hello hello good morning everyone today it is a pleasure to me to introduce the speaker dr. Andy Kumar he's one of my senior colleague in the division of Plant Pathology Indian Agricultural Research Institute he did his BSC and MSC from Tamil Nadu Agricultural University and he obtained his PhD degree from this prestigious Institute in the year 1997 then he joined a scientist in in Indian Institute of spice research and after that he moved to IRI as a principal scientist in the year 2010 he did a postdoctoral research work in was in Indian University in Netherland he is a very good teacher in our division is one of the finest teacher and you will able to see his teaching ability today and he is teaching several course in our division the one of the important course is molecular host-pathogen interaction and he guided six MSC student and five PhD students and presently he is guiding for PhD students he's having research paper in different international journals of repute with high impact factor and his eye index is 18 and I 20 index is 27 and total citation is 1105 with this brief introduction I welcome dr. Kumar to deliver a talk on application of this genome editing technology to mitigate the biotic stresses particularly fungus and bacteria thank you thank you doctor Anil band for a nice word about me welcome all the students this lecture is all about genome editing technology for fungal and bacterial diseases and management what I'm going to do here is let you know how this plant really interact with the pathogen or how the pathogens really interact with the plants this is so important for all of you to make a choice because we have a technology right now you can edit anything which is DNA right so technology part is well set anybody can do this genome editing now and that is really witnessed in number of publications which is coming on a daily basis right so the focal point of this lecture is I will try to make you understand how pathogen really interact with the plants in the process how many plant Jeets are really affected by the pathogen invasion so perhaps those genes are so important for genome editing right so let's move into the lecture now the title is the application of the editing technology for enhancing crop resistance against fungal and bacterial diseases in crop plants and I just would like to have a quick recap of what you have done in the last ten days so maybe in few slides the quick recap of the tip definitions and various g-e-t tools that would be followed by my own pet subject the threatening diseases of crop plants what we need to really focus now and why genome editing tool is important or vital for crop protection may be in the agriculture version too and what is it cellular genetic and molecular basis of susceptibility and assistance these two are two contrasting terminologies in plant ecology right so you should know what is the basis of susceptibility which eventually culminate in a phenotype what is called as symptoms and you also should know what the basis of for assistance which is the most Dre accepted our most disabled trait in agriculture because if a crop is resistant to pathogens that definitely you can secure the so called for security and the choices of genomic lows a or genes for editing that may lead to the desired phenotype so in this case it is no disease situation is the one we really want by doing all editing technologies and of course number of success stories I am really spellbound after seeing all those success stories which is coming on a daily basis right I will take you to the two rougher series of slides where people have confirmed using some kind of editing in some plant genes they have come out with a kind of product which is ready in terms of you know achievements so let's say take a tour of those achievements and of course let's make a kind of comparison between the genome editing technology and also the conventional technology where we really stand in terms of the force and also the end product so this is a flow of this lecture perhaps it might take an hour or so let us deal one by one just to recall once again I think all of you are quite familiar now what is the genome editing this is a very apt definition I could see it's a very broad-based definition a collection of advanced molecular biological techniques we can put it as a genome with techniques also to facilitate precise efficient and targeted modification of genomic low sale so the genomic low say could be anything it could be a kind of coding sequence or including the promoters or it can be anything which you would like to muddle with right and what's happening in those three loose a that's the second page that is insertions maybe deletions or point mutations you think of a kind of type of mutation you can really inflict those things in the genome right so this is I just borrowed from this particular author holla at all 2018 and of course you must might have been given a lot of input by the trainee urban and Co the genesis of the whole technology what we have right now I think perhaps we cannot ignore the zinc finger nucleotide that was to start with and it is a kind of early years of genome editing so we have a missing finger nucleus that bear bound to DNA I think they are all exposed to all these things I'm sure right yeah and this is something like you know the DNA binding domain at the n-terminus there is a zinc finger protein and there is a C terminal there is a kind of enzyme what is called as folk 1 the folk 1 stands for bacterial enzyme that is a flavor bacterium okey no quotes so this is a nucleus is borrowed from this particular prokaryote so what's happening here is the targeted sequence of zinc finger neatly a spear or typically 18 to 36 base pair in length excluding the spaces right so this the the cartoon of how it really looks like and we have the parallel e another one came to literature what is called as talents and that is a transcription transcription activator like effector nucleuses and again these two are almost look alike so you have a kind of folk one enzyme on the C terminal and we have on n terminal you have T so this is a kind of configuration of this particular protein again it does the same thing like the zinc finger protein and I also come across with that we come across another kind of nucleus as what is called as mega nucleus and again there is a great deal of literature's available I appeal to all the students to keep abreast of this particular terminology mega nucleuses and this is a very very unique enzymes it has got a kind of very long recognition site and there are two different kinds one is called intron into nucleus and the intein endonucleases please enlarge your knowledge on this particular subject and the focal point of the training program and also my own focal point here is the Christmas that is clustered regularly interspaced palindromic repeats right and this is creating values in the subject biology now in any field of biology so this is a the cartoon how it looks like and the key feature is the crystals were originally discovered in the late 80s is she know at all I think dr. Newman might've given the historical account of this particular discovery and originally discovered in streptococcus pyogenes and I presently it is making a lot of impact in biology in particular agriculture and human medicine so this is a nutshell about the CRISPR Casa nucleus it's a boundary a synthetic guide RNA is trna complimentary to 200 base pair our 'get in the in the nuclear genome it can be anything it can be a coding sequence or it can be any non coding or promoter the base pairing between the DNA sequence and sgrna after proto space are adjacent motif what is called as a prime motive recognition allows the DNA cleavage at the target site by kazna a nucleus so I think you must have already understood the Pam's are so important they only attract or the cast nine protein and that takes RNA to the complementary sequences in the genome ik region where it inflicts a kind of what he called restriction you make it may be a kind of double cut or single cut whatever all those things you might have quite familiar now so the question here is what this caste 9 is really capable of that is very very important for you to understand it can create any kind of mutations which I already told you so the caste name or the modified versions of caste name what I'm want to make a point here is already the modifications have come in caste 9 protein they are all tailor-made for a specific applications in crop sciences the modified reakless variant can inflict changes in the arrangement of nucleotide bases in the genes or genomic low say for that matter right so what kind of changes it can inflict indels the single thing a single kind of orang a kaznian complex can induce a double-standard break the famous Adeus beast on the target locus so in the process what happens there DSV is a primarily with any host the DNA is attacked they have a inherent mechanism to repair it right that in one such inherent mechanism is what is called as the quiet is a very nice acronym that is nhej a that is non-homologous end joining method this is one of the most important the inherent mechanism in any very biological system to repair the damage to DNA so this is exploited here to inflict or to trigger the mutations in those genomes OC right and which can lead to a kind of it can lead to a frameshift it can if the frameshift is there definitely is going to alter the expression the protein composition of the the one which is translated out of that particular loosely so this is a simple principle behind this and it can create even deletions for example if the casting is going to cut both stands and you can the entire strand is removed this is what is called as excision of the target fragment that is also quite a reality under this kaznian protein and it can create insertions you can you can make the whole system to insert something in the genome so that is the power of this particular technology though it is called as insertion why are non non-homologous in joining that any nhej a mediated repair of the double-stranded break can be exploited to insert a donor fragment if you have the donor fragment designed for your your own applications you can supply that and that's going to get you integrated into the low say of your every interest so this is another option and insertion via HDR is another option so if you have the homologous sequences which is already tailor-made in your target in your in your what you call donor you can even inflict what is called as HDR that is homology directed repair so this is another reality so and the variants i was talking about i I stumbled upon two variants one is the D casts that is deactivated casts or some it is also called as dead casts that means the enzyme lost stability to cleave the nucleic