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Sep 20
InterConnections: Get to Know Jacob Botkin

A Reference Genome Sequence Resource for the Sugar Beet Root Rot Pathogen Aphanomyces cochlioides

Annie Harvieux, UMN Plant Pathology Communications and Relations Coordinator

Jacob Botkin, graduate research assistant

From his undergraduate plant pathology internship to his work assembling and annotating the Aphanomyces cochlioides genome, Jacob Botkin's plant pathology career thus far has been a testament to versatility and embracing the unknown.

While interning in the University of Minnesota Plant Disease Clinic (PDC) during his bachelor's degree program, Botkin discovered a love for examining plant samples and studying plant–microbe interactions. This great fit led to a subsequent job at the Forest Service research lab in St. Paul, MN, that was doing similar diagnostic work.

Botkin points out that what surprised him most when transitioning from coursework to the PDC was how much is still left to discover about plant health and plant genetics in particular. This theme of discovery held true as he pursued his master's degree in plant pathology at the University of Minnesota under the guidance of Drs. Ashok Chanda and Cory Hirsch. During his master's program Botkin picked up more skills on genome assembly and annotation, optimizing soil DNA isolations and qPCR-based detection of soilborne pathogens.

Minnesota is number one in the nation for sugar beet production, and sugar beet production is consistently challenged by A. cochlioides, especially during wet years. To sequence and annotate the A. cochlioides genome, Botkin unlocked an entirely new skill set through on-the-go learning and collaboration: computation and coding. Despite his lack of experience in this side of the work, Botkin was encouraged not to worry about it and to take on the new challenge. Botkin credits Dr. Hirsch, assistant professor of plant pathology, with giving him regular, detailed, and ongoing lessons in coding skills, as well as Hirsch's plant genomics coursework. Spending summers at the Northwest Research and Outreach Center and pursuing opportunities to present this research to sugar beet stakeholders were also rewarding experiences for Botkin.

When the COVID-19 pandemic began and universities sent staff and students home, Botkin's work continued. With his DNA sequence data in hand, Botkin was able to work from home and do the computational portion of the project utilizing the Minnesota Supercomputing Institute's computing power. Botkin identifies this as the steepest part of the learning curve, particularly installing and configuring new software being used for plant pathogen genome assembly and annotation.

This growth has all paid off by adding versatility and adaptability to Botkin's skills and career options. Beyond going from being a mild technophobe to being his new lab's bioinformatic troubleshooter, Botkin now has a variety of skills that can take him from the computer desk to the lab to the greenhouse in a single project. This ability to work in a variety of environments and to pick up new skills and bring them into any subsequent environment has helped Botkin break the assumption that plant pathology is too niche of a career path and move into embracing the variety of skills, settings, and job options available for him.

Mar 21
InterConnections: Get to Know Gongjun Shi

​A Conserved Hypothetic​al Gene Is Required but Not Sufficient for Ptr ToxC Production in Pyrenophora tritici-repentis​

Gongjun Shi

Current Position: Research Specialist, Department of Plant Pathology, North Dakota State University, ND, USA

Education: Ph.D. degree in genomics and bioinformatics at North Dakota State University, USA; Ph.D. and M.S. degrees in olericulture at Nanjing Agricultural University, China; and B.S. degree, with honors, in olericulture at Shandong Agricultural University, China

Nonscientific Interests: Hiking, running, photography, cooking, and volunteering

Brief Bio: I was born in a small village in Shandong Province, China, and had a dream to be a medical doctor one day. However, I was not accepted into medical school, which led me to pursue degrees in olericulture and then genomics and bioinformatics. Now, I am proud to be a plant pathologist. During my years working with Brassica, I was fascinated with the sophisticated mechanisms by which plants fertilize at the molecular level. How plants recognize self and non-self pollen particularly drew my attention. Joining the Key Lab of Southern Vegetable Crop Genetic Improvement led by Dr. Xilin Hou allowed me to pursue this project. At the same time, how plants distinguish self and non-self molecules captured my eye for understanding how plants can effectively balance energy for both growth and defense processes.

