The oomycete pathogen Phytophthora infestans causes potato late blight, and as a potato and tomato specialist pathogen, is seemingly poorly adapted to infect plants outside the Solanaceae. Here, we report the unexpected finding that P. infestans can infect Arabidopsis thaliana when another oomycete pathogen, Albugo laibachii, has colonized the host plant. The behaviour and speed of P. infestans infection in Arabidopsis pre-infected with A. laibachii resemble P. infestans infection of susceptible potato plants. Transcriptional profiling of P. infestans genes during infection revealed a significant overlap in the sets of secreted-protein genes that are induced in P. infestans upon colonization of potato and susceptible Arabidopsis, suggesting major similarities in P. infestans gene expression dynamics on the two plant species. Furthermore, we found haustoria of A. laibachii and P. infestans within the same Arabidopsis cells. This Arabidopsis - A. laibachii - P. infestans tripartite interaction opens up various possibilities to dissect the molecular mechanisms of P. infestans infection and the processes occurring in co-infected Arabidopsis cells.
In February 2016, a new fungal disease was spotted in wheat fields across eight districts in Bangladesh. The epidemic spread to an estimated 15,741 hectares, about 16% of cultivated wheat area in Bangladesh, with yield losses reaching up to 100%. Within weeks of the onset of the epidemic, we performed transcriptome sequencing of symptomatic leaf samples collected directly from Bangladeshi fields. Population genomics analyses revealed that the outbreak was caused by a wheat-infecting South American lineage of the blast fungus Magnaporthe oryzae. We show that genomic surveillance can be rapidly applied to monitor plant disease outbreaks and provide valuable information regarding the identity and origin of the infectious agent.
This week on the blog, Professor Sophien Kamoun describes his work on plant–pathogen interactions at The Sainsbury Lab, UK, and discusses the future of plant disease.
Could you begin by describing the focus of your research on plant pathogens?
We study several aspects of plant–pathogen interactions, ranging from genome-level analyses to mechanistic investigations focused on individual proteins. Our projects are driven by some of the major questions in the field: how do plant pathogens evolve? How do they adapt and specialize to their hosts? How do plant pathogen effectors co-opt host processes?
One personal aim is to narrow the gap between research on the mechanisms and evolution of these processes. We hope to demonstrate how mechanistic research benefits from a robust phylogenetic framework to test specific hypotheses about how evolution has shaped molecular mechanisms of pathogenicity and immunity.
Tree diseases such as sudden oak death, ash dieback and olive quick decline syndrome have been making the news a lot recently. Are diseases like these becoming more common, and if so, why?
It’s well documented that the scale and frequency of emerging plant diseases has increased. There are many factors to blame. Increased global trade is one. Climate change is another. There is no question that we need to increase our surveillance and diagnostics efforts. We’re nowhere near having coordinated responses to new disease outbreaks in plant pathology, especially when it comes to deploying the latest genomics methods. We really need to remedy this.
The wheat blast fungus recently hit Bangladesh. Could you briefly outline how it is being tackled by plant pathologists?
Wheat blast has just emerged this last February in Bangladesh – its first report in Asia. It could spread to neighboring countries and become a major threat to wheat production in South Asia. Thus, we had to act fast. We used an Open Science approach to mobilize collaborators in Bangladesh and the wider blast fungus community, and managed to identify the pathogen strain in just a few weeks. It turned out that the Bangladeshi outbreak was caused by a clone related to the South American lineage of the pathogen. Now that we know the enemy, we can proceed to put in place an informed response plan. It’s challenging but at least we know the nature of the pathogen – a first step in any response plan to a disease outbreak.
Which emerging diseases do you foresee having a large impact on food security in the future?
Obviously, any disease outbreak in the major food crops would be of immediate concern, but we shouldn’t neglect the smaller crops, which are so critical to agriculture in the developing world. This is one of the challenges of plant pathology: how to handle the numerous plants and their many pathogens.