acid and we have another one what is called as our cast it is RNA yes so these two are the new additions our new variants that is going to create kind of applications in in biological sciences so what it can capable of doing when is transcriptional repression for example a dead casts guide RNA complex can be targeted to a promoter region of the gene in the process you can replace the transcription this is another option and it another option is a silencing silencing via our cache because a new variant what is called as RNA binding variant of cast 9 what is called as our cast 9 it can bind to a specific sequence of the messenger rna's leading to a translational silencing what I'm trying to tell here is the the silencing can be a transcriptional level it can be at even translational level all of them are going to assist you in editing the genome one way or other okay and to other applications of this particular cast named protein perhaps it is again going to make a big impact in biological science one is the base editing so base editing is something like you know a modified cast 9 for example cast 9 is cited in a deaminase fusion protein can be targeted to a specific region leading to deamination of cytosine so if the cytokine is deaminated what happens is the the daughter nucleic acid what is going to be biosynthesized will have a T so that is the modification what you are going to do so this is another application and at another one is epigenetic editing is also a reality using castile protein so here the D cast the one what what I already told a transcriptional repression is possible with the case of with the help of D castmate the D caste 9 can be fused to a traditional protein to mines to allow for epigenetic editing so that is it is done through the two mechanisms one is this site in the middle is that a stand 11 translocation the oxygen is one and a historian acetyl transferase fusion protein can be targeted to very specific sites to enhance a gene expression this is very interesting so if you want to enhance even gene expression that's that's possible using this technology and the opposite of that for example the transcriptional repression is also possible using another caste variant the caste name with the cited in methyl is that is the DNA methyl is transferase 1 or histone methyl is that leads to transcriptional repression so students what I just highlighted in few slides is the the different options oat is available right now for us to edit the genome right so with that background let us move on to the topic for today that is why genome editing technology is needed for reproduction so I would definitely make some justifications for doing this genome editing in crop crop lines for crop protection so these are all some of their what do you call everyone knows about these plants are talked by too many pathogens all right too much of abiotic stress they are always reducing the genetic potential of the crop lens to yield more and more or to give you the best quality product for us so pathogens can cause substantial losses they threaten the food security and of course we know pretty well that the existing technologies are very insufficient to sustain the agriculture production so all of you must be knowing the two best technology in crop production especially in plant disease management is Andrew chemicals and followed by hostile resistance these two are thoroughly exploited to sustain agriculture production all these days right but the question here is can we really depend on these two technologies forever right here comes the the question of they're either the durability or they're the safety issues associated with the chemical so that the world is quite aware of all those things the host resistance is remember is not durable right although we claim that we have the best of the best technology to control plant diseases this is only a short lived right there is nothing like a really durable coastal resistance so somewhere in some part of the world some new variant emerges and that makes you were the so called resistant variety or they call a useless there's a reality we have number of examples I'll just show you in the next slide and chemicals not to talk much about it for example one reason incident is any chemical residues on your finished product which is meant for export now it is under scanners now right so you should not have any residues on the produce if you want to really market your product so the best technology is what we are posting off in agriculture especially for crop disease management the host resistance is not durable and chemical based disease management is always under scanner and somewhere people are going to oppose it right so therefore I would say in my opinion you need to have innovative crop protection test strategies to sustain the agriculture production by reducing the crop losses by crop the plant pathogens so again to justify why this novel methods are needed to protect economically important crops right this is very important so the reason incidence I would take two examples one is in North Africa a crop like banana there was no record of Xanthomonas alright so suddenly there is an outbreak of a new kind of disease in Tanzania and other places and that disease it was it is caused by a completely different pathogen what is called a Xanthomonas till that time there was no record of a bonafide Xanthomonas species affecting banana so this is what is called as the pathogen somehow found its way into banana crop and started affecting it it become a serious problem right note is called as XPW that isn't ominous bacterial wilt right few years ago and asian country - it created a kind of waves in in agriculture fraternity there is outbreak of wait blast in Bangladesh right and there was no record of a serious crop loss in wheat by a blast disease so until we experienced it in our neighborhood that is in Bangladesh what is called as the weed block weed blasts outbreak in Asia the these two incidents give us it gives us say enough of evidence to say that the crops are not really safe it is only a time that is going to decide whether the crop is going to be free from the pathogens or not so what that statement here is the pathogen they continuously evolve and adapt and that set reality to overcome the host immunity the so called the resistant plant becoming susceptible across a different completely different species for example the pathogen is known in rice and it is recorded in beet right so what is the link between these two I would say that it's a horse to jump right so the pathogen can emerge on any crop right and another example I think pathology students if anybody sitting here they would understand this there is a strain of wheat rust fungi what is called as a ug99 it created so much of her work in again some part of Africa and and these two examples are enough to highlight pathogens always a wall and they overcome host immunity sooner or later right so that means the host resistance cannot be dependent forever to mitigate crop losses right and agro chemicals are not not accepted because they are not only the sense not they are not environment friendly they may pose a risk for exports and the classical its example if the tricyclics ole in rice it is creating so much a problem for our farmers right the elevation of amoral hundredfold in European countries and middle-east countries is a serious issue now because we know that rice I close all is an integral part of rice forming especially in the basmati growing region because where the blast is regular occurrence we need tricyclics ole to control rice blast right and the residues what is left on those produce is a serious problem now the exports are being rejected by the importing countries so that means these two technology what we have it cannot be rely upon for long so this is just to tell you about the different kind of diseases what is really affecting crop plants this is a kind of just to tell you what kind of symptoms that this is the pathogen and costs so those who are not too familiar with the terminologies here we have different type of pathogens that it reflect their lifestyle for example in our necro troops we have hemi by troops and we have bio drops right and what i want to see here is the different kind of lifestyle adopted by these different pathogens can really create a serious yield loss in several crops right the classical example desert in your salon in rice and we have magna poopy and fighter for us in rice and potatoes and her bio troops you have three major diseases in the world one is the pack senior rustman era safely for humility with bloom area podium elders and we have downing will use and so and so on so these pathogens there are two divers and two different kind of lifestyles they can really create a serious yield loss next few slides I'll just tell you some of the top diseases which the world is really focusing on now and to start with the fungal disease so among all the pathogens the fungal diseases are the one they're the most dominant one because there are 8,000 fungal species they are capable of causing plant diseases right that's too many numbers right so we have starting from vaccinia virus Fusarium septoria bloom area again wilt caused by Fusarium oxes flora Magna put the rising number of serials including beet and we have bought right in scenario this information I could just borrow it from another flagship journal in Plant Pathology that is Molecular Plant Pathology they have come out with the top ten diseases in the world the fungal diseases I think all of them are highlighted here right and come to another group of pathogens they are equally important in fact a subject when pathology awal from one of one of these pathogens that is fight or infestans it's a serious problem in potatoes we have fighter brackets ec in vegetables and we have plasma for avicii cola in the case of grape wine and we have a vital opposite kind number of orchard crops in particular citrus and we have Albergo Candida inclusively plants all of them are very serious problems and they are capable of reducing the potential of these crops and again I just borrowed it from molecular climatology journal and come to the bacterial diseases there are small numbers but they can really create an explosive epidemics in number of crops right and the threatening bacterial diseases of crop plants they can cause riot II of symptoms for example sports and blights can cause wills and rods and the top ten bacterial pathogen which we need to really focus our research effort should go on that's listed here Pseudomonas syringae arrives to nestle in a serum there was bacterial built-in so lineage which doubles in ginger we have agrobacterium tumefaciens in stone fruits we have Xanthomonas rice a plethora of racing in rice Xanthomonas campestris number of petrol