To identify the RB CC domain that does not interact with IPI-O4, Hari Karki et al. explored natural variations in the RB CC domain from different Solanaceae species. The study provides a clue about engineering new variants of known R genes that can further expand the resistance spectrum.
Also in this issue...
In recently published research, Maria Laura Malvino et al. studied Xanthomonas bacteria and how they have evolved to evade recognition by host plants. They found some Xanthomonas species demonstrated a responsive memory, supporting previous work showing that bacteria deploy different strategies to improve their long-term fitness under fluctuating environmental conditions.
Compared to their gram-negative peers, actinobacterial pathogens are greatly understudied, in part due to the limited number of genetic and biochemical tools. Work recently published in MPMI by Danielle Stevens et al. highlights new genetic tools adapted and developed for Clavibacter with potential application in orphan systems.
This project explored the development and pathogenesis of an understudied phytopathogen, Sclerotinia sclerotiorum. Although forward genetic approaches are often utilized to screen for mutants after random mutagenesis, Yan Xu et al. had to devise their own protocol for successful targeted gene disruption. Overcoming several obstacles, they created a forward genetic pipeline for studying a nonmodel fungal species.
IS-MPMI is looking forward to the new year and four new virtual seminars! This series is freely available to all. Register today!
Abstracts from the July 12–13 and September 15–16 IS-MPMI eSymposia Series are now available in MPMI. This is a great opportunity to learn about the emerging science presented at the 2021 IS-MPMI online meeting.
Associate Professor of Molecular Virology Hernán García-Ruiz, University of Nebraska-Lincoln, discusses how the Top 10 Unanswered Questions in MPMI are used to inspire students in his introductory graduate-level course.
ROOT & SHOOT is seeking organizations, individuals, or sets of individuals (partnerships) to work with them to achieve their goals for inclusion, diversity, equity, and accessibility within their organizations and the community.
Explore the Editor's Picks from the September, October, and November issues of MPMI.
We are always looking for content for Interactions. Please contact Interactions Editor-in-Chief Dennis Halterman with questions or article ideas.
Name: Hari Karki
Current Position: Molecular breeder (tomato) at Lipman Family Farms, Florida, USA
Education: M.S. and Ph.D. degrees in plant health at Louisiana State University, Baton Rouge, LA, USA
Brief Bio: Over the years, I have conducted research in the field of plant pathology, genetics, genomics, and molecular biology at Louisiana State University (LSU), The Sainsbury Laboratory (TSL) and U.S. Department of Agriculture (USDA). I was always attracted to different aspects of agriculture, which eventually led to my enrollment at the agriculture institute of Nepal. After completion of my undergraduate degree, I joined the Department of Plant Pathology and Crop Physiology at LSU to pursue a master's degree, studying the bacterial pathogen
Burkholderia glumae. After completion of a M.S. degree in plant health, I continued studying for a Ph.D. degree and worked on understanding the virulence mechanism and population diversities of
B. glumae through targeted sequencing and mutagenesis of pathogenic and nonpathogenic isolates. At TSL, I worked on a capture-based next-generation sequencing method, resistant gene enrichment and sequencing (RenSeq), and gene enrichment and sequencing (GenSeq) to map and clone resistance genes against late blight of potato caused by
Phytophthora infestans. At the USDA, I worked on the molecular dissection of
RB (also known as
Rpi-blb1) mediated late blight resistance in potato.
RB is a broad-spectrum late blight resistance gene cloned from
Solanum bulbocastanum, which recognizes
P. infestans effector IPI-O (in planta–induced gene O), also known as
IPI-O is a multigene effector family that has been divided into three major classes. IPI-O class I and class II variants detect
RB and initiate resistance activation; however, with class III variants, IPI-O4 not only escapes recognition by
RB but is also capable of inhibiting the hypersensitive response (HR) by directly binding the
RB CC domain. To identify the
RB CC domain that does not interact with IPI-O4, we explored natural variations in the
RB CC domain from different Solanaceae species and identified the
RB CC domain from
S. pinnatisectum (pnt) that does not interact directly with IPI-O4. We identified crucial amino acids in the
RB CC domain that play an important role in the avoidance of suppression activity of IPI-O4 and, thus, could enable resistance activation even in the presence of this suppressor. We further modified these amino acids in a wild-type
RB gene and concluded that modification of single amino acids within the
RB CC domain can either diminish or increase the resistance capability of the
RB gene. Our study provides a clue about engineering new variants of known
R genes that can further expand the resistance spectrum.