After enrolling in the Department of Plant Pathology at North Dakota State University, I worked in Dr. Justin Faris' lab and focused on the cloning of wheat sensitivity genes interacting with necrotrophic effectors produced by Parastagnospora nodorum. Collaborating with Dr. Tim Friesen's group, we found that necrotrophic specialists could hijack both PAMP-triggered immunity and the effector-triggered immunity pathway to cause disease. As a postdoc, I entered Dr. Daniel Kliebenstein's lab at the University of California, Davis, to work on a necrotrophic generalist, Botrytis cinerea, to understand its virulence across the plant kingdom. Currently, I am working on wheat tan spot disease and bacteria leaf streak research led by Dr. Zhaohui Liu. I continue to leverage my plant breeding background, together with my expertise in plant pathology, to unveil many more exciting stories of phytopathogens and plant immunity.​

Dec 20
InterConnections: Get to Know Hari Karki

Natural Genetic Diversity in the Potato Resistance Gene RB Confers Suppression Avoidance from Phytophthora Effector IPI-O4

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 Avrblb1.

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.

Dec 20
InterConnections: Get to Know Maria Malvino

Influence of Flagellin Polymorphisms, Gene Regulation, and Responsive Memory on the Motility of Xanthomonas Species That Cause Bacterial Spot Disease of Solanaceous Plants

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.​

Dec 20
InterConnections: Get to Know Danielle Stevens

​​A Genetic Toolkit for Investigating Clavibacter Species: Markerless Deletion, Permissive Site Identification, and an Integrative Plasmid

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.

Twitter: @Dani_M_Stevens

Personal website:

Dec 20
InterConnections: Get to Know Yan Xu

A Forward Genetic Screen in Sclerotinia sclerotiorum Revealed the Transcriptional Regulation of Its Sclerotial Melanisation Pathway​​​

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, S. sclerotiorum.

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.​​

Sep 16
InterConnections: Get to Know Valentyna Klymiuk

Tandem Protein Kinases Emerge as New Regulators of Plant Immunity

Name: Valentyna Klymiuk

Current Position: Postdoctoral researcher, Crop Development Centre and Department of Plant Sciences, University of Saskatchewan, Saskatoon, Canada.

Education: M.S. and Ph.D. degrees in hydrobiology at Donetsk National University, Donetsk, Ukraine; Ph.D. degree in plant genomics and host-parasite interactions at the University of Haifa, Haifa, Israel.

Non-scientific Interest: Hiking, playing piano, cross-stitching.

Brief Bio: I obtained my B.S., M.S., and one of two Ph.D. degrees from Donetsk National University, Ukraine. These degrees were in the area of hydrobiology, in which I focused on biodiversity and ecology of microalgae communities of continental salt lakes. Because of my growing interest in genetics and genomics, I decided to continue my studies, and I completed a second Ph.D. degree from the University of Haifa, Israel, where my studies focused on plant genomics and host-parasite interactions. Currently, I am a postdoctoral research fellow studying the genetic basis of disease resistance in wheat and its wild relatives. More specifically, I have studied innate resistance to wheat diseases, with an emphasis on identification, gene cloning, and functional characterization of tandem kinase proteins (TKP). Decades of research on canonical immune receptors, exhibiting nucleotide-binding leucine-rich repeat (NBS-LRR) or receptor-like protein (RLP)/receptor-like kinase (RLK) architectures, have firmed their established role in plant immune response. However, there is a general lack of focus on other receptor types, such as TKPs, and my interest lies in shedding light on the role of this important protein family in plant immune response. Currently, one barley and four wheat TKP genes have been functionally validated, but many more have yet to be discovered because TKPs are widespread and diverse across the plant kingdom. To bring more attention to TKPs and highlight their role in plant immunity, together with other co-authors from this research field, I published a review article in MPMI that provides the first comprehensive summary of information for all functionally validated TKPs. A detailed literature review also allowed us to propose a model of TKP evolution through duplication or fusion event and model of molecular function, in which the pseudokinase domain is suggested to serve as a decoy for pathogen effector, while the kinase domain is essential for downstream signaling. I believe that this work provides a deeper investigation of TKPs and will pave the way for future gene manipulation and synthetic engineering of novel plant resistance genes.

