Fungal Effectors and Plant Susceptibility

Fungal Effectors and Plant Susceptibility

Annual Review of Plant Biology

Vol. 66:513-545 (Volume publication date April 2015)
https://doi.org/10.1146/annurev-arplant-043014-114623

Abstract

Plants can be colonized by fungi that have adopted highly diverse lifestyles, ranging from symbiotic to necrotrophic. Colonization is governed in all systems by hundreds of secreted fungal effector molecules. These effectors suppress plant defense responses and modulate plant physiology to accommodate fungal invaders and provide them with nutrients. Fungal effectors either function in the interaction zone between the fungal hyphae and host or are transferred to plant cells. This review describes the effector repertoires of 84 plant-colonizing fungi. We focus on the mechanisms that allow these fungal effectors to promote virulence or compatibility, discuss common plant nodes that are targeted by effectors, and provide recent insights into effector evolution. In addition, we address the issue of effector uptake in plant cells and highlight open questions and future challenges.

Keywords

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Figure 1  Plant colonization by fungi with different lifestyles. (a) Necrotrophic fungi such as Botrytis cinerea and Sclerotinia sclerotiorum generally grow subcuticularly and kill epidermal cells by secreting toxic metabolites and proteins. Their hyphae eventually replace large parts of the plant epidermis. Both early and late developmental stages are shown. (b) Obligate biotrophic pathogens such as rust fungi (Uromyces viciae-fabae) and powdery mildew (Blumeria graminis f. sp. hordei) undergo a complex series of developmental steps and eventually form a haustorial mother cell from which the haustorium, a balloon-shaped feeding structure, develops. After initial intracellular growth, the biotrophic maize pathogen Ustilago maydis switches to predominantly intercellular growth at late stages, when massive fungal proliferation occurs and large plant tumors are induced. The biotrophic tomato pathogen Cladosporium fulvum colonizes the extracellular compartment of tomato leaves and later produces large numbers of conidiophores that block stomata and cause chlorosis or cell death (necrosis). (c) The obligate arbuscular mycorrhizal root symbiont Rhizophagus irregularis colonizes individual cortical cells with highly branched feeding structures called arbuscules. The ectomycorrhizal fungus Laccaria bicolor grows exclusively intercellularly; colonizes roots by forming a mantle or sheath of hyphae, which covers the root epidermis; and grows between cortical cells, generating the so-called Hartig net. Endophytes can colonize either plant roots (Piriformospora indica) or the aerial plant organs (Epichloë festuca) and can grow either intracellularly (P. indica) or intercellularly (E. festuca). (d) Hemibiotrophic fungi such as Colletotrichum spp. and Magnaporthe oryzae initially develop bulged biotrophic invasive hyphae that later change into thin necrotrophic hyphae. Both biotrophic and necrotrophic phases are shown. Hyphae are shown in blue (nonpathogenic fungi) or violet (pathogenic fungi), photosynthetic tissue in green, and root tissue in brown; solid green or brown lines indicate living tissue, and dashed green or brown lines indicate dead tissue. Note that all intracellular structures are encased by the plant plasma membrane, indicated by a solid gray line; a dashed gray line surrounding the hyphae indicates a switch to necrotrophy. The membranous biotrophic interfacial complex structure in M. oryzae–infected cells is shown in pink.

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Figure 2  Disease symptoms caused by phytopathogenic fungi with different lifestyles: (a) Botrytis cinerea infecting tomato (image courtesy of D. Blancard), (b) Sclerotinia sclerotiorum infecting rapeseed (image reproduced with permission from Paysan Breton; http://www.paysan-breton.fr), (c) Uromyces viciae-fabae infecting bean (image courtesy of K.D. Zinnert), (d) Blumeria graminis infecting barley (image courtesy of P. Spanu), (e) Ustilago maydis infecting maize, (f) Cladosporium fulvum infecting tomato (image courtesy of D. Blancard), (g) Colletotrichum higginsianum infecting mustard spinach (image courtesy of H. Horie; http://www.boujo.net), and (h) Magnaporthe oryzae infecting rice (image courtesy of N.J. Talbot).