wasps and Xanthomonas acts in the buddhist several pepper was in our country we have some de Mulas acts in a buddhist path of our puny key in pomegranate bacterial blanket of number one disease and we have a venial amela war of course we don't have this problem in other country but again it's internationally well recognized a plant pathogen xyla love has to do is fine dakea back to bacterium and so and so forth so what i want what i am trying to highlight here is although we have 200 bacterial species are known to cause plant diseases among the 200 the top 10 what was highlighted that's here so they can attack both above ground and above ground below ground portion and that can really create devastation so those slides just to impress upon you the seriousness at which these pathogens damages the crop right so i make it a point here for bacterial disease management the the only option which is a time-tested and proven is hosted resistance right and again it comes with the risk of losing the RO system the sooner or later because you know the bacteria they can short generation time they can really come out the huge numbers in the infection for site and few of them are already a mutant that can really overcome the resistance the pathogenic variability is so much in bacterial pathogens and any resistance what you bring it is very very shortly and it is going to the disease is going to come back to you again right to with that all those background so let us take the class forward and there is a strong justification because we have a too many problems from the pathogen side the technologies are although rated as very high-end technologies but they have their own deficiencies so we need new kind of approaches in the next agricultural scenario right so but before you venture into such an effort in my opinion what is more needed here is you should really understand what is the basis of the disease in the first place right and what is the basis of resistance that is also you need to know and what is the mechanisms really operating in either side right definitely there is interaction there are two entities are involved involved here one is the pathogen another one is the host when they interact something is happening on either side and definitely it is if something is happening the world definitely it is something related to the gene and the gene products right so before you decide to apply the GT for Disease Control or crop protection you must understand the cellular genetic and molecular basis of the so called disease and the new disease the disease is what is called susceptibility and the no disease is nothing but there are systems right in the next few slides it is typically host-pathogen interactions related subject you pay attention to the slides let's come to the first point how this power plant is recognizing a micro that's very very important right if you if you if you take any plant pot foliar you take rhizosphere you just split it in a symbol nutrient rich media you can easily count ten to seven to ten to eight bacterial species per gram oh that's too much number right right and on foliar you would easily score ten to six to ten to seven bacteria and I'm not talking about other microflora what is associated in those initials right just one microbial and IT that is bacteria is living in hundred millions in the rhizosphere maybe around a million to ten millions on phyllo sphere the question is are they in invert on the plant part or is there any communication which is happening between these two that's very very important right so the understanding what we have based on the information what is available in the literature maybe it accumulated over the last twenty twenty-five years the plant recognizes the pathogens are it put it like this the plant recognizes the microorganisms by that's kind of factor what is called as the receptors and those receptors are popularly called as P are ours P are ours it is an acronym for a pattern recognition the receptors so what I am trying to make make a point here is plants the award this is a subtype all right exclusively to recognize what is coming on to that sphere right the P are ours are highly conserved proteins in plants that means it is something which is so intimately associated with the plant life right and it's a great degree of conservation of these fear R's in plants right if you take a family why's a specific fear or is there in all members of the particular family right we'll take some examples in the future slides what's happening here when the microbe come to the plant side the P are ours are the first to want to really interact with the microbes right so what's happening here the P ours interact with the microbes the question here is what part of the microbe is interacting with the p RR that's very very important so those part is now called us that's right it is pathogen associated molecular patterns that means much similar to the receptors what is present on the plant or a world by the plant this microbes also evolved something like terms in in their life right these two I would say the factors are extremely concerned in the respective organisms the conservation of here are is there in the plan it is much similar to the consideration of pans in microorganisms are targets so what's happening here so it's a picture these PRS are anchored in the cell membrane right you can imagine something like this so what happens they are just anchored on the cell membrane they do not do anything understand until the plant encounters a micro lens right when they encounter a micro organism so what's happening here is the bacteria has come to the immediate vicinity of the plan and it started negotiating with the plant so what's happening this is a imagine this is a pathogen this is a PRRS so the moment it comes near to the plant they interact what I mean there is a physical interaction between these two components right literally there is a physical interaction because the proteins are configured by the plant in such a way that PRRS and pants can undergo kind of something like a lock-and-key configuration so this is so important so what's happening once it is they are interacting with each other the plant open up a completely different kind of metabolism in the host right otherwise it is silent so that is what is called as the difference signaling pathways this different signaling pathway is popularly called as a Pam triggered immunity or simply called as a PGI right I hope you understand this this is a first step in plan microbe interaction right remember this PTI is good enough for the plant to stop most of the microorganisms right they will not cross this boundary provided the microbe happened to be a pathogen right that means PTA is a powerful inherent ability of the plant to keep the microorganism away from Miss Indus fear right this it works all the time because I already told you have a hundred million bacteria which is living in the rhizosphere not all of them are entering into the plant right we have ten are a million or ten million bacteria which is living on the fully earth we have only one or two Xanthomonas which is causing disease in rice right that's the extreme minority which is capable of causing disease the rest all kept away from the plants fear or rather it's they are kept away from exploiting the plant metabolism for their own lifecycle completion right try to understand this PT is good enough to stop most of the microorganisms right but the question here is what is it PRA can we is there any are they characterized one what they are chemically composed off the literature what it says we have four different types of receptors of course different classifications are there you will read it from literature's and in my perception these four are based on their chemical nature we can classify these pr's into four different categories number one in the chart is our L case that is the receptor like kinases and receptor like proteins right and third and fourth is it to be characterized but the reports are already started coming in literature one is called intracellular soluble receptor erroneous extracellular soluble receptors these four are the major classes of receptors they are anchored in this site of a swift membrane of the plant and they negotiate with the micro organisms right and this is how it looks like right the sceptre like a kinase we have a kind of L or R domain which is going to perceive the micro organism or the pants of micro organism and we have a transmembrane domain which gives encourage for this molecule or the compound on the membrane and we have there is something like a nice to mine which is the cytoplasmic side of the the whole setup right this is a receptor like - and this is the receptor like protein the difference is there is no kind of divine in our LK if there is no kindness to mine the same protein assort is called us or L piece a septa like proteins and is there any known or L case in the literature or in plants yes we have number of our L case which is already well documented and we know their function as well right this the list we have FL us - that is a flagel in sensitive to is the classical p RR and it interact with a pathogen molecule what is called as flood chilling and it's not then there flagyl in what is going to interact with this FL us - it is only that 22 i mean an asset of this flagellin is going to interact with chemistry that that is called flag 22 right so you have flagyl in - is a classical or lk followed by EF R that is elongation factor receptor in our f1 and in our of - that is a node factor receptors we have xa21 exit 26 it's a well known we are using this in agriculture for mitigation of rice bacterial blight and PID - that is resistant rice blast barley RPG - tomato SR 160 and the pep or one these are all well characterized we know the function of these receptors they are all classified as oral case let's see some of the are LPS the classic alone is the one which is going to give resistance against rice glass that is C bit chitin elicits are binding protein right this protein is and curd in the rice cytoplasmic membrane and that is going to interact with the pathogen associated molecular pattern in this particular case it is catching because we know that most of the fungal pathogen they have chitin in their cell wall right chitin which is completely absent in the plant right so the plant can interact with the chitin which is not native to the plant so this is a well-characterized one Elliott EAX one that is ethylene inducing xylene is in tomatoes and we have CF 2 4 & 9 the classical one that is a cladosporium form proteins in tomatoes they are all class paradise RL peace let's move on to the peers right I was talking about the receptors and these receptors where they are going to interact on the pathogen side so the well-known