Mar 19
InterConnections: Get to Know Kelley Clark

​​The December 2020 Editor’s Pick for MPMI is “Sec-Delivered Effector 1 (SDE1) of ‘Candidatus Liberibacter asiaticus’ Promotes Citrus Huanglongbing,” in which Kelley Clark and co-authors demonstrate the effect that the SDE1 protein from the citrus greening (huanglongbing) pathogen can have on plants. Their results show that the effector is an important virulence factor that induces premature senescence-like responses in both Arabidopsis and citrus host plants.


Sec-Delivered Effector 1 (SDE1) of ‘Candidatus Liberibacter asiaticus’ Promotes Citrus Huanglongbing

03Clark_Image1.jpgName: Kelley J. Clark

Current position: Postdoctoral researcher, University of Arkansas (located at USDA-ARS Salinas, CA).

Education: Ph.D. degree in microbiology and plant pathology at the University of California, Riverside, and B.S. degree in plant sciences at the University of Arizona.

Non-scientific Interests: Gardening, traveling to national parks, hiking, walking my cat.

Brief Bio: Currently, I am a postdoctoral researcher for the University of Arkansas, but stationed in Salinas, CA, at the USDA-ARS facilities. My research project is on spinach downy mildew, and I am located in the Salinas Valley because it is the “salad bowl of the world,” producing the majority of the leafy greens we consume! The research recently published in MPMI is the final chapter of my Ph.D. thesis from my time at UC Riverside under the supervision of Prof. Wenbo Ma. Our overarching goal was to understand how an effector of Candidatus Liberibacter asiaticus contributes to huanglongbing (HLB) disease progression. More specifically, for this publication we wanted to understand how the effector SDE1 contributes to leaf yellowing in Arabidopsis and how this relates to HLB yellowing symptoms in citrus.

This project challenged me on many levels, both intellectually and emotionally, especially as my passion for research progressed and I grew as a scientist. The HLB-associated pathogen, Ca. L. asiaticus, is obligate, which presents many obstacles, but also opportunities, for novel research. During my Ph.D. studies, I was fortunate to learn several new techniques, have access to state-of-the art technology, and collaborate with distinguished scientists. For this project, we had access to SDE1-transgenic citrus, which would not have been possible without help from our collaborators Prof. Nian Wang and Dr. Zhiqian Pang at the University of Florida. Additionally, we implemented NanoString technology to directly measure the transcript quantity of specific genes in citrus. Although this technology is widely used in medical research, it holds tremendous potential for plant–microbe interaction research, as well as other fields of study.

Giving a presentation on citrus pathogens to visitors at the California Citrus State Historic Park.
In addition to gaining a technical skill set, I grew passionate about citriculture from studying its history in Riverside, CA. When I moved to Riverside to pursue my Ph.D. research, I volunteered at the California Citrus State Historic Park. The park consists of more than 250 acres of citrus groves showcasing more than 80 different citrus varieties and includes a museum highlighting the history of citrus in California. Did you know that Riverside is home to the parent Navel orange tree planted by Eliza Tibbets in 1873? The tree is still alive today, and you can visit it on the corner of Magnolia and Arlington Streets, but due to the threat of HLB, the tree is covered with a mesh tent to keep out the insect vector that transmits Ca. L. asiaticus. Volunteering at the park gave me the opportunity to immerse myself in the rich culture of citrus and see others admire it is as well, which drove my research efforts, since HLB continues to threaten not only the citrus industry, but our connection to its past, present, and future.

I look forward to working on more challenging and insightful projects in the future, incorporating both the knowledge I gained from this research and the inspiration I drew from learning about the agricultural history of a specific crops.


Twitter: @KelleyJ_Clark

Sep 10
InterConnections: Get to Know Member Laura Bacete

The May 2020 Editor’s pick for MPMI is “Arabidopsis Response Regulator 6 (ARR6) Modulates Plant Cell-Wall Composition and Disease Resistance.” The first author is Laura Bacete, a graduate student in the lab of Antonio Molina at the Universidad Politécnica de Madrid. To read more about Laura, you can find her bio here​. Laura is now a postdoc at the Institute for Biology at the Norwegian University of Science and Technology. Antonio recently presented this work in a What’s New in MPMI? Seminar. You can find a recording of his seminar here.

