The MPMI Editor’s pick for March 2020 is “Comparative Genomics Screen Identifies Microbe-Associated Molecular Patterns from ‘Candidatus Liberibacter’ spp. That Elicit Immune Responses in Plants” with first author Yuan Chen, a postdoctoral researcher in the lab of Jennifer Lewis at the USDA Plant Gene Expression Center and UC-Berkeley. Lewis shared some background for their work and how collaborations and feedback from colleagues can really help to bring a project together.

Submitted by Jennifer Lewis

Citrus greening, or Huanglongbing, caused by ‘Candidatus Liberibacter asiaticus,’ has decimated citrus groves in Florida and is spreading through other citrus-growing areas of the United States. Citrus greening causes blotchy mottle (random yellowing) on leaves; bitter, misshapen, and discolored (green) fruit; and tree decline—all of which affects the quality and flavor of oranges.

Citrus greening is a difficult scientific problem to investigate, because it is caused by an unculturable phloem-restricted pathogen and because it affects a woody species (which means plants grow very slowly and take a lot of time to produce the next generation). I was familiar with the work done by Honour McCann, formerly at the University of Toronto and now at the Max Planck Institute in Tübingen, and David Guttman, at the University of Toronto, to identify microbe-/pathogen-associated molecular patterns (MAMPs/PAMPs) by bioinformatic approaches (McCann et al., 2012). Work from Cyril Zipfel, University of Zurich, and others had shown that applying purified MAMPs to Arabidopsis induces immune responses, such as the production of reactive oxygen species (ROS), and that this so-called pattern-triggered immunity (PTI) response protects plants from infection (Lacombe et al., 2010; Zipfel et al., 2004). We therefore thought that we could boost the immune response in citrus if we could identify endogenous MAMPs in ‘Ca. L. asiaticus.’

MAMPs are highly conserved peptides and therefore believed to be essential for the microbe. However, individual amino acid residues in a MAMP might be under positive selection to avoid recognition by the plant’s defenses. Positive selection leads to an increase in variants in the population, because the variants are beneficial to the pathogen. In contrast, deleterious variants are subject to negative selection, because they impair fitness.

Claire Bendix, a former USDA postdoc in my lab, carried out computational analyses to identify putative MAMPs with signatures of positive selection from ‘Ca. L. asiaticus’ genomes. We hypothesized that putative MAMPs should be specific to strains that infect citrus—not found in the nonpathogenic relative L. crescens and possibly also absent in ‘Ca. L. solanacearum,’ which infects solanaceous plants.

Yuan Chen, a University of California, Berkeley postdoc in my lab, tested the role of putative MAMPs in eliciting immune responses, starting first in Arabidopsis and Nicotiana benthamiana, as these plants are commonly used to look at ROS production, which is a feature of the immune response. These assays gave exciting results, showing that two of the ‘Ca. L. asiaticus’ peptides could elicit ROS production. However, it was well known that peptides could be contaminated with flagellin peptide (flg22), which is a very strong elicitor of PTI, and therefore give a false-positive result. To ensure that the ‘Ca. L. asiaticus’ peptides were not contaminated, Yuan tested for ROS production in the Arabidopsis flagellin receptor (fls2) mutant, as well as in the Ws ecotype of Arabidopsis, which lacks FLS2. She found that both ‘Ca. L. asiaticus’ peptides still elicited ROS responses, indicating that they were not contaminated with flg22. This control was very important in convincing us the results were real. In addition, Yuan read about some MAMPs that could induce a second ROS burst (Segonzac et al., 2011; Shang-Guan et al., 2018), which might lead to longer immune responses. She tested all of our peptides, but none was able to induce a second ROS burst.

Yuan then turned to optimizing ROS assays in citrus, first using flg22 as the elicitor. This proved to be quite challenging, as citrus has a waxy cuticle on the leaves and it was difficult to deliver the peptides. Yuan tried many different approaches (including different surfactants, inoculation methods, and nanoparticle delivery systems) before developing a reliable and reproducible protocol using vacuum infiltration of new flush (leaves) and luminescence-based detection of ROS production. She then tested our peptides on several Citrus species and found one (pksG) that elicited strong ROS production.

