Podcasts about Agrobacterium

With (sort of) special appearances by Tobin Bell, Rosalind Franklin, and a 19th-century Dutch scientist whose name I certainly did not get right. Music by James Milor from Pixabay Information provided by: Tobacco (Nicotiana tabacum L.) - A model system for tissue culture interventions and genetic engineering by Thumballi R. Ganapathi, et al. (2004) [Abstract] In vitro transformation of cultured cells from Nicotiana tabacum by Agrobacterium tumefaciens by L. Marton, et al. (1979) [Abstract] https://www.pmiscience.com/en/smoke-free/nicotine/tobacco-plant-research/ On the historical significance of Beijerinck and his contagium vivum fluidum for modern virology by Neeraja Sankaran (2018) Therapeutic potential and phytoremediation capabilities of the tobacco plant: Advancements through genetic engineering and cultivation techniques by Nidhi Selwal, et al. (2023) https://doi.org/10.1016/j.bcab.2023.102845 PLANT vs. PATHOGEN: Enlisting Tobacco in the Fight Against Anthrax by Graeme Stemp-Morlock (2006) https://doi.org/10.1289%2Fehp.114-a364 “Tobacco Research and Its Relevance to Science, Medicine and Industry” by T.C. Tso (2006) DOI: 10.2478/cttr-2013-0824 Phytochemicals derived from Nicotiana tabacum L. plant contribute to pharmaceutical development by Wenji Zhang, et al. (2024) https://doi.org/10.3389%2Ffphar.2024.1372456

Today my former undergraduate student Lauren Augusta, currently in a PhD program in Microbiology at the University of Indiana, joins the podcast to chat about how she chose her career path in the microbial sciences, and her future path. Host: Mark O. Martin Guest: Lauren Augusta Subscribe: Apple Podcasts, Spotify Become a patron of Matters Microbial! Links for this episode The program for creating the maze on this session's thumbnail image is here. Here is the website for Micropia, the microbiology museum in Amsterdam in the Netherlands.  SO WORTH YOUR TIME.  I wish that I worked there! Here is a description of Micropia's “tardigrade chair” which is kind of a tourist destination! Lauren Augusta, today's guest on the podcast, did a wonderful video advertisement for my institution, the University of Puget Sound. An introduction for beginning micronauts about Agrobacterium, and why you should care about this natural genetic engineer, as well as a more advanced review.  Plus another fine review from Dr. Clay Fuqua and coworkers.   Lovely overviews of the global signaling molecule of bacteria that Lauren studies, cyclic-di-GMP, can be found here, here, and here.   The Microbiology Department website at the University of Indiana where Lauren is working on her Ph.D. The faculty webpage of Lauren's Ph.D. supervisor, Dr. Clay Fuqua. Intro music is by Reber Clark Send your questions and comments to mattersmicrobial@gmail.com