Plant Cell Wall Composition and Disease Resistance: A Journey across Novel Mechanisms of Plant Immunity

Submitted by Laura Bacete and Antonio Molina

Fig​ure 1. Schema of Arabidopsis thaliana cell wall composition and wall roles in disease resistance responses. Primary cell walls are composed of cellulose and different pectins and hemicelluloses. Secondary cell walls are reinforced with lignin. Cell wall function in disease resistance to pathogens as a first passive physical barrier that pathogens need to overcome for infection progression, but also as a constantly monitored dynamic structure whose integrity is perceived by different molecular systems. The plant cell wall is also a source of metabolites and proteins with direct activity against pathogens and of signaling molecules, such as damage-associated molecular patterns (DAMPs), that trigger immunity responses.

Traditionally, the plant cell wall has been considered simply a physical defensive barrier against pathogens. However, this outdated view has evolved to a novel concept that considers the plant cell wall as a dynamic structure regulating different processes of plant immunity and development (Figure 1​) (Bacete et al. 2018). Recently, we have published in Molecular Plant-Microbe Interactions (MPMI) our last findings about the impact of the alteration of the cytokinin-responsive Arabidopsis Response Regulator 6 (ARR6) gene expression on the modulation of plant cell wall composition and disease resistance responses (Bacete et al. 2020). Here, we describe the story of how we reached this fascinating discovery, and how our research group, initially focused on A. thaliana resistance to necrotrophic fungi, started a journey that led us to identify a novel mechanism of plant immunity and to determine the relevance of plant cell wall composition in disease resistance. This journey led us to the conviction that plant cell wall-mediated immunity is a key and dynamic component of plant disease resistance against necrotrophic fungi—our initial pathogens of interest—but also against all the plant pathogens we have studied.

The complexity of the plant immune system

The complexity of the plant immunity system, comprising different mechanisms of resistance, was well known at the beginning of this century. These mechanisms include diverse molecular monitoring systems that perceive stresses-derived signals, as well as microbe-associated molecular patterns (MAMPs) and effectors (avirulent proteins) from pathogens, which trigger specific resistance responses upon perception by specific plant receptors (Jones and Dangl 2006). The evolution of such monitoring systems has enabled plants to fine-tune their defensive responses and to adapt their physiological response to environmental condition changes. Also, it is well known that plant defensive responses are mediated by phytohormones, like salicylic acid (SA), ethylene (ET), and jasmonic acid (JA), which were initially described as mainly required for plant resistance to biotrophic (SA) and nectrotrophic (ET and JA) pathogens, respectively (Robert-Seilaniantz et al. 2011). In recent years, other phytohormones have been added to this list of “defensive hormones.” These include abscisic acid (ABA), brassinosteroids, gibberellins, auxins, and more recently cytokinin, as shown in recent articles and in our MPMI paper (Bacete et al. 2020; Argueso et al. 2012; Gupta et al. 2020).

Two decades ago, the plant cell wall was considered in the plant immunity field to be simply a structure displaying a physical defensive role—a sort of passive barrier with no essential function in a complex plant immune system. Nevertheless, it had been demonstrated by several groups that the plant cell wall is a dynamic and highly regulated structure with several important functions for plant growth and development. All plant cells have a primary plant cell wall that is mainly composed of cellulose—which is the principal load-bearing component—pectins, hemicelluloses, and structural glycoproteins. In addition, cells that have completed their cellular expansion and need to strengthen their structure for functional reasons (e.g., to form vessel or fiber cells) generate a secondary cell wall that also includes lignin. The plant cell wall is a prominent structure to manage mechanical stresses caused by either internal (e.g., due to osmotic pressure) or external (e.g., caused by pathogen attack) physiological/environmental changes. Therefore, an important question arose several years ago: how do plants perceive these changes in their cell walls? In recent years, the status of the plant cell wall has been shown to be constantly monitored through a series of cell wall integrity (CWI) surveillance mechanisms (Bacete and Hamann 2020), and the wall has been found to be a source of damage-associated molecular patterns (DAMPs), mainly of carbohydrate-based compositions, that trigger immune responses (Bacete et al. 2018, 2020).