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Figure 3  The secretome composition of fungi with different lifestyles. The secretomes of 84 plant-colonizing fungi and 5 saprophytic fungi were sorted into secreted plant cell wall–degrading enzymes (PCWDEs; dark blue), secreted proteins with a functional annotation except PCWDEs (blue), and secreted proteins without a functional annotation (light blue). To define the secretomes, we discarded pseudogenes from the set of predicted gene models and defined secreted proteins based on the presence of an N-terminal signal peptide as predicted by SignalP 4.0 (113) and on the absence of transmembrane domains as predicted by TMHMM 2.0c (TMHMM score <2) (71). We then used the Pfam database (http://pfam.xfam.org) to assign functional domains to the determined set of secreted proteins, as described recently (180). We used the CAZymes Analysis Toolkit (http://mothra.ornl.gov/cgi-bin/cat/cat.cgi) to filter out proteins with Pfam annotations corresponding to CAZymes that are PCWDEs. To define the set of PCWDEs, we extracted from the Carbohydrate-Active Enzymes (CAZy) database (80) all glycoside hydrolase families that contain cellulases (EC 3.2.1.4 and 3.2.1.91) and xylanases (EC 3.2.1.8 and 3.2.1.37) based on the EC numbers (67). Similarly, we identified all polysaccharide lyase and carbohydrate esterase families that contain pectinolytic enzymes, as previously defined (52). As a result, the following Pfam IDs were considered (the corresponding CAZy families are in parentheses): Glyco_hydro (GH)_1, 3, 4, 6, 7, 8, 9, 10, 11, 12, 16, 26, 28, 30, 39, 43, 44, 45, 48, 51, 52, 54, 61 (AA9), 62, 88, 105, and 116; Cellulase (GH5); Pec_lyase_C (PL1); Pectate_lyase_2 (PL2); Pectate_lyase (PL3); Pec_lyase (PL10); and Pectate_lyase22 (PL22). Note that the following CAZy families were not present in the analyzed secretomes: GH4, 8, 48, 52, and 116, and PL2, 10, and 22. Proteins that contain at least one of the Pfam domains that define a PCWDE were grouped as secreted PCWDEs. Proteins that exclusively possess Pfam domains of unknown function or contain no Pfam annotation were grouped as secreted proteins without a functional annotation. All other proteins were grouped as secreted proteins with functional annotation except PCWDEs. Based on information in the literature (Supplemental Table 1), we grouped all sequenced fungi according to their lifestyle during plant colonization. The five saprotrophic fungi serve as a contrasting set. If the genome sequence for more than one isolate of a species was publicly available, then we separately analyzed each isolate and displayed the average value of all isolates; species for which this applies are indicated by an asterisk. Supplemental Table 1 lists all isolates used for this analysis. Abbreviations: A, ascomycete; B, basidiomycete; G, glomeromycete.

Figure Locations

...secreted proteins with functional annotation except PCWDEs, and secreted proteins without functional annotation (Figure 3)....

...we grouped the fungi according to their feeding strategies (Figure 3)....

...Secretomes were analyzed and categorized for each fungus separately as described in Figure 3, ...

...which has a set of carbohydrate-degrading enzymes that is more similar to those of necrotrophs and hemibiotrophs (20) (Figure 3). C. fulvum is a close relative of the hemibiotroph Dothistroma septosporum and may have only recently adapted to a new host in which its lifestyle changed from hemibiotrophic to biotrophic, ...

...which has a reduced set of secreted PCWDEs (45) (Figure 3); this fungus instead expresses a large number of secreted proteases, ...

...We observe that fungi with the highest total number of secreted proteins are overrepresented in the hemibiotroph group (Figure 3), ...

...the obligate biotrophic rust fungi feature an exceptionally large set of secreted proteins (Figures 3...

...and the proportion of secreted proteins without functional annotation is particularly high within this group (Figures 3...

...The symbiotic fungi show large variations in both numbers of secreted proteins and composition, which may not be immediately apparent from Figure 3...

... owing to ascertainment bias (there are ten related Epichloë species included in this group) (Figures 3...

...with a relatively large set of secreted proteins among the symbionts (Figures 3...

...such as rust fungi as well as the most relevant plant-growth-promoting AM fungi, are obligate biotrophs (Figure 3)....

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Figure 4  Lifestyle-specific trends within the secretome composition of plant-colonizing fungi. Secretomes were analyzed and categorized for each fungus separately as described in Figure 3, and for each category the values from fungi with the same lifestyle were combined. Results are shown in the form of box plots, where the top and bottom of the boxes indicate the 25% and 75% quartiles, respectively, and the thick middle line indicates the 50% quartile (median). The whiskers correspond to the lowest and highest data points within the 1.5 interquartile range of the lower and upper quartiles, respectively. Outliers are indicated by open circles. (a) Number of secreted proteins relative to the total proteome. Outliers are as follows: saprotrophs, Saccharomyces cerevisiae (4.2%); hemibiotrophs, Magnaporthe oryzae (13.3%), Colletotrichum orbiculare (12.7%), Pyrenochaeta lycopersici (4.9%), and Moniliophthora perniciosa (4.2%); obligate biotrophs, Melampsora larici-populina (10.6%) and Melampsora lini (3.5%); symbionts, Periglandula ipomoeae (9.4%) and Rhizophagus irregularis (2.3%). (b) Number of secreted plant cell wall–degrading enzymes (PCWDEs) relative to the total number of secreted proteins. The outliers are as follows: saprotrophs, Saccharomyces cerevisiae (2.8%); necrotrophs, Rhizoctonia solani (13.5%), Alternaria brassicicola (11.0%); facultative biotrophs, Cladosporium fulvum (6.5%), Aciculosporium take (3.9%), Balansia obtecta (3.7%); obligate biotrophs, Melampsora lini (3.7%); symbionts, Piriformospora indica (11.3%) and Rhizophagus irregularis (0%). (c) Number of secreted proteins without functional annotation relative to the total number of secreted proteins. The outliers are as follows: hemibiotrophs, Pyrenochaeta lycopersici (65.8%); obligate biotrophs, Melampsora lini (66.0%); symbionts, Rhizophagus irregularis (69.5%), Laccaria bicolor (63.1%), and Tuber melanosporum (42.3%).