For the last few years, we have been collaborating with Bill Dawson, University of Florida, to test our peptides in sweet orange. Bill and his colleagues developed a viral delivery system using Citrus tristeza virus (CTV) that allows them to deliver novel molecules to the phloem of citrus trees (Dawson et al., 2015). They are testing whether CTV-mediated delivery of our peptides reduces symptoms from ‘Ca. L. asiaticus’ in the field.

Our work shows how fundamental work can be applied to an important agricultural problem. It combined bioinformatics, evolutionary biology, genomics, plant pathology, and cell biology to find ways of fighting against a devastating disease. Our work also illustrates the importance of delving into the literature and having key controls to have confidence in your data. This work also raises many interesting questions regarding the evolution of phloem-restricted pathogens. For example, perhaps ‘Ca. L. asiaticus’ adapted to colonize the phloem in part to avoid PTI.

We were fortunate to have great discussions with lab members; excellent greenhouse support from greenhouse manager Lia Poasa; nursery technicians Shawna Kelley, Julie Calfas, and Christopher Tucker; and a great collaboration with Bill Dawson and members of the Dawson lab, Choaa El–Mohtar and Cecile Robertson.

Front left to right: Ilea Chau, Jamie Calma, Yuritzy Rodriguez, Yuan Chen, Karl Schreiber

Back left to right: Jana Hassan, Hunter Thornton, Jennifer Lewis, Maël Baudin, Jacob Carroll-Johnson, Jack Kim.​

Refere​​nces

Dawson, W. O., Bar-Joseph, M., Garnsey, S. M., and Moreno, P. 2015. Citrus tristeza virus: Making an ally from an enemy. Annu. Rev. Phytopathol. 53:137-155.

Lacombe, S., Rougon-Cardoso, A., Sherwood, E., Peeters, N., Dahlbeck, D., van Esse, H. P., Smoker, M., Rallapalli, G., Thomma, B., Staskawicz, B. J., Jones, J. D. G., and Zipfel, C. 2010. Interfamily transfer of a plant pattern-recognition receptor confers broad-spectrum bacterial resistance. Nat. Biotechnol. 28:365-369.

McCann, H. C., Nahal, H., Thakur, S., and Guttman, D. S. 2012. Identification of innate immunity elicitors using molecular signatures of natural selection. Proc. Natl. Acad. Sci. U.S.A. 109:4215-4220.

Segonzac, C., Feike, D., Gimenez-Ibanez, S., Hann, D. R., Zipfel, C., and Rathjen, J. P. 2011. Hierarchy and roles of pathogen-associated molecular pattern-induced responses in Nicotiana benthamiana. Plant Physiol. 156:687-699.

Shang-Guan, K., Wang, M., Htwe, N., Li, P., Li, Y., Qi, F., Zhang, D., Cao, M., Kim, C., and Weng, H. 2018. Lipopolysaccharides trigger two successive bursts of reactive oxygen species at distinct cellular locations. Plant Physiol. 176:2543-2556.

Zipfel, C., Robatzek, S., Navarro, L., Oakeley, E. J., Jones, J. D. G., Felix, G., and Boller, T. 2004. Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428:764-767.

The April 2020 Editor’s pick for MPMI is “Arabidopsis EDR1 Protein Kinase Regulates the Association of EDS1 and PAD4 to Inhibit Cell Death”. First author Matt Neubauer shared a summary of how the project came about, and a little about the trials and tribulations that eventually led to publication of his graduate work while in the lab of Roger Innes at Indiana University. Matt has now gone on to a postdoctoral position at North Carolina State University. Matt recently presented the first What’s New in MPMI! virtual seminar. You can watch his seminar here​.

After many years, it is great to have our work on the role of EDR1 in the regulation of PAD4 and EDS1 published in MPMI. This project was initially intended to be a short investigation, however, it evolved greatly over time. The winding story of this project highlights how accidental discoveries and observations can enable important scientific discoveries.

I originally became interested in plant biology and biotechnology as a high school student, which motivated me to pursue a degree in biology. After finishing my BS in biology at Loyola University Chicago, I joined the Genome, Cell, and Developmental Biology PhD program at Indiana University. I became a member of Roger Innes’s lab in early 2014 and began working on a few projects focused on investigating the role of EDR1 in regulating plant defenses. In my second year, I took up the project that we ultimately published in MPMI.