As far as new problems, I view insect pests as being a particular challenge. Our basic understanding of insect–plant interactions is not as well developed as it is for microbial pathogens, and research has somewhat neglected the impact of plant immunity. The range of many insect pests is expanding because of climate change, and we are moving to ban many of the widely used insecticides. This is an area of research I would recommend for an early career scientist.
What advice would you give to a young researcher in this area?
Ask the right questions and look beyond the current trends. Think big. Be ambitious. Don’t shy away from embracing the latest technologies and methods. It’s important to work on real world systems. Thanks to technological advances, genomics, genome editing etc., the advantages of working on model systems are not as obvious as they were in the past.
How can we mitigate the risks to crops from plant diseases in the future?
My general take is to be suspicious of silver bullets. I like to say “Don’t bet against the pathogen”. I believe that for truly sustainable solutions, we need to continuously alter the control methods, for example by regularly releasing new resistant crop varieties. Only then we can keep up with rapidly evolving pathogens. One analogy would be the flu jab, which has a different formulation every year depending on the make-up of the flu virus population.
Is there anything else you’d like to add?
I read that public and private funding of plant science is less than one tenth of biomedical research. Not a great state of affairs when one considers that we will add another two billion people to the planet in the next 30 years. As one of my colleagues once said: “medicine might save you one day; but plants keep you alive everyday”.
The plant pathogenic fungus Fusarium oxysporum secretes an effector that is similar to a plant peptide hormone, underscoring the variety of mechanisms that plant pathogens have evolved to tamper with host physiology.
Plant pathogens cause devastating diseases of crop plants and threaten food security in an era of continuous population growth. Annual losses due to fungal and oomycete diseases amount to enough food calories to feed at least half a billion people. Understanding how plant pathogens infect and colonize plants should help to develop disease-resistant crops. It appears that plant pathogens are sophisticated manipulators of their hosts. They secrete effector molecules that alter host biological processes in a variety of ways, generally promoting the pathogen lifestyle. A new study by Masachis, Segorbe and colleagues describes a new mechanism by which plant pathogens interfere with plant physiology. They discovered that the root-infecting fungus F. oxysporum secretes a peptide similar to the plant regulatory peptide RALF (rapid alkalinization factor) to induce host tissue alkalinization and enhance plant colonization. This study demonstrates that in addition to secreting classical plant hormones (or mimics thereof), fungi have also evolved functional homologues of plant peptides to alter host cellular processes.
A team of scientists in the UK and Bangladesh are turning to the combined knowledge of the global scientific community to address an emerging threat to Asian agriculture.
The target is the fearsome fungal disease wheat blast. The pathogen was spotted in Bangladesh in February this year—its first report in Asia. Wheat is the second major food source in Bangladesh, after rice. The blast disease has, so far, caused up to 90% yield losses in more than 15,000 hectares. Scientists fear that the pathogen could spread further to other wheat growing areas in South Asia.
The UK and Bangladeshi teams are making raw genetic data for the wheat blast pathogen available on a new website—http://www.wheatblast.net—and inviting others to do the same. Professor Sophien Kamoun, of The Sainsbury Laboratory in Norwich, who is leading the project, said that a wide cultural change is needed for scientists to optimally address new threats to food security.
"I have a beef with the way that research is typically done. We need a fundamentally new approach to sharing genetic data for emerging plant diseases," he said. "We need to generate and make data public more rapidly and seek input from a larger crowd because, collectively, we are better able to answer questions."
Professor Kamoun, with colleagues at The Genome Analysis Centre and John Innes Centre in Norwich, and with Professor Tofazzal Islam's Team of Bangabndhu Sheikh Mujubur Rahman Agricultural University (BSMRAU) in Bangladesh, is hoping that the wheatblast.net website, together with an accompanying Facebook page, will provide a hub for information, collaboration and comment. They are basing the site on their successful Open Ash Dieback website, which brought scientists together in the fight against ash dieback disease.
The blast fungus normally infects rice and over 50 types of grasses. Occasionally, a blast fungus strain would jump from one host to another resulting in a new disease. Such a "host jump" to wheat has happened in Brazil in the 1980s. The wheat blast pathogen is now rife in South America, where it infects up to 3 million hectares and causes serious crop losses.