RNA Interference, known as RNAi, is a biological process that leads to the silencing of gene expression.  A lot of plant viruses are RNA viruses including grapevine leafroll-associated virus and grapevine red blotch virus. Yen-Wen Kuo, Assistant Project Scientist in the Department of Plant Pathology at the University of California, Davis is researching ways to induce RNAi in grapevines to target virus. Growers may have heard of double-stranded RNA sprays which are intended to initiate RNAi. The challenge has been that double-stranded RNA breaks down quickly in the elements. The Kou lab is working to improve this process and look for alternatives that will have little impact on the ecology. Resources: 71: New Techniques to Detect Grapevine Leafroll Disease 131: Virus Detection in Grapevines Abstract: Development of Agrobacterium tumefaciens Infiltration of Infectious Clones of Grapevine Geminivirus A Directly into Greenhouse-Grown Grapevine and Nicotiana benthamiana Plants Kuo Laboratory – Plant Virology Maher Al Rwahnih, Foundation plant services RNA-Based Vaccination of Plants for Control of Viruses Yen-wen Kuo's Google Scholar page Vineyard Team Programs: Juan Nevarez Memorial Scholarship - Donate SIP Certified – Show your care for the people and planet   Sustainable Ag Expo – The premiere winegrowing event of the year Sustainable Winegrowing Education On-Demand (Western SARE) – Sign Up! Vineyard Team – Become a Member Get More Subscribe wherever you listen so you never miss an episode on the latest science and research with the Sustainable Winegrowing Podcast. Since 1994, Vineyard Team has been your resource for workshops and field demonstrations, research, and events dedicated to the stewardship of our natural resources. Learn more at www.vineyardteam.org.   Transcript Craig Macmillan  0:00  Our guest today is Yen-Wen Kuo. And she is Assistant Professor in the Department of Plant Pathology at UC Davis. I'm Craig Macmillan, your host, and I'm very excited to have Dr. Koh here with us today. Welcome.   Yen-Wen Kuo  0:11  Thank you for having me.   Craig Macmillan  0:13  So you've been doing some interesting work the lab on interference RNA, and also how it affects plant viruses and possibly insects in the future. Can you explain for those of us that did not take genetics like we were supposed to in college, what interference RNA is and how it works?   Yen-Wen Kuo  0:29  Sure. So RNA interference is a biological process in which certain types of RNA RNAs can trigger RNA interference. And then once it's triggered, it will produce specifics more RNAs, that can regulate gene expression, by degrading or binding to the target RNAs containing a homologous sequence containing a similar sequence of those small RNAs. So this is a general concept of RNA interference, we also call it RNAi is very complicated the whole process. And there are different pathways and mechanisms included in the RNA interference. RNAi is a primary and effective antiviral defense in plants, but also found in some fungi and insects and lower eukaryotes. And because of all these different mechanisms, scientists and researchers, they they work on different aspects of this mechanism for either plants or animals. And they're also looking for different potential and better ways to use RNAi for different applications.   Craig Macmillan  1:45  So if I understand correctly, you have cell and there is DNA in that cell, and there's genes that code for certain things. And so the RNA is was transmitting or was carrying information from that's encoded with the gene out into the world to do something, is that a fair explanation?   Yen-Wen Kuo  2:05  So the genome there in plants or animals and human is their DNA genomes is DNA, and then the DNA will transcribed into RNA. And those RNA, some of the messenger RNAs can translate into proteins. So it's a how the central dogma from DNA makes RNA and then RNA makes protein. In the old days, we thought that oh, the protein is the important things because the protein can have different functional, different functions in different ways to to regulate everything in the body or in different organisms. But then afterwards, we found that actually RNAs they have many different forms and they can function at the RNA level. So it can interfere with gene expressions and many different things.   Craig Macmillan  3:03  And how does this apply to plant viruses because you've done some really exciting work with Gemini viruses, I believe with grapevine virus a Tell me a little bit about that work and how that works.   Yen-Wen Kuo  3:15  A lot of plant viruses, they are RNA viruses, a lot of those devastating viruses in grapevines, for example, grapevine leaf roll associated virus or grapevine red blotch virus they. So grapevine leaf roll associated viruses and RNA virus and grapevine red blotch is DNA virus. So there are different types of viruses. And so my work is trying to use different viruses making them into viral vectors to induce RNAi in Grapevine plants, to target those important viruses causing diseases in the field for the grapevines. And because so for example, when the viruses they are infecting plants, they will trigger RNAi in the plant, so that plants can protect themselves from virus infection. And because of that, we're trying to develop viral vectors can trigger RNA interference to target those viruses that's causing diseases. The work I have on the grapevine Gemini virus A that GGVA is to either develop the virus into viral vectors to target RNA virus first. So that's the initial plan for us to use. GGVA the grapevine Gemini virus A target grapevine leaf roll associated viruses. So before we eventually target that virus, we have to do a lot of different tests. We need to know if the clones the constructs or DNA constructs we have of this, GGVA can actually affect Gravelines plants, so we have to do that. And then we want to see if we can develop it into viral vector to carry the sequence we want them to express in grapevines to do the work we want them to do. So then we use it to target genes in the plants to see if they can silence the genes in the plants. So then we did that, we found that yes, we can use that viral vector to silence genes in plants. And then now we try to see that if we can use this viral vector to target other RNA viruses, or other grapevine RNA viruses, because we are actually at the same time developing different viral vectors, and one of them is GBA, is grapevine virus, a another's name, it can be very confusing. GGVA is a DNA virus. GVA is an RNA virus totally different to viruses. So since we have both viruses in the lab, so first, we try to prove the concept. We use the GGVA, the DNA virus, to target the GBA wild type virus, to see if we can see any effects. The GBA infection viral titers in the infected grapevines. So this is what we're working on right now. And so eventually, we want to use this viral vector, and potentially other viral vectors to to target grapevine leaf roll associated virus. And maybe we can use it to target mealybugs too.   