerged conditions we looked and we isolated endophytes from the roots from the stem from the leaves and a different growth phase because as we as we hear there's a lot of parameters how you grow your plant where you grow it uh where you select your endophyte some endophytes will get into the root and stay there some endophytes will get up to the stem some endophytes will get up to the leaves so we try to do we try to cover as much as possible so we did several samples and and of course we looked at also the microbiome and we in roots uh dry submerged leaves again we have we have published all this data and we see some differences but what i want to talk about here is that we actually have a collection one thousand thirteen isolated this is the work of it is it is per tiny and now postdoc philx moron moronto is also has a poster uh is doing now so we have a very large collection trust me working with one thousand three hundred eighteen bacteria is a lot of work so we have this collection and we started doing some classification but uh again to make a long story short we cannot work with one thousand thirteen one thousand three hundred eighteen isolates it's impossible big companies can do that we can't so we threw a series of uh uh shoot shooting in the dark and some semi-logical uh you know ways to reduce the number we classified and then we did some in vitro tests you know it's easy in vitro you can check so many things doesn't make compounds doesn't make oxen does it swarm does it swim but again i find those rather limiting because you know you have a bacteria that doesn't have these phenotypes in the lab then it might have them all in the in the plant right so you can do that but again you have to uh we we were rather um flexible in in in uh then you know choosing the ones we want to continue with so we have we had a set of two hundred and eighteen isolates which we after we did in vitro we rolled down to forty eight and these forty eight are other different species different infinotypic profiling and these forty eight we started to do uh work with the plant again and with some we can get the mid with 21 endophytes we can we can then uh re put them inside the plant by single inoculations 27 we couldn't i'll come back to that and then some of them had plant growth promoting effect others didn't but again working all the time with single inoculums we are together with the joint genome institute in in in walnut creek in california we have a community project that will sequence all these forty eight we're now sequencing all these forty eight genomes so we're gonna have the sequence of these but what we're really interested is of these forty eight in terms of signaling and that i'm interested in is do they have interspecies do they communicate with each other we're trying to reconstituting uh microbiome so we're doing this work right now we're working with these forty eight trying to think about communication and community as well as well as applications uh and what what felix is doing in my lab which i i think we just started to do that is is the concept of a simplified microbiome so we take rather than we've done all the single inoculations and now personally i don't like that we're now doing inoculations as a group okay so we can take 10 10 to 30 strains that of these forty eight and inoculate them together to the plan and then we can look at plan growth promotion or we can then look at community structure how which ones get in which one forms community and the message i want to finish it with is that yes the consortium is also having a beneficial effect at least in the lab the plants grow a bit more there's more dry weight weight on that we haven't done any challenging with pathogens yet but the message i want to leave with is that we get different results from the single inoculums so an end of that which resolute it when we affect the plant alone we cannot get it inside but when we infect them as a group initial experiments are telling us clearly that the profile is completely different so i think i think again we have to be very careful you know a single bacteria that can go in on its own and uh and bacteria which cannot go in on their own but in the end when we put them together with others they can get inside so i think this is a very important point that we again working with purified single scenarios could be the wrong way to go and again we have to always think what happens out there when these endophytes get into plants in nature they are surrounded by so many other species and even entry can be affected by this community other in communication maybe some bacteria don't have the mechanisms to get in but they can benefit from the mechanism of others maybe bacteria can stick to each other and one guy can get in and the other guy can follow in lots of different scenarios so we're very interested in that and it's not going to be easy to study but again the message i want to give again let's think about the nature think about what happens out there and try to design our experiments as closely as possible to to the real scenario okay i think i finished so oops so i i try to to mention everybody along the way the here is my group in bold and they're all here so i hope you can interact with them we also Felix also is a poster about this last point that i made so he'll be able to to talk to you about this and i i have collaborated you know very important collaborations with which i you know the field is so multidisciplinary if we don't collaborate it's difficult to make good progress and and the funding is is is is coming along and it is not easy i mean to get funding for this kind of work but we so far we we are managing to to stay above water okay that's it thank you thank you