Exploring Arabidopsis thaliana disease resistance to necrotrophic fungi: The initials

Early in the foundation of our lab at the Technical University of Madrid (UPM, Spain), we performed several screenings of A. thaliana mutant collections and quantitative trait loci (QTL) analyses of ecotypes to identify novel genetic components of plant resistance to necrotrophic fungi. The reason for this initial objective was that the genetic determinants of plant resistance to this type of fungi were understudied, despite the fact that necrotrophic fungi cause important yield loses in agriculture. We selected for these initial screenings several strains from different necrotrophic fungi species, but we particularly focused on one strain that had been serendipitously isolated by Brigitte Mauch-Mani (Neuchatel University, Switzerland) from Arabidopsis plants growing under her lab conditions (Ton and Mauch-Mani 2004). This necrotrophic fungal strain was an ascomycete from Plectosphaerella cucumerina, which was very easy to handle in the lab and, more importantly, gave very reproducible necrotrophic symptoms in different A. thaliana genotypes. We named this isolate PcBMM to recognize the contribution of Brigitte Mauch-Mani to its discovery. PcBMM transformed our scientific goals, changed our view of the genetic determinants of plants resistance to necrotrophic fungi, and revealed an unexpected and relevant contribution of the plant cell wall to immunity. This exciting journey with Plectospherella has recently reached an important milestone with the publication in MPMI of the first sequence and annotation of the genomes and transcriptomes of three Plectospherella strains (including PcBMM) with different lifestyles on A. thaliana genotypes (Muñoz-Barrios et al. 2020).

In our early screenings with PcBMM we identified several A. thaliana cell wall mutants, like ern1/irx1/lew2 (impaired in AtCESA8 required for secondary cell wall cellulose synthesis), displaying broad-spectrum resistance to PcBMM and other necrotrophic and biotrophic pathogens and enhanced resistance to abiotic stresses. This initial finding was shocking, but exciting, since it was not in accordance with the classical view of plant disease resistance to necrotrophic pathogens. Intriguingly, the molecular bases of irx1 resistance did not seem to be dependent on canonical defensive pathways (e.g., the expected ET and JA for necrotrophic fungi), but instead it relied on novel mechanisms of immunity involving ABA signaling and antimicrobial compounds like tryptophan-derived metabolites and peptides (Hernandez-Blanco et al. 2007). Moreover, in additional screening aimed at deciphering PcBMM genetic resistance, we frequently found A. thaliana mutants with enhanced susceptibility to PcBMM and additional pathogens, which showed alterations in their plant cell wall composition. Among these mutants were erecta (er), impaired in a receptor-like protein kinase, and agb1, defective in the beta-subunit of Arabidopsis heterotrimeric G protein, that display different biochemical alterations in their cell wall composition compared with that of wild-type plants (Delgado-Cerezo et al. 2012; Llorente et al., 2005; Sánchez-Rodríguez et al. 2009; Torres et al. 2013). These and additional exciting results suggested that ER and heterotrimeric G proteins play roles in regulating novel mechanisms of disease resistance mediated by the cell wall in addition to their function in plant development (Sánchez-Rodríguez et al. 2009). The function of ER-mediated pathway in immunity was further corroborated by the characterization of the role in plant immunity of YODA, a mitogen-activated protein kinase kinase kinase (MAPK3) functioning downstream of ER in plant development (Bergmann 2004). YODA has been found to regulate broad-spectrum disease resistance through noncanonical defensive mechanisms involving cell wall-mediated resistance and the up-regulation of the expression of specific protein receptors and peptidic DAMPs (Sopeña-Torres et al. 2018; Téllez et al. 2020).