Craig Macmillan  6:35  How are these vectors introduced to the plant?   Yen-Wen Kuo  6:38  We modify from the previous reports how people try to deliver those constructs the plasmids into grapevines. Most of the experiments or the assays, from before, they needed to have grapevine plants grown from in vitro, on media or from embryos. But that's really a lot of work. And it will be harder to have applications in the field. So then we develop vacuuming filtration method that we can directly vacuum infiltrate those plasmids that those DNA construct plasmids directly into the greenhouse grown grapevine plants. So those plants are propagated from the cuttings and then those plants, they are usually maybe 12 to 19 inches high above the soil when we infiltrated those plasmids into those grow vine plants. So this is an we got pretty good results, we successfully introduced those DNA constructs into the grapevine plans and those constructs can be infectious and initiate the whole the virus replicate in the grapevine.   Craig Macmillan  7:50  So is this something that can be done in a nursery then with new plants? And basically, they then would come with the vector or is it something you could do in the field?   Yen-Wen Kuo  7:57  Yes, I think the plan is that we can introduce those plasmas in the nursery in greenhouse plants before we plant them into the field. So then the plants that's planted into the field, they can have this viral vector to protect the plants from specific viruses.   Craig Macmillan  8:18  Got it. That's really neat. That's a great idea. And it's pretty cool. So that's fantastic. And in the work that you're doing so far, it sounds really exciting. And it sounds like the direction that you're kind of going in the future is with leaf roll virus that you mentioned. And then also, interaction with mealybugs you mentioned. Can you tell me more about that? What's that work all about?   Yen-Wen Kuo  8:39  Because this virus does GGVA and other viral vectors we're working on to a lot of viruses infecting grape vines, their phloem limited virus, so this GGVA is also phloem limited, meaning that the virus is can only infect the tissues around or in the phloem  is restricted. It doesn't go to like mesophyll cells or epidermal cells in infected plants, because mealybugs they feed on phloems. So we think if they can pick up those RNA interference signals, may be those RNA interference signals those small RNAs can target mealybugs too. So we can choose different target sequences in mealybugs. Hopefully you can see some effects for many bucks to to prevent that from transmitting viruses or have lethal effects for mealybugs. That's the plan. Hopefully we can do that. But we have to do tests to see how the efficacy and everything though it can have mealybugs, because there are previously they are different studies they use RNAi on insects, and many people prove that they can see some effects. We hope that the viral vector approach can also use for really apply this into the field for grapevine plants.   Craig Macmillan  10:00  What kind of index on insects are we talking about?   Yen-Wen Kuo  10:03  Depends on what target genes or sequences we choose. For my first choice, I would like to have a target that can prevent the transmission of the virus by mealybug, that will be my choice. I'm not sure if it's good to kill the insects, if it's going to affect the ecology too much. So if we can make the mealybug not transmitting the virus or other diseases, I think there will be a very good first step if we can see a lower transmission rate. And and then we can see if we need to adjust from there.   Craig Macmillan  10:40  That is amazing. And we haven't, yeah, the little bit of research that I did we have we do have proof of concept basically on this in other cropping systems. Is that right?   Yen-Wen Kuo  10:55  Yes,   Craig Macmillan  10:55  Can you tell me a little bit more about that, because that might give us some some vision of where we might go in the vineyard industry.   Yen-Wen Kuo  11:01  So, the RNAi applications, people are already trying to do some of those works. So, one example is that before people can spray double stranded RNA into the field. So, let me talk a little bit about the introduction of why using double stranded RNA. So, there are different types of RNAs that can induce RNA interference, certain types, one of them is double stranded RNA, either double stranded RNA or the single stranded RNA, they can form into a secondary structure in folding into a structure like a hairpin RNA, those are found to be able to induce RNA interference. And there's also other things like artificial micro RNAs, there are different types of RNAs that can induce RNAi and most convenient ways to make double stranded RNA. And people have been synthesizing the double stranded RNA or using bacteria to produce those double stranded RNA and then spraying to the field to get some protection for the plants. It worked at some level, but it's just not stable enough. Although double stranded RNA is more stable compared to single stranded RNA, steroids and RNA can be degraded in the field with the sun and everything the whole environment it can be degraded, people started to look for ways like bio clay to protect the RNA, and then so, they can spray in the field. So, the RNA can last longer and cause the effects. So, those double stranded RNAs can be absorbed by the insects, they can pick up from the surface of the plant