In this review, various molecular tools used in filamentous fungi are compared and discussed, including methods for genetic transformation (e.g., protoplast transformation, electroporation, and microinjection), the construction of random mutant libraries (e.g., restriction enzyme mediated integration, transposon arrayed gene knockout, and Agrobacterium tumefaciens mediated transformation), and the analysis of gene function (e.g., RNA interference and transcription activator-like effector nucleases). We also focused on practical strategies that could enhance the efficiency of genetic manipulation in filamentous fungi, such as choosing a proper screening system and marker genes, assembling target-cassettes or vectors effectively, and transforming into strains that are deficient in the nonhomologous end joining pathway.
Mitogen-activated protein kinases (MAPKs) target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3 and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses) is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g. WRKY transcription factors and proteins encoded by the genes from the “PEN” pathway required for penetration resistance to filamentous pathogens). Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org).
Powdery mildew fungi (Ascomycota phylum) are obligate biotrophic plant pathogens that can only grow and reproduce on living host cells. They infect a wide range of plants, including many crops and the diseases they cause are common, easily recognizable and widespread. Although functional investigations in these genetically intractable organisms have been hampered by their obligate biotrophic nature, recent advances in genomics and transcriptomics have contributed tremendously to our understanding of powdery mildew biology. Comparative genomics was a powerful tool to pinpoint what distinguishes powdery mildew fungi from other filamentous plant pathogens and helped us to better understand how obligate biotrophy evolved. Comparative genome analyses among isolates in both the wheat and the barley powdery mildew lineages revealed isolate-specific mosaic genome structures of evolutionary young and old haplogroups. In addition to providing hints into the evolutionary origin of powdery mildew fungi, the observed mosaic genome structure also reflects the reproductive mode of these pathogens and explains how the large standing genetic variation is generated in powdery mildew populations. In this chapter, I discuss how the revolution in genomics has contributed and will contribute in the future to better understand the obligate biotrophic lifestyle, the virulence arsenal, the reproductive mode and the evolutionary history of powdery mildew fungi
This review focuses on plant peptides involved in defense against pathogen infection and those involved in the regulation of growth and development. Defense peptides, defensins, cyclotides and anti-microbial peptides are compared and contrasted. Signaling peptides are classified according to their major sites of activity. Finally, a network approach to creating an interactomic peptide map is described.
Plants use a range of mechanisms to respond to challenge by plant pathogens and, in turn, plant pathogens use a range of mechanisms to interfere with and evade these responses. Plant defence responses include those that are mediated by salicylic acid and jasmonic acid–ethylene but, so far, few phytopathogen effectors that interfere with these hormone-based systems have been identified. A study in PLoS Biology now shows that an effector from the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis attenuates the salicylic acid response, thus enhancing biotrophy.
Many oomycete effectors contain an amino-terminal RXLR (where X is any amino acid) motif that correlates with entry into host cells. Previous characterization of the RXLR effector repertoire of H. arabidopsidis identified a subset of effectors that localize to the plant cell nucleus and interact with components of the Mediator complex — a multisubunit transcriptional regulation complex that is present in all eukaryotes. In plants, Mediator has been shown to influence key processes, including plant development and, recently, plant immunity.
Caillaud and colleagues present an in-depth characterization of one of the H. arabidopsidis RXLR nuclear effectors, HaRxL44, which interacts with the MED19a subunit of the Mediator complex. Analysis of transgenic Arabidopsis thaliana lines showed that those in which MED19a was non-functional were more susceptible than the wild type to infection with H. arabidopsidsis. By contrast, transgenic lines in which MED19a was overproduced were more resistant to infection than the wild type. This indicates that MED19a is a positive regulator of A. thaliana immunity to H. arabidopsidsisinfection. Confocal microscopy to monitor the subcellular localization of MED19a-containing and HaRxL44-containing fusion proteins showed that both localize to the plant nucleolus and nucleoplasm. However, colocalization analysis showed that when HaRxL44 was present in the nucleoplasm MED19a could not be detected. Further investigations revealed that HaRxL44 degrades MED19a in a proteasome-dependent manner.
To pinpoint the mechanism by which HaRxL44-mediated MED19a degradation affects the immune response to H. arabidopsidsis infection, the authors used quantitative reverse transcription (qRT)-PCR analysis and gene expression profiling. They found that, in the presence of HaRxL44, marker genes that were consistent with jasmonic acid–ethylene signalling were upregulated, whereas marker genes that were consistent with salicylic acid signalling — which has a key role in the response to biotrophic pathogens — were downregulated. Finally, the authors investigated the cell-specific expression patterns of marker genes that are associated with the salicylic acid response and found that H. arabidopsidsis infection only suppresses the salicylic acid response in those cells that have been parasitized by oomycete haustoria.