Exploring the contribution of the plant cell wall to A. thaliana immunity: The ARR6 example

Figure 2. Selection of cell wall mutants and biased resistance screening performed. Collection of cell wall mutants tested included well-known mutants and putative cell wall mutants impaired in either genes encoding proteins implied in the biosynthesis or remodeling of plant cell wall or genes expressed during cell wall biogenesis processes (Pesquet et al. 2005). The resistance of these mutants to four different type of pathogens with different colonization styles was determined. A significant number of mutants showed altered susceptibility/resistance to one or more pathogens compared with wild-type plants (additional details provided in Molina et al. 2020).​

The findings described above led us to the conviction that plant cell wall composition and integrity were essential components of A. thaliana immunity. To explore this regulatory effect of the plant cell wall on A. thaliana immunity and resistance to different type of pathogens, we decided to follow a biased mutant screening approach and to perform a detailed analysis of the resistance to different pathogens of a collection of selected Arabidopsis mutants impaired in either the primary or secondary cell wall (Molina et al. 2020). In this biased screening (Figure 2), an astonishingly high number of cell wall mutants showed altered susceptibility/resistance to one or more of the pathogens tested compared with wild-type plants, further supporting the key contribution of the plant cell wall to disease resistance (for further details are provided in Molina et al. 2020).

One of the cell wall mutants with disease resistance alterations was impaired in the ARR6 gene (arr6), and it is characterized in our MPMI paper (Bacete et al. 2020). Our first observations on two mutant alleles (arr6-3 and arr6-2) of ARR6 indicated that they both had alterations in their cell wall composition and in their resistance to different pathogens with different colonization styles. ARR proteins have been described as components of the cytokinin signaling pathway, which has previously been involved in the modulation of some disease-resistance responses (Argueso et al. 2012; Gupta et al. 2020). In our work recently published in MPMI (Bacete et al. 2020), we describe a previously unknown function of ARR6 by showing that ARR6 is actually a regulator of cell wall composition and of disease resistance responses against different pathogens causing important diseases, like the necrotrophic fungus PcBMM and the vascular bacterium Ralstonia solanacearum. arr6 mutants, which do not have functional versions of the ARR6 gene, are more resistant to PcBMM fungus but more rapidly and intensely develop the disease symptoms caused by the vascular bacterium R. solanacearum. In contrast, plants that display higher levels of ARR6 expression (by transgenic overexpression) than wild-type plants or arr6-3 (e.g., overexpressor and complementation lines, respectively) are more resistant to the bacteria but more susceptible to the fungus. Transcriptomic and metabolomic analyses revealed that, in arr6 plants, canonical disease-resistance pathways, like those activated by defensive phytohormones, were not altered, whereas immune responses triggered by microbe-associated molecular patterns were slightly enhanced. As in previous research approaches performed in the lab, our findings of the bases of the resistance were again original and out of the canons, which is something that always triggers researchers’ curiosity, making our work even more intriguing and exciting, but also risky for publication. Moreover, the characterization of ARR6-mediated resistance reinforced our view of plant cell wall relevance in the modulation of specific immune responses and confirmed the opportunities provided by plant cell wall mutants for the identification of novel and uncharacterized mechanisms of plant immunity.

We next hypothesized that some cell wall component could be released from arr6 walls due to their observed alteration in composition and that this compound might function as DAMP that will be recognized by a plant receptor, triggering immunity. However, cell walls are very complex, so we had to obtain simpler cell wall fractions enriched in main biochemical components. Remarkably, pectin-enriched cell wall fractions from arr6 plants activated more intense immune responses than similar wall fractions from wild-type plants, suggesting that the arr6 pectin fraction is enriched in wall-related DAMPs. The next step we performed in this research area was the purification of these putative DAMP molecules from arr6 pectin fractions. Actually, we have recently described the characterization of the immune-active pectin fractions of arr6 by further fractionation of it by chromatographic means (Mélida et al. 2020). These analyses pointed to the role of pentose-based oligosaccharides in triggering plant immune responses in arr6. Specifically, we have identified pentose-based oligosaccharide structures, such as beta-1,4-xylooligosaccharides, with specific degrees of polymerization carrying arabinose decorations. Remarkably, these novel DAMPs, which trigger immune responses in Arabidopsis, also activate immune responses in crops and confer enhanced disease resistance to pathogens, including necrotrophic fungi (Mélida et al. 2020). The characterization of these new cell wall-derived plant DAMPs represents the culmination of a long journey across novel mechanisms of plant immunity in our lab that led us to determine the significant and specific contribution of plant cell wall composition in disease resistance. This has been a journey that we initiated with the necrotrophic fungus PcBMM and that has taken us to the identification of novel, noncanonical, cell wall-mediated mechanisms of immunity of relevance for different sets of pathogens. We sincerely guess that our research can contribute to the development of innovative crop protection technologies to reach the desire goal of more sustainable agriculture that will feed the growing human population.


