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.
Ralstonia solanacearum, the causal agent of bacterial wilt, exerts its pathogenicity through more than a hundred secreted proteins, many of them depending directly on the functionality of a type 3 secretion system (T3SS). To date, only few type 3 effectors (T3Es) have been identified as required for bacterial pathogenicity, notably due to redundancy among the large R. solanacearum effector repertoire. In order to identify groups of effectors collectively promoting disease on susceptible hosts, we investigated the role of putative post-translational regulators in the control of type 3 secretion. A shotgun secretome analysis with label-free quantification using tandem mass spectrometry was performed on the R. solanacearum GMI1000 strain. 228 proteins were identified, among which a large proportion of T3Es, called Rip (Ralstonia injected proteins). Thanks to this proteomic approach, RipBJ was identified as a new effector specifically secreted through T3SS and translocated into plant cells. A focused Rip secretome analysis using hpa (hypersensitive response and pathogenicity associated) mutants revealed a fine secretion regulation and specific subsets of Rips with different secretion patterns. We showed that a set of Rips (RipF1, RipW, RipX, RipAB and RipAM) are secreted in an Hpa-independent manner. We hypothesize that these Rips could be preferentially involved in the first stages of type 3 secretion. In addition, the secretion of about thirty other Rips is controlled by HpaB and HpaG. HpaB, a candidate chaperone was demonstrated to positively control secretion of numerous Rips, while HpaG was shown to act as a negative regulator of secretion. To evaluate the impact of altered T3E secretion on plant pathogenesis, the hpa mutants were assayed on several host plants. HpaB was required for bacterial pathogenicity on multiple hosts whereas HpaG was found to be specifically required for full R. solanacearum pathogenicity on the legume plant Medicago truncatula.
Plants are sessile organisms that have evolved exquisite and sophisticated mechanisms to adapt to their biotic and abiotic environment. Plants deploy receptors and vast signalling networks to detect, transmit and respond to a given biotic threat by inducing properly dosed defence responses. Genetic analyses and, more recently, next-generation -omics approaches have allowed unprecedented insights into the mechanisms that drive immunity. Similarly, functional genomics and the emergence of pathogen genomes have allowed reciprocal studies on the mechanisms governing pathogen virulence and host susceptibility, collectively allowing more comprehensive views on the processes that govern disease and resistance. Among others, the identification of secreted pathogen molecules (effectors) that modify immunity-associated processes has changed the plant–microbe interactions conceptual landscape. Effectors are now considered both important factors facilitating disease and novel probes, suited to study immunity in plants. In this review, we will describe the various mechanisms and processes that take place in the nucleus and help regulate immune responses in plants. Based on the premise that any process required for immunity could be targeted by pathogen effectors, we highlight and describe a number of functional assays that should help determine effector functions and their impact on immune-related processes. The identification of new effector functions that modify nuclear processes will help dissect nuclear signalling further and assist us in our bid to bolster immunity in crop plants.
Next to numerous abiotic stresses, plants are constantly exposed to a variety of pathogens within their environment. Thus, their ability to survive and prosper during the course of evolution was strongly dependent on adapting efficient strategies to perceive and to respond to such potential threats. It is therefore not surprising that modern plants have a highly sophisticated immune repertoire consisting of diverse signal perception and intracellular signaling pathways. This signaling network is intricate and deeply interconnected, probably reflecting the diverse lifestyles and infection strategies used by the multitude of invading phytopathogens. Moreover it allows signal communication between developmental and defense programs thereby ensuring that plant growth and fitness are not significantly retarded. How plants integrate and prioritize the incoming signals and how this information is transduced to enable appropriate immune responses is currently a major research area. An important finding has been that pathogen-triggered cellular responses involve massive transcriptional reprogramming within the host. Additional key observations emerging from such studies are that transcription factors (TFs) are often sites of signal convergence and that signal-regulated TFs act in concert with other context-specific TFs and transcriptional co-regulators to establish sensory transcription regulatory networks required for plant immunity.
