Maize smut caused by the fungus Ustilago maydis is a widespread disease characterized by the development of large plant tumours. U. maydis is a biotrophic pathogen that requires living plant tissue for its development and establishes an intimate interaction zone between fungal hyphae and the plant plasma membrane. U. maydis actively suppresses plant defence responses by secreted protein effectors1, 2. Its effector repertoire comprises at least 386 genes mostly encoding proteins of unknown function1, 3, 4 and expressed exclusively during the biotrophic stage3. The U. maydis secretome also contains about 150 proteins with probable roles in fungal nutrition, fungal cell wall modification and host penetration as well as proteins unlikely to act in the fungal-host interface4 like a chorismate mutase. Chorismate mutases are key enzymes of the shikimate pathway and catalyse the conversion of chorismate to prephenate, the precursor for tyrosine and phenylalanine synthesis. Root-knot nematodes inject a secreted chorismate mutase into plant cells likely to affect development5, 6. Here we show that the chorismate mutase Cmu1 secreted by U. maydis is a virulence factor. The enzyme is taken up by plant cells, can spread to neighbouring cells and changes the metabolic status of these cells through metabolic priming. Secreted chorismate mutases are found in many plant-associated microbes and might serve as general tools for host manipulation.
The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host innate immune systems. Recognition of flagellin monomers occurs by flagellin-specific pattern-recognition receptors, such as Toll-like receptor 5 (TLR5) in mammals and flagellin-sensitive 2 (FLS2) in plants. Activation of these immune systems via flagellin leads eventually to elimination of the bacterium from the host. In order to prevent immune activation and thus favor survival in the host, bacteria secrete many proteins that hamper such recognition. In our search for Toll like receptor (TLR) antagonists, we screened bacterial supernatants and identified alkaline protease (AprA) of Pseudomonas aeruginosa as a TLR5 signaling inhibitor as evidenced by a marked reduction in IL-8 production and NF-κB activation. AprA effectively degrades the TLR5 ligand monomeric flagellin, while polymeric flagellin (involved in bacterial motility) and TLR5 itself resist degradation. The natural occurring alkaline protease inhibitor AprI of P. aeruginosa blocked flagellin degradation by AprA. P. aeruginosa aprA mutants induced an over 100-fold enhanced activation of TLR5 signaling, because they fail to degrade excess monomeric flagellin in their environment. Interestingly, AprA also prevents flagellin-mediated immune responses (such as growth inhibition and callose deposition) in Arabidopsis thaliana plants. This was due to decreased activation of the receptor FLS2 and clearly demonstrated by delayed stomatal closure with live bacteria in plants. Thus, by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition by the innate immune systems of both mammals and plants.
Insect herbivores have highly diverse life cycles and feeding behaviors. They establish close interactions with their plant hosts and suppress plant defenses. Chewing herbivores evoke characteristic defense responses distinguishable from general mechanical damage. In addition, piercing-sucking hemipteran insects display typical feeding behavior that suggests active suppression of plant defense responses. Effectors that modulate plant defenses have been identified in the saliva of these insects. Tools for high-throughput effector identification and functional characterization have been developed. In addition, in some insect species it is possible to silence gene expression by RNAi. Together, this technological progress has enabled the identification of insect herbivore effectors and their targets that will lead to the development of novel strategies for pest resistances in plants.
Time lapse of infection by Phytophthora infestans on two different potato varieties. The second one is the potato variety that has actually been sequenced for the potato genome project. /via Remco Stam, Huitema Lab.