plant receptors participating in the host pathogen interaction the pants with a known destructors I always tell this says five examples one is a flag 22 is interacting with their fellows true is documented in number of question right and we have L of 18 L of 26 that is elongation factor 18 and elongation factor 28 these two are receptors these two are what I call a paths they interact with a receptor wrote is called as e F that is elongation factor receptor that is documented in Brassica family in particular arrived offices and we have another one Alber signs a heptagon cosine there is a receptor that is interacting with the pathogen molecule that is in fight up from your gas burma in so our build system that is extracellular glue finish and we have another one ethylene inducing xylene is that is exe ix2 in tomato it is interacting and the ethylene index is really nice is a kind of receptor that is interacting with what a caller it is a Pam it is interacting with the x2 that is a receptor in tomato tomato system and the Titan that is interacting with the C babe so these are well characterized one and they participate in a host pathogen interaction and the outcome of this interaction in the first step is always spam triggered immunity right so the interaction between Pam and receptor leads to Pam triggered immunity or PGI and this is also called as horizontal or our systems right why it is called horizontal resistance let us not forget it is not FL s to flag 22 alone is present in the pathogen side you have so many other fans also so those fans also can theoretically can interact with another receptor in the same plant so it is always polygenic so what I am trying to say here is there are many PRRS which is present in the planet they can technically interact with many Pam's in a particular pathogen right are microorganisms and for that reason we always consider this a PTA as a polygenic and also called as horizontal resistance and this is this is a point that take-home message is the interaction between parents and PR R which leads to a Pam degrade immunity or PTA or horizontal resistance right but if that is the end of the story then they there should not be any planned disease at all right but the diseases are coming all the time so that means something else is really operating beyond this PTI right so that the point here is the PT is a very very short lead or rather it is going to be a call nullified by the microbe which happen to be a pathogen right so the pathogen can overcome this base eliminate it because pathogen also would like to be warned would like to complete its life cycle would like to enlarge its population in a system so they need resources and the resources are from the plant side that means they a world mechanism to make this PTI null and void that's what going to happen and that is possible finally the pathogen where completely different mechanisms right let's see what is that the successful pathogen which defeat the pitch line and cause infection in the plant right and this is done with the help of a powerful pathogen secreted protein and those proteins are often called as in factors in other terms a microbe which is capable of secreting effectors are all called as pathogens right and the effectors are coded by a specific genetic low say in pathogens and they are popularly called as Rulon's genes if you read literature they were originally termed the C Evelyn's genes right now we know that they are they are the most potential Willens factor of most of the pathogens and this effect is what is its function this effect is would try to stop this PTA from happening in the host it has to do that then only it can make further inroads in the host system to exploit the host metabolism right so effector can interfere with ap a signaling and the make the plane it's acceptable right and effector target plant cellular hubs and Gino genomic regions or client proteins the cost here is what is the effective function in the host what it does it targets some plant cellular hubs number one and some genomic regions in the plant genome or some plant X or virus and the size two proteins so what's happening here the selective plant targeting which leads to boil synthesis of our accumulation of factors which is needed by the pathogen to undergo the so called a developmental transition from one stage to another stage in the case of fungal pathogens or to enlarge its population size in the case of bacterial pathogens so they need energy this energy is obtained from those host factors and the host factors are specifically targeted by the effectors right so this is the most important point the whole step right from effector delivery into the pack last are effected delivery in the cytoplasm till they reach the cellular hubs or the plant proteins to convert what is available there for something which is palatable for the pathogen the entire step is popularly called as the pathogenesis even soft pathogenesis pathway the culmination of this pathogenesis pathway is what is called as the pathogen colonization and visibly this is also called as a life cycle completion of a pathogen and how this is possible when the spore lands on the foliar part it is a single spore in the case of rice plus after ten days a lesion is going to release the 30,000 connealy so how is it possible in a matter of ten days a single Commedia is going to release at 30,000 KU Nydia so we need resources we need energy space everything is provided by the plant and this is possible with the help of the so called powerful pathogen factors they are called as effectors it is the handiwork of effectors that make the plant associated factors into something which is palatable by the to undergo a developmental transition from one stage to another stage in the case of fungal pathogen it is the transition is from a vegetative phase to your reproductive phase in particular the so called a sexual reproductive phase right a single Canadia is going to produce 30,000 clonally produce to Canadia that is a real threat in agriculture because at the 30,000 colony i can create theoretically 30,000 independent infections in the field right that is a that is how this pathogen would recall the a wall and they they increase enlarge its population in the ecosystems right and what happens in the process the plant react the plant react say it is not going to be silent because it has to react and this reaction is perceived by all of us in the form of symptoms right so plant react to the cellular events which is happening much against its wish but it cannot stop it because effectors are having a free ride into the system and it is going to give convert what is available in the plant part to something which is palatable by this this pathogen and the colonization is ensured there the reproductive structures are formed and plant is reacting to that in the form of symptoms and remember the whole event is end of the day you start seeing the symptoms or you start seeing the pathogen directly on the plant part in the case of rust and powdery mildews and this whole phenomenon is called us affected triggered susceptibility right this is the second stage in the plant pathogen interaction right in plant microbe interaction the first stage is the I would say the first and last stage the air but the pathogen happened to be the microbe happened to be a pathogen the second stage is triggered and that leads to a phenomenon what is called as effector trigger sucked ability right this effective trigger it's a separate susceptibility has got very unique phenotype that phenotype is what is called as this is it varies from pathogen to pathogen wilt pathogen causes wilt right foliar pathogens leaf spot pathogens they caught leaf spots some causes blight right it varies from pathogen to pathogen and system to system right so one important point here is effectors are always expressed when the microbe the pathogen pathogen is already there in the host system right so really the effectors are expressed in the pathogen side right so the point here is effectors act outside the pathogen cell what I mean to say here is as long as the pathogen is not interacting with the plant the effector genes are not going to be expressed right they will express only when they already in contact with the plant system the PTA is already triggered the next stage they are expressed either they are in the site the upper class or they are released into the cytoplasm right this effector to great susceptibility is the one which is we know that I will be communicated to you that is a one what is perceived as a disease symptoms or science but this affected triggered susceptibility can be stopped how it can be stopped it can be stopped very nicely but we have been doing it for many many years this is stopped just by entering into a filter a program what is called as rust breeding for disease resistance what you do you bring another factor into the host system which can stop this effector from entering you'll be affected to get susceptibility right so affected to get susceptibility can be stopped by deliberate introduction of functional or proteins encoded by our genes this is one of the most successful research activities worldwide people have done and come out with pumping success in many crops right all our breeding program for resistance breeding for assistance program is based on this principle what you do you bring a resistant gene into the cultivar which is going to stop the effector from entering into defector to good it's a separate susceptibility or which is going to stop the CTS from happening in the hostile system right let us see how this is possible right the plant breeders have developed a resistant cultivars by introgression of our genes so what is this our genes after all this our genes are in my opinion they are activated our proteins perceived and physically interact with the effectors I was talking about the PR are physically interact with maps much similar to the story here the effectors are physically interact with the so called or proteins for the benefit of the students you must understand our proteins are nothing but intracellular receptors right the most of the intracellular receptors or cytoplasmic receptors what we call now they are all the product of this orgies and they interact physically when this or protein physically interact with the effectors what is going to happen here the plant disease resistant gene encode proteins are intracellular receptors that so to sense the invasion of the pathogens so the point here is the function of PR the function of affect our genes are technically same right well PR ours are the general perception of all microbes what is coming to the plants fear but the our proteins are a specific receptors they can interact with those pathogens which entered into the plant system you got it so that is