or the plant can absorb those double stranded RNA into the plants. So, those are different ways and people started to see some effects on that, but still, we have to improve those different methods delivering double stranded RNA or other types of RNA to induce RNA interference in the plant. So, they are different different approaches. So, one of that is now we are trying using virus to introduce the RNAi to induce the RNAi in the plants. So, people are trying different ways to deliver those specific RNAs to induce RNAi to target specific diseases, sometimes not just viral diseases, that they will try to target fungal disease or something else and insects. This is what many different groups they are trying to do also previously, another way is to try to make transgenic plants. So if we can make plants to express those RNAs that can induce RNAi targeting to specific diseases, then you don't need to really use any tool to the deliver because the transgenic plants itself can produce those RNAs doing to induce RNAi plants. So that's also another way that people are trying to do we call that host induced gene silencing HIGS, and the virus induced gene silencing is the way my group is working on and we call it VIGs vigs. So there are different ways that which we would use to introduce those RNAs to induce RNAi in the plants.   Craig Macmillan  14:31  And right now you are at the greenhouse stage, if I understand correctly.   Yen-Wen Kuo  14:35  Yes.   Craig Macmillan  14:36  Have you introduced mealybug into your experiments into your work yet?   Yen-Wen Kuo  14:40  Not yet. We are just working on targeting grapevine virus first to see the effects. So where we have to continue monitoring those tested plants to see if the effects can last long, and the efficacy and how good they can be. So now we're at four for five months, so it's still we can see the targeted virus is being suppressed in a very, very low titer. So GVA can cause some symptoms in the grapevine plants when they see the plans are infected. But we have to peel off the bark to see the symptoms, we want to see that after targeting to the GBA virus, we saw that the viral titer is very low, if we can see that, also, the symptoms is not there anymore, is now like wild type, when when the virus was infecting in the plants alone, if we can see the difference, we don't even see the symptoms there will be really great. And this part, hopefully I can collaborate with the collaborators, Maher, he's run the foundation plan services, he can help my group on this, to see that how good the effects can be using this GGVA viral vector. So after that, if we can successfully target two different viruses, then we will start to work to change the target sequence in this viral vector to target mealybugs. So that's after the virus work.   Craig Macmillan  16:12  Yeah, well, that's very exciting. This is a really fascinating idea, and obviously is still relatively new. And I think it's really great that you and everybody else is working on this sounds like there's tremendous potential, and I hope that you folks continue on are able to continue on, is there one thing really related to this topic, you would tell growers one thing that you would advise them or you would educate them with?   Yen-Wen Kuo  16:34  I understand that there could be some concerns and maybe doubts, questioning RNAi applications in the field, because before, they already probably heard about the spray of double stranded RNA or other methods, and they saw some effects but not stable enough. So they may have some concerns or doubts, I think many scientists are trying different delivery methods that can be applied efficiently in the field. And we will do different types of tests and trials to make sure we work on any potential issues of this technology before applying them in the field and try not to affect the whole ecology or anything in the field too. And obviously, the current approaches we have are not enough to keep certain grapevine diseases, at low enough incidence. So we have to explore more potential control approaches before those diseases get worse, and adjust the ways to manage those different grapevine diseases with this changing environment. And I think hopefully, we can all work together to achieve this same goal. And I understand this is something new, I hope everyone can keep an open mind and willing to work with us to do different trials and see if we can improve different approaches to control different diseases.   Craig Macmillan  17:58  Well, I hope so too. grape growers are very creative. And they're always looking for solutions to their problems that very much fit what you're describing. And it sounds to me, this could be another tool in the IPM toolbox that may not be the single solution may not be a silver bullet. But it sounds very exciting that it may play a very important role to improve the efficacy of other techniques we have, which is great. Where can people find out more about you?   Yen-Wen Kuo  18:22  So because I will, setting up my lab, so hopefully I can have a lab website soon. I don't have accounts at Twitter or Instagram.   Craig Macmillan  18:34  Neither do I.   Yen-Wen Kuo  18:36  I don't use social media a lot. So my email that people can reach me through the email. And hopefully, when this is up or in your podcast, I will have my lab website set up so people can find us our work, my lab website.   Craig Macmillan  18:53  And we will have links and everything else that we can find posted on the episode page at the Vineyard Team podcast website. I want to thank you for being on the program. This was really, really interesting and is a kind of a view into the future of what's possible. Yeah. Our guest today was Dr. Yen-Wen Kuo. She is with the Department of Plant Pathology at the University of California Davis. And I want to thank you for being on the podcast.   Yen-Wen Kuo  19:20  Thank you for having me on the show. I really appreciate this opportunity to talk about research to explain some details about our work to the course and hopefully, I answer some questions that growers might have. I look forward to in the future maybe collaborating with different people to make this thing to work.   Nearly Perfect Transcription by https://otter.ai