The oomycete pathogen Phytophthora infestans causes potato late blight, one of the most economically damaging plant diseases worldwide. P. infestans produces AVR3a, an essential modular virulence effector with an N-terminal RXLR domain that is required for host-cell entry. In host cells, AVR3a stabilizes and inhibits the function of the E3 ubiquitin ligase CMPG1, a key factor in host immune responses including cell death triggered by the pathogen-derived elicitor protein INF1 elicitin. To elucidate the molecular basis of AVR3a effector function, we determined the structure of Phytophthora capsici AVR3a4, a close homolog of P. infestans AVR3a. Our structural and functional analyses reveal that the effector domain of AVR3a contains a conserved, positively charged patch and that this region, rather than the RXLR domain, is required for binding to phosphatidylinositol monophosphates (PIPs) in vitro. Mutations affecting PIP binding do not abolish AVR3a recognition by the resistance protein R3a but reduce its ability to suppress INF1-triggered cell death in planta. Similarly, stabilization of CMPG1 in planta is diminished by these mutations. The steady-state levels of non–PIP-binding mutant proteins in planta are reduced greatly, although these proteins are stable in vitro. Furthermore, overexpression of a phosphatidylinositol phosphate 5-kinase results in reduction of AVR3a levels in planta. Our results suggest that the PIP-binding ability of the AVR3a effector domain is essential for its accumulation inside host cells to suppress CMPG1-dependent immunity.
The in planta association of the Hyaloperonospora arabidopsidis effector ATR1 with the cognate Arabidopsis thaliana RPP1 immune receptor activates a disease-resistance signaling pathway that inhibits pathogen growth. To define the molecular events specifying effector recognition by RPP1, we determined the crystal structure of ATR1 and assayed in planta the effects of surface polymorphisms that are critical to activating plant immunity. ATR1 adopts an elongated, all-helical, two-domain, seahorse-like structure with an overall architecture unlike any previously described fold. Structural comparisons highlight a tandemly duplicated, five-helix motif in the C-terminal domain that creates a structural framework for rapid diversification. Identification and mapping of critical recognition sites suggest that ATR1 detection by the RPP1 resistance protein is mediated by several distinct protein surfaces that allow the effectors to escape recognition through diverse surface polymorphisms. ATR1 gain-of-recognition mutants demonstrate that multiple amino acid substitutions are necessary for recognition and that surface polymorphisms exert additive effects. These results suggest that ATR1 is a modular repeat protein belonging to an ancient family of oomycete effectors that rapidly evolves to escape host detection and adopt diverse virulence functions.
Stem rust (Puccinia graminis f. sp. tritici; Pgt) is a devastating fungal disease of wheat and barley. Pgt race TTKSK (isolate Ug99) is a serious threat to these Triticeae grain crops because resistance is rare. In barley, the complex Rpg-TTKSK locus on chromosome 5H is presently the only known source of qualitative resistance to this aggressive Pgt race. Segregation for resistance observed on seedlings of the Q21861 × SM89010 (QSM) doubled-haploid (DH) population was found to be predominantly qualitative, with little of the remaining variance explained by loci other than Rpg-TTKSK. In contrast, analysis of adult QSM DH plants infected by field inoculum of Pgt race TTKSK in Njoro, Kenya, revealed several additional quantitative trait loci that contribute to resistance. To molecularly characterize these loci, Barley1 GeneChips were used to measure the expression of 22,792 genes in the QSM population after inoculation with Pgt race TTKSK or mock-inoculation. Comparison of expression Quantitative Trait Loci (eQTL) between treatments revealed an inoculation-dependent expression polymorphism implicating Actin depolymerizing factor3 (within the Rpg-TTKSK locus) as a candidate susceptibility gene. In parallel, we identified a chromosome 2H trans-eQTL hotspot that co-segregates with an enhancer of Rpg-TTKSK-mediated, adult plant resistance discovered through the Njoro field trials. Our genome-wide eQTL studies demonstrate that transcript accumulation of 25% of barley genes is altered following challenge by Pgt race TTKSK, but that few of these genes are regulated by the qualitative Rpg-TTKSK on chromosome 5H. It is instead the chromosome 2H trans-eQTL hotspot that orchestrates the largest inoculation-specific responses, where enhanced resistance is associated with transcriptional suppression of hundreds of genes scattered throughout the genome. Hence, the present study associates the early suppression of genes expressed in this host–pathogen interaction with enhancement of R-gene mediated resistance.