Sep 10
InterConnections: Get to Know Member Dawei Xin

The June 2020 Editor’s pick for MPMI is “RNA Sequencing-Associated Study Identifies GmDRR1 as Positively Regulating the Establishment of Symbiosis in Soybean” with corresponding authors Dawei Xin and Qingshan Chen from the Northeast Agricultural University in Harbin, China. To read more about Dawei you can find his bio here.​

RNA Sequencing-Associated Study Identifies GmDRR1 as Positively Regulating the Establishment of Symbiosis in Soybean

Submitted by Dawei Xin and Qingshan Chen

Soybean is one of the most important crops in the world, supplying protein and oil to humans and animals. Symbiosis is a special characteristic of legumes that allows them to fix nitrogen from the air. However, chemical nitrogen fertilization is still the main source utilized in legume crops, which causes serious pollution in the environment. Too little is understood about the mechanism of symbiosis, which impedes utilization of symbiosis in agriculture. The benefits of symbiosis encourages us to become more familiar with the molecular mechanism of legume–Rhizobium interaction. The genes of Rhizobium sp. and host both play a pivotal role in symbiosis establishment.

In recent decades, type effector (T3E) was found and identified as playing a pivotal role in nodule formation. To date, there is no gene has been identified in a legume host that directly interacts with T3E. Our lab has been working to identify the genes that might interact with T3E and the soybean response mechanism to Rhizobium spp. Considering the complex genetic background of soybean, we selected a genetic population to identify the genes underlying symbiosis and the response to T3E. Chromosome segment substituted lines (CSSL) with wild soybean genomic sequences are an ideal genetic material to locate quantitative trait loci (QTL) and mining genes in the target chromosome regions.

Identifying special soybean lines

To identify the chromosome region that might underlie symbiosis and the response to T3E during symbiosis establishment, we screened the CSSL population, first to compare the nodule-related phenotype and genotype of CSSL. After inoculation with wild-type Rhizobium sp., two lines of CSSL were identified. One line can form more nodules than the recurrent parent, and other can form fewer nodules than the recurrent parent. This supports the hypothesis that substituted chromosome segments play a role in the identified phenotype. The substituted segments on the chromosome were detected by resequencing the genome of two identified lines of CSSL and the recurrent parent.

Mining the response of candidate genes to Rhizobium sp. and T3E

As there are no single substituted segments on the chromosome, we needed to identify the target region to reduce our workload. To accomplish this, we used CSSL to map the QTL underlying nodule number after inoculation with a wild Rhizobium sp. and derived T3E mutant. At the same time, RNA sequencing was performed to detect the gene expression pattern located in the substituted segment of chromosome. We used a wild-type Rhizobium sp. and T3E mutant strain to inoculate the two identified CSSL lines and the recurrent parent. Many different expression genes were found. To delimitate the region on the chromosome, we used the QTL assistant to find the chromosome region. Because the length of substituted segments can be identified by genomic resequencing and molecular analysis, we can narrow down the chromosome region to a shortened region. This was a great help to us in identifying the candidate for further work. Now, several candidate genes that can interact with T3E have been identified, and we have designed a more detailed experiment to elucidate the interaction mechanism.

We are pleased that our work was accepted for publication by MPMI and that we could share our findings with other researchers who we followed during manuscript preparation.

We duly acknowledge funding from the Nature Science Foundation of China and the graduate students of our lab at Northeast Agricultural University.

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