A project trying to potentially thwart the spread of a disease-causing bacterium, or Agrobacterium.

Dr. Jeff Changdescribed a project where he is trying to potentially thwart the spread of a disease-causing bacterium

Jumping Agrobacterium

Dr. Jeff Chang, a researcher and professor at Oregon State University, described a project

One of the major objections to genetic engineering is that a DNA segment is transferred to the crop, and lands in a somewhat random location.  That’s because genetic engineering largely uses Agrobacterium to perform the [...] The post 294 – Widespread GMOs in Nature first appeared on Talking Biotech Podcast.

Dr. Jeff Chang, a researcher at Oregon State University described a project where he is trying to potentially thwart the spread of a disease-causing bacterium

Crystal reviews 2013 research about the role of Agrobacterium in Morgellons and other human diseases. --- Send in a voice message: https://anchor.fm/more-morgellons/message Support this podcast: https://anchor.fm/more-morgellons/support

Thwarting the spread of a disease-causing bacterium.

Agrobakterien sind wichtige Genfähren zu Herstellung gentechnisch veränderter Pflanzen. Doch auch die natürliche Lebensweise dieser Bakterien ist hochinteressant. Wir betrachten heute, wie aus tumorauslösenden Schädlingen hocheffziente Werkzeuge der Gentechnik wurden. Weitere Infos auf BiOfunk.net

Now we will tour through a survey of some sequenced genomes. All three domains of life will be represented, but the Bacteria and Archaea will get the lion’s share. For each genome, we will learn why scientists are interested in the organism, some basic data about the genome, its genes and encoded proteins, a few surprises from the genome sequence, and an example of how scientists took the next step past having the genome sequence. Our first sequence is my personal favorite, the soil bacterium and plant pathogen and biotechnology agent Agrobacterium tumefaciens C58.

How does an engineer approach microbial genetics? cworks with microbes of all kinds to optimize metabolic and agricultural systems. Here he discusses his work with Rhodobacter to make biofuels and for membrane protein expression, with Agrobacterium and plant pathogenic viruses to make drought-resistant plants, and with Clostridium and yeast cocultures for lignocellulose digestion. Take the listener survey at asm.org/mtmpoll Full shownotes at asm.org/mtm Links for this Episode: Wayne Curtis Lab site at Penn State University PLoS One: Molecular Cloning, Overexpression, and Characerization of a Novel Water Channel protein from Rhodobacter sphaeroides Protein Expression and Purification: Advancing Rhodobacter sphaeroides as a Platform for Expression of Functional Membrane Proteins Biotechnology for Biofuels: Consortia-Mediated Bioprocessing of Cellulose to Ethanol with a symbiotic Clostridium phytofermentans/Yeast Co-Culture HOM Tidbit: Genentech “Cloning Insulin” blog HOM Tidbit: Genentech press release announcing insulin cloning  