Potato late blight, caused by the destructive Irish famine pathogen Phytophthora infestans, is a major threat to global food security1,2. All late blight resistance genes identified to date belong to the coiled-coil, nucleotide-binding, leucine-rich repeat class of intracellular immune receptors3. However, virulent races of the pathogen quickly evolved to evade recognition by these cytoplasmic immune receptors4. Here we demonstrate that the receptor-like protein ELR (elicitin response) from the wild potato Solanum microdontum mediates extracellular recognition of the elicitin domain, a molecular pattern that is conserved in Phytophthora species. ELR associates with the immune co-receptor BAK1/SERK3 and mediates broad-spectrum recognition of elicitin proteins from several Phytophthora species, including four diverse elicitins from P. infestans. Transfer of ELR into cultivated potato resulted in enhanced resistance to P. infestans. Pyramiding cell surface pattern recognition receptors with intracellular immune receptors could maximize the potential of generating a broader and potentially more durable resistance to this devastating plant pathogen.
by Adam D. Steinbrenner, Sandra Goritschnig, Brian J. Staskawicz In plants, specific recognition of pathogen effector proteins by nucleotide-binding leucine-rich repeat (NLR) receptors leads to activation of immune responses.
Summary Fungal plant pathogens such as Zymoseptoria tritici (formerly known as Mycosphaerella graminicola) secrete repertoires of effectors facilitating infection or triggering host defence mechanisms.
Highlight Botrytis-infected grapes are able to initiate a basal defence response by reprogramming the transcriptome and metabolome but lack important defences that are naturally established in healthy ripening berries.
Our conceptual and mechanistic understanding of how plant nucleotide-binding leucine-rich repeat (NLR or NB-LRR) proteins perceive pathogens continues to advance. NLRs are intracellular multidomain proteins that recognize pathogen-derived effectors either directly or indirectly (Jones and Dangl, 2006; van der Hoorn and Kamoun, 2008; Dodds and Rathjen, 2010; Cesari et al., 2014). In the direct model, the NLR protein binds a pathogen effector or serves as a substrate for the effector’s enzymatic activity. In the indirect model, the NLR recognizes modifications of additional host protein(s) targeted by the effector. Such intermediate host protein(s) are often called effector targets (ETs). However, given that effectors can act on multiple host targets, the specific protein that mediates recognition by the NLR may not be the effector’s operative target and may have evolved to function as a decoy dedicated to pathogen detection. This “decoy” model contrasts with the “guard” model in which the NLR perceives the effector via its action on its operative target (van der Hoorn and Kamoun, 2008).
In a recent article, Cesari et al. (2014) elegantly synthesized the literature to propose a novel model of how NLRs recognise effectors termed the “integrated decoy” hypothesis. Based on new data from several pathosystems, it appears that some NLRs recognize pathogen effectors through extraneous domains that have evolved by duplication of an ET followed by fusion into the NLR. This NLR-integrated domain mimics the effector binding/substrate property of the original ET to enable pathogen detection. In addition, these “receptor” or “sensor” NLRs typically partner with NLR proteins with a classic architecture that function as signalling partners required for the resistance response (Eitas and Dangl, 2010; Cesari et al., 2013; Cesari et al., 2014; Williams et al., 2014).
Here, we expand on the Cesari et al. (2014) model and introduce the possibility that NLR-integrated domains do not have to be decoys (as in defective mimics) of the effector’s operative target. Indeed, in addition to binding effectors or serving as their substrates, operative targets carry a biochemical activity that is modulated by the effector. The perturbation of this activity by the effector leads to effector-triggered susceptibility, an activity often related to immunity (Boller and He, 2009; Dodds and Rathjen, 2010; Win et al., 2012). Clearly NLR-integrated domains must retain the “sensor” activity of the ancestral ET, but they could also retain their biochemical activity, continuing to function in the effector-targeted pathway even as an extraneous domain within a classic NLR architecture. At present, this possibility cannot be discounted given that the biochemical activities of the ancestral ETs and their NLR-integrated counterparts are generally unknown. Additionally, when NLR-fusions occurred recently, there may not have been enough time for the integrated ET to lose its original function and evolve into a decoy. We therefore propose to refer to the extraneous domains of classic NLR proteins described by Cesari et al. (2014) as sensor domains (SD), a term that is agnostic to any potential biochemical activities of the integrated module.