The project began when a postdoc in our lab, Irene Serrano, discovered that the edr1-1 mutants she was working with actually carried a second mutation, located in the PAD4 gene (pad4-13). PAD4 is a well-known regulator of plant disease resistance, so we worried that some of our previous experiments using edr1-1 plants may have been influenced by the presence of this mutation. Irene sought to determine what effect pad4-13 has on edr1-1 plants, and whether it had been present in any of our previous seed stocks. Her results indicated that pad4-13 results in a gain-of-function, enhancing plant resistance and cell death. Interestingly, it did not affect the primary edr1 phenotype, enhanced resistance to powdery mildew.

At this point, we felt we had a duty to report these results to the scientific community, particularly those who had studied edr1-1 mutants. We determined that our original edr1-1 stocks did not contain pad4-13, however, we wanted to inform others about this mutation and alert them to the possibility that it was present in their lines. Due to the nature of this discovery, we felt this work would best be suited for a lower impact journal. However, we had a few interesting ideas that we wanted to test which we felt might enhance our work.

One of my objectives when I became involved in the project was to investigate how the pad4-13 mutation enhances PAD4 signaling. This was a long process, with many dead ends. Ultimately, we were unable to demonstrate how pad4-13 enhances PAD4 activity. It did not alter PAD4 accumulation or localization, formation of the PAD4/EDS1 complex, or block an important modification site. We concluded that pad4-13 likely affects an unknown PAD4 signaling mechanism.

Despite these setbacks, we had another idea we wanted to pursue whether EDR1 directly regulates PAD4. This hypothesis was based upon the observation that pad4-13 enhances some, but not all, edr1 phenotypes; as well as previously published data showing that loss of PAD4 suppresses edr1 phenotypes. These observations indicate that EDR1 and PAD4 may function in a common pathway. Since the interaction between PAD4 and its partner EDS1 is important for downstream defense signaling, I tested whether expression of EDR1 affects the formation of the EDS1/PAD4 complex. We were excited to find that, indeed, expression of EDR1 inhibited the formation of the complex. This discovery prompted us to further investigate the interaction between EDR1 and EDS1/PAD4, a slow process that occurred over many years.

We were initially disappointed and worried when we found the pad4-13 mutation in our edr1-1 stocks. In fact, we thought it was appropriate that pad4-13, which had proved so difficult to understand, was the thirteenth PAD4 allele to be discovered. Despite being an unlucky discovery, pad4-13 led us to important insights into the function of EDR1. Our decision to pursue the hypothesis that EDR1 directly interacts with PAD4 and EDS1 was a fortunate one which led to a new discovery about plant defense signaling.

In order to include the viewpoints of our members-in-training in society decision making and congress planning, the IS-MPMI Board of Directors (BOD) has added two junior members. The BOD received applications from several impressive candidates from around the globe and evaluated each based on excellence in scholarship and training, collaboration within the society, leadership at his or her institution, and outreach within his or her community. We are pleased to welcome Patricia Baldrich and Charles Roussin-Léveilléeto the board! Read further for more about Charles and Patricia.

Patricia Baldrich 

Dear IS-MPMI members,

I am a postdoctoral researcher in the lab of Blake Meyers, at the Donald Danforth Plant Science Center (Saint Louis, Missouri, United States). As a scientist who has worked in three different continents, I have witnessed the ability that diverse perspectives have in creating great interdisciplinary and international collaborations, which I believe are the driving forces that move science forward. My passion for plant-microbe interactions and learning how organisms from different kingdoms communicate has guided my career journey. The focus of my research is on small RNAs and their use as communication tools in the interaction of plants and their associated microbes.

I believe that communication is essential for successful interactions, not only for plants and microbes, but also for humans. I would love to serve as a reliable bridge between junior members and the BOD. My goal is to establish effective and sustainable avenues to bring the voice of the junior members to the society. Together, with your input and support, we will introduce innovative and beneficial ideas into our congress, meetings, and journals. I am here to listen and understand.