Prof Kamoun and colleagues are working with Professor Tofazzal Islam's team, of the Department of Biotechnology of BSMRAU in Gazipur, Bangladesh. They hope that the genetic data will help determine whether the Bangladeshi wheat-infecting strain has evolved independently from local grass-infecting fungi or was somehow introduced into the country.
Professor Tofazzal Islam said "This pathogen causes a destructive disease on rice and it would be disastrous if the same situation arises now in wheat. Genomic and post-genomic research should clarify the origin of the wheat strain and guide measures for disease management. Prompt responses are needed from the scientific community and the government of Bangladesh for addressing this national crisis to ensure increasing wheat production, which is linked with future food and nutritional security of the nation."
The group of scientists includes Dr Diane Saunders at The Genome Analysis Centre and John Innes Centre who developed a technique last year, known as Field Pathogenomics. To date, Field Pathogenomics has been applied to track another fungal crop disease - yellow rust. The method generates highly-specific genetic information directly from diseased wheat samples to determine the identity of the pathogen strain that's associated with an epidemic. Application of this method to wheat blast should unmask the pathogen in Bangladesh and contribute to a response plan.
The recent wheat blast epidemic in Bangladesh has prompted Professor Nick Talbot, University of Exeter, to post on the wheatblast.net website a set of genetic data generated by his group from worldwide populations of the wheat and rice blast fungus. Prof Talbot said "In an emergency like this one, the community must come together to share data and compare notes. Only then, we will determine the true identity of the pathogen and put in place effective measures in a timely fashion."
Professor Neil Hall, Director of The Genome Analysis Centre said: "It is critical in emerging crises like this that scientific data is rapidly generated and made available as soon as possible. Having an open-access site has already galvanized open exchange of information for the ash dieback disease. The scientific community needs to rally behind open science to respond to recurrent threats to global food security."
Rust fungal pathogens of wheat (Triticum spp.) affect crop yields worldwide. The molecular mechanisms underlying the virulence of these pathogens remain elusive, due to the limited availability of suitable molecular genetic research tools. Notably, the inability to perform high-throughput analyses of candidate virulence proteins (also known as effectors) impairs progress. We previously established a pipeline for the fast-forward screens of rust fungal candidate effectors in the model plant Nicotiana benthamiana. This pipeline involves selecting candidate effectors in silico and performing cell biology and protein-protein interaction assays in planta to gain insight into the putative functions of candidate effectors. In this study, we used this pipeline to identify and characterize sixteen candidate effectors from the wheat yellow rust fungal pathogen Puccinia striiformis f sp tritici. Nine candidate effectors targeted a specific plant subcellular compartment or protein complex, providing valuable information on their putative functions in plant cells. One candidate effector, PST02549, accumulated in processing bodies (P-bodies), protein complexes involved in mRNA decapping, degradation, and storage. PST02549 also associates with the P-body-resident ENHANCER OF mRNA DECAPPING PROTEIN 4 (EDC4) from N. benthamiana and wheat. We propose that P-bodies are a novel plant cell compartment targeted by pathogen effectors.
Plants use autophagy to safeguard against infectious diseases. However, how plant pathogens interfere with autophagy related processes is unknown. Here we show that PexRD54, an effector from the Irish potato famine pathogen Phytophthora infestans, binds host autophagy protein ATG8CL to stimulate autophagosome formation. PexRD54 depletes the autophagy cargo receptor Joka2 out of ATG8CL complexes and interferes with Joka2's positive effect on pathogen defense. Thus a plant pathogen effector has evolved to antagonize a host autophagy cargo receptor in order to counteract host defenses.