How to test whether or not SDs are decoys? We propose a straightforward genetic test that can reject the decoy hypothesis. Isogenic plants either carrying or lacking the NLR-SD can be challenged with a pathogen strain that lacks the matching avirulence effector (Figure 1). There are several possible outcomes. If the NLR-SD isogenic lines do not differ in their response to the pathogen without the matching effector, the result is inconclusive and the null decoy hypothesis cannot be rejected. If the presence of NLR-SD without the known matching effector shows higher levels of resistance, and there are no signs of typical effector-triggered immunity, then the SD is likely to have retained the ET biochemical activity and contributes to basal immunity in a manner analogous to the ancestral ET. An even more interesting result would be if in the absence of the matching effector, the NLR-SD line is more susceptible as has been shown for several ETs (van Schie and Takken, 2014). In this scenario, another (unrecognized) effector might still be targeting the original biochemical activity of the SD domain. It would be conceptually fascinating if an NLR that functions as a resistance (R) gene against certain strains of a pathogen becomes a susceptibility (S) gene when exposed to other strains. Once again, this concept emphasizes how the outcome of plant-pathogen interactions is so critically dependent on the genotypes of the interacting organisms – a gene that has a certain impact in a particular genetic combination can have the exact opposite effect in another (Jones and Dangl, 2006; van der Hoorn and Kamoun, 2008; Dodds and Rathjen, 2010; Win et al., 2012).
Our goal is not to engage in an exercise in semantics. However, we wish to avoid conceptually restrictive terminology and urge the plant-microbe interactions community to test a rich spectrum of models and hypotheses. The proposed sensor domain terminology would accommodate this breadth of ideas. Ultimately, it may very well turn out that the majority, if not all, of the NLR integrated domains have lost their biochemical activities and have evolved into decoys. Also, it is possible that the sensor domain has already evolved into a decoy prior to recombination into a NLR. Nonetheless, further genetic and biochemical experiments are required to determine whether sensor domains of NLR-SDs are decoys or biochemically functional duplicates of their ancestral ETs.
In nature plants are colonised by beneficial and pathogenic microbes. While plants benefit from the interactions with beneficial microbes (mutualists), pathogenic microbes cause diseases and arrest plant growth. Both mutualistic and pathogenic microbes are initially confronted with a highly effective immune system, which they have to overcome in order to colonise their hosts. Hence, despite the different outcomes of mutualistic and pathogenic interactions, both microbial groups face the same hurdles to establish their accommodation in the plant. A plethora of recent studies indicated that mutualists and pathogens secrete effectors, mainly proteins but also small interfering RNAs, with the purpose not only to manipulate host immunity but also to modify host metabolism in order to create an environment suitable for microbial reproduction.
The session will highlight the current knowledge of how mutualistic and pathogenic microbes employ effectors to successfully establish their respective interactions with plants. The aim is to bring together a group of experts in plant microbe interactions to identify commonalities and discrepancies in the mode of action of mutualistic and pathogenic effectors. Improving our understanding of effector biology will enable us to uncover the molecular principles governing mutualism and disease outbreaks and to synergistically apply this knowledge to sustainably enhance stress adaptation in crops.
The gene-for-gene hypothesis states that for each gene controlling resistance in the host, there is a corresponding, specific gene controlling avirulence in the pathogen. Allelic series of the cereal mildew resistance genes Pm3 and Mla provide an excellent system for genetic and molecular analysis of resistance specificity. Despite this opportunity for molecular research, avirulence genes in mildews remain underexplored. Earlier work in barley powdery mildew (B.g. hordei) has shown that the reaction to some Mla resistance alleles is controlled by multiple genes. Similarly, several genes are involved in the specific interaction of wheat mildew (B.g. tritici) with the Pm3 allelic series. We found that two mildew genes control avirulence on Pm3f: one gene is involved in recognition by the resistance protein as demonstrated by functional studies in wheat and the heterologous host Nicotiana benthamiana. A second gene is a suppressor, and resistance is only observed in mildew genotypes combining the inactive suppressor and the recognized Avr. We propose that such suppressor/avirulence gene combinations provide the basis of specificity in mildews. Depending on the particular gene combinations in a mildew race, different genes will be genetically identified as the “avirulence” gene. Additionally, the observation of two LINE retrotransposon-encoded avirulence genes in B.g. hordei further suggests that the control of avirulence in mildew is more complex than a canonical gene-for-gene interaction. To fully understand the mildew-cereal interactions, more knowledge on avirulence determinants is needed and we propose ways how this can be achieved based on recent advances in the field.
