Here is a quick roundup of the Roundtable discussion I was involved in on Microbiomes in Food and Agriculture run by the IFAL at UC Davis.
Was informed of this by Titus Brown, one of the instructors. Info copied from here.
Microbial genomics and transcriptomics hands-on, Sep 24-25
Who: Ben Johnson (Michigan State University); Tracy Teal (Data Carpentry); C. Titus Brown (UC Davis).
Host: C. Titus Brown
When: Sep 24 and 25, 2015
Times: 9am-3pm on both days
Where: TBD (UC Davis campus).
Cost: there is no fee.
This workshop is open to everyone, including graduate students, postdocs, staff, faculty, and community members. We have extra space for UC Davis VetMed affiliates; contact the host if you are an SVM affiliate.
This two-day hands-on workshop will introduce biologists to microbial genomics and transcriptomics. The primary focus will be on genome assembly and annotation, and subsequent transcriptome analysis, of bacteria.
We will be analyzing a stock data set, and we will be using the Amazon cloud.
- Logging into the Amazon Cloud
- Short read quality and trimming
- Genome assembly (with SPAdes or MEGAHIT)
- Genome annotation (with Prokka)
- RNAseq analysis
- Differential expression analysis
Attendees will need to bring a computer with a Web browser, an Internet connection, and an ssh client; Windows users should install MobaXterm before the workshop.
LICENSE: This documentation and all textual/graphic site content is licensed under the Creative Commons – 0 License (CC0) — fork @ github. Presentations (PPT/PDF) and PDFs are the property of their respective owners and are under the terms indicated within the presentation.
This is a post in my continuing series of the “Story Behind the Paper.” series. This post is from Benjamin Schwessinger, Pamela Ronald, Rory Pruitt, Anna Joe, and Ofir Bahar.
A Phoenix Rises from the Ashes: A new discovery emerges from the 2009 retraction.
|A phoenix depicted in a book of legendary creatures by FJ Bertuch (1747–1822).
Via Wikipedia Commons – based on this
This is the story behind our report published today in Science Advances.
In Science Advances we report that one class of bacteria produces a previously undescribed, and long sought after, molecule recognized by plants carrying a specific receptor.
The story began in the 1970s, when Professor Gurdev Khush and colleagues demonstrated that a wild species of rice was immune to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), causal agent of a serious disease of rice globally. In the 1990s Ronald began studying the rice/Xoo interaction. Because both rice and Xoo are genetically tractable, the rice/Xoo biological system proved to be an excellent system for studies of the molecular mechanisms governing the plant immune response. In 1995, two postdoctoral fellows in Ronald’s lab at the University of California, Davis- Guoliang Wang and Wenyuan Song-reported that this rice immune response was controlled by a single receptor kinase, called XA21.
The predicted structure of the XA21 protein, with a predicted leucine rich repeat extracellular domain and an intracellular kinase domain, suggested that XA21 could sense a secreted microbial molecule and then activate an immune response.
A few years after the discovery of the XA21 receptor, the fly Toll and mouse Toll-like receptors (Tlr4) were shown to share striking structural similarities with XA21 and other plant receptors. The animal receptors also recognized and responded to microbial molecules. Together these discoveries demonstrated that plants and animal use similar mechanisms to protect against infection. Professors Bruce Beutler and Jules Hoffman were awarded the 2011 Nobel Prize in Physiology or Medicine for their important work.
The Ronald laboratory then spent twenty years trying to identify the microbial molecule that is recognized by XA21. The research led to the identification of a number of microbial genes that are required for activation of XA21-mediated immunity (rax genes). These genes encode a tyrosine sulfotransferase, RaxST, and three components of a predicted type 1 secretion system: a membrane fusion protein, RaxA; an ATP-binding cassette transporter, RaxB; and an outer membrane protein, RaxC. raxST, raxA, and raxB are located in a single operon (raxSTAB). Based on these findings, we hypothesized that the activator of XA21-mediated immunity is a tyrosine sulfated, type 1-secreted protein.
We were excited about this idea because sulfation has emerged as an important posttranslational modification controlling receptor-ligand interactions. It is a common posttranslational modification of eukaryotic proteins and plays important roles in regulating development and immune responses. The importance of this area of research to biology and medicine is reflected in the recent report of a novel drug that blocks HIV infection. To achieve this breakthrough, the researchers exploited the observation that HIV binds tyrosine sulfated amino acids for cell entry (Gardner et al., 2015).