Charles Roussin-Léveillée 

Bonjour/Hi!

My name is Charles and I am thrilled to join the IS-MPMI BOD as a junior member. I am currently a first year PhD candidate in the research group of Peter Moffett at the Université de Sherbrooke, in Quebec, Canada. I investigate how microbes manipulate their host cells to induce an ideal living space in the apoplast. I discovered the field of plant-microbe interaction as an undergraduate and have travelled around the world to learn more about this topic through amazing internships. Outside the lab, I am a passionate hiker who loves to participate in discussions about anthropology, diversity and inclusion, as well as science in general (anything that can help advance society and address inequalities).

The scientific wealth that has been generated by humankind since our beginning is staggering. To push even further, science will require a richness of diversifying ideas. Therefore, collaboration between every nation and the breakdown of diversity barriers will send scientific knowledge to unseen heights. During my time on the IS-MPMI BOD, my goals will be to address diversity and inclusion issues as well as to propose ideas to increase collaboration between developed and less developed countries to enrich our scientific community. Our society has made big jumps in these topics over the last few years. However, much more has yet to be done and understanding each others’ issues will help build stronger bonds between our current and future members.

As such, in collaboration with other IS-MPMI members, I would like to determine the ‘top 10’ issues impacting our society. By aiming the spotlight at these issues, I believe that we will be able to discuss more in-depth about the solutions that could be undertaken to resolve them. I intend to promote the sharing of scientific knowledge between continents by suggesting the creation of scientific summer schools in molecular plant-microbe interactions. I can’t be more excited to discuss varying topics with our members worldwide.

I look forward to participating in the advancement of the large and vibrant research society that is the IS-MPMI.

Regine Kahmann’s election as Foreign Member of the Royal Society constitutes recognition of her outstanding scientific achievements. She served as director of the Max Planck Institute for Terrestrial Microbiology until 2019, and also served as the IS-MPMI president from July 2016 to July 2019.

What area(s) of molecular plant–microbe interactions do you feel your research has impacted most? 

I believe the demonstration that the connection between mating and virulence in smut fungi is achieved by combinatorial control of two homeodomain proteins which dimerize only when they originate from different alleles was a major breakthrough, because it meant that this complex acts as central regulator also of virulence. The second area concerns novel secreted effector proteins which we ended up working with after I spotted that many of the respective genes are arranged in clusters in the genome and for which we subsequently showed by generating deletions that many of these clusters impacted on virulence. And the third area is effector translocation to the host, where we think we have identified the machinery for this that is used by smut fungi (and still struggle to publish this).

What advice do you have for young scientists aspiring to achieve the level of science that has major impact? 

Do not only thrive for low hanging fruits and do not give up too quickly if something does not work.

When you were a post-doc, what had the largest influence on your decision to enter your specific research area in your permanent position? Was this a “hot topic” at the time, or did you choose to go in a different direction? 

I switched fields after my postdoc when I had my first independent position and moved from phage work and Escherichia coli to plant microbe interactions. I picked the Ustilago maydis–maize system in the early eighties definitely not because it was a hot topic. The system had been studied by several excellent geneticists but had not advanced to the molecular area. At the time I felt that the phage work would not sustain my scientific career and I found U. maydis and its ability to induce plant tumors simply fascinating.

Giles Oldroyd, the Russell R Geiger professor of crop science and director of the Crop Science Centre and group leader at the Sainsbury Laboratory, University of Cambridge, has been recognized for his outstanding contributions to science in plant-microbe interactions with his election as a fellow of the Royal Society.

What area(s) of molecular plant–microbe interactions do you feel your research has impacted most?

My research over the last 20 years has focused on beneficial interactions that plants form with microorganisms, in particular those associations leading to intracellular colonization. I started work on the nitrogen-fixing association between legumes and bacteria, but have since diversified, especially the arbuscular mycorrhizal symbiosis. Recent work has revealed that the signaling processes we have dissected in legumes, most likely are used in all intracellular symbioses in plants.

What advice do you have for young scientists aspiring to achieve the level of science that has major impact?

Follow your heart and your passion. You have to love what you are doing. A science career is not always the easy path. One has to deal frequently with rejection, papers or grant applications, and often one needs to work long hours. It is the passion and enthusiasm for one’s subject that sees you through the tough times.