Convergence towards a similar genome architecture in phylogenetically unrelated plant pathogens. The fl anking distance between neighboring genes provides a measurement of local gene density and is displayed as a color-coded heat map based on a whole genome analysis of the fungus Leptosphaeria maculans and the oomycete Phytophthora infestans. In addition, the figure displays the distribution of Avr effector genes of L. maculans and P. infestans according to the length of their 50 and 30 flanking intergenic regions. Note how in both cases the Avr effector genes primarily occupy the gene sparse regions of the genome. (See the paper of Suomeng Dong, Sylvain Raffaele and Sophien Kamoun.)
Secreted peroxidases are well-known components of damage-induced defense responses in plants. A recent study in Nature ( Turrà et al., 2015) has revealed that these enzymes can inadvertently serve as reporters of wounded sites and constitute an “Achilles heel,” allowing adapted pathogens to track and enter host tissue.
Rust fungal pathogens of wheat (Triticum spp.) affect crop yields worldwide. The molecular mechanisms underlying the virulence of these pathogens remain elusive, due to the limited availability of suitable molecular genetic research tools. Notably, the inability to perform high-throughput analyses of candidate virulence proteins (also known as effectors) impairs progress. We previously established a pipeline for the fast-forward screens of rust fungal effectors in the model plant Nicotiana benthamiana. This pipeline involves selecting candidate effectors in silico and performing cell biology and protein-protein interaction assays in planta to gain insight into the putative functions of candidate effectors. In this study, we used this pipeline to identify and characterize sixteen candidate effectors from the wheat yellow rust fungal pathogen Puccinia striiformis f sp tritici. Nine candidate effectors targeted a specific plant subcellular compartment or protein complex, providing valuable information on their putative functions in plant cells. One candidate effector, PST02549, accumulated in processing bodies (P-bodies), protein complexes involved in mRNA decapping, degradation, and storage. PST02549 also associates with the P-body-resident ENHANCER OF mRNA DECAPPING PROTEIN 4 (EDC4) from N. benthamiana and wheat. Our work identifies P-bodies as a novel plant cell compartment targeted by pathogen effectors.
Background. Downy mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily.
Results. Here a high-quality draft genome of Plasmopara halstedii is reported and analysed with respect to various aspects, including genome organisation, secondary metabolism, effector proteins and comparative genomics with other sequenced oomycetes. Interestingly, the present analyses revealed further variation of the RxLR motif, suggesting an important role of the conservation of the dEER-motif. Orthology analyses revealed the conservation of 28 RxLR-like core effectors among Phytophthora species. Only six putative RxLR-like effectors were shared by the two sequenced downy mildews, highlighting the fast and largely independent evolution of two of the three major downy mildew lineages. This is seemingly supported by phylogenomic results, in which downy mildews did not appear to be monophyletic.
Conclusions. The genome resource will be useful for developing markers for monitoring the pathogen population and might provide the basis for new approaches to fight Phytophthora and downy mildew pathogens by targeting core pathogenicity effectors.
Location: Norwich, United Kingdom Current job/title: Senior Group Leader One word that describes how you work: Starbucks Favorite thing you do at work: Tweet Favorite plant: “benth” aka Nicotiana benthamiana One interesting project you have been working on: Open wheat blast http://wheatblast.net
I studied genetics at Pierre and Marie Curie University in Paris and UC Davis, before working at Wageningen University, The Ohio State University, and The Sainsbury Laboratory (TSL) where I served as Head from 2009-2014. At TSL, my group studies several aspects of plant-pathogen interactions, ranging from genome-level analyses to mechanistic research focused on individual proteins. Our projects are driven by some of the major questions in the field: How do plant pathogens evolve? How do they adapt and specialize on their hosts? How do plant pathogen effectors co-opt host processes? One aim is to narrow the gap between mechanistic and evolutionary research by testing specific hypotheses about how evolution has shaped molecular mechanisms of pathogenicity and immunity.
*What is your workspace setup like?
A standing desk surrounded by piles of papers and stuff, or a sofa in some coffee shop.
*What are some tools, apps, or websites that you use or visit every day? Do you have a favorite resource?
Twitter has become the primary source for all knowledge.
If a magical scientific genie appeared from an erlenmeyer flask in your lab, what would you ask for?
3D structures of protein complexes. Loads of them!
