5 Biotechnology and plant disease management-case studies
Transcript
Welcome! This is the last and final talk of this week and here in this particular talk we will be seeing some successful cases where biotechnological tools were applied for bringing out some commercial cultivars of crop plants.
Let us take the example of pathogen derived resistance which was employed in the papaya crop and problem was in Hawaii Island of United States. Papaya was a most commercial cultivated crop in Hawaii Island and it was devastated due to a pathogen that is Papaya Ring Spot Virus (PRSV) and you can see that the whole orchard was devastated because of this infestation and there was a goal or there was an objective or there was a challenge for the scientists to tackle this problem because papaya was the most commercially significant crop of that particular area. It is the coat protein gene of Papaya Ring Spot Virus that was cloned in 1986 and transgenic lines were developed through transformation of embryogenic cultures of papaya using Biolistic approach that was done in 1988.
So this was the approach and this was the target that is the coat protein gene and this was done by around 1988. The transformation was done on commercial cultivars of ‘Sunrise’ and its siblings known as ‘Sunset’, and ‘Kapoho’. Kapoho was the most popular cultivar that was grown extensively in the Puna district of Hawaii Island. So there were seventeen independently transformed plants were obtained and the line 55-1 was when tested against the pathogenic strain of the virus proved to be highly resistant. Here you can see that that line 55-1 was growing healthy and there was no symptom produced whereas the traditional varieties of non-transgenic origin were severely affected by the pathogen. This gave hope to the researchers to go further and they develop a resistant line from the already existing line 55-1and they named it as ‘Sunset’ which was the much less desirable than the yellow-fleshed ‘Kapoho’ because ‘Kapoho’ the fruit quality of Kapoho that is yellow-fleshed and it was more preferred by the local people but the Sunset was originated from the red-fleshed papaya. So there was again a problem that although it is resistant to the Papaya Ring Spot Virus but the variety or quality of the fruits were not appreciated by the local growers.
So two news transgenic cultivars were developed and they were named ‘SunUp’ and ‘Rainbow’. ‘SunUp’ is the transgenic red-fleshed ‘Sunset’ that is homozygous to the coat protein gene and rainbow is a yellow-fleshed F1 hybrid that was developed by crossing ‘SunUp’ and non[1]transgenic ‘Kapoho’ . If we just look into the entire strategy sunrise had two siblings ‘Sunset’ and ‘Kapoho’ all were used for transformation and a line from sunset that is 55-1 was found to be highly resistant against the Papaya Ring Spot Virus. Then since this is a not preferred variety of papaya in that area the ‘SunUp’ was then crossed with ‘Kapoho’ the most preferred variety and the resultant variety that transgenic line that came out that was named as rainbow which was yellow[1]fleshed and preferred by the local growers. So rainbow was the cultivar that was finally released for commercial cultivation in that area and you can see that the field testing level this was successfully grown besides the non-transgenic plants where the non transgenic plants were completely infected and destroyed by the Papaya Ring Spot Virus but these transgenic plants were Virus free.
This is a large-scale field trial and the aerial view again showed that this is the transgenic ‘Rainbow’ plants which was in the center of the field again which was surrounded by the non[1]transgenic ‘Sunrise’ plants and you can see the devastation of these non transgenic plants in the field and how the transgenic ‘Rainbow’ plants were protected against the pathogen under field condition. This was a successful experiment under field condition that has led to development of resistance against the ‘Papaya Ring Spot Virus’ and it was then later commercially released for cultivation. So this is the commercial cultivation of papaya in this area and the same technology was transferred to even countries like Thailand where the Papaya plants were grown which were resistant against Papaya Ring Spot Viruses.
Then another strategy which was adopted in potato because the cultivated species of potato does not harbor any resistant gene or source against the most dreaded pathogen that is Phytophthora infestans. So wild potatoes were explored and there were some resistant sources that were identified in their wild potatoes and those wild potatoes were then utilized for transmission of genes from those wild sources to the cultivated species. The concept was that because of the non availability of the specific recognition PRRs in the cultivated species so there was no resistance in the cultivated crop species. But once a resistant protein was a resistant source was identified in a wild source which was then incorporated into the genome of the cultivated species they were able to identify the pathogen effector molecules and there was resistance development. But we know that this pathogen is revolves very quickly so the pathogen can modify or they have modified its effector in a way that this particular R-gene product was unable to identify this effector molecule. So the new strategy is to incorporate more than one resistant genes from or pyramid the resistant gene sources from wild species so that different R-genes will be able to identify at least one of the different effectors that are produced by the Phytophthora species so that resistance response can be generated or defense can be generated in the host.