When you were a post-doc, what had the largest influence on your decision to enter your specific research area in your permanent position? Was this a “hot topic” at the time, or did you choose to go in a different direction?

I started working in nodulation because I thought it a fascinating area: a bacterial signal that can promote plant development. But I also recognized that the skills I had learnt as a PhD student in molecular genetics were invaluable for understanding nodulation. At the time I started my postdoc at Stanford University, the model legumes had just been developed, mutant populations existed, and genetic maps were available. It was a field that my skills could be readily applied. I was lucky to join this field at an exciting time as the process of nodulation, symbiosis signaling in particular, was genetically dissected. It was incredibly exciting to be part of the discovery of this signaling pathway.

Barbara Valent, distinguished professor in the Department of Plant Pathology at Kansas State University, has earned membership in the prestigious U.S. National Academy of Sciences. The National Academy of Sciences is considered the country’s leading authority on matters related to science and technology. As a member, Valent joins a group of scholars that is often sought out to provide independent, objective advice to national leaders on problems where scientific insights are critical.

What area(s) of molecular plant–microbe interactions do you feel your research has impacted most?

New understandings of hemibiotrophy: I’m most proud of our work to elucidate the repeated live-cell invasion strategy executed by the blast fungus to cause disease. Except for pioneering Japanese scientists who established rice blast fungal genetics, there were few people working with rice blast when I began. I’m also proud of how many laboratories worldwide are studying the system and how much the rice blast research community has learned about fungal pathogenesis and host specificity over the past 40 years. I can’t claim to have triggered all that, but I’m proud to have played a part.

What advice do you have for young scientists aspiring to achieve the level of science that has major impact?

It’s important to choose a research problem that matters to the world and also excites your passion. It’s critical to stay in touch with the field biology of your organism (pathosystem) in order for your research to be relevant in the real world. Fungi are microbes that have the ability to rapidly adapt to life in laboratories, and it is critical to maintain and study the fungus in its original form out there in nature.

When you were a post-doc, what had the largest influence on your decision to enter your specific research area in your permanent position? Was this a “hot topic” at the time, or did you choose to go in a different direction? 

As a graduate student studying biochemical Oomycete-plant interactions, I was inspired by a small book entitled “Genetics of host-parasite interactions” written by Peter Day. I applied to post-doc in Gerry Fink’s lab, which had just transformed yeast, adding new power to fungal molecular genetics. In Gerry’s lab, I chose to devote my career to the rice blast fungus based on many features that suggested it could become a good model system: the sexual stage for the fungus had just been described and it was an Ascomycete like yeast and Neurospora; the fungus could be cultured away from its host; field pathologists reported extreme genetic instability in race structure; and the disease has real-world importance on rice, wheat and other cereal crops.

In order to include the viewpoints of our members-in-training in society decision making and congress planning, the IS-MPMI Board of Directors (BOD) has added two junior members. The BOD received applications from several impressive candidates from around the globe and evaluated each based on excellence in scholarship and training, collaboration within the society, leadership at his or her institution, and outreach within his or her community. We are pleased to welcome Patricia Baldrich and Charles Roussin-Léveilléeto the board! Read further for more about Charles and Patricia.

Patricia Baldrich

Dear IS-MPMI members,

I am a postdoctoral researcher in the lab of Blake Meyers, at the Donald Danforth Plant Science Center (Saint Louis, Missouri, United States). As a scientist who has worked in three different continents, I have witnessed the ability that diverse perspectives have in creating great interdisciplinary and international collaborations, which I believe are the driving forces that move science forward. My passion for plant-microbe interactions and learning how organisms from different kingdoms communicate has guided my career journey. The focus of my research is on small RNAs and their use as communication tools in the interaction of plants and their associated microbes.

I believe that communication is essential for successful interactions, not only for plants and microbes, but also for humans. I would love to serve as a reliable bridge between junior members and the BOD. My goal is to establish effective and sustainable avenues to bring the voice of the junior members to the society. Together, with your input and support, we will introduce innovative and beneficial ideas into our congress, meetings, and journals. I am here to listen and understand.