Plant immune receptors involved in disease resistance and crop protection are related to the animal Nod-like receptor (NLR) class, and recognise the virulence effectors of plant pathogens, whereby they arm the plant’s defensive response. Although plant NLRs mainly contain three protein domains, about 10 % of these receptors identified by extensive cross-plant species data base searches have now been shown to include novel and highly variable integrated domains, some of which have been shown to detect pathogen effectors by direct interaction. Sarris et al. have identified a large number of integrated domains that can be used to detect effector targets in host plant proteomes and identify unknown pathogen effectors.
Since the time of writing, a closely related paper has been released: Kroj T, Chanclud E, Michel-Romiti C, Grand X, Morel J-B. Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread. New Phytol. 2016
The Oomycota include many economically significant microbial pathogens of crop species. Understanding the mechanisms by which oomycetes infect plants and identifying methods to provide durable resistance are major research goals. Over the last few years, many elicitors that trigger plant immunity have been identified, as well as host genes that mediate susceptibility to oomycete pathogens. The mechanisms behind these processes have subsequently been investigated and many new discoveries made, marking a period of exciting research in the oomycete pathology field. This review provides an introduction to our current knowledge of the pathogenic mechanisms used by oomycetes, including elicitors and effectors, plus an overview of the major principles of host resistance: the established R gene hypothesis and the more recently defined susceptibility (S) gene model. Future directions for development of oomycete-resistant plants are discussed, along with ways that recent discoveries in the field of oomycete-plant interactions are generating novel means of studying how pathogen and symbiont colonizations overlap.
Salicylic acid (SA) is an important phytohormone that plays a vital role in a number of physiological responses, including plant defense. The last two decades have witnessed a number of breakthroughs related to biosynthesis, transport, perception and signaling mediated by SA. These findings demonstrate that SA plays a crictical role in both local and systemic defense responses. Systemic acquired resistance (SAR) is one such SA-dependent response. SAR is a long distance signaling mechanism that provides broad spectrum and long-lasting resistance to secondary infections throughout the plant. This unique feature makes SAR a highly desirable trait in crop production. This review summarizes the recent advances in the role of SA in SAR and discusses its relationship to other SAR inducers.
Plant secondary metabolites carry out numerous functions in interactions between plants and a broad range of other organisms. Experimental evidence strongly supports the indispensable contribution of many constitutive and pathogen-inducible phytochemicals to plant innate immunity. Extensive studies on model plant species, particularly Arabidopsis thaliana, have brought significant advances in our understanding of the molecular mechanisms underpinning pathogen-triggered biosynthesis and activation of defensive secondary metabolites. However, despite the proven significance of secondary metabolites in plant response to pathogenic microorganisms, little is known about the precise mechanisms underlying their contribution to plant immunity. This insufficiency concerns information on the dynamics of cellular and subcellular localization of defensive phytochemicals during the encounters with microbial pathogens and precise knowledge on their mode of action. As many secondary metabolites are characterized by their in vitro antimicrobial activity, these compounds were commonly considered to function in plant defense as in planta antibiotics. Strikingly, recent experimental evidence suggests that at least some of these compounds alternatively may be involved in controlling several immune responses that are evolutionarily conserved in the plant kingdom, including callose deposition and programmed cell death.
Bacterial blight of rice is caused by the γ-proteobacterium Xanthomonas oryzae pv. oryzae, which utilizes a group of type III TAL (transcription activator-like) effectors to induce host gene expression and condition host susceptibility. Five SWEET genes are functionally redundant to support bacterial disease, but only two were experimentally proven targets of natural TAL effectors. Here, we report the identification of the sucrose transporter gene OsSWEET13 as the disease susceptibility gene for PthXo2 and the existence of cryptic recessive resistance to PthXo2-dependent X. oryzae pv. oryzae due to promoter variations of OsSWEET13 in japonica rice. PthXo2-containing strains induce OsSWEET13 in indica rice IR24 due to the presence of an unpredicted and undescribed effector binding site not present in the alleles in japonica rice Nipponbare and Kitaake. The specificity of effector-associated gene induction and disease susceptibility is attributable to a single nucleotide polymorphism (SNP), which is also found in a polymorphic allele of OsSWEET13 known as the recessive resistance gene xa25 from the rice cultivar Minghui 63. The mutation of OsSWEET13 with CRISPR/Cas9 technology further corroborates the requirement of OsSWEET13 expression for the state of PthXo2-dependent disease susceptibility to X. oryzae pv. oryzae. Gene profiling of a collection of 104 strains revealed OsSWEET13 induction by 42 isolates of X. oryzae pv. oryzae. Heterologous expression of OsSWEET13 in Nicotiana benthamiana leaf cells elevates sucrose concentrations in the apoplasm. The results corroborate a model whereby X. oryzae pv. oryzae enhances the release of sucrose from host cells in order to exploit the host resources.