the most important point here so they are very specific receptors they are capable of interacting with pathogen included proteins those proteins are called effector pockets right this is happening either in the apple astok side or it is happening in the cytoplasmic side so what is happening here the intracellular receptors there so to sense invasion of pathogen and subsequently triggered a series of downstream immune responses I was talking about the same thing in PGI when PR are interact with the Pam's the plant open up a completely different signaling pathway in the plant system to stop the microbe from entering into the plant site right same thing happens here so when these all proteins are expressed functionally viable or protein they have a protein protein interaction of the effectors when they interact what's happening in the plant side is the plant will not allow the effectors to gain access to those cellular hubs which otherwise would have supported the pathogen to undergo life cycle completion the effectors will not do allow that so rather it opened up another pathway that pathway is what is called as a difference a signaling pathway this seek defense signaling pathway is quite strong different signaling pathway this pathway has got several biochemical and phenotypic phenotype for example ro is production salicylic acid production HR we have a PR proteins and overall what is called as SAR that is systemic acquire a resistance to keep the rest of the cells rest of the tissues free from pathogen this is a cascade of even which is operating in the plant cell and who opened up this it is the interaction between our protein and the effector protein is opening this particular pathway to keep the pathogen from gaining access to those cellular cups which otherwise would have contributed for the effector triggered susceptibility right so the intracellular receptors are two major classes right now very popular only is the TIR NBS olara that stands for toll interleukin nucleotide binding site leucine-rich repeat and coil the coil nucleotide binding site leucine-rich repeat these two are well characterized one let us take some examples the TIR class is well characterized in the case of linseed that is mallams or a linear it is having a specific interaction with the effector remember and I talked about this intracellular receptors or cytoplasmic receptor what is called as our proteins they have a very specific effector molecule in a very specific pathogens right it's not general at all it's very very specific for example yellow 5 l 6 + l 7 or cytoplasmic resistant proteins or intracellular receptors they have very specific binding ability on effector which is produced by MLM seralini that is the AVR l 5 6 & 7 right a classical example of TIR another example cytoplasmic resistant protein M which mediate the recognition of Melhem seralini a effector protein we are young let's take some example for c c NB s lraa the classical example is rise notice AVR Peto AVR pita is a effector proteins that interact with the effector receptor in the rice that receptor is rice else LC c c and b lraa these are all excuse me these are all the well-documented one and tomato i too is a receptor which mediate the recognition of an effector coded by Fusarium oxes forum in tomato that is six3 these these pairs are well characterized and the effector and receptor pairs when they interact remember the host is going to open up a different signaling pathway right and that difference signaling pathway is called as the affected triggered immunity so here comes the so called on to diagonally opposite phenotypes i triggered by the same effect right the effector in the absence of receptor can trigger effector triggered susceptibility but in the presence of if a receptor a completely opposite phenotype in the plant what is called as the effector triggered immunity so these two are the one i was talking about the molecular basis of susceptibility and the molecular basis of resistance so what is happening here the interaction between the effector and a functional or protein also called the cytoplasmic receptor or intra cellular receptor by enlarge they belong to a class of protein and BS lrr when they interact with the effector the outcome of this interaction is immunity in the host and that immunity is ETI very popularly called as vertical resistance remember vertical resistance is the one which is thoroughly exploited in agriculture to remove the varieties which is resistant to number of plant pathogens is so successful in V building programs so successful in rice breeding programs and many other crop which is so important for food security right this is what is called as vertical resistance just to some of the story what we have been discussing in the last few slides to start with the PTI we entered into ETS and finally with the help of breeders we triggered et I just to sum up I take you to 1940s 1945 to 1950 there is a very famous hypothesis proposed nobody really took note of that that hypothesis was gene for gene hypothesis like very few people paid attention to that it all done by a passionate worker hi sketch floor a series of four or five papers all single other papers and he came out with this theory now we realized whatever he hypothesized in 1945 the other thing is true literally there is no no mistakes whatever he he proposed let's see what is that that what he told at that time there is no concept of gene in those case he told something like host conditioning factors for resistance he recalled the terminology what is called as host conditioning factors and pathogen side he called again the pathogen conditioning factors the host conditioning factors hypothesized five-floor is nothing but the receptors or functional or proteins or functional or gene whatever right our product of host origins the the corresponding pathogen conditioning factors beautifully we told there is a corresponding what pathogen conditioning factor that means these two are matching the host conditioning factor and the corresponding pathogen conditioning factor that is the thing but the product of the so called pathogen genes there are effectors right so there is a receptor on one side in the host we have effector on the other side in the pathogen when they interact the interaction always leads to no disease condition so people thought there is something like a virulence factor which is operating in the pathogen they call it a savior genes because there is no proper explanation those two now we know much better the AVR genes and virulence genes they are only the same there is no difference it is a situational if there is a receptor then it is a ABR is insured or no disease is insured if there is no receptor then the susceptibility is ensured right so it is virulence our agency is not decided by the pathogen but rather it is decided by the presence or absence of receptors on the host side we try to take this find is the most important point right and when the host or gene product and pathogen ABR gene product participate in food protein-protein interaction and the outcome is accelerated defense all the time provided the our protein is having the right configuration to come to a lock-and-key conformation with the effective protein that's more important so here comes the pathogen play the tricks they modify the effector and become out of the different protein so our protein becomes functionless when we call it as the resistance is broken down is a simple mutation human which is triggered in the AVR genes it come out with a completely different protein which cannot pair with the so called a receptor protein remember our protein is still there but it become non-functional right the mutation has taken place in the pathogen side so the outcome is accelerated defense or the host resistance by a cascade of signaling events which I already told you different series of events there are a number of other genes are also participating in that particular even right and several other plant gene also participating in the the whole even it's not only the protein and the effect of protein in the interact of course they start the reaction but it is guided by so many other genes from the plant side okay this is a nutshell how plant and pathogen really interact and what are all the factors they guide that interaction if you have any doubt please clarify before I move on to the next slide anybody is having any clarification please okay great it's a nice question I already told pants are extremely concerned on the microbial side it is a general general molecule which is present in most of the microorganisms right effectors are something which is a speciality proteins produced by a few elite microorganisms those elite microorganisms are called pathogens you got my point so we have more than 99.9% of the microbes cannot gain access to the plants fear to take the plant metabolism for right no because we have PTA that's good enough but with a few the mini school of microorganisms they acquire that capability to produce effective through deeds only those microbes they can negotiate with the host metabolism because the effectors are so powerful they are delivered right in the perp last and cytoplasm and they can manipulate the planets right they have axis right into cytoplasm and they have access right into nucleus also because we know the talents that transcriptional activated effectors are delivered in the nucleus so you can imagine so parents they cannot enter into the plant system at all their interaction is only at the surface level right so that's a big difference right times our general effectors are very very specific effect are produced by own pathogen is it cannot be the given I spy yeah or gene of another host or gene of a complete a closely related cost also not possible right they are very specific that two major difference on is highly conserved another one is highly polymorphic right effectors coding genes are highly polymorphic they are vulnerable for any mutations right there is a point mutation in effector coding genes relates to emergence of new race is it clear to all of you this is a concept I wanted to communicate to you right so let's move on in the absence of functional or protein I mean it here there is nothing like no origins our genes are always there right the question is whether it is functional or not now what is the what is the meaning of functional here functional means a protein which is capable of interacting with the effector protein that is what is called as functional right there is a correct protein conformation on either side they can physically interact with each other I am NOT taking you to a tour of different hypotheses now proposed to explain how this effector and the receptors are really interacting so these are beyond the scope of this class but you take the message there