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Figure 5  Coevolutionary principles driving effector and plant target evolution. Population-wide allele frequencies of a pathogen-derived effector molecule (red line) and a host-derived interactor (green line) can follow (a) the arms race model or (b) the trench warfare model. Allele fixation (selective sweeps) and recurrent development of new alleles (indicated by light-colored lines) in the arms race model contrast with the fluctuation of allele frequencies in the trench warfare model.

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Figure 6  Effector genes residing in distinct genome compartments. (a) Effector genes are located in repeat-rich, gene-sparse regions in Leptosphaeria, Magnaporthe, and Phytophthora spp. (b) Effector genes are located on mobile, conditionally dispensable chromosomes consisting mainly of repeat-rich DNA in Fusarium spp. (c) Effector genes are located at chromosomal breakpoints of highly rearranged chromosomes in Verticillium spp. (two nonhomologous chromosomes are depicted in white and yellow before and after rearrangement). (d) Effector genes are located in gene clusters in smut fungi.

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Figure 7  Mode and site of action of fungal effectors. An intracellular fungal structure that secretes effectors is shown in yellow. This structure could be the tip of a biotrophic hyphae, part of a haustorium, or part of an arbuscule. Plant plasma membranes are shown in gray, the fungal plasma membrane is shown in black, the fungal cytoplasm is shown in brown, the plant cytoplasm is shown in light green, and the plant cell wall is shown in dark green. The plant membrane surrounding the arbuscules is also called the periarbuscular membrane or extrahaustorial membrane (in fungi that form haustoria). The apoplastic space between the fungal hypha and the plant plasma membrane has been widened and is shown as a light blue area; in reality, these membranes tightly encompass fungal structures. Fungal effectors and targeted plant proteins are shown in various colors and are surrounded by black lines and dark green lines, respectively. Effectors with a known mode of function are depicted here with their plant proteins or plant-derived substances as interaction partners. Note that the PtrToxA-ToxABP1 interaction may not directly induce plant cell death (108).

Figure Locations

...These are mostly protein effectors but also include protein toxins and other metabolites that interfere with or induce certain plant processes (Figure 7)....

...Pep1 is a secreted effector of U. maydis and related smut fungi that accumulates in the apoplast (Figure 7)....

...a secreted maize peroxidase that is a conserved component of the plant reactive oxygen species (ROS)–generating system (50) (Figure 7)....

...Pit2 directly inhibits a set of apoplastic maize cysteine proteases whose activity promotes salicylic acid–associated plant defenses (96) (Figure 7)....

...which can serve as a precursor for the synthesis of salicylic acid in plastids, thereby promoting virulence (Figure 7)....

...where it interacts with and stabilizes the maize cytoplasmic protein kinase ZmTTK1 (Figure 7)....

...Avr4 binds to chitin in the fungal cell wall, thereby protecting against hydrolysis by plant chitinases (163) (Figure 7)....

...Ecp6 of C. fulvum sequesters chitin oligosaccharides that are released from the cell walls of invading hyphae to prevent the elicitation of host immunity (18) (Figure 7)....

...possibly by interfering with dimerization of the plant chitin receptor (135) (Figure 7)....

...Avr2 selectively inhibits the apoplastic proteases PIP1 and Rcr3 (130, 140) (Figure 7)....

...C. fulvum secretes Tom1—a tomatinase that degrades α-tomatine into the less toxic compounds β-tomatine and tomatidine—into the apoplast (Figure 7). α-Tomatine is an antifungal glycoalkaloid that provides a basal defense against C. fulvum in tomato. tom1 mutants are more sensitive to α-tomatine and display reduced virulence on tomato (106)....

...which are secreted upon biotic stress, suggesting a role in suppressing defense (176) (Figure 7)....

...ToxA localizes to the chloroplast and interacts with ToxABP1 (Figure 7), ...

...hijack the host RNA interference machinery by binding to A. thaliana ARGONAUTE 1 (AGO1) (Figure 7) and selectively silence host immunity genes that show complementarity to these RNAs....

...Slp1 specifically binds chitin (Figure 7) and is able to suppress chitin-triggered PTI....

...the rice PRR chitin elicitor–binding protein that together with OsCERK plays a key role in the perception and transduction of the chitin oligosaccharide signal (58, 92) (Figure 7). M. oryzae Δslp1 strains were affected in virulence, ...

...AvrPiz-t interacts with and inhibits the rice RING E3 ubiquitin ligase APIP6 (Figure 7), ...

...a host ethylene-responsive transcription factor that regulates the expression of several defense-related genes in Medicago truncatula (69) (Figure 7)....

...A recent report found that MiSSP7 localizes to the plant nucleus and interacts with PtJAZ6 (Figure 7), ...

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