Charles Roussin-Léveillée 

Bonjour/Hi!

My name is Charles and I am thrilled to join the IS-MPMI BOD as a junior member. I am currently a first year PhD candidate in the research group of Peter Moffett at the Université de Sherbrooke, in Quebec, Canada. I investigate how microbes manipulate their host cells to induce an ideal living space in the apoplast. I discovered the field of plant-microbe interaction as an undergraduate and have travelled around the world to learn more about this topic through amazing internships. Outside the lab, I am a passionate hiker who loves to participate in discussions about anthropology, diversity and inclusion, as well as science in general (anything that can help advance society and address inequalities).

The scientific wealth that has been generated by humankind since our beginning is staggering. To push even further, science will require a richness of diversifying ideas. Therefore, collaboration between every nation and the breakdown of diversity barriers will send scientific knowledge to unseen heights. During my time on the IS-MPMI BOD, my goals will be to address diversity and inclusion issues as well as to propose ideas to increase collaboration between developed and less developed countries to enrich our scientific community. Our society has made big jumps in these topics over the last few years. However, much more has yet to be done and understanding each others’ issues will help build stronger bonds between our current and future members.

As such, in collaboration with other IS-MPMI members, I would like to determine the ‘top 10’ issues impacting our society. By aiming the spotlight at these issues, I believe that we will be able to discuss more in-depth about the solutions that could be undertaken to resolve them. I intend to promote the sharing of scientific knowledge between continents by suggesting the creation of scientific summer schools in molecular plant-microbe interactions. I can’t be more excited to discuss varying topics with our members worldwide.

I look forward to participating in the advancement of the large and vibrant research society that is the IS-MPMI.

Molecular Plant-Microbe Interactions and the Phytobiomes Journal​ are initiating two-year assistant feature editor positions for postdoctoral scientists. We are looking for creative individuals with innovative communication skills and who are eager to volunteer their time to engage with both the scientific and non-scientific community. Assistant feature editors will have the opportunity to be part of a journal team, see the inner workings of journals, and interact with the scientific community for Molecular Plant-Microbe Interactions and the Phytobiomes Journal

These positions will be suitable for highly engaged postdoctoral fellows who would like to gain experience and behind the scenes knowledge of publishing in Molecular-Plant Microbe Interactions and the Phytobiomes Journal, develop their writing and communication skills and have the opportunity to be mentored outside their current lab environment. We anticipate a commitment of approximately 5-7 hours per month, realizing that there will be some variability each month, depending on the projects the assistant features editor has taken on.

A key role of the assistant feature editor will be to amplify the impact of our research publications by writing at multiple levels and targeting different audiences. We envision that assistant feature editors will create their own niche based on their interests and ideas. Some possible areas for engagement are: writing a blog or online research summary, interviews with authors, participation in the Microgreens ​podcast, working with our staff amplification specialist to write news stories for a non-scientific audience, writing Twitter threads or posting on other social media, writing a mini-review or highlight to accompany a paper in an issue, and occasional manuscript review. We imagine that assistant feature editors may expand on our ideas, initiating other activities beyond what we have envisioned.

Assistant feature editors will work closely with the editors-in-chief as well as with other senior editors and members of the journal staff. These interactions as well as interactions with authors and the greater community will provide additional opportunities for networking with scientists beyond the connections formed based on their research.

Applications for assistant feature editor positions will be handled jointly by the editor-in-chiefs for Molecular Plant-Microbe Interactions and the Phytobiomes Journal. Indicate in your cover letter your journal preference or whether you would be interested in working with both journals.  To apply, provide the following to Jeanne Harris and Carolyn Young.

• One-page cover letter outlining your research focus area, expertise and interest in the position. Highlight your experience with social media, writing, and your engagement with aspects of science beyond your own research.
• Current CV
• A first-author paper that you were responsible for writing
• Contact details for two professional referees

We are looking for people who will be creative about ways to engage the scientific and non-scientific communities. Provide us with an example of your writing about a scientific topic of your choice (on MPMI or Phytobiomes topics) for either a lay audience or your scientific peers (i.e. blog post, feature article, etc.) (500 word limit).

Applications will continue to be reviewed until all positions are filled.