Thousands of plane trees along the banks of the Canal du Midi, a Unesco heritage site, will have to be cut down because of a deadly fungus - Ceratocystis platani
Current models of plant–pathogen interactions stipulate that pathogens secrete effector proteins that disable plant defense components known as virulence targets. Occasionally, the perturbations caused by these effectors trigger innate immunity via plant disease resistance proteins as described by the “guard hypothesis.” This model is nicely illustrated by the interaction between the fungal plant pathogen Cladosporium fulvum and tomato. C. fulvum secretes a protease inhibitor Avr2 that targets the tomato cysteine protease Rcr3pim. In plants that carry the resistance protein Cf2, Rcr3pim is required for resistance to C. fulvum strains expressing Avr2, thus fulfilling one of the predictions of the guard hypothesis. Another prediction of the guard hypothesis has not yet been tested. Considering that virulence targets are important components of defense, different effectors from unrelated pathogens are expected to evolve to disable the same host target. In this study we confirm this prediction using a different pathogen of tomato, the oomycete Phytophthora infestans that is distantly related to fungi such as C. fulvum. This pathogen secretes an array of protease inhibitors including EPIC1 and EPIC2B that inhibit tomato cysteine proteases. Here we show that, similar to Avr2, EPIC1 and EPIC2B bind and inhibit Rcr3pim. However, unlike Avr2, EPIC1 and EPIC2B do not trigger hypersensitive cell death or defenses on Cf-2/Rcr3pim tomato. We also found that the rcr3–3 mutant of tomato that carries a premature stop codon in the Rcr3 gene exhibits enhanced susceptibility to P. infestans, suggesting a role for Rcr3pim in defense. In conclusion, our findings fulfill a key prediction of the guard hypothesis and suggest that the effectors Avr2, EPIC1, and EPIC2B secreted by two unrelated pathogens of tomato target the same defense protease Rcr3pim. In contrast to C. fulvum, P. infestans appears to have evolved stealthy effectors that carry inhibitory activity without triggering plant innate immunity.
Already reeling from a 20-year losing battle with a devastating disease, the banana variety eaten in the United States is now threatened by a new—but old—enemy.
Protease inhibitors (PIs) are involved in diverse biological processes in many organisms due to their function in the precise regulation of proteases. Here, we studied the functions of Arabidopsis UNUSUAL SERINE PROTEASE INHIBITOR (UPI) encoding an 8.8 kDa protein of atypical sequence composition relative to other PIs. Plants harboring a loss of function UPI allele display enhanced susceptibility to the necrotrophic fungi Botrytis cinerea and Alternaria brassicicola as well as the generalist herbivore Trichoplusia ni. Further, ectopic expression confers increased resistance to B. cinerea and T. ni. By contrast, the mutant has wild-type responses to virulent, avirulent, and non-pathogenic strains of Pseudomonas syringae limiting the defense function of UPI to necrotrophic fungal infection and insect herbivory. UPI is significantly induced by jasmonate (JA), salicylate (SA), and abscisic acid (ABA) but repressed by ethylene (ET) indicating complex phytohormone regulation of UPI expression. upi also has significantly delayed flowering associated with decreased SOC1 expression and elevated levels of MAF1, two regulators of floral transition. Recombinant UPI strongly inhibits the serine protease chymotrypsin but also weakly blocks the cysteine protease papain. Interestingly, JA induces intra- and extracellular UPI accumulation suggesting possible function in intercellular signaling or extracellular targets. Overall, our results show UPI is a dual-specificity PI that functions in plant growth and defense, likely through the regulation of endogenous proteases and/or those of biotic invaders.