Despite a clear model and diverse supporting data suggesting that Xoo secretes a sulfated peptide, the identity of this molecule remained elusive.
In 2009, the Ronald laboratory reported that XA21 recognized a sulfated peptide. However we later discovered major errors in this work and in 2013, we retracted the paper. We discussed these mistakes in several lectures, post and articles including a Keystone symposium, Scientific American, Nature, and Schwessinger’s blog (here and here). The process with which we addressed the problems was highlighted as “Doing the right thing” by Retraction Watch, a blog that reports on retractions of scientific papers. The retraction was included as one of the top 10 retractions of 2013.
The new Discovery
Today, in Science Advances, we are delighted to report the identification of the microbial molecule that activates XA21-mediated immunity. As predicted, it is a tyrosine-sulfated protein. We named this microbial protein RaxX.
|The rice immune receptor recognizes the bacterial molecule RaxX and initiates an appropriate immune response. Illustration by Kelsey Wood.|
To isolate this molecule, postdoctoral fellow Rory Pruitt systematically created bacterial mutants carrying deletions near the RaxSTAB operon. He showed that one of the deletion mutants lost the ability to activate the XA21-mediated immune response. The deleted region encodes a small open reading frame that we named RaxX. Xoo strains lacking RaxX and Xoo strains that carry mutations in the single RaxX tyrosine residue (Y41) are able to evade XA21-mediated immunity. Postdoctoral fellow Anna Joe, together with collaborators at the University of Texas, Austin and at the Joint Bioenergy Institute in Emeryville, showed that Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Postdoctoral fellow Benjamin Schwessinger, graduate student Nick Thomas and collaborators showed that sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21–amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, demonstrating the co-evolution of host and pathogen. RaxX is highly conserved in many Xanthomonas species.
Our results indicate that the presence or absence of sulfation is decisive for the ability of RaxX to trigger XA21-mediated immunity.
The new insights gained from the discovery and characterization of RaxX may be useful for the engineering of resistant crop varieties and for the development of therapeutic reagents that can block microbial infection of both plants and animals.
Notes on the publication process
“The scientific life is the most complex of all to write about. In the case of scientists, impulse becomes compulsion”. — Carol Shields
After we discovered mistakes in our previous paper, we spent several years correcting the scientific literature both by retracting the original Science paper (Lee et al. 2009) and by following up with publications to further correct the literature (Bahar et al. 2014). We made extra efforts to control the results in this current report.
Wrestling with the retraction and discovering the new molecule in rapid succession was an enormous challenge. Here we share some of the lessons learned.
Pamela Ronald, Professor, Department Plant Pathology and the Genome Center, UC Davis; Director of Grass Genetics, the Joint Bioenergy Institute:
I would not wish a retraction on anyone. Scientists are supposed to catch their mistakes before publication. Still, I am astonished to conclude that the process has in some ways been positive.
On an administrative level, the lab is running more efficiently. I have instituted new practices for the lab: created duplicate stocks of key strains (validated and maintained by the lab manager), mandated electronic notebooks for each lab member and required that all new assays be independently validated by three independent researchers before publication.
But the best part of this bad situation has been working with this particular team. It has been an immense privilege to watch each person work through the situation in their own way, collaborate, and make new discoveries. Respect for each other and for the scientific process was paramount. After figuring out what went wrong (no easy task), they tried not to look back. They did not give up, even when it would have made sense to do so. Their persistence and optimism in face of this daunting challenge buoyed all of our spirits. I will always be in awe of their work and will always be grateful.
Equally stunning was the supportive and kind response from the scientific community. We received many letters of encouragement – even from complete strangers. It helped us keep going.
There are still hills to climb. Some scientists may be extra skeptical of results from my lab for a long time to come. For example, in a critique of our submission, one of reviewer’s asked, “how do we know the strains weren’t mixed up again this time?”
Rory Pruitt, postdoctoral scholar in the Ronald lab.
I was only a few months into my postdoc when I became convinced that the majority of the Ax21 story was incorrect (Ax21 was the proposed elicitor of XA21-mediated immunity in the retracted papers). My mind was filled with questions. How could this happen? What results can I believe? Admittedly, the biggest question that hounded me was “Should I be looking for a new job?” There were a few key factors that led to my decision to stay in the lab. I think these factors were also critical to this story working out as a “success.”