*What have been the biggest productivity tools you’ve been using either for a long time or recently adopted?
Typing or dictating notes on my phone and being able to do so at any time.
*What’s some of the best advice you’ve ever received?
Never worry about things you cannot control.
*What’s the best thing you’ve ever learned?
Doing some basic bioinformatics on my MacBook.
*Music, silence, white noise - what works for you?
Music! My zen playlist.
What do you do when the pipette is down and the computer is powered off?
Travel, food, and sports.
*What do you spend time thinking about that’s not your next proposal, publication, or project deadline?
I’m really excited about the travel plans for this summer.
Plant biology has long been a field of pioneering discoveries with broad impacts. What’s the next pioneering discovery in plant biology?
Synthetic immune receptors.
*If you’re OK sharing, what’s one way readers can get in touch or follow along with your work (email, blog, twitter, etc.)?
A recent study by Kroj et al. (New Phytologist, 2016) surveyed nucleotide binding-leucine rich repeat (NLR) proteins from plant genomes for the presence of extraneous integrated domains that may serve as decoys or sensors for pathogen effectors. They reported that a FAM75 domain of unknown function occurs near the C-terminus of the potato late blight NLR protein R3a. Here, we investigated in detail the domain architecture of the R3a protein, its potato paralog R3b, and their tomato ortholog I2. We conclude that the R3a, R3b, and I2 proteins do not carry additional domains besides the classic NLR modules, and that the FAM75 domain match is likely a false positive among computationally predicted NLR-integrated domains.
Background. Magnaporthe oryzae (anamorph Pyricularia oryzae) is the causal agent of blast disease of Poaceae crops and their wild relatives. To understand the genetic mechanisms that drive host specialization of M. oryzae, we carried out whole genome resequencing of four M. oryzae isolates from rice (Oryza sativa), one from foxtail millet (Setaria italica), three from wild foxtail millet S. viridis, and one isolate each from finger millet (Eleusine coracana), wheat (Triticum aestivum) and oat (Avena sativa), in addition to an isolate of a sister species M. grisea, that infects the wild grass Digitaria sanguinalis.
Results. Whole genome sequence comparison confirmed that M. oryzae Oryza and Setaria isolates form a monophyletic and close to another monophyletic group consisting of isolates from Triticum and Avena. This supports previous phylogenetic analysis based on a small number of genes and molecular markers. When comparing the host specific subgroups, 1.2–3.5 % of genes showed presence/absence polymorphisms and 0–6.5 % showed an excess of non-synonymous substitutions. Most of these genes encoded proteins whose functional domains are present in multiple copies in each genome. Therefore, the deleterious effects of these mutations could potentially be compensated by functional redundancy. Unlike the accumulation of nonsynonymous nucleotide substitutions, gene loss appeared to be independent of divergence time. Interestingly, the loss and gain of genes in pathogens from the Oryza and Setaria infecting lineages occurred more frequently when compared to those infecting Triticum and Avena even though the genetic distance between Oryza and Setaria lineages was smaller than that between Triticum and Avena lineages. In addition, genes showing gain/loss and nucleotide polymorphisms are linked to transposable elements highlighting the relationship between genome position and gene evolution in this pathogen species.
Conclusion. Our comparative genomics analyses of host-specific M. oryzae isolates revealed gain and loss of genes as a major evolutionary mechanism driving specialization to Oryza and Setaria. Transposable elements appear to facilitate gene evolution possibly by enhancing chromosomal rearrangements and other forms of genetic variation.
Each year, staple crops around the world suffer massive losses in yield owing to the destruc- tive effects of pathogens. Improving the disease resistance of crops by boosting their immunity has been a key objective of agricultural bio- tech ever since the discovery of plant immune receptors in the 1990s. Nucleotide-binding leucine-rich repeat (NLR) proteins, a family of intracellular immune receptors that recog- nize pathogen molecules, are promising targets for enhancing pathogen resistance. In a recent paper in Science, Kim et al.1 describe a clever twist on this approach in which the host target protein for the pathogen effector is engineered rather than the NLR protein itself (Fig. 1).
The cell re-entry assay is widely used to evaluate pathogen effector protein uptake into plant cells. The assay is based on the premise that effector proteins secreted out of a leaf cell would translocate back into the cytosol of the same cell via a yet unknown host-derived uptake mechanism. Here, we critically assess this assay by expressing domains of the effector proteins AvrM-A of Melampsora lini and AVR3a of Phytophthora infestans fused to a signal peptide and fluorescent proteins in Nicotiana benthamiana. We found that the secreted fusion proteins do not re-enter plant cells from the apoplast and that the assay is prone to false-positives. We therefore emit a cautionary note on the use of the cell re-entry assay for protein trafficking studies.
Plant cells secrete a wide range of proteins in extracellular spaces in response to pathogen attack. The poplar Rust-Induced Secreted Protein (RISP) is a small cationic protein of unknown function that was identified as the most induced gene in poplar leaves during immune responses to the leaf rust pathogen Melampsora larici-populina, an obligate biotrophic parasite. Here, we combined in planta and in vitro molecular biology approaches to tackle the function of RISP. Using a RISP-mCherry fusion transiently expressed in Nicotiana benthamiana leaves, we demonstrated that RISP is secreted into the apoplast. A recombinant RISP specifically binds to M. larici-populina urediniospores and inhibits their germination. It also arrests the growth of the fungus in vitro and on poplar leaves. Interestingly, RISP also triggers poplar cell culture alkalinisation and is cleaved at the C-terminus by a plant-encoded mechanism. Altogether our results indicate that RISP is an antifungal protein that has the ability to trigger cellular responses.
The oomycete pathogen Phytophthora infestans causes potato late blight, and as a potato and tomato specialist pathogen, is seemingly poorly adapted to infect plants outside the Solanaceae. Here, we report the unexpected finding that P. infestans can infect Arabidopsis thaliana when another oomycete pathogen, Albugo laibachii, has colonized the host plant. The behaviour and speed of P. infestans infection in Arabidopsis pre-infected with A. laibachii resemble P. infestans infection of susceptible potato plants. Transcriptional profiling of P. infestans genes during infection revealed a significant overlap in the sets of secreted-protein genes that are induced in P. infestans upon colonisation of potato and susceptible Arabidopsis, suggesting major similarities in P. infestans gene expression dynamics on the two plant species. Furthermore, we found haustoria of A. laibachii and P. infestanswithin in the same Arabidopsis cells. This Arabidopsis - A. laibachii - P. infestanstripartite interaction opens up various possibilities to dissect the molecular mechanisms of P. infestans infection and the processes occurring in co-infected Arabidopsis cells.
The 28th Fungal Genetics Conference was held March 17−22, 2015, at the Asilomar Conference Center in Pacific Grove, California (http://www.genetics-gsa.org/fungal/2015/index.shtml). Arguably the most popular of international fungal genetics conferences, the Asilomar meeting reached its registration cap 2 days before the early bird deadline, with 910 participants from 35 countries.
One striking feature of this year’s meeting was the high level of Twitter participation. On the basis of analytics from the health care social media analytics company Symplur (http://www.symplur.com/healthcare-hashtags) the hashtag #Fungal15 racked up 3456 tweets from 349 participants, and tweets were seen by more than 3 million others.
As is traditional, the meeting co-organizers have been asked to summarize highlights of the conference. As is also traditional, such a summary is a nearly impossible task with 20 plenary talks, 216 concurrent talks, and 662 posters to be considered. In recognition of the high level of social media participation and to give greater coverage, scientific co-chairs Michelle Momany (University of Georgia) and Antonio Di Pietro (University of Cordoba, Spain) invited the top tweeters to join us in picking highlights of the 28th Fungal Genetics Conference. Even so, these highlights were not able to cover all the terrific science at the meeting.