These data show that the H. arabidopsidsis HaRxL44 effector targets MED19a to modulate the balance between jasmonic acid–ethylene and salicylic acid signalling, such that the salicylic acid response is attenuated and biotrophic infection is favoured. It will be interesting to follow future studies that characterize the functions of the other H. arabidopsidsis RXLR nuclear effectors.
Caillaud, M.-C. et al. A downy mildew effector attenuates salicylic acid-triggered immunity inArabidopsis by interacting with the host mediator complex. PLoS Biol. 11, e1001732 (2013)
Genes encoding plant nucleotide-binding leucine-rich repeat (NB-LRR) proteins confer dominant resistance to diverse pathogens. The wild-type potato NB-LRR protein Rx confers resistance against a single strain of potato virus X (PVX), whereas LRR mutants protect against both a second PVX strain and the distantly related poplar mosaic virus (PopMV). In one of the Rx mutants there was a cost to the broad-spectrum resistance because the response to PopMV was transformed from a mild disease on plants carrying wild-type Rx to a trailing necrosis that killed the plant. To explore the use of secondary mutagenesis to eliminate this cost of broad-spectrum resistance, we performed random mutagenesis of the N-terminal domains of this broad-recognition version of Rx and isolated four mutants with a stronger response against the PopMV coat protein due to enhanced activation sensitivity. These mutations are located close to the nucleotide-binding pocket, a highly conserved structure that likely controls the “switch” between active and inactive NB-LRR conformations. Stable transgenic plants expressing one of these versions of Rx are resistant to the strains of PVX and the PopMV that previously caused trailing necrosis. We conclude from this work that artificial evolution of NB-LRR disease resistance genes in crops can be enhanced by modification of both activation and recognition phases, to both accentuate the positive and eliminate the negative aspects of disease resistance.
Live-cell imaging assisted by fluorescent markers has been fundamental to understanding the focused secretory 'warfare' that occurs between plants and biotrophic pathogens that feed on living plant cells. Pathogens succeed through the spatiotemporal deployment of a remarkably diverse range of effector proteins to control plant defences and cellular processes. Some effectors can be secreted by appressoria even before host penetration, many enter living plant cells where they target diverse subcellular compartments and others move into neighbouring cells to prepare them before invasion. This Review summarizes the latest advances in our understanding of the cell biology of biotrophic interactions between plants and their eukaryotic filamentous pathogens based on in planta analyses of effectors.
Plants posses an intricate innate immune system that enables them to fight off most invading pathogens. Around the world, agriculture relies on robust disease resistance to ensure adequate food and feed production. Researchers and breeders are constantly generating new resistant crop varieties mostly employing the lengthy process of conventional breeding. Nonetheless, crop losses due to plant pathogens are estimated to be over 15% every year - the main cause of such losses is rapid evolution of new virulent races. In order to keep up with emerging pathogens, we need to gain a deeper and more systematic understanding of the immune system of our crops. During the past two decades, molecular understanding of plant innate immune signaling has been greatly expanded using dicotyledonous model systems such as Arabidopsis thaliana. Now, it is time to connect this volume of knowledge with the immune system of the crop species.
In this Research Topic we aim to collect manuscripts covering the current knowledge of the immune systems of major crop species. Specifically, we encourage the submission of manuscripts (Original Research, Hypothesis & Theory, Methods, Reviews, Mini Reviews, Perspective and Opinion) covering the following topics:
a. Manuscripts describing our current understanding of the plant immune system with a focus on crop species or comparative analyses between model systems and crops.
b. Manuscripts exploring how to best exploit our insight into genomes of plant pathogens and molecular understanding of effector function. c. Manuscripts debating (novel) strategies of how to generate more resistant crop varieties. These might include biotechnological, social and economical aspects of crop improvement.
We anticipate that this Research Topic will become an important resource for plant immunologists especially those interested in comparative studies of plant innate immune systems of model systems and crop species.
Benjamin Schwessinger UC Davis Davis, USA
Rebecca Bart Donald Danforth Plant Science Center St. Louis, USA
Gitta Coaker University of California, Davis Davis, USA
Ksenia V Krasileva University of California Davis Davis, USA
Species in ecological communities build complex webs of interaction. Although revealing the architecture of these networks is fundamental to understanding ecological and evolutionary dynamics in nature, it has been difficult to characterize the structure of most species-rich ecological systems. By overcoming this limitation through next-generation sequencing technology, we herein uncover the network architecture of below-ground plant–fungus symbioses, which are ubiquitous to terrestrial ecosystems. The examined symbiotic network of a temperate forest in Japan includes 33 plant species and 387 functionally and phylogenetically diverse fungal taxa, and the overall network architecture differs fundamentally from that of other ecological networks. In contrast to results for other ecological networks and theoretical predictions for symbiotic networks, the plant–fungus network shows moderate or relatively low levels of interaction specialization and modularity and an unusual pattern of ‘nested’ network architecture. These results suggest that species-rich ecological networks are more architecturally diverse than previously recognized.