Fluorescent tagging has become the strategy of choice for examining the subcellular localisation of proteins. To develop a versatile community resource for this method in oomycetes, plasmids were constructed that allow the expression of either of four spectrally distinct proteins (CFP, GFP, YFP, and mCherry), alone or fused at their N- or C-termini to sequences of interest. Equivalent sets of plasmids were made using neomycin or hygromycin phosphotransferases (nptII, hpt) as selectable markers, to facilitate double-labelling and aid work in diverse species. The fluorescent proteins and drug-resistance markers were fused to transcriptional regulatory sequences from the oomycete Bremia lactucae, which are known to function in diverse oomycetes, although the promoter in the fluorescence cassette (ham34) can be replaced easily by a promoter of interest. The function of each plasmid was confirmed in Phytophthora infestans. Moreover, fusion proteins were generated using targeting sequences for the endoplasmic reticulum, Golgi, mitochondria, nuclei, and peroxisomes. Studies of the distribution of the fusions in mycelia and sporangia provided insight into cellular organisation at different stages of development. This toolbox of vectors should advance studies of gene function and cell biology in Phytophthora and other oomycetes.
Resistance in tomato to infection by Pseudomonas syringae involves both detection of PAMPs and recognition by the host Pto kinase of pathogen effector AvrPtoB which is translocated into the host cell and interferes with PAMP-triggered immunity (PTI). The N-terminal portion of AvrPtoB is sufficient for its virulence activity and for recognition by Pto. An amino acid substitution in this protein, F173A, abolishes these activities. To investigate the mechanisms of AvrPtoB virulence, we screened for tomato proteins that interact with AvrPtoB and identified Bti9, a LysM receptor-like kinase. Bti9 has the highest amino acid similarity to Arabidopsis CERK1 among the tomato LysM-RLKs and belongs to a clade containing three other tomato proteins, SlLyk11, SlLyk12, and SlLyk13 all of which interact with AvrPtoB. The F173A substitution disrupts the interaction of AvrPtoB with Bti9 and SlLyk13 suggesting these LysM-RLKs are its virulence targets. Two independent tomato lines with RNAi-mediated reduced expression of Bti9 and SlLyk13 were more susceptible to P. syringae. Bti9 kinase activity was inhibited in vitro by the N-terminal domain of AvrPtoB in an F173-dependent manner. These results indicate Bti9/SlLyk13 play a role in plant immunity and the N-terminal domain of AvrPtoB may have evolved to interfere with their kinase activity. Finally, we found that Bti9 and Pto interact with AvrPtoB in a structurally similar although not identical fashion suggesting Pto may have evolved as a molecular mimic of LysM-RLK kinase domains.
Phytoplasma research begins to bloom. Indeed, this review shows that substantial progress has been made with the identification of phytoplasma effectors that alter flower development, induce witches' broom, affect leaf shape, and modify plant-insect interactions. Phytoplasmas have a unique life cycle among pathogens, as they invade organisms of two distinct kingdoms, namely plants (Plantae) and insects (Animalia), and replicate intracellularly in both. Phytoplasmas release effectors into host cells of plants and insects to target host molecules, and in plants these effectors unload from the phloem to access distal tissues and alter basic developmental processes. The effectors provide phytoplasmas with a fitness advantage by modulating their plant and insect hosts. We expect that further research on the functional characterization of phytoplasma effectors will generate new knowledge that is relevant to fundamental aspects of plant sciences and entomology, and for agriculture by improving yields of crops affected by phytoplasma diseases.
Phytoplasmas are insect-transmitted bacterial plant pathogens and cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches’ Broom (AY-WB) readily infects the model plant Arabidopsis thaliana ecotype Col-0, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches' broom). We found that the majority of 56 genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein SAP54 that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen, and reports the first example of a microbial pathogen effector protein that targets flower development in a host.
A new technology for genome editing may put the zinc finger nuclease franchise out of business, some believe. Not so fast, say the finger people. Laura DeFrancesco reports.
In June, Life Technologies of Carlsbad, California, received an exclusive license from the Two Blades Foundation of Evanston, Illinois, and a group of plant biologists from Martin Luther University in Halle, Germany, to commercialize a new genome editing technology—transcription activator-like (TAL) effectors. With the newly announced license spurring on Life Technologies, researchers may soon be able to choose from several commercial sources for designer TAL effectors...