is a kind of physical interaction which is taking place between receptor and effector that interaction leads to a phenomenon what is called as PGI right so but in the absence of a functional or protein what I mean a functional or protein is so important here the pathogen coded protein also called as effectors can trigger host to susceptibility right which I already told via many other host factors the cellular hops I was talking about the the plant genes or clan gene associated factors they are all what I call going to interact with the effectors or effector has got direct access to those domains and when they have direct access to those domains the outcome is always susceptibility who is going to block it a functional or protein if there is no functional or protein the effector will have direct access to those domains which is going to contribute for a pathogen life cycle completion is it clear to all of you let's move on to the next one now we come back to the genome editing now right now the story is out here how the pathogen really interact with the host who are all the main players here right x and p are ours on one side we have effectors and receptors on other side and the third one is so many other associated plant genes they participate in plan signaling pathways so right now we have three plant associated genes or gene products to deal with only surface receptors what is called as PR us another one is specific receptors they are called our proteins are intracellular receptors and third one is a grouper plant genes they do ancillary role here in the signaling pathways there are so many genes are there they participate in signaling pathway was talking about our ways production how are we this produce it assign handiwork of some genes there right there is our charge induction right there is a lagina fication cell wall reinforcement kalos accumulation right so meaning phenol accumulation fight relaxing biosynthesis right they're all somewhere some genes are expressing something that is playing a role there this group of genes are called signaling pathway associated genes so now the choice is you have three different choice at your disposal on this surface receptors a neuron is intracellular receptors or cytoplasmic receptors and third one is a group of genes they have an ancillary role right let's see the four genome editing for this is resistance the choice of gene or genomically low say may be any one of these plant receptors they participate in either pta ret i right so how many different types and any of the affected targets playing a role in ETS I was talking about in the absence of functional or proteins the effectors will have direct access to certain cellular hubs or Kline gene associated factors right if the plant gene which is very defined and the function is well characterized having a direct involvement in ETS and all those genes can be targeted here right and planned cellular proteins are planned genomic regions coding for susceptibility factor right and this susceptibility factor is now called as yes genes yes gene means the genes plan genes which is assisting the pathogen to take nourishment from the plant very interesting this plant genes are also doing the job against the plant right those genes are called ability cheats right so third one is any one of the different signaling susceptibility a different signaling or susceptible susceptibility signaling pathway associated practice and modification of different signaling populace these are all the choices let's see what will happen if you try to edit or if you try to meddle with those receptors we have to see the pros and cons of doing that that's very very important here right this is clear to all of you either PTA or a TA associated receptors can be a potential target no doubt and it can be those cellular hubs are proteins or the genomic region of the plant they somehow assist the pathogen to undergo the transition from one phase to another phase in the process the pathogen is colonizing there establishing the symptom via right those Jim's right they are popularly called as yes chips right we will come to that discussion so third one is anyone of this signaling pathway associated genes these are the potential target let us not forget we are trying to manipulate the plant genes right and in my opinion let us not meddle with the pathogen genes at all because you cannot control them right you may control the isolate what you have but what about the rest of the isolated they are out enjoying the environment outside you can't do that it's very difficult so what is manageable here is the plant genome that is right in your disposal right I think I hope I answered your question so the editing of genes which is coding for plant receptors this is a kind of in my opinion I was when I was reading this my mind was going to different directions the direction one is it really feasible number one if you do that what will what are all the consequences let us have some discussion here this I have borrowed from one famous paper appeared in there current opinion in plant biology if I'm wrong please search this the author is Bart Ouma from bargaining it so he proposed the receptors in particular our LPS and our case are the potential targets for bringing broad resistance in several hosts let's take that example now let's see editing of receptors right so how many types of receptors are there we have what is called as general receptors they are on the cytoplasmic membrane they negotiate with Pam's pathogen associated molecular patterns and we have intracellular receptors there are two kinds I told CC NB s lraa and TIR and BS lraa they negotiate with a powerful pathogen molecule what is called as effectors right they are specific receptors remember general receptors are highly conserved mutations are not tolerated please you must understand conservation means mutations are not tolerated if there is a mutation there would be written as cost come to the specific receptors they are extremely polymorphic much like the polymer be some what you see in or proteins or G's we know that our genes are highly polymorphic right much similar to that on pathogen side also the vectors are highly polymorphic let's forget about the effector now it will come to the receptor here they are very specific right and base modifications our genome editing may be performed in pathogen recognition sites what I what I want to make a mention here in those receptors see please remember I was talking about e lr r in the case of plants in the case of P R R and L R are in the case of our proteins remember they are the place where the pathogen associated molecules are going to interact what is called as pathogen recognition sites p RS right in the surplus to enhance pathogen perception so has to trigger innate immune response this is one hypothesis you can bring a specific basis in those lr r so that you can increase the perception capabilities of those receptors it's very interesting and a revolutionary thinking right so what Mark Toma really highlighted here was you can bring a specific base modifications that's a reality in genome editing so that would in the process it would enhance the perception remember and Hanasi enhancing the perception would always lead to immunity it may be a PTA or it may be a TI right say perception is not happening it might lead to susceptibility I already told if there is a change in the pathogen effector protein what do you mean by that the perception is lost the Ark routine is unable to see the effector because the protein is different protein it comes with a different pattern so it is unable to perceive what is coming inside that ensures ETS right so what this paper says you change the perception capabilities of those effectors not only that those are proteins non-functional or proteins you can bring life to the non-functional or proteins just by changing the basis there it's a really revolutionary thinking in my opinion it is a quite a reality let's number one so this approach is being explored to mount multiple pathogen perception that's also wonderful thinking because most of the our genes cloned on sequence right now they have the same backbone and BS lrr right if that is the case definitely you can bring a subtle changes in the perception PRS that would bring capability to this receptor to recognize many pathogens so that is also another option right but in my opinion this need to be done with a great clear we know pretty well if they a small amino acid change in the effector makes the or protein non-functional if you do the same thing in the receptor site the same thing can happen there as well right this need to be done very carefully as the receptor modification must not lead to the effector non-perception by edited receptors I try to understand this statement right if you edit the receptor in the process whatever protein is going to come out of this receptor should we should be able to retain the perception capability of the effectors right this is one of the most important point otherwise the ETS would be insured here now whatever effort you are doing is going to be a counterproductive operation right and therefore the correct identification and modification of PRS assumes crucial here for any success right this is one option right and this is the broad options available for us right now receptor or resistant gene editing and what is suggested here is what is called as synthetic resistant plan where you try to modify the receptors to bring more and more PRS into the same receptor in the process the plant become resistant to diverse group of pathogens right that's what it is here genome editing for single resistance and genome editing for multiple resistance if you deal with the PRS you just modify the non-functional orgy base modification that leads to your functional or protein then that is going to trigger ETI in the host when the eighty is the most desired trait because we need that more and more in the crop systems so that the pathogen can be controlled very easily right but in the case of multiple what what what you were suggesting is a potential use of editing technology is engineering and normal synthetic or proteins or genes able to mount resistant to several pathogens by combining PRS from different origins right here comes the resources what is available at your disposal here most of the genomes are led out and we know what is there right and getting a kind of diverse or protein from a particular host is quite a reality now right so if you know the PRS and you can do the modification in one or protein RG you try to bring modification the PRS which is going to perceive a diverse pathogens so that means what a single or protein would do multiple pathogen perception and it would evoke a similar stereotypic response in the form of ETA so that is what it is