The TWiV team reviews the first FDA approved gene therapy, accidental exposure to poliovirus type 2 in a manufacturing plant, and production of a candidate poliovirus vaccine in plants. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler Become a patron of TWiV! Links for this episode ASM Conference on Viral Manipulation of Nuclear Processes ASM Public Outreach Fellowship Kymriah approved (PennMed) CAR T cells (NCBI) Cost of Kymriah (NYTimes) Accidental exposure to poliovirus type 2 (Eurosurveill) GAPIII (WHO) Poliovaccine candidate in plants (Nat Commun) Vertical vaccine farm (TWiV 47) All picornaviruses, all the time (TWiV 425) Image credit Letters read on TWiV 459 This episode is brought to you by the Defense Threat Reduction Agency. Part of the U.S. Department of Defense, the Agency’s Chemical and Biological Technologies Department hosts the 2017 Chemical and Biological Defense Science & Technology Conference to exchange information on the latest and most dynamic developments for countering chemical and biological weapons of mass destruction. Find out more at http://www.cbdstconference.com Weekly Science Picks Kathy - Cassini photos Dickson - Caliber Biotherapeutics Alan - Grav Rich - Google street view of the International Space Station (article) Vincent - the bioinformatics chat Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Marc Van Montagu, President of the European Federation of Biotechnology (EFB) and former Professor of Molecular Biology at the University of Ghent, talks about his life and career with Joanne Chory, Professor of Plant Molecular and Cellular Biology at the Salk Institute for Biological Studies. Dr. Van Montagu recounts how he went from studying chemistry to discovering the gene transfer mechanism from Agrobacterium to plants, which opened the door to gene engineering and the creation of transgenic plants. Through the EFB and the Institute of Plant Biotechnology Outreach, of which he is the founder and chairman, Dr. Montagu is now dedicated to educating the general public and informing political leaders about the necessity of using science and plant engineering to prepare a sustainable future for the planet and its growing population.

This episode: Scientists find a way to possibly make an HIV vaccine using bacteria! Download Episode (2.8 MB, 3 minutes)Show notes:News item/Journal Paper Other interesting stories: Bacteria may help to store excess carbon dioxide underground Bacteriophage needles that penetrate bacteria have iron tip Pathogen could be useful, produces very strong glue Yeast made to respond to magnets, useful for biotech Exploring the ocean, microscopically Improving knowledge of an already well-known useful bacterium Cold-loving yeasts: where they are and what they (could) do (paper) Post questions or comments here or email to bacteriofiles at gmail dot com . Thanks for listening! Subscribe at iTunes, check out the show at Twitter or SciencePodcasters.org

Abstract In this study, two topics causing major public concern related to transgenic plants were investigated: The possibility of a horizontal gene transfer from plant to bacteria and the impact of transgenic plants after herbicide treatment on root associated bacteria. The transgenic plant chosen for this study was Roundup Ready® (RR) soybean, which is tolerant to the herbicide glyphosate and is the most commonly used genetically modified crop worldwide. Glyphosate, the active ingredient of Roundup Ready®, inhibits the EPSPS enzyme (5-enolpyruvylshikimate-3-phosphate synthase). EPSPS is an enzyme involved in the shikimic acid pathway leading to the aromatic amino acid biosynthesis and its inhibition leads to growth reduction of plants and microorganisms. RR crops are glyphosate tolerant due to the introduction of the CP4-EPSPS gene coding for a glyphosate insensitive EPSPS enzyme. The transgenic construct is under expression of a CaMV 35S promoter a nos transcriptional termination element from Agrobacterium tumefaciens. Horizontal gene transfer experiments with the EPSPS gene of the RR soybean were performed under controlled laboratory conditions and were targeted to the nitrogen fixing symbiont of soybean Bradyrhizobium japonicum. This bacterium comprises the requirements of a possible receptor for the glyphosate resistance trait, as it is sensitive to the herbicide and thus the acquirement of glyphosate resistance would signify a positive adaptation to glyphosate accumulated in the roots after herbicide application. Two key conditions for gene transfer from the CP4-EPSPS gene from the RR soybean to B. japonicum were evaluated in this study: The required specific conditions for B. japonicum to undergo natural transformation and the expression of the CP4-EPSPS gene in B. japonicum. For that purpose, the CP4-EPSPS gene was cloned into a B. japonicum chromosomal integration vector and was transferred by biparental mating into the B. japonicum genome. Subsequently, the expression of the CP4-EPSPS gene in B. japonicum was tested under increasing glyphosate selection pressure. Results of these experiments indicated that B. japonicum is not naturally transformable under any conditions known from the more than 40 so far reported naturally transformable bacteria. Furthermore, the CP4-EPSPS genetic construct, as contained in RR soybean, has been shown in this study to be not active in B. japonicum. Consequently, if there would be a gene transfer of the plant CP4-EPSPS to B. japonicum, this genetic construct does not confer glyphosate resistance to B. japonicum and does not constitute any adaptive advantage to the bacterium under glyphosate selection pressure. As the genetic trait of glyphosate resistance has been found in several bacteria, it would be more probable that the common mating exchange between bacterial groups could disperse the glyphosate resistance within an environment. Moreover, in the specific case of B. japonicum, a high spontaneous mutation rate for glyphosate resistance was observed, suggesting that B. japonicum can also adapt to the glyphosate selection pressure by mutation under natural conditions. The impact of transgenic plants with their respective herbicide treatments on root associated bacteria was investigated in a greenhouse experiment. The composition and diversity of bacterial communities of RR soybean rhizospheres were analyzed and compared between glyphosate-treated and untreated plants. Samples from five harvests with two glyphosate applications were analysed by 16S rRNA gene T-RFLP analysis complemented with the evaluation of three clone libraries. Multivariate statistical analysis of the data was used to visualize changes in the microbial populations in response to glyphosate applications and in order to find groups of organisms responsible for the observed community shifts. A comparison of the rhizosphere communities revealed that a Burkholderia related group was significantly inhibited by glyphosate application, while the abundance of a group of Gemmatimonadetes related sequences increased significantly after the herbicide treatment. The significant increment of Gemmatimonadetes abundance after glyphosate application could indicate that these organisms are able to metabolize the herbicide. Shannon diversity indices were calculated based on the T-RFLP results with the aim to compare bacterial diversity in the rhizosphere of glyphosate-treated and non treated RR soybeans. Interestingly, the bacterial community associated to RR soybean roots after glyphosate application not only demonstrated effective resilience after the disturbance but in addition the bacterial diversity also increased in comparison to the untreated control samples. It is possible, that in an environment with organisms which are able to metabolize glyphosate, the key for enhancing diversity could be the succession of metabolites, which can be further utilized by a diverse range of bacteria.