A gene that is known as Rpi-amr3i was transferred from non-tuber forming potato plants known as Solanum americanum and when it was incorporated into the cultivated species of potato that is Solanum tuberosum then it conferred full resistance against Phytophthora infestans. So here you can see that this is the transgenic line that is expressing the Rpi-amr3i gene which is showing complete resistance against the related pathogen Phytopthora infestans. This is the control plants which are carrying a non-functional Candidate gene of Rpi-amr3a. So here you can see that entire leaf is diseased the pathogen growth is evident in all parts of the plant whereas there is no disease or the pathogen was unable to grow on these Transgenic lines. So this is a strategy that has been successfully adopted in potato for bringing wild resources as resistance source and incorporating them on the cultivated species. This is a field trial of the same crop against the same pathogens where you can see that these are non transgenic plants which are completely devastated whereas the transgenic plants which are grown adjacent to the non transgenic plants and they are showing resistance against the pathogen.
Then there is another mechanism where we can incorporate a particular set of genes which are not directly toxic to the pathogen rather they can strengthen the host defense mechanisms by incorporating multiple copies or more than one copies of those genes in the cultivated species, say for example utilizing Cytosolic ascorbate peroxidase and copper or Zinc superoxide dismutase. If we over express these genes in a particular plant then we can expect better resistance against its pathogens.
Here you can see that transgenic tobacco plants which were over expressing the cytosolicsod (Cysod) that is superoxide dismutase that was cloned from spinach and cytosolic ascorbate peroxidase this was cloned from pea and incorporated in tobacco. There were 17 lines that were developed and the line 17 carried two copies of a , superoxide dismutase line 51 carried two copies of ascorbate peroxidase whereas lines 35 and 39 carried both sod and apx in a single copy. when these transgenic lines were then subjected to pathogens like Pseudomonas syringae pv. tabaci then this is the control plant showing severe symptoms of the disease and in comparison to these we can see that lesser symptoms that are produced on these lines at the same time when the plant was introduced with another pathogen that is Agrobacterium tumefaciens, this is the control plant you can see that this is the crown gall that is developed or lesion developed in the control plant in comparison to these others have lesser amount of disease and this is the line 51 that showed minimum necrotic lesions. so it was further investigated and the mechanism for lesser necrotic lesion development was found and it was established.
As when the pathogen was challenged to the pathogen with to the plant then the NADPH oxidase was activated which then led to synthesis of superoxide and that is in the Apoplastic space and then superoxide then gets converted to hydrogen peroxide with the use of superoxide dismutase. some of this superoxide along with hydrogen peroxide they can migrate to the Cytosol of the plant through the plasma membrane and this superoxide generation or hydrogen peroxide generation can also takes place through plant mediated activities but they can also become higher because this Apoplastic superoxide hydrogen peroxide can also move to the cytosolic space. So altogether this excess super oxide and hydrogen peroxide in the cytosol lead to cell death or necrosis that is manifested in terms of HR hypersensitive response or Necrotization. But when we have additional copies of Cytosolic sod Cytosolic ascorbate peroxidase we lower down this concentration from superoxide to hydrogen peroxide and finally from hydrogen peroxide to water.So in this way we make the load lower on the plant cell and thereby the HR response or necrotization is minimized. That is what manifested in this particular case where you can see that over expression of both the copies has led to minimum disease symptom development in this particular line in comparison to the other lines which had single copies of these genes.
So these are different strategies which can be used for management of plant diseases under biotechnological era and this has promised as we can see that some of those technologies were so successful that commercial varieties were developed out of it and some of these technologies are at the verge of development of new commercial varieties based on their successful experimentation in the lab in the glass house and under field conditions. so with this we come to an end of this particular course. I congratulate all the students and all the participants who have attended this course successfully and I wish and I believe that you must have gained a significant insight into the understanding of particularly plant pathogen interaction, then principles of plant disease development, then principles of plant disease management along with biotechnological and genomic application of plant disease management as well. So I thank you all for participating in this particular course and I wish you good luck.
Thank you.