Name: Maria Laura Malvino
Current Position: Ph.D. candidate, crop sciences, University of Illinois at Urbana-Champaign, IL, USA
Education: B.S. degree in food science and technology and M.S. degree in biotechnology at the Universidad de Buenos Aires, Argentina; M.S. and Ph.D. degrees in crop sciences at the University of Illinois at Urbana-Champaign, IL, USA
Non-scientific Interest: Ice hockey, running, roller blading
Brief Bio: While I was wrapping up my studies in the food science and technology program, I realized that what I really wanted to do was to improve our food through altering its genetics, as I thought that was the way to make a bigger impact and lead to real change. Therefore, shortly after I finished my bachelor's degree, I pursued an M.S. degree in biotechnology. I also was fortunate enough to work for some years at a seed company performing biotechnology-related research. After a while, out of curiosity, I applied for a Fulbright Scholarship, and I won! This opportunity allowed me to fulfill my dreams of coming to the United States and studying what I love the most—how to improve the genetics of our crop plants. In my recently published research, I navigated the fascinating world of
Xanthomonas bacteria and how they have evolved to evade recognition by their host plants. When I first found polymorphisms in the flagellin proteins that form the bacterial flagellum, I thought there could be a correlation between the different variants and bacterial motility, but this was not the case. Interestingly, I found some
Xanthomonas species demonstrated a responsive memory, which is a phenomenon that has been observed in other bacterial species in response to different stimuli. This evidence supports previous work showing that bacteria deploy different strategies to improve their long-term fitness under constantly fluctuating environmental conditions. In terms of agricultural production, it is important to have a clear understanding of plant pathogens in order to defeat them.
Name: Danielle Stevens
Current Position: Integrative genetics and genomics Ph.D. candidate and USDA NIFA predoctoral fellow, University of California Davis, California, USA
Education: B.S. degree in biochemistry and biophysics at Oregon State University, OR, USA
Non-scientific Interest: Traveling, tech, hiking
Brief Bio: Often we hear of scientists who were driven by their passions as children. Growing up, I enjoyed science but nothing in particular perked my interests. At Oregon State University, I worked toward a B.S. degree in biochemistry and biophysics thinking I would work in the medical field to make a difference in people's lives. However, an accidental introduction to gram-positive actinobacterial plant pathogens and molecular plant–microbe interactions thanks to
Dr. Jeff Chang changed much of my perspective and goals.
As an undergraduate student in his lab, I investigated the contribution of bacterial virulence loci to disease in plant-associated
Rhodococcus and worked collaboratively in a team that elucidated the misdiagnosis of beneficial
Rhodococcus bacteria as a pathogen in pistachio. During those formative years, I learned and loved what it meant to do science. I also witnessed the economic implications of the misdiagnosis on both pistachio growers and in the loss of time for many research groups as we tried to repeat incorrect findings. Since then, I have been committed to making my research, both data and code, accessible to others.
Now, I am fortunate to continue studying actinobacterial pathogens, focusing on important crop pathogens of the
Clavibacter genus under the mentorship of
Dr. Gitta Coaker. Using large-scale genomics and functional biology, I am investigating effector-driven host range, which has been a question in
Clavibacter biology for over a decade. Additionally, I am investigating how these bacteria interact with the plant immune system, an area which has been relatively unexplored in the context of pattern-triggered immunity.
Compared to their gram-negative peers, actinobacterial pathogens are greatly understudied, in part, due to the limited number of genetic and biochemical tools. Thus, I wanted to first generate a genetic toolkit for
Clavibacter during my Ph.D. studies, which could expand the type of questions that could be investigated. The work I published in
MPMI highlights new genetic tools we have adapted and developed for
Clavibacter with potential application in orphan systems. We have a vector designed for markerless deletion and another that can be combined with an R package, permissR, which aids in targeted integrated expression. These vectors build on tools designed for other actinobacterial pathogens, while taking advantage of the growing genomics-focused era of plant–microbe research today.
In the long run, I hope to continue combining computational and functional approaches to unravel how actinobacterial pathogens evolve and adapt to their hosts. In turn, this can help us develop long-term, sustainable solutions to managing actinobacterial pathogens.
Name: Yan Xu
Current Position: Botany, University of British Columbia, Vancouver, BC, Canada
Brief Bio: I am excited to have our paper, "A Forward Genetic Screen in
Sclerotinia sclerotiorum Revealed the Transcriptional Regulation of Its Sclerotial Melanisation Pathway," published in
MPMI. This project was initiated by me four years ago when I became a Ph.D. student in
Dr. Xin Li's lab at the University of British Columbia. The goal of my Ph.D. thesis was to explore the development and pathogenesis of a notorious, but understudied, phytopathogen,
If you visit our lab's website, you will find that we mainly study the molecular mechanisms of plant innate immunity using the model plant
Arabidopsis thaliana. We are basically a plant lab without any other lab members who had previous experience with this pathogen, except for my supervisor, who studied
S. minor during her Ph.D. program. You can image how hard it was and how many setbacks I have encountered during my research.