During evolution of photoautotrophic eukaryotes, the nucleus has gained a dominant role in the coordination of the integrated genetic system of the cell consisting of three specifically coevolved genetic compartments. The photosynthetic machinery is encoded by the chloroplast and nuclear genomes. Therefore, biosynthesis and assembly of stochiometric amounts of subunits as well as association of the proteins with corresponding cofactors need to be managed and precisely regulated. To identify novel nuclear-encoded factors involved in the regulation of chloroplast gene expression at different levels, 12 nuclear mutants with high chlorophyll fluorescence (hcf) phenotypes denoting quite diverse defects in the photosynthetic apparatus were selected. Three of them, hcf145, hcf109 and hcf101, were analysed and the affected genes were characterized in more detail. Spectroscopic, fluorimetric and immunological studies have revealed that hcf145 and hcf101 were predominantly affected in photosystem I (PSI), while hcf109 had pleiotropic deficiencies. Remarkably, the dramatic reduction of PSI core complex accumulation in hcf145 was not accompanied by corresponding deficiencies of the outer light-harvesting antenna complex. A comparison of stationary transcript levels with rates of transcription, as estimated by Northern and chloroplast run-on transcription analysis, revealed that the hcf145 mutant is primarily and specifically characterised by a reduced stability of tricistronic chloroplast psaA-psaB-rps14 transcripts. The corresponding operon encodes the two large PSI polypeptides PsaA and PsaB, which form the heterodimeric PSI reaction centre, and the ribosomal protein S14. Chloroplast translation inhibition experiments excluded translational defects as the primary cause of impaired mRNA stability. Defined intervals of the tricistronic transcript were quantified by real-time RT-PCR which established that the psaA region is less stable than the rps14 region in hcf145. Therefore, although up to date, no 5'-3' exoribonucleases have been found in eubacteria (including the ancestors of plants), factor HCF145 appears to be required for the protection of the psaA-psaB-rps14 mRNA against progressive ribonucleolytic degradation starting at the 5' end. In the hcf109 mutant, exclusively plastid transcripts containing UGA stop codons are unstable. The affected gene encodes the first described chloroplast peptide chain release factor AtprfB. Its full-length cDNA, introduced into hcf109 via Agrobacterium-mediated transformation, could functionally complement the mutant. Homology of AtprfB to eubacterial release factors indicates that processes of translational termination in chloroplasts resemble those in eubacteria. The mutant phenotype revealed that translation of all plastid mRNAs containing UGA stop codons is exclusively terminated by AtprfB. However, besides its peptide chain release function, AtprfB appears to acquire yet unknown roles in regulating the stability and translation of the chloroplast mRNAs containing UGA stop codons. These additional regulatory functions could reflect evolutionary constraints which keep the number of plastid TGA stop codons high in vascular plant organelles in contrast to those of algae, mosses and ferns. In contrast to hcf145, steady-state levels and translation of photosynthetic transcripts are not altered in the PSI mutant hcf101. Separation of thylakoid membrane complexes by sucrose gradient centrifugation has uncovered that, similar to hcf145, accumulation of the outer antenna of PSI is not changed in hcf101. Therefore, hcf101 is affected in the assembly of the PSI core complexes. Expression of the HCF101 full-length cDNA in the hcf101 genetic background functionally complemented the mutant. The HCF101 protein encodes a very ancient and universally conserved protein of P-loop ATPases. HCF101 is plastid-localised and represents the first described factor essentially required for the assembly of PSI and other [4Fe-4S]-containing protein complexes in the chloroplast. Relatives of HCF101 are divided into four classes present in all organisms and in all cellular compartments. The antiquity of HCF101 points to the importance of Fe-S cluster biogenesis during the earliest phases of cell evolution. The ubiquity of HCF101 indicates that it is essential for all free-living cells.