Phytopathogens deliver effector proteins inside host plant cells to promote infection. These proteins can also be sensed by the plant immune system, leading to restriction of pathogen growth. Effector genes can display signatures of positive selection and rapid evolution, presumably a consequence of their co-evolutionary arms race with plants. The molecular mechanisms underlying how effectors evolve to gain new virulence functions and/or evade the plant immune system are poorly understood. Here, we report the crystal structures of the effector domains from two oomycete RXLR proteins, Phytophthora capsici AVR3a11 and Phytophthora infestans PexRD2. Despite sharing < 20% sequence identity in their effector domains they display a conserved core α-helical fold. Bioinformatic analyses suggest the core fold occurs in ~44% of annotated Phytophthora RXLR effectors, both as a single domain and in tandem repeats of up to 11 units. Functionally important and polymorphic residues map to the surface of the structures and PexRD2, but not AVR3a11, oligomerizes in planta. We conclude that the core α-helical fold enables functional adaptation of these fast-evolving effectors through (i) insertion/deletions in loop regions between α-helices, (ii) extensions to the N- and C-termini, (iii) amino acid replacements in surface residues, (iv) tandem domain duplications and (v) oligomerization. We hypothesize that the molecular stability provided by this core fold, combined with considerable potential for plasticity, underlies the evolution of effectors that maintain their virulence activities while evading recognition by the plant immune system.
Little is known about the function of host factors involved in disease susceptibility. The barley (Hordeum vulgare) ROP (RHO of plants) G-protein RACB is required for full susceptibility of the leaf epidermis to invasion by the biotrophic fungus Blumeria graminis f. sp hordei. Stable transgenic knockdown of RACB reduced the ability of barley to accommodate haustoria of B. graminis in intact epidermal leaf cells and to form hairs on the root epidermis, suggesting that RACB is a common element of root hair outgrowth and ingrowth of haustoria in leaf epidermal cells. We further identified a barley MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN (MAGAP1) interacting with RACB in yeast and in planta. Fluorescent MAGAP1 decorated cortical microtubules and was recruited by activated RACB to the cell periphery. Under fungal attack, MAGAP1-labeled microtubules built a polarized network at sites of successful defense. By contrast, microtubules loosened where the fungus succeeded in penetration. Genetic evidence suggests a function of MAGAP1 in limiting susceptibility to penetration by B. graminis. Additionally, MAGAP1 influenced the polar organization of cortical microtubules. These results add to our understanding of how intact plant cells accommodate fungal infection structures and suggest that RACB and MAGAP1 might be antagonistic players in cytoskeleton organization for fungal entry.
Mutualistic interactions of plants with true fungi are a well-known and widespread phenomenon, which includes mycorrhiza and non-mycorrhizal endophytes like species of Epichloë. Despite the fact that these organisms intrude into plants, neither strong defence reactions nor the onset of symptoms of disease can be observed in most or even all infested plants, in contrast to endophytic pathogens. Oomycetes are fungal-like organisms belonging to the kingdom Straminipila, which includes diatoms and seaweeds. Although having evolved many convergent traits with true fungi and occupying similar evolutionary niches, widespread oomycete endophytes are not known to date, although more than 500 endophytic pathogens, including species of the obligate biotrophic genus Albugo, have been described. Here, we report that oomycetes of the genus Albugo are widespread in siliques of natural host populations. A total of 759 plants, encompassing four genera with rare reports of white blister incidents and one with common incidents, were collected from 25 sites in Germany. Nested PCR with species-specific primers revealed that 5–27% of the hosts with rare disease incidence carried asymptomatic Albugo in their siliques, although only on a single plant of 583 individuals, an isolated pustule on a single leaf could be observed. Control experiments confirmed that these results were not because of attached spores, but because of endophytic mycelium. Vertical inheritance of oomycete infections has been reported for several plant pathogens, and it seems likely that in nature this way of transmission plays an important role in the persistence of asymptomatic endophytic Albugo species.