needed here right and this is a paper current opinion in plant biology if you have time it isn't easy to read nicely written and he gives a complete account of receptor modifications and receptor editing of receptor coding genes as a solution for mitigating more than one pathogen in a crop plants right so I'll move on to next important target perhaps lot of work is done now what is called as editing of the genes which is coding for susceptibility factors the success whatever we have in the form of product in the form of success stories are all from this particular manipulation right s gene manipulation so editing of effector targets or effect s genes using modern omec platforms several susceptibility genes have been identified now providing many potential targets for improving crop protection and there if you see the last five years of papers you see more and more of genome edited crops in particular with the help of CRISPR Castile and the products are led out I'll I will show a couple of them here the unprecedented fishin see of genome editing techniques in editing the specific sequence of his genes which represent the best candidate for engineering resistance right now right what's happening here is you edit those plant genes which is going to interact with the effectors in the absence of our proteins and if you edit them and make the factor which is not palatable for the pathogen it's a very simple concept right so it has conferred resistance in various crops I think number of references are there so these are all some of the success stories sergey's already there and different crops I'm not going to read it here I will come to some specific cases here and these are all the success stories in different crops like you have tomatoes grapes apple vie and rice and tomatoes and citrus a lot of success stories right and a success story with this gene or promoter sequence editing let's take of three examples here the affected target the plant cellular cups just to rewind what's really happening and these susceptibility factors are the potential targets and the classical one which is already there and a number of obligation is ml whoa right I think you please put this as a search word that will take you to a world of Emmylou lo site in different crop plants right so ml or locust is the best studied one and there are seven cleats and played four and played five were so important because this clade for group of s genes they they assist a pod room will be pathogen to cause the disease in the plants especially in the case of monocots remember or durability of greatest number one problem in many places right portability of weight will marry and Grameen is a form of special is pretty see right and the create five is for dicots we have powdery mildew a number of dicot plants all's right so this is a cleat or somehow the clade four and five it evolved in those plants to assist the five bottom will be pathogen to cause disease and they are bonafide susceptibility factors in those monocots and dicots right and people have attempted to manipulate this ml lolo site and the phylogenetic analysis of three hundred and forty-one Emmylou proteins from different host plants to uncover the evolutionary history and origin of him a low family living evolution the family has diversified into seven phylogenetic plates with a clay plate four and five are so important for us because they are directly involved in assisting the portability pathogen to cause disease and very important agriculturally important crops like feet and also so many diverse crops in dicots as well right and they help the portability pathogen to infect and this is typical Oracle ml whoa the gene was mapped into the genome of higher plants omalo encodes a plasma membrane associated protein that binds a column a calmodulin with it's a C terminal cytoplasmic tail here is a called model in binding a domain that is relatively well conserved in the many of mallow family and this protein is structurally related to g-protein coupled receptor in meta zones and the function in susceptibility towards waldron will do a number of plants right so this is a typical OD column a lo characterized one so some some information about this ml whoa this is mildew-resistant locus and one of the best-known s genes and it's a prominent example of robustness in durable pathogen resistance program the wild-type allele saw Emma Luo was first discovered in barley to confer susceptibility in barley against a powdery mildew and that is plumeria grameen is foremost special is howdy I am a low-end Corsa membrane associated protein with the seven transmembrane domains so you can count there are seven here so it is conserved throughout monocots and dicots and it is allocated for mildew fungal penetration under the host epidermal cell I was talking about there is some function they confer to the pathogen here it is a penetration of host epidermal cells the mutation in a Milwaukee that CRISPR cast nine has also confirmed mildew resistance in wheat and tomato in a non-transgenic systems so this is inertial I don't want to take the too much of time on this you know it very well they have edited this ml low low site and they have confirmed it through number of approaches and they have sequenced it and they found that around 48 to 49 base pair deletion chromosomal deletion in the same a low-low site that gave a complete resistance to powdery mildew that's what it is highlighted here and they named this particular variety Isetta melo the tremolo is a variety which is editor tomato line which gives a complete resistance to powdery mildews okay they have edited the ml will lose sight right the leaves of editor tomato line infected with oil neo like apostasy showing the full resistance towards this pathogen to wild-type leaves right so next example is an has enhanced disease resistant sort it also called a CDR one that is in feet again for quorum we'll do a wrapped like mutagen associate activated protein they have been targeted by CRISPR cast nine and significant reduction in will do in in which the thumping success what is reported in the recent time several groups is in the case of rice so I will take you to few slides on this success story in rice in rice there is a group of plant genes they help the pathogen to sustain this ETS in the process the symptom appears and those genes are called sweet genes right the pathogen Xanthomonas or IC path of our i say it secretes one or more six known transcription activator effectors though it is called as a tails these tails has got a very specific target in the host genome those targets are called sweet genes remember sweet genes are hosts associated genes and those genes products are specifically targeted by these effectors the effectors are transcriptional activator like effectors and these effectors they bind to the specific promoter sequences and induce the disease blight in the rice leaf there are three genes are well documented sweet eleven thirteen and fourteen and people have touched those genes thirteen eleven thirteen and fourteen CRISPR casts nine mediated genome editing has been done to induce mutations in all three sweet gene promoters remember they have touched the promoter region and editing was further confirmed by sequence analysis of what we call they have confirmed it and and the trials they have conducted the edited line and they found that there is a broad-spectrum resistance against the die was Xanthomonas isolates so that is I think they took around 63 in place and they found that the editor lines are resistant to 63 different Xanthomonas that is something we need to look into because the ATA based resistance program is extremely vulnerable i was talking about a single point mutation make that ETA functionless but in this case just imagine a small mutation in the host side a sweet gene is conferring a broad resistance against the diverse isolates of the same pathogen that's a power because I was talking about bacterial pathogens are so diverse and they are variable all the time so this approach perhaps one of the potential approach for bringing a broad resistance in crop like rice now this is a nutshell the regions and the effector in question here is tails and the effector target is sweet genes and interaction which leads to ETS and the genome editing option has prevented the ETS from happening in the host that's what they did in nutshell right they edited the sweet genes in the process what happens the interaction is broken there is no interaction between the tails and also the sweet gene products so the ETS is stopped abruptly so the mutation gene makes the plant completely resistant that's what the story so the genome editor zoo lines they have they didn't stop it there they took it to the mega where it is like iír 64 and chicka rang a sub one and the same thing they did here and they come out with the so-called completely resistance against bacterial blight in rice right and they also did a lot of other agronomic traits yield potential quality and everything seems to be same as the kind of wild-type one here I must make one important point here this is a slide what I borrowed from that particular paper and you can see here there's a complete resistant on both edited sweet gene edited lines in rice and against divers Xanthomonas isolates right so I will move on to the third approach not too many papers are there in the literature tackling or editing the genes which is associated in the signaling pathway I just brought a couple of them in PR one is a kind of gene which is participating in number of signaling events in the plan you can think of something like this and the TGA transcription factor we know it very well they participate in SAR and salicylic acid mediated and Vickie 70s another transcription factor can be tackled these are all some options and I could not find a kind of thumping success like the one what we found in SGS so I'll just stop it here and these are all some of the success stories in agriculture in particular fungal and bacterial disease management using genome editing route so the list is there don't try to read it here you all peoples are in public domain you can read at any time and I just would like to make a point here this particular table what is dominating here is that the CRISPR casts so that is that is the power right so although the talents are here two talents are here but most of the success stories are coming from Christopher Cass that is the point and the low say what they tackle here is also here and again if you see all of them are associated with the susceptibility factors right so the the product what we have or what people witnessed in the form of success stories most of them are from the so called ETS associated growth plan factors and popularly called as susceptibility genes right whether it is ml 1 ml low again ml o one