Early on, I went to Pam with some of my doubts. It was terrifying to approach my new boss and I say I didn’t believe some of her published work (including a Science paper!). But I needed to know that I could be honest with her and not feel pressured into only showing results that fit the established model. Pam listened to my concerns and those of others in the lab. Most importantly, she showed that she was committed to getting the story right and correcting the literature if need be.
In addition to Pam, there was a great team of postdocs and graduate students who were equally devoted to correcting the science. At times it seemed a long, painful process with little reward (there’s not a good space on a CV for working towards a retraction). Nevertheless, it needed to be done so that we and other labs could move forward. I was encouraged by Ofir, Ben, and others who worked persistently on this.
A final factor in my decision to stay is the prospect of new discovery. If Ax21 isn’t the activator of XA21-mediated immunity, what is? Maybe we can find it! It’s that hope of new discovery that keeps us coming back to the lab bench. My postdoctoral experience has had some highs and lows, but I am glad I stuck it out. With persistence, enthusiasm, and a good team committed to reliable science, we were able to not only correct earlier mistakes but also move forward.
Benjamin Schwessinger, former Ronald Laboratory postdoctoral scholar and now independent research fellow in Australia, at the Australian National University in Canberra.
You have much to lose as an early career researcher if you are thrust into a situation where results cannot be reproduced. In a hyper competitive environment irreproducible results you are trying to build on are a big problem, no matter how smart, privileged, and gifted you are. Lengthy delays in publishing as a postdoc can cause great harm to a career. Here are the main factors that made us successful in the face of adversity.
(Be lucky) have your own funding
Your own funding makes you financially and also scientifically more independent. It ensures your academic freedom. I was grateful to have been supported independently by the Human Frontier Science Program. It made me bolder and braver in speaking out. I was able to choose to stay or go. Because of the team I believed in I decided to stay!
Get confidential outside advice
Getting some outside confidential impartial advice on how to approach this problem is very important. Many senior figures have most likely seen similar cases in the past and have more insight. Following through with this advice is a total different matter. I decided to stay!
Work through it together as a team. Build on each other’s strength and talk about all possibilities. Repeat each other’s experiments with all required controls. Invite well respected figures in the field to independently test (and confirm) core experiments.
Admit mistakes and retract
Everyone makes mistakes. They are part of the scientific discovery and science has to be self-correcting. Retractions are an integral part of this process. Not to retract is NOT an option! It obstructs all future progress in the subject matter.
Follow the data
Do controls, repeats, and repetitions of conclusive experiments. Seeing is better than believing.
Ofir Bahar, former Ronald Laboratory postdoctoral scholar and now principal investigator, Plant-Microbe Interaction Research Group, the Volcani Center, Israel,
I remember the day, early 2013, when we were driving back to Davis from a happy and relaxed baby shower at Benjamin’s place in Oakland, Rory mentioned to me “you know, I deleted an upstream and a downstream region to raxSTAB. The downstream mutant was no different than wild type, but the upstream mutant forms long lesions on XA21 plants…”
This was the turning point; I immediately knew this was a big discovery and a major break through for the lab.
But before that moment, we were a bunch of enthusiastic post docs that just loved doing science. We wrote these nice proposals to get our fellowships, based on the amazing story of the rice immune receptor XA21 and its (thought to be) elicitor Ax21.
It was a fascinating story we were all so excited about having read it in Science. Of course we joined the Ronald lab to follow up on this initial discovery, but well… the building upon part did not work as we all might have wished. We had to dig deep, real deep, to figure out what was going on and what went wrong before our arrival to the lab. So, a year….. year-and-a-half in our new positions we finally reached the ultimate conclusion that there was a big hole in the model – there’s no elicitor! Or, there is, but it’s not Ax21 and we don’t have a clue what the identity of this molecule might be. It felt like we were thrown back 10 years, to 2004 with the da Silva paper just published describing the requirement of the three Xanthomonas genes RaxST, RaxA and RaxB for XA21 immune activation.
Those were ‘dark ages’ and difficult times. Understanding that most of the time you invested so far was, at least in practical terms (e.g. publications), for nothing, and that there is no biological model to work on, but that it needs total reboot. To be honest I was feeling a bit worried at that time for my scientific career. But then, a series of exciting discoveries (including some that are not published yet) gave me hope again. Well… isn’t this how science goes, bad, bad, bad, bad, good, bad, bad, bad, good and so on. I remember Pam telling me: “you know why I love a big group? There has got to be some positive results coming all the time”
Later, a few months after Rory shared with me his finding, we already knew what it was, and we were very certain, this is the ONE. Unfortunately, or luckily, I got a position offered at my home country and I gladly accepted it. So I actually wasn’t there for the flower stage (you know… the decorations), but I was very happy to have been there when the bud of this beautiful flower to be emerged. Every time I think of this story its like, WOW, can you believe all this has happened in just 3-4 years, unbelievable.