Plants generate effective responses to infection by recognizing both conserved and variable pathogen-encoded molecules. Pathogens deploy virulence effector proteins into host cells, where they interact physically with host proteins to modulate defense. We generated an interaction network of plant-pathogen effectors from two pathogens spanning the eukaryote-eubacteria divergence, three classes of Arabidopsis immune system proteins, and ~8000 other Arabidopsis proteins. We noted convergence of effectors onto highly interconnected host proteins and indirect, rather than direct, connections between effectors and plant immune receptors. We demonstrated plant immune system functions for 15 of 17 tested host proteins that interact with effectors from both pathogens. Thus, pathogens from different kingdoms deploy independently evolved virulence proteins that interact with a limited set of highly connected cellular hubs to facilitate their diverse life-cycle strategies.
The PPIN-1 network contains 3,148 interactions among 926 immune related proteins. It was generated using a high-quality Y2H screening pipeline detailed in Mukhtar et al. The 926 immune related proteins include 83 combined effectors from the bacterium Pseudomonas syringae (gold nodes) and obligate biotrophic oomycete Hyaloperonospora arabidopsidis (purple nodes), 170 known Arabidopsis immune receptors and response proteins (N-termini of NB-LRR disease resistance proteins, cytoplasmic domains of LRR-RLKs and other known defense proteins; red, pink, and blue nodes respectively) and 673 newly identified interacting Arabidopsis proteins (grey nodes). Direct interactions derived from the PPIN-1 screen are shown as grey lines (immune edges). Interactions between the 926 immune proteins and Arabidopsis proteins identified in an independent screen (AI1) are shown as green edges. Interactions between proteins found in the literature (LCI) are shown as blue edges. Interactions identified in multiple datasets are indicated by different colored edges as marked.
Bacterial pathogens employ the type III secretion system to secrete and translocate effector proteins into their hosts. The primary function of these effector proteins is believed to be the suppression of host defence responses or innate immunity. However, some effector proteins may be recognized by the host and consequently trigger a targeted immune response. The YopJ/HopZ/AvrRxv family of bacterial effector proteins is a widely distributed and evolutionarily diverse family, found in both animal and plant pathogens, as well as plant symbionts. How can an effector family effectively promote the virulence of pathogens on hosts from two separate kingdoms? Our understanding of the evolutionary relationships among the YopJ superfamily members provides an excellent opportunity to address this question and to investigate the functions and virulence strategies of a diverse type III effector family in animal and plant hosts. In this work, we briefly review the literature on YopJ, the archetypal member from Yersinia pestis, and discuss members of the superfamily in species of Pseudomonas, Xanthomonas, Ralstonia and Rhizobium. We review the molecular and cellular functions, if known, of the YopJ homologues in plants, and highlight the diversity of responses in different plant species, with a particular focus on the Pseudomonas syringae HopZ family. The YopJ superfamily provides an excellent foundation for the study of effector diversification in the context of wide-ranging, co-evolutionary interactions.
Reliance of biotrophic pathogens on living plant tissues to propagate implies strong interdependence between host metabolism and nutrient uptake by the pathogen. However, factors determining host suitability and establishment of infection are largely unknown. We describe a loss-of-inhibition allele of ASPARTATE KINASE2 and a loss-of-function allele of DIHYDRODIPICOLINATE SYNTHASE2 identified in a screen for Arabidopsis thaliana mutants with increased resistance to the obligate biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa). Through different molecular mechanisms, these mutations perturb amino acid homeostasis leading to overaccumulation of the Asp-derived amino acids Met, Thr, and Ile. Although detrimental for the plant, the mutations do not cause defense activation, and both mutants retain full susceptibility to the adapted obligate biotrophic fungus Golovinomyces orontii (Go). Chemical treatments mimicking the mutants’ metabolic state identified Thr as the amino acid suppressing Hpa but not Go colonization. We conclude that perturbations in amino acid homeostasis render the mutant plants unsuitable as an infection substrate for Hpa. This may be explained by deployment of the same amino acid biosynthetic pathways by oomycetes and plants. Our data show that the plant host metabolic state can, in specific ways, influence the ability of adapted biotrophic strains to cause disease.
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