The first obstacle I had was how to obtain mutants with phenotypes of interest. Forward genetic approaches are often utilized to screen for mutants after random mutagenesis. For most fungal research, asexual conidia are used to conduct genetic screens. However, this fungus does not produce conidia. Meanwhile, the multinucleate feature of its asexual tissues rendered the problem worse. After several failed attempts with mutagenizing sclerotia, we ended up using sexual, haploid ascospores, which turned out to be ideal for mutagenesis. The next question was selection of a suitable mutagen. We first tried EMS, which is broadly applied in
Arabidopsis studies. However, this chemical was problematic, because it killed all ascospores after mutagenesis and washes. Finally, we settled on a relatively mild mutagen, UV irradiation, and were able to acquire many mutants with the desired phenotypic defects.
Next, we sequenced many of our mutants using next generation sequencing (NGS) since the expense of NGS has decreased drastically over the past few years. After analyzing the NGS data, I was able to find several candidates for each of my mutants. The biggest problem I had at the time was to knock out the candidate genes to determine which in the mutation is responsible for my mutant phenotype. Targeted gene replacement by homologous recombination has been applied in many fungi with relatively high efficiency. However, this method did not help me obtain any knockout mutants during six months of attempts. After modifying the protocol many times using modifications from relevant literature, we ultimately set up our own protocol for successful targeted gene disruption.
Looking back, with every step I moved forward, I encountered unpredicted difficulties. Although sometimes frustrating, I really enjoyed identifying the problems and solving them. I hope that the forward genetic pipeline mentioned in this paper can be applied to facilitate in-depth studies of other nonmodel fungal species in the future.
Associate professor of molecular virology at the
University of Nebraska-Lincoln
Hernán García-Ruiz used the
Top 10 Unanswered Questions in MPMI to inspire the students in his introductory graduate-level course. Learn more about his process in the interview below.
1) How did you incorporate the Top 10 Unanswered Questions into your class and teachings?
At the University of Nebraska-Lincoln, PLPT 801"Biology of Plant Pathogens" is our introductory graduate-level course. Under the leadership of myself and with collaboration from
Peter Mullin, and
Tom Powers, we use an inquiry-based learning approach, and we incorporated the
MPMI journal's "Top 10 Unanswered Questions in MPMI." At the start of the semester, students used
this review to learn plant pathology terminology, experimental techniques and approaches, and current research topics in plant-microbe interactions.
During the second week of the semester, students read a cutting-edge research paper or review that addressed one of the "Top 10 Unanswered Questions in MPMI." This activity allowed students to establish a connection between a knowledge gap and a publication that discussed that knowledge gap. Additionally, students used their selected paper as a model to structure and format a term paper on a current topic of interest in plant pathology.
Students chose a current topic in plant pathology and prepared literature review manuscripts that provided novel insights. The standard of quality is that these reviews must be suitable for publication in
At the end of the course, students are evaluated based on several criteria:
- Critical assessment of the research paper or literature review they read in week
- A literature review manuscript formatted for
- The Plant Pathology Symposium that is open to the Department of Plant Pathology and the entire university, as well as the general public. This semester it was held on December 16, 2021 at 8:05 AM.
2) What made the Top 10 Unanswered Questions a helpful teaching tool?
In our course, we want our students to understand molecular plant pathology from the basics all the way to the cutting edge.
MPMI's "Top 10 Unanswered Questions in MPMI" wholly summarizes the cutting edge as defined by society members at the 2019 IS-MPMI Congress. It also provided an outstanding framework for students to learn the current research topics of interest worldwide.
3) Anything else you’d like to share about this experience?
Our inquiry-based learning approach is an effective and innovative method of instruction. Molecular plant pathology is an expansive field that covers many researchers and topics and defies the constraints of traditional lecture-based instruction. By focusing on the unanswered questions of plant pathology, we can go beyond simple memorization of known facts and direct the interest of beginning graduate students to the areas where they can contribute the most. Interestingly, by learning about the "Top 10 Unanswered Questions in MPMI
," students naturally scrutinize their own thesis project and have made significant adjustments.
Dr. García-Ruiz would also like to recognize Samuel Eastman, a 4th-year graduate student who has helped with his class two years in a row, first as a TA then as a co-instructor. Samuel participated in the IS-MPMI Congress and enthusiastically embraced using the Top 10 Unanswered Questions as a teaching tool.
A group of nonprofit plant science organizations and societies have assembled to Root Out Oppression Together and Share Our Outcomes Transparently (ROOT & SHOOT), through a Research Coordination Network Grant funded by the National Science Foundation. Our overarching goal is to transform our discipline by getting to the root of the systemic oppressions that exist within our organizations (and ourselves) so we can regrow as inclusive, liberated, spaces.
We are seeking an organization, individual, or set of individuals (partnerships) who will work with us to achieve our goal. We need help to cocreate equitable and inclusive practices for our community; provide sustainable, inclusive, equitable, anti-racist/anti-oppression leadership training; guide power and equity assessments within our participating organizations; and consult for intervention programs. More detail on these projects can be found
We will start reviewing applications on January 14, 2022. We aim to start working with successful applicant(s) in April 2022.