There is considerable evidence in the literature that beneficial rhizospheric microbes can alter plant morphology, enhance plant growth, and increase mineral content. Of late, there is a surge to understand the impact of the microbiome on plant health. Recent research shows the utilization of novel sequencing techniques to identify the microbiome in model systems such as Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). However, it is not known how the community of microbes identified may play a role to improve plant health and fitness. There are very few detailed studies with isolated beneficial microbes showing the importance of the functional microbiome in plant fitness and disease protection. Some recent work on the cultivated microbiome in rice (Oryza sativa) shows that a wide diversity of bacterial species is associated with the roots of field-grown rice plants. However, the biological significance and potential effects of the microbiome on the host plants are completely unknown. Work performed with isolated strains showed various genetic pathways that are involved in the recognition of host-specific factors that play roles in beneficial host-microbe interactions. The composition of the microbiome in plants is dynamic and controlled by multiple factors. In the case of the rhizosphere, temperature, pH, and the presence of chemical signals from bacteria, plants, and nematodes all shape the environment and influence which organisms will flourish. This provides a basis for plants and their microbiomes to selectively associate with one another. This Update addresses the importance of the functional microbiome to identify phenotypes that may provide a sustainable and effective strategy to increase crop yield and food security.
Plant roots are host to a multitude of filamentous microorganisms. Among these, arbuscular mycorrhizal fungi provide benefits to plants, while pathogens trigger diseases resulting in significant crop yield losses. It is therefore imperative to study processes which allow plants to discriminate detrimental and beneficial interactions in order to protect crops from diseases while retaining the ability for sustainable bio-fertilisation strategies. Accumulating evidence suggests that some symbiosis processes also affect plant–pathogen interactions. A large part of this overlap likely constitutes plant developmental processes. Moreover, microbes utilise effector proteins to interfere with plant development. Here we list relevant recent findings on how plant–microbe interactions intersect with plant development and highlight future research leads.
In plants, cell-surface receptors control immunity and development through the recognition of extracellular ligands. Leucine-rich repeat receptor-like proteins (LRR-RLPs) constitute a large multigene family of cell-surface receptors. Although this family has been intensively studied, a limited number of ligands has been identified so far, mostly because methods used for their identification and characterisation are complex and fastidious. In this study, we combined genome and transcriptome analyses to describe the LRR-RLP gene family in the model tree poplar (Populus trichocarpa). In total, 82 LRR-RLP genes have been identified in P. trichocarpa genome, among which 66 are organised in clusters of up to seven members. In these clusters, LRR-RLP genes are interspersed by orphan, poplar-specific genes encoding small proteins of unknown function (SPUFs). In particular, the nine largest clusters of LRR-RLP genes (47 LRR-RLPs) include 71 SPUF genes that account for 59% of the non-LRR-RLP gene content within these clusters. Forty-four LRR-RLP and fifty-five SPUF genes are expressed in poplar leaves, mostly at low levels, except for members of some clusters that show higher and sometimes coordinated expression levels. Notably, wounding of poplar leaves strongly induced the expression of a defense SPUF gene named Rust-Induced Secreted protein (RISP) that has been previously reported as a marker of poplar defense responses. Interestingly, we show that the RISP-associated LRR-RLP gene is highly expressed in poplar leaves and slightly induced by wounding. Both gene promoters share a highly conserved region of approx. 300 nucleotides. This led us to hypothesize that the corresponding pair of proteins could be involved in poplar immunity, possibly as a ligand/receptor couple.In conclusion, we speculate that some poplar SPUFs, such as RISP, represent candidate endogenous peptide ligands of the associated LRR-RLPs and we discuss how to investigate further this hypothesis.
Plants are members of complex communities and interact both with antagonists and beneficial organisms. An important question in plant defense-signaling research is how plants integrate signals induced by pathogens, insect herbivores and beneficial microbes into the most appropriate adaptive response. Molecular and genomic tools are now being used to uncover the complexity of the induced defense signaling networks that have evolved during the arms races between plants and the other organisms with which they intimately interact. To understand the functioning of the complex defense signaling network in nature, molecular biologists and ecologists have joined forces to place molecular mechanisms of induced plant defenses in an ecological perspective. In this Research Topic, we aim to provide an on-line, open-access snapshot of the current state of the art of the field of induced plant responses to microbes and insects, with a special focus on the translation of molecular mechanisms to ecology and vice versa. We will collect Original Research and Review papers on the topic, but also other article types, such as Methods and Opinions are welcome.
GAINESVILLE, Fla. --- Researchers from the Institute of Food and Agricultural Sciences at the University of Florida are closer to finding a possible cure for citrus canker after identifying a gene that makes citrus trees susceptible to the bacterial pathogen