My lesson is, never lose hope, be critical, believe it when you see it, work on multiple projects, enjoy science and openly share science
Anna Joe, postdoctoral scholar in the Ronald lab.
I was in my final year grad school and looking for a postdoc position in early 2013. The Ronald lab was on the top of my wish list because I was fascinated by the Ax21 story in Science 2009. But just before I applied for a position in the Ronald lab I learned that something went wrong with Ax21 and that the original paper would be retracted. Many thoughts crossed my mind. Main one was “Do I still want to join the Ronald lab?”. Actually it was easy to answer the question once I spoke with Pam about it and talked with her lab members during the visit for my formal interview. “Yes, I’d like to work in the lab which just retracted two papers”. This for sure sounds crazy to most people. However, the whole experience of my visit gave my many reasons to join the Ronald lab. Correction of errors is a part of science (I knew this because I also had difficult time to track down a mix up plants problem before) but not many people are brave enough to admit mistakes. Pam and all lab members honestly, clearly stated to me what the errors were and how they verified the problems. They communicated well with each other, shared idea freely and respected other’s opinions. Their open mind and transparency attracted me.
On top of that I was very curious about the unexplored, new Xa21 activator. All other lab members might have felt the same curiosity and channeled its energy to continuously work through the problems during last several years. Although I did not share the “dark period”, I could see everybody in the lab was persistent with the common effort to correct the science. I experienced incredibly good teamwork and great collaboration. All of those are the driving force of our success. Finally, I’d like to mention that we could not make it without the support and encouragement from the scientific community. Many scientists shared their thoughts and advice and were rooting for us. Most collaborators unhesitatingly complied with our requests for assistance. They helped us not only “do the right thing”, but also do better science.
So I saw this Tweet earlier today
— Gena Hoffman (@GenaEHoffman) March 24, 2015
And that sounded very interesting. So I clicked on the link to check out the Plant Breeding for Food Security: The Global Impact of Plant Genetics in Rice Production A symposium honoring Dr. Gurdev Khush symposium. And, then I went to the program. And sadly I saw something there that was not to my liking. The speakers were almost all male (men labelled in yellow, women in green)
- Welcome to the Khush Symposium (Alan Bennett)
- The Plant Breeding Center (Charles Brummer)
- The Confucius Institute (Glenn Young)
- Global food production – challenges and opportunities (Ken Cassman) Food production, technology and climate (David Lobell)
- Panel – Impact of Gurdev Khush on plant genetics and food security Tomato genetics
- (Dani Zamir)
- (Pam Ronald)
- (Gary Toenniessen)
- (Gurdev Khush)
- Lunch; The California Rice Industry (Kent McKenzie)
- The rice theory of culture (Thomas Talhelm)
- Recent advances in rice productivity and the future (David MacKill)
- Hybrid rice technology contributions to global food security (Sant Virmani)
- Super green rice (Qifa Zhang)
- Tackling the wheat yield barrier (Matthew Reynolds)
- African Orphan Crops – inspiration and execution (Howard Shapiro/Allen Van Deynze)
If this was a symposium outside UC Davis the first thing I would do would be to post about it. To Twitter or my blog or both. And to critique them. Why? Because there is a bad history in STEM fields of having meetings and conferences have under-representation of women as speakers. And this has become a passion of mine and I write about it a lot. But I hesitated. Why? Because this was from UC Davis and many of the people involved are friends / colleagues. I did not want to anger them, or embarrass them. And I don’t think there is any intentional bias here by any means. But, if I am going to critique people outside UC Davis, it seems like I should also apply the same standards to people inside UC Davis and to colleagues and friends.
So I posted to Twitter a response:
But that did not seem sufficient. So I wrote up this post. Underrepresentation of women as speakers is a serious issue in STEM fields. And it is solvable (e.g., see Some suggestions for having diverse speakers at meetings by myself and the wonderful Ten Simple Rules to Achieve Conference Speaker Gender Balance by Jennifer Martin).