Tue, 22 Mar 2005 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/3390/ https://edoc.ub.uni-muenchen.de/3390/1/Hoeppner_Christoph.pdf Höppner, Christoph

Agrobacterium tumefaciens ist ein Gram-negatives Bodenbakterium, das dikotyle Pflanzen infiziert. Mittels eines Typ IV Sekretionssystems werden dabei ein Teil der bakteriellen DNA (T-DNA), sowie die Virulenzproteine VirE2, VirE3, VirD2 und VirF in die pflanzliche Zelle transferiert. Das Typ IV Sekretionssystem von A. tumefaciens besteht aus den 12 Vir-Proteinen VirB1-VirB11 und VirD4, die zu einem die äußere und innere Membran durchspannenden Proteinkomplex und einem Pilus (T-Pilus) assemblieren. Im Rahmen dieser Arbeit wurden Protein-Protein-Interaktionen im VirB/D4-Sekretionssystem von A. tumefaciens C58 analysiert, mit dem Ziel einer Aufklärung der Assemblierung von Transporterstruktur und T-Pilus. Ergebnisse: 1) Unter Verwendung des milden nicht-ionischen Detergenz n-Dodecyl-b-D-maltosid wurde eine potentielle Vorstufe der T-Pilus Assemblierung solubilisiert. In dem ca. 100 kDa großen Proteinkomplex wurden die Vir-Proteine VirB2, VirB3, VirB5, VirB6, VirB7, VirB8 und geringe Mengen VirB9 detektiert. Die T-Pilus Proteine VirB2, VirB5 und VirB7 zeigten bei Analyse mittels BN-PAGE und 2D-Gelelektrophorese eine Kofraktionierung. Unter Berücksichtigung bereits bekannter Fakten wurde ein Modell der T-Pilus Assemblierung wurde erstellt. 2) Ein 27 kDa großes, mit VirB5 interagierendes Protein wurde entdeckt und nach Aufreinigung und N-terminaler Sequenzierung als „trans-Zeatin produzierendes Protein“ (Tzs) identifiziert. Die Untersuchung der enzymatischen Aktivität von Tzs ergab eine Umsetzung von 4-Hydroxy-3-methyl-2-(E)-butenyldiphosphat (HMBPP) mit AMP zu Zeatinribosid-5´-monophosphat (ZMP), einem Phytohormon der Cytokinin-Klasse. HMBPP ist ein Zwischenprodukt des alternativen Isoprenoid-Biosynthese Weges (DXP-Weg) Gram-negativer Bakterien. 3) Eine Interaktion von VirB5 mit VirB5, sowie VirB5 mit VirE2 konnte gezeigt werden. Im Rahmen der Analysen wurde neben den Piluskomponenten VirB2, VirB5 und VirB7 auch VirB1 in isolierten T-Pili detektiert.