Highlight Botrytis-infected grapes are able to initiate a basal defence response by reprogramming the transcriptome and metabolome but lack important defences that are naturally established in healthy ripening berries.
Rust fungi are devastating crop pathogens that deliver effector proteins into infected tissues to modulate plant functions and promote parasitic growth. The genome of the poplar leaf rust fungus Melampsora larici-populina revealed a large catalogue of secreted proteins, some of which have been considered candidate effectors. Unravelling how these proteins function in host cells is key to understanding pathogenicity mechanisms and developing resistant plants. In this study, we used an effectoromics pipeline to select, clone, and express 20 candidate effectors in Nicotiana benthamiana leaf cells to determine their subcellular localisation and identify the plant proteins they interact with. Confocal microscopy revealed that six candidate effectors target the nucleus, nucleoli, chloroplasts, mitochondria and discrete cellular bodies. We also used coimmunoprecipitation and mass spectrometry to identify 606 N. benthamiana proteins that associate with the candidate effectors. Five candidate effectors specifically associated with a small set of plant proteins that may represent biologically relevant interactors. We confirmed the interaction between the candidate effector MLP124017 and the TOPLESS-Related Protein 4 from poplar by in planta coimmunoprecipitation. Altogether, our data enable us to validate effector proteins from M. larici-populina and reveal that these proteins may target multiple compartments and processes in plant cells. It also shows that N. benthamiana can be a powerful heterologous system to study effectors of obligate biotrophic pathogens.
Alternaria species are mainly saprophytic fungi but some are plant pathogens. Seven pathotypes of Alternaria alternata use secondary metabolites of host-specific toxins as pathogenicity factors. These toxins kill host cells prior to colonization.
Perception of pathogen associated molecular patterns (PAMPs) by cell surface localized pattern recognition receptors (PPRs), activates plant basal defense responses in a process known as PAMP/PRR–triggered immunity (PTI). In turn, pathogens deploy effector proteins that interfere with different steps in PTI signaling. However, our knowledge of PTI suppression by filamentous plant pathogens, i.e. fungi and oomycetes, remains fragmentary. Previous work revealed that BAK1/SERK3, a regulatory receptor of several PRRs, contributes to basal immunity against the Irish potato famine pathogen Phytophthora infestans. Moreover BAK1/SERK3 is required for the cell death induced by P. infestans elicitin INF1, a protein with characteristics of PAMPs. The P. infestans host-translocated RXLR-WY effector AVR3a is known to supress INF1-mediated defense by binding the E3 ligase CMPG1. In contrast, AVR3aKI-Y147del, a deletion mutant of the C-terminal tyrosine of AVR3a, fails to bind CMPG1 and suppress INF1 cell death. Here we studied the extent to which AVR3a and its variants perturb additional BAK1/SERK3 dependent PTI responses using the plant PRR FLAGELLIN SENSING 2 (FLS2). We found that all tested variants of AVR3a, including AVR3aKI-Y147del, suppress early defense responses triggered by the bacterial flagellin-derived peptide flg22 and reduce internalization of activated FLS2 from the plasma membrane without disturbing its nonactivated localization. Consistent with this effect of AVR3a on FLS2 endocytosis, we discovered that AVR3a associates with the Dynamin-Related Protein DRP2, a plant GTPase implicated in receptor-mediated endocytosis. Interestingly, DRP2 is required for ligand-induced FLS2 internalization but does not affect internalization of the growth receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1). Furthermore, overexpression of DRP2 suppressed accumulation of reactive oxygen species triggered by PAMP treatment. We conclude that AVR3a associates with a key cellular trafficking and membrane-remodeling complex involved in immune receptor-mediated endocytosis and signaling. AVR3a is a multifunctional effector that can suppress BAK1/SERK3 mediated immunity through at least two different pathways.
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