Plants defend against pathogens using both cell surface and intracellular immune receptors (Dodds & Rathjen, 2010; Win et al., 2012). Plant cell surface receptors include receptor-like kinases (RLKs) and receptor-like proteins (RLPs), which respond to pathogen-derived apoplastic molecules (Boller & Felix, 2009; Thomma et al., 2011). By contrast, plant intracellular immune receptors are typically nucleotide-binding leucine-rich repeat (NB-LRR or NLR) proteins, which respond to translocated effectors from a diversity of pathogens (Eitas & Dangl, 2010; Bonardi et al., 2012). These receptors engage in microbial perception either by directly binding pathogen molecules or indirectly by sensing pathogen-induced perturbations (Win et al., 2012). However, signaling events downstream of pathogen recognition remain poorly understood.
In addition to their role in microbial recognition, some NLR proteins contribute to signal transduction and/or amplification (Gabriels et al., 2007; Bonardi et al., 2011; Cesari et al., 2014). An emerging model is that NLR proteins often function in pairs, with ‘helper’ proteins required for the activity of ‘sensors’ that mediate pathogen recognition (Bonardi et al., 2011, 2012). Among previously reported NLR helpers, NRC1 (NB-LRR protein required for hypersensitive-response (HR)-associated cell death 1) stands out for having been reported as a signaling hub required for the cell death mediated by both cell surface immune receptors such as Cf-4, Cf-9, Ve1 and LeEix2, as well as intracellular immune receptors, namely Pto, Rx and Mi-1.2 (Gabriels et al., 2006, 2007; Sueldo, 2014; Sueldo et al., 2015). However, these studies did not take into account the Nicotiana benthamiana genome sequence, and it remains questionable whether NRC1 is indeed required for the reported phenotypes.
Functional analyses of NRC1 were performed using virus-induced gene silencing (VIGS) (Gabriels et al., 2007), a method that is popular for genetic analyses in several plant systems, particularly the model solanaceous plant N. benthamiana (Burch-Smith et al., 2004). However, interpretation of VIGS can be problematic as the experiment can result in off-target silencing (Senthil-Kumar & Mysore, 2011). In addition, heterologous gene fragments from other species (e.g. tomato) have been frequently used to silence homologs in N. benthamiana, particularly in studies that predate the sequencing of the N. benthamiana genome (Burton et al., 2000; Liu et al., 2002b; Lee et al., 2003; Gabriels et al., 2006, 2007; Senthil-Kumar et al., 2007; Oh et al., 2010). In the NRC1 study, a fragment of a tomato gene corresponding to the LRR domain was used for silencing in N. benthamiana (Gabriels et al., 2007). Given that a draft genome sequence of N. benthamiana has been generated (Bombarely et al., 2012) and silencing prediction tools have become available (Fernandez-Pozo et al., 2015), we can now design better VIGS experiments and revisit previously published studies.
Two questions arise about the NRC1 study. First, is there a NRC1 ortholog in N. benthamiana? Second, are the reported phenotypes caused by silencing of NRC1 in N. benthamiana? In this study, we investigated NRC1-like genes in solanaceous plants using a combination of genome annotation, phylogenetics, gene silencing and genetic complementation experiments. We discovered three paralogs of NRC1, which we termed NRC2a, NRC2b and NRC3, are required for hypersensitive cell death and resistance mediated by Pto, but are not essential for the cell death triggered by Rx and Mi-1.2. NRC2a/b and NRC3 weakly contribute to the hypersensitive cell death triggered by Cf-4. Our results highlight the importance of applying genetic complementation assays to validate gene function in RNA silencing experiments.
CRISPR/Cas has recently been transferred to plants to make them resistant to geminiviruses, a damaging family of DNA viruses. We discuss the potential and the limitations of this method.
Geminiviridae are a family of DNA viruses that infect a diversity of plants. These insect-transmitted viruses can cause destructive diseases in crop plants and have been described as a curse to food security. Until now, limited progress has been made with developing crop varieties resistant to geminiviruses. In the current issue of Genome Biology, Ali et al. report on a new strategy towards improving plant resistance to geminiviruses using the bacterial CRISPR/Cas system.
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