from Triticum aestivum from solanum lycopersicum vita gain and there is another low say here a CRF in the case of rice so they they they have given a broad-spectrum resistance against all those paths this is another again the list what is what is being reported and another interesting story is so most of the reports so what we are witnessing now is coming from 2016 onwards that means there's a great deal of work is really going on in the last two three four years to come out with a product right so that is all about the success and promise what we have witnessed using the revolutionary genome editing technology but it comes with a lot of the flip side also it is all is not well all the time right so if you're lucky yes you are agronomic traits are really good and in addition to the dated line that is the most to decide one you try to edit for pathogen resistance okay that the general assistance is done but in the process the plant is not growing then it is of no use right so the one of the most important highlight of this genome editing in particular with the help of SG is the fitness cost let us not forget there may be a multifunctional s genes they have more than one function in addition to guiding the pathogen they may have some vital role in the very developmental process of the plant itself if you touch them yes you get a resistant line but in the process the plant may not look like the plant again it may be a maybe something else some handicap may be some Fitness cost so that choice of s gene becomes so important right so there comes the role of individuals who is touching this kind of work they need to do a thorough literature search they need to go for maybe a different isoform it is not having multi function and they they need to touch the one which is having exclusive role in the so-called rulings or the susceptibility and then the success is in short right so this is a kind of a slide the comparison I think you make you can make your own points here it's not my points here of course the traditional one is very slow the big efforts are needed in terms of money and time and only our genes are addressed the classical breeding program we address only the our genes and undecide rates and phenotypes you need to be removed through different kind of back cross methods and that delay the arrival of your variety and in my opinion not they are not really durable if you put the time scale of a decade they are not durable they may walk for maybe a couple of years or so but if you put a decade it may not work right and in the classical I always tell this point s our 32:31 in the case of vite it was resistant for 30 years on fine morning ug99 came and make that particular resistant gene non-functional right that means it is always in the timescale any cultivar may become redundant or become what you call not so useful in agriculture so in the case of s genes perhaps that a promise such an approach such a lacuna may not be there because the paper says that switching editing gives a resistant to 60-plus isolates of Xanthomonas is a very strong message we the durability solidity right so that is the important point so edited lines editing option is the rapid quick no restrictions in either on our genes or s genes choices use and precisely controlled lists of or non-target because you are specifically modifying that particular gene the precision and also already call them no off target is ensuring yeah durability is also there in the case of s gene based one so I think that's the last one I think that's somebody's written here definitely this CRISPR Canaan system become a gold standard method for genome editing for various reasons I think different speakers might have highlighted this in the same platform and a simple efficient and versatility and genome editing has been applied to enhance parameter systems against number of pathogens I showed several slides here and susceptibility genes of the prime focus right now provided they are having very narrow spectrum of activity only to guide the pathogen to cause disease they do not have any other multi function those multi functions should not be related to the planned developmental process and the current legal framework of course we may be yesterday there was a talk I think he met all listen to the talk and definitely in future the bio edited disease resistant crops will become a standard tool in plant breeding programs I think the afford by an HP cast in IRA is a small step to instigate the N Minds to initiate such a program in the years to come so with this a thank you all of you for it's a pretty extended lecture I do not know whether it really benefited all of you so with that I close my lecture and I thank organisers in particular dr. Vishal happen Ram Sharan Bhattacharya and Daniel band for giving me a platform to share my thought process and if you have any quick questions I am ready to answer curious thank you sir for your lecture it was good for those who do not in this area how the things are working so the mechanism was very nice I exactly want to know how this effector molecules are produced infection they are produced good it's a nice question for effector genes in the pathogen they are not really needed for the pathogen in the true sense and there are so many other microbes they live happily outside the plant system they do not have effectors or other functional effectors are not there still they lead a happy life but why few microbes evolved effectors that is no evolution they evolved this gene clusters only to take advantage of the hosts for their perpetuation number one their multiplication colonization and the very disease what is after all disease enlarging its population so that the progenies are sustained in the environment for long time the effectors are typically expressed when the pathogen is in the interior of the plant system right remember effector genes are in the pathogen their expression is always happens when they interact with the planet endogenously or wherever the pathogen is adapted may be a powdery mildew epidermal so when they interact with the pathogens it's a physical interaction maybe the PPA signal is somewhat linked to the effect of gene expression right there must be a trigger for the effector gene to express remember number one once it is expressed they are there are endoplasmic reticulum in the pathogen by enlarge they are produced there and it is transported outside the pathogen system into the plant body in the bacterial system it is thoroughly worked out most of the plant pathogenic bacteria they have a syringe kind of stuff on their surface what is called as type 3 secretion system the type 3 secretion system you see the electron micrographic photo it is typically like a syringe right is exploited so the type 3 secretion system and effective secretion is all coordinated Lee expressed when the effector is released a bias on the size in the pathogen upon some trigger which is coming from the host perhaps it is from VTA we are not very sure the effective is ready because effector is not needed for the pathogen at all right it need to be transported outside the pathogen into the plant that is mediated with the help of type 3 secretion system in the case of bacteria in the case of fringe' all of you must be knowing the fine G produce a speciality structure what is called as historia this historia is kind of invagination in the cytoplasmic side of the plant cell you just imagine the textbooks there is a historia which is coming into the plant site o+ and in that the site o plus the historial boundaries that is a transaction point between the pathogen and the plant right the effectors are released into the plant side through a Surya the mechanism still to be worked out but that is the transaction point between the pathogen and the plant cell the historia is the one which delivers the factor into the plants a 2 plus so in the case of nematodes we know that all stylet born a motors are planned pathogenic one it is something like a syringe that Stylites are the one which releases the effectors into the plant system right this is how you know how the effectors is delivered like it's a fungal disease back viral diseases you know it there is a insect which is injecting the virus right into the cytoplasm yes if at all any virus coding effectors they are directly delivered into the cytoplasm right so there are too many ways of delivering the effectors the pathogen your world in their lifetime they know how to negotiate with the plant cytoplasm and the nucleus also to take the advantage for them right yeah the so-called R Gean based wound is that only so what happens is it is not blocking the effector biosynthesis rather we need more effective sexually to trigger ata right if you need effectors the third effect on ETA will not be triggered right and there is no point in manipulating the fact regarding genus it's not needed actually right let the effector be there because I already told many PRS you need more and more perception by the our genes we need effectors because when they interact then only they et is going to be triggered in the plan system so they need to be ineffective I was talking about ata and ETS but for effectors there is no ETA there is no ETS we need effectors definitely no doubt and equally what we need is the receptors which is able to interact with effector in the process the ETA is triggered in the plant system yes please so as you mentioned that if production is there any one can fight that in some cases if you see the if you working in if so if we have or Express the some of the gene that can fight with our OS and all that thing's and minimize the generation of our OS like although some amount of RS need for signaling all that so my question is that if you working and over expressing any of the DISA biotic stress gene and in that case if you can minimize the ro s production implant so how can we check whether this gene having like also resistance to some of the this biotic stress also so in that case yeah I'll sum it up I think you are you want to reduce always production in the plant so that the biotic stress let us not forget always a difference signal a difference molecule is needed by the factor small amount it it will be idea it is needed by the planet definitely yeah so otherwise the our voice is not a component of this different secondary pathway because they are very powerful antioxidants I would say antimicrobials they play a dual role first they themselves are antibiotic number one and number two is they can participate in signaling events there they do both the functions but in your question I don't think I will be able to answer that how to manipulate or regulate always production in the pan system III I don't think I am competent to answer this question but yes if anybody who has some knowledge you can interfere here