Now – do I know who the possible speakers were for this symposium? No – I don’t really know the field. Is it possible that there just are no women in the field? Sure. But I would bet anything that is not the case here. Having a meeting where the ratio of speakers is 16:1 male: female sets a bad example. UC Davis and the organizers of this meeting can do better. And though this will possibly hurt me in various ways (I already got grief from one person who I will not name for the Tweet), I think it is critical that we call out examples such as this.
And finally I note – I have taken on the issue of women at STEM conferences and meetings because, well, it is easy to identify cases where the numbers are anomalous and it is relatively easy to solve. But it is also important that we consider other aspects of diversity of speakers (age, ethnicity, career stage, etc). It is important to have diversity of speakers at meetings for many many reasons. Speaking is a career building opportunity. Speakers serve as role models for others. Diverse points of view are important to have represented. Bias – whether simplicity or explicit damages the whole practice of science. And more. Yes, we need to work on many aspects of diversity in STEM fields. Improving the diversity of speakers at meetings is but one part of this. But it is an important part and it is relatively easy to do. So just do it. And call attention to it. Even if it hurts.
UPDATE 3/25 11:29 AM
The meeting organizers have responded on Twitter
Storify of some responses here
The University invites comments on the proposed draft Presidential Policy on Open Access, which is based on the Academic Senate Open Access Policy for all Academic Senate members adopted on July 24, 2013.
The proposed new policy extends open access rights and responsibilities to all non-Senate members of the UC community who are authors of scholarly articles, including faculty, other academic personnel, students, administrators, and staff. The policy allows non-Senate authors of scholarly articles to maintain legal control over their research articles while making their work freely available to the public. In addition, the proposed policy outlines procedures for implementing the policy for all UC authors, both Senate and non-Senate. Although the policy assumes all authors will make their scholarly articles available to the public, there is a procedure, which authors must undertake proactively, to opt out of the open access process.
The proposal is located on the UCOP Academic Personnel and Programs website, “Policies under review,” under the “Systemwide Review” tab at http://www.ucop.edu/academic-personnel-programs/academic-personnel-policy/policies-under-review/index.html. If you prefer these documents as attachments, please let me know.
Quikc post here. A month or so ago I went to the UC Davis Medical School in Sacramento for a meeting and got to see this amazing new sculpture for the first time.
I just love things like this: The Aggie Transcript | An undergraduate life sciences journal at UC Davis. From their site:
The Aggie Transcript is is a forum for undergraduate UC Davis students to share news, original writing, and art related to the life sciences.
Here are some recent posts:
- Mapping neurons through online gaming by Jenny Cade
- Evolution of Tooth Ename by Marisa Sanchez
- New Target For Alzheimer’s Treatment by David Ivanov
- Is your spit making you fat? by Daniel Friedman
- Viral Evolution by Ashlee Chang
- The hunter-gatherer gut microbiome by Jenny Cade
Note – written in 2014 – in need of update
So – getting lots of calls and emails and having local meetings with people interested in microbiology at UC Davis. Rather than continuing to answer each person separately I am just going to put everything together on this page. Departments and groups with a major focus on microbiology.
- Microbiology and Molecular Genetics in the College of Biological Sciences
- Pathology, Microbiology and Immunology in the School of Veterinary Medicine
- Medical Microbiology and Immunology in the School of Medicine
- Plant Pathology
- Department of Pathology in the School of Medicine
- Department of Internal Medicine Division of Infectious Diseases in the School of Medicine
- USDA ARS Crop Pathology and Genetics Group
Departments with a lot of people working on microbiology
Phoebe Ayers, librarian extraordinaire at UC Davis is running another Wikipedia Editathon on women in science and academia. See Wikipedia:GLAM/University of California Davis Libraries. It will be May 23. The last one went quite well. I had posted a few announcements here and there (e.g., Wanted – participants and helpers for a “Women in Science Editathon) about the previous one that was inspired by Dawn Sumner and run by Phoebe. These are good ways not only to help promote women in science but also to learn a bit about Wikipedia and about some female scientists.
I wrote a post on the UC DAVIS ADVANCE Blog recruiting people to participate in a Wikipedia Editathon regarding Women in Science: Wanted – participants and helpers for a “Women in Science” Wikipedia Editathon at #UCDavis March 4 – UC Davis ADVANCE
And Phoebe Ayers from the UC Davis Physical Sciences and Engineering Library has volunteered to host the event there.
See her post about this. Please consider signing up to